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Complete Genome of the Starch-Degrading Myxobacteria
Sandaracinus amylolyticus DSM 53668T
Gaurav Sharma Indu Khatri and Srikrishna Subramanian
Protein Science and Engineering CSIR-Institute of Microbial Technology Sector-39A Chandigarh India
Corresponding author E-mail krishnaimtechresin
Accepted June 14 2016
Data deposition The complete genome sequence of Sandaracinus amylolyticus DSM 53668T and its annotations are deposited at DDBJENA
GenBank under accession number CP011125
Abstract
Myxobacteria are members of d-proteobacteria and are typified by large genomes well-coordinated social behavior gliding motility
and starvation-induced fruiting body formation Here we report the 1033 Mb whole genome ofa starch-degrading myxobacterium
Sandaracinus amylolyticus DSM 53668T that encodes 8962 proteins 56 tRNA and two rRNA operons Phylogenetic analysis in
silico DNA-DNA hybridization and average nucleotide identity reveal its divergence from other myxobacterial species and support its
taxonomic characterization into a separate family Sandaracinaceae within the suborder Sorangiineae Sequence similarity searches
using the Carbohydrate-active enzymes (CAZyme) database help identify the enzyme repertoire of S amylolyticus involved in starch
agar chitin andcellulosedegradation We identified16a-amylasesand twog-amylases in the S amylolyticusgenomethat likelyplay
a role in starch degradation While many of the amylases are seen conserved in other d-proteobacteria we notice several novel
amylases acquired via horizontal transfer from members belonging to phylum Deinococcus-Thermus Acidobacteria and
Cyanobacteria No agar degrading enzyme(s) were identified in the S amylolyticus genome Interestingly several putative b-gluco-
sidases and endoglucanases proteins involved in cellulose degradation were identified However the absence of cellobiohydrolases
exoglucanases corroborates with the lack of cellulose degradation by this bacteria
Key words CAZyme amylase agarase cellulase methylome phylogeny
Introduction
Myxobacteria [order Myxococcales] are Gram-negative and
aerobic (except Anaeromyxobacter) d-proteobacteria These
bacteria possess large genomes and a complex life cycle
wherein they coordinate within swarms and hunt other mi-
crobes (Shimkets 1990Whitworth 2008) Myxobacterial spe-
cies have been of industrial interest due to their significant role
in secondary metabolites production (Weissman and Muller
2010 Steinmetz et al 2012) Order Myxococcales at present
comprises of more than 27 taxonomically classified genera
and 60 species This order has been classified in three subor-
ders Cystobacterineae (four families) Nannocystineae (two
families) and Sorangiineae (four families) The members of
suborder Sorangiineae are cellulolytic bacteriolytic and pro-
teolytic as compared with other family members and form a
separate clade in myxobacterial 16S rRNA-based phylogeny
(Reichenbach 2005 Garcia and Muller 2014) Till date the
largest complete bacterial genomes Sorangium cellulosum
strain So0157-2 (1478 Mb) (Han et al 2013) and S cellulo-
sum strain Soce56 (1303 Mb) (Schneiker et al 2007) have
been reported from the suborder Sorangiineae These mem-
bers are known to degrade diverse complex polysaccharides
such as cellulose starch casein agar chitin gelatin dextrin
xylan agarose etc (Garcia and Muller 2014) They have also
been reported as microbial predators to organisms such as
Escherichia coli Bacillus subtilis Mycobacterium chitae
Cyanobacteria yeast etc (Garcia and Muller 2014) Among
the four characterized families of the suborder Sorangiineae
genomic data are available only for Sorangium and
Chondromyces that belong to Polyangiaceae family which
are the largest source of myxobacterial secondary metabolites
(Schneiker et al 2007Weissman and Muller 2010Han et al
2013)
Within suborder Sorangiineae Sandaracinus amylolyticus
strain DSM 53668T is the type strain of S amylolyticus gen
nov sp nov proposed as a novel species in the family
GBE
The Author 2016 Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (httpcreativecommonsorglicensesby40) which permits unrestricted reuse
distribution and reproduction in any medium provided the original work is properly cited
2520 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
Sandaracinaceae within order Myxococcales (Mohr et al
2012) Morphologically and biochemically the strain is a
Gram-negative aerobic rod-shaped during vegetative
states catalase-negative oxidase-positive and pigment form-
ing myxobacteria (Mohr et al 2012) This strain was isolated in
1996 from a gray-brown soil sample (having plant residues)
collection by Helmholtz Zentrums fur Infektionsforschung
[HZI] during 1995 from a botanical garden in Lucknow
Uttar Pradesh India (Reichenbach and Dworkin 1992 Mohr
et al 2012) Sandaracinus amylolyticus possesses typical myx-
obacterial characteristics including gliding motility secondary
metabolite production (Steinmetz et al 2012) and spore gen-
eration during harsh environmental conditions Here we
report the complete genome of S amylolyticus DSM 53668T
with special emphasis on the starch degrading properties
along with the protein repertoire involved in starch agarose
chitin and cellulose degradation
Materials and Methods
Culturing and Genomic DNA Isolation
Actively growing plate cultures of S amylolyticus was pro-
cured from Deutsche Sammlung von Mikroorganismen und
Zellkulturen (DSMZ) as strain number DSM 53668T [NOSO-4T]
Sandaracinus amylolyticus DSM 53668T was grown aerobi-
cally on CY agar plate culture (DSM Medium 67 as prescribed
by DSMZ culture collection) at 28ndash30 C for a minimum of 5
days The culture was yellow-orange in color attributed to the
presence of carotenoids (Mohr et al 2012) DNA for sequenc-
ing was obtained using both the Zymogen Research Bacterial
fungal DNA isolation kit and PhenolndashChloroformndashIsoamyl
Alcohol-based manual method The quantity and quality of
the extraction were checked by gel electrophoresis along with
Nanodrop method and followed by Qubit quantification
Genome Sequencing Assembly and Annotation
Sandaracinus amylolyticus was sequenced on four single mol-
ecule real-time (SMRT) cells using P6 polymerase and C4
chemistry (P6C4) on a Pacific Biosciences RSII instrument at
McGill University and Genome Quebec Innovation Center
Montreal (Quebec) Canada DNA sample was sheared and
concentrated using AMPure magnetic beads and treated by
ExoVII to remove single stranded ends SMRTbell long libraries
were constructed with 10 mg whole genomic DNA using
ldquoProcedure and Checklistndash20 kb Template Preparation Using
BluePippinTM Size Selection System protocolrdquo (httpwww
pacbcomwp-contentuploads201509Shared-Protocol-20-
kb-Template-Preparation-Using-BluePippin-Size -Selection-
System-15-kb-Size-Cutoffpdf last accessed July 5 2016)
Blunt ligation reactions were prepared and SMRTbell tem-
plates were purified using AMPure magnetic beads
BluePippinTM size selection was performed to retain long
reads (gt20000) for sequencing The size-selected SMRTbell
templates were annealed and then loaded onto four SMRT
cells and sequenced using P6C4 chemistry with 180-min
movie times Sequencing yielded a total of 241674 reads
with a mean read length of 467 kb totaling
1129177817 bp (~85 coverage) De novo assembly was
carried out using the hierarchical genome assembly process
(HGAP) protocol in SMRT Analysis v20 including consensus
polishing with Quiver (Chin et al 2013) Since the
BluePippinTM Size Selection (gt20000) protocol filters out
small DNA fragments such as plasmids Illumina paired-end
(PE) data were also obtained using the Illumina HiSeq1000
sequencing platform at the C-CAMP Bangalore India
Sequencing resulted in 13103763 PE reads (insert size 350
bp and read length 101 bp) out of which 11663870 PE
reads were selected after quality filtering [PHRED quality
score 20 minimum read length 70] using NGSQC Toolkit
v223 (Patel and Jain 2012) To trace the presence of any
plasmid the filtered Illumina reads were mapped using
CLCbio wb80 (wwwclcbiocom last accessed July 8 2016)
to the bacterial plasmid database (httpwwwebiacukge-
nomesplasmidhtml last accessed July 8 2016)
Gene prediction and functional annotation were per-
formed by Rapid Annotation using Subsystem Technology
(RAST) (Aziz et al 2008) RNAmmer 12 (Lagesen et al
2007) and tRNAscan-SE-123 (Lowe and Eddy 1997) were
used to predict rRNA and tRNA genes respectively
Phylogenetic Analysis Genome-Genome Distance andAverage Nucleotide Identity
16S rRNA genes from various strains of suborder Sorangiineae
along with neighboring families were extracted from the
National Centre for Biotechnology Information (NCBI) and
were aligned using ClustalW module of BIOEDIT sequence
alignment tool (version 7130 Hall 1999) The resulting align-
ment was used as an input in Molecular Evolutionary Genetics
Analysis (MEGA) v606 (Tamura et al 2013) to generate a max-
imum likelihood (ML) tree [model Tamura 3-param bootstrap
100] Initial tree(s) for the heuristic search were obtained by
applying the Neighbor-Joining method to a matrix of pairwise
distances estimated using the Maximum Composite Likelihood
approach In silico DNA-DNA hybridization (DDH) values among
the members of suborder Sorangiineae and other species mem-
bers of order Myxococcales were calculated using Genome-To-
Genome Distance Calculator (GGDC) server (Auch et al 2010)
Average Nucleotide Identity (ANI) matrix was also generated
among the genomes studied (Richter and Rossello-Mora 2009)
Genome and Proteome Analysis
Sandaracinus amylolyticus proteome was subjected to
hmmscan (Eddy 2011) against Hidden Markov Model
(HMM) profiles of Carbohydrate-Active enZYmes (CAZY)
database (dbCAN HMMs 30 Yin et al 2012Lombard et al
2014) The functional domains and other known sequence
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2521
motifs involved in starch cellulose chitin and agar degrada-
tion were retrieved on the basis of EC number for each
enzyme category Also the proteins of S amylolyticus were
mapped against the CAZY protein database using Basic-Local
Alignment Search Tool (BLASTp) (E-value cutoff of 1e5 min-
imum query coverage of 50 and minimum percent identity
of 35) (Altschul et al 1990) NCBI BLAST + (v 2226+) and
nonredundant protein (NR) database downloaded on
September 6 2014 was used throughout the study
Clustered regularly interspaced short palindromic repeat
(CRISPR) elements and insertion elements were identified
using CRISPRfinder (Grissa et al 2007) and ISfinder (Siguier
et al 2006) respectively
Sandaracinus amylolyticus Amylases
The a-amylases and g-amylases from S amylolyticus were sub-
jected to the BLASTp against amylase sequences from the
CAZY database (clustered at 70 sequence identity) and
top hits were retrieved The amylases from S amylolyticus
were cut into the domains and were aligned using MUltiple
Sequence Comparison by Log-Expectation (MUSCLE) (Edgar
2004) incorporated in MEGA v606 Further the evolutionary
tree was generated by ML method [model Jones Taylor and
Thorton bootstrap 100] using MEGA v606 Initial tree(s) for
the heuristic search were obtained by applying the Maximum
Parsimony method to a matrix of pairwise distances estimated
using the Maximum Composite Likelihood approach Further
the S amylolyticus GH13 amylases along with the closely re-
lated members of each phylogenetic clade were aligned using
PROfile Multiple Alignment with predicted Local Structures
and 3D constraints (PROMALS3D) with Taka-Amylase A of
Aspergillus oryzae [2TAA] (Matsuura et al 1984) as a struc-
tural template (Pei et al 2008)
Results and Discussion
Sandaracinus amylolyticus Genome Properties
Sandaracinus amylolyticus genome was assembled de novo by
HGAP v20 (Chin et al 2013) of the SMRT portal as a single
contig of 1033 Mb with no plasmid and representing the
complete genome with approximately 85 coverage and
72 G+C content (fig 1) No complete plasmid could be
retrieved from the mapping of Illumina PE reads against the
bacterial plasmid database Plasmids are rare in the order
Myxococcales and till date only one plasmid pMF1 from
Myxococcus fulvus 124B02 has been reported (Zhao et al
2008) RAST-based annotation predicted 8962 coding
genes out of which 4939 proteins (5516) were function-
ally annotated while the remaining (4484) were hypothet-
ical proteins (supplementary table S1 Supplementary Material
online) Sandaracinus amylolyticus genome has two rRNA op-
erons and 56 aminoacyl-tRNA synthetase genes
This genome encodes two sequentially identical 16S rRNA
which shows 100 identity with previously reported 16S rRNA
sequence of S amylolyticus strain NOSO-4T [NCBI Reference
Sequence NR_1180011] followed by 8798 identity with
Chondromyces apiculatus strain Cm a14 [NR_0253441]
Among the myxobacteria with sequenced genomes it shows
the maximum identity (8789) to the 16S rRNA gene of C
apiculatus DSM 436 (strain Cm a2) [GenBank M942742] fol-
lowed by 875 identity to S cellulosum 16S rRNA (Soce0157-
2 and Soce56) The 16S rRNA-based phylogeny of
Sandaracinus and related myxobacteria suggest its classification
as a separate family within the suborder Sorangiineae (supple-
mentary fig S1 Supplementary Material online) Sandaracinus
amylolyticus 16S rRNA forms a separate branch distinct from
other families in the suborder Sorangiineae phylogenetic tree
representing family Sandaracinaceae We also calculated in
silico DDH values for all available myxobacterial genomes
using the GGDC server (Auch et al 2010) Sandaracinus
amylolyticus is not closely related to other Myxococcales as
the maximum DDH value was identified to be 181 with S
cellulosum followed by 174 with C apiculatus DSM 436 sug-
gesting a distant relation to Polyangiaceae members which
further confirms its placement in a separate family
Sandaracinaceae ANI calculations likewise support the above
conclusions (supplementary table S2 Supplementary Material
online)
The members of suborder Sorangiineae such as the S cel-
lulosum species have been well characterized for degrading
cellulose and other complex polysaccharides (Schneiker et al
2007 Han et al 2013) Similarly S amylolyticus has been
reported as the only myxobacteria capable of degrading
starch (Mohr et al 2012) Thus in order to gain insights into
the genes involved in the degradation of complex polysaccha-
rides especially starch the complete genome of S amylolyticus
was determined and investigated for the presence of carbo-
hydrate active enzymes
Sandaracinus amylolyticus CAZome
Two-hundred and forty-three proteins (~27 of total prote-
ome) of S amylolyticus were annotated with diverse combi-
nations of CAZY domains that consist of 72 GT domains 72
CE domains 58 GH domains 19 AA domains 4 PL domains
and 26 CBM domains (fig 2A) Fourteen out of these 243
proteins contained more than one CAZY domain whereas
the rest had only a single CAZY domain In multiple CAZY-
domain containing proteins various carbohydrate binding
modules such as CBM48 CBM50 and CBM21 are pre-
sent along with other CAZY category proteins for assisting in
their functions The CAZyme distribution observed in
S amylolyticus indicates its ability to degrade various
types of biomass Here we examine the S amylolyticus pro-
teome involved in starch agar chitin and cellulose
degradation
Sharma et al GBE
2522 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
Evolutionary History of S amylolyticus Amylases
Amylases belonging to glycoside hydrolase (GH) enzymatic
category degrade starch into sugar moieties by the
hydrolysis of a-14- and a-16-glycosidic bond (Van der
Maarel et al 2002) Based on the site of action they are di-
vided into three categories a-amylase (EC3211 GH13
GH57 GH119 GH126) b-amylase (EC3212 GH14) and
-amylase (EC3213 GH15 GH97) Sandaracinus
amylolyticus genome contains both a- and -amylases
whereas no b-amylases could be found Among the 4 a-am-
ylases GH families 15 GH13 proteins and 1 GH57 protein
were found We could identify two -amylases belonging to
Sandaracinus amylolyticusTDSM 53668
CP01112510328378 bp
FIG 1mdashCircular representation of the Sandaracinus amylolyticus DSM 53668T complete genome Circles (from inside to outside) 1 and 2 (GC content
black line and GC skew magenta and green lines) circle 3 (CAZome Proteins annotated as carbohydrate active enzymes Red) circle 4 5 and 6 (Amylases
Cellulases and Chitinases Black) circle 7 (encoded RNA Magenta) circle 8 (CDS present on negative strand Blue) circle 9 (CDS present on positive strand
Green) circle 10 (S amylolyticus DSM 53668T complete genome Red) BRIG 095 was used to build the circular representation (Alikhan et al 2011)
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2523
GH15 category (fig 2B) The starch degrading proteins have
been reported to have CBM20 CBM21 CBM25 CBM26
CBM34 CBM41 CBM45 CBM48 CBM53 and CBM58
modules that help in glycogenstarch binding truncation of
which reduces its enzymatic activity (Noguchi et al 2011
Janecek et al 2014) GH15 and GH57 domains are present
as single domain proteins in S amylolyticus Interestingly in 5
out of 15 GH13 S amylolyticus proteins (AKF03796
AKF03860 AKF06952 AKF07413 and AKF11765) the a-
amylase GH13 domain was present along with a CBM48
module at the N-termini The CBM48 module assists in the
GH13 function by serving as starch-binding domains
The GH13 family include the a-amylase domains with an (b
a)8 barrel catalytic domain These proteins have 4ndash7 conserved
sequence regions (CSRs) and a specific catalytic triad consisting
of a catalytic nucleophile (aspartic acid) on strand b4 proton
donor (glutamic acid) on strand b5 and a transition state sta-
bilizer (aspartic acid) on strand b7 (Janecek et al 2014) GH13
members show extremely low sequence identity with their ho-
mologs but the catalytic triad is highly conserved GH57 family
a-amylases have a (ba)7 barrel structure and have five CSRs as
compared with the GH13 family However only two of the
catalytic residues viz the nucleophile on strand b4 and the
proton donor on strand b7 are conserved (Blesak and Janecek
2012) Irrespective of these differences GH13 and GH57 both
employ the same retaining mechanism of action (Janecek and
Kuchtova 2012) Sandaracinus amylolyticus GH13 proteins
were retrieved and the structure of Taka amylase of A
oryzae (PDBid 2TAA) was used to perform a structure-based
alignment using PROMALS3D (Matsuura et al 1984 Pei et al
2008) All the seven CSR regions and the three catalytic
residues were conserved in all S amylolyticus GH13 amylases
(supplementary figs S2 and S3 Supplementary Material
online) Similarly S amylolyticus GH57 (AKF06951) was
aligned with its homologs using PROMALS3D based on the
4-a-glucanotransferase structure of Thermococcus litoralis
(PDBid 1K1W) In the GH57 alignment all five CSRs and
both catalytic residues were conserved (supplementary figS4
Supplementary Material online)
In order to understand the provenance of these a- and g-
amylases they were subjected to BLASTp against the NR data-
base To include amylases across the entire bacterial phylum
the CAZY-NR database for GH13 and GH57 domains were
retrieved and subjected to BLASTClust at 70 identity and
70 length coverage that reduced the domain sequence
space from 19590 to 4038 domains Further amylase do-
mains from S amylolyticus were subjected to BLASTp against
these 4038 domains and their top hits were retrieved and
included in the phylogenetic analysis All these GH13 and
GH57 domains along with S amylolyticus amylase domains
were aligned using MUSCLE as incorporated in MEGAv606
and ML-based phylogeny with 100 bootstrap values was
drawn (fig 3) The ML tree obtained was divided into three
major clades where the clade at root composed of all the GH57
domains and the other two major clades further composed of
all the GH13 sequences The representative bacterial amylases
from various clades show close relations with amylases of di-
verse bacterial groups suggesting the multiple gain and loss of
these domains As all the amylase domains of S amylolyticus
are scattered across the tree the phylogenetic position of these
domains could provide us insights about their provenance and
reveal possible cases of horizontal transfers
19
26
72
58
72
4
0
10
20
30
40
50
60
70
80
AA CBM CE GH GT PL
A B
Num
ber o
f pro
tein
s
GH13
GH13
CBM48 GH13
GH13CBM48
GH13
GH57
GH13CBM48
GH13GH13CBM48
GH13
GH13
GH13
GH15
GH13CBM48
GH13
AKF03605AKF03642AKF03796
AKF03860AKF03861AKF03862AKF04017AKF06951AKF06952
AKF07406AKF07413AKF07527AKF09634AKF09643AKF10340AKF10487AKF11765AKF11772
GH13
GH13
GH15
FIG 2mdashSandaracinus amylolyticus DSM 53668T CAZome distribution (A) Sandaracinus amylolyticus DSM 53668T CAZY families presented in a bar
graph x axis represents the CAZY categories and y axis represents the number of proteins (B) a- and g-amylases along with their CAZome domain
architecture The domains have been colored as GH13 sky blue GH15 dark red GH57 green and CBM48 orange
Sharma et al GBE
2524 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
Taking into account the GH57 clade the sole GH57
domain of S amylolyticus (GenBank ID AKF06951) shares
the clade with anaerobic Deltaproteobacteria Among the
GH13 domains of S amylolyticus AKF03862 AKF07527
AKF09643 AKF11765 and AKF11772 shares clade with
other myxobacteria Among these AKF11772 and
AKF07527 share sister branches and form sister clades with
other myxobacterial species such as S cellulosum Soce56 and
Plesiocystis pacifica SIR-1 and are further related to the clade
containing Gammaproteobacteria Apart from these two am-
ylases AKF03860 and AKF06952 are similar to each other and
group with Candidatus accumulibacter phosphtis amylase
(YP_0031672861) from the phylum Acidobacteria
Interestingly AKF03860 and AKF06952 group with members
GH13 ZP 021632211 Kordia algicida OT-1
GH13 ZP 067516041 Parascardovia denticolens F0305
GH13 YP 0035709041 Salinibacter ruber M8
GH13 YP 0032500041 Fibrobacter succinogenes subsp succinogenes S85
GH13 AKF03862 Sandaracinus amylolyticus DSM 53668
GH13 YP 0039537801 Stigmatella aurantiaca
GH13 CAJ738231 Candidatus Kuenenia stuttgartiensis
GH13 YP 0228461 Picrophilus torridus DSM 9790GH13 ZP 029306261 Verrucomicrobium spinosum DSM 4136
GH13 CAX841261 uncultured bacterium
GH13 YP 0031678771 Candidatus Accumulibacter phosphatis clade IIA str UW-1GH13 ZP 031321321 Chthoniobacter flavus Ellin428
GH13 ZP 018759791 Lentisphaera araneosa HTCC2155GH13 YP 0033894351 Spirosoma linguale DSM 74GH13 NP 7734061 Bradyrhizobium diazoefficiens USDA 110
GH13 YP 0037757321 Herbaspirillum seropedicae SmR1GH13 YP 4266911 Rhodospirillum rubrum ATCC 11170GH13 ZP 035079501 Rhizobium etli Brasil 5
GH13 ZP 057930691 Butyrivibrio crossotus DSM 2876
GH13 ZP 066231321 Turicibacter sanguinis PC909
GH13 ZP 044499741 Catonella morbi ATCC 51271
GH13 EGE534241 Streptococcus parauberis NCFD 2020
GH13 196049865 Lactobacillus Plantarum
GH13 YP 0042196261 Granulicella tundricola MP5ACTX9
GH13 YP 0011935611 Flavobacterium johnsoniae UW101
GH13 YP 0032910851 Rhodothermus marinus DSM 4252GH13 YP 0040967591 Bacillus cellulosilyticus DSM 2522GH13 ZP 037030351 Flavobacteria bacterium MS024-2A
GH13 XP 0025352531 Ricinus communis
GH13 YP 7579561 Maricaulis maris MCS10GH13 ZP 010417351 Erythrobacter sp NAP1GH13 YP 0018177571 Opitutus terrae PB90-1GH13 EFR587141 Alistipes sp HGB5
GH13 CBK627851 Alistipes shahii WAL 8301GH13 EFR582631 Alistipes sp HGB5
GH13 YP 0034042011 Haloterrigena turkmenica DSM 5511
GH13 YP 0035357541 Haloferax volcanii DS2
GH13 YP 0035357541 Haloferax volcanii DS2
GH13 YP 0025661621 Halorubrum lacusprofundi ATCC 49239
GH13 YP 1368761 Haloarcula marismortui ATCC 43049
GH13 ZP 080436741 Haladaptatus paucihalophilus DX253
GH13 YP 0025652161 Halorubrum lacusprofundi ATCC 49239
GH13 YP 0035346301 Haloferax volcanii DS2
GH13 YP 0033225661 Thermobaculum terrenum ATCC BAA-798
GH13 YP 0021353421 Anaeromyxobacter sp K
GH13 AKF09643 Sandaracinus amylolyticus DSM 53668
GH13 YP 0016137031 Sorangium cellulosum So ce56
GH1
3 99
0316
21 H
alot
herm
othr
ix or
enii
GH1
3 AA
D051
991
Pae
niba
cillu
s po
lymyx
a
GH1
3 YP
003
6999
601
Bac
illus
sele
nitir
educ
ens
MLS
10
GH1
3 ZP
022
0799
81
Copr
ococ
cus
euta
ctus
ATC
C 27
759
GH
13 B
AA07
401
1 Xa
ntho
mon
as c
ampe
stris
GH
13 Y
P 00
4052
835
1 M
ariv
irga
tract
uosa
DSM
412
6
GH1
3 ZP
037
0954
51
Cory
neba
cter
ium
mat
ruch
otii A
TCC
3380
6
GH1
3 NP
267
649
1 La
ctoc
occu
s la
ctis
subs
p la
ctis
Il140
3
GH1
3 YP
001
3023
201
Par
abac
tero
ides
dist
ason
is AT
CC 8
503
GH1
3 YP
001
2276
361
Syn
echo
cocc
us s
p R
CC30
7
GH1
3 YP
002
5652
591
Hal
orub
rum
lacu
spro
fund
i ATC
C 49
239
GH13
ZP
0709
0550
1 C
oryn
ebac
teriu
m g
enita
lium
ATC
C 33
030
GH13
YP 0
0283
4100
1 C
oryn
ebac
teriu
m a
urim
ucos
um A
TCC
7009
75
GH13 A
KF03
861
Sand
arac
inus a
mylo
lyticu
s DSM
536
68
GH13
ZP 0
0050
865
1 M
agne
tosp
irillum
mag
neto
tacti
cum
MS-
1
GH13 Y
P 00
1568
811
1 Pe
troto
ga m
obilis
SJ9
5
GH13 Y
P 02
2847
1 P
icrop
hilus
torri
dus D
SM 9
790
GH13 E
FT25
5011
Pro
pionib
acter
ium ac
nes H
L110
PA3
GH13 E
FA80
1951
Poly
spho
ndyli
um pa
llidum
PN50
0
GH13 A
BD4655
81 V
ermam
oeba
verm
iform
is
GH13 Z
P 0350
5050
1 Rhiz
obium
etli B
rasil
5
GH13 YP 00
1348
4651
Pseud
omon
as ae
rugino
sa PA7
GH13 YP 00
2238
3511
Klebsie
lla pn
eumon
iae 34
2
GH13 YP 00
1437
9781
Cron
obac
ter sa
kaza
kii ATCC B
AA-894
GH13 Y
P 0011
7103
71 P
seud
omon
as st
utzeri
A1501
GH13 Y
P 0019
7240
81 S
tenotr
opho
monas
malt
ophil
ia K27
9a
GH13 Y
P 0043
1901
11 S
phing
obac
terium
sp 2
1
GH13 YP 0013127791 Sinorhizobium m
edicae W
SM419
GH13 ZP 035028921 Rhizobium etli K
im 5
GH13 YP 67
6219
1 Che
lativo
rans s
p BNC1
GH13 YP 00
2376
8861
Cya
nothe
ce sp
PCC 74
24
GH13 YP 0036383061 Cellulomonas flavig
ena DSM 20109
GH13 YP 0040977291 Intrasporangium ca
lvum DSM 43043
GH13 YP 0011691691 Rhodobacter s
phaeroides ATCC 17025
GH13 ZP 011580901 Oceanicola granulosus H
TCC2516
GH13 YP 0038382651 Micromonospora aurantiaca ATCC 27029
GH13 YP 0040977251 Intrasporangium calvu
m DSM 43043
GH13 YP 0036954761 Starkeya novella DSM 506
GH13 ZP 035166441 Rhizobium etli IE4771
GH13 ZP 031273131 Chthoniobacter flavus Ellin
428
GH13 AKF03642 Sandaracinus amylolyticus DSM 53668
GH13 YP 0031252671 Chitinophaga pinensis DSM 2588
GH13 YP 0030850521 Dyadobacter fermentans DSM 18053
GH13 YP 0033879241 Spirosoma linguale DSM 74
GH13 YP 0034301801 Streptococcus gallolyticus UCN34
GH13 ZP 078184041 Eremococcus coleocola ACS-139-V-Col8
GH13 YP 0034774501 Thermoanaerobacter italicus Ab9
GH13 ZP 048517861 Paenibacillus sp oral taxon 786 str D14
GH13 YP 0038414241 Caldicellulosiruptor obsidiansis OB47
GH13 YP 0024279661 Desulfurococcus kamchatkensis 1221n
GH13 YP 0034262831 Bacillus pseudofirmus OF4
GH13 ZP 076854311 Oscillochloris trichoides DG-6GH13 YP 0016174961 Sorangium cellulosum So ce56
GH13 ZP 019113371 Plesiocystis pacifica SIR-1
GH13 YP 6318811 Myxococcus xanthus DK 1622
GH13 AKF11765 Sandaracinus amylolyticus DSM 53668
GH13 ZP 036771121 Bacteroides cellulosilyticus DSM 14838
GH13 YP 0037157841 Croceibacter atlanticus HTCC2559
GH13 ZP 012015681 Flavobacteria bacterium BBFL7
GH13 ZP 019113321 Plesiocystis pacifica SIR-1GH13 YP 0031962511 Robiginitalea biformata HTCC2501
GH13 YP 0040531921 Marivirga tractuosa DSM 4126
GH13 ZP 021817881 Flavobacteriales bacterium ALC-1
GH13 CBL150401 Ruminococcus bromii L2-63
GH13 ZP 080051001 Bacillus sp 2 A 57 CT2
GH13 CBK911271 Eubacterium rectale DSM 17629
GH13 YP 0029369771 Eubacterium rectale ATCC 33656
GH13 YP 0021366011 Anaeromyxobacter sp K
GH13 ZP 014604541 Stigmatella aurantiaca
GH
13 728851 Streptomyces lividans
GH
13 BAJ281541 Kitasatospora setae KM-6054
GH
13 YP 0033390201 Streptosporangium roseum
DSM
43021
GH
13 YP 9214291 Nocardioides sp JS614
GH
13 AEB462451 Verrucosispora maris AB-18-032
GH
13 CAJ810301 Actinoplanes sp
GH
13 NP 9387401 C
orynebacterium diphtheriae N
CTC
13129
GH
13 YP 0036976141 Arcanobacterium haem
olyticum D
SM 20595
GH
13 ZP 039259261 Actinomyces coleocanis D
SM 15436
GH
13 YP 0040999421 Intrasporangium calvum
DSM
43043
GH
13 YP 0030989701 Actinosynnema m
irum D
SM 43827
GH
13 YP 0036376081 Cellulom
onas flavigena DSM
20109
GH
13 ZP 009938561 Janibacter sp HTC
C2649
GH13 YP 0030608421 Hirschia baltica ATCC 49814
GH13 YP 7579631 M
aricaulis maris M
CS10
GH13 YP 0016857121 Caulobacter sp K31
GH13 ZP 013016731 Sphingom
onas sp SKA58
GH13 YP 0038198341 Brevundim
onas subvibrioides ATCC 15264
GH
13 YP 4971641 Novosphingobium
aromaticivorans D
SM 12444
GH13 YP 6609551 Pseudoalterom
onas atlantica T6c
GH13 ZP 011126451 Reinekea blandensis M
ED297
GH13 YP 2677341 Colwellia psychrerythraea 34H
GH13 ZP 011126461 Reinekea blandensis M
ED297
GH13 AKF11772 Sandaracinus amylolyticus DSM
53668
GH13 AKF07527 Sandaracinus amylolyticus DSM
53668
GH13 YP 0016184291 Sorangium cellulosum
So ce56
GH13 ZP 019113391 Plesiocystis pacifica SIR-1
GH13 ZP 036315591 Pedosphaera parvula Ellin514
GH13 YP 0016604791 Microcystis aeruginosa NIES-843
GH13 YP 0043416831 Archaeoglobus veneficus SNP6
GH13 ZP 073351521 Desulfovibrio fructosovorans JJ
GH
13 AKF03605 Sandaracinus amylolyticus D
SM 53668
GH
13 YP 0027853821 Deinococcus deserti VC
D115
GH
13 NP 2951951 D
einococcus radiodurans R1
GH
13 YP 004171
1212
M
SD
sisn
epoc
ira
m su
ccoc
onie
D 1
661
1
GH
13 YP 0041715811 Deinococcus m
aricopensis DSM
21211
GH
13 YP 0037038171 Truepera radiovictrix DSM
17093
GH
13 YP 6052101 Deinococcus geotherm
alis DSM
11300
GH
13 YP 0027555491 A
cidobacterium capsulatum
ATCC
51196
GH
13 Y
P9
XTC
A5P
M alocirdnut allecilunarG 1860612400
GH
13 YP 6335241 M
yxococcus xanthus DK
1622G
H13 Y
PB
S su
cihp
ortid
ica
suhp
ortny
S 1
9412
64
GH
13 A
AW32
490
1 iiklahcsttog acnarborean
A
GH
13 Y
P 00
2316
532
1 An
oxyb
acillu
s fla
vith
erm
us W
K1
GH
13 Y
P 00
3252
634
1 G
eoba
cillu
s sp
Y41
2MC
61G
H13
ZP
0117
0936
1 B
acillu
s sp
NR
RL
B-14
911
GH
13 Z
P 07
7074
981
Bac
illus
sp m
3-13
GH
13 Z
P1-
GS ps sullica
B 116916810
GH
13 Y
P 00
1813
251
1 Ex
iguo
bact
eriu
m s
ibiri
cum
255
-15
GH
13 Y
P 00
2886
871
1 Ex
iguo
bact
eriu
m s
p A
T1b
GH
13 Y
P 00
1568
454
1 Pe
troto
ga m
obilis
SJ9
5
GH
13 Y
P 00
2940
188
1 Ko
smot
oga
olea
ria T
BF 1
95
1G
H13
ZP
0125
8420
1 V
ibrio
alg
inol
ytic
us 1
2G01
GH
13 1
0911
18 T
herm
oact
inom
yces
vul
garis
GH
13 Z
P 06
9705
751
Kte
dono
bact
er ra
cem
ifer D
SM 4
4963
GH
13 Y
P 00
1545
735
1 H
erpe
tosi
phon
aur
antia
cus
DSM
785
GH
13 E
FY95
778
1 M
etar
hizi
um ro
berts
ii AR
SEF
23
GH
13 N
P 58
8205
1 S
chiz
osac
char
omyc
es p
ombe
972
h-
GH
13 N
P 59
4401
1 S
chiz
osac
char
omyc
es p
ombe
972
h-
GH
13 X
P 00
1908
940
1 Po
dosp
ora
anse
rina
S m
at+
GH
13 4
1016
824
Schw
anni
omyc
es o
ccid
enta
lis
GH
13 B
AF98
616
1 H
ypho
pich
ia b
urto
nii
GH13 AKF07406 Sandaracinus amylolyticus DSM 53668
GH13 YP 0043692881 Desulfobacca acetoxidans DSM 11109
GH13 YP 0023716111 Cyanothece sp PCC 8801GH13 YP 3219451 Anabaena variabilis ATCC 29413
GH13 YP 4738811 Synechococcus sp JA-3-3AbGH13 YP 8464671 Syntrophobacter fumaroxidans MPOBGH13 ZP 063702061 Desulfovibrio sp FW1012B
GH13 YP 0033193471 Sphaerobacter thermophilus DSM 20745GH13 YP 0037057361 Truepera radiovictrix DSM 17093GH13 YP 4120971 Nitrosospira multiformis ATCC 25196GH13 YP 6431071 Rubrobacter xylanophilus DSM 9941
GH13 BAD389831 Rhodobacter sphaeroides f sp denitrificans
GH13 YP 3175601 Nitrobacter winogradskyi Nb-255
GH13 YP 8215181 Candidatus Solibacter usitatus Ellin6076
GH13 YP 0034506361 Azospirillum sp B510
GH13 ZP 070169471 Desulfonatronospira thiodismutans ASO3-1
GH13 YP 0041842351 Terriglobus saanensis SP1PR4
GH13 ZP 070315011 Granulicella mallensis MP5ACTX8
GH13 YP 0029438961 Variovorax paradoxus S110
GH13 YP 5918381 Candidatus Koribacter versatilis Ellin345
GH13 YP 7445681 Granulibacter bethesdensis CGDNIH1
GH13 YP 0039098001 Burkholderia sp CCGE1003
GH13 ZP 024903661 Burkholderia pseudomallei NCTC 13177
GH13 YP 0016309761 Bordetella petrii DSM 12804
GH13 YP 0022527351 Ralstonia solanacearum MolK2
GH13 YP 0037759061 Herbaspirillum seropedicae SmR1
GH13 YP 5455251 Methylobacillus flagellatus KT
GH13 YP 0013485041 Pseudomonas aeruginosa PA7
GH13 YP 0016008321 Gluconacetobacter diazotrophicus PA1 5
GH13 AEB584851 Pseudomonas mendocina NK-01
GH13 YP 0027996471 Azotobacter vinelandii DJ
GH
13 N
P 63
5804
1 X
anth
omon
as c
ampe
stris
pv
cam
pest
ris s
tr A
TCC
339
13
GH
13 Y
P 00
3188
581
1 Ac
etob
acte
r pas
teur
ianu
s IF
O 3
283-
01
GH
13 Y
P 74
4570
1 G
ranu
libac
ter b
ethe
sden
sis
CG
DN
IH1
GH
13 Y
P 00
3052
329
1 M
ethy
lovo
rus
gluc
oset
roph
us S
IP3-
4
GH
13 Z
P 06
8981
631
Ros
eom
onas
cer
vica
lis A
TCC
499
57
GH
13 Y
P 00
2943
895
1 Va
riovo
rax
para
doxu
s S1
10
GH
13 Y
P 31
4931
1 T
hiob
acillu
s de
nitri
fican
s AT
CC
252
59
GH
13 Y
P 42
5597
1 R
hodo
spiri
llum
rubr
um A
TCC
111
70
GH
13 Y
P 93
3302
1 A
zoar
cus
sp B
H72
GH
13 N
P 43
7816
1 S
inor
hizo
bium
mel
iloti
1021
GH
13 Z
P 01
1553
281
Oce
anic
ola
gran
ulos
us H
TCC
2516
GH
13 Y
P 00
1167
979
1 R
hodo
bact
er s
phae
roid
es A
TCC
170
25
GH
13 Y
P 00
1440
575
1 C
rono
bact
er s
akaz
akii A
TCC
BAA
-894
GH
13 Z
P 01
4401
991
Ful
vim
arin
a pe
lagi
HTC
C25
06
GH
13 Y
P 00
4234
793
1 Ac
idov
orax
ave
nae
subs
p a
vena
e AT
CC
198
60
GH
13 Y
P 00
1818
894
1 O
pitu
tus
terra
e PB
90-1
GH
13 Y
P 00
3154
808
1 Br
achy
bact
eriu
m fa
eciu
m D
SM 4
810
GH
13 ZP 010940301 Blastopirellula marina D
SM 3645
GH
13 ZP 027320551 Gem
mata obscuriglobus U
QM
2246
GH
13 YP 4273821 Rhodospirillum
rubrum ATC
C 11170
GH
13 YP 0021361131 Anaeromyxobacter sp K
GH
13 YP 0039532711 Stigmatella aurantiaca
GH
13 YP 0021384951 Geobacter bem
idjiensis BemG
H13 Y
P23
-C
RF ii
notl
ad r
etca
boe
G 1
3868
3520
0 G
H13 Y
P65
ec o
S mu
solu
llec
mu
igna
roS
191
8716
100
GH
13 ZPB2
101
WF
ps o
irbi
vofl
use
D 1
5398
6360
GH
13 Y
P 82
1516
1 C
andi
datu
s S
olib
acte
r usi
tatu
s E
llin6
076
GH
13 Y
P24
NFC ilte
muibozihR 1077074
GH
13
86635 M
SD sucityloly
ma sunicaradnaS 43690F
KA
GH
13 Y
P 00
1820
541
1 O
pitu
tus
terra
e P
B90
-1G
H13
ZP
0313
0066
1 C
htho
niob
acte
r fla
vus
Ellin
428
GH
13 YP 0035707081 Salinibacter ruber M8
GH
13 YP 0037057351 Truepera radiovictrix DSM
17093
GH
13 YP 0032689441 Haliangium
ochraceum D
SM 14365
GH
13 YP 0016130311 Sorangium cellulosum
So ce56
GH
13 YP 6288101 Myxococcus xanthus D
K 1622
GH
13 62738967 Deinococcus radiodurans
GH
13 AKF04017 Sandaracinus amylolyticus D
SM 53668
GH
13 YP 0027878991 Deinococcus deserti VC
D115
GH13 YP 5893201 Candidatus Koribacter versatilis Ellin345
GH13 ZP 063702081 Desulfovibrio sp FW
1012B
GH13 YP 0041842341 Terriglobus saanensis SP1PR4
GH13 YP 8731321 Acidotherm
us cellulolyticus 11B
GH13 ZP 070169501 Desulfonatronospira thiodism
utans ASO3-1
GH13 ADX845721 Sulfolobus islandicus REY15A
GH13 ZP 027318151 G
emm
ata obscuriglobus UQM
2246
GH13 YP 0016347811 Chloroflexus aurantiacus J-10-fl
GH
13 NP 9534051 G
eobacter sulfurreducens PCA
GH
13 YP 0025366761 Geobacter daltonii FR
C-32
GH
13 YP 0012121361 Pelotomaculum
thermopropionicum
SI
GH
13 YP 0041781661 Isosphaera pallida ATCC
43644
GH
13 ZP 036310341 Pedosphaera parvula Ellin514
GH13 ZP 031287911 Chthoniobacter flavus Ellin428
GH13 NP 6913271 Oceanobacillus ih
eyensis HTE831
GH13 YP 0025601851 Macro
coccus c
aseolyticus J
CSC5402
GH13 YP 0030110001 Paenibacill
us sp J
DR-2
GH13 CAJ384141 Anaerobranca
gottsch
alkii
GH13 YP 00
1988
1311
Lacto
bacill
us ca
sei B
L23
GH13 YP 00
2459
6561
Des
ulfito
bacte
rium ha
fnien
se D
CB-2
GH13 YP 00
3947
0781
Paenib
acillu
s poly
myxa S
C2
GH13 Y
P 0013
1197
11 C
lostrid
ium be
ijerin
ckii N
CIMB 80
52
GH13 Y
P 0039
6683
31 Ily
obac
ter po
lytrop
us D
SM 2926
GH13 Z
P 0539
1277
1 Clos
tridium
carb
oxidi
vora
ns P
7
GH13 Y
P 0013
9686
01 C
lostrid
ium kl
uyve
ri DSM 55
5
GH13 ZP 02
2128
451
Clostrid
ium ba
rtlettii
DSM 16
795
GH13 ZP 04
4331
841
Bacillu
s coa
gulan
s 36D
1
GH13 C
BL37
8151
Ana
eros
tipes
hadr
us
GH13 Z
P 04
4519
311
Abiotro
phia
defec
tiva A
TCC 49
176
GH13
YP
0031
4407
51
Slac
kia h
eliot
rinire
duce
ns D
SM 2
0476
GH13
ZP 0
2025
300
1 Eu
bacte
rium
vent
riosu
m A
TCC
2756
0
GH1
3 YP
004
3093
801
Clo
strid
ium
lent
ocel
lum
DSM
542
7
GH13
YP 5
1565
71
Chlam
ydop
hila
felis
GH13
ZP 0
6299
217
1 Pa
rach
lamyd
ia ac
anth
amoe
bae
str H
allrsquos
cocc
us
GH13
CBL1
5412
1 R
umino
cocc
us b
rom
ii L2-
63
GH13 Y
P 00
1692
347
1 Fi
nego
ldia
mag
na AT
CC 293
28
GH13 Z
P 07
5262
211
Pep
tostr
epto
cocc
us st
omat
is DSM
176
78
GH1
3 YP
003
4751
021
Clo
strid
iale
s ge
nom
osp
BVA
B3 s
tr U
PII9
-5
GH1
3 YP
004
1716
241
Dei
noco
ccus
mar
icope
nsis
DSM
212
11
GH1
3 YP
022
845
1 Pi
crop
hilu
s to
rridu
s DS
M 9
790
GH1
3 YP
001
8207
181
Opi
tutu
s te
rrae
PB90
-1
GH1
3 YP
003
1672
861
Can
dida
tus A
ccum
ulib
acte
r pho
spha
tis c
lade
IIA
str
UW-1
GH1
3 AK
F038
60 S
anda
racin
us a
mylo
lyticu
s DS
M 5
3668
GH1
3 AK
F069
52 S
anda
racin
us a
mylo
lyticu
s DS
M 5
3668
GH
13 Y
P 00
2762
222
1 G
emm
atim
onas
aur
antia
ca T
-27
GH1
3 ZP
082
0671
01
Gor
doni
a ne
ofel
ifaec
is NR
RL B
-593
95
GH
13 Y
P 00
3964
374
1 Ke
togu
loni
cige
nium
vul
gare
Y25
GH13 ZP 045772811 Oxalobacter fo
rmigenes H
OxBLS
GH13 YP 0033856211 Spirosoma linguale DSM 74
GH13 AEA650561 Burkholderia gladioli B
SR3
GH13 YP 0030725361 Teredinibacter turnerae T7901
GH13 YP 4255961 Rhodospirillum ru
brum ATCC 11170
GH13 YP 0010427851 Rhodobacter sphaeroides ATCC 17029
GH13 ZP 075890671 Sinorhizobium meliloti BL225C
GH13 YP 0030504181 Methylovorus glucosetrophus SIP3-4
GH13 YP 4224271 Magnetospirillum magneticum AMB-1
GH13 YP 0042554001 Deinococcus proteolyticus MRP
GH13 ZP 029300551 Verrucomicrobium spinosum DSM 4136
GH13 ZP 080190951 Lautropia mirabilis ATCC 51599
GH13 YP 5335351 Rhodopseudomonas palustris BisB18
GH13 YP 8654291 Magnetococcus marinus MC-1
GH13 YP 0028938741 Tolumonas auensis DSM 9187
GH13 YP 6612131 Pseudoalteromonas atlantica T6c
GH13 YP 0040654571 Pseudoalteromonas sp SM9913
GH13 YP 8302311 Arthrobacter sp FB24
GH13 YP 0042347941 Acidovorax avenae subsp avenae ATCC 19860
GH13 YP 0016390191 Methylobacterium extorquens PA1
GH13 ZP 068060481 Brevibacterium mcbrellneri ATCC 49030
GH13 YP 0031655431 Candidatus Accumulibacter phosphatis clade IIA str UW-1
GH13 YP 9813411 Polaromonas naphthalenivorans CJ2
GH13 ZP 029305921 Verrucomicrobium spinosum DSM 4136
GH13 YP 8283661 Candidatus Solibacter usitatus Ellin6076
GH13 YP 0015687661 Petrotoga mobilis SJ95
GH13 YP 2560581 Sulfolobus acidocaldarius DSM 639
GH13 YP 0023732201 Cyanothece sp PCC 8801
GH13 AKF07413 Sandaracinus amylolyticus DSM 53668
GH13 AKF03796 Sandaracinus amylolyticus DSM 53668
GH13 EFS357291 Propionibacterium acnes HL013PA1
GH13 YP 0033827501 Kribbella flavida DSM 17836
GH13 YP 9230111 Nocardioides sp JS614
GH13 AEA239681 Pseudonocardia dioxanivorans CB1190
GH13 YP 0031037201 Actinosynnema mirum DSM 43827
GH57 YP 0018695221 Nostoc punctiforme PCC 73102
GH57 YP 0041847961 Terriglobus saanensis SP1PR4
GH57 YP 0033170621 Thermanaerovibrio acidaminovorans DSM 6589
GH57 YP 0025231331 Thermomicrobium roseum DSM 5159
GH57 CBL286501 Fretibacterium fastidiosum
GH57 YP 0041781671 Isosphaera pallida ATCC 43644
GH57 YP 0027549471 Acidobacterium capsulatum ATCC 51196
GH57 ZP 070292791 Granulicella mallensis MP5ACTX8
GH57 YP 0018749261 Elusimicrobium minutum Pei191
GH57 ADL259941 Fibrobacter succinogenes subsp succinogenes S85
GH57 YP 4773871 Synechococcus sp JA-2-3B
GH57 YP 8454221 Syntrophobacter fumaroxidans MPOB
GH57 NP 9534062 Geobacter sulfurreducens PCA
GH57 YP 5020321 Methanospirillum hungatei JF-1
GH57 YP 0019958521 Chloroherpeton thalassium ATCC 35110
GH57 YP 5937791 Candidatus Koribacter versatilis Ellin345
GH57 YP 0010471761 Methanoculleus marisnigri JR1
GH57 YP 0014309471 Roseiflexus castenholzii DSM 13941
GH57 YP 0029516361 Desulfovibrio magneticus RS-1
GH57 YP 0038064671 Desulfarculus baarsii DSM 2075
GH57 YP 0013789531 Anaeromyxobacter sp Fw109-5
GH57 ZP 063702071 Desulfovibrio sp FW1012B
GH57 YP 0035531061 Aminobacterium colombiense DSM 12261
GH57 ZP 064391271 Anaerobaculum hydrogeniformans ATCC BAA-1850
GH57 AKF06951 Sandaracinus amylolyticus DSM 53668
GH57 ZP 063915611 Dethiosulfovibrio peptidovorans DSM 11002
GH57 YP 0024634161 Chloroflexus aggregans DSM 9485
GH57 YP 0043692891 Desulfobacca acetoxidans DSM 11109
GH57 YP 8464661 Syntrophobacter fumaroxidans M
POB
GH57 CAJ738171 Candidatus Kuenenia stuttgartiensis
GH57 YP 0031969181 Desulfohalobium
retbaense DSM 5692
FIG 3mdashPhylogeny of Sandaracinus amylolyticus GH13 and GH57 a-amylases Sandaracinus amylolyticus a-amylases (GH13 and GH57) are colored in
pink along with their domain architecture displayed as Domain Color Shape [GH13 Red rectangle GH57 Black rectangle GH77 dark pink rectangle
CBM48 Dark blue circle CBM20 green circle CBM41 cyan circle] Myxobacterial homologs are colored fluorescent green Bootstrap values correspond-
ing to the tree nodes are provided
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2525
of the phyla Betaproteobacteria suggestive of a possible hor-
izontal transfer
AKF04017 and AKF03605 have close homologs in the ex-
tremophile Deinococcus desserti VCD115 and shares clade
with certain myxobacterial amylases belonging to
Myxococcus xanthus S cellulosum and Haliangium ocraceum
which further share clades with Deinococcus amylases
AKF04017 and AKF03605 share the highest sequence identity
with D desserti VCD115 plasmid protein (YP_002787899
533) and chromosomal protein (YP_002785382 456)
respectively that might point to its plasmid-borne horizontal
transfer across the clade (fig 3) The sequence conservation of
CSRs and the high percentage identity of these amylases to
Deinococcus support the inference for the putative horizontal
transfers among these members AKF09634 does not share its
sister branch with any member but the clade contains mem-
bers from diverse phyla that is Rhizobium from alphaproteo-
bacteria Opitutus from Chlamydae Chthonibacter from
Verrucomicrobia suggesting the multiple gain and loss of
this amylase among prokaryotes Similarly AKF07413 shares
the clade with members of Cyanobacteria AKF07406 shares
the clade with members of Deltaproteobacteria AKF03861
with members of Alphaproteobacteria and AKF03642 with
members of Bacteroidetes Taken together we can suggest
that many of the S amylolyticus amylases have been acquired
via horizontal gene transfer events from other bacterial species
belonging to the phylum Deinococcus-Thermus
Acidobacteria Cyanobacteria and Alphaproteobacteria
To further support these horizontal transfer events we
checked the sequence conservation between S amylolyticus
amylases and closely related members in the phylogenetic tree
and aligned using PROMALS-3D with Taka-Amylase A of A
oryzae (PDBid 2TAA) as a structural template We also per-
formed synteny studies analyzing proteins present in the vicin-
ity of all identified S amylolyticus GH13 and GH57 amylases
and their closely related homologs as revealed by phylogenetic
studies We could not trace any conserved syntenic regions in
these genomes whereas in the case of AKF03862 and
AKF09634 we identified glycogen debranching enzymes in
Stigmatella aurantiaca DW43-1 [56 identity and 98
query coverage of AKF03860 with WP_0133760911] and
Opitutus terrae PB90-1 [57 identity and 97 query cover-
age of AKF09635 YP_0018205421] which further support
their putative horizontal transfer in S amylolyticus
Similarly the phylogenetic inference was drawn from two
representative domains of g-amylases using the same meth-
odology as followed for a-amylases (fig 4) We found that
one of the g-amylases AKF10340 lies closer to M xanthus
amylase whereas the other AKF10487 shares its sister
branch with Nitrosococcus watsonii a Gammaproteobacteria
This suggests that while some of the a- and g-amylases
are present in many myxobacterial species a majority of the
a- and g-amylases are present only in S amylolyticus and not
in other myxobacterial genomes possibly due to the result of
horizontal transfer events from diverse bacterial phylum such
as Deinococcus-Thermus Cyanobacteria Alphaproteobac-
teria Gammaproteobacteria and Acidobacteria
Agar Degrading Enzymes
Some bacteria are known to degrade agar and use it as a
carbon source and possess agarase enzymes that help in hy-
drolyzing agar in oligosaccharides (Chi et al 2012) Depending
on the site of action they can be of two types a-agarase
(agarose 3-glycanohydrolase 321158 GH96) and
b-agarase (agarose 4-glycanohydrolase 32181 GH16
GH50 GH86 GH118) (table 1) Both families of agarases
GH15 EFV841771 Achrom
obacter xylosoxidans C54
GH
15 ZP 011267341 Nitrococcus m
obilis Nb-231
GH15 YP 4129921 Nitrosospira m
ultiformis ATCC 25196
GH15 YP 5716301 Nitrobacter ham
burgensis X14 plasmid
GH15 ZP 012260811 Aurantim
onas manganoxydans SI85-9A1
GH15 YP 0016032801 Gluconacetobacter diazotrophicus PA1 5
GH15 YP 7451371 Granulibacter bethesdensis CGDNIH1
GH15 AEA616211 Burkholderia gladioli BSR3
GH15 YP 0018344311 Beijerinckia indica subsp indica ATCC 9039
GH15 YP 0027560251 Acidobacterium capsulatum
ATCC 51196
GH15 CBJ387091 Ralstonia solanacearum CMR15
GH15 YP 3189151 Nitrobacter winogradskyi Nb-255
GH15 YP 0012344011 Acidiphilium cryptum JF-5
GH15 YP 0040115781 Rhodomicrobium vannielii ATCC 17100
GH15 YP 0016419391 Methylobacterium extorquens PA1GH15 YP 5252641 Rhodoferax ferrireducens T118
GH15 YP 0041811661 Terriglobus saanensis SP1PR4
GH15 ZP 031333281 Chthoniobacter flavus Ellin428
GH15 CCA537261 Streptomyces venezuelae ATCC 10712
GH15 YP 0015065621 Frankia sp EAN1pec
GH15 YP 0024892701 Arthrobacter chlorophenolicus A6
GH15 YP 0018202091 Opitutus terrae PB90-1
GH15 YP 8729011 Acidothermus cellulolyticus 11B
GH15 YP 0013795591 Anaeromyxobacter sp Fw109-5
GH15 YP 0024929091 Anaeromyxobacter dehalogenans 2CP-1
GH15 YP 6355121 Myxococcus xanthus DK 1622
GH15 YP 0011047051 Saccharopolyspora erythraea NRRL 2338
GH15 YP 0011092191 Saccharopolyspora erythraea NRRL 2338
GH15 AKF10340 Sandaracinus amylolyticus DSM 53668
GH15 YP 6342991 Myxococcus xanthus DK 1622
GH15 YP 0031091351 Acidimicrobium ferrooxidans DSM 10331
GH15 YP 0035703361 Salinibacter ruber M8
GH15 YP 0025241401 Thermomicrobium roseum DSM 5159 plasmid
GH15 YP 0033214441 Sphaerobacter thermophilus DSM 20745
GH15 NP 7336051 Streptomyces coelicolor A3-2
GH15 YP 0035282991 Nitrosococcus halophilus Nc 4
GH15 YP 0043676121 Marinithermus hydrothermalis DSM 14884
GH15 YP 0043687811 Marinithermus hydrothermalis DSM 14884
GH15 YP 0034042871 Haloterrigena turkmenica DSM 5511
GH15 ZP 080461001 Haladaptatus paucihalophilus DX253
GH1
5 YP
023
376
1 Pi
crop
hilu
s to
rrid
us D
SM 9
790
GH1
5 ZP
055
7131
11
Ferr
opla
sma
acid
arm
anus
fer1
GH1
5 BA
B604
801
The
rmop
lasm
a vo
lcan
ium
GSS
1
GH
15 Y
P 00
3323
761
1 Th
erm
obac
ulum
terr
enum
ATC
C B
AA
-798
GH
15 Y
P 00
3899
615
1 C
yano
thec
e sp
PC
C 7
822
plas
mid
GH
15 Y
P 00
3705
927
1 Tr
uepe
ra ra
diov
ictr
ix D
SM 1
7093
GH
15 Y
P 00
2478
443
1 A
rthr
obac
ter c
hlor
ophe
nolic
us A
6 pl
asm
idG
H15
AK
F104
87 S
anda
raci
nus
amyl
olyt
icus
DSM
536
68G
H15
YP
0037
6113
81
Nitr
osoc
occu
s w
atso
nii C
-113
GH1
5 ZP
069
7134
91
Kted
onob
acte
r rac
emife
r DSM
449
63
GH15
ZP
0697
6049
1 K
tedo
noba
cter
race
mife
r DSM
449
63
GH15
YP
0033
9335
81
Cone
xiba
cter
woe
sei D
SM 1
4684
GH15
YP
0042
3629
11 A
cido
vora
x av
enae
sub
sp a
vena
e ATC
C 19
860
GH15 Z
P 03
6273
271
Pedo
spha
era p
arvu
la El
lin51
4
GH15 Y
P 00
1618
7001
Sor
angi
um ce
llulo
sum
So
ce56
GH15 ZP
0145
9561
1 Stig
matella
aura
ntiac
a DW
4-3-1
GH15 ZP
0503
8177
1 Syn
echo
cocc
us sp
PCC 73
35
GH15 YP 64
4038
1 Rubrobac
ter xy
lanophilu
s DSM 99
41
GH15 ZP 064746651 Frankia symbiont of D
atisca glomerata
GH15 ZP 069744041 Ktedonobacter racemife
r DSM 44963
GH15 ZP 069653261 Ktedonobacter racemifer D
SM 44963
GH15 ZP 069661381 Ktedonobacter racemifer D
SM 44963
FIG 4mdashPhylogeny of Sandaracinus amylolyticus GH15 g-amylases
Sandaracinus amylolyticus g-amylases are colored dark pink and myxobac-
terial homologs are colored fluorescent green Bootstrap values corre-
sponding to the tree nodes are provided
Sharma et al GBE
2526 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
were absent in S amylolyticus genome which could be related
to its agar nondegrading property (Mohr et al 2012) as sup-
ported by colony morphology where agar was not penetrated
or liquefied by the bacteria (supplementary fig S5
Supplementary Material online)
Cellulose Degrading Enzymes
We identified only GH5 and GH74 in S amylolyticus proteome
in two and three proteins respectively GH5 domain was pre-
sent as a single unit in AKF04963 and AKF09194 whereas
GH74 was identified in AKF04859 AKF06963 and
AKF09775 associated with a GH74 domain CAZY domains
for cellobiohydrolaseexoglucanases (represented by GH6
GH7 GH9 and GH48) were not present in S amylolyticus
Cellobiohydrolaseexoglucanases plays a significant role in cel-
lulose degradation as they cut two to four sugar subunits from
the exposed ends formed by the endoglucanases which are
used as initial substrates for b-glucosidase (Mba Medie et al
2012) The absence of cellobiohydrolase or exoglucanases
could be a possible reason for the lack of cellulose degradation
in S amylolyticus Out of b-glucosidase representative CAZY
families GH1 GH3 GH5 GH9 GH30 and GH116 only GH1
GH3 and GH5 families were found in three two and two
proteins in S amylolyticus respectively AKF03843 AKF0
6028 and AKF10711 have a GH1 domain whereas
AKF09078 and AKF10875 have only the GH3 domain
Chitin Degrading Enzymes
Chitinases (EC32114) are involved in degradation of chitin
a component of the cell wall of fungi and exoskeleton of
arthropods GH18 GH19 GH23 and GH48 families have
been reported in the CAZY database as chitinases that hydro-
lyse b-14 linkages between N-acetylglucosamine units GH18
is a chitinase family with a characteristic ldquo[LIVM
FY][DN]G[LIVMF][DN][LIVMF][DN]Erdquo motif and is expressed
in archaea prokaryotes and eukaryotes (Funkhouser and
Aronson 2007) GH18 chitinases were not found in S amylo-
lyticus genome while members of GH19 (AKF03006 and
AKF10423) and GH23 (AKF04575 AKF05533 AKF06346
AKF08835 and AKF11377) could be identified GH19
family chitinases are mostly reported to be present in plants
with only a few members in prokaryotes (Kasprzewska 2003
Funkhouser and Aronson 2007) Among the two identified
GH19 homologs we found that AKF10423 is approximately
65 identical (~68 query coverage) with cyanobacteria
such as Nodosilinea nodulosa and Limnoraphis robusta
along with another myxobacterium Chondromyces crocatus
Besides these homologs we also found that AKF10423 shows
maximum similarity with eukaryotic GH19 chitinases counter-
parts as present in Acacia mangium Festuca arundinacea
Triticum aestivum and Leucaena leucocephala (55ndash59 iden-
tity with 70 query coverage) We identified that another
chitinase AKF06346 has GH23 domain along with three
copies of CBM50 which is generally present in the enzymes
cleaving either chitin or peptidoglycan
Conclusion
The complete 1033 Mb circular genome of S amylolyticus
type strain DSM 53668T was determined CAZy analysis re-
vealed the presence of 72 GT domains 72 CE domains 58 GH
domains 19 AA domains 4 PL domains and 26 CBM do-
mains Sixteen S amylolyticus proteins were identified of
which 15 are a-amylases of class GH13 and 1 GH57 Two
g-amylases of GH15 family were also identified Phylogeny
analysis revealed that ten GH13 a-amylases and one GH15
g-amylase have been aquired via horizontal gene transfer
from other bacterial phyla such as Deinococcus-Thermus
Cyanobacteria Alphaproteobacteria Gammaproteobacteria
and Acidobacteria
Table 1
Cellulose Starch Chitin and Agar Degrading Enzymes and Their Representative CAZY Domains Distribution
Substrate Enzyme Category Enzyme Name EC Number CAZY Families (protein repertoire in brackets)
Starch Amylase a-Amylase 3211 GH13 (15) GH57 (1) GH119 GH126
Amylase b-Amylase 3212 GH14
Amylase g-Amylase 3213 GH15 (2) GH97
Chitin Chitinase Chitinase 32114 GH18 GH19 (2) GH23 (5) GH48
Agar a-Agarase Agarose 3-glycanohydrolase 321158 GH96
b-Agarase Agarose 4-glycanohydrolase 32181 GH16 GH50 GH86 GH118
Cellulose Cellulase b-Glucosidase 32121 GH1 (3) GH3 (2) GH5 (2) GH9 GH30 GH116
Cellulase Cellobiohydrolase or Exoglucanase 321176 GH48 GH6 GH7 GH9
Cellulase Endoglucanase 3214 GH5 (2) GH6 GH7 GH8 GH9 GH12 GH44
GH45 GH48 GH51 GH74 (3) GH124
NOTEmdashEC number enzyme category and CAZY families for proteins involved in starch chitin agar and cellulose degradation are shown The number of Sandaracinusamylolyticus proteins identified having CAZY domains are represented in bold fonts in respective families
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2527
Supplementary Material
Supplementary figures S1ndashS5 and tables S1 and S2 are avail-
able at Genome Biology and Evolution online (httpwww
gbeoxfordjournalsorg)
Acknowledgments
This work is supported by a project ldquoExpansion and modern-
ization of Microbial Type Culture Collection and Gene Bank
(MTCC) jointly supported by Council of Scientific and
Industrial Research (CSIR) Grant No BSC0402 and
Department of Biotechnology (DBT) Govt of India Grant No
BTPR7368INF221772012rdquo The authors thank MTCC for
providing help in procuring the cultures from DSMZ They
acknowledge Dr Ramya TNC for providing workbench for cul-
turing myxobacteria GS and IK acknowledge the Council
of Scientific and Industrial Research (CSIR) and University
Grant Commission (UGC) for the research fellowship respec-
tively They also thank the Genome Quebec Innovation
Centre McGill University Canada and C-CAMP next-genera-
tion genomics facility Bangalore India for help in obtaining
PacBio and Illumina NGS data respectively
Literature CitedAlikhan NF Petty NK Ben Zakour NL Beatson SA 2011 BLAST Ring
Image Generator (BRIG) simple prokaryote genome comparisons
BMC Genomics 12402
Altschul SF Gish W Miller W Myers EW Lipman DJ 1990 Basic local
alignment search tool J Mol Biol 215403ndash410
Auch AF Von Jan M Klenk HP Goker M 2010 Digital DNA-DNA hybrid-
ization for microbial species delineation by means of genome-to-
genome sequence comparison Stand Genomic Sci 2117ndash134
Aziz RK et al 2008 The RAST Server rapid annotations using subsystems
technology BMC Genomics 975
Blesak K Janecek S 2012 Sequence fingerprints of enzyme specificities
from the glycoside hydrolase family GH57 Extremophiles 16497ndash
506
Chi WJ Chang YK Hong SK 2012 Agar degradation by microorganisms
and agar-degrading enzymes Appl Microbiol Biotechnol 94917ndash
930
Chin CS et al 2013 Nonhybrid finished microbial genome assemblies
from long-read SMRT sequencing data Nat Methods 10563ndash569
Eddy SR 2011 Accelerated profile HMM searches PLoS Comput Biol
7e1002195
Edgar RC 2004 MUSCLE multiple sequence alignment with high accu-
racy and high throughput Nucleic Acids Res 321792ndash1797
Funkhouser JD Aronson NN Jr 2007 Chitinase family GH18 evolutionary
insights from the genomic history of a diverse protein family BMC Evol
Biol 796
Garcia R Muller R 2014 The family Polyangiaceae In Rosenberg E
Delong E Lory S Stackebrandt E Thompson F editors The prokary-
otes Berlin Heidelberg Springer p 247ndash279
Grissa I Vergnaud G Pourcel C 2007 CRISPRFinder a web tool to identify
clustered regularly interspaced short palindromic repeats Nucleic
Acids Res 35W52ndashW57
Hall TA 1999 BioEdit a user-friendly biological sequence alignment editor
and analysis program for Windows 9598NT Nucleic Acids Symp Ser
4195ndash98
Han K et al 2013 Extraordinary expansion of a Sorangium cellulosum
genome from an alkaline milieu Sci Rep 32101
Janecek S Kuchtova A 2012 In silico identification of catalytic residues
and domain fold of the family GH119 sharing the catalytic machinery
with the alpha-amylase family GH57 FEBS Lett 5863360ndash3366
Janecek S Svensson B Macgregor EA 2014 alpha-Amylase an enzyme
specificity found in various families of glycoside hydrolases Cell Mol
Life Sci 711149ndash1170
Kasprzewska A 2003 Plant chitinasesndashregulation and function Cell Mol
Biol Lett 8809ndash824
Lagesen K et al 2007 RNAmmer consistent and rapid annotation of
ribosomal RNA genes Nucleic Acids Res 353100ndash3108
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B
2014 The carbohydrate-active enzymes database (CAZy) in 2013
Nucleic Acids Res 42D490ndashD495
Lowe TM Eddy SR 1997 tRNAscan-SE a program for improved detection
of transfer RNA genes in genomic sequence Nucleic Acids Res
25955ndash964
Matsuura Y Kusunoki M Harada W Kakudo M 1984 Structure
and possible catalytic residues of Taka-amylase A J Biochem
95697ndash702
Mba Medie F Davies GJ Drancourt M Henrissat B 2012 Genome anal-
yses highlight the different biological roles of cellulases Nat Rev
Microbiol 10227ndash234
Mohr KI Garcia RO Gerth K Irschik H Muller R 2012
Sandaracinus amylolyticus gen nov sp nov a starch-degrading
soil myxobacterium and description of Sandaracinaceae fam
nov Int J Syst Evol Microbiol 621191ndash1198
Noguchi J et al 2011 Crystal structure of the branching enzyme I
(BEI) from Oryza sativa L with implications for catalysis and sub-
strate binding Glycobiology 211108ndash1116
Patel RK Jain M 2012 NGS QC Toolkit a toolkit for quality control of next
generation sequencing data PLoS One 7e30619
Pei J Kim BH Grishin NV PROMALS3D a tool for multiple protein se-
quence and structure alignments Nucleic Acids Res 36 2295ndash2300
Reichenbach H Dworkin M 1992 The myxobacteria In Balows A
Truper HG Dworkin M Harder W Schleifer KH editors The pro-
karyotes a handbook on the biology of bacteria ecophysiology
isolation identification applications New York Springer New
York p 3416ndash3487
Reichenbach H 2005 ldquoOrder VIII Myxococcalesrdquo Bergeyrsquos Manual of
Systematic Bacteriology In Brenner DJ Krieg NR Staley JT Garrity
GM editors Vol 2 2nd ed part C New York Springer-Verlag New
York Inc pp 1074ndash1079
Richter M Rossello-Mora R 2009 Shifting the genomic gold standard for
the prokaryotic species definition Proc Natl Acad Sci U S A 106
19126ndash19131
Schneiker S et al 2007 Complete genome sequence of the myxobacter-
ium Sorangium cellulosum Nat Biotechnol 25 1281ndash1289
Shimkets LJ 1990 Social and developmental biology of the myxobacteria
Microbiol Rev 54 473ndash501
Siguier P Perochon J Lestrade L Mahillon J Chandler M 2006 ISfinder
the reference centre for bacterial insertion sequences Nucleic Acids
Res 34 D32ndashD36
Steinmetz H et al 2012 Indiacens A and B prenyl indoles from the
myxobacterium Sandaracinus amylolyticus J Nat Prod 75 1803ndash
1805
Tamura K Stecher G Peterson D Filipski A Kumar S 2013 MEGA6
molecular evolutionary genetics analysis version 60 Mol Biol Evol
30 2725ndash2729
Van Der Maarel MJ Van Der Veen B Uitdehaag JC Leemhuis H Dijkhuizen
L 2002 Properties and applications of starch-converting enzymes of
the alpha-amylase family J Biotechnol 94 137ndash155
Sharma et al GBE
2528 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
Weissman KJ Muller R 2010 Myxobacterial secondary metabolites
bioactivities and modes-of-action Nat Prod Rep 27 1276ndash1295
Whitworth DE 2008 Myxobacteria multicellularity and differentiation
Washington DC ASM Press American Society for Microbiology
Yin Y et al 2012 dbCAN a web resource for automated carbohydrate-
active enzyme annotation Nucleic Acids Res 40 W445ndashW451
Zhao JY et al 2008 Discovery of the autonomously replicating plasmid
pMF1 from Myxococcus fulvus and development of a gene cloning
system in Myxococcus xanthus Appl Environ Microbiol 74 1980ndash
1987
Associate editor Howard Ochman
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2529
Sandaracinaceae within order Myxococcales (Mohr et al
2012) Morphologically and biochemically the strain is a
Gram-negative aerobic rod-shaped during vegetative
states catalase-negative oxidase-positive and pigment form-
ing myxobacteria (Mohr et al 2012) This strain was isolated in
1996 from a gray-brown soil sample (having plant residues)
collection by Helmholtz Zentrums fur Infektionsforschung
[HZI] during 1995 from a botanical garden in Lucknow
Uttar Pradesh India (Reichenbach and Dworkin 1992 Mohr
et al 2012) Sandaracinus amylolyticus possesses typical myx-
obacterial characteristics including gliding motility secondary
metabolite production (Steinmetz et al 2012) and spore gen-
eration during harsh environmental conditions Here we
report the complete genome of S amylolyticus DSM 53668T
with special emphasis on the starch degrading properties
along with the protein repertoire involved in starch agarose
chitin and cellulose degradation
Materials and Methods
Culturing and Genomic DNA Isolation
Actively growing plate cultures of S amylolyticus was pro-
cured from Deutsche Sammlung von Mikroorganismen und
Zellkulturen (DSMZ) as strain number DSM 53668T [NOSO-4T]
Sandaracinus amylolyticus DSM 53668T was grown aerobi-
cally on CY agar plate culture (DSM Medium 67 as prescribed
by DSMZ culture collection) at 28ndash30 C for a minimum of 5
days The culture was yellow-orange in color attributed to the
presence of carotenoids (Mohr et al 2012) DNA for sequenc-
ing was obtained using both the Zymogen Research Bacterial
fungal DNA isolation kit and PhenolndashChloroformndashIsoamyl
Alcohol-based manual method The quantity and quality of
the extraction were checked by gel electrophoresis along with
Nanodrop method and followed by Qubit quantification
Genome Sequencing Assembly and Annotation
Sandaracinus amylolyticus was sequenced on four single mol-
ecule real-time (SMRT) cells using P6 polymerase and C4
chemistry (P6C4) on a Pacific Biosciences RSII instrument at
McGill University and Genome Quebec Innovation Center
Montreal (Quebec) Canada DNA sample was sheared and
concentrated using AMPure magnetic beads and treated by
ExoVII to remove single stranded ends SMRTbell long libraries
were constructed with 10 mg whole genomic DNA using
ldquoProcedure and Checklistndash20 kb Template Preparation Using
BluePippinTM Size Selection System protocolrdquo (httpwww
pacbcomwp-contentuploads201509Shared-Protocol-20-
kb-Template-Preparation-Using-BluePippin-Size -Selection-
System-15-kb-Size-Cutoffpdf last accessed July 5 2016)
Blunt ligation reactions were prepared and SMRTbell tem-
plates were purified using AMPure magnetic beads
BluePippinTM size selection was performed to retain long
reads (gt20000) for sequencing The size-selected SMRTbell
templates were annealed and then loaded onto four SMRT
cells and sequenced using P6C4 chemistry with 180-min
movie times Sequencing yielded a total of 241674 reads
with a mean read length of 467 kb totaling
1129177817 bp (~85 coverage) De novo assembly was
carried out using the hierarchical genome assembly process
(HGAP) protocol in SMRT Analysis v20 including consensus
polishing with Quiver (Chin et al 2013) Since the
BluePippinTM Size Selection (gt20000) protocol filters out
small DNA fragments such as plasmids Illumina paired-end
(PE) data were also obtained using the Illumina HiSeq1000
sequencing platform at the C-CAMP Bangalore India
Sequencing resulted in 13103763 PE reads (insert size 350
bp and read length 101 bp) out of which 11663870 PE
reads were selected after quality filtering [PHRED quality
score 20 minimum read length 70] using NGSQC Toolkit
v223 (Patel and Jain 2012) To trace the presence of any
plasmid the filtered Illumina reads were mapped using
CLCbio wb80 (wwwclcbiocom last accessed July 8 2016)
to the bacterial plasmid database (httpwwwebiacukge-
nomesplasmidhtml last accessed July 8 2016)
Gene prediction and functional annotation were per-
formed by Rapid Annotation using Subsystem Technology
(RAST) (Aziz et al 2008) RNAmmer 12 (Lagesen et al
2007) and tRNAscan-SE-123 (Lowe and Eddy 1997) were
used to predict rRNA and tRNA genes respectively
Phylogenetic Analysis Genome-Genome Distance andAverage Nucleotide Identity
16S rRNA genes from various strains of suborder Sorangiineae
along with neighboring families were extracted from the
National Centre for Biotechnology Information (NCBI) and
were aligned using ClustalW module of BIOEDIT sequence
alignment tool (version 7130 Hall 1999) The resulting align-
ment was used as an input in Molecular Evolutionary Genetics
Analysis (MEGA) v606 (Tamura et al 2013) to generate a max-
imum likelihood (ML) tree [model Tamura 3-param bootstrap
100] Initial tree(s) for the heuristic search were obtained by
applying the Neighbor-Joining method to a matrix of pairwise
distances estimated using the Maximum Composite Likelihood
approach In silico DNA-DNA hybridization (DDH) values among
the members of suborder Sorangiineae and other species mem-
bers of order Myxococcales were calculated using Genome-To-
Genome Distance Calculator (GGDC) server (Auch et al 2010)
Average Nucleotide Identity (ANI) matrix was also generated
among the genomes studied (Richter and Rossello-Mora 2009)
Genome and Proteome Analysis
Sandaracinus amylolyticus proteome was subjected to
hmmscan (Eddy 2011) against Hidden Markov Model
(HMM) profiles of Carbohydrate-Active enZYmes (CAZY)
database (dbCAN HMMs 30 Yin et al 2012Lombard et al
2014) The functional domains and other known sequence
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2521
motifs involved in starch cellulose chitin and agar degrada-
tion were retrieved on the basis of EC number for each
enzyme category Also the proteins of S amylolyticus were
mapped against the CAZY protein database using Basic-Local
Alignment Search Tool (BLASTp) (E-value cutoff of 1e5 min-
imum query coverage of 50 and minimum percent identity
of 35) (Altschul et al 1990) NCBI BLAST + (v 2226+) and
nonredundant protein (NR) database downloaded on
September 6 2014 was used throughout the study
Clustered regularly interspaced short palindromic repeat
(CRISPR) elements and insertion elements were identified
using CRISPRfinder (Grissa et al 2007) and ISfinder (Siguier
et al 2006) respectively
Sandaracinus amylolyticus Amylases
The a-amylases and g-amylases from S amylolyticus were sub-
jected to the BLASTp against amylase sequences from the
CAZY database (clustered at 70 sequence identity) and
top hits were retrieved The amylases from S amylolyticus
were cut into the domains and were aligned using MUltiple
Sequence Comparison by Log-Expectation (MUSCLE) (Edgar
2004) incorporated in MEGA v606 Further the evolutionary
tree was generated by ML method [model Jones Taylor and
Thorton bootstrap 100] using MEGA v606 Initial tree(s) for
the heuristic search were obtained by applying the Maximum
Parsimony method to a matrix of pairwise distances estimated
using the Maximum Composite Likelihood approach Further
the S amylolyticus GH13 amylases along with the closely re-
lated members of each phylogenetic clade were aligned using
PROfile Multiple Alignment with predicted Local Structures
and 3D constraints (PROMALS3D) with Taka-Amylase A of
Aspergillus oryzae [2TAA] (Matsuura et al 1984) as a struc-
tural template (Pei et al 2008)
Results and Discussion
Sandaracinus amylolyticus Genome Properties
Sandaracinus amylolyticus genome was assembled de novo by
HGAP v20 (Chin et al 2013) of the SMRT portal as a single
contig of 1033 Mb with no plasmid and representing the
complete genome with approximately 85 coverage and
72 G+C content (fig 1) No complete plasmid could be
retrieved from the mapping of Illumina PE reads against the
bacterial plasmid database Plasmids are rare in the order
Myxococcales and till date only one plasmid pMF1 from
Myxococcus fulvus 124B02 has been reported (Zhao et al
2008) RAST-based annotation predicted 8962 coding
genes out of which 4939 proteins (5516) were function-
ally annotated while the remaining (4484) were hypothet-
ical proteins (supplementary table S1 Supplementary Material
online) Sandaracinus amylolyticus genome has two rRNA op-
erons and 56 aminoacyl-tRNA synthetase genes
This genome encodes two sequentially identical 16S rRNA
which shows 100 identity with previously reported 16S rRNA
sequence of S amylolyticus strain NOSO-4T [NCBI Reference
Sequence NR_1180011] followed by 8798 identity with
Chondromyces apiculatus strain Cm a14 [NR_0253441]
Among the myxobacteria with sequenced genomes it shows
the maximum identity (8789) to the 16S rRNA gene of C
apiculatus DSM 436 (strain Cm a2) [GenBank M942742] fol-
lowed by 875 identity to S cellulosum 16S rRNA (Soce0157-
2 and Soce56) The 16S rRNA-based phylogeny of
Sandaracinus and related myxobacteria suggest its classification
as a separate family within the suborder Sorangiineae (supple-
mentary fig S1 Supplementary Material online) Sandaracinus
amylolyticus 16S rRNA forms a separate branch distinct from
other families in the suborder Sorangiineae phylogenetic tree
representing family Sandaracinaceae We also calculated in
silico DDH values for all available myxobacterial genomes
using the GGDC server (Auch et al 2010) Sandaracinus
amylolyticus is not closely related to other Myxococcales as
the maximum DDH value was identified to be 181 with S
cellulosum followed by 174 with C apiculatus DSM 436 sug-
gesting a distant relation to Polyangiaceae members which
further confirms its placement in a separate family
Sandaracinaceae ANI calculations likewise support the above
conclusions (supplementary table S2 Supplementary Material
online)
The members of suborder Sorangiineae such as the S cel-
lulosum species have been well characterized for degrading
cellulose and other complex polysaccharides (Schneiker et al
2007 Han et al 2013) Similarly S amylolyticus has been
reported as the only myxobacteria capable of degrading
starch (Mohr et al 2012) Thus in order to gain insights into
the genes involved in the degradation of complex polysaccha-
rides especially starch the complete genome of S amylolyticus
was determined and investigated for the presence of carbo-
hydrate active enzymes
Sandaracinus amylolyticus CAZome
Two-hundred and forty-three proteins (~27 of total prote-
ome) of S amylolyticus were annotated with diverse combi-
nations of CAZY domains that consist of 72 GT domains 72
CE domains 58 GH domains 19 AA domains 4 PL domains
and 26 CBM domains (fig 2A) Fourteen out of these 243
proteins contained more than one CAZY domain whereas
the rest had only a single CAZY domain In multiple CAZY-
domain containing proteins various carbohydrate binding
modules such as CBM48 CBM50 and CBM21 are pre-
sent along with other CAZY category proteins for assisting in
their functions The CAZyme distribution observed in
S amylolyticus indicates its ability to degrade various
types of biomass Here we examine the S amylolyticus pro-
teome involved in starch agar chitin and cellulose
degradation
Sharma et al GBE
2522 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
Evolutionary History of S amylolyticus Amylases
Amylases belonging to glycoside hydrolase (GH) enzymatic
category degrade starch into sugar moieties by the
hydrolysis of a-14- and a-16-glycosidic bond (Van der
Maarel et al 2002) Based on the site of action they are di-
vided into three categories a-amylase (EC3211 GH13
GH57 GH119 GH126) b-amylase (EC3212 GH14) and
-amylase (EC3213 GH15 GH97) Sandaracinus
amylolyticus genome contains both a- and -amylases
whereas no b-amylases could be found Among the 4 a-am-
ylases GH families 15 GH13 proteins and 1 GH57 protein
were found We could identify two -amylases belonging to
Sandaracinus amylolyticusTDSM 53668
CP01112510328378 bp
FIG 1mdashCircular representation of the Sandaracinus amylolyticus DSM 53668T complete genome Circles (from inside to outside) 1 and 2 (GC content
black line and GC skew magenta and green lines) circle 3 (CAZome Proteins annotated as carbohydrate active enzymes Red) circle 4 5 and 6 (Amylases
Cellulases and Chitinases Black) circle 7 (encoded RNA Magenta) circle 8 (CDS present on negative strand Blue) circle 9 (CDS present on positive strand
Green) circle 10 (S amylolyticus DSM 53668T complete genome Red) BRIG 095 was used to build the circular representation (Alikhan et al 2011)
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2523
GH15 category (fig 2B) The starch degrading proteins have
been reported to have CBM20 CBM21 CBM25 CBM26
CBM34 CBM41 CBM45 CBM48 CBM53 and CBM58
modules that help in glycogenstarch binding truncation of
which reduces its enzymatic activity (Noguchi et al 2011
Janecek et al 2014) GH15 and GH57 domains are present
as single domain proteins in S amylolyticus Interestingly in 5
out of 15 GH13 S amylolyticus proteins (AKF03796
AKF03860 AKF06952 AKF07413 and AKF11765) the a-
amylase GH13 domain was present along with a CBM48
module at the N-termini The CBM48 module assists in the
GH13 function by serving as starch-binding domains
The GH13 family include the a-amylase domains with an (b
a)8 barrel catalytic domain These proteins have 4ndash7 conserved
sequence regions (CSRs) and a specific catalytic triad consisting
of a catalytic nucleophile (aspartic acid) on strand b4 proton
donor (glutamic acid) on strand b5 and a transition state sta-
bilizer (aspartic acid) on strand b7 (Janecek et al 2014) GH13
members show extremely low sequence identity with their ho-
mologs but the catalytic triad is highly conserved GH57 family
a-amylases have a (ba)7 barrel structure and have five CSRs as
compared with the GH13 family However only two of the
catalytic residues viz the nucleophile on strand b4 and the
proton donor on strand b7 are conserved (Blesak and Janecek
2012) Irrespective of these differences GH13 and GH57 both
employ the same retaining mechanism of action (Janecek and
Kuchtova 2012) Sandaracinus amylolyticus GH13 proteins
were retrieved and the structure of Taka amylase of A
oryzae (PDBid 2TAA) was used to perform a structure-based
alignment using PROMALS3D (Matsuura et al 1984 Pei et al
2008) All the seven CSR regions and the three catalytic
residues were conserved in all S amylolyticus GH13 amylases
(supplementary figs S2 and S3 Supplementary Material
online) Similarly S amylolyticus GH57 (AKF06951) was
aligned with its homologs using PROMALS3D based on the
4-a-glucanotransferase structure of Thermococcus litoralis
(PDBid 1K1W) In the GH57 alignment all five CSRs and
both catalytic residues were conserved (supplementary figS4
Supplementary Material online)
In order to understand the provenance of these a- and g-
amylases they were subjected to BLASTp against the NR data-
base To include amylases across the entire bacterial phylum
the CAZY-NR database for GH13 and GH57 domains were
retrieved and subjected to BLASTClust at 70 identity and
70 length coverage that reduced the domain sequence
space from 19590 to 4038 domains Further amylase do-
mains from S amylolyticus were subjected to BLASTp against
these 4038 domains and their top hits were retrieved and
included in the phylogenetic analysis All these GH13 and
GH57 domains along with S amylolyticus amylase domains
were aligned using MUSCLE as incorporated in MEGAv606
and ML-based phylogeny with 100 bootstrap values was
drawn (fig 3) The ML tree obtained was divided into three
major clades where the clade at root composed of all the GH57
domains and the other two major clades further composed of
all the GH13 sequences The representative bacterial amylases
from various clades show close relations with amylases of di-
verse bacterial groups suggesting the multiple gain and loss of
these domains As all the amylase domains of S amylolyticus
are scattered across the tree the phylogenetic position of these
domains could provide us insights about their provenance and
reveal possible cases of horizontal transfers
19
26
72
58
72
4
0
10
20
30
40
50
60
70
80
AA CBM CE GH GT PL
A B
Num
ber o
f pro
tein
s
GH13
GH13
CBM48 GH13
GH13CBM48
GH13
GH57
GH13CBM48
GH13GH13CBM48
GH13
GH13
GH13
GH15
GH13CBM48
GH13
AKF03605AKF03642AKF03796
AKF03860AKF03861AKF03862AKF04017AKF06951AKF06952
AKF07406AKF07413AKF07527AKF09634AKF09643AKF10340AKF10487AKF11765AKF11772
GH13
GH13
GH15
FIG 2mdashSandaracinus amylolyticus DSM 53668T CAZome distribution (A) Sandaracinus amylolyticus DSM 53668T CAZY families presented in a bar
graph x axis represents the CAZY categories and y axis represents the number of proteins (B) a- and g-amylases along with their CAZome domain
architecture The domains have been colored as GH13 sky blue GH15 dark red GH57 green and CBM48 orange
Sharma et al GBE
2524 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
Taking into account the GH57 clade the sole GH57
domain of S amylolyticus (GenBank ID AKF06951) shares
the clade with anaerobic Deltaproteobacteria Among the
GH13 domains of S amylolyticus AKF03862 AKF07527
AKF09643 AKF11765 and AKF11772 shares clade with
other myxobacteria Among these AKF11772 and
AKF07527 share sister branches and form sister clades with
other myxobacterial species such as S cellulosum Soce56 and
Plesiocystis pacifica SIR-1 and are further related to the clade
containing Gammaproteobacteria Apart from these two am-
ylases AKF03860 and AKF06952 are similar to each other and
group with Candidatus accumulibacter phosphtis amylase
(YP_0031672861) from the phylum Acidobacteria
Interestingly AKF03860 and AKF06952 group with members
GH13 ZP 021632211 Kordia algicida OT-1
GH13 ZP 067516041 Parascardovia denticolens F0305
GH13 YP 0035709041 Salinibacter ruber M8
GH13 YP 0032500041 Fibrobacter succinogenes subsp succinogenes S85
GH13 AKF03862 Sandaracinus amylolyticus DSM 53668
GH13 YP 0039537801 Stigmatella aurantiaca
GH13 CAJ738231 Candidatus Kuenenia stuttgartiensis
GH13 YP 0228461 Picrophilus torridus DSM 9790GH13 ZP 029306261 Verrucomicrobium spinosum DSM 4136
GH13 CAX841261 uncultured bacterium
GH13 YP 0031678771 Candidatus Accumulibacter phosphatis clade IIA str UW-1GH13 ZP 031321321 Chthoniobacter flavus Ellin428
GH13 ZP 018759791 Lentisphaera araneosa HTCC2155GH13 YP 0033894351 Spirosoma linguale DSM 74GH13 NP 7734061 Bradyrhizobium diazoefficiens USDA 110
GH13 YP 0037757321 Herbaspirillum seropedicae SmR1GH13 YP 4266911 Rhodospirillum rubrum ATCC 11170GH13 ZP 035079501 Rhizobium etli Brasil 5
GH13 ZP 057930691 Butyrivibrio crossotus DSM 2876
GH13 ZP 066231321 Turicibacter sanguinis PC909
GH13 ZP 044499741 Catonella morbi ATCC 51271
GH13 EGE534241 Streptococcus parauberis NCFD 2020
GH13 196049865 Lactobacillus Plantarum
GH13 YP 0042196261 Granulicella tundricola MP5ACTX9
GH13 YP 0011935611 Flavobacterium johnsoniae UW101
GH13 YP 0032910851 Rhodothermus marinus DSM 4252GH13 YP 0040967591 Bacillus cellulosilyticus DSM 2522GH13 ZP 037030351 Flavobacteria bacterium MS024-2A
GH13 XP 0025352531 Ricinus communis
GH13 YP 7579561 Maricaulis maris MCS10GH13 ZP 010417351 Erythrobacter sp NAP1GH13 YP 0018177571 Opitutus terrae PB90-1GH13 EFR587141 Alistipes sp HGB5
GH13 CBK627851 Alistipes shahii WAL 8301GH13 EFR582631 Alistipes sp HGB5
GH13 YP 0034042011 Haloterrigena turkmenica DSM 5511
GH13 YP 0035357541 Haloferax volcanii DS2
GH13 YP 0035357541 Haloferax volcanii DS2
GH13 YP 0025661621 Halorubrum lacusprofundi ATCC 49239
GH13 YP 1368761 Haloarcula marismortui ATCC 43049
GH13 ZP 080436741 Haladaptatus paucihalophilus DX253
GH13 YP 0025652161 Halorubrum lacusprofundi ATCC 49239
GH13 YP 0035346301 Haloferax volcanii DS2
GH13 YP 0033225661 Thermobaculum terrenum ATCC BAA-798
GH13 YP 0021353421 Anaeromyxobacter sp K
GH13 AKF09643 Sandaracinus amylolyticus DSM 53668
GH13 YP 0016137031 Sorangium cellulosum So ce56
GH1
3 99
0316
21 H
alot
herm
othr
ix or
enii
GH1
3 AA
D051
991
Pae
niba
cillu
s po
lymyx
a
GH1
3 YP
003
6999
601
Bac
illus
sele
nitir
educ
ens
MLS
10
GH1
3 ZP
022
0799
81
Copr
ococ
cus
euta
ctus
ATC
C 27
759
GH
13 B
AA07
401
1 Xa
ntho
mon
as c
ampe
stris
GH
13 Y
P 00
4052
835
1 M
ariv
irga
tract
uosa
DSM
412
6
GH1
3 ZP
037
0954
51
Cory
neba
cter
ium
mat
ruch
otii A
TCC
3380
6
GH1
3 NP
267
649
1 La
ctoc
occu
s la
ctis
subs
p la
ctis
Il140
3
GH1
3 YP
001
3023
201
Par
abac
tero
ides
dist
ason
is AT
CC 8
503
GH1
3 YP
001
2276
361
Syn
echo
cocc
us s
p R
CC30
7
GH1
3 YP
002
5652
591
Hal
orub
rum
lacu
spro
fund
i ATC
C 49
239
GH13
ZP
0709
0550
1 C
oryn
ebac
teriu
m g
enita
lium
ATC
C 33
030
GH13
YP 0
0283
4100
1 C
oryn
ebac
teriu
m a
urim
ucos
um A
TCC
7009
75
GH13 A
KF03
861
Sand
arac
inus a
mylo
lyticu
s DSM
536
68
GH13
ZP 0
0050
865
1 M
agne
tosp
irillum
mag
neto
tacti
cum
MS-
1
GH13 Y
P 00
1568
811
1 Pe
troto
ga m
obilis
SJ9
5
GH13 Y
P 02
2847
1 P
icrop
hilus
torri
dus D
SM 9
790
GH13 E
FT25
5011
Pro
pionib
acter
ium ac
nes H
L110
PA3
GH13 E
FA80
1951
Poly
spho
ndyli
um pa
llidum
PN50
0
GH13 A
BD4655
81 V
ermam
oeba
verm
iform
is
GH13 Z
P 0350
5050
1 Rhiz
obium
etli B
rasil
5
GH13 YP 00
1348
4651
Pseud
omon
as ae
rugino
sa PA7
GH13 YP 00
2238
3511
Klebsie
lla pn
eumon
iae 34
2
GH13 YP 00
1437
9781
Cron
obac
ter sa
kaza
kii ATCC B
AA-894
GH13 Y
P 0011
7103
71 P
seud
omon
as st
utzeri
A1501
GH13 Y
P 0019
7240
81 S
tenotr
opho
monas
malt
ophil
ia K27
9a
GH13 Y
P 0043
1901
11 S
phing
obac
terium
sp 2
1
GH13 YP 0013127791 Sinorhizobium m
edicae W
SM419
GH13 ZP 035028921 Rhizobium etli K
im 5
GH13 YP 67
6219
1 Che
lativo
rans s
p BNC1
GH13 YP 00
2376
8861
Cya
nothe
ce sp
PCC 74
24
GH13 YP 0036383061 Cellulomonas flavig
ena DSM 20109
GH13 YP 0040977291 Intrasporangium ca
lvum DSM 43043
GH13 YP 0011691691 Rhodobacter s
phaeroides ATCC 17025
GH13 ZP 011580901 Oceanicola granulosus H
TCC2516
GH13 YP 0038382651 Micromonospora aurantiaca ATCC 27029
GH13 YP 0040977251 Intrasporangium calvu
m DSM 43043
GH13 YP 0036954761 Starkeya novella DSM 506
GH13 ZP 035166441 Rhizobium etli IE4771
GH13 ZP 031273131 Chthoniobacter flavus Ellin
428
GH13 AKF03642 Sandaracinus amylolyticus DSM 53668
GH13 YP 0031252671 Chitinophaga pinensis DSM 2588
GH13 YP 0030850521 Dyadobacter fermentans DSM 18053
GH13 YP 0033879241 Spirosoma linguale DSM 74
GH13 YP 0034301801 Streptococcus gallolyticus UCN34
GH13 ZP 078184041 Eremococcus coleocola ACS-139-V-Col8
GH13 YP 0034774501 Thermoanaerobacter italicus Ab9
GH13 ZP 048517861 Paenibacillus sp oral taxon 786 str D14
GH13 YP 0038414241 Caldicellulosiruptor obsidiansis OB47
GH13 YP 0024279661 Desulfurococcus kamchatkensis 1221n
GH13 YP 0034262831 Bacillus pseudofirmus OF4
GH13 ZP 076854311 Oscillochloris trichoides DG-6GH13 YP 0016174961 Sorangium cellulosum So ce56
GH13 ZP 019113371 Plesiocystis pacifica SIR-1
GH13 YP 6318811 Myxococcus xanthus DK 1622
GH13 AKF11765 Sandaracinus amylolyticus DSM 53668
GH13 ZP 036771121 Bacteroides cellulosilyticus DSM 14838
GH13 YP 0037157841 Croceibacter atlanticus HTCC2559
GH13 ZP 012015681 Flavobacteria bacterium BBFL7
GH13 ZP 019113321 Plesiocystis pacifica SIR-1GH13 YP 0031962511 Robiginitalea biformata HTCC2501
GH13 YP 0040531921 Marivirga tractuosa DSM 4126
GH13 ZP 021817881 Flavobacteriales bacterium ALC-1
GH13 CBL150401 Ruminococcus bromii L2-63
GH13 ZP 080051001 Bacillus sp 2 A 57 CT2
GH13 CBK911271 Eubacterium rectale DSM 17629
GH13 YP 0029369771 Eubacterium rectale ATCC 33656
GH13 YP 0021366011 Anaeromyxobacter sp K
GH13 ZP 014604541 Stigmatella aurantiaca
GH
13 728851 Streptomyces lividans
GH
13 BAJ281541 Kitasatospora setae KM-6054
GH
13 YP 0033390201 Streptosporangium roseum
DSM
43021
GH
13 YP 9214291 Nocardioides sp JS614
GH
13 AEB462451 Verrucosispora maris AB-18-032
GH
13 CAJ810301 Actinoplanes sp
GH
13 NP 9387401 C
orynebacterium diphtheriae N
CTC
13129
GH
13 YP 0036976141 Arcanobacterium haem
olyticum D
SM 20595
GH
13 ZP 039259261 Actinomyces coleocanis D
SM 15436
GH
13 YP 0040999421 Intrasporangium calvum
DSM
43043
GH
13 YP 0030989701 Actinosynnema m
irum D
SM 43827
GH
13 YP 0036376081 Cellulom
onas flavigena DSM
20109
GH
13 ZP 009938561 Janibacter sp HTC
C2649
GH13 YP 0030608421 Hirschia baltica ATCC 49814
GH13 YP 7579631 M
aricaulis maris M
CS10
GH13 YP 0016857121 Caulobacter sp K31
GH13 ZP 013016731 Sphingom
onas sp SKA58
GH13 YP 0038198341 Brevundim
onas subvibrioides ATCC 15264
GH
13 YP 4971641 Novosphingobium
aromaticivorans D
SM 12444
GH13 YP 6609551 Pseudoalterom
onas atlantica T6c
GH13 ZP 011126451 Reinekea blandensis M
ED297
GH13 YP 2677341 Colwellia psychrerythraea 34H
GH13 ZP 011126461 Reinekea blandensis M
ED297
GH13 AKF11772 Sandaracinus amylolyticus DSM
53668
GH13 AKF07527 Sandaracinus amylolyticus DSM
53668
GH13 YP 0016184291 Sorangium cellulosum
So ce56
GH13 ZP 019113391 Plesiocystis pacifica SIR-1
GH13 ZP 036315591 Pedosphaera parvula Ellin514
GH13 YP 0016604791 Microcystis aeruginosa NIES-843
GH13 YP 0043416831 Archaeoglobus veneficus SNP6
GH13 ZP 073351521 Desulfovibrio fructosovorans JJ
GH
13 AKF03605 Sandaracinus amylolyticus D
SM 53668
GH
13 YP 0027853821 Deinococcus deserti VC
D115
GH
13 NP 2951951 D
einococcus radiodurans R1
GH
13 YP 004171
1212
M
SD
sisn
epoc
ira
m su
ccoc
onie
D 1
661
1
GH
13 YP 0041715811 Deinococcus m
aricopensis DSM
21211
GH
13 YP 0037038171 Truepera radiovictrix DSM
17093
GH
13 YP 6052101 Deinococcus geotherm
alis DSM
11300
GH
13 YP 0027555491 A
cidobacterium capsulatum
ATCC
51196
GH
13 Y
P9
XTC
A5P
M alocirdnut allecilunarG 1860612400
GH
13 YP 6335241 M
yxococcus xanthus DK
1622G
H13 Y
PB
S su
cihp
ortid
ica
suhp
ortny
S 1
9412
64
GH
13 A
AW32
490
1 iiklahcsttog acnarborean
A
GH
13 Y
P 00
2316
532
1 An
oxyb
acillu
s fla
vith
erm
us W
K1
GH
13 Y
P 00
3252
634
1 G
eoba
cillu
s sp
Y41
2MC
61G
H13
ZP
0117
0936
1 B
acillu
s sp
NR
RL
B-14
911
GH
13 Z
P 07
7074
981
Bac
illus
sp m
3-13
GH
13 Z
P1-
GS ps sullica
B 116916810
GH
13 Y
P 00
1813
251
1 Ex
iguo
bact
eriu
m s
ibiri
cum
255
-15
GH
13 Y
P 00
2886
871
1 Ex
iguo
bact
eriu
m s
p A
T1b
GH
13 Y
P 00
1568
454
1 Pe
troto
ga m
obilis
SJ9
5
GH
13 Y
P 00
2940
188
1 Ko
smot
oga
olea
ria T
BF 1
95
1G
H13
ZP
0125
8420
1 V
ibrio
alg
inol
ytic
us 1
2G01
GH
13 1
0911
18 T
herm
oact
inom
yces
vul
garis
GH
13 Z
P 06
9705
751
Kte
dono
bact
er ra
cem
ifer D
SM 4
4963
GH
13 Y
P 00
1545
735
1 H
erpe
tosi
phon
aur
antia
cus
DSM
785
GH
13 E
FY95
778
1 M
etar
hizi
um ro
berts
ii AR
SEF
23
GH
13 N
P 58
8205
1 S
chiz
osac
char
omyc
es p
ombe
972
h-
GH
13 N
P 59
4401
1 S
chiz
osac
char
omyc
es p
ombe
972
h-
GH
13 X
P 00
1908
940
1 Po
dosp
ora
anse
rina
S m
at+
GH
13 4
1016
824
Schw
anni
omyc
es o
ccid
enta
lis
GH
13 B
AF98
616
1 H
ypho
pich
ia b
urto
nii
GH13 AKF07406 Sandaracinus amylolyticus DSM 53668
GH13 YP 0043692881 Desulfobacca acetoxidans DSM 11109
GH13 YP 0023716111 Cyanothece sp PCC 8801GH13 YP 3219451 Anabaena variabilis ATCC 29413
GH13 YP 4738811 Synechococcus sp JA-3-3AbGH13 YP 8464671 Syntrophobacter fumaroxidans MPOBGH13 ZP 063702061 Desulfovibrio sp FW1012B
GH13 YP 0033193471 Sphaerobacter thermophilus DSM 20745GH13 YP 0037057361 Truepera radiovictrix DSM 17093GH13 YP 4120971 Nitrosospira multiformis ATCC 25196GH13 YP 6431071 Rubrobacter xylanophilus DSM 9941
GH13 BAD389831 Rhodobacter sphaeroides f sp denitrificans
GH13 YP 3175601 Nitrobacter winogradskyi Nb-255
GH13 YP 8215181 Candidatus Solibacter usitatus Ellin6076
GH13 YP 0034506361 Azospirillum sp B510
GH13 ZP 070169471 Desulfonatronospira thiodismutans ASO3-1
GH13 YP 0041842351 Terriglobus saanensis SP1PR4
GH13 ZP 070315011 Granulicella mallensis MP5ACTX8
GH13 YP 0029438961 Variovorax paradoxus S110
GH13 YP 5918381 Candidatus Koribacter versatilis Ellin345
GH13 YP 7445681 Granulibacter bethesdensis CGDNIH1
GH13 YP 0039098001 Burkholderia sp CCGE1003
GH13 ZP 024903661 Burkholderia pseudomallei NCTC 13177
GH13 YP 0016309761 Bordetella petrii DSM 12804
GH13 YP 0022527351 Ralstonia solanacearum MolK2
GH13 YP 0037759061 Herbaspirillum seropedicae SmR1
GH13 YP 5455251 Methylobacillus flagellatus KT
GH13 YP 0013485041 Pseudomonas aeruginosa PA7
GH13 YP 0016008321 Gluconacetobacter diazotrophicus PA1 5
GH13 AEB584851 Pseudomonas mendocina NK-01
GH13 YP 0027996471 Azotobacter vinelandii DJ
GH
13 N
P 63
5804
1 X
anth
omon
as c
ampe
stris
pv
cam
pest
ris s
tr A
TCC
339
13
GH
13 Y
P 00
3188
581
1 Ac
etob
acte
r pas
teur
ianu
s IF
O 3
283-
01
GH
13 Y
P 74
4570
1 G
ranu
libac
ter b
ethe
sden
sis
CG
DN
IH1
GH
13 Y
P 00
3052
329
1 M
ethy
lovo
rus
gluc
oset
roph
us S
IP3-
4
GH
13 Z
P 06
8981
631
Ros
eom
onas
cer
vica
lis A
TCC
499
57
GH
13 Y
P 00
2943
895
1 Va
riovo
rax
para
doxu
s S1
10
GH
13 Y
P 31
4931
1 T
hiob
acillu
s de
nitri
fican
s AT
CC
252
59
GH
13 Y
P 42
5597
1 R
hodo
spiri
llum
rubr
um A
TCC
111
70
GH
13 Y
P 93
3302
1 A
zoar
cus
sp B
H72
GH
13 N
P 43
7816
1 S
inor
hizo
bium
mel
iloti
1021
GH
13 Z
P 01
1553
281
Oce
anic
ola
gran
ulos
us H
TCC
2516
GH
13 Y
P 00
1167
979
1 R
hodo
bact
er s
phae
roid
es A
TCC
170
25
GH
13 Y
P 00
1440
575
1 C
rono
bact
er s
akaz
akii A
TCC
BAA
-894
GH
13 Z
P 01
4401
991
Ful
vim
arin
a pe
lagi
HTC
C25
06
GH
13 Y
P 00
4234
793
1 Ac
idov
orax
ave
nae
subs
p a
vena
e AT
CC
198
60
GH
13 Y
P 00
1818
894
1 O
pitu
tus
terra
e PB
90-1
GH
13 Y
P 00
3154
808
1 Br
achy
bact
eriu
m fa
eciu
m D
SM 4
810
GH
13 ZP 010940301 Blastopirellula marina D
SM 3645
GH
13 ZP 027320551 Gem
mata obscuriglobus U
QM
2246
GH
13 YP 4273821 Rhodospirillum
rubrum ATC
C 11170
GH
13 YP 0021361131 Anaeromyxobacter sp K
GH
13 YP 0039532711 Stigmatella aurantiaca
GH
13 YP 0021384951 Geobacter bem
idjiensis BemG
H13 Y
P23
-C
RF ii
notl
ad r
etca
boe
G 1
3868
3520
0 G
H13 Y
P65
ec o
S mu
solu
llec
mu
igna
roS
191
8716
100
GH
13 ZPB2
101
WF
ps o
irbi
vofl
use
D 1
5398
6360
GH
13 Y
P 82
1516
1 C
andi
datu
s S
olib
acte
r usi
tatu
s E
llin6
076
GH
13 Y
P24
NFC ilte
muibozihR 1077074
GH
13
86635 M
SD sucityloly
ma sunicaradnaS 43690F
KA
GH
13 Y
P 00
1820
541
1 O
pitu
tus
terra
e P
B90
-1G
H13
ZP
0313
0066
1 C
htho
niob
acte
r fla
vus
Ellin
428
GH
13 YP 0035707081 Salinibacter ruber M8
GH
13 YP 0037057351 Truepera radiovictrix DSM
17093
GH
13 YP 0032689441 Haliangium
ochraceum D
SM 14365
GH
13 YP 0016130311 Sorangium cellulosum
So ce56
GH
13 YP 6288101 Myxococcus xanthus D
K 1622
GH
13 62738967 Deinococcus radiodurans
GH
13 AKF04017 Sandaracinus amylolyticus D
SM 53668
GH
13 YP 0027878991 Deinococcus deserti VC
D115
GH13 YP 5893201 Candidatus Koribacter versatilis Ellin345
GH13 ZP 063702081 Desulfovibrio sp FW
1012B
GH13 YP 0041842341 Terriglobus saanensis SP1PR4
GH13 YP 8731321 Acidotherm
us cellulolyticus 11B
GH13 ZP 070169501 Desulfonatronospira thiodism
utans ASO3-1
GH13 ADX845721 Sulfolobus islandicus REY15A
GH13 ZP 027318151 G
emm
ata obscuriglobus UQM
2246
GH13 YP 0016347811 Chloroflexus aurantiacus J-10-fl
GH
13 NP 9534051 G
eobacter sulfurreducens PCA
GH
13 YP 0025366761 Geobacter daltonii FR
C-32
GH
13 YP 0012121361 Pelotomaculum
thermopropionicum
SI
GH
13 YP 0041781661 Isosphaera pallida ATCC
43644
GH
13 ZP 036310341 Pedosphaera parvula Ellin514
GH13 ZP 031287911 Chthoniobacter flavus Ellin428
GH13 NP 6913271 Oceanobacillus ih
eyensis HTE831
GH13 YP 0025601851 Macro
coccus c
aseolyticus J
CSC5402
GH13 YP 0030110001 Paenibacill
us sp J
DR-2
GH13 CAJ384141 Anaerobranca
gottsch
alkii
GH13 YP 00
1988
1311
Lacto
bacill
us ca
sei B
L23
GH13 YP 00
2459
6561
Des
ulfito
bacte
rium ha
fnien
se D
CB-2
GH13 YP 00
3947
0781
Paenib
acillu
s poly
myxa S
C2
GH13 Y
P 0013
1197
11 C
lostrid
ium be
ijerin
ckii N
CIMB 80
52
GH13 Y
P 0039
6683
31 Ily
obac
ter po
lytrop
us D
SM 2926
GH13 Z
P 0539
1277
1 Clos
tridium
carb
oxidi
vora
ns P
7
GH13 Y
P 0013
9686
01 C
lostrid
ium kl
uyve
ri DSM 55
5
GH13 ZP 02
2128
451
Clostrid
ium ba
rtlettii
DSM 16
795
GH13 ZP 04
4331
841
Bacillu
s coa
gulan
s 36D
1
GH13 C
BL37
8151
Ana
eros
tipes
hadr
us
GH13 Z
P 04
4519
311
Abiotro
phia
defec
tiva A
TCC 49
176
GH13
YP
0031
4407
51
Slac
kia h
eliot
rinire
duce
ns D
SM 2
0476
GH13
ZP 0
2025
300
1 Eu
bacte
rium
vent
riosu
m A
TCC
2756
0
GH1
3 YP
004
3093
801
Clo
strid
ium
lent
ocel
lum
DSM
542
7
GH13
YP 5
1565
71
Chlam
ydop
hila
felis
GH13
ZP 0
6299
217
1 Pa
rach
lamyd
ia ac
anth
amoe
bae
str H
allrsquos
cocc
us
GH13
CBL1
5412
1 R
umino
cocc
us b
rom
ii L2-
63
GH13 Y
P 00
1692
347
1 Fi
nego
ldia
mag
na AT
CC 293
28
GH13 Z
P 07
5262
211
Pep
tostr
epto
cocc
us st
omat
is DSM
176
78
GH1
3 YP
003
4751
021
Clo
strid
iale
s ge
nom
osp
BVA
B3 s
tr U
PII9
-5
GH1
3 YP
004
1716
241
Dei
noco
ccus
mar
icope
nsis
DSM
212
11
GH1
3 YP
022
845
1 Pi
crop
hilu
s to
rridu
s DS
M 9
790
GH1
3 YP
001
8207
181
Opi
tutu
s te
rrae
PB90
-1
GH1
3 YP
003
1672
861
Can
dida
tus A
ccum
ulib
acte
r pho
spha
tis c
lade
IIA
str
UW-1
GH1
3 AK
F038
60 S
anda
racin
us a
mylo
lyticu
s DS
M 5
3668
GH1
3 AK
F069
52 S
anda
racin
us a
mylo
lyticu
s DS
M 5
3668
GH
13 Y
P 00
2762
222
1 G
emm
atim
onas
aur
antia
ca T
-27
GH1
3 ZP
082
0671
01
Gor
doni
a ne
ofel
ifaec
is NR
RL B
-593
95
GH
13 Y
P 00
3964
374
1 Ke
togu
loni
cige
nium
vul
gare
Y25
GH13 ZP 045772811 Oxalobacter fo
rmigenes H
OxBLS
GH13 YP 0033856211 Spirosoma linguale DSM 74
GH13 AEA650561 Burkholderia gladioli B
SR3
GH13 YP 0030725361 Teredinibacter turnerae T7901
GH13 YP 4255961 Rhodospirillum ru
brum ATCC 11170
GH13 YP 0010427851 Rhodobacter sphaeroides ATCC 17029
GH13 ZP 075890671 Sinorhizobium meliloti BL225C
GH13 YP 0030504181 Methylovorus glucosetrophus SIP3-4
GH13 YP 4224271 Magnetospirillum magneticum AMB-1
GH13 YP 0042554001 Deinococcus proteolyticus MRP
GH13 ZP 029300551 Verrucomicrobium spinosum DSM 4136
GH13 ZP 080190951 Lautropia mirabilis ATCC 51599
GH13 YP 5335351 Rhodopseudomonas palustris BisB18
GH13 YP 8654291 Magnetococcus marinus MC-1
GH13 YP 0028938741 Tolumonas auensis DSM 9187
GH13 YP 6612131 Pseudoalteromonas atlantica T6c
GH13 YP 0040654571 Pseudoalteromonas sp SM9913
GH13 YP 8302311 Arthrobacter sp FB24
GH13 YP 0042347941 Acidovorax avenae subsp avenae ATCC 19860
GH13 YP 0016390191 Methylobacterium extorquens PA1
GH13 ZP 068060481 Brevibacterium mcbrellneri ATCC 49030
GH13 YP 0031655431 Candidatus Accumulibacter phosphatis clade IIA str UW-1
GH13 YP 9813411 Polaromonas naphthalenivorans CJ2
GH13 ZP 029305921 Verrucomicrobium spinosum DSM 4136
GH13 YP 8283661 Candidatus Solibacter usitatus Ellin6076
GH13 YP 0015687661 Petrotoga mobilis SJ95
GH13 YP 2560581 Sulfolobus acidocaldarius DSM 639
GH13 YP 0023732201 Cyanothece sp PCC 8801
GH13 AKF07413 Sandaracinus amylolyticus DSM 53668
GH13 AKF03796 Sandaracinus amylolyticus DSM 53668
GH13 EFS357291 Propionibacterium acnes HL013PA1
GH13 YP 0033827501 Kribbella flavida DSM 17836
GH13 YP 9230111 Nocardioides sp JS614
GH13 AEA239681 Pseudonocardia dioxanivorans CB1190
GH13 YP 0031037201 Actinosynnema mirum DSM 43827
GH57 YP 0018695221 Nostoc punctiforme PCC 73102
GH57 YP 0041847961 Terriglobus saanensis SP1PR4
GH57 YP 0033170621 Thermanaerovibrio acidaminovorans DSM 6589
GH57 YP 0025231331 Thermomicrobium roseum DSM 5159
GH57 CBL286501 Fretibacterium fastidiosum
GH57 YP 0041781671 Isosphaera pallida ATCC 43644
GH57 YP 0027549471 Acidobacterium capsulatum ATCC 51196
GH57 ZP 070292791 Granulicella mallensis MP5ACTX8
GH57 YP 0018749261 Elusimicrobium minutum Pei191
GH57 ADL259941 Fibrobacter succinogenes subsp succinogenes S85
GH57 YP 4773871 Synechococcus sp JA-2-3B
GH57 YP 8454221 Syntrophobacter fumaroxidans MPOB
GH57 NP 9534062 Geobacter sulfurreducens PCA
GH57 YP 5020321 Methanospirillum hungatei JF-1
GH57 YP 0019958521 Chloroherpeton thalassium ATCC 35110
GH57 YP 5937791 Candidatus Koribacter versatilis Ellin345
GH57 YP 0010471761 Methanoculleus marisnigri JR1
GH57 YP 0014309471 Roseiflexus castenholzii DSM 13941
GH57 YP 0029516361 Desulfovibrio magneticus RS-1
GH57 YP 0038064671 Desulfarculus baarsii DSM 2075
GH57 YP 0013789531 Anaeromyxobacter sp Fw109-5
GH57 ZP 063702071 Desulfovibrio sp FW1012B
GH57 YP 0035531061 Aminobacterium colombiense DSM 12261
GH57 ZP 064391271 Anaerobaculum hydrogeniformans ATCC BAA-1850
GH57 AKF06951 Sandaracinus amylolyticus DSM 53668
GH57 ZP 063915611 Dethiosulfovibrio peptidovorans DSM 11002
GH57 YP 0024634161 Chloroflexus aggregans DSM 9485
GH57 YP 0043692891 Desulfobacca acetoxidans DSM 11109
GH57 YP 8464661 Syntrophobacter fumaroxidans M
POB
GH57 CAJ738171 Candidatus Kuenenia stuttgartiensis
GH57 YP 0031969181 Desulfohalobium
retbaense DSM 5692
FIG 3mdashPhylogeny of Sandaracinus amylolyticus GH13 and GH57 a-amylases Sandaracinus amylolyticus a-amylases (GH13 and GH57) are colored in
pink along with their domain architecture displayed as Domain Color Shape [GH13 Red rectangle GH57 Black rectangle GH77 dark pink rectangle
CBM48 Dark blue circle CBM20 green circle CBM41 cyan circle] Myxobacterial homologs are colored fluorescent green Bootstrap values correspond-
ing to the tree nodes are provided
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2525
of the phyla Betaproteobacteria suggestive of a possible hor-
izontal transfer
AKF04017 and AKF03605 have close homologs in the ex-
tremophile Deinococcus desserti VCD115 and shares clade
with certain myxobacterial amylases belonging to
Myxococcus xanthus S cellulosum and Haliangium ocraceum
which further share clades with Deinococcus amylases
AKF04017 and AKF03605 share the highest sequence identity
with D desserti VCD115 plasmid protein (YP_002787899
533) and chromosomal protein (YP_002785382 456)
respectively that might point to its plasmid-borne horizontal
transfer across the clade (fig 3) The sequence conservation of
CSRs and the high percentage identity of these amylases to
Deinococcus support the inference for the putative horizontal
transfers among these members AKF09634 does not share its
sister branch with any member but the clade contains mem-
bers from diverse phyla that is Rhizobium from alphaproteo-
bacteria Opitutus from Chlamydae Chthonibacter from
Verrucomicrobia suggesting the multiple gain and loss of
this amylase among prokaryotes Similarly AKF07413 shares
the clade with members of Cyanobacteria AKF07406 shares
the clade with members of Deltaproteobacteria AKF03861
with members of Alphaproteobacteria and AKF03642 with
members of Bacteroidetes Taken together we can suggest
that many of the S amylolyticus amylases have been acquired
via horizontal gene transfer events from other bacterial species
belonging to the phylum Deinococcus-Thermus
Acidobacteria Cyanobacteria and Alphaproteobacteria
To further support these horizontal transfer events we
checked the sequence conservation between S amylolyticus
amylases and closely related members in the phylogenetic tree
and aligned using PROMALS-3D with Taka-Amylase A of A
oryzae (PDBid 2TAA) as a structural template We also per-
formed synteny studies analyzing proteins present in the vicin-
ity of all identified S amylolyticus GH13 and GH57 amylases
and their closely related homologs as revealed by phylogenetic
studies We could not trace any conserved syntenic regions in
these genomes whereas in the case of AKF03862 and
AKF09634 we identified glycogen debranching enzymes in
Stigmatella aurantiaca DW43-1 [56 identity and 98
query coverage of AKF03860 with WP_0133760911] and
Opitutus terrae PB90-1 [57 identity and 97 query cover-
age of AKF09635 YP_0018205421] which further support
their putative horizontal transfer in S amylolyticus
Similarly the phylogenetic inference was drawn from two
representative domains of g-amylases using the same meth-
odology as followed for a-amylases (fig 4) We found that
one of the g-amylases AKF10340 lies closer to M xanthus
amylase whereas the other AKF10487 shares its sister
branch with Nitrosococcus watsonii a Gammaproteobacteria
This suggests that while some of the a- and g-amylases
are present in many myxobacterial species a majority of the
a- and g-amylases are present only in S amylolyticus and not
in other myxobacterial genomes possibly due to the result of
horizontal transfer events from diverse bacterial phylum such
as Deinococcus-Thermus Cyanobacteria Alphaproteobac-
teria Gammaproteobacteria and Acidobacteria
Agar Degrading Enzymes
Some bacteria are known to degrade agar and use it as a
carbon source and possess agarase enzymes that help in hy-
drolyzing agar in oligosaccharides (Chi et al 2012) Depending
on the site of action they can be of two types a-agarase
(agarose 3-glycanohydrolase 321158 GH96) and
b-agarase (agarose 4-glycanohydrolase 32181 GH16
GH50 GH86 GH118) (table 1) Both families of agarases
GH15 EFV841771 Achrom
obacter xylosoxidans C54
GH
15 ZP 011267341 Nitrococcus m
obilis Nb-231
GH15 YP 4129921 Nitrosospira m
ultiformis ATCC 25196
GH15 YP 5716301 Nitrobacter ham
burgensis X14 plasmid
GH15 ZP 012260811 Aurantim
onas manganoxydans SI85-9A1
GH15 YP 0016032801 Gluconacetobacter diazotrophicus PA1 5
GH15 YP 7451371 Granulibacter bethesdensis CGDNIH1
GH15 AEA616211 Burkholderia gladioli BSR3
GH15 YP 0018344311 Beijerinckia indica subsp indica ATCC 9039
GH15 YP 0027560251 Acidobacterium capsulatum
ATCC 51196
GH15 CBJ387091 Ralstonia solanacearum CMR15
GH15 YP 3189151 Nitrobacter winogradskyi Nb-255
GH15 YP 0012344011 Acidiphilium cryptum JF-5
GH15 YP 0040115781 Rhodomicrobium vannielii ATCC 17100
GH15 YP 0016419391 Methylobacterium extorquens PA1GH15 YP 5252641 Rhodoferax ferrireducens T118
GH15 YP 0041811661 Terriglobus saanensis SP1PR4
GH15 ZP 031333281 Chthoniobacter flavus Ellin428
GH15 CCA537261 Streptomyces venezuelae ATCC 10712
GH15 YP 0015065621 Frankia sp EAN1pec
GH15 YP 0024892701 Arthrobacter chlorophenolicus A6
GH15 YP 0018202091 Opitutus terrae PB90-1
GH15 YP 8729011 Acidothermus cellulolyticus 11B
GH15 YP 0013795591 Anaeromyxobacter sp Fw109-5
GH15 YP 0024929091 Anaeromyxobacter dehalogenans 2CP-1
GH15 YP 6355121 Myxococcus xanthus DK 1622
GH15 YP 0011047051 Saccharopolyspora erythraea NRRL 2338
GH15 YP 0011092191 Saccharopolyspora erythraea NRRL 2338
GH15 AKF10340 Sandaracinus amylolyticus DSM 53668
GH15 YP 6342991 Myxococcus xanthus DK 1622
GH15 YP 0031091351 Acidimicrobium ferrooxidans DSM 10331
GH15 YP 0035703361 Salinibacter ruber M8
GH15 YP 0025241401 Thermomicrobium roseum DSM 5159 plasmid
GH15 YP 0033214441 Sphaerobacter thermophilus DSM 20745
GH15 NP 7336051 Streptomyces coelicolor A3-2
GH15 YP 0035282991 Nitrosococcus halophilus Nc 4
GH15 YP 0043676121 Marinithermus hydrothermalis DSM 14884
GH15 YP 0043687811 Marinithermus hydrothermalis DSM 14884
GH15 YP 0034042871 Haloterrigena turkmenica DSM 5511
GH15 ZP 080461001 Haladaptatus paucihalophilus DX253
GH1
5 YP
023
376
1 Pi
crop
hilu
s to
rrid
us D
SM 9
790
GH1
5 ZP
055
7131
11
Ferr
opla
sma
acid
arm
anus
fer1
GH1
5 BA
B604
801
The
rmop
lasm
a vo
lcan
ium
GSS
1
GH
15 Y
P 00
3323
761
1 Th
erm
obac
ulum
terr
enum
ATC
C B
AA
-798
GH
15 Y
P 00
3899
615
1 C
yano
thec
e sp
PC
C 7
822
plas
mid
GH
15 Y
P 00
3705
927
1 Tr
uepe
ra ra
diov
ictr
ix D
SM 1
7093
GH
15 Y
P 00
2478
443
1 A
rthr
obac
ter c
hlor
ophe
nolic
us A
6 pl
asm
idG
H15
AK
F104
87 S
anda
raci
nus
amyl
olyt
icus
DSM
536
68G
H15
YP
0037
6113
81
Nitr
osoc
occu
s w
atso
nii C
-113
GH1
5 ZP
069
7134
91
Kted
onob
acte
r rac
emife
r DSM
449
63
GH15
ZP
0697
6049
1 K
tedo
noba
cter
race
mife
r DSM
449
63
GH15
YP
0033
9335
81
Cone
xiba
cter
woe
sei D
SM 1
4684
GH15
YP
0042
3629
11 A
cido
vora
x av
enae
sub
sp a
vena
e ATC
C 19
860
GH15 Z
P 03
6273
271
Pedo
spha
era p
arvu
la El
lin51
4
GH15 Y
P 00
1618
7001
Sor
angi
um ce
llulo
sum
So
ce56
GH15 ZP
0145
9561
1 Stig
matella
aura
ntiac
a DW
4-3-1
GH15 ZP
0503
8177
1 Syn
echo
cocc
us sp
PCC 73
35
GH15 YP 64
4038
1 Rubrobac
ter xy
lanophilu
s DSM 99
41
GH15 ZP 064746651 Frankia symbiont of D
atisca glomerata
GH15 ZP 069744041 Ktedonobacter racemife
r DSM 44963
GH15 ZP 069653261 Ktedonobacter racemifer D
SM 44963
GH15 ZP 069661381 Ktedonobacter racemifer D
SM 44963
FIG 4mdashPhylogeny of Sandaracinus amylolyticus GH15 g-amylases
Sandaracinus amylolyticus g-amylases are colored dark pink and myxobac-
terial homologs are colored fluorescent green Bootstrap values corre-
sponding to the tree nodes are provided
Sharma et al GBE
2526 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
were absent in S amylolyticus genome which could be related
to its agar nondegrading property (Mohr et al 2012) as sup-
ported by colony morphology where agar was not penetrated
or liquefied by the bacteria (supplementary fig S5
Supplementary Material online)
Cellulose Degrading Enzymes
We identified only GH5 and GH74 in S amylolyticus proteome
in two and three proteins respectively GH5 domain was pre-
sent as a single unit in AKF04963 and AKF09194 whereas
GH74 was identified in AKF04859 AKF06963 and
AKF09775 associated with a GH74 domain CAZY domains
for cellobiohydrolaseexoglucanases (represented by GH6
GH7 GH9 and GH48) were not present in S amylolyticus
Cellobiohydrolaseexoglucanases plays a significant role in cel-
lulose degradation as they cut two to four sugar subunits from
the exposed ends formed by the endoglucanases which are
used as initial substrates for b-glucosidase (Mba Medie et al
2012) The absence of cellobiohydrolase or exoglucanases
could be a possible reason for the lack of cellulose degradation
in S amylolyticus Out of b-glucosidase representative CAZY
families GH1 GH3 GH5 GH9 GH30 and GH116 only GH1
GH3 and GH5 families were found in three two and two
proteins in S amylolyticus respectively AKF03843 AKF0
6028 and AKF10711 have a GH1 domain whereas
AKF09078 and AKF10875 have only the GH3 domain
Chitin Degrading Enzymes
Chitinases (EC32114) are involved in degradation of chitin
a component of the cell wall of fungi and exoskeleton of
arthropods GH18 GH19 GH23 and GH48 families have
been reported in the CAZY database as chitinases that hydro-
lyse b-14 linkages between N-acetylglucosamine units GH18
is a chitinase family with a characteristic ldquo[LIVM
FY][DN]G[LIVMF][DN][LIVMF][DN]Erdquo motif and is expressed
in archaea prokaryotes and eukaryotes (Funkhouser and
Aronson 2007) GH18 chitinases were not found in S amylo-
lyticus genome while members of GH19 (AKF03006 and
AKF10423) and GH23 (AKF04575 AKF05533 AKF06346
AKF08835 and AKF11377) could be identified GH19
family chitinases are mostly reported to be present in plants
with only a few members in prokaryotes (Kasprzewska 2003
Funkhouser and Aronson 2007) Among the two identified
GH19 homologs we found that AKF10423 is approximately
65 identical (~68 query coverage) with cyanobacteria
such as Nodosilinea nodulosa and Limnoraphis robusta
along with another myxobacterium Chondromyces crocatus
Besides these homologs we also found that AKF10423 shows
maximum similarity with eukaryotic GH19 chitinases counter-
parts as present in Acacia mangium Festuca arundinacea
Triticum aestivum and Leucaena leucocephala (55ndash59 iden-
tity with 70 query coverage) We identified that another
chitinase AKF06346 has GH23 domain along with three
copies of CBM50 which is generally present in the enzymes
cleaving either chitin or peptidoglycan
Conclusion
The complete 1033 Mb circular genome of S amylolyticus
type strain DSM 53668T was determined CAZy analysis re-
vealed the presence of 72 GT domains 72 CE domains 58 GH
domains 19 AA domains 4 PL domains and 26 CBM do-
mains Sixteen S amylolyticus proteins were identified of
which 15 are a-amylases of class GH13 and 1 GH57 Two
g-amylases of GH15 family were also identified Phylogeny
analysis revealed that ten GH13 a-amylases and one GH15
g-amylase have been aquired via horizontal gene transfer
from other bacterial phyla such as Deinococcus-Thermus
Cyanobacteria Alphaproteobacteria Gammaproteobacteria
and Acidobacteria
Table 1
Cellulose Starch Chitin and Agar Degrading Enzymes and Their Representative CAZY Domains Distribution
Substrate Enzyme Category Enzyme Name EC Number CAZY Families (protein repertoire in brackets)
Starch Amylase a-Amylase 3211 GH13 (15) GH57 (1) GH119 GH126
Amylase b-Amylase 3212 GH14
Amylase g-Amylase 3213 GH15 (2) GH97
Chitin Chitinase Chitinase 32114 GH18 GH19 (2) GH23 (5) GH48
Agar a-Agarase Agarose 3-glycanohydrolase 321158 GH96
b-Agarase Agarose 4-glycanohydrolase 32181 GH16 GH50 GH86 GH118
Cellulose Cellulase b-Glucosidase 32121 GH1 (3) GH3 (2) GH5 (2) GH9 GH30 GH116
Cellulase Cellobiohydrolase or Exoglucanase 321176 GH48 GH6 GH7 GH9
Cellulase Endoglucanase 3214 GH5 (2) GH6 GH7 GH8 GH9 GH12 GH44
GH45 GH48 GH51 GH74 (3) GH124
NOTEmdashEC number enzyme category and CAZY families for proteins involved in starch chitin agar and cellulose degradation are shown The number of Sandaracinusamylolyticus proteins identified having CAZY domains are represented in bold fonts in respective families
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2527
Supplementary Material
Supplementary figures S1ndashS5 and tables S1 and S2 are avail-
able at Genome Biology and Evolution online (httpwww
gbeoxfordjournalsorg)
Acknowledgments
This work is supported by a project ldquoExpansion and modern-
ization of Microbial Type Culture Collection and Gene Bank
(MTCC) jointly supported by Council of Scientific and
Industrial Research (CSIR) Grant No BSC0402 and
Department of Biotechnology (DBT) Govt of India Grant No
BTPR7368INF221772012rdquo The authors thank MTCC for
providing help in procuring the cultures from DSMZ They
acknowledge Dr Ramya TNC for providing workbench for cul-
turing myxobacteria GS and IK acknowledge the Council
of Scientific and Industrial Research (CSIR) and University
Grant Commission (UGC) for the research fellowship respec-
tively They also thank the Genome Quebec Innovation
Centre McGill University Canada and C-CAMP next-genera-
tion genomics facility Bangalore India for help in obtaining
PacBio and Illumina NGS data respectively
Literature CitedAlikhan NF Petty NK Ben Zakour NL Beatson SA 2011 BLAST Ring
Image Generator (BRIG) simple prokaryote genome comparisons
BMC Genomics 12402
Altschul SF Gish W Miller W Myers EW Lipman DJ 1990 Basic local
alignment search tool J Mol Biol 215403ndash410
Auch AF Von Jan M Klenk HP Goker M 2010 Digital DNA-DNA hybrid-
ization for microbial species delineation by means of genome-to-
genome sequence comparison Stand Genomic Sci 2117ndash134
Aziz RK et al 2008 The RAST Server rapid annotations using subsystems
technology BMC Genomics 975
Blesak K Janecek S 2012 Sequence fingerprints of enzyme specificities
from the glycoside hydrolase family GH57 Extremophiles 16497ndash
506
Chi WJ Chang YK Hong SK 2012 Agar degradation by microorganisms
and agar-degrading enzymes Appl Microbiol Biotechnol 94917ndash
930
Chin CS et al 2013 Nonhybrid finished microbial genome assemblies
from long-read SMRT sequencing data Nat Methods 10563ndash569
Eddy SR 2011 Accelerated profile HMM searches PLoS Comput Biol
7e1002195
Edgar RC 2004 MUSCLE multiple sequence alignment with high accu-
racy and high throughput Nucleic Acids Res 321792ndash1797
Funkhouser JD Aronson NN Jr 2007 Chitinase family GH18 evolutionary
insights from the genomic history of a diverse protein family BMC Evol
Biol 796
Garcia R Muller R 2014 The family Polyangiaceae In Rosenberg E
Delong E Lory S Stackebrandt E Thompson F editors The prokary-
otes Berlin Heidelberg Springer p 247ndash279
Grissa I Vergnaud G Pourcel C 2007 CRISPRFinder a web tool to identify
clustered regularly interspaced short palindromic repeats Nucleic
Acids Res 35W52ndashW57
Hall TA 1999 BioEdit a user-friendly biological sequence alignment editor
and analysis program for Windows 9598NT Nucleic Acids Symp Ser
4195ndash98
Han K et al 2013 Extraordinary expansion of a Sorangium cellulosum
genome from an alkaline milieu Sci Rep 32101
Janecek S Kuchtova A 2012 In silico identification of catalytic residues
and domain fold of the family GH119 sharing the catalytic machinery
with the alpha-amylase family GH57 FEBS Lett 5863360ndash3366
Janecek S Svensson B Macgregor EA 2014 alpha-Amylase an enzyme
specificity found in various families of glycoside hydrolases Cell Mol
Life Sci 711149ndash1170
Kasprzewska A 2003 Plant chitinasesndashregulation and function Cell Mol
Biol Lett 8809ndash824
Lagesen K et al 2007 RNAmmer consistent and rapid annotation of
ribosomal RNA genes Nucleic Acids Res 353100ndash3108
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B
2014 The carbohydrate-active enzymes database (CAZy) in 2013
Nucleic Acids Res 42D490ndashD495
Lowe TM Eddy SR 1997 tRNAscan-SE a program for improved detection
of transfer RNA genes in genomic sequence Nucleic Acids Res
25955ndash964
Matsuura Y Kusunoki M Harada W Kakudo M 1984 Structure
and possible catalytic residues of Taka-amylase A J Biochem
95697ndash702
Mba Medie F Davies GJ Drancourt M Henrissat B 2012 Genome anal-
yses highlight the different biological roles of cellulases Nat Rev
Microbiol 10227ndash234
Mohr KI Garcia RO Gerth K Irschik H Muller R 2012
Sandaracinus amylolyticus gen nov sp nov a starch-degrading
soil myxobacterium and description of Sandaracinaceae fam
nov Int J Syst Evol Microbiol 621191ndash1198
Noguchi J et al 2011 Crystal structure of the branching enzyme I
(BEI) from Oryza sativa L with implications for catalysis and sub-
strate binding Glycobiology 211108ndash1116
Patel RK Jain M 2012 NGS QC Toolkit a toolkit for quality control of next
generation sequencing data PLoS One 7e30619
Pei J Kim BH Grishin NV PROMALS3D a tool for multiple protein se-
quence and structure alignments Nucleic Acids Res 36 2295ndash2300
Reichenbach H Dworkin M 1992 The myxobacteria In Balows A
Truper HG Dworkin M Harder W Schleifer KH editors The pro-
karyotes a handbook on the biology of bacteria ecophysiology
isolation identification applications New York Springer New
York p 3416ndash3487
Reichenbach H 2005 ldquoOrder VIII Myxococcalesrdquo Bergeyrsquos Manual of
Systematic Bacteriology In Brenner DJ Krieg NR Staley JT Garrity
GM editors Vol 2 2nd ed part C New York Springer-Verlag New
York Inc pp 1074ndash1079
Richter M Rossello-Mora R 2009 Shifting the genomic gold standard for
the prokaryotic species definition Proc Natl Acad Sci U S A 106
19126ndash19131
Schneiker S et al 2007 Complete genome sequence of the myxobacter-
ium Sorangium cellulosum Nat Biotechnol 25 1281ndash1289
Shimkets LJ 1990 Social and developmental biology of the myxobacteria
Microbiol Rev 54 473ndash501
Siguier P Perochon J Lestrade L Mahillon J Chandler M 2006 ISfinder
the reference centre for bacterial insertion sequences Nucleic Acids
Res 34 D32ndashD36
Steinmetz H et al 2012 Indiacens A and B prenyl indoles from the
myxobacterium Sandaracinus amylolyticus J Nat Prod 75 1803ndash
1805
Tamura K Stecher G Peterson D Filipski A Kumar S 2013 MEGA6
molecular evolutionary genetics analysis version 60 Mol Biol Evol
30 2725ndash2729
Van Der Maarel MJ Van Der Veen B Uitdehaag JC Leemhuis H Dijkhuizen
L 2002 Properties and applications of starch-converting enzymes of
the alpha-amylase family J Biotechnol 94 137ndash155
Sharma et al GBE
2528 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
Weissman KJ Muller R 2010 Myxobacterial secondary metabolites
bioactivities and modes-of-action Nat Prod Rep 27 1276ndash1295
Whitworth DE 2008 Myxobacteria multicellularity and differentiation
Washington DC ASM Press American Society for Microbiology
Yin Y et al 2012 dbCAN a web resource for automated carbohydrate-
active enzyme annotation Nucleic Acids Res 40 W445ndashW451
Zhao JY et al 2008 Discovery of the autonomously replicating plasmid
pMF1 from Myxococcus fulvus and development of a gene cloning
system in Myxococcus xanthus Appl Environ Microbiol 74 1980ndash
1987
Associate editor Howard Ochman
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2529
motifs involved in starch cellulose chitin and agar degrada-
tion were retrieved on the basis of EC number for each
enzyme category Also the proteins of S amylolyticus were
mapped against the CAZY protein database using Basic-Local
Alignment Search Tool (BLASTp) (E-value cutoff of 1e5 min-
imum query coverage of 50 and minimum percent identity
of 35) (Altschul et al 1990) NCBI BLAST + (v 2226+) and
nonredundant protein (NR) database downloaded on
September 6 2014 was used throughout the study
Clustered regularly interspaced short palindromic repeat
(CRISPR) elements and insertion elements were identified
using CRISPRfinder (Grissa et al 2007) and ISfinder (Siguier
et al 2006) respectively
Sandaracinus amylolyticus Amylases
The a-amylases and g-amylases from S amylolyticus were sub-
jected to the BLASTp against amylase sequences from the
CAZY database (clustered at 70 sequence identity) and
top hits were retrieved The amylases from S amylolyticus
were cut into the domains and were aligned using MUltiple
Sequence Comparison by Log-Expectation (MUSCLE) (Edgar
2004) incorporated in MEGA v606 Further the evolutionary
tree was generated by ML method [model Jones Taylor and
Thorton bootstrap 100] using MEGA v606 Initial tree(s) for
the heuristic search were obtained by applying the Maximum
Parsimony method to a matrix of pairwise distances estimated
using the Maximum Composite Likelihood approach Further
the S amylolyticus GH13 amylases along with the closely re-
lated members of each phylogenetic clade were aligned using
PROfile Multiple Alignment with predicted Local Structures
and 3D constraints (PROMALS3D) with Taka-Amylase A of
Aspergillus oryzae [2TAA] (Matsuura et al 1984) as a struc-
tural template (Pei et al 2008)
Results and Discussion
Sandaracinus amylolyticus Genome Properties
Sandaracinus amylolyticus genome was assembled de novo by
HGAP v20 (Chin et al 2013) of the SMRT portal as a single
contig of 1033 Mb with no plasmid and representing the
complete genome with approximately 85 coverage and
72 G+C content (fig 1) No complete plasmid could be
retrieved from the mapping of Illumina PE reads against the
bacterial plasmid database Plasmids are rare in the order
Myxococcales and till date only one plasmid pMF1 from
Myxococcus fulvus 124B02 has been reported (Zhao et al
2008) RAST-based annotation predicted 8962 coding
genes out of which 4939 proteins (5516) were function-
ally annotated while the remaining (4484) were hypothet-
ical proteins (supplementary table S1 Supplementary Material
online) Sandaracinus amylolyticus genome has two rRNA op-
erons and 56 aminoacyl-tRNA synthetase genes
This genome encodes two sequentially identical 16S rRNA
which shows 100 identity with previously reported 16S rRNA
sequence of S amylolyticus strain NOSO-4T [NCBI Reference
Sequence NR_1180011] followed by 8798 identity with
Chondromyces apiculatus strain Cm a14 [NR_0253441]
Among the myxobacteria with sequenced genomes it shows
the maximum identity (8789) to the 16S rRNA gene of C
apiculatus DSM 436 (strain Cm a2) [GenBank M942742] fol-
lowed by 875 identity to S cellulosum 16S rRNA (Soce0157-
2 and Soce56) The 16S rRNA-based phylogeny of
Sandaracinus and related myxobacteria suggest its classification
as a separate family within the suborder Sorangiineae (supple-
mentary fig S1 Supplementary Material online) Sandaracinus
amylolyticus 16S rRNA forms a separate branch distinct from
other families in the suborder Sorangiineae phylogenetic tree
representing family Sandaracinaceae We also calculated in
silico DDH values for all available myxobacterial genomes
using the GGDC server (Auch et al 2010) Sandaracinus
amylolyticus is not closely related to other Myxococcales as
the maximum DDH value was identified to be 181 with S
cellulosum followed by 174 with C apiculatus DSM 436 sug-
gesting a distant relation to Polyangiaceae members which
further confirms its placement in a separate family
Sandaracinaceae ANI calculations likewise support the above
conclusions (supplementary table S2 Supplementary Material
online)
The members of suborder Sorangiineae such as the S cel-
lulosum species have been well characterized for degrading
cellulose and other complex polysaccharides (Schneiker et al
2007 Han et al 2013) Similarly S amylolyticus has been
reported as the only myxobacteria capable of degrading
starch (Mohr et al 2012) Thus in order to gain insights into
the genes involved in the degradation of complex polysaccha-
rides especially starch the complete genome of S amylolyticus
was determined and investigated for the presence of carbo-
hydrate active enzymes
Sandaracinus amylolyticus CAZome
Two-hundred and forty-three proteins (~27 of total prote-
ome) of S amylolyticus were annotated with diverse combi-
nations of CAZY domains that consist of 72 GT domains 72
CE domains 58 GH domains 19 AA domains 4 PL domains
and 26 CBM domains (fig 2A) Fourteen out of these 243
proteins contained more than one CAZY domain whereas
the rest had only a single CAZY domain In multiple CAZY-
domain containing proteins various carbohydrate binding
modules such as CBM48 CBM50 and CBM21 are pre-
sent along with other CAZY category proteins for assisting in
their functions The CAZyme distribution observed in
S amylolyticus indicates its ability to degrade various
types of biomass Here we examine the S amylolyticus pro-
teome involved in starch agar chitin and cellulose
degradation
Sharma et al GBE
2522 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
Evolutionary History of S amylolyticus Amylases
Amylases belonging to glycoside hydrolase (GH) enzymatic
category degrade starch into sugar moieties by the
hydrolysis of a-14- and a-16-glycosidic bond (Van der
Maarel et al 2002) Based on the site of action they are di-
vided into three categories a-amylase (EC3211 GH13
GH57 GH119 GH126) b-amylase (EC3212 GH14) and
-amylase (EC3213 GH15 GH97) Sandaracinus
amylolyticus genome contains both a- and -amylases
whereas no b-amylases could be found Among the 4 a-am-
ylases GH families 15 GH13 proteins and 1 GH57 protein
were found We could identify two -amylases belonging to
Sandaracinus amylolyticusTDSM 53668
CP01112510328378 bp
FIG 1mdashCircular representation of the Sandaracinus amylolyticus DSM 53668T complete genome Circles (from inside to outside) 1 and 2 (GC content
black line and GC skew magenta and green lines) circle 3 (CAZome Proteins annotated as carbohydrate active enzymes Red) circle 4 5 and 6 (Amylases
Cellulases and Chitinases Black) circle 7 (encoded RNA Magenta) circle 8 (CDS present on negative strand Blue) circle 9 (CDS present on positive strand
Green) circle 10 (S amylolyticus DSM 53668T complete genome Red) BRIG 095 was used to build the circular representation (Alikhan et al 2011)
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2523
GH15 category (fig 2B) The starch degrading proteins have
been reported to have CBM20 CBM21 CBM25 CBM26
CBM34 CBM41 CBM45 CBM48 CBM53 and CBM58
modules that help in glycogenstarch binding truncation of
which reduces its enzymatic activity (Noguchi et al 2011
Janecek et al 2014) GH15 and GH57 domains are present
as single domain proteins in S amylolyticus Interestingly in 5
out of 15 GH13 S amylolyticus proteins (AKF03796
AKF03860 AKF06952 AKF07413 and AKF11765) the a-
amylase GH13 domain was present along with a CBM48
module at the N-termini The CBM48 module assists in the
GH13 function by serving as starch-binding domains
The GH13 family include the a-amylase domains with an (b
a)8 barrel catalytic domain These proteins have 4ndash7 conserved
sequence regions (CSRs) and a specific catalytic triad consisting
of a catalytic nucleophile (aspartic acid) on strand b4 proton
donor (glutamic acid) on strand b5 and a transition state sta-
bilizer (aspartic acid) on strand b7 (Janecek et al 2014) GH13
members show extremely low sequence identity with their ho-
mologs but the catalytic triad is highly conserved GH57 family
a-amylases have a (ba)7 barrel structure and have five CSRs as
compared with the GH13 family However only two of the
catalytic residues viz the nucleophile on strand b4 and the
proton donor on strand b7 are conserved (Blesak and Janecek
2012) Irrespective of these differences GH13 and GH57 both
employ the same retaining mechanism of action (Janecek and
Kuchtova 2012) Sandaracinus amylolyticus GH13 proteins
were retrieved and the structure of Taka amylase of A
oryzae (PDBid 2TAA) was used to perform a structure-based
alignment using PROMALS3D (Matsuura et al 1984 Pei et al
2008) All the seven CSR regions and the three catalytic
residues were conserved in all S amylolyticus GH13 amylases
(supplementary figs S2 and S3 Supplementary Material
online) Similarly S amylolyticus GH57 (AKF06951) was
aligned with its homologs using PROMALS3D based on the
4-a-glucanotransferase structure of Thermococcus litoralis
(PDBid 1K1W) In the GH57 alignment all five CSRs and
both catalytic residues were conserved (supplementary figS4
Supplementary Material online)
In order to understand the provenance of these a- and g-
amylases they were subjected to BLASTp against the NR data-
base To include amylases across the entire bacterial phylum
the CAZY-NR database for GH13 and GH57 domains were
retrieved and subjected to BLASTClust at 70 identity and
70 length coverage that reduced the domain sequence
space from 19590 to 4038 domains Further amylase do-
mains from S amylolyticus were subjected to BLASTp against
these 4038 domains and their top hits were retrieved and
included in the phylogenetic analysis All these GH13 and
GH57 domains along with S amylolyticus amylase domains
were aligned using MUSCLE as incorporated in MEGAv606
and ML-based phylogeny with 100 bootstrap values was
drawn (fig 3) The ML tree obtained was divided into three
major clades where the clade at root composed of all the GH57
domains and the other two major clades further composed of
all the GH13 sequences The representative bacterial amylases
from various clades show close relations with amylases of di-
verse bacterial groups suggesting the multiple gain and loss of
these domains As all the amylase domains of S amylolyticus
are scattered across the tree the phylogenetic position of these
domains could provide us insights about their provenance and
reveal possible cases of horizontal transfers
19
26
72
58
72
4
0
10
20
30
40
50
60
70
80
AA CBM CE GH GT PL
A B
Num
ber o
f pro
tein
s
GH13
GH13
CBM48 GH13
GH13CBM48
GH13
GH57
GH13CBM48
GH13GH13CBM48
GH13
GH13
GH13
GH15
GH13CBM48
GH13
AKF03605AKF03642AKF03796
AKF03860AKF03861AKF03862AKF04017AKF06951AKF06952
AKF07406AKF07413AKF07527AKF09634AKF09643AKF10340AKF10487AKF11765AKF11772
GH13
GH13
GH15
FIG 2mdashSandaracinus amylolyticus DSM 53668T CAZome distribution (A) Sandaracinus amylolyticus DSM 53668T CAZY families presented in a bar
graph x axis represents the CAZY categories and y axis represents the number of proteins (B) a- and g-amylases along with their CAZome domain
architecture The domains have been colored as GH13 sky blue GH15 dark red GH57 green and CBM48 orange
Sharma et al GBE
2524 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
Taking into account the GH57 clade the sole GH57
domain of S amylolyticus (GenBank ID AKF06951) shares
the clade with anaerobic Deltaproteobacteria Among the
GH13 domains of S amylolyticus AKF03862 AKF07527
AKF09643 AKF11765 and AKF11772 shares clade with
other myxobacteria Among these AKF11772 and
AKF07527 share sister branches and form sister clades with
other myxobacterial species such as S cellulosum Soce56 and
Plesiocystis pacifica SIR-1 and are further related to the clade
containing Gammaproteobacteria Apart from these two am-
ylases AKF03860 and AKF06952 are similar to each other and
group with Candidatus accumulibacter phosphtis amylase
(YP_0031672861) from the phylum Acidobacteria
Interestingly AKF03860 and AKF06952 group with members
GH13 ZP 021632211 Kordia algicida OT-1
GH13 ZP 067516041 Parascardovia denticolens F0305
GH13 YP 0035709041 Salinibacter ruber M8
GH13 YP 0032500041 Fibrobacter succinogenes subsp succinogenes S85
GH13 AKF03862 Sandaracinus amylolyticus DSM 53668
GH13 YP 0039537801 Stigmatella aurantiaca
GH13 CAJ738231 Candidatus Kuenenia stuttgartiensis
GH13 YP 0228461 Picrophilus torridus DSM 9790GH13 ZP 029306261 Verrucomicrobium spinosum DSM 4136
GH13 CAX841261 uncultured bacterium
GH13 YP 0031678771 Candidatus Accumulibacter phosphatis clade IIA str UW-1GH13 ZP 031321321 Chthoniobacter flavus Ellin428
GH13 ZP 018759791 Lentisphaera araneosa HTCC2155GH13 YP 0033894351 Spirosoma linguale DSM 74GH13 NP 7734061 Bradyrhizobium diazoefficiens USDA 110
GH13 YP 0037757321 Herbaspirillum seropedicae SmR1GH13 YP 4266911 Rhodospirillum rubrum ATCC 11170GH13 ZP 035079501 Rhizobium etli Brasil 5
GH13 ZP 057930691 Butyrivibrio crossotus DSM 2876
GH13 ZP 066231321 Turicibacter sanguinis PC909
GH13 ZP 044499741 Catonella morbi ATCC 51271
GH13 EGE534241 Streptococcus parauberis NCFD 2020
GH13 196049865 Lactobacillus Plantarum
GH13 YP 0042196261 Granulicella tundricola MP5ACTX9
GH13 YP 0011935611 Flavobacterium johnsoniae UW101
GH13 YP 0032910851 Rhodothermus marinus DSM 4252GH13 YP 0040967591 Bacillus cellulosilyticus DSM 2522GH13 ZP 037030351 Flavobacteria bacterium MS024-2A
GH13 XP 0025352531 Ricinus communis
GH13 YP 7579561 Maricaulis maris MCS10GH13 ZP 010417351 Erythrobacter sp NAP1GH13 YP 0018177571 Opitutus terrae PB90-1GH13 EFR587141 Alistipes sp HGB5
GH13 CBK627851 Alistipes shahii WAL 8301GH13 EFR582631 Alistipes sp HGB5
GH13 YP 0034042011 Haloterrigena turkmenica DSM 5511
GH13 YP 0035357541 Haloferax volcanii DS2
GH13 YP 0035357541 Haloferax volcanii DS2
GH13 YP 0025661621 Halorubrum lacusprofundi ATCC 49239
GH13 YP 1368761 Haloarcula marismortui ATCC 43049
GH13 ZP 080436741 Haladaptatus paucihalophilus DX253
GH13 YP 0025652161 Halorubrum lacusprofundi ATCC 49239
GH13 YP 0035346301 Haloferax volcanii DS2
GH13 YP 0033225661 Thermobaculum terrenum ATCC BAA-798
GH13 YP 0021353421 Anaeromyxobacter sp K
GH13 AKF09643 Sandaracinus amylolyticus DSM 53668
GH13 YP 0016137031 Sorangium cellulosum So ce56
GH1
3 99
0316
21 H
alot
herm
othr
ix or
enii
GH1
3 AA
D051
991
Pae
niba
cillu
s po
lymyx
a
GH1
3 YP
003
6999
601
Bac
illus
sele
nitir
educ
ens
MLS
10
GH1
3 ZP
022
0799
81
Copr
ococ
cus
euta
ctus
ATC
C 27
759
GH
13 B
AA07
401
1 Xa
ntho
mon
as c
ampe
stris
GH
13 Y
P 00
4052
835
1 M
ariv
irga
tract
uosa
DSM
412
6
GH1
3 ZP
037
0954
51
Cory
neba
cter
ium
mat
ruch
otii A
TCC
3380
6
GH1
3 NP
267
649
1 La
ctoc
occu
s la
ctis
subs
p la
ctis
Il140
3
GH1
3 YP
001
3023
201
Par
abac
tero
ides
dist
ason
is AT
CC 8
503
GH1
3 YP
001
2276
361
Syn
echo
cocc
us s
p R
CC30
7
GH1
3 YP
002
5652
591
Hal
orub
rum
lacu
spro
fund
i ATC
C 49
239
GH13
ZP
0709
0550
1 C
oryn
ebac
teriu
m g
enita
lium
ATC
C 33
030
GH13
YP 0
0283
4100
1 C
oryn
ebac
teriu
m a
urim
ucos
um A
TCC
7009
75
GH13 A
KF03
861
Sand
arac
inus a
mylo
lyticu
s DSM
536
68
GH13
ZP 0
0050
865
1 M
agne
tosp
irillum
mag
neto
tacti
cum
MS-
1
GH13 Y
P 00
1568
811
1 Pe
troto
ga m
obilis
SJ9
5
GH13 Y
P 02
2847
1 P
icrop
hilus
torri
dus D
SM 9
790
GH13 E
FT25
5011
Pro
pionib
acter
ium ac
nes H
L110
PA3
GH13 E
FA80
1951
Poly
spho
ndyli
um pa
llidum
PN50
0
GH13 A
BD4655
81 V
ermam
oeba
verm
iform
is
GH13 Z
P 0350
5050
1 Rhiz
obium
etli B
rasil
5
GH13 YP 00
1348
4651
Pseud
omon
as ae
rugino
sa PA7
GH13 YP 00
2238
3511
Klebsie
lla pn
eumon
iae 34
2
GH13 YP 00
1437
9781
Cron
obac
ter sa
kaza
kii ATCC B
AA-894
GH13 Y
P 0011
7103
71 P
seud
omon
as st
utzeri
A1501
GH13 Y
P 0019
7240
81 S
tenotr
opho
monas
malt
ophil
ia K27
9a
GH13 Y
P 0043
1901
11 S
phing
obac
terium
sp 2
1
GH13 YP 0013127791 Sinorhizobium m
edicae W
SM419
GH13 ZP 035028921 Rhizobium etli K
im 5
GH13 YP 67
6219
1 Che
lativo
rans s
p BNC1
GH13 YP 00
2376
8861
Cya
nothe
ce sp
PCC 74
24
GH13 YP 0036383061 Cellulomonas flavig
ena DSM 20109
GH13 YP 0040977291 Intrasporangium ca
lvum DSM 43043
GH13 YP 0011691691 Rhodobacter s
phaeroides ATCC 17025
GH13 ZP 011580901 Oceanicola granulosus H
TCC2516
GH13 YP 0038382651 Micromonospora aurantiaca ATCC 27029
GH13 YP 0040977251 Intrasporangium calvu
m DSM 43043
GH13 YP 0036954761 Starkeya novella DSM 506
GH13 ZP 035166441 Rhizobium etli IE4771
GH13 ZP 031273131 Chthoniobacter flavus Ellin
428
GH13 AKF03642 Sandaracinus amylolyticus DSM 53668
GH13 YP 0031252671 Chitinophaga pinensis DSM 2588
GH13 YP 0030850521 Dyadobacter fermentans DSM 18053
GH13 YP 0033879241 Spirosoma linguale DSM 74
GH13 YP 0034301801 Streptococcus gallolyticus UCN34
GH13 ZP 078184041 Eremococcus coleocola ACS-139-V-Col8
GH13 YP 0034774501 Thermoanaerobacter italicus Ab9
GH13 ZP 048517861 Paenibacillus sp oral taxon 786 str D14
GH13 YP 0038414241 Caldicellulosiruptor obsidiansis OB47
GH13 YP 0024279661 Desulfurococcus kamchatkensis 1221n
GH13 YP 0034262831 Bacillus pseudofirmus OF4
GH13 ZP 076854311 Oscillochloris trichoides DG-6GH13 YP 0016174961 Sorangium cellulosum So ce56
GH13 ZP 019113371 Plesiocystis pacifica SIR-1
GH13 YP 6318811 Myxococcus xanthus DK 1622
GH13 AKF11765 Sandaracinus amylolyticus DSM 53668
GH13 ZP 036771121 Bacteroides cellulosilyticus DSM 14838
GH13 YP 0037157841 Croceibacter atlanticus HTCC2559
GH13 ZP 012015681 Flavobacteria bacterium BBFL7
GH13 ZP 019113321 Plesiocystis pacifica SIR-1GH13 YP 0031962511 Robiginitalea biformata HTCC2501
GH13 YP 0040531921 Marivirga tractuosa DSM 4126
GH13 ZP 021817881 Flavobacteriales bacterium ALC-1
GH13 CBL150401 Ruminococcus bromii L2-63
GH13 ZP 080051001 Bacillus sp 2 A 57 CT2
GH13 CBK911271 Eubacterium rectale DSM 17629
GH13 YP 0029369771 Eubacterium rectale ATCC 33656
GH13 YP 0021366011 Anaeromyxobacter sp K
GH13 ZP 014604541 Stigmatella aurantiaca
GH
13 728851 Streptomyces lividans
GH
13 BAJ281541 Kitasatospora setae KM-6054
GH
13 YP 0033390201 Streptosporangium roseum
DSM
43021
GH
13 YP 9214291 Nocardioides sp JS614
GH
13 AEB462451 Verrucosispora maris AB-18-032
GH
13 CAJ810301 Actinoplanes sp
GH
13 NP 9387401 C
orynebacterium diphtheriae N
CTC
13129
GH
13 YP 0036976141 Arcanobacterium haem
olyticum D
SM 20595
GH
13 ZP 039259261 Actinomyces coleocanis D
SM 15436
GH
13 YP 0040999421 Intrasporangium calvum
DSM
43043
GH
13 YP 0030989701 Actinosynnema m
irum D
SM 43827
GH
13 YP 0036376081 Cellulom
onas flavigena DSM
20109
GH
13 ZP 009938561 Janibacter sp HTC
C2649
GH13 YP 0030608421 Hirschia baltica ATCC 49814
GH13 YP 7579631 M
aricaulis maris M
CS10
GH13 YP 0016857121 Caulobacter sp K31
GH13 ZP 013016731 Sphingom
onas sp SKA58
GH13 YP 0038198341 Brevundim
onas subvibrioides ATCC 15264
GH
13 YP 4971641 Novosphingobium
aromaticivorans D
SM 12444
GH13 YP 6609551 Pseudoalterom
onas atlantica T6c
GH13 ZP 011126451 Reinekea blandensis M
ED297
GH13 YP 2677341 Colwellia psychrerythraea 34H
GH13 ZP 011126461 Reinekea blandensis M
ED297
GH13 AKF11772 Sandaracinus amylolyticus DSM
53668
GH13 AKF07527 Sandaracinus amylolyticus DSM
53668
GH13 YP 0016184291 Sorangium cellulosum
So ce56
GH13 ZP 019113391 Plesiocystis pacifica SIR-1
GH13 ZP 036315591 Pedosphaera parvula Ellin514
GH13 YP 0016604791 Microcystis aeruginosa NIES-843
GH13 YP 0043416831 Archaeoglobus veneficus SNP6
GH13 ZP 073351521 Desulfovibrio fructosovorans JJ
GH
13 AKF03605 Sandaracinus amylolyticus D
SM 53668
GH
13 YP 0027853821 Deinococcus deserti VC
D115
GH
13 NP 2951951 D
einococcus radiodurans R1
GH
13 YP 004171
1212
M
SD
sisn
epoc
ira
m su
ccoc
onie
D 1
661
1
GH
13 YP 0041715811 Deinococcus m
aricopensis DSM
21211
GH
13 YP 0037038171 Truepera radiovictrix DSM
17093
GH
13 YP 6052101 Deinococcus geotherm
alis DSM
11300
GH
13 YP 0027555491 A
cidobacterium capsulatum
ATCC
51196
GH
13 Y
P9
XTC
A5P
M alocirdnut allecilunarG 1860612400
GH
13 YP 6335241 M
yxococcus xanthus DK
1622G
H13 Y
PB
S su
cihp
ortid
ica
suhp
ortny
S 1
9412
64
GH
13 A
AW32
490
1 iiklahcsttog acnarborean
A
GH
13 Y
P 00
2316
532
1 An
oxyb
acillu
s fla
vith
erm
us W
K1
GH
13 Y
P 00
3252
634
1 G
eoba
cillu
s sp
Y41
2MC
61G
H13
ZP
0117
0936
1 B
acillu
s sp
NR
RL
B-14
911
GH
13 Z
P 07
7074
981
Bac
illus
sp m
3-13
GH
13 Z
P1-
GS ps sullica
B 116916810
GH
13 Y
P 00
1813
251
1 Ex
iguo
bact
eriu
m s
ibiri
cum
255
-15
GH
13 Y
P 00
2886
871
1 Ex
iguo
bact
eriu
m s
p A
T1b
GH
13 Y
P 00
1568
454
1 Pe
troto
ga m
obilis
SJ9
5
GH
13 Y
P 00
2940
188
1 Ko
smot
oga
olea
ria T
BF 1
95
1G
H13
ZP
0125
8420
1 V
ibrio
alg
inol
ytic
us 1
2G01
GH
13 1
0911
18 T
herm
oact
inom
yces
vul
garis
GH
13 Z
P 06
9705
751
Kte
dono
bact
er ra
cem
ifer D
SM 4
4963
GH
13 Y
P 00
1545
735
1 H
erpe
tosi
phon
aur
antia
cus
DSM
785
GH
13 E
FY95
778
1 M
etar
hizi
um ro
berts
ii AR
SEF
23
GH
13 N
P 58
8205
1 S
chiz
osac
char
omyc
es p
ombe
972
h-
GH
13 N
P 59
4401
1 S
chiz
osac
char
omyc
es p
ombe
972
h-
GH
13 X
P 00
1908
940
1 Po
dosp
ora
anse
rina
S m
at+
GH
13 4
1016
824
Schw
anni
omyc
es o
ccid
enta
lis
GH
13 B
AF98
616
1 H
ypho
pich
ia b
urto
nii
GH13 AKF07406 Sandaracinus amylolyticus DSM 53668
GH13 YP 0043692881 Desulfobacca acetoxidans DSM 11109
GH13 YP 0023716111 Cyanothece sp PCC 8801GH13 YP 3219451 Anabaena variabilis ATCC 29413
GH13 YP 4738811 Synechococcus sp JA-3-3AbGH13 YP 8464671 Syntrophobacter fumaroxidans MPOBGH13 ZP 063702061 Desulfovibrio sp FW1012B
GH13 YP 0033193471 Sphaerobacter thermophilus DSM 20745GH13 YP 0037057361 Truepera radiovictrix DSM 17093GH13 YP 4120971 Nitrosospira multiformis ATCC 25196GH13 YP 6431071 Rubrobacter xylanophilus DSM 9941
GH13 BAD389831 Rhodobacter sphaeroides f sp denitrificans
GH13 YP 3175601 Nitrobacter winogradskyi Nb-255
GH13 YP 8215181 Candidatus Solibacter usitatus Ellin6076
GH13 YP 0034506361 Azospirillum sp B510
GH13 ZP 070169471 Desulfonatronospira thiodismutans ASO3-1
GH13 YP 0041842351 Terriglobus saanensis SP1PR4
GH13 ZP 070315011 Granulicella mallensis MP5ACTX8
GH13 YP 0029438961 Variovorax paradoxus S110
GH13 YP 5918381 Candidatus Koribacter versatilis Ellin345
GH13 YP 7445681 Granulibacter bethesdensis CGDNIH1
GH13 YP 0039098001 Burkholderia sp CCGE1003
GH13 ZP 024903661 Burkholderia pseudomallei NCTC 13177
GH13 YP 0016309761 Bordetella petrii DSM 12804
GH13 YP 0022527351 Ralstonia solanacearum MolK2
GH13 YP 0037759061 Herbaspirillum seropedicae SmR1
GH13 YP 5455251 Methylobacillus flagellatus KT
GH13 YP 0013485041 Pseudomonas aeruginosa PA7
GH13 YP 0016008321 Gluconacetobacter diazotrophicus PA1 5
GH13 AEB584851 Pseudomonas mendocina NK-01
GH13 YP 0027996471 Azotobacter vinelandii DJ
GH
13 N
P 63
5804
1 X
anth
omon
as c
ampe
stris
pv
cam
pest
ris s
tr A
TCC
339
13
GH
13 Y
P 00
3188
581
1 Ac
etob
acte
r pas
teur
ianu
s IF
O 3
283-
01
GH
13 Y
P 74
4570
1 G
ranu
libac
ter b
ethe
sden
sis
CG
DN
IH1
GH
13 Y
P 00
3052
329
1 M
ethy
lovo
rus
gluc
oset
roph
us S
IP3-
4
GH
13 Z
P 06
8981
631
Ros
eom
onas
cer
vica
lis A
TCC
499
57
GH
13 Y
P 00
2943
895
1 Va
riovo
rax
para
doxu
s S1
10
GH
13 Y
P 31
4931
1 T
hiob
acillu
s de
nitri
fican
s AT
CC
252
59
GH
13 Y
P 42
5597
1 R
hodo
spiri
llum
rubr
um A
TCC
111
70
GH
13 Y
P 93
3302
1 A
zoar
cus
sp B
H72
GH
13 N
P 43
7816
1 S
inor
hizo
bium
mel
iloti
1021
GH
13 Z
P 01
1553
281
Oce
anic
ola
gran
ulos
us H
TCC
2516
GH
13 Y
P 00
1167
979
1 R
hodo
bact
er s
phae
roid
es A
TCC
170
25
GH
13 Y
P 00
1440
575
1 C
rono
bact
er s
akaz
akii A
TCC
BAA
-894
GH
13 Z
P 01
4401
991
Ful
vim
arin
a pe
lagi
HTC
C25
06
GH
13 Y
P 00
4234
793
1 Ac
idov
orax
ave
nae
subs
p a
vena
e AT
CC
198
60
GH
13 Y
P 00
1818
894
1 O
pitu
tus
terra
e PB
90-1
GH
13 Y
P 00
3154
808
1 Br
achy
bact
eriu
m fa
eciu
m D
SM 4
810
GH
13 ZP 010940301 Blastopirellula marina D
SM 3645
GH
13 ZP 027320551 Gem
mata obscuriglobus U
QM
2246
GH
13 YP 4273821 Rhodospirillum
rubrum ATC
C 11170
GH
13 YP 0021361131 Anaeromyxobacter sp K
GH
13 YP 0039532711 Stigmatella aurantiaca
GH
13 YP 0021384951 Geobacter bem
idjiensis BemG
H13 Y
P23
-C
RF ii
notl
ad r
etca
boe
G 1
3868
3520
0 G
H13 Y
P65
ec o
S mu
solu
llec
mu
igna
roS
191
8716
100
GH
13 ZPB2
101
WF
ps o
irbi
vofl
use
D 1
5398
6360
GH
13 Y
P 82
1516
1 C
andi
datu
s S
olib
acte
r usi
tatu
s E
llin6
076
GH
13 Y
P24
NFC ilte
muibozihR 1077074
GH
13
86635 M
SD sucityloly
ma sunicaradnaS 43690F
KA
GH
13 Y
P 00
1820
541
1 O
pitu
tus
terra
e P
B90
-1G
H13
ZP
0313
0066
1 C
htho
niob
acte
r fla
vus
Ellin
428
GH
13 YP 0035707081 Salinibacter ruber M8
GH
13 YP 0037057351 Truepera radiovictrix DSM
17093
GH
13 YP 0032689441 Haliangium
ochraceum D
SM 14365
GH
13 YP 0016130311 Sorangium cellulosum
So ce56
GH
13 YP 6288101 Myxococcus xanthus D
K 1622
GH
13 62738967 Deinococcus radiodurans
GH
13 AKF04017 Sandaracinus amylolyticus D
SM 53668
GH
13 YP 0027878991 Deinococcus deserti VC
D115
GH13 YP 5893201 Candidatus Koribacter versatilis Ellin345
GH13 ZP 063702081 Desulfovibrio sp FW
1012B
GH13 YP 0041842341 Terriglobus saanensis SP1PR4
GH13 YP 8731321 Acidotherm
us cellulolyticus 11B
GH13 ZP 070169501 Desulfonatronospira thiodism
utans ASO3-1
GH13 ADX845721 Sulfolobus islandicus REY15A
GH13 ZP 027318151 G
emm
ata obscuriglobus UQM
2246
GH13 YP 0016347811 Chloroflexus aurantiacus J-10-fl
GH
13 NP 9534051 G
eobacter sulfurreducens PCA
GH
13 YP 0025366761 Geobacter daltonii FR
C-32
GH
13 YP 0012121361 Pelotomaculum
thermopropionicum
SI
GH
13 YP 0041781661 Isosphaera pallida ATCC
43644
GH
13 ZP 036310341 Pedosphaera parvula Ellin514
GH13 ZP 031287911 Chthoniobacter flavus Ellin428
GH13 NP 6913271 Oceanobacillus ih
eyensis HTE831
GH13 YP 0025601851 Macro
coccus c
aseolyticus J
CSC5402
GH13 YP 0030110001 Paenibacill
us sp J
DR-2
GH13 CAJ384141 Anaerobranca
gottsch
alkii
GH13 YP 00
1988
1311
Lacto
bacill
us ca
sei B
L23
GH13 YP 00
2459
6561
Des
ulfito
bacte
rium ha
fnien
se D
CB-2
GH13 YP 00
3947
0781
Paenib
acillu
s poly
myxa S
C2
GH13 Y
P 0013
1197
11 C
lostrid
ium be
ijerin
ckii N
CIMB 80
52
GH13 Y
P 0039
6683
31 Ily
obac
ter po
lytrop
us D
SM 2926
GH13 Z
P 0539
1277
1 Clos
tridium
carb
oxidi
vora
ns P
7
GH13 Y
P 0013
9686
01 C
lostrid
ium kl
uyve
ri DSM 55
5
GH13 ZP 02
2128
451
Clostrid
ium ba
rtlettii
DSM 16
795
GH13 ZP 04
4331
841
Bacillu
s coa
gulan
s 36D
1
GH13 C
BL37
8151
Ana
eros
tipes
hadr
us
GH13 Z
P 04
4519
311
Abiotro
phia
defec
tiva A
TCC 49
176
GH13
YP
0031
4407
51
Slac
kia h
eliot
rinire
duce
ns D
SM 2
0476
GH13
ZP 0
2025
300
1 Eu
bacte
rium
vent
riosu
m A
TCC
2756
0
GH1
3 YP
004
3093
801
Clo
strid
ium
lent
ocel
lum
DSM
542
7
GH13
YP 5
1565
71
Chlam
ydop
hila
felis
GH13
ZP 0
6299
217
1 Pa
rach
lamyd
ia ac
anth
amoe
bae
str H
allrsquos
cocc
us
GH13
CBL1
5412
1 R
umino
cocc
us b
rom
ii L2-
63
GH13 Y
P 00
1692
347
1 Fi
nego
ldia
mag
na AT
CC 293
28
GH13 Z
P 07
5262
211
Pep
tostr
epto
cocc
us st
omat
is DSM
176
78
GH1
3 YP
003
4751
021
Clo
strid
iale
s ge
nom
osp
BVA
B3 s
tr U
PII9
-5
GH1
3 YP
004
1716
241
Dei
noco
ccus
mar
icope
nsis
DSM
212
11
GH1
3 YP
022
845
1 Pi
crop
hilu
s to
rridu
s DS
M 9
790
GH1
3 YP
001
8207
181
Opi
tutu
s te
rrae
PB90
-1
GH1
3 YP
003
1672
861
Can
dida
tus A
ccum
ulib
acte
r pho
spha
tis c
lade
IIA
str
UW-1
GH1
3 AK
F038
60 S
anda
racin
us a
mylo
lyticu
s DS
M 5
3668
GH1
3 AK
F069
52 S
anda
racin
us a
mylo
lyticu
s DS
M 5
3668
GH
13 Y
P 00
2762
222
1 G
emm
atim
onas
aur
antia
ca T
-27
GH1
3 ZP
082
0671
01
Gor
doni
a ne
ofel
ifaec
is NR
RL B
-593
95
GH
13 Y
P 00
3964
374
1 Ke
togu
loni
cige
nium
vul
gare
Y25
GH13 ZP 045772811 Oxalobacter fo
rmigenes H
OxBLS
GH13 YP 0033856211 Spirosoma linguale DSM 74
GH13 AEA650561 Burkholderia gladioli B
SR3
GH13 YP 0030725361 Teredinibacter turnerae T7901
GH13 YP 4255961 Rhodospirillum ru
brum ATCC 11170
GH13 YP 0010427851 Rhodobacter sphaeroides ATCC 17029
GH13 ZP 075890671 Sinorhizobium meliloti BL225C
GH13 YP 0030504181 Methylovorus glucosetrophus SIP3-4
GH13 YP 4224271 Magnetospirillum magneticum AMB-1
GH13 YP 0042554001 Deinococcus proteolyticus MRP
GH13 ZP 029300551 Verrucomicrobium spinosum DSM 4136
GH13 ZP 080190951 Lautropia mirabilis ATCC 51599
GH13 YP 5335351 Rhodopseudomonas palustris BisB18
GH13 YP 8654291 Magnetococcus marinus MC-1
GH13 YP 0028938741 Tolumonas auensis DSM 9187
GH13 YP 6612131 Pseudoalteromonas atlantica T6c
GH13 YP 0040654571 Pseudoalteromonas sp SM9913
GH13 YP 8302311 Arthrobacter sp FB24
GH13 YP 0042347941 Acidovorax avenae subsp avenae ATCC 19860
GH13 YP 0016390191 Methylobacterium extorquens PA1
GH13 ZP 068060481 Brevibacterium mcbrellneri ATCC 49030
GH13 YP 0031655431 Candidatus Accumulibacter phosphatis clade IIA str UW-1
GH13 YP 9813411 Polaromonas naphthalenivorans CJ2
GH13 ZP 029305921 Verrucomicrobium spinosum DSM 4136
GH13 YP 8283661 Candidatus Solibacter usitatus Ellin6076
GH13 YP 0015687661 Petrotoga mobilis SJ95
GH13 YP 2560581 Sulfolobus acidocaldarius DSM 639
GH13 YP 0023732201 Cyanothece sp PCC 8801
GH13 AKF07413 Sandaracinus amylolyticus DSM 53668
GH13 AKF03796 Sandaracinus amylolyticus DSM 53668
GH13 EFS357291 Propionibacterium acnes HL013PA1
GH13 YP 0033827501 Kribbella flavida DSM 17836
GH13 YP 9230111 Nocardioides sp JS614
GH13 AEA239681 Pseudonocardia dioxanivorans CB1190
GH13 YP 0031037201 Actinosynnema mirum DSM 43827
GH57 YP 0018695221 Nostoc punctiforme PCC 73102
GH57 YP 0041847961 Terriglobus saanensis SP1PR4
GH57 YP 0033170621 Thermanaerovibrio acidaminovorans DSM 6589
GH57 YP 0025231331 Thermomicrobium roseum DSM 5159
GH57 CBL286501 Fretibacterium fastidiosum
GH57 YP 0041781671 Isosphaera pallida ATCC 43644
GH57 YP 0027549471 Acidobacterium capsulatum ATCC 51196
GH57 ZP 070292791 Granulicella mallensis MP5ACTX8
GH57 YP 0018749261 Elusimicrobium minutum Pei191
GH57 ADL259941 Fibrobacter succinogenes subsp succinogenes S85
GH57 YP 4773871 Synechococcus sp JA-2-3B
GH57 YP 8454221 Syntrophobacter fumaroxidans MPOB
GH57 NP 9534062 Geobacter sulfurreducens PCA
GH57 YP 5020321 Methanospirillum hungatei JF-1
GH57 YP 0019958521 Chloroherpeton thalassium ATCC 35110
GH57 YP 5937791 Candidatus Koribacter versatilis Ellin345
GH57 YP 0010471761 Methanoculleus marisnigri JR1
GH57 YP 0014309471 Roseiflexus castenholzii DSM 13941
GH57 YP 0029516361 Desulfovibrio magneticus RS-1
GH57 YP 0038064671 Desulfarculus baarsii DSM 2075
GH57 YP 0013789531 Anaeromyxobacter sp Fw109-5
GH57 ZP 063702071 Desulfovibrio sp FW1012B
GH57 YP 0035531061 Aminobacterium colombiense DSM 12261
GH57 ZP 064391271 Anaerobaculum hydrogeniformans ATCC BAA-1850
GH57 AKF06951 Sandaracinus amylolyticus DSM 53668
GH57 ZP 063915611 Dethiosulfovibrio peptidovorans DSM 11002
GH57 YP 0024634161 Chloroflexus aggregans DSM 9485
GH57 YP 0043692891 Desulfobacca acetoxidans DSM 11109
GH57 YP 8464661 Syntrophobacter fumaroxidans M
POB
GH57 CAJ738171 Candidatus Kuenenia stuttgartiensis
GH57 YP 0031969181 Desulfohalobium
retbaense DSM 5692
FIG 3mdashPhylogeny of Sandaracinus amylolyticus GH13 and GH57 a-amylases Sandaracinus amylolyticus a-amylases (GH13 and GH57) are colored in
pink along with their domain architecture displayed as Domain Color Shape [GH13 Red rectangle GH57 Black rectangle GH77 dark pink rectangle
CBM48 Dark blue circle CBM20 green circle CBM41 cyan circle] Myxobacterial homologs are colored fluorescent green Bootstrap values correspond-
ing to the tree nodes are provided
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2525
of the phyla Betaproteobacteria suggestive of a possible hor-
izontal transfer
AKF04017 and AKF03605 have close homologs in the ex-
tremophile Deinococcus desserti VCD115 and shares clade
with certain myxobacterial amylases belonging to
Myxococcus xanthus S cellulosum and Haliangium ocraceum
which further share clades with Deinococcus amylases
AKF04017 and AKF03605 share the highest sequence identity
with D desserti VCD115 plasmid protein (YP_002787899
533) and chromosomal protein (YP_002785382 456)
respectively that might point to its plasmid-borne horizontal
transfer across the clade (fig 3) The sequence conservation of
CSRs and the high percentage identity of these amylases to
Deinococcus support the inference for the putative horizontal
transfers among these members AKF09634 does not share its
sister branch with any member but the clade contains mem-
bers from diverse phyla that is Rhizobium from alphaproteo-
bacteria Opitutus from Chlamydae Chthonibacter from
Verrucomicrobia suggesting the multiple gain and loss of
this amylase among prokaryotes Similarly AKF07413 shares
the clade with members of Cyanobacteria AKF07406 shares
the clade with members of Deltaproteobacteria AKF03861
with members of Alphaproteobacteria and AKF03642 with
members of Bacteroidetes Taken together we can suggest
that many of the S amylolyticus amylases have been acquired
via horizontal gene transfer events from other bacterial species
belonging to the phylum Deinococcus-Thermus
Acidobacteria Cyanobacteria and Alphaproteobacteria
To further support these horizontal transfer events we
checked the sequence conservation between S amylolyticus
amylases and closely related members in the phylogenetic tree
and aligned using PROMALS-3D with Taka-Amylase A of A
oryzae (PDBid 2TAA) as a structural template We also per-
formed synteny studies analyzing proteins present in the vicin-
ity of all identified S amylolyticus GH13 and GH57 amylases
and their closely related homologs as revealed by phylogenetic
studies We could not trace any conserved syntenic regions in
these genomes whereas in the case of AKF03862 and
AKF09634 we identified glycogen debranching enzymes in
Stigmatella aurantiaca DW43-1 [56 identity and 98
query coverage of AKF03860 with WP_0133760911] and
Opitutus terrae PB90-1 [57 identity and 97 query cover-
age of AKF09635 YP_0018205421] which further support
their putative horizontal transfer in S amylolyticus
Similarly the phylogenetic inference was drawn from two
representative domains of g-amylases using the same meth-
odology as followed for a-amylases (fig 4) We found that
one of the g-amylases AKF10340 lies closer to M xanthus
amylase whereas the other AKF10487 shares its sister
branch with Nitrosococcus watsonii a Gammaproteobacteria
This suggests that while some of the a- and g-amylases
are present in many myxobacterial species a majority of the
a- and g-amylases are present only in S amylolyticus and not
in other myxobacterial genomes possibly due to the result of
horizontal transfer events from diverse bacterial phylum such
as Deinococcus-Thermus Cyanobacteria Alphaproteobac-
teria Gammaproteobacteria and Acidobacteria
Agar Degrading Enzymes
Some bacteria are known to degrade agar and use it as a
carbon source and possess agarase enzymes that help in hy-
drolyzing agar in oligosaccharides (Chi et al 2012) Depending
on the site of action they can be of two types a-agarase
(agarose 3-glycanohydrolase 321158 GH96) and
b-agarase (agarose 4-glycanohydrolase 32181 GH16
GH50 GH86 GH118) (table 1) Both families of agarases
GH15 EFV841771 Achrom
obacter xylosoxidans C54
GH
15 ZP 011267341 Nitrococcus m
obilis Nb-231
GH15 YP 4129921 Nitrosospira m
ultiformis ATCC 25196
GH15 YP 5716301 Nitrobacter ham
burgensis X14 plasmid
GH15 ZP 012260811 Aurantim
onas manganoxydans SI85-9A1
GH15 YP 0016032801 Gluconacetobacter diazotrophicus PA1 5
GH15 YP 7451371 Granulibacter bethesdensis CGDNIH1
GH15 AEA616211 Burkholderia gladioli BSR3
GH15 YP 0018344311 Beijerinckia indica subsp indica ATCC 9039
GH15 YP 0027560251 Acidobacterium capsulatum
ATCC 51196
GH15 CBJ387091 Ralstonia solanacearum CMR15
GH15 YP 3189151 Nitrobacter winogradskyi Nb-255
GH15 YP 0012344011 Acidiphilium cryptum JF-5
GH15 YP 0040115781 Rhodomicrobium vannielii ATCC 17100
GH15 YP 0016419391 Methylobacterium extorquens PA1GH15 YP 5252641 Rhodoferax ferrireducens T118
GH15 YP 0041811661 Terriglobus saanensis SP1PR4
GH15 ZP 031333281 Chthoniobacter flavus Ellin428
GH15 CCA537261 Streptomyces venezuelae ATCC 10712
GH15 YP 0015065621 Frankia sp EAN1pec
GH15 YP 0024892701 Arthrobacter chlorophenolicus A6
GH15 YP 0018202091 Opitutus terrae PB90-1
GH15 YP 8729011 Acidothermus cellulolyticus 11B
GH15 YP 0013795591 Anaeromyxobacter sp Fw109-5
GH15 YP 0024929091 Anaeromyxobacter dehalogenans 2CP-1
GH15 YP 6355121 Myxococcus xanthus DK 1622
GH15 YP 0011047051 Saccharopolyspora erythraea NRRL 2338
GH15 YP 0011092191 Saccharopolyspora erythraea NRRL 2338
GH15 AKF10340 Sandaracinus amylolyticus DSM 53668
GH15 YP 6342991 Myxococcus xanthus DK 1622
GH15 YP 0031091351 Acidimicrobium ferrooxidans DSM 10331
GH15 YP 0035703361 Salinibacter ruber M8
GH15 YP 0025241401 Thermomicrobium roseum DSM 5159 plasmid
GH15 YP 0033214441 Sphaerobacter thermophilus DSM 20745
GH15 NP 7336051 Streptomyces coelicolor A3-2
GH15 YP 0035282991 Nitrosococcus halophilus Nc 4
GH15 YP 0043676121 Marinithermus hydrothermalis DSM 14884
GH15 YP 0043687811 Marinithermus hydrothermalis DSM 14884
GH15 YP 0034042871 Haloterrigena turkmenica DSM 5511
GH15 ZP 080461001 Haladaptatus paucihalophilus DX253
GH1
5 YP
023
376
1 Pi
crop
hilu
s to
rrid
us D
SM 9
790
GH1
5 ZP
055
7131
11
Ferr
opla
sma
acid
arm
anus
fer1
GH1
5 BA
B604
801
The
rmop
lasm
a vo
lcan
ium
GSS
1
GH
15 Y
P 00
3323
761
1 Th
erm
obac
ulum
terr
enum
ATC
C B
AA
-798
GH
15 Y
P 00
3899
615
1 C
yano
thec
e sp
PC
C 7
822
plas
mid
GH
15 Y
P 00
3705
927
1 Tr
uepe
ra ra
diov
ictr
ix D
SM 1
7093
GH
15 Y
P 00
2478
443
1 A
rthr
obac
ter c
hlor
ophe
nolic
us A
6 pl
asm
idG
H15
AK
F104
87 S
anda
raci
nus
amyl
olyt
icus
DSM
536
68G
H15
YP
0037
6113
81
Nitr
osoc
occu
s w
atso
nii C
-113
GH1
5 ZP
069
7134
91
Kted
onob
acte
r rac
emife
r DSM
449
63
GH15
ZP
0697
6049
1 K
tedo
noba
cter
race
mife
r DSM
449
63
GH15
YP
0033
9335
81
Cone
xiba
cter
woe
sei D
SM 1
4684
GH15
YP
0042
3629
11 A
cido
vora
x av
enae
sub
sp a
vena
e ATC
C 19
860
GH15 Z
P 03
6273
271
Pedo
spha
era p
arvu
la El
lin51
4
GH15 Y
P 00
1618
7001
Sor
angi
um ce
llulo
sum
So
ce56
GH15 ZP
0145
9561
1 Stig
matella
aura
ntiac
a DW
4-3-1
GH15 ZP
0503
8177
1 Syn
echo
cocc
us sp
PCC 73
35
GH15 YP 64
4038
1 Rubrobac
ter xy
lanophilu
s DSM 99
41
GH15 ZP 064746651 Frankia symbiont of D
atisca glomerata
GH15 ZP 069744041 Ktedonobacter racemife
r DSM 44963
GH15 ZP 069653261 Ktedonobacter racemifer D
SM 44963
GH15 ZP 069661381 Ktedonobacter racemifer D
SM 44963
FIG 4mdashPhylogeny of Sandaracinus amylolyticus GH15 g-amylases
Sandaracinus amylolyticus g-amylases are colored dark pink and myxobac-
terial homologs are colored fluorescent green Bootstrap values corre-
sponding to the tree nodes are provided
Sharma et al GBE
2526 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
were absent in S amylolyticus genome which could be related
to its agar nondegrading property (Mohr et al 2012) as sup-
ported by colony morphology where agar was not penetrated
or liquefied by the bacteria (supplementary fig S5
Supplementary Material online)
Cellulose Degrading Enzymes
We identified only GH5 and GH74 in S amylolyticus proteome
in two and three proteins respectively GH5 domain was pre-
sent as a single unit in AKF04963 and AKF09194 whereas
GH74 was identified in AKF04859 AKF06963 and
AKF09775 associated with a GH74 domain CAZY domains
for cellobiohydrolaseexoglucanases (represented by GH6
GH7 GH9 and GH48) were not present in S amylolyticus
Cellobiohydrolaseexoglucanases plays a significant role in cel-
lulose degradation as they cut two to four sugar subunits from
the exposed ends formed by the endoglucanases which are
used as initial substrates for b-glucosidase (Mba Medie et al
2012) The absence of cellobiohydrolase or exoglucanases
could be a possible reason for the lack of cellulose degradation
in S amylolyticus Out of b-glucosidase representative CAZY
families GH1 GH3 GH5 GH9 GH30 and GH116 only GH1
GH3 and GH5 families were found in three two and two
proteins in S amylolyticus respectively AKF03843 AKF0
6028 and AKF10711 have a GH1 domain whereas
AKF09078 and AKF10875 have only the GH3 domain
Chitin Degrading Enzymes
Chitinases (EC32114) are involved in degradation of chitin
a component of the cell wall of fungi and exoskeleton of
arthropods GH18 GH19 GH23 and GH48 families have
been reported in the CAZY database as chitinases that hydro-
lyse b-14 linkages between N-acetylglucosamine units GH18
is a chitinase family with a characteristic ldquo[LIVM
FY][DN]G[LIVMF][DN][LIVMF][DN]Erdquo motif and is expressed
in archaea prokaryotes and eukaryotes (Funkhouser and
Aronson 2007) GH18 chitinases were not found in S amylo-
lyticus genome while members of GH19 (AKF03006 and
AKF10423) and GH23 (AKF04575 AKF05533 AKF06346
AKF08835 and AKF11377) could be identified GH19
family chitinases are mostly reported to be present in plants
with only a few members in prokaryotes (Kasprzewska 2003
Funkhouser and Aronson 2007) Among the two identified
GH19 homologs we found that AKF10423 is approximately
65 identical (~68 query coverage) with cyanobacteria
such as Nodosilinea nodulosa and Limnoraphis robusta
along with another myxobacterium Chondromyces crocatus
Besides these homologs we also found that AKF10423 shows
maximum similarity with eukaryotic GH19 chitinases counter-
parts as present in Acacia mangium Festuca arundinacea
Triticum aestivum and Leucaena leucocephala (55ndash59 iden-
tity with 70 query coverage) We identified that another
chitinase AKF06346 has GH23 domain along with three
copies of CBM50 which is generally present in the enzymes
cleaving either chitin or peptidoglycan
Conclusion
The complete 1033 Mb circular genome of S amylolyticus
type strain DSM 53668T was determined CAZy analysis re-
vealed the presence of 72 GT domains 72 CE domains 58 GH
domains 19 AA domains 4 PL domains and 26 CBM do-
mains Sixteen S amylolyticus proteins were identified of
which 15 are a-amylases of class GH13 and 1 GH57 Two
g-amylases of GH15 family were also identified Phylogeny
analysis revealed that ten GH13 a-amylases and one GH15
g-amylase have been aquired via horizontal gene transfer
from other bacterial phyla such as Deinococcus-Thermus
Cyanobacteria Alphaproteobacteria Gammaproteobacteria
and Acidobacteria
Table 1
Cellulose Starch Chitin and Agar Degrading Enzymes and Their Representative CAZY Domains Distribution
Substrate Enzyme Category Enzyme Name EC Number CAZY Families (protein repertoire in brackets)
Starch Amylase a-Amylase 3211 GH13 (15) GH57 (1) GH119 GH126
Amylase b-Amylase 3212 GH14
Amylase g-Amylase 3213 GH15 (2) GH97
Chitin Chitinase Chitinase 32114 GH18 GH19 (2) GH23 (5) GH48
Agar a-Agarase Agarose 3-glycanohydrolase 321158 GH96
b-Agarase Agarose 4-glycanohydrolase 32181 GH16 GH50 GH86 GH118
Cellulose Cellulase b-Glucosidase 32121 GH1 (3) GH3 (2) GH5 (2) GH9 GH30 GH116
Cellulase Cellobiohydrolase or Exoglucanase 321176 GH48 GH6 GH7 GH9
Cellulase Endoglucanase 3214 GH5 (2) GH6 GH7 GH8 GH9 GH12 GH44
GH45 GH48 GH51 GH74 (3) GH124
NOTEmdashEC number enzyme category and CAZY families for proteins involved in starch chitin agar and cellulose degradation are shown The number of Sandaracinusamylolyticus proteins identified having CAZY domains are represented in bold fonts in respective families
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2527
Supplementary Material
Supplementary figures S1ndashS5 and tables S1 and S2 are avail-
able at Genome Biology and Evolution online (httpwww
gbeoxfordjournalsorg)
Acknowledgments
This work is supported by a project ldquoExpansion and modern-
ization of Microbial Type Culture Collection and Gene Bank
(MTCC) jointly supported by Council of Scientific and
Industrial Research (CSIR) Grant No BSC0402 and
Department of Biotechnology (DBT) Govt of India Grant No
BTPR7368INF221772012rdquo The authors thank MTCC for
providing help in procuring the cultures from DSMZ They
acknowledge Dr Ramya TNC for providing workbench for cul-
turing myxobacteria GS and IK acknowledge the Council
of Scientific and Industrial Research (CSIR) and University
Grant Commission (UGC) for the research fellowship respec-
tively They also thank the Genome Quebec Innovation
Centre McGill University Canada and C-CAMP next-genera-
tion genomics facility Bangalore India for help in obtaining
PacBio and Illumina NGS data respectively
Literature CitedAlikhan NF Petty NK Ben Zakour NL Beatson SA 2011 BLAST Ring
Image Generator (BRIG) simple prokaryote genome comparisons
BMC Genomics 12402
Altschul SF Gish W Miller W Myers EW Lipman DJ 1990 Basic local
alignment search tool J Mol Biol 215403ndash410
Auch AF Von Jan M Klenk HP Goker M 2010 Digital DNA-DNA hybrid-
ization for microbial species delineation by means of genome-to-
genome sequence comparison Stand Genomic Sci 2117ndash134
Aziz RK et al 2008 The RAST Server rapid annotations using subsystems
technology BMC Genomics 975
Blesak K Janecek S 2012 Sequence fingerprints of enzyme specificities
from the glycoside hydrolase family GH57 Extremophiles 16497ndash
506
Chi WJ Chang YK Hong SK 2012 Agar degradation by microorganisms
and agar-degrading enzymes Appl Microbiol Biotechnol 94917ndash
930
Chin CS et al 2013 Nonhybrid finished microbial genome assemblies
from long-read SMRT sequencing data Nat Methods 10563ndash569
Eddy SR 2011 Accelerated profile HMM searches PLoS Comput Biol
7e1002195
Edgar RC 2004 MUSCLE multiple sequence alignment with high accu-
racy and high throughput Nucleic Acids Res 321792ndash1797
Funkhouser JD Aronson NN Jr 2007 Chitinase family GH18 evolutionary
insights from the genomic history of a diverse protein family BMC Evol
Biol 796
Garcia R Muller R 2014 The family Polyangiaceae In Rosenberg E
Delong E Lory S Stackebrandt E Thompson F editors The prokary-
otes Berlin Heidelberg Springer p 247ndash279
Grissa I Vergnaud G Pourcel C 2007 CRISPRFinder a web tool to identify
clustered regularly interspaced short palindromic repeats Nucleic
Acids Res 35W52ndashW57
Hall TA 1999 BioEdit a user-friendly biological sequence alignment editor
and analysis program for Windows 9598NT Nucleic Acids Symp Ser
4195ndash98
Han K et al 2013 Extraordinary expansion of a Sorangium cellulosum
genome from an alkaline milieu Sci Rep 32101
Janecek S Kuchtova A 2012 In silico identification of catalytic residues
and domain fold of the family GH119 sharing the catalytic machinery
with the alpha-amylase family GH57 FEBS Lett 5863360ndash3366
Janecek S Svensson B Macgregor EA 2014 alpha-Amylase an enzyme
specificity found in various families of glycoside hydrolases Cell Mol
Life Sci 711149ndash1170
Kasprzewska A 2003 Plant chitinasesndashregulation and function Cell Mol
Biol Lett 8809ndash824
Lagesen K et al 2007 RNAmmer consistent and rapid annotation of
ribosomal RNA genes Nucleic Acids Res 353100ndash3108
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B
2014 The carbohydrate-active enzymes database (CAZy) in 2013
Nucleic Acids Res 42D490ndashD495
Lowe TM Eddy SR 1997 tRNAscan-SE a program for improved detection
of transfer RNA genes in genomic sequence Nucleic Acids Res
25955ndash964
Matsuura Y Kusunoki M Harada W Kakudo M 1984 Structure
and possible catalytic residues of Taka-amylase A J Biochem
95697ndash702
Mba Medie F Davies GJ Drancourt M Henrissat B 2012 Genome anal-
yses highlight the different biological roles of cellulases Nat Rev
Microbiol 10227ndash234
Mohr KI Garcia RO Gerth K Irschik H Muller R 2012
Sandaracinus amylolyticus gen nov sp nov a starch-degrading
soil myxobacterium and description of Sandaracinaceae fam
nov Int J Syst Evol Microbiol 621191ndash1198
Noguchi J et al 2011 Crystal structure of the branching enzyme I
(BEI) from Oryza sativa L with implications for catalysis and sub-
strate binding Glycobiology 211108ndash1116
Patel RK Jain M 2012 NGS QC Toolkit a toolkit for quality control of next
generation sequencing data PLoS One 7e30619
Pei J Kim BH Grishin NV PROMALS3D a tool for multiple protein se-
quence and structure alignments Nucleic Acids Res 36 2295ndash2300
Reichenbach H Dworkin M 1992 The myxobacteria In Balows A
Truper HG Dworkin M Harder W Schleifer KH editors The pro-
karyotes a handbook on the biology of bacteria ecophysiology
isolation identification applications New York Springer New
York p 3416ndash3487
Reichenbach H 2005 ldquoOrder VIII Myxococcalesrdquo Bergeyrsquos Manual of
Systematic Bacteriology In Brenner DJ Krieg NR Staley JT Garrity
GM editors Vol 2 2nd ed part C New York Springer-Verlag New
York Inc pp 1074ndash1079
Richter M Rossello-Mora R 2009 Shifting the genomic gold standard for
the prokaryotic species definition Proc Natl Acad Sci U S A 106
19126ndash19131
Schneiker S et al 2007 Complete genome sequence of the myxobacter-
ium Sorangium cellulosum Nat Biotechnol 25 1281ndash1289
Shimkets LJ 1990 Social and developmental biology of the myxobacteria
Microbiol Rev 54 473ndash501
Siguier P Perochon J Lestrade L Mahillon J Chandler M 2006 ISfinder
the reference centre for bacterial insertion sequences Nucleic Acids
Res 34 D32ndashD36
Steinmetz H et al 2012 Indiacens A and B prenyl indoles from the
myxobacterium Sandaracinus amylolyticus J Nat Prod 75 1803ndash
1805
Tamura K Stecher G Peterson D Filipski A Kumar S 2013 MEGA6
molecular evolutionary genetics analysis version 60 Mol Biol Evol
30 2725ndash2729
Van Der Maarel MJ Van Der Veen B Uitdehaag JC Leemhuis H Dijkhuizen
L 2002 Properties and applications of starch-converting enzymes of
the alpha-amylase family J Biotechnol 94 137ndash155
Sharma et al GBE
2528 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
Weissman KJ Muller R 2010 Myxobacterial secondary metabolites
bioactivities and modes-of-action Nat Prod Rep 27 1276ndash1295
Whitworth DE 2008 Myxobacteria multicellularity and differentiation
Washington DC ASM Press American Society for Microbiology
Yin Y et al 2012 dbCAN a web resource for automated carbohydrate-
active enzyme annotation Nucleic Acids Res 40 W445ndashW451
Zhao JY et al 2008 Discovery of the autonomously replicating plasmid
pMF1 from Myxococcus fulvus and development of a gene cloning
system in Myxococcus xanthus Appl Environ Microbiol 74 1980ndash
1987
Associate editor Howard Ochman
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2529
Evolutionary History of S amylolyticus Amylases
Amylases belonging to glycoside hydrolase (GH) enzymatic
category degrade starch into sugar moieties by the
hydrolysis of a-14- and a-16-glycosidic bond (Van der
Maarel et al 2002) Based on the site of action they are di-
vided into three categories a-amylase (EC3211 GH13
GH57 GH119 GH126) b-amylase (EC3212 GH14) and
-amylase (EC3213 GH15 GH97) Sandaracinus
amylolyticus genome contains both a- and -amylases
whereas no b-amylases could be found Among the 4 a-am-
ylases GH families 15 GH13 proteins and 1 GH57 protein
were found We could identify two -amylases belonging to
Sandaracinus amylolyticusTDSM 53668
CP01112510328378 bp
FIG 1mdashCircular representation of the Sandaracinus amylolyticus DSM 53668T complete genome Circles (from inside to outside) 1 and 2 (GC content
black line and GC skew magenta and green lines) circle 3 (CAZome Proteins annotated as carbohydrate active enzymes Red) circle 4 5 and 6 (Amylases
Cellulases and Chitinases Black) circle 7 (encoded RNA Magenta) circle 8 (CDS present on negative strand Blue) circle 9 (CDS present on positive strand
Green) circle 10 (S amylolyticus DSM 53668T complete genome Red) BRIG 095 was used to build the circular representation (Alikhan et al 2011)
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2523
GH15 category (fig 2B) The starch degrading proteins have
been reported to have CBM20 CBM21 CBM25 CBM26
CBM34 CBM41 CBM45 CBM48 CBM53 and CBM58
modules that help in glycogenstarch binding truncation of
which reduces its enzymatic activity (Noguchi et al 2011
Janecek et al 2014) GH15 and GH57 domains are present
as single domain proteins in S amylolyticus Interestingly in 5
out of 15 GH13 S amylolyticus proteins (AKF03796
AKF03860 AKF06952 AKF07413 and AKF11765) the a-
amylase GH13 domain was present along with a CBM48
module at the N-termini The CBM48 module assists in the
GH13 function by serving as starch-binding domains
The GH13 family include the a-amylase domains with an (b
a)8 barrel catalytic domain These proteins have 4ndash7 conserved
sequence regions (CSRs) and a specific catalytic triad consisting
of a catalytic nucleophile (aspartic acid) on strand b4 proton
donor (glutamic acid) on strand b5 and a transition state sta-
bilizer (aspartic acid) on strand b7 (Janecek et al 2014) GH13
members show extremely low sequence identity with their ho-
mologs but the catalytic triad is highly conserved GH57 family
a-amylases have a (ba)7 barrel structure and have five CSRs as
compared with the GH13 family However only two of the
catalytic residues viz the nucleophile on strand b4 and the
proton donor on strand b7 are conserved (Blesak and Janecek
2012) Irrespective of these differences GH13 and GH57 both
employ the same retaining mechanism of action (Janecek and
Kuchtova 2012) Sandaracinus amylolyticus GH13 proteins
were retrieved and the structure of Taka amylase of A
oryzae (PDBid 2TAA) was used to perform a structure-based
alignment using PROMALS3D (Matsuura et al 1984 Pei et al
2008) All the seven CSR regions and the three catalytic
residues were conserved in all S amylolyticus GH13 amylases
(supplementary figs S2 and S3 Supplementary Material
online) Similarly S amylolyticus GH57 (AKF06951) was
aligned with its homologs using PROMALS3D based on the
4-a-glucanotransferase structure of Thermococcus litoralis
(PDBid 1K1W) In the GH57 alignment all five CSRs and
both catalytic residues were conserved (supplementary figS4
Supplementary Material online)
In order to understand the provenance of these a- and g-
amylases they were subjected to BLASTp against the NR data-
base To include amylases across the entire bacterial phylum
the CAZY-NR database for GH13 and GH57 domains were
retrieved and subjected to BLASTClust at 70 identity and
70 length coverage that reduced the domain sequence
space from 19590 to 4038 domains Further amylase do-
mains from S amylolyticus were subjected to BLASTp against
these 4038 domains and their top hits were retrieved and
included in the phylogenetic analysis All these GH13 and
GH57 domains along with S amylolyticus amylase domains
were aligned using MUSCLE as incorporated in MEGAv606
and ML-based phylogeny with 100 bootstrap values was
drawn (fig 3) The ML tree obtained was divided into three
major clades where the clade at root composed of all the GH57
domains and the other two major clades further composed of
all the GH13 sequences The representative bacterial amylases
from various clades show close relations with amylases of di-
verse bacterial groups suggesting the multiple gain and loss of
these domains As all the amylase domains of S amylolyticus
are scattered across the tree the phylogenetic position of these
domains could provide us insights about their provenance and
reveal possible cases of horizontal transfers
19
26
72
58
72
4
0
10
20
30
40
50
60
70
80
AA CBM CE GH GT PL
A B
Num
ber o
f pro
tein
s
GH13
GH13
CBM48 GH13
GH13CBM48
GH13
GH57
GH13CBM48
GH13GH13CBM48
GH13
GH13
GH13
GH15
GH13CBM48
GH13
AKF03605AKF03642AKF03796
AKF03860AKF03861AKF03862AKF04017AKF06951AKF06952
AKF07406AKF07413AKF07527AKF09634AKF09643AKF10340AKF10487AKF11765AKF11772
GH13
GH13
GH15
FIG 2mdashSandaracinus amylolyticus DSM 53668T CAZome distribution (A) Sandaracinus amylolyticus DSM 53668T CAZY families presented in a bar
graph x axis represents the CAZY categories and y axis represents the number of proteins (B) a- and g-amylases along with their CAZome domain
architecture The domains have been colored as GH13 sky blue GH15 dark red GH57 green and CBM48 orange
Sharma et al GBE
2524 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
Taking into account the GH57 clade the sole GH57
domain of S amylolyticus (GenBank ID AKF06951) shares
the clade with anaerobic Deltaproteobacteria Among the
GH13 domains of S amylolyticus AKF03862 AKF07527
AKF09643 AKF11765 and AKF11772 shares clade with
other myxobacteria Among these AKF11772 and
AKF07527 share sister branches and form sister clades with
other myxobacterial species such as S cellulosum Soce56 and
Plesiocystis pacifica SIR-1 and are further related to the clade
containing Gammaproteobacteria Apart from these two am-
ylases AKF03860 and AKF06952 are similar to each other and
group with Candidatus accumulibacter phosphtis amylase
(YP_0031672861) from the phylum Acidobacteria
Interestingly AKF03860 and AKF06952 group with members
GH13 ZP 021632211 Kordia algicida OT-1
GH13 ZP 067516041 Parascardovia denticolens F0305
GH13 YP 0035709041 Salinibacter ruber M8
GH13 YP 0032500041 Fibrobacter succinogenes subsp succinogenes S85
GH13 AKF03862 Sandaracinus amylolyticus DSM 53668
GH13 YP 0039537801 Stigmatella aurantiaca
GH13 CAJ738231 Candidatus Kuenenia stuttgartiensis
GH13 YP 0228461 Picrophilus torridus DSM 9790GH13 ZP 029306261 Verrucomicrobium spinosum DSM 4136
GH13 CAX841261 uncultured bacterium
GH13 YP 0031678771 Candidatus Accumulibacter phosphatis clade IIA str UW-1GH13 ZP 031321321 Chthoniobacter flavus Ellin428
GH13 ZP 018759791 Lentisphaera araneosa HTCC2155GH13 YP 0033894351 Spirosoma linguale DSM 74GH13 NP 7734061 Bradyrhizobium diazoefficiens USDA 110
GH13 YP 0037757321 Herbaspirillum seropedicae SmR1GH13 YP 4266911 Rhodospirillum rubrum ATCC 11170GH13 ZP 035079501 Rhizobium etli Brasil 5
GH13 ZP 057930691 Butyrivibrio crossotus DSM 2876
GH13 ZP 066231321 Turicibacter sanguinis PC909
GH13 ZP 044499741 Catonella morbi ATCC 51271
GH13 EGE534241 Streptococcus parauberis NCFD 2020
GH13 196049865 Lactobacillus Plantarum
GH13 YP 0042196261 Granulicella tundricola MP5ACTX9
GH13 YP 0011935611 Flavobacterium johnsoniae UW101
GH13 YP 0032910851 Rhodothermus marinus DSM 4252GH13 YP 0040967591 Bacillus cellulosilyticus DSM 2522GH13 ZP 037030351 Flavobacteria bacterium MS024-2A
GH13 XP 0025352531 Ricinus communis
GH13 YP 7579561 Maricaulis maris MCS10GH13 ZP 010417351 Erythrobacter sp NAP1GH13 YP 0018177571 Opitutus terrae PB90-1GH13 EFR587141 Alistipes sp HGB5
GH13 CBK627851 Alistipes shahii WAL 8301GH13 EFR582631 Alistipes sp HGB5
GH13 YP 0034042011 Haloterrigena turkmenica DSM 5511
GH13 YP 0035357541 Haloferax volcanii DS2
GH13 YP 0035357541 Haloferax volcanii DS2
GH13 YP 0025661621 Halorubrum lacusprofundi ATCC 49239
GH13 YP 1368761 Haloarcula marismortui ATCC 43049
GH13 ZP 080436741 Haladaptatus paucihalophilus DX253
GH13 YP 0025652161 Halorubrum lacusprofundi ATCC 49239
GH13 YP 0035346301 Haloferax volcanii DS2
GH13 YP 0033225661 Thermobaculum terrenum ATCC BAA-798
GH13 YP 0021353421 Anaeromyxobacter sp K
GH13 AKF09643 Sandaracinus amylolyticus DSM 53668
GH13 YP 0016137031 Sorangium cellulosum So ce56
GH1
3 99
0316
21 H
alot
herm
othr
ix or
enii
GH1
3 AA
D051
991
Pae
niba
cillu
s po
lymyx
a
GH1
3 YP
003
6999
601
Bac
illus
sele
nitir
educ
ens
MLS
10
GH1
3 ZP
022
0799
81
Copr
ococ
cus
euta
ctus
ATC
C 27
759
GH
13 B
AA07
401
1 Xa
ntho
mon
as c
ampe
stris
GH
13 Y
P 00
4052
835
1 M
ariv
irga
tract
uosa
DSM
412
6
GH1
3 ZP
037
0954
51
Cory
neba
cter
ium
mat
ruch
otii A
TCC
3380
6
GH1
3 NP
267
649
1 La
ctoc
occu
s la
ctis
subs
p la
ctis
Il140
3
GH1
3 YP
001
3023
201
Par
abac
tero
ides
dist
ason
is AT
CC 8
503
GH1
3 YP
001
2276
361
Syn
echo
cocc
us s
p R
CC30
7
GH1
3 YP
002
5652
591
Hal
orub
rum
lacu
spro
fund
i ATC
C 49
239
GH13
ZP
0709
0550
1 C
oryn
ebac
teriu
m g
enita
lium
ATC
C 33
030
GH13
YP 0
0283
4100
1 C
oryn
ebac
teriu
m a
urim
ucos
um A
TCC
7009
75
GH13 A
KF03
861
Sand
arac
inus a
mylo
lyticu
s DSM
536
68
GH13
ZP 0
0050
865
1 M
agne
tosp
irillum
mag
neto
tacti
cum
MS-
1
GH13 Y
P 00
1568
811
1 Pe
troto
ga m
obilis
SJ9
5
GH13 Y
P 02
2847
1 P
icrop
hilus
torri
dus D
SM 9
790
GH13 E
FT25
5011
Pro
pionib
acter
ium ac
nes H
L110
PA3
GH13 E
FA80
1951
Poly
spho
ndyli
um pa
llidum
PN50
0
GH13 A
BD4655
81 V
ermam
oeba
verm
iform
is
GH13 Z
P 0350
5050
1 Rhiz
obium
etli B
rasil
5
GH13 YP 00
1348
4651
Pseud
omon
as ae
rugino
sa PA7
GH13 YP 00
2238
3511
Klebsie
lla pn
eumon
iae 34
2
GH13 YP 00
1437
9781
Cron
obac
ter sa
kaza
kii ATCC B
AA-894
GH13 Y
P 0011
7103
71 P
seud
omon
as st
utzeri
A1501
GH13 Y
P 0019
7240
81 S
tenotr
opho
monas
malt
ophil
ia K27
9a
GH13 Y
P 0043
1901
11 S
phing
obac
terium
sp 2
1
GH13 YP 0013127791 Sinorhizobium m
edicae W
SM419
GH13 ZP 035028921 Rhizobium etli K
im 5
GH13 YP 67
6219
1 Che
lativo
rans s
p BNC1
GH13 YP 00
2376
8861
Cya
nothe
ce sp
PCC 74
24
GH13 YP 0036383061 Cellulomonas flavig
ena DSM 20109
GH13 YP 0040977291 Intrasporangium ca
lvum DSM 43043
GH13 YP 0011691691 Rhodobacter s
phaeroides ATCC 17025
GH13 ZP 011580901 Oceanicola granulosus H
TCC2516
GH13 YP 0038382651 Micromonospora aurantiaca ATCC 27029
GH13 YP 0040977251 Intrasporangium calvu
m DSM 43043
GH13 YP 0036954761 Starkeya novella DSM 506
GH13 ZP 035166441 Rhizobium etli IE4771
GH13 ZP 031273131 Chthoniobacter flavus Ellin
428
GH13 AKF03642 Sandaracinus amylolyticus DSM 53668
GH13 YP 0031252671 Chitinophaga pinensis DSM 2588
GH13 YP 0030850521 Dyadobacter fermentans DSM 18053
GH13 YP 0033879241 Spirosoma linguale DSM 74
GH13 YP 0034301801 Streptococcus gallolyticus UCN34
GH13 ZP 078184041 Eremococcus coleocola ACS-139-V-Col8
GH13 YP 0034774501 Thermoanaerobacter italicus Ab9
GH13 ZP 048517861 Paenibacillus sp oral taxon 786 str D14
GH13 YP 0038414241 Caldicellulosiruptor obsidiansis OB47
GH13 YP 0024279661 Desulfurococcus kamchatkensis 1221n
GH13 YP 0034262831 Bacillus pseudofirmus OF4
GH13 ZP 076854311 Oscillochloris trichoides DG-6GH13 YP 0016174961 Sorangium cellulosum So ce56
GH13 ZP 019113371 Plesiocystis pacifica SIR-1
GH13 YP 6318811 Myxococcus xanthus DK 1622
GH13 AKF11765 Sandaracinus amylolyticus DSM 53668
GH13 ZP 036771121 Bacteroides cellulosilyticus DSM 14838
GH13 YP 0037157841 Croceibacter atlanticus HTCC2559
GH13 ZP 012015681 Flavobacteria bacterium BBFL7
GH13 ZP 019113321 Plesiocystis pacifica SIR-1GH13 YP 0031962511 Robiginitalea biformata HTCC2501
GH13 YP 0040531921 Marivirga tractuosa DSM 4126
GH13 ZP 021817881 Flavobacteriales bacterium ALC-1
GH13 CBL150401 Ruminococcus bromii L2-63
GH13 ZP 080051001 Bacillus sp 2 A 57 CT2
GH13 CBK911271 Eubacterium rectale DSM 17629
GH13 YP 0029369771 Eubacterium rectale ATCC 33656
GH13 YP 0021366011 Anaeromyxobacter sp K
GH13 ZP 014604541 Stigmatella aurantiaca
GH
13 728851 Streptomyces lividans
GH
13 BAJ281541 Kitasatospora setae KM-6054
GH
13 YP 0033390201 Streptosporangium roseum
DSM
43021
GH
13 YP 9214291 Nocardioides sp JS614
GH
13 AEB462451 Verrucosispora maris AB-18-032
GH
13 CAJ810301 Actinoplanes sp
GH
13 NP 9387401 C
orynebacterium diphtheriae N
CTC
13129
GH
13 YP 0036976141 Arcanobacterium haem
olyticum D
SM 20595
GH
13 ZP 039259261 Actinomyces coleocanis D
SM 15436
GH
13 YP 0040999421 Intrasporangium calvum
DSM
43043
GH
13 YP 0030989701 Actinosynnema m
irum D
SM 43827
GH
13 YP 0036376081 Cellulom
onas flavigena DSM
20109
GH
13 ZP 009938561 Janibacter sp HTC
C2649
GH13 YP 0030608421 Hirschia baltica ATCC 49814
GH13 YP 7579631 M
aricaulis maris M
CS10
GH13 YP 0016857121 Caulobacter sp K31
GH13 ZP 013016731 Sphingom
onas sp SKA58
GH13 YP 0038198341 Brevundim
onas subvibrioides ATCC 15264
GH
13 YP 4971641 Novosphingobium
aromaticivorans D
SM 12444
GH13 YP 6609551 Pseudoalterom
onas atlantica T6c
GH13 ZP 011126451 Reinekea blandensis M
ED297
GH13 YP 2677341 Colwellia psychrerythraea 34H
GH13 ZP 011126461 Reinekea blandensis M
ED297
GH13 AKF11772 Sandaracinus amylolyticus DSM
53668
GH13 AKF07527 Sandaracinus amylolyticus DSM
53668
GH13 YP 0016184291 Sorangium cellulosum
So ce56
GH13 ZP 019113391 Plesiocystis pacifica SIR-1
GH13 ZP 036315591 Pedosphaera parvula Ellin514
GH13 YP 0016604791 Microcystis aeruginosa NIES-843
GH13 YP 0043416831 Archaeoglobus veneficus SNP6
GH13 ZP 073351521 Desulfovibrio fructosovorans JJ
GH
13 AKF03605 Sandaracinus amylolyticus D
SM 53668
GH
13 YP 0027853821 Deinococcus deserti VC
D115
GH
13 NP 2951951 D
einococcus radiodurans R1
GH
13 YP 004171
1212
M
SD
sisn
epoc
ira
m su
ccoc
onie
D 1
661
1
GH
13 YP 0041715811 Deinococcus m
aricopensis DSM
21211
GH
13 YP 0037038171 Truepera radiovictrix DSM
17093
GH
13 YP 6052101 Deinococcus geotherm
alis DSM
11300
GH
13 YP 0027555491 A
cidobacterium capsulatum
ATCC
51196
GH
13 Y
P9
XTC
A5P
M alocirdnut allecilunarG 1860612400
GH
13 YP 6335241 M
yxococcus xanthus DK
1622G
H13 Y
PB
S su
cihp
ortid
ica
suhp
ortny
S 1
9412
64
GH
13 A
AW32
490
1 iiklahcsttog acnarborean
A
GH
13 Y
P 00
2316
532
1 An
oxyb
acillu
s fla
vith
erm
us W
K1
GH
13 Y
P 00
3252
634
1 G
eoba
cillu
s sp
Y41
2MC
61G
H13
ZP
0117
0936
1 B
acillu
s sp
NR
RL
B-14
911
GH
13 Z
P 07
7074
981
Bac
illus
sp m
3-13
GH
13 Z
P1-
GS ps sullica
B 116916810
GH
13 Y
P 00
1813
251
1 Ex
iguo
bact
eriu
m s
ibiri
cum
255
-15
GH
13 Y
P 00
2886
871
1 Ex
iguo
bact
eriu
m s
p A
T1b
GH
13 Y
P 00
1568
454
1 Pe
troto
ga m
obilis
SJ9
5
GH
13 Y
P 00
2940
188
1 Ko
smot
oga
olea
ria T
BF 1
95
1G
H13
ZP
0125
8420
1 V
ibrio
alg
inol
ytic
us 1
2G01
GH
13 1
0911
18 T
herm
oact
inom
yces
vul
garis
GH
13 Z
P 06
9705
751
Kte
dono
bact
er ra
cem
ifer D
SM 4
4963
GH
13 Y
P 00
1545
735
1 H
erpe
tosi
phon
aur
antia
cus
DSM
785
GH
13 E
FY95
778
1 M
etar
hizi
um ro
berts
ii AR
SEF
23
GH
13 N
P 58
8205
1 S
chiz
osac
char
omyc
es p
ombe
972
h-
GH
13 N
P 59
4401
1 S
chiz
osac
char
omyc
es p
ombe
972
h-
GH
13 X
P 00
1908
940
1 Po
dosp
ora
anse
rina
S m
at+
GH
13 4
1016
824
Schw
anni
omyc
es o
ccid
enta
lis
GH
13 B
AF98
616
1 H
ypho
pich
ia b
urto
nii
GH13 AKF07406 Sandaracinus amylolyticus DSM 53668
GH13 YP 0043692881 Desulfobacca acetoxidans DSM 11109
GH13 YP 0023716111 Cyanothece sp PCC 8801GH13 YP 3219451 Anabaena variabilis ATCC 29413
GH13 YP 4738811 Synechococcus sp JA-3-3AbGH13 YP 8464671 Syntrophobacter fumaroxidans MPOBGH13 ZP 063702061 Desulfovibrio sp FW1012B
GH13 YP 0033193471 Sphaerobacter thermophilus DSM 20745GH13 YP 0037057361 Truepera radiovictrix DSM 17093GH13 YP 4120971 Nitrosospira multiformis ATCC 25196GH13 YP 6431071 Rubrobacter xylanophilus DSM 9941
GH13 BAD389831 Rhodobacter sphaeroides f sp denitrificans
GH13 YP 3175601 Nitrobacter winogradskyi Nb-255
GH13 YP 8215181 Candidatus Solibacter usitatus Ellin6076
GH13 YP 0034506361 Azospirillum sp B510
GH13 ZP 070169471 Desulfonatronospira thiodismutans ASO3-1
GH13 YP 0041842351 Terriglobus saanensis SP1PR4
GH13 ZP 070315011 Granulicella mallensis MP5ACTX8
GH13 YP 0029438961 Variovorax paradoxus S110
GH13 YP 5918381 Candidatus Koribacter versatilis Ellin345
GH13 YP 7445681 Granulibacter bethesdensis CGDNIH1
GH13 YP 0039098001 Burkholderia sp CCGE1003
GH13 ZP 024903661 Burkholderia pseudomallei NCTC 13177
GH13 YP 0016309761 Bordetella petrii DSM 12804
GH13 YP 0022527351 Ralstonia solanacearum MolK2
GH13 YP 0037759061 Herbaspirillum seropedicae SmR1
GH13 YP 5455251 Methylobacillus flagellatus KT
GH13 YP 0013485041 Pseudomonas aeruginosa PA7
GH13 YP 0016008321 Gluconacetobacter diazotrophicus PA1 5
GH13 AEB584851 Pseudomonas mendocina NK-01
GH13 YP 0027996471 Azotobacter vinelandii DJ
GH
13 N
P 63
5804
1 X
anth
omon
as c
ampe
stris
pv
cam
pest
ris s
tr A
TCC
339
13
GH
13 Y
P 00
3188
581
1 Ac
etob
acte
r pas
teur
ianu
s IF
O 3
283-
01
GH
13 Y
P 74
4570
1 G
ranu
libac
ter b
ethe
sden
sis
CG
DN
IH1
GH
13 Y
P 00
3052
329
1 M
ethy
lovo
rus
gluc
oset
roph
us S
IP3-
4
GH
13 Z
P 06
8981
631
Ros
eom
onas
cer
vica
lis A
TCC
499
57
GH
13 Y
P 00
2943
895
1 Va
riovo
rax
para
doxu
s S1
10
GH
13 Y
P 31
4931
1 T
hiob
acillu
s de
nitri
fican
s AT
CC
252
59
GH
13 Y
P 42
5597
1 R
hodo
spiri
llum
rubr
um A
TCC
111
70
GH
13 Y
P 93
3302
1 A
zoar
cus
sp B
H72
GH
13 N
P 43
7816
1 S
inor
hizo
bium
mel
iloti
1021
GH
13 Z
P 01
1553
281
Oce
anic
ola
gran
ulos
us H
TCC
2516
GH
13 Y
P 00
1167
979
1 R
hodo
bact
er s
phae
roid
es A
TCC
170
25
GH
13 Y
P 00
1440
575
1 C
rono
bact
er s
akaz
akii A
TCC
BAA
-894
GH
13 Z
P 01
4401
991
Ful
vim
arin
a pe
lagi
HTC
C25
06
GH
13 Y
P 00
4234
793
1 Ac
idov
orax
ave
nae
subs
p a
vena
e AT
CC
198
60
GH
13 Y
P 00
1818
894
1 O
pitu
tus
terra
e PB
90-1
GH
13 Y
P 00
3154
808
1 Br
achy
bact
eriu
m fa
eciu
m D
SM 4
810
GH
13 ZP 010940301 Blastopirellula marina D
SM 3645
GH
13 ZP 027320551 Gem
mata obscuriglobus U
QM
2246
GH
13 YP 4273821 Rhodospirillum
rubrum ATC
C 11170
GH
13 YP 0021361131 Anaeromyxobacter sp K
GH
13 YP 0039532711 Stigmatella aurantiaca
GH
13 YP 0021384951 Geobacter bem
idjiensis BemG
H13 Y
P23
-C
RF ii
notl
ad r
etca
boe
G 1
3868
3520
0 G
H13 Y
P65
ec o
S mu
solu
llec
mu
igna
roS
191
8716
100
GH
13 ZPB2
101
WF
ps o
irbi
vofl
use
D 1
5398
6360
GH
13 Y
P 82
1516
1 C
andi
datu
s S
olib
acte
r usi
tatu
s E
llin6
076
GH
13 Y
P24
NFC ilte
muibozihR 1077074
GH
13
86635 M
SD sucityloly
ma sunicaradnaS 43690F
KA
GH
13 Y
P 00
1820
541
1 O
pitu
tus
terra
e P
B90
-1G
H13
ZP
0313
0066
1 C
htho
niob
acte
r fla
vus
Ellin
428
GH
13 YP 0035707081 Salinibacter ruber M8
GH
13 YP 0037057351 Truepera radiovictrix DSM
17093
GH
13 YP 0032689441 Haliangium
ochraceum D
SM 14365
GH
13 YP 0016130311 Sorangium cellulosum
So ce56
GH
13 YP 6288101 Myxococcus xanthus D
K 1622
GH
13 62738967 Deinococcus radiodurans
GH
13 AKF04017 Sandaracinus amylolyticus D
SM 53668
GH
13 YP 0027878991 Deinococcus deserti VC
D115
GH13 YP 5893201 Candidatus Koribacter versatilis Ellin345
GH13 ZP 063702081 Desulfovibrio sp FW
1012B
GH13 YP 0041842341 Terriglobus saanensis SP1PR4
GH13 YP 8731321 Acidotherm
us cellulolyticus 11B
GH13 ZP 070169501 Desulfonatronospira thiodism
utans ASO3-1
GH13 ADX845721 Sulfolobus islandicus REY15A
GH13 ZP 027318151 G
emm
ata obscuriglobus UQM
2246
GH13 YP 0016347811 Chloroflexus aurantiacus J-10-fl
GH
13 NP 9534051 G
eobacter sulfurreducens PCA
GH
13 YP 0025366761 Geobacter daltonii FR
C-32
GH
13 YP 0012121361 Pelotomaculum
thermopropionicum
SI
GH
13 YP 0041781661 Isosphaera pallida ATCC
43644
GH
13 ZP 036310341 Pedosphaera parvula Ellin514
GH13 ZP 031287911 Chthoniobacter flavus Ellin428
GH13 NP 6913271 Oceanobacillus ih
eyensis HTE831
GH13 YP 0025601851 Macro
coccus c
aseolyticus J
CSC5402
GH13 YP 0030110001 Paenibacill
us sp J
DR-2
GH13 CAJ384141 Anaerobranca
gottsch
alkii
GH13 YP 00
1988
1311
Lacto
bacill
us ca
sei B
L23
GH13 YP 00
2459
6561
Des
ulfito
bacte
rium ha
fnien
se D
CB-2
GH13 YP 00
3947
0781
Paenib
acillu
s poly
myxa S
C2
GH13 Y
P 0013
1197
11 C
lostrid
ium be
ijerin
ckii N
CIMB 80
52
GH13 Y
P 0039
6683
31 Ily
obac
ter po
lytrop
us D
SM 2926
GH13 Z
P 0539
1277
1 Clos
tridium
carb
oxidi
vora
ns P
7
GH13 Y
P 0013
9686
01 C
lostrid
ium kl
uyve
ri DSM 55
5
GH13 ZP 02
2128
451
Clostrid
ium ba
rtlettii
DSM 16
795
GH13 ZP 04
4331
841
Bacillu
s coa
gulan
s 36D
1
GH13 C
BL37
8151
Ana
eros
tipes
hadr
us
GH13 Z
P 04
4519
311
Abiotro
phia
defec
tiva A
TCC 49
176
GH13
YP
0031
4407
51
Slac
kia h
eliot
rinire
duce
ns D
SM 2
0476
GH13
ZP 0
2025
300
1 Eu
bacte
rium
vent
riosu
m A
TCC
2756
0
GH1
3 YP
004
3093
801
Clo
strid
ium
lent
ocel
lum
DSM
542
7
GH13
YP 5
1565
71
Chlam
ydop
hila
felis
GH13
ZP 0
6299
217
1 Pa
rach
lamyd
ia ac
anth
amoe
bae
str H
allrsquos
cocc
us
GH13
CBL1
5412
1 R
umino
cocc
us b
rom
ii L2-
63
GH13 Y
P 00
1692
347
1 Fi
nego
ldia
mag
na AT
CC 293
28
GH13 Z
P 07
5262
211
Pep
tostr
epto
cocc
us st
omat
is DSM
176
78
GH1
3 YP
003
4751
021
Clo
strid
iale
s ge
nom
osp
BVA
B3 s
tr U
PII9
-5
GH1
3 YP
004
1716
241
Dei
noco
ccus
mar
icope
nsis
DSM
212
11
GH1
3 YP
022
845
1 Pi
crop
hilu
s to
rridu
s DS
M 9
790
GH1
3 YP
001
8207
181
Opi
tutu
s te
rrae
PB90
-1
GH1
3 YP
003
1672
861
Can
dida
tus A
ccum
ulib
acte
r pho
spha
tis c
lade
IIA
str
UW-1
GH1
3 AK
F038
60 S
anda
racin
us a
mylo
lyticu
s DS
M 5
3668
GH1
3 AK
F069
52 S
anda
racin
us a
mylo
lyticu
s DS
M 5
3668
GH
13 Y
P 00
2762
222
1 G
emm
atim
onas
aur
antia
ca T
-27
GH1
3 ZP
082
0671
01
Gor
doni
a ne
ofel
ifaec
is NR
RL B
-593
95
GH
13 Y
P 00
3964
374
1 Ke
togu
loni
cige
nium
vul
gare
Y25
GH13 ZP 045772811 Oxalobacter fo
rmigenes H
OxBLS
GH13 YP 0033856211 Spirosoma linguale DSM 74
GH13 AEA650561 Burkholderia gladioli B
SR3
GH13 YP 0030725361 Teredinibacter turnerae T7901
GH13 YP 4255961 Rhodospirillum ru
brum ATCC 11170
GH13 YP 0010427851 Rhodobacter sphaeroides ATCC 17029
GH13 ZP 075890671 Sinorhizobium meliloti BL225C
GH13 YP 0030504181 Methylovorus glucosetrophus SIP3-4
GH13 YP 4224271 Magnetospirillum magneticum AMB-1
GH13 YP 0042554001 Deinococcus proteolyticus MRP
GH13 ZP 029300551 Verrucomicrobium spinosum DSM 4136
GH13 ZP 080190951 Lautropia mirabilis ATCC 51599
GH13 YP 5335351 Rhodopseudomonas palustris BisB18
GH13 YP 8654291 Magnetococcus marinus MC-1
GH13 YP 0028938741 Tolumonas auensis DSM 9187
GH13 YP 6612131 Pseudoalteromonas atlantica T6c
GH13 YP 0040654571 Pseudoalteromonas sp SM9913
GH13 YP 8302311 Arthrobacter sp FB24
GH13 YP 0042347941 Acidovorax avenae subsp avenae ATCC 19860
GH13 YP 0016390191 Methylobacterium extorquens PA1
GH13 ZP 068060481 Brevibacterium mcbrellneri ATCC 49030
GH13 YP 0031655431 Candidatus Accumulibacter phosphatis clade IIA str UW-1
GH13 YP 9813411 Polaromonas naphthalenivorans CJ2
GH13 ZP 029305921 Verrucomicrobium spinosum DSM 4136
GH13 YP 8283661 Candidatus Solibacter usitatus Ellin6076
GH13 YP 0015687661 Petrotoga mobilis SJ95
GH13 YP 2560581 Sulfolobus acidocaldarius DSM 639
GH13 YP 0023732201 Cyanothece sp PCC 8801
GH13 AKF07413 Sandaracinus amylolyticus DSM 53668
GH13 AKF03796 Sandaracinus amylolyticus DSM 53668
GH13 EFS357291 Propionibacterium acnes HL013PA1
GH13 YP 0033827501 Kribbella flavida DSM 17836
GH13 YP 9230111 Nocardioides sp JS614
GH13 AEA239681 Pseudonocardia dioxanivorans CB1190
GH13 YP 0031037201 Actinosynnema mirum DSM 43827
GH57 YP 0018695221 Nostoc punctiforme PCC 73102
GH57 YP 0041847961 Terriglobus saanensis SP1PR4
GH57 YP 0033170621 Thermanaerovibrio acidaminovorans DSM 6589
GH57 YP 0025231331 Thermomicrobium roseum DSM 5159
GH57 CBL286501 Fretibacterium fastidiosum
GH57 YP 0041781671 Isosphaera pallida ATCC 43644
GH57 YP 0027549471 Acidobacterium capsulatum ATCC 51196
GH57 ZP 070292791 Granulicella mallensis MP5ACTX8
GH57 YP 0018749261 Elusimicrobium minutum Pei191
GH57 ADL259941 Fibrobacter succinogenes subsp succinogenes S85
GH57 YP 4773871 Synechococcus sp JA-2-3B
GH57 YP 8454221 Syntrophobacter fumaroxidans MPOB
GH57 NP 9534062 Geobacter sulfurreducens PCA
GH57 YP 5020321 Methanospirillum hungatei JF-1
GH57 YP 0019958521 Chloroherpeton thalassium ATCC 35110
GH57 YP 5937791 Candidatus Koribacter versatilis Ellin345
GH57 YP 0010471761 Methanoculleus marisnigri JR1
GH57 YP 0014309471 Roseiflexus castenholzii DSM 13941
GH57 YP 0029516361 Desulfovibrio magneticus RS-1
GH57 YP 0038064671 Desulfarculus baarsii DSM 2075
GH57 YP 0013789531 Anaeromyxobacter sp Fw109-5
GH57 ZP 063702071 Desulfovibrio sp FW1012B
GH57 YP 0035531061 Aminobacterium colombiense DSM 12261
GH57 ZP 064391271 Anaerobaculum hydrogeniformans ATCC BAA-1850
GH57 AKF06951 Sandaracinus amylolyticus DSM 53668
GH57 ZP 063915611 Dethiosulfovibrio peptidovorans DSM 11002
GH57 YP 0024634161 Chloroflexus aggregans DSM 9485
GH57 YP 0043692891 Desulfobacca acetoxidans DSM 11109
GH57 YP 8464661 Syntrophobacter fumaroxidans M
POB
GH57 CAJ738171 Candidatus Kuenenia stuttgartiensis
GH57 YP 0031969181 Desulfohalobium
retbaense DSM 5692
FIG 3mdashPhylogeny of Sandaracinus amylolyticus GH13 and GH57 a-amylases Sandaracinus amylolyticus a-amylases (GH13 and GH57) are colored in
pink along with their domain architecture displayed as Domain Color Shape [GH13 Red rectangle GH57 Black rectangle GH77 dark pink rectangle
CBM48 Dark blue circle CBM20 green circle CBM41 cyan circle] Myxobacterial homologs are colored fluorescent green Bootstrap values correspond-
ing to the tree nodes are provided
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2525
of the phyla Betaproteobacteria suggestive of a possible hor-
izontal transfer
AKF04017 and AKF03605 have close homologs in the ex-
tremophile Deinococcus desserti VCD115 and shares clade
with certain myxobacterial amylases belonging to
Myxococcus xanthus S cellulosum and Haliangium ocraceum
which further share clades with Deinococcus amylases
AKF04017 and AKF03605 share the highest sequence identity
with D desserti VCD115 plasmid protein (YP_002787899
533) and chromosomal protein (YP_002785382 456)
respectively that might point to its plasmid-borne horizontal
transfer across the clade (fig 3) The sequence conservation of
CSRs and the high percentage identity of these amylases to
Deinococcus support the inference for the putative horizontal
transfers among these members AKF09634 does not share its
sister branch with any member but the clade contains mem-
bers from diverse phyla that is Rhizobium from alphaproteo-
bacteria Opitutus from Chlamydae Chthonibacter from
Verrucomicrobia suggesting the multiple gain and loss of
this amylase among prokaryotes Similarly AKF07413 shares
the clade with members of Cyanobacteria AKF07406 shares
the clade with members of Deltaproteobacteria AKF03861
with members of Alphaproteobacteria and AKF03642 with
members of Bacteroidetes Taken together we can suggest
that many of the S amylolyticus amylases have been acquired
via horizontal gene transfer events from other bacterial species
belonging to the phylum Deinococcus-Thermus
Acidobacteria Cyanobacteria and Alphaproteobacteria
To further support these horizontal transfer events we
checked the sequence conservation between S amylolyticus
amylases and closely related members in the phylogenetic tree
and aligned using PROMALS-3D with Taka-Amylase A of A
oryzae (PDBid 2TAA) as a structural template We also per-
formed synteny studies analyzing proteins present in the vicin-
ity of all identified S amylolyticus GH13 and GH57 amylases
and their closely related homologs as revealed by phylogenetic
studies We could not trace any conserved syntenic regions in
these genomes whereas in the case of AKF03862 and
AKF09634 we identified glycogen debranching enzymes in
Stigmatella aurantiaca DW43-1 [56 identity and 98
query coverage of AKF03860 with WP_0133760911] and
Opitutus terrae PB90-1 [57 identity and 97 query cover-
age of AKF09635 YP_0018205421] which further support
their putative horizontal transfer in S amylolyticus
Similarly the phylogenetic inference was drawn from two
representative domains of g-amylases using the same meth-
odology as followed for a-amylases (fig 4) We found that
one of the g-amylases AKF10340 lies closer to M xanthus
amylase whereas the other AKF10487 shares its sister
branch with Nitrosococcus watsonii a Gammaproteobacteria
This suggests that while some of the a- and g-amylases
are present in many myxobacterial species a majority of the
a- and g-amylases are present only in S amylolyticus and not
in other myxobacterial genomes possibly due to the result of
horizontal transfer events from diverse bacterial phylum such
as Deinococcus-Thermus Cyanobacteria Alphaproteobac-
teria Gammaproteobacteria and Acidobacteria
Agar Degrading Enzymes
Some bacteria are known to degrade agar and use it as a
carbon source and possess agarase enzymes that help in hy-
drolyzing agar in oligosaccharides (Chi et al 2012) Depending
on the site of action they can be of two types a-agarase
(agarose 3-glycanohydrolase 321158 GH96) and
b-agarase (agarose 4-glycanohydrolase 32181 GH16
GH50 GH86 GH118) (table 1) Both families of agarases
GH15 EFV841771 Achrom
obacter xylosoxidans C54
GH
15 ZP 011267341 Nitrococcus m
obilis Nb-231
GH15 YP 4129921 Nitrosospira m
ultiformis ATCC 25196
GH15 YP 5716301 Nitrobacter ham
burgensis X14 plasmid
GH15 ZP 012260811 Aurantim
onas manganoxydans SI85-9A1
GH15 YP 0016032801 Gluconacetobacter diazotrophicus PA1 5
GH15 YP 7451371 Granulibacter bethesdensis CGDNIH1
GH15 AEA616211 Burkholderia gladioli BSR3
GH15 YP 0018344311 Beijerinckia indica subsp indica ATCC 9039
GH15 YP 0027560251 Acidobacterium capsulatum
ATCC 51196
GH15 CBJ387091 Ralstonia solanacearum CMR15
GH15 YP 3189151 Nitrobacter winogradskyi Nb-255
GH15 YP 0012344011 Acidiphilium cryptum JF-5
GH15 YP 0040115781 Rhodomicrobium vannielii ATCC 17100
GH15 YP 0016419391 Methylobacterium extorquens PA1GH15 YP 5252641 Rhodoferax ferrireducens T118
GH15 YP 0041811661 Terriglobus saanensis SP1PR4
GH15 ZP 031333281 Chthoniobacter flavus Ellin428
GH15 CCA537261 Streptomyces venezuelae ATCC 10712
GH15 YP 0015065621 Frankia sp EAN1pec
GH15 YP 0024892701 Arthrobacter chlorophenolicus A6
GH15 YP 0018202091 Opitutus terrae PB90-1
GH15 YP 8729011 Acidothermus cellulolyticus 11B
GH15 YP 0013795591 Anaeromyxobacter sp Fw109-5
GH15 YP 0024929091 Anaeromyxobacter dehalogenans 2CP-1
GH15 YP 6355121 Myxococcus xanthus DK 1622
GH15 YP 0011047051 Saccharopolyspora erythraea NRRL 2338
GH15 YP 0011092191 Saccharopolyspora erythraea NRRL 2338
GH15 AKF10340 Sandaracinus amylolyticus DSM 53668
GH15 YP 6342991 Myxococcus xanthus DK 1622
GH15 YP 0031091351 Acidimicrobium ferrooxidans DSM 10331
GH15 YP 0035703361 Salinibacter ruber M8
GH15 YP 0025241401 Thermomicrobium roseum DSM 5159 plasmid
GH15 YP 0033214441 Sphaerobacter thermophilus DSM 20745
GH15 NP 7336051 Streptomyces coelicolor A3-2
GH15 YP 0035282991 Nitrosococcus halophilus Nc 4
GH15 YP 0043676121 Marinithermus hydrothermalis DSM 14884
GH15 YP 0043687811 Marinithermus hydrothermalis DSM 14884
GH15 YP 0034042871 Haloterrigena turkmenica DSM 5511
GH15 ZP 080461001 Haladaptatus paucihalophilus DX253
GH1
5 YP
023
376
1 Pi
crop
hilu
s to
rrid
us D
SM 9
790
GH1
5 ZP
055
7131
11
Ferr
opla
sma
acid
arm
anus
fer1
GH1
5 BA
B604
801
The
rmop
lasm
a vo
lcan
ium
GSS
1
GH
15 Y
P 00
3323
761
1 Th
erm
obac
ulum
terr
enum
ATC
C B
AA
-798
GH
15 Y
P 00
3899
615
1 C
yano
thec
e sp
PC
C 7
822
plas
mid
GH
15 Y
P 00
3705
927
1 Tr
uepe
ra ra
diov
ictr
ix D
SM 1
7093
GH
15 Y
P 00
2478
443
1 A
rthr
obac
ter c
hlor
ophe
nolic
us A
6 pl
asm
idG
H15
AK
F104
87 S
anda
raci
nus
amyl
olyt
icus
DSM
536
68G
H15
YP
0037
6113
81
Nitr
osoc
occu
s w
atso
nii C
-113
GH1
5 ZP
069
7134
91
Kted
onob
acte
r rac
emife
r DSM
449
63
GH15
ZP
0697
6049
1 K
tedo
noba
cter
race
mife
r DSM
449
63
GH15
YP
0033
9335
81
Cone
xiba
cter
woe
sei D
SM 1
4684
GH15
YP
0042
3629
11 A
cido
vora
x av
enae
sub
sp a
vena
e ATC
C 19
860
GH15 Z
P 03
6273
271
Pedo
spha
era p
arvu
la El
lin51
4
GH15 Y
P 00
1618
7001
Sor
angi
um ce
llulo
sum
So
ce56
GH15 ZP
0145
9561
1 Stig
matella
aura
ntiac
a DW
4-3-1
GH15 ZP
0503
8177
1 Syn
echo
cocc
us sp
PCC 73
35
GH15 YP 64
4038
1 Rubrobac
ter xy
lanophilu
s DSM 99
41
GH15 ZP 064746651 Frankia symbiont of D
atisca glomerata
GH15 ZP 069744041 Ktedonobacter racemife
r DSM 44963
GH15 ZP 069653261 Ktedonobacter racemifer D
SM 44963
GH15 ZP 069661381 Ktedonobacter racemifer D
SM 44963
FIG 4mdashPhylogeny of Sandaracinus amylolyticus GH15 g-amylases
Sandaracinus amylolyticus g-amylases are colored dark pink and myxobac-
terial homologs are colored fluorescent green Bootstrap values corre-
sponding to the tree nodes are provided
Sharma et al GBE
2526 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
were absent in S amylolyticus genome which could be related
to its agar nondegrading property (Mohr et al 2012) as sup-
ported by colony morphology where agar was not penetrated
or liquefied by the bacteria (supplementary fig S5
Supplementary Material online)
Cellulose Degrading Enzymes
We identified only GH5 and GH74 in S amylolyticus proteome
in two and three proteins respectively GH5 domain was pre-
sent as a single unit in AKF04963 and AKF09194 whereas
GH74 was identified in AKF04859 AKF06963 and
AKF09775 associated with a GH74 domain CAZY domains
for cellobiohydrolaseexoglucanases (represented by GH6
GH7 GH9 and GH48) were not present in S amylolyticus
Cellobiohydrolaseexoglucanases plays a significant role in cel-
lulose degradation as they cut two to four sugar subunits from
the exposed ends formed by the endoglucanases which are
used as initial substrates for b-glucosidase (Mba Medie et al
2012) The absence of cellobiohydrolase or exoglucanases
could be a possible reason for the lack of cellulose degradation
in S amylolyticus Out of b-glucosidase representative CAZY
families GH1 GH3 GH5 GH9 GH30 and GH116 only GH1
GH3 and GH5 families were found in three two and two
proteins in S amylolyticus respectively AKF03843 AKF0
6028 and AKF10711 have a GH1 domain whereas
AKF09078 and AKF10875 have only the GH3 domain
Chitin Degrading Enzymes
Chitinases (EC32114) are involved in degradation of chitin
a component of the cell wall of fungi and exoskeleton of
arthropods GH18 GH19 GH23 and GH48 families have
been reported in the CAZY database as chitinases that hydro-
lyse b-14 linkages between N-acetylglucosamine units GH18
is a chitinase family with a characteristic ldquo[LIVM
FY][DN]G[LIVMF][DN][LIVMF][DN]Erdquo motif and is expressed
in archaea prokaryotes and eukaryotes (Funkhouser and
Aronson 2007) GH18 chitinases were not found in S amylo-
lyticus genome while members of GH19 (AKF03006 and
AKF10423) and GH23 (AKF04575 AKF05533 AKF06346
AKF08835 and AKF11377) could be identified GH19
family chitinases are mostly reported to be present in plants
with only a few members in prokaryotes (Kasprzewska 2003
Funkhouser and Aronson 2007) Among the two identified
GH19 homologs we found that AKF10423 is approximately
65 identical (~68 query coverage) with cyanobacteria
such as Nodosilinea nodulosa and Limnoraphis robusta
along with another myxobacterium Chondromyces crocatus
Besides these homologs we also found that AKF10423 shows
maximum similarity with eukaryotic GH19 chitinases counter-
parts as present in Acacia mangium Festuca arundinacea
Triticum aestivum and Leucaena leucocephala (55ndash59 iden-
tity with 70 query coverage) We identified that another
chitinase AKF06346 has GH23 domain along with three
copies of CBM50 which is generally present in the enzymes
cleaving either chitin or peptidoglycan
Conclusion
The complete 1033 Mb circular genome of S amylolyticus
type strain DSM 53668T was determined CAZy analysis re-
vealed the presence of 72 GT domains 72 CE domains 58 GH
domains 19 AA domains 4 PL domains and 26 CBM do-
mains Sixteen S amylolyticus proteins were identified of
which 15 are a-amylases of class GH13 and 1 GH57 Two
g-amylases of GH15 family were also identified Phylogeny
analysis revealed that ten GH13 a-amylases and one GH15
g-amylase have been aquired via horizontal gene transfer
from other bacterial phyla such as Deinococcus-Thermus
Cyanobacteria Alphaproteobacteria Gammaproteobacteria
and Acidobacteria
Table 1
Cellulose Starch Chitin and Agar Degrading Enzymes and Their Representative CAZY Domains Distribution
Substrate Enzyme Category Enzyme Name EC Number CAZY Families (protein repertoire in brackets)
Starch Amylase a-Amylase 3211 GH13 (15) GH57 (1) GH119 GH126
Amylase b-Amylase 3212 GH14
Amylase g-Amylase 3213 GH15 (2) GH97
Chitin Chitinase Chitinase 32114 GH18 GH19 (2) GH23 (5) GH48
Agar a-Agarase Agarose 3-glycanohydrolase 321158 GH96
b-Agarase Agarose 4-glycanohydrolase 32181 GH16 GH50 GH86 GH118
Cellulose Cellulase b-Glucosidase 32121 GH1 (3) GH3 (2) GH5 (2) GH9 GH30 GH116
Cellulase Cellobiohydrolase or Exoglucanase 321176 GH48 GH6 GH7 GH9
Cellulase Endoglucanase 3214 GH5 (2) GH6 GH7 GH8 GH9 GH12 GH44
GH45 GH48 GH51 GH74 (3) GH124
NOTEmdashEC number enzyme category and CAZY families for proteins involved in starch chitin agar and cellulose degradation are shown The number of Sandaracinusamylolyticus proteins identified having CAZY domains are represented in bold fonts in respective families
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2527
Supplementary Material
Supplementary figures S1ndashS5 and tables S1 and S2 are avail-
able at Genome Biology and Evolution online (httpwww
gbeoxfordjournalsorg)
Acknowledgments
This work is supported by a project ldquoExpansion and modern-
ization of Microbial Type Culture Collection and Gene Bank
(MTCC) jointly supported by Council of Scientific and
Industrial Research (CSIR) Grant No BSC0402 and
Department of Biotechnology (DBT) Govt of India Grant No
BTPR7368INF221772012rdquo The authors thank MTCC for
providing help in procuring the cultures from DSMZ They
acknowledge Dr Ramya TNC for providing workbench for cul-
turing myxobacteria GS and IK acknowledge the Council
of Scientific and Industrial Research (CSIR) and University
Grant Commission (UGC) for the research fellowship respec-
tively They also thank the Genome Quebec Innovation
Centre McGill University Canada and C-CAMP next-genera-
tion genomics facility Bangalore India for help in obtaining
PacBio and Illumina NGS data respectively
Literature CitedAlikhan NF Petty NK Ben Zakour NL Beatson SA 2011 BLAST Ring
Image Generator (BRIG) simple prokaryote genome comparisons
BMC Genomics 12402
Altschul SF Gish W Miller W Myers EW Lipman DJ 1990 Basic local
alignment search tool J Mol Biol 215403ndash410
Auch AF Von Jan M Klenk HP Goker M 2010 Digital DNA-DNA hybrid-
ization for microbial species delineation by means of genome-to-
genome sequence comparison Stand Genomic Sci 2117ndash134
Aziz RK et al 2008 The RAST Server rapid annotations using subsystems
technology BMC Genomics 975
Blesak K Janecek S 2012 Sequence fingerprints of enzyme specificities
from the glycoside hydrolase family GH57 Extremophiles 16497ndash
506
Chi WJ Chang YK Hong SK 2012 Agar degradation by microorganisms
and agar-degrading enzymes Appl Microbiol Biotechnol 94917ndash
930
Chin CS et al 2013 Nonhybrid finished microbial genome assemblies
from long-read SMRT sequencing data Nat Methods 10563ndash569
Eddy SR 2011 Accelerated profile HMM searches PLoS Comput Biol
7e1002195
Edgar RC 2004 MUSCLE multiple sequence alignment with high accu-
racy and high throughput Nucleic Acids Res 321792ndash1797
Funkhouser JD Aronson NN Jr 2007 Chitinase family GH18 evolutionary
insights from the genomic history of a diverse protein family BMC Evol
Biol 796
Garcia R Muller R 2014 The family Polyangiaceae In Rosenberg E
Delong E Lory S Stackebrandt E Thompson F editors The prokary-
otes Berlin Heidelberg Springer p 247ndash279
Grissa I Vergnaud G Pourcel C 2007 CRISPRFinder a web tool to identify
clustered regularly interspaced short palindromic repeats Nucleic
Acids Res 35W52ndashW57
Hall TA 1999 BioEdit a user-friendly biological sequence alignment editor
and analysis program for Windows 9598NT Nucleic Acids Symp Ser
4195ndash98
Han K et al 2013 Extraordinary expansion of a Sorangium cellulosum
genome from an alkaline milieu Sci Rep 32101
Janecek S Kuchtova A 2012 In silico identification of catalytic residues
and domain fold of the family GH119 sharing the catalytic machinery
with the alpha-amylase family GH57 FEBS Lett 5863360ndash3366
Janecek S Svensson B Macgregor EA 2014 alpha-Amylase an enzyme
specificity found in various families of glycoside hydrolases Cell Mol
Life Sci 711149ndash1170
Kasprzewska A 2003 Plant chitinasesndashregulation and function Cell Mol
Biol Lett 8809ndash824
Lagesen K et al 2007 RNAmmer consistent and rapid annotation of
ribosomal RNA genes Nucleic Acids Res 353100ndash3108
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B
2014 The carbohydrate-active enzymes database (CAZy) in 2013
Nucleic Acids Res 42D490ndashD495
Lowe TM Eddy SR 1997 tRNAscan-SE a program for improved detection
of transfer RNA genes in genomic sequence Nucleic Acids Res
25955ndash964
Matsuura Y Kusunoki M Harada W Kakudo M 1984 Structure
and possible catalytic residues of Taka-amylase A J Biochem
95697ndash702
Mba Medie F Davies GJ Drancourt M Henrissat B 2012 Genome anal-
yses highlight the different biological roles of cellulases Nat Rev
Microbiol 10227ndash234
Mohr KI Garcia RO Gerth K Irschik H Muller R 2012
Sandaracinus amylolyticus gen nov sp nov a starch-degrading
soil myxobacterium and description of Sandaracinaceae fam
nov Int J Syst Evol Microbiol 621191ndash1198
Noguchi J et al 2011 Crystal structure of the branching enzyme I
(BEI) from Oryza sativa L with implications for catalysis and sub-
strate binding Glycobiology 211108ndash1116
Patel RK Jain M 2012 NGS QC Toolkit a toolkit for quality control of next
generation sequencing data PLoS One 7e30619
Pei J Kim BH Grishin NV PROMALS3D a tool for multiple protein se-
quence and structure alignments Nucleic Acids Res 36 2295ndash2300
Reichenbach H Dworkin M 1992 The myxobacteria In Balows A
Truper HG Dworkin M Harder W Schleifer KH editors The pro-
karyotes a handbook on the biology of bacteria ecophysiology
isolation identification applications New York Springer New
York p 3416ndash3487
Reichenbach H 2005 ldquoOrder VIII Myxococcalesrdquo Bergeyrsquos Manual of
Systematic Bacteriology In Brenner DJ Krieg NR Staley JT Garrity
GM editors Vol 2 2nd ed part C New York Springer-Verlag New
York Inc pp 1074ndash1079
Richter M Rossello-Mora R 2009 Shifting the genomic gold standard for
the prokaryotic species definition Proc Natl Acad Sci U S A 106
19126ndash19131
Schneiker S et al 2007 Complete genome sequence of the myxobacter-
ium Sorangium cellulosum Nat Biotechnol 25 1281ndash1289
Shimkets LJ 1990 Social and developmental biology of the myxobacteria
Microbiol Rev 54 473ndash501
Siguier P Perochon J Lestrade L Mahillon J Chandler M 2006 ISfinder
the reference centre for bacterial insertion sequences Nucleic Acids
Res 34 D32ndashD36
Steinmetz H et al 2012 Indiacens A and B prenyl indoles from the
myxobacterium Sandaracinus amylolyticus J Nat Prod 75 1803ndash
1805
Tamura K Stecher G Peterson D Filipski A Kumar S 2013 MEGA6
molecular evolutionary genetics analysis version 60 Mol Biol Evol
30 2725ndash2729
Van Der Maarel MJ Van Der Veen B Uitdehaag JC Leemhuis H Dijkhuizen
L 2002 Properties and applications of starch-converting enzymes of
the alpha-amylase family J Biotechnol 94 137ndash155
Sharma et al GBE
2528 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
Weissman KJ Muller R 2010 Myxobacterial secondary metabolites
bioactivities and modes-of-action Nat Prod Rep 27 1276ndash1295
Whitworth DE 2008 Myxobacteria multicellularity and differentiation
Washington DC ASM Press American Society for Microbiology
Yin Y et al 2012 dbCAN a web resource for automated carbohydrate-
active enzyme annotation Nucleic Acids Res 40 W445ndashW451
Zhao JY et al 2008 Discovery of the autonomously replicating plasmid
pMF1 from Myxococcus fulvus and development of a gene cloning
system in Myxococcus xanthus Appl Environ Microbiol 74 1980ndash
1987
Associate editor Howard Ochman
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2529
GH15 category (fig 2B) The starch degrading proteins have
been reported to have CBM20 CBM21 CBM25 CBM26
CBM34 CBM41 CBM45 CBM48 CBM53 and CBM58
modules that help in glycogenstarch binding truncation of
which reduces its enzymatic activity (Noguchi et al 2011
Janecek et al 2014) GH15 and GH57 domains are present
as single domain proteins in S amylolyticus Interestingly in 5
out of 15 GH13 S amylolyticus proteins (AKF03796
AKF03860 AKF06952 AKF07413 and AKF11765) the a-
amylase GH13 domain was present along with a CBM48
module at the N-termini The CBM48 module assists in the
GH13 function by serving as starch-binding domains
The GH13 family include the a-amylase domains with an (b
a)8 barrel catalytic domain These proteins have 4ndash7 conserved
sequence regions (CSRs) and a specific catalytic triad consisting
of a catalytic nucleophile (aspartic acid) on strand b4 proton
donor (glutamic acid) on strand b5 and a transition state sta-
bilizer (aspartic acid) on strand b7 (Janecek et al 2014) GH13
members show extremely low sequence identity with their ho-
mologs but the catalytic triad is highly conserved GH57 family
a-amylases have a (ba)7 barrel structure and have five CSRs as
compared with the GH13 family However only two of the
catalytic residues viz the nucleophile on strand b4 and the
proton donor on strand b7 are conserved (Blesak and Janecek
2012) Irrespective of these differences GH13 and GH57 both
employ the same retaining mechanism of action (Janecek and
Kuchtova 2012) Sandaracinus amylolyticus GH13 proteins
were retrieved and the structure of Taka amylase of A
oryzae (PDBid 2TAA) was used to perform a structure-based
alignment using PROMALS3D (Matsuura et al 1984 Pei et al
2008) All the seven CSR regions and the three catalytic
residues were conserved in all S amylolyticus GH13 amylases
(supplementary figs S2 and S3 Supplementary Material
online) Similarly S amylolyticus GH57 (AKF06951) was
aligned with its homologs using PROMALS3D based on the
4-a-glucanotransferase structure of Thermococcus litoralis
(PDBid 1K1W) In the GH57 alignment all five CSRs and
both catalytic residues were conserved (supplementary figS4
Supplementary Material online)
In order to understand the provenance of these a- and g-
amylases they were subjected to BLASTp against the NR data-
base To include amylases across the entire bacterial phylum
the CAZY-NR database for GH13 and GH57 domains were
retrieved and subjected to BLASTClust at 70 identity and
70 length coverage that reduced the domain sequence
space from 19590 to 4038 domains Further amylase do-
mains from S amylolyticus were subjected to BLASTp against
these 4038 domains and their top hits were retrieved and
included in the phylogenetic analysis All these GH13 and
GH57 domains along with S amylolyticus amylase domains
were aligned using MUSCLE as incorporated in MEGAv606
and ML-based phylogeny with 100 bootstrap values was
drawn (fig 3) The ML tree obtained was divided into three
major clades where the clade at root composed of all the GH57
domains and the other two major clades further composed of
all the GH13 sequences The representative bacterial amylases
from various clades show close relations with amylases of di-
verse bacterial groups suggesting the multiple gain and loss of
these domains As all the amylase domains of S amylolyticus
are scattered across the tree the phylogenetic position of these
domains could provide us insights about their provenance and
reveal possible cases of horizontal transfers
19
26
72
58
72
4
0
10
20
30
40
50
60
70
80
AA CBM CE GH GT PL
A B
Num
ber o
f pro
tein
s
GH13
GH13
CBM48 GH13
GH13CBM48
GH13
GH57
GH13CBM48
GH13GH13CBM48
GH13
GH13
GH13
GH15
GH13CBM48
GH13
AKF03605AKF03642AKF03796
AKF03860AKF03861AKF03862AKF04017AKF06951AKF06952
AKF07406AKF07413AKF07527AKF09634AKF09643AKF10340AKF10487AKF11765AKF11772
GH13
GH13
GH15
FIG 2mdashSandaracinus amylolyticus DSM 53668T CAZome distribution (A) Sandaracinus amylolyticus DSM 53668T CAZY families presented in a bar
graph x axis represents the CAZY categories and y axis represents the number of proteins (B) a- and g-amylases along with their CAZome domain
architecture The domains have been colored as GH13 sky blue GH15 dark red GH57 green and CBM48 orange
Sharma et al GBE
2524 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
Taking into account the GH57 clade the sole GH57
domain of S amylolyticus (GenBank ID AKF06951) shares
the clade with anaerobic Deltaproteobacteria Among the
GH13 domains of S amylolyticus AKF03862 AKF07527
AKF09643 AKF11765 and AKF11772 shares clade with
other myxobacteria Among these AKF11772 and
AKF07527 share sister branches and form sister clades with
other myxobacterial species such as S cellulosum Soce56 and
Plesiocystis pacifica SIR-1 and are further related to the clade
containing Gammaproteobacteria Apart from these two am-
ylases AKF03860 and AKF06952 are similar to each other and
group with Candidatus accumulibacter phosphtis amylase
(YP_0031672861) from the phylum Acidobacteria
Interestingly AKF03860 and AKF06952 group with members
GH13 ZP 021632211 Kordia algicida OT-1
GH13 ZP 067516041 Parascardovia denticolens F0305
GH13 YP 0035709041 Salinibacter ruber M8
GH13 YP 0032500041 Fibrobacter succinogenes subsp succinogenes S85
GH13 AKF03862 Sandaracinus amylolyticus DSM 53668
GH13 YP 0039537801 Stigmatella aurantiaca
GH13 CAJ738231 Candidatus Kuenenia stuttgartiensis
GH13 YP 0228461 Picrophilus torridus DSM 9790GH13 ZP 029306261 Verrucomicrobium spinosum DSM 4136
GH13 CAX841261 uncultured bacterium
GH13 YP 0031678771 Candidatus Accumulibacter phosphatis clade IIA str UW-1GH13 ZP 031321321 Chthoniobacter flavus Ellin428
GH13 ZP 018759791 Lentisphaera araneosa HTCC2155GH13 YP 0033894351 Spirosoma linguale DSM 74GH13 NP 7734061 Bradyrhizobium diazoefficiens USDA 110
GH13 YP 0037757321 Herbaspirillum seropedicae SmR1GH13 YP 4266911 Rhodospirillum rubrum ATCC 11170GH13 ZP 035079501 Rhizobium etli Brasil 5
GH13 ZP 057930691 Butyrivibrio crossotus DSM 2876
GH13 ZP 066231321 Turicibacter sanguinis PC909
GH13 ZP 044499741 Catonella morbi ATCC 51271
GH13 EGE534241 Streptococcus parauberis NCFD 2020
GH13 196049865 Lactobacillus Plantarum
GH13 YP 0042196261 Granulicella tundricola MP5ACTX9
GH13 YP 0011935611 Flavobacterium johnsoniae UW101
GH13 YP 0032910851 Rhodothermus marinus DSM 4252GH13 YP 0040967591 Bacillus cellulosilyticus DSM 2522GH13 ZP 037030351 Flavobacteria bacterium MS024-2A
GH13 XP 0025352531 Ricinus communis
GH13 YP 7579561 Maricaulis maris MCS10GH13 ZP 010417351 Erythrobacter sp NAP1GH13 YP 0018177571 Opitutus terrae PB90-1GH13 EFR587141 Alistipes sp HGB5
GH13 CBK627851 Alistipes shahii WAL 8301GH13 EFR582631 Alistipes sp HGB5
GH13 YP 0034042011 Haloterrigena turkmenica DSM 5511
GH13 YP 0035357541 Haloferax volcanii DS2
GH13 YP 0035357541 Haloferax volcanii DS2
GH13 YP 0025661621 Halorubrum lacusprofundi ATCC 49239
GH13 YP 1368761 Haloarcula marismortui ATCC 43049
GH13 ZP 080436741 Haladaptatus paucihalophilus DX253
GH13 YP 0025652161 Halorubrum lacusprofundi ATCC 49239
GH13 YP 0035346301 Haloferax volcanii DS2
GH13 YP 0033225661 Thermobaculum terrenum ATCC BAA-798
GH13 YP 0021353421 Anaeromyxobacter sp K
GH13 AKF09643 Sandaracinus amylolyticus DSM 53668
GH13 YP 0016137031 Sorangium cellulosum So ce56
GH1
3 99
0316
21 H
alot
herm
othr
ix or
enii
GH1
3 AA
D051
991
Pae
niba
cillu
s po
lymyx
a
GH1
3 YP
003
6999
601
Bac
illus
sele
nitir
educ
ens
MLS
10
GH1
3 ZP
022
0799
81
Copr
ococ
cus
euta
ctus
ATC
C 27
759
GH
13 B
AA07
401
1 Xa
ntho
mon
as c
ampe
stris
GH
13 Y
P 00
4052
835
1 M
ariv
irga
tract
uosa
DSM
412
6
GH1
3 ZP
037
0954
51
Cory
neba
cter
ium
mat
ruch
otii A
TCC
3380
6
GH1
3 NP
267
649
1 La
ctoc
occu
s la
ctis
subs
p la
ctis
Il140
3
GH1
3 YP
001
3023
201
Par
abac
tero
ides
dist
ason
is AT
CC 8
503
GH1
3 YP
001
2276
361
Syn
echo
cocc
us s
p R
CC30
7
GH1
3 YP
002
5652
591
Hal
orub
rum
lacu
spro
fund
i ATC
C 49
239
GH13
ZP
0709
0550
1 C
oryn
ebac
teriu
m g
enita
lium
ATC
C 33
030
GH13
YP 0
0283
4100
1 C
oryn
ebac
teriu
m a
urim
ucos
um A
TCC
7009
75
GH13 A
KF03
861
Sand
arac
inus a
mylo
lyticu
s DSM
536
68
GH13
ZP 0
0050
865
1 M
agne
tosp
irillum
mag
neto
tacti
cum
MS-
1
GH13 Y
P 00
1568
811
1 Pe
troto
ga m
obilis
SJ9
5
GH13 Y
P 02
2847
1 P
icrop
hilus
torri
dus D
SM 9
790
GH13 E
FT25
5011
Pro
pionib
acter
ium ac
nes H
L110
PA3
GH13 E
FA80
1951
Poly
spho
ndyli
um pa
llidum
PN50
0
GH13 A
BD4655
81 V
ermam
oeba
verm
iform
is
GH13 Z
P 0350
5050
1 Rhiz
obium
etli B
rasil
5
GH13 YP 00
1348
4651
Pseud
omon
as ae
rugino
sa PA7
GH13 YP 00
2238
3511
Klebsie
lla pn
eumon
iae 34
2
GH13 YP 00
1437
9781
Cron
obac
ter sa
kaza
kii ATCC B
AA-894
GH13 Y
P 0011
7103
71 P
seud
omon
as st
utzeri
A1501
GH13 Y
P 0019
7240
81 S
tenotr
opho
monas
malt
ophil
ia K27
9a
GH13 Y
P 0043
1901
11 S
phing
obac
terium
sp 2
1
GH13 YP 0013127791 Sinorhizobium m
edicae W
SM419
GH13 ZP 035028921 Rhizobium etli K
im 5
GH13 YP 67
6219
1 Che
lativo
rans s
p BNC1
GH13 YP 00
2376
8861
Cya
nothe
ce sp
PCC 74
24
GH13 YP 0036383061 Cellulomonas flavig
ena DSM 20109
GH13 YP 0040977291 Intrasporangium ca
lvum DSM 43043
GH13 YP 0011691691 Rhodobacter s
phaeroides ATCC 17025
GH13 ZP 011580901 Oceanicola granulosus H
TCC2516
GH13 YP 0038382651 Micromonospora aurantiaca ATCC 27029
GH13 YP 0040977251 Intrasporangium calvu
m DSM 43043
GH13 YP 0036954761 Starkeya novella DSM 506
GH13 ZP 035166441 Rhizobium etli IE4771
GH13 ZP 031273131 Chthoniobacter flavus Ellin
428
GH13 AKF03642 Sandaracinus amylolyticus DSM 53668
GH13 YP 0031252671 Chitinophaga pinensis DSM 2588
GH13 YP 0030850521 Dyadobacter fermentans DSM 18053
GH13 YP 0033879241 Spirosoma linguale DSM 74
GH13 YP 0034301801 Streptococcus gallolyticus UCN34
GH13 ZP 078184041 Eremococcus coleocola ACS-139-V-Col8
GH13 YP 0034774501 Thermoanaerobacter italicus Ab9
GH13 ZP 048517861 Paenibacillus sp oral taxon 786 str D14
GH13 YP 0038414241 Caldicellulosiruptor obsidiansis OB47
GH13 YP 0024279661 Desulfurococcus kamchatkensis 1221n
GH13 YP 0034262831 Bacillus pseudofirmus OF4
GH13 ZP 076854311 Oscillochloris trichoides DG-6GH13 YP 0016174961 Sorangium cellulosum So ce56
GH13 ZP 019113371 Plesiocystis pacifica SIR-1
GH13 YP 6318811 Myxococcus xanthus DK 1622
GH13 AKF11765 Sandaracinus amylolyticus DSM 53668
GH13 ZP 036771121 Bacteroides cellulosilyticus DSM 14838
GH13 YP 0037157841 Croceibacter atlanticus HTCC2559
GH13 ZP 012015681 Flavobacteria bacterium BBFL7
GH13 ZP 019113321 Plesiocystis pacifica SIR-1GH13 YP 0031962511 Robiginitalea biformata HTCC2501
GH13 YP 0040531921 Marivirga tractuosa DSM 4126
GH13 ZP 021817881 Flavobacteriales bacterium ALC-1
GH13 CBL150401 Ruminococcus bromii L2-63
GH13 ZP 080051001 Bacillus sp 2 A 57 CT2
GH13 CBK911271 Eubacterium rectale DSM 17629
GH13 YP 0029369771 Eubacterium rectale ATCC 33656
GH13 YP 0021366011 Anaeromyxobacter sp K
GH13 ZP 014604541 Stigmatella aurantiaca
GH
13 728851 Streptomyces lividans
GH
13 BAJ281541 Kitasatospora setae KM-6054
GH
13 YP 0033390201 Streptosporangium roseum
DSM
43021
GH
13 YP 9214291 Nocardioides sp JS614
GH
13 AEB462451 Verrucosispora maris AB-18-032
GH
13 CAJ810301 Actinoplanes sp
GH
13 NP 9387401 C
orynebacterium diphtheriae N
CTC
13129
GH
13 YP 0036976141 Arcanobacterium haem
olyticum D
SM 20595
GH
13 ZP 039259261 Actinomyces coleocanis D
SM 15436
GH
13 YP 0040999421 Intrasporangium calvum
DSM
43043
GH
13 YP 0030989701 Actinosynnema m
irum D
SM 43827
GH
13 YP 0036376081 Cellulom
onas flavigena DSM
20109
GH
13 ZP 009938561 Janibacter sp HTC
C2649
GH13 YP 0030608421 Hirschia baltica ATCC 49814
GH13 YP 7579631 M
aricaulis maris M
CS10
GH13 YP 0016857121 Caulobacter sp K31
GH13 ZP 013016731 Sphingom
onas sp SKA58
GH13 YP 0038198341 Brevundim
onas subvibrioides ATCC 15264
GH
13 YP 4971641 Novosphingobium
aromaticivorans D
SM 12444
GH13 YP 6609551 Pseudoalterom
onas atlantica T6c
GH13 ZP 011126451 Reinekea blandensis M
ED297
GH13 YP 2677341 Colwellia psychrerythraea 34H
GH13 ZP 011126461 Reinekea blandensis M
ED297
GH13 AKF11772 Sandaracinus amylolyticus DSM
53668
GH13 AKF07527 Sandaracinus amylolyticus DSM
53668
GH13 YP 0016184291 Sorangium cellulosum
So ce56
GH13 ZP 019113391 Plesiocystis pacifica SIR-1
GH13 ZP 036315591 Pedosphaera parvula Ellin514
GH13 YP 0016604791 Microcystis aeruginosa NIES-843
GH13 YP 0043416831 Archaeoglobus veneficus SNP6
GH13 ZP 073351521 Desulfovibrio fructosovorans JJ
GH
13 AKF03605 Sandaracinus amylolyticus D
SM 53668
GH
13 YP 0027853821 Deinococcus deserti VC
D115
GH
13 NP 2951951 D
einococcus radiodurans R1
GH
13 YP 004171
1212
M
SD
sisn
epoc
ira
m su
ccoc
onie
D 1
661
1
GH
13 YP 0041715811 Deinococcus m
aricopensis DSM
21211
GH
13 YP 0037038171 Truepera radiovictrix DSM
17093
GH
13 YP 6052101 Deinococcus geotherm
alis DSM
11300
GH
13 YP 0027555491 A
cidobacterium capsulatum
ATCC
51196
GH
13 Y
P9
XTC
A5P
M alocirdnut allecilunarG 1860612400
GH
13 YP 6335241 M
yxococcus xanthus DK
1622G
H13 Y
PB
S su
cihp
ortid
ica
suhp
ortny
S 1
9412
64
GH
13 A
AW32
490
1 iiklahcsttog acnarborean
A
GH
13 Y
P 00
2316
532
1 An
oxyb
acillu
s fla
vith
erm
us W
K1
GH
13 Y
P 00
3252
634
1 G
eoba
cillu
s sp
Y41
2MC
61G
H13
ZP
0117
0936
1 B
acillu
s sp
NR
RL
B-14
911
GH
13 Z
P 07
7074
981
Bac
illus
sp m
3-13
GH
13 Z
P1-
GS ps sullica
B 116916810
GH
13 Y
P 00
1813
251
1 Ex
iguo
bact
eriu
m s
ibiri
cum
255
-15
GH
13 Y
P 00
2886
871
1 Ex
iguo
bact
eriu
m s
p A
T1b
GH
13 Y
P 00
1568
454
1 Pe
troto
ga m
obilis
SJ9
5
GH
13 Y
P 00
2940
188
1 Ko
smot
oga
olea
ria T
BF 1
95
1G
H13
ZP
0125
8420
1 V
ibrio
alg
inol
ytic
us 1
2G01
GH
13 1
0911
18 T
herm
oact
inom
yces
vul
garis
GH
13 Z
P 06
9705
751
Kte
dono
bact
er ra
cem
ifer D
SM 4
4963
GH
13 Y
P 00
1545
735
1 H
erpe
tosi
phon
aur
antia
cus
DSM
785
GH
13 E
FY95
778
1 M
etar
hizi
um ro
berts
ii AR
SEF
23
GH
13 N
P 58
8205
1 S
chiz
osac
char
omyc
es p
ombe
972
h-
GH
13 N
P 59
4401
1 S
chiz
osac
char
omyc
es p
ombe
972
h-
GH
13 X
P 00
1908
940
1 Po
dosp
ora
anse
rina
S m
at+
GH
13 4
1016
824
Schw
anni
omyc
es o
ccid
enta
lis
GH
13 B
AF98
616
1 H
ypho
pich
ia b
urto
nii
GH13 AKF07406 Sandaracinus amylolyticus DSM 53668
GH13 YP 0043692881 Desulfobacca acetoxidans DSM 11109
GH13 YP 0023716111 Cyanothece sp PCC 8801GH13 YP 3219451 Anabaena variabilis ATCC 29413
GH13 YP 4738811 Synechococcus sp JA-3-3AbGH13 YP 8464671 Syntrophobacter fumaroxidans MPOBGH13 ZP 063702061 Desulfovibrio sp FW1012B
GH13 YP 0033193471 Sphaerobacter thermophilus DSM 20745GH13 YP 0037057361 Truepera radiovictrix DSM 17093GH13 YP 4120971 Nitrosospira multiformis ATCC 25196GH13 YP 6431071 Rubrobacter xylanophilus DSM 9941
GH13 BAD389831 Rhodobacter sphaeroides f sp denitrificans
GH13 YP 3175601 Nitrobacter winogradskyi Nb-255
GH13 YP 8215181 Candidatus Solibacter usitatus Ellin6076
GH13 YP 0034506361 Azospirillum sp B510
GH13 ZP 070169471 Desulfonatronospira thiodismutans ASO3-1
GH13 YP 0041842351 Terriglobus saanensis SP1PR4
GH13 ZP 070315011 Granulicella mallensis MP5ACTX8
GH13 YP 0029438961 Variovorax paradoxus S110
GH13 YP 5918381 Candidatus Koribacter versatilis Ellin345
GH13 YP 7445681 Granulibacter bethesdensis CGDNIH1
GH13 YP 0039098001 Burkholderia sp CCGE1003
GH13 ZP 024903661 Burkholderia pseudomallei NCTC 13177
GH13 YP 0016309761 Bordetella petrii DSM 12804
GH13 YP 0022527351 Ralstonia solanacearum MolK2
GH13 YP 0037759061 Herbaspirillum seropedicae SmR1
GH13 YP 5455251 Methylobacillus flagellatus KT
GH13 YP 0013485041 Pseudomonas aeruginosa PA7
GH13 YP 0016008321 Gluconacetobacter diazotrophicus PA1 5
GH13 AEB584851 Pseudomonas mendocina NK-01
GH13 YP 0027996471 Azotobacter vinelandii DJ
GH
13 N
P 63
5804
1 X
anth
omon
as c
ampe
stris
pv
cam
pest
ris s
tr A
TCC
339
13
GH
13 Y
P 00
3188
581
1 Ac
etob
acte
r pas
teur
ianu
s IF
O 3
283-
01
GH
13 Y
P 74
4570
1 G
ranu
libac
ter b
ethe
sden
sis
CG
DN
IH1
GH
13 Y
P 00
3052
329
1 M
ethy
lovo
rus
gluc
oset
roph
us S
IP3-
4
GH
13 Z
P 06
8981
631
Ros
eom
onas
cer
vica
lis A
TCC
499
57
GH
13 Y
P 00
2943
895
1 Va
riovo
rax
para
doxu
s S1
10
GH
13 Y
P 31
4931
1 T
hiob
acillu
s de
nitri
fican
s AT
CC
252
59
GH
13 Y
P 42
5597
1 R
hodo
spiri
llum
rubr
um A
TCC
111
70
GH
13 Y
P 93
3302
1 A
zoar
cus
sp B
H72
GH
13 N
P 43
7816
1 S
inor
hizo
bium
mel
iloti
1021
GH
13 Z
P 01
1553
281
Oce
anic
ola
gran
ulos
us H
TCC
2516
GH
13 Y
P 00
1167
979
1 R
hodo
bact
er s
phae
roid
es A
TCC
170
25
GH
13 Y
P 00
1440
575
1 C
rono
bact
er s
akaz
akii A
TCC
BAA
-894
GH
13 Z
P 01
4401
991
Ful
vim
arin
a pe
lagi
HTC
C25
06
GH
13 Y
P 00
4234
793
1 Ac
idov
orax
ave
nae
subs
p a
vena
e AT
CC
198
60
GH
13 Y
P 00
1818
894
1 O
pitu
tus
terra
e PB
90-1
GH
13 Y
P 00
3154
808
1 Br
achy
bact
eriu
m fa
eciu
m D
SM 4
810
GH
13 ZP 010940301 Blastopirellula marina D
SM 3645
GH
13 ZP 027320551 Gem
mata obscuriglobus U
QM
2246
GH
13 YP 4273821 Rhodospirillum
rubrum ATC
C 11170
GH
13 YP 0021361131 Anaeromyxobacter sp K
GH
13 YP 0039532711 Stigmatella aurantiaca
GH
13 YP 0021384951 Geobacter bem
idjiensis BemG
H13 Y
P23
-C
RF ii
notl
ad r
etca
boe
G 1
3868
3520
0 G
H13 Y
P65
ec o
S mu
solu
llec
mu
igna
roS
191
8716
100
GH
13 ZPB2
101
WF
ps o
irbi
vofl
use
D 1
5398
6360
GH
13 Y
P 82
1516
1 C
andi
datu
s S
olib
acte
r usi
tatu
s E
llin6
076
GH
13 Y
P24
NFC ilte
muibozihR 1077074
GH
13
86635 M
SD sucityloly
ma sunicaradnaS 43690F
KA
GH
13 Y
P 00
1820
541
1 O
pitu
tus
terra
e P
B90
-1G
H13
ZP
0313
0066
1 C
htho
niob
acte
r fla
vus
Ellin
428
GH
13 YP 0035707081 Salinibacter ruber M8
GH
13 YP 0037057351 Truepera radiovictrix DSM
17093
GH
13 YP 0032689441 Haliangium
ochraceum D
SM 14365
GH
13 YP 0016130311 Sorangium cellulosum
So ce56
GH
13 YP 6288101 Myxococcus xanthus D
K 1622
GH
13 62738967 Deinococcus radiodurans
GH
13 AKF04017 Sandaracinus amylolyticus D
SM 53668
GH
13 YP 0027878991 Deinococcus deserti VC
D115
GH13 YP 5893201 Candidatus Koribacter versatilis Ellin345
GH13 ZP 063702081 Desulfovibrio sp FW
1012B
GH13 YP 0041842341 Terriglobus saanensis SP1PR4
GH13 YP 8731321 Acidotherm
us cellulolyticus 11B
GH13 ZP 070169501 Desulfonatronospira thiodism
utans ASO3-1
GH13 ADX845721 Sulfolobus islandicus REY15A
GH13 ZP 027318151 G
emm
ata obscuriglobus UQM
2246
GH13 YP 0016347811 Chloroflexus aurantiacus J-10-fl
GH
13 NP 9534051 G
eobacter sulfurreducens PCA
GH
13 YP 0025366761 Geobacter daltonii FR
C-32
GH
13 YP 0012121361 Pelotomaculum
thermopropionicum
SI
GH
13 YP 0041781661 Isosphaera pallida ATCC
43644
GH
13 ZP 036310341 Pedosphaera parvula Ellin514
GH13 ZP 031287911 Chthoniobacter flavus Ellin428
GH13 NP 6913271 Oceanobacillus ih
eyensis HTE831
GH13 YP 0025601851 Macro
coccus c
aseolyticus J
CSC5402
GH13 YP 0030110001 Paenibacill
us sp J
DR-2
GH13 CAJ384141 Anaerobranca
gottsch
alkii
GH13 YP 00
1988
1311
Lacto
bacill
us ca
sei B
L23
GH13 YP 00
2459
6561
Des
ulfito
bacte
rium ha
fnien
se D
CB-2
GH13 YP 00
3947
0781
Paenib
acillu
s poly
myxa S
C2
GH13 Y
P 0013
1197
11 C
lostrid
ium be
ijerin
ckii N
CIMB 80
52
GH13 Y
P 0039
6683
31 Ily
obac
ter po
lytrop
us D
SM 2926
GH13 Z
P 0539
1277
1 Clos
tridium
carb
oxidi
vora
ns P
7
GH13 Y
P 0013
9686
01 C
lostrid
ium kl
uyve
ri DSM 55
5
GH13 ZP 02
2128
451
Clostrid
ium ba
rtlettii
DSM 16
795
GH13 ZP 04
4331
841
Bacillu
s coa
gulan
s 36D
1
GH13 C
BL37
8151
Ana
eros
tipes
hadr
us
GH13 Z
P 04
4519
311
Abiotro
phia
defec
tiva A
TCC 49
176
GH13
YP
0031
4407
51
Slac
kia h
eliot
rinire
duce
ns D
SM 2
0476
GH13
ZP 0
2025
300
1 Eu
bacte
rium
vent
riosu
m A
TCC
2756
0
GH1
3 YP
004
3093
801
Clo
strid
ium
lent
ocel
lum
DSM
542
7
GH13
YP 5
1565
71
Chlam
ydop
hila
felis
GH13
ZP 0
6299
217
1 Pa
rach
lamyd
ia ac
anth
amoe
bae
str H
allrsquos
cocc
us
GH13
CBL1
5412
1 R
umino
cocc
us b
rom
ii L2-
63
GH13 Y
P 00
1692
347
1 Fi
nego
ldia
mag
na AT
CC 293
28
GH13 Z
P 07
5262
211
Pep
tostr
epto
cocc
us st
omat
is DSM
176
78
GH1
3 YP
003
4751
021
Clo
strid
iale
s ge
nom
osp
BVA
B3 s
tr U
PII9
-5
GH1
3 YP
004
1716
241
Dei
noco
ccus
mar
icope
nsis
DSM
212
11
GH1
3 YP
022
845
1 Pi
crop
hilu
s to
rridu
s DS
M 9
790
GH1
3 YP
001
8207
181
Opi
tutu
s te
rrae
PB90
-1
GH1
3 YP
003
1672
861
Can
dida
tus A
ccum
ulib
acte
r pho
spha
tis c
lade
IIA
str
UW-1
GH1
3 AK
F038
60 S
anda
racin
us a
mylo
lyticu
s DS
M 5
3668
GH1
3 AK
F069
52 S
anda
racin
us a
mylo
lyticu
s DS
M 5
3668
GH
13 Y
P 00
2762
222
1 G
emm
atim
onas
aur
antia
ca T
-27
GH1
3 ZP
082
0671
01
Gor
doni
a ne
ofel
ifaec
is NR
RL B
-593
95
GH
13 Y
P 00
3964
374
1 Ke
togu
loni
cige
nium
vul
gare
Y25
GH13 ZP 045772811 Oxalobacter fo
rmigenes H
OxBLS
GH13 YP 0033856211 Spirosoma linguale DSM 74
GH13 AEA650561 Burkholderia gladioli B
SR3
GH13 YP 0030725361 Teredinibacter turnerae T7901
GH13 YP 4255961 Rhodospirillum ru
brum ATCC 11170
GH13 YP 0010427851 Rhodobacter sphaeroides ATCC 17029
GH13 ZP 075890671 Sinorhizobium meliloti BL225C
GH13 YP 0030504181 Methylovorus glucosetrophus SIP3-4
GH13 YP 4224271 Magnetospirillum magneticum AMB-1
GH13 YP 0042554001 Deinococcus proteolyticus MRP
GH13 ZP 029300551 Verrucomicrobium spinosum DSM 4136
GH13 ZP 080190951 Lautropia mirabilis ATCC 51599
GH13 YP 5335351 Rhodopseudomonas palustris BisB18
GH13 YP 8654291 Magnetococcus marinus MC-1
GH13 YP 0028938741 Tolumonas auensis DSM 9187
GH13 YP 6612131 Pseudoalteromonas atlantica T6c
GH13 YP 0040654571 Pseudoalteromonas sp SM9913
GH13 YP 8302311 Arthrobacter sp FB24
GH13 YP 0042347941 Acidovorax avenae subsp avenae ATCC 19860
GH13 YP 0016390191 Methylobacterium extorquens PA1
GH13 ZP 068060481 Brevibacterium mcbrellneri ATCC 49030
GH13 YP 0031655431 Candidatus Accumulibacter phosphatis clade IIA str UW-1
GH13 YP 9813411 Polaromonas naphthalenivorans CJ2
GH13 ZP 029305921 Verrucomicrobium spinosum DSM 4136
GH13 YP 8283661 Candidatus Solibacter usitatus Ellin6076
GH13 YP 0015687661 Petrotoga mobilis SJ95
GH13 YP 2560581 Sulfolobus acidocaldarius DSM 639
GH13 YP 0023732201 Cyanothece sp PCC 8801
GH13 AKF07413 Sandaracinus amylolyticus DSM 53668
GH13 AKF03796 Sandaracinus amylolyticus DSM 53668
GH13 EFS357291 Propionibacterium acnes HL013PA1
GH13 YP 0033827501 Kribbella flavida DSM 17836
GH13 YP 9230111 Nocardioides sp JS614
GH13 AEA239681 Pseudonocardia dioxanivorans CB1190
GH13 YP 0031037201 Actinosynnema mirum DSM 43827
GH57 YP 0018695221 Nostoc punctiforme PCC 73102
GH57 YP 0041847961 Terriglobus saanensis SP1PR4
GH57 YP 0033170621 Thermanaerovibrio acidaminovorans DSM 6589
GH57 YP 0025231331 Thermomicrobium roseum DSM 5159
GH57 CBL286501 Fretibacterium fastidiosum
GH57 YP 0041781671 Isosphaera pallida ATCC 43644
GH57 YP 0027549471 Acidobacterium capsulatum ATCC 51196
GH57 ZP 070292791 Granulicella mallensis MP5ACTX8
GH57 YP 0018749261 Elusimicrobium minutum Pei191
GH57 ADL259941 Fibrobacter succinogenes subsp succinogenes S85
GH57 YP 4773871 Synechococcus sp JA-2-3B
GH57 YP 8454221 Syntrophobacter fumaroxidans MPOB
GH57 NP 9534062 Geobacter sulfurreducens PCA
GH57 YP 5020321 Methanospirillum hungatei JF-1
GH57 YP 0019958521 Chloroherpeton thalassium ATCC 35110
GH57 YP 5937791 Candidatus Koribacter versatilis Ellin345
GH57 YP 0010471761 Methanoculleus marisnigri JR1
GH57 YP 0014309471 Roseiflexus castenholzii DSM 13941
GH57 YP 0029516361 Desulfovibrio magneticus RS-1
GH57 YP 0038064671 Desulfarculus baarsii DSM 2075
GH57 YP 0013789531 Anaeromyxobacter sp Fw109-5
GH57 ZP 063702071 Desulfovibrio sp FW1012B
GH57 YP 0035531061 Aminobacterium colombiense DSM 12261
GH57 ZP 064391271 Anaerobaculum hydrogeniformans ATCC BAA-1850
GH57 AKF06951 Sandaracinus amylolyticus DSM 53668
GH57 ZP 063915611 Dethiosulfovibrio peptidovorans DSM 11002
GH57 YP 0024634161 Chloroflexus aggregans DSM 9485
GH57 YP 0043692891 Desulfobacca acetoxidans DSM 11109
GH57 YP 8464661 Syntrophobacter fumaroxidans M
POB
GH57 CAJ738171 Candidatus Kuenenia stuttgartiensis
GH57 YP 0031969181 Desulfohalobium
retbaense DSM 5692
FIG 3mdashPhylogeny of Sandaracinus amylolyticus GH13 and GH57 a-amylases Sandaracinus amylolyticus a-amylases (GH13 and GH57) are colored in
pink along with their domain architecture displayed as Domain Color Shape [GH13 Red rectangle GH57 Black rectangle GH77 dark pink rectangle
CBM48 Dark blue circle CBM20 green circle CBM41 cyan circle] Myxobacterial homologs are colored fluorescent green Bootstrap values correspond-
ing to the tree nodes are provided
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2525
of the phyla Betaproteobacteria suggestive of a possible hor-
izontal transfer
AKF04017 and AKF03605 have close homologs in the ex-
tremophile Deinococcus desserti VCD115 and shares clade
with certain myxobacterial amylases belonging to
Myxococcus xanthus S cellulosum and Haliangium ocraceum
which further share clades with Deinococcus amylases
AKF04017 and AKF03605 share the highest sequence identity
with D desserti VCD115 plasmid protein (YP_002787899
533) and chromosomal protein (YP_002785382 456)
respectively that might point to its plasmid-borne horizontal
transfer across the clade (fig 3) The sequence conservation of
CSRs and the high percentage identity of these amylases to
Deinococcus support the inference for the putative horizontal
transfers among these members AKF09634 does not share its
sister branch with any member but the clade contains mem-
bers from diverse phyla that is Rhizobium from alphaproteo-
bacteria Opitutus from Chlamydae Chthonibacter from
Verrucomicrobia suggesting the multiple gain and loss of
this amylase among prokaryotes Similarly AKF07413 shares
the clade with members of Cyanobacteria AKF07406 shares
the clade with members of Deltaproteobacteria AKF03861
with members of Alphaproteobacteria and AKF03642 with
members of Bacteroidetes Taken together we can suggest
that many of the S amylolyticus amylases have been acquired
via horizontal gene transfer events from other bacterial species
belonging to the phylum Deinococcus-Thermus
Acidobacteria Cyanobacteria and Alphaproteobacteria
To further support these horizontal transfer events we
checked the sequence conservation between S amylolyticus
amylases and closely related members in the phylogenetic tree
and aligned using PROMALS-3D with Taka-Amylase A of A
oryzae (PDBid 2TAA) as a structural template We also per-
formed synteny studies analyzing proteins present in the vicin-
ity of all identified S amylolyticus GH13 and GH57 amylases
and their closely related homologs as revealed by phylogenetic
studies We could not trace any conserved syntenic regions in
these genomes whereas in the case of AKF03862 and
AKF09634 we identified glycogen debranching enzymes in
Stigmatella aurantiaca DW43-1 [56 identity and 98
query coverage of AKF03860 with WP_0133760911] and
Opitutus terrae PB90-1 [57 identity and 97 query cover-
age of AKF09635 YP_0018205421] which further support
their putative horizontal transfer in S amylolyticus
Similarly the phylogenetic inference was drawn from two
representative domains of g-amylases using the same meth-
odology as followed for a-amylases (fig 4) We found that
one of the g-amylases AKF10340 lies closer to M xanthus
amylase whereas the other AKF10487 shares its sister
branch with Nitrosococcus watsonii a Gammaproteobacteria
This suggests that while some of the a- and g-amylases
are present in many myxobacterial species a majority of the
a- and g-amylases are present only in S amylolyticus and not
in other myxobacterial genomes possibly due to the result of
horizontal transfer events from diverse bacterial phylum such
as Deinococcus-Thermus Cyanobacteria Alphaproteobac-
teria Gammaproteobacteria and Acidobacteria
Agar Degrading Enzymes
Some bacteria are known to degrade agar and use it as a
carbon source and possess agarase enzymes that help in hy-
drolyzing agar in oligosaccharides (Chi et al 2012) Depending
on the site of action they can be of two types a-agarase
(agarose 3-glycanohydrolase 321158 GH96) and
b-agarase (agarose 4-glycanohydrolase 32181 GH16
GH50 GH86 GH118) (table 1) Both families of agarases
GH15 EFV841771 Achrom
obacter xylosoxidans C54
GH
15 ZP 011267341 Nitrococcus m
obilis Nb-231
GH15 YP 4129921 Nitrosospira m
ultiformis ATCC 25196
GH15 YP 5716301 Nitrobacter ham
burgensis X14 plasmid
GH15 ZP 012260811 Aurantim
onas manganoxydans SI85-9A1
GH15 YP 0016032801 Gluconacetobacter diazotrophicus PA1 5
GH15 YP 7451371 Granulibacter bethesdensis CGDNIH1
GH15 AEA616211 Burkholderia gladioli BSR3
GH15 YP 0018344311 Beijerinckia indica subsp indica ATCC 9039
GH15 YP 0027560251 Acidobacterium capsulatum
ATCC 51196
GH15 CBJ387091 Ralstonia solanacearum CMR15
GH15 YP 3189151 Nitrobacter winogradskyi Nb-255
GH15 YP 0012344011 Acidiphilium cryptum JF-5
GH15 YP 0040115781 Rhodomicrobium vannielii ATCC 17100
GH15 YP 0016419391 Methylobacterium extorquens PA1GH15 YP 5252641 Rhodoferax ferrireducens T118
GH15 YP 0041811661 Terriglobus saanensis SP1PR4
GH15 ZP 031333281 Chthoniobacter flavus Ellin428
GH15 CCA537261 Streptomyces venezuelae ATCC 10712
GH15 YP 0015065621 Frankia sp EAN1pec
GH15 YP 0024892701 Arthrobacter chlorophenolicus A6
GH15 YP 0018202091 Opitutus terrae PB90-1
GH15 YP 8729011 Acidothermus cellulolyticus 11B
GH15 YP 0013795591 Anaeromyxobacter sp Fw109-5
GH15 YP 0024929091 Anaeromyxobacter dehalogenans 2CP-1
GH15 YP 6355121 Myxococcus xanthus DK 1622
GH15 YP 0011047051 Saccharopolyspora erythraea NRRL 2338
GH15 YP 0011092191 Saccharopolyspora erythraea NRRL 2338
GH15 AKF10340 Sandaracinus amylolyticus DSM 53668
GH15 YP 6342991 Myxococcus xanthus DK 1622
GH15 YP 0031091351 Acidimicrobium ferrooxidans DSM 10331
GH15 YP 0035703361 Salinibacter ruber M8
GH15 YP 0025241401 Thermomicrobium roseum DSM 5159 plasmid
GH15 YP 0033214441 Sphaerobacter thermophilus DSM 20745
GH15 NP 7336051 Streptomyces coelicolor A3-2
GH15 YP 0035282991 Nitrosococcus halophilus Nc 4
GH15 YP 0043676121 Marinithermus hydrothermalis DSM 14884
GH15 YP 0043687811 Marinithermus hydrothermalis DSM 14884
GH15 YP 0034042871 Haloterrigena turkmenica DSM 5511
GH15 ZP 080461001 Haladaptatus paucihalophilus DX253
GH1
5 YP
023
376
1 Pi
crop
hilu
s to
rrid
us D
SM 9
790
GH1
5 ZP
055
7131
11
Ferr
opla
sma
acid
arm
anus
fer1
GH1
5 BA
B604
801
The
rmop
lasm
a vo
lcan
ium
GSS
1
GH
15 Y
P 00
3323
761
1 Th
erm
obac
ulum
terr
enum
ATC
C B
AA
-798
GH
15 Y
P 00
3899
615
1 C
yano
thec
e sp
PC
C 7
822
plas
mid
GH
15 Y
P 00
3705
927
1 Tr
uepe
ra ra
diov
ictr
ix D
SM 1
7093
GH
15 Y
P 00
2478
443
1 A
rthr
obac
ter c
hlor
ophe
nolic
us A
6 pl
asm
idG
H15
AK
F104
87 S
anda
raci
nus
amyl
olyt
icus
DSM
536
68G
H15
YP
0037
6113
81
Nitr
osoc
occu
s w
atso
nii C
-113
GH1
5 ZP
069
7134
91
Kted
onob
acte
r rac
emife
r DSM
449
63
GH15
ZP
0697
6049
1 K
tedo
noba
cter
race
mife
r DSM
449
63
GH15
YP
0033
9335
81
Cone
xiba
cter
woe
sei D
SM 1
4684
GH15
YP
0042
3629
11 A
cido
vora
x av
enae
sub
sp a
vena
e ATC
C 19
860
GH15 Z
P 03
6273
271
Pedo
spha
era p
arvu
la El
lin51
4
GH15 Y
P 00
1618
7001
Sor
angi
um ce
llulo
sum
So
ce56
GH15 ZP
0145
9561
1 Stig
matella
aura
ntiac
a DW
4-3-1
GH15 ZP
0503
8177
1 Syn
echo
cocc
us sp
PCC 73
35
GH15 YP 64
4038
1 Rubrobac
ter xy
lanophilu
s DSM 99
41
GH15 ZP 064746651 Frankia symbiont of D
atisca glomerata
GH15 ZP 069744041 Ktedonobacter racemife
r DSM 44963
GH15 ZP 069653261 Ktedonobacter racemifer D
SM 44963
GH15 ZP 069661381 Ktedonobacter racemifer D
SM 44963
FIG 4mdashPhylogeny of Sandaracinus amylolyticus GH15 g-amylases
Sandaracinus amylolyticus g-amylases are colored dark pink and myxobac-
terial homologs are colored fluorescent green Bootstrap values corre-
sponding to the tree nodes are provided
Sharma et al GBE
2526 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
were absent in S amylolyticus genome which could be related
to its agar nondegrading property (Mohr et al 2012) as sup-
ported by colony morphology where agar was not penetrated
or liquefied by the bacteria (supplementary fig S5
Supplementary Material online)
Cellulose Degrading Enzymes
We identified only GH5 and GH74 in S amylolyticus proteome
in two and three proteins respectively GH5 domain was pre-
sent as a single unit in AKF04963 and AKF09194 whereas
GH74 was identified in AKF04859 AKF06963 and
AKF09775 associated with a GH74 domain CAZY domains
for cellobiohydrolaseexoglucanases (represented by GH6
GH7 GH9 and GH48) were not present in S amylolyticus
Cellobiohydrolaseexoglucanases plays a significant role in cel-
lulose degradation as they cut two to four sugar subunits from
the exposed ends formed by the endoglucanases which are
used as initial substrates for b-glucosidase (Mba Medie et al
2012) The absence of cellobiohydrolase or exoglucanases
could be a possible reason for the lack of cellulose degradation
in S amylolyticus Out of b-glucosidase representative CAZY
families GH1 GH3 GH5 GH9 GH30 and GH116 only GH1
GH3 and GH5 families were found in three two and two
proteins in S amylolyticus respectively AKF03843 AKF0
6028 and AKF10711 have a GH1 domain whereas
AKF09078 and AKF10875 have only the GH3 domain
Chitin Degrading Enzymes
Chitinases (EC32114) are involved in degradation of chitin
a component of the cell wall of fungi and exoskeleton of
arthropods GH18 GH19 GH23 and GH48 families have
been reported in the CAZY database as chitinases that hydro-
lyse b-14 linkages between N-acetylglucosamine units GH18
is a chitinase family with a characteristic ldquo[LIVM
FY][DN]G[LIVMF][DN][LIVMF][DN]Erdquo motif and is expressed
in archaea prokaryotes and eukaryotes (Funkhouser and
Aronson 2007) GH18 chitinases were not found in S amylo-
lyticus genome while members of GH19 (AKF03006 and
AKF10423) and GH23 (AKF04575 AKF05533 AKF06346
AKF08835 and AKF11377) could be identified GH19
family chitinases are mostly reported to be present in plants
with only a few members in prokaryotes (Kasprzewska 2003
Funkhouser and Aronson 2007) Among the two identified
GH19 homologs we found that AKF10423 is approximately
65 identical (~68 query coverage) with cyanobacteria
such as Nodosilinea nodulosa and Limnoraphis robusta
along with another myxobacterium Chondromyces crocatus
Besides these homologs we also found that AKF10423 shows
maximum similarity with eukaryotic GH19 chitinases counter-
parts as present in Acacia mangium Festuca arundinacea
Triticum aestivum and Leucaena leucocephala (55ndash59 iden-
tity with 70 query coverage) We identified that another
chitinase AKF06346 has GH23 domain along with three
copies of CBM50 which is generally present in the enzymes
cleaving either chitin or peptidoglycan
Conclusion
The complete 1033 Mb circular genome of S amylolyticus
type strain DSM 53668T was determined CAZy analysis re-
vealed the presence of 72 GT domains 72 CE domains 58 GH
domains 19 AA domains 4 PL domains and 26 CBM do-
mains Sixteen S amylolyticus proteins were identified of
which 15 are a-amylases of class GH13 and 1 GH57 Two
g-amylases of GH15 family were also identified Phylogeny
analysis revealed that ten GH13 a-amylases and one GH15
g-amylase have been aquired via horizontal gene transfer
from other bacterial phyla such as Deinococcus-Thermus
Cyanobacteria Alphaproteobacteria Gammaproteobacteria
and Acidobacteria
Table 1
Cellulose Starch Chitin and Agar Degrading Enzymes and Their Representative CAZY Domains Distribution
Substrate Enzyme Category Enzyme Name EC Number CAZY Families (protein repertoire in brackets)
Starch Amylase a-Amylase 3211 GH13 (15) GH57 (1) GH119 GH126
Amylase b-Amylase 3212 GH14
Amylase g-Amylase 3213 GH15 (2) GH97
Chitin Chitinase Chitinase 32114 GH18 GH19 (2) GH23 (5) GH48
Agar a-Agarase Agarose 3-glycanohydrolase 321158 GH96
b-Agarase Agarose 4-glycanohydrolase 32181 GH16 GH50 GH86 GH118
Cellulose Cellulase b-Glucosidase 32121 GH1 (3) GH3 (2) GH5 (2) GH9 GH30 GH116
Cellulase Cellobiohydrolase or Exoglucanase 321176 GH48 GH6 GH7 GH9
Cellulase Endoglucanase 3214 GH5 (2) GH6 GH7 GH8 GH9 GH12 GH44
GH45 GH48 GH51 GH74 (3) GH124
NOTEmdashEC number enzyme category and CAZY families for proteins involved in starch chitin agar and cellulose degradation are shown The number of Sandaracinusamylolyticus proteins identified having CAZY domains are represented in bold fonts in respective families
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2527
Supplementary Material
Supplementary figures S1ndashS5 and tables S1 and S2 are avail-
able at Genome Biology and Evolution online (httpwww
gbeoxfordjournalsorg)
Acknowledgments
This work is supported by a project ldquoExpansion and modern-
ization of Microbial Type Culture Collection and Gene Bank
(MTCC) jointly supported by Council of Scientific and
Industrial Research (CSIR) Grant No BSC0402 and
Department of Biotechnology (DBT) Govt of India Grant No
BTPR7368INF221772012rdquo The authors thank MTCC for
providing help in procuring the cultures from DSMZ They
acknowledge Dr Ramya TNC for providing workbench for cul-
turing myxobacteria GS and IK acknowledge the Council
of Scientific and Industrial Research (CSIR) and University
Grant Commission (UGC) for the research fellowship respec-
tively They also thank the Genome Quebec Innovation
Centre McGill University Canada and C-CAMP next-genera-
tion genomics facility Bangalore India for help in obtaining
PacBio and Illumina NGS data respectively
Literature CitedAlikhan NF Petty NK Ben Zakour NL Beatson SA 2011 BLAST Ring
Image Generator (BRIG) simple prokaryote genome comparisons
BMC Genomics 12402
Altschul SF Gish W Miller W Myers EW Lipman DJ 1990 Basic local
alignment search tool J Mol Biol 215403ndash410
Auch AF Von Jan M Klenk HP Goker M 2010 Digital DNA-DNA hybrid-
ization for microbial species delineation by means of genome-to-
genome sequence comparison Stand Genomic Sci 2117ndash134
Aziz RK et al 2008 The RAST Server rapid annotations using subsystems
technology BMC Genomics 975
Blesak K Janecek S 2012 Sequence fingerprints of enzyme specificities
from the glycoside hydrolase family GH57 Extremophiles 16497ndash
506
Chi WJ Chang YK Hong SK 2012 Agar degradation by microorganisms
and agar-degrading enzymes Appl Microbiol Biotechnol 94917ndash
930
Chin CS et al 2013 Nonhybrid finished microbial genome assemblies
from long-read SMRT sequencing data Nat Methods 10563ndash569
Eddy SR 2011 Accelerated profile HMM searches PLoS Comput Biol
7e1002195
Edgar RC 2004 MUSCLE multiple sequence alignment with high accu-
racy and high throughput Nucleic Acids Res 321792ndash1797
Funkhouser JD Aronson NN Jr 2007 Chitinase family GH18 evolutionary
insights from the genomic history of a diverse protein family BMC Evol
Biol 796
Garcia R Muller R 2014 The family Polyangiaceae In Rosenberg E
Delong E Lory S Stackebrandt E Thompson F editors The prokary-
otes Berlin Heidelberg Springer p 247ndash279
Grissa I Vergnaud G Pourcel C 2007 CRISPRFinder a web tool to identify
clustered regularly interspaced short palindromic repeats Nucleic
Acids Res 35W52ndashW57
Hall TA 1999 BioEdit a user-friendly biological sequence alignment editor
and analysis program for Windows 9598NT Nucleic Acids Symp Ser
4195ndash98
Han K et al 2013 Extraordinary expansion of a Sorangium cellulosum
genome from an alkaline milieu Sci Rep 32101
Janecek S Kuchtova A 2012 In silico identification of catalytic residues
and domain fold of the family GH119 sharing the catalytic machinery
with the alpha-amylase family GH57 FEBS Lett 5863360ndash3366
Janecek S Svensson B Macgregor EA 2014 alpha-Amylase an enzyme
specificity found in various families of glycoside hydrolases Cell Mol
Life Sci 711149ndash1170
Kasprzewska A 2003 Plant chitinasesndashregulation and function Cell Mol
Biol Lett 8809ndash824
Lagesen K et al 2007 RNAmmer consistent and rapid annotation of
ribosomal RNA genes Nucleic Acids Res 353100ndash3108
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B
2014 The carbohydrate-active enzymes database (CAZy) in 2013
Nucleic Acids Res 42D490ndashD495
Lowe TM Eddy SR 1997 tRNAscan-SE a program for improved detection
of transfer RNA genes in genomic sequence Nucleic Acids Res
25955ndash964
Matsuura Y Kusunoki M Harada W Kakudo M 1984 Structure
and possible catalytic residues of Taka-amylase A J Biochem
95697ndash702
Mba Medie F Davies GJ Drancourt M Henrissat B 2012 Genome anal-
yses highlight the different biological roles of cellulases Nat Rev
Microbiol 10227ndash234
Mohr KI Garcia RO Gerth K Irschik H Muller R 2012
Sandaracinus amylolyticus gen nov sp nov a starch-degrading
soil myxobacterium and description of Sandaracinaceae fam
nov Int J Syst Evol Microbiol 621191ndash1198
Noguchi J et al 2011 Crystal structure of the branching enzyme I
(BEI) from Oryza sativa L with implications for catalysis and sub-
strate binding Glycobiology 211108ndash1116
Patel RK Jain M 2012 NGS QC Toolkit a toolkit for quality control of next
generation sequencing data PLoS One 7e30619
Pei J Kim BH Grishin NV PROMALS3D a tool for multiple protein se-
quence and structure alignments Nucleic Acids Res 36 2295ndash2300
Reichenbach H Dworkin M 1992 The myxobacteria In Balows A
Truper HG Dworkin M Harder W Schleifer KH editors The pro-
karyotes a handbook on the biology of bacteria ecophysiology
isolation identification applications New York Springer New
York p 3416ndash3487
Reichenbach H 2005 ldquoOrder VIII Myxococcalesrdquo Bergeyrsquos Manual of
Systematic Bacteriology In Brenner DJ Krieg NR Staley JT Garrity
GM editors Vol 2 2nd ed part C New York Springer-Verlag New
York Inc pp 1074ndash1079
Richter M Rossello-Mora R 2009 Shifting the genomic gold standard for
the prokaryotic species definition Proc Natl Acad Sci U S A 106
19126ndash19131
Schneiker S et al 2007 Complete genome sequence of the myxobacter-
ium Sorangium cellulosum Nat Biotechnol 25 1281ndash1289
Shimkets LJ 1990 Social and developmental biology of the myxobacteria
Microbiol Rev 54 473ndash501
Siguier P Perochon J Lestrade L Mahillon J Chandler M 2006 ISfinder
the reference centre for bacterial insertion sequences Nucleic Acids
Res 34 D32ndashD36
Steinmetz H et al 2012 Indiacens A and B prenyl indoles from the
myxobacterium Sandaracinus amylolyticus J Nat Prod 75 1803ndash
1805
Tamura K Stecher G Peterson D Filipski A Kumar S 2013 MEGA6
molecular evolutionary genetics analysis version 60 Mol Biol Evol
30 2725ndash2729
Van Der Maarel MJ Van Der Veen B Uitdehaag JC Leemhuis H Dijkhuizen
L 2002 Properties and applications of starch-converting enzymes of
the alpha-amylase family J Biotechnol 94 137ndash155
Sharma et al GBE
2528 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
Weissman KJ Muller R 2010 Myxobacterial secondary metabolites
bioactivities and modes-of-action Nat Prod Rep 27 1276ndash1295
Whitworth DE 2008 Myxobacteria multicellularity and differentiation
Washington DC ASM Press American Society for Microbiology
Yin Y et al 2012 dbCAN a web resource for automated carbohydrate-
active enzyme annotation Nucleic Acids Res 40 W445ndashW451
Zhao JY et al 2008 Discovery of the autonomously replicating plasmid
pMF1 from Myxococcus fulvus and development of a gene cloning
system in Myxococcus xanthus Appl Environ Microbiol 74 1980ndash
1987
Associate editor Howard Ochman
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2529
Taking into account the GH57 clade the sole GH57
domain of S amylolyticus (GenBank ID AKF06951) shares
the clade with anaerobic Deltaproteobacteria Among the
GH13 domains of S amylolyticus AKF03862 AKF07527
AKF09643 AKF11765 and AKF11772 shares clade with
other myxobacteria Among these AKF11772 and
AKF07527 share sister branches and form sister clades with
other myxobacterial species such as S cellulosum Soce56 and
Plesiocystis pacifica SIR-1 and are further related to the clade
containing Gammaproteobacteria Apart from these two am-
ylases AKF03860 and AKF06952 are similar to each other and
group with Candidatus accumulibacter phosphtis amylase
(YP_0031672861) from the phylum Acidobacteria
Interestingly AKF03860 and AKF06952 group with members
GH13 ZP 021632211 Kordia algicida OT-1
GH13 ZP 067516041 Parascardovia denticolens F0305
GH13 YP 0035709041 Salinibacter ruber M8
GH13 YP 0032500041 Fibrobacter succinogenes subsp succinogenes S85
GH13 AKF03862 Sandaracinus amylolyticus DSM 53668
GH13 YP 0039537801 Stigmatella aurantiaca
GH13 CAJ738231 Candidatus Kuenenia stuttgartiensis
GH13 YP 0228461 Picrophilus torridus DSM 9790GH13 ZP 029306261 Verrucomicrobium spinosum DSM 4136
GH13 CAX841261 uncultured bacterium
GH13 YP 0031678771 Candidatus Accumulibacter phosphatis clade IIA str UW-1GH13 ZP 031321321 Chthoniobacter flavus Ellin428
GH13 ZP 018759791 Lentisphaera araneosa HTCC2155GH13 YP 0033894351 Spirosoma linguale DSM 74GH13 NP 7734061 Bradyrhizobium diazoefficiens USDA 110
GH13 YP 0037757321 Herbaspirillum seropedicae SmR1GH13 YP 4266911 Rhodospirillum rubrum ATCC 11170GH13 ZP 035079501 Rhizobium etli Brasil 5
GH13 ZP 057930691 Butyrivibrio crossotus DSM 2876
GH13 ZP 066231321 Turicibacter sanguinis PC909
GH13 ZP 044499741 Catonella morbi ATCC 51271
GH13 EGE534241 Streptococcus parauberis NCFD 2020
GH13 196049865 Lactobacillus Plantarum
GH13 YP 0042196261 Granulicella tundricola MP5ACTX9
GH13 YP 0011935611 Flavobacterium johnsoniae UW101
GH13 YP 0032910851 Rhodothermus marinus DSM 4252GH13 YP 0040967591 Bacillus cellulosilyticus DSM 2522GH13 ZP 037030351 Flavobacteria bacterium MS024-2A
GH13 XP 0025352531 Ricinus communis
GH13 YP 7579561 Maricaulis maris MCS10GH13 ZP 010417351 Erythrobacter sp NAP1GH13 YP 0018177571 Opitutus terrae PB90-1GH13 EFR587141 Alistipes sp HGB5
GH13 CBK627851 Alistipes shahii WAL 8301GH13 EFR582631 Alistipes sp HGB5
GH13 YP 0034042011 Haloterrigena turkmenica DSM 5511
GH13 YP 0035357541 Haloferax volcanii DS2
GH13 YP 0035357541 Haloferax volcanii DS2
GH13 YP 0025661621 Halorubrum lacusprofundi ATCC 49239
GH13 YP 1368761 Haloarcula marismortui ATCC 43049
GH13 ZP 080436741 Haladaptatus paucihalophilus DX253
GH13 YP 0025652161 Halorubrum lacusprofundi ATCC 49239
GH13 YP 0035346301 Haloferax volcanii DS2
GH13 YP 0033225661 Thermobaculum terrenum ATCC BAA-798
GH13 YP 0021353421 Anaeromyxobacter sp K
GH13 AKF09643 Sandaracinus amylolyticus DSM 53668
GH13 YP 0016137031 Sorangium cellulosum So ce56
GH1
3 99
0316
21 H
alot
herm
othr
ix or
enii
GH1
3 AA
D051
991
Pae
niba
cillu
s po
lymyx
a
GH1
3 YP
003
6999
601
Bac
illus
sele
nitir
educ
ens
MLS
10
GH1
3 ZP
022
0799
81
Copr
ococ
cus
euta
ctus
ATC
C 27
759
GH
13 B
AA07
401
1 Xa
ntho
mon
as c
ampe
stris
GH
13 Y
P 00
4052
835
1 M
ariv
irga
tract
uosa
DSM
412
6
GH1
3 ZP
037
0954
51
Cory
neba
cter
ium
mat
ruch
otii A
TCC
3380
6
GH1
3 NP
267
649
1 La
ctoc
occu
s la
ctis
subs
p la
ctis
Il140
3
GH1
3 YP
001
3023
201
Par
abac
tero
ides
dist
ason
is AT
CC 8
503
GH1
3 YP
001
2276
361
Syn
echo
cocc
us s
p R
CC30
7
GH1
3 YP
002
5652
591
Hal
orub
rum
lacu
spro
fund
i ATC
C 49
239
GH13
ZP
0709
0550
1 C
oryn
ebac
teriu
m g
enita
lium
ATC
C 33
030
GH13
YP 0
0283
4100
1 C
oryn
ebac
teriu
m a
urim
ucos
um A
TCC
7009
75
GH13 A
KF03
861
Sand
arac
inus a
mylo
lyticu
s DSM
536
68
GH13
ZP 0
0050
865
1 M
agne
tosp
irillum
mag
neto
tacti
cum
MS-
1
GH13 Y
P 00
1568
811
1 Pe
troto
ga m
obilis
SJ9
5
GH13 Y
P 02
2847
1 P
icrop
hilus
torri
dus D
SM 9
790
GH13 E
FT25
5011
Pro
pionib
acter
ium ac
nes H
L110
PA3
GH13 E
FA80
1951
Poly
spho
ndyli
um pa
llidum
PN50
0
GH13 A
BD4655
81 V
ermam
oeba
verm
iform
is
GH13 Z
P 0350
5050
1 Rhiz
obium
etli B
rasil
5
GH13 YP 00
1348
4651
Pseud
omon
as ae
rugino
sa PA7
GH13 YP 00
2238
3511
Klebsie
lla pn
eumon
iae 34
2
GH13 YP 00
1437
9781
Cron
obac
ter sa
kaza
kii ATCC B
AA-894
GH13 Y
P 0011
7103
71 P
seud
omon
as st
utzeri
A1501
GH13 Y
P 0019
7240
81 S
tenotr
opho
monas
malt
ophil
ia K27
9a
GH13 Y
P 0043
1901
11 S
phing
obac
terium
sp 2
1
GH13 YP 0013127791 Sinorhizobium m
edicae W
SM419
GH13 ZP 035028921 Rhizobium etli K
im 5
GH13 YP 67
6219
1 Che
lativo
rans s
p BNC1
GH13 YP 00
2376
8861
Cya
nothe
ce sp
PCC 74
24
GH13 YP 0036383061 Cellulomonas flavig
ena DSM 20109
GH13 YP 0040977291 Intrasporangium ca
lvum DSM 43043
GH13 YP 0011691691 Rhodobacter s
phaeroides ATCC 17025
GH13 ZP 011580901 Oceanicola granulosus H
TCC2516
GH13 YP 0038382651 Micromonospora aurantiaca ATCC 27029
GH13 YP 0040977251 Intrasporangium calvu
m DSM 43043
GH13 YP 0036954761 Starkeya novella DSM 506
GH13 ZP 035166441 Rhizobium etli IE4771
GH13 ZP 031273131 Chthoniobacter flavus Ellin
428
GH13 AKF03642 Sandaracinus amylolyticus DSM 53668
GH13 YP 0031252671 Chitinophaga pinensis DSM 2588
GH13 YP 0030850521 Dyadobacter fermentans DSM 18053
GH13 YP 0033879241 Spirosoma linguale DSM 74
GH13 YP 0034301801 Streptococcus gallolyticus UCN34
GH13 ZP 078184041 Eremococcus coleocola ACS-139-V-Col8
GH13 YP 0034774501 Thermoanaerobacter italicus Ab9
GH13 ZP 048517861 Paenibacillus sp oral taxon 786 str D14
GH13 YP 0038414241 Caldicellulosiruptor obsidiansis OB47
GH13 YP 0024279661 Desulfurococcus kamchatkensis 1221n
GH13 YP 0034262831 Bacillus pseudofirmus OF4
GH13 ZP 076854311 Oscillochloris trichoides DG-6GH13 YP 0016174961 Sorangium cellulosum So ce56
GH13 ZP 019113371 Plesiocystis pacifica SIR-1
GH13 YP 6318811 Myxococcus xanthus DK 1622
GH13 AKF11765 Sandaracinus amylolyticus DSM 53668
GH13 ZP 036771121 Bacteroides cellulosilyticus DSM 14838
GH13 YP 0037157841 Croceibacter atlanticus HTCC2559
GH13 ZP 012015681 Flavobacteria bacterium BBFL7
GH13 ZP 019113321 Plesiocystis pacifica SIR-1GH13 YP 0031962511 Robiginitalea biformata HTCC2501
GH13 YP 0040531921 Marivirga tractuosa DSM 4126
GH13 ZP 021817881 Flavobacteriales bacterium ALC-1
GH13 CBL150401 Ruminococcus bromii L2-63
GH13 ZP 080051001 Bacillus sp 2 A 57 CT2
GH13 CBK911271 Eubacterium rectale DSM 17629
GH13 YP 0029369771 Eubacterium rectale ATCC 33656
GH13 YP 0021366011 Anaeromyxobacter sp K
GH13 ZP 014604541 Stigmatella aurantiaca
GH
13 728851 Streptomyces lividans
GH
13 BAJ281541 Kitasatospora setae KM-6054
GH
13 YP 0033390201 Streptosporangium roseum
DSM
43021
GH
13 YP 9214291 Nocardioides sp JS614
GH
13 AEB462451 Verrucosispora maris AB-18-032
GH
13 CAJ810301 Actinoplanes sp
GH
13 NP 9387401 C
orynebacterium diphtheriae N
CTC
13129
GH
13 YP 0036976141 Arcanobacterium haem
olyticum D
SM 20595
GH
13 ZP 039259261 Actinomyces coleocanis D
SM 15436
GH
13 YP 0040999421 Intrasporangium calvum
DSM
43043
GH
13 YP 0030989701 Actinosynnema m
irum D
SM 43827
GH
13 YP 0036376081 Cellulom
onas flavigena DSM
20109
GH
13 ZP 009938561 Janibacter sp HTC
C2649
GH13 YP 0030608421 Hirschia baltica ATCC 49814
GH13 YP 7579631 M
aricaulis maris M
CS10
GH13 YP 0016857121 Caulobacter sp K31
GH13 ZP 013016731 Sphingom
onas sp SKA58
GH13 YP 0038198341 Brevundim
onas subvibrioides ATCC 15264
GH
13 YP 4971641 Novosphingobium
aromaticivorans D
SM 12444
GH13 YP 6609551 Pseudoalterom
onas atlantica T6c
GH13 ZP 011126451 Reinekea blandensis M
ED297
GH13 YP 2677341 Colwellia psychrerythraea 34H
GH13 ZP 011126461 Reinekea blandensis M
ED297
GH13 AKF11772 Sandaracinus amylolyticus DSM
53668
GH13 AKF07527 Sandaracinus amylolyticus DSM
53668
GH13 YP 0016184291 Sorangium cellulosum
So ce56
GH13 ZP 019113391 Plesiocystis pacifica SIR-1
GH13 ZP 036315591 Pedosphaera parvula Ellin514
GH13 YP 0016604791 Microcystis aeruginosa NIES-843
GH13 YP 0043416831 Archaeoglobus veneficus SNP6
GH13 ZP 073351521 Desulfovibrio fructosovorans JJ
GH
13 AKF03605 Sandaracinus amylolyticus D
SM 53668
GH
13 YP 0027853821 Deinococcus deserti VC
D115
GH
13 NP 2951951 D
einococcus radiodurans R1
GH
13 YP 004171
1212
M
SD
sisn
epoc
ira
m su
ccoc
onie
D 1
661
1
GH
13 YP 0041715811 Deinococcus m
aricopensis DSM
21211
GH
13 YP 0037038171 Truepera radiovictrix DSM
17093
GH
13 YP 6052101 Deinococcus geotherm
alis DSM
11300
GH
13 YP 0027555491 A
cidobacterium capsulatum
ATCC
51196
GH
13 Y
P9
XTC
A5P
M alocirdnut allecilunarG 1860612400
GH
13 YP 6335241 M
yxococcus xanthus DK
1622G
H13 Y
PB
S su
cihp
ortid
ica
suhp
ortny
S 1
9412
64
GH
13 A
AW32
490
1 iiklahcsttog acnarborean
A
GH
13 Y
P 00
2316
532
1 An
oxyb
acillu
s fla
vith
erm
us W
K1
GH
13 Y
P 00
3252
634
1 G
eoba
cillu
s sp
Y41
2MC
61G
H13
ZP
0117
0936
1 B
acillu
s sp
NR
RL
B-14
911
GH
13 Z
P 07
7074
981
Bac
illus
sp m
3-13
GH
13 Z
P1-
GS ps sullica
B 116916810
GH
13 Y
P 00
1813
251
1 Ex
iguo
bact
eriu
m s
ibiri
cum
255
-15
GH
13 Y
P 00
2886
871
1 Ex
iguo
bact
eriu
m s
p A
T1b
GH
13 Y
P 00
1568
454
1 Pe
troto
ga m
obilis
SJ9
5
GH
13 Y
P 00
2940
188
1 Ko
smot
oga
olea
ria T
BF 1
95
1G
H13
ZP
0125
8420
1 V
ibrio
alg
inol
ytic
us 1
2G01
GH
13 1
0911
18 T
herm
oact
inom
yces
vul
garis
GH
13 Z
P 06
9705
751
Kte
dono
bact
er ra
cem
ifer D
SM 4
4963
GH
13 Y
P 00
1545
735
1 H
erpe
tosi
phon
aur
antia
cus
DSM
785
GH
13 E
FY95
778
1 M
etar
hizi
um ro
berts
ii AR
SEF
23
GH
13 N
P 58
8205
1 S
chiz
osac
char
omyc
es p
ombe
972
h-
GH
13 N
P 59
4401
1 S
chiz
osac
char
omyc
es p
ombe
972
h-
GH
13 X
P 00
1908
940
1 Po
dosp
ora
anse
rina
S m
at+
GH
13 4
1016
824
Schw
anni
omyc
es o
ccid
enta
lis
GH
13 B
AF98
616
1 H
ypho
pich
ia b
urto
nii
GH13 AKF07406 Sandaracinus amylolyticus DSM 53668
GH13 YP 0043692881 Desulfobacca acetoxidans DSM 11109
GH13 YP 0023716111 Cyanothece sp PCC 8801GH13 YP 3219451 Anabaena variabilis ATCC 29413
GH13 YP 4738811 Synechococcus sp JA-3-3AbGH13 YP 8464671 Syntrophobacter fumaroxidans MPOBGH13 ZP 063702061 Desulfovibrio sp FW1012B
GH13 YP 0033193471 Sphaerobacter thermophilus DSM 20745GH13 YP 0037057361 Truepera radiovictrix DSM 17093GH13 YP 4120971 Nitrosospira multiformis ATCC 25196GH13 YP 6431071 Rubrobacter xylanophilus DSM 9941
GH13 BAD389831 Rhodobacter sphaeroides f sp denitrificans
GH13 YP 3175601 Nitrobacter winogradskyi Nb-255
GH13 YP 8215181 Candidatus Solibacter usitatus Ellin6076
GH13 YP 0034506361 Azospirillum sp B510
GH13 ZP 070169471 Desulfonatronospira thiodismutans ASO3-1
GH13 YP 0041842351 Terriglobus saanensis SP1PR4
GH13 ZP 070315011 Granulicella mallensis MP5ACTX8
GH13 YP 0029438961 Variovorax paradoxus S110
GH13 YP 5918381 Candidatus Koribacter versatilis Ellin345
GH13 YP 7445681 Granulibacter bethesdensis CGDNIH1
GH13 YP 0039098001 Burkholderia sp CCGE1003
GH13 ZP 024903661 Burkholderia pseudomallei NCTC 13177
GH13 YP 0016309761 Bordetella petrii DSM 12804
GH13 YP 0022527351 Ralstonia solanacearum MolK2
GH13 YP 0037759061 Herbaspirillum seropedicae SmR1
GH13 YP 5455251 Methylobacillus flagellatus KT
GH13 YP 0013485041 Pseudomonas aeruginosa PA7
GH13 YP 0016008321 Gluconacetobacter diazotrophicus PA1 5
GH13 AEB584851 Pseudomonas mendocina NK-01
GH13 YP 0027996471 Azotobacter vinelandii DJ
GH
13 N
P 63
5804
1 X
anth
omon
as c
ampe
stris
pv
cam
pest
ris s
tr A
TCC
339
13
GH
13 Y
P 00
3188
581
1 Ac
etob
acte
r pas
teur
ianu
s IF
O 3
283-
01
GH
13 Y
P 74
4570
1 G
ranu
libac
ter b
ethe
sden
sis
CG
DN
IH1
GH
13 Y
P 00
3052
329
1 M
ethy
lovo
rus
gluc
oset
roph
us S
IP3-
4
GH
13 Z
P 06
8981
631
Ros
eom
onas
cer
vica
lis A
TCC
499
57
GH
13 Y
P 00
2943
895
1 Va
riovo
rax
para
doxu
s S1
10
GH
13 Y
P 31
4931
1 T
hiob
acillu
s de
nitri
fican
s AT
CC
252
59
GH
13 Y
P 42
5597
1 R
hodo
spiri
llum
rubr
um A
TCC
111
70
GH
13 Y
P 93
3302
1 A
zoar
cus
sp B
H72
GH
13 N
P 43
7816
1 S
inor
hizo
bium
mel
iloti
1021
GH
13 Z
P 01
1553
281
Oce
anic
ola
gran
ulos
us H
TCC
2516
GH
13 Y
P 00
1167
979
1 R
hodo
bact
er s
phae
roid
es A
TCC
170
25
GH
13 Y
P 00
1440
575
1 C
rono
bact
er s
akaz
akii A
TCC
BAA
-894
GH
13 Z
P 01
4401
991
Ful
vim
arin
a pe
lagi
HTC
C25
06
GH
13 Y
P 00
4234
793
1 Ac
idov
orax
ave
nae
subs
p a
vena
e AT
CC
198
60
GH
13 Y
P 00
1818
894
1 O
pitu
tus
terra
e PB
90-1
GH
13 Y
P 00
3154
808
1 Br
achy
bact
eriu
m fa
eciu
m D
SM 4
810
GH
13 ZP 010940301 Blastopirellula marina D
SM 3645
GH
13 ZP 027320551 Gem
mata obscuriglobus U
QM
2246
GH
13 YP 4273821 Rhodospirillum
rubrum ATC
C 11170
GH
13 YP 0021361131 Anaeromyxobacter sp K
GH
13 YP 0039532711 Stigmatella aurantiaca
GH
13 YP 0021384951 Geobacter bem
idjiensis BemG
H13 Y
P23
-C
RF ii
notl
ad r
etca
boe
G 1
3868
3520
0 G
H13 Y
P65
ec o
S mu
solu
llec
mu
igna
roS
191
8716
100
GH
13 ZPB2
101
WF
ps o
irbi
vofl
use
D 1
5398
6360
GH
13 Y
P 82
1516
1 C
andi
datu
s S
olib
acte
r usi
tatu
s E
llin6
076
GH
13 Y
P24
NFC ilte
muibozihR 1077074
GH
13
86635 M
SD sucityloly
ma sunicaradnaS 43690F
KA
GH
13 Y
P 00
1820
541
1 O
pitu
tus
terra
e P
B90
-1G
H13
ZP
0313
0066
1 C
htho
niob
acte
r fla
vus
Ellin
428
GH
13 YP 0035707081 Salinibacter ruber M8
GH
13 YP 0037057351 Truepera radiovictrix DSM
17093
GH
13 YP 0032689441 Haliangium
ochraceum D
SM 14365
GH
13 YP 0016130311 Sorangium cellulosum
So ce56
GH
13 YP 6288101 Myxococcus xanthus D
K 1622
GH
13 62738967 Deinococcus radiodurans
GH
13 AKF04017 Sandaracinus amylolyticus D
SM 53668
GH
13 YP 0027878991 Deinococcus deserti VC
D115
GH13 YP 5893201 Candidatus Koribacter versatilis Ellin345
GH13 ZP 063702081 Desulfovibrio sp FW
1012B
GH13 YP 0041842341 Terriglobus saanensis SP1PR4
GH13 YP 8731321 Acidotherm
us cellulolyticus 11B
GH13 ZP 070169501 Desulfonatronospira thiodism
utans ASO3-1
GH13 ADX845721 Sulfolobus islandicus REY15A
GH13 ZP 027318151 G
emm
ata obscuriglobus UQM
2246
GH13 YP 0016347811 Chloroflexus aurantiacus J-10-fl
GH
13 NP 9534051 G
eobacter sulfurreducens PCA
GH
13 YP 0025366761 Geobacter daltonii FR
C-32
GH
13 YP 0012121361 Pelotomaculum
thermopropionicum
SI
GH
13 YP 0041781661 Isosphaera pallida ATCC
43644
GH
13 ZP 036310341 Pedosphaera parvula Ellin514
GH13 ZP 031287911 Chthoniobacter flavus Ellin428
GH13 NP 6913271 Oceanobacillus ih
eyensis HTE831
GH13 YP 0025601851 Macro
coccus c
aseolyticus J
CSC5402
GH13 YP 0030110001 Paenibacill
us sp J
DR-2
GH13 CAJ384141 Anaerobranca
gottsch
alkii
GH13 YP 00
1988
1311
Lacto
bacill
us ca
sei B
L23
GH13 YP 00
2459
6561
Des
ulfito
bacte
rium ha
fnien
se D
CB-2
GH13 YP 00
3947
0781
Paenib
acillu
s poly
myxa S
C2
GH13 Y
P 0013
1197
11 C
lostrid
ium be
ijerin
ckii N
CIMB 80
52
GH13 Y
P 0039
6683
31 Ily
obac
ter po
lytrop
us D
SM 2926
GH13 Z
P 0539
1277
1 Clos
tridium
carb
oxidi
vora
ns P
7
GH13 Y
P 0013
9686
01 C
lostrid
ium kl
uyve
ri DSM 55
5
GH13 ZP 02
2128
451
Clostrid
ium ba
rtlettii
DSM 16
795
GH13 ZP 04
4331
841
Bacillu
s coa
gulan
s 36D
1
GH13 C
BL37
8151
Ana
eros
tipes
hadr
us
GH13 Z
P 04
4519
311
Abiotro
phia
defec
tiva A
TCC 49
176
GH13
YP
0031
4407
51
Slac
kia h
eliot
rinire
duce
ns D
SM 2
0476
GH13
ZP 0
2025
300
1 Eu
bacte
rium
vent
riosu
m A
TCC
2756
0
GH1
3 YP
004
3093
801
Clo
strid
ium
lent
ocel
lum
DSM
542
7
GH13
YP 5
1565
71
Chlam
ydop
hila
felis
GH13
ZP 0
6299
217
1 Pa
rach
lamyd
ia ac
anth
amoe
bae
str H
allrsquos
cocc
us
GH13
CBL1
5412
1 R
umino
cocc
us b
rom
ii L2-
63
GH13 Y
P 00
1692
347
1 Fi
nego
ldia
mag
na AT
CC 293
28
GH13 Z
P 07
5262
211
Pep
tostr
epto
cocc
us st
omat
is DSM
176
78
GH1
3 YP
003
4751
021
Clo
strid
iale
s ge
nom
osp
BVA
B3 s
tr U
PII9
-5
GH1
3 YP
004
1716
241
Dei
noco
ccus
mar
icope
nsis
DSM
212
11
GH1
3 YP
022
845
1 Pi
crop
hilu
s to
rridu
s DS
M 9
790
GH1
3 YP
001
8207
181
Opi
tutu
s te
rrae
PB90
-1
GH1
3 YP
003
1672
861
Can
dida
tus A
ccum
ulib
acte
r pho
spha
tis c
lade
IIA
str
UW-1
GH1
3 AK
F038
60 S
anda
racin
us a
mylo
lyticu
s DS
M 5
3668
GH1
3 AK
F069
52 S
anda
racin
us a
mylo
lyticu
s DS
M 5
3668
GH
13 Y
P 00
2762
222
1 G
emm
atim
onas
aur
antia
ca T
-27
GH1
3 ZP
082
0671
01
Gor
doni
a ne
ofel
ifaec
is NR
RL B
-593
95
GH
13 Y
P 00
3964
374
1 Ke
togu
loni
cige
nium
vul
gare
Y25
GH13 ZP 045772811 Oxalobacter fo
rmigenes H
OxBLS
GH13 YP 0033856211 Spirosoma linguale DSM 74
GH13 AEA650561 Burkholderia gladioli B
SR3
GH13 YP 0030725361 Teredinibacter turnerae T7901
GH13 YP 4255961 Rhodospirillum ru
brum ATCC 11170
GH13 YP 0010427851 Rhodobacter sphaeroides ATCC 17029
GH13 ZP 075890671 Sinorhizobium meliloti BL225C
GH13 YP 0030504181 Methylovorus glucosetrophus SIP3-4
GH13 YP 4224271 Magnetospirillum magneticum AMB-1
GH13 YP 0042554001 Deinococcus proteolyticus MRP
GH13 ZP 029300551 Verrucomicrobium spinosum DSM 4136
GH13 ZP 080190951 Lautropia mirabilis ATCC 51599
GH13 YP 5335351 Rhodopseudomonas palustris BisB18
GH13 YP 8654291 Magnetococcus marinus MC-1
GH13 YP 0028938741 Tolumonas auensis DSM 9187
GH13 YP 6612131 Pseudoalteromonas atlantica T6c
GH13 YP 0040654571 Pseudoalteromonas sp SM9913
GH13 YP 8302311 Arthrobacter sp FB24
GH13 YP 0042347941 Acidovorax avenae subsp avenae ATCC 19860
GH13 YP 0016390191 Methylobacterium extorquens PA1
GH13 ZP 068060481 Brevibacterium mcbrellneri ATCC 49030
GH13 YP 0031655431 Candidatus Accumulibacter phosphatis clade IIA str UW-1
GH13 YP 9813411 Polaromonas naphthalenivorans CJ2
GH13 ZP 029305921 Verrucomicrobium spinosum DSM 4136
GH13 YP 8283661 Candidatus Solibacter usitatus Ellin6076
GH13 YP 0015687661 Petrotoga mobilis SJ95
GH13 YP 2560581 Sulfolobus acidocaldarius DSM 639
GH13 YP 0023732201 Cyanothece sp PCC 8801
GH13 AKF07413 Sandaracinus amylolyticus DSM 53668
GH13 AKF03796 Sandaracinus amylolyticus DSM 53668
GH13 EFS357291 Propionibacterium acnes HL013PA1
GH13 YP 0033827501 Kribbella flavida DSM 17836
GH13 YP 9230111 Nocardioides sp JS614
GH13 AEA239681 Pseudonocardia dioxanivorans CB1190
GH13 YP 0031037201 Actinosynnema mirum DSM 43827
GH57 YP 0018695221 Nostoc punctiforme PCC 73102
GH57 YP 0041847961 Terriglobus saanensis SP1PR4
GH57 YP 0033170621 Thermanaerovibrio acidaminovorans DSM 6589
GH57 YP 0025231331 Thermomicrobium roseum DSM 5159
GH57 CBL286501 Fretibacterium fastidiosum
GH57 YP 0041781671 Isosphaera pallida ATCC 43644
GH57 YP 0027549471 Acidobacterium capsulatum ATCC 51196
GH57 ZP 070292791 Granulicella mallensis MP5ACTX8
GH57 YP 0018749261 Elusimicrobium minutum Pei191
GH57 ADL259941 Fibrobacter succinogenes subsp succinogenes S85
GH57 YP 4773871 Synechococcus sp JA-2-3B
GH57 YP 8454221 Syntrophobacter fumaroxidans MPOB
GH57 NP 9534062 Geobacter sulfurreducens PCA
GH57 YP 5020321 Methanospirillum hungatei JF-1
GH57 YP 0019958521 Chloroherpeton thalassium ATCC 35110
GH57 YP 5937791 Candidatus Koribacter versatilis Ellin345
GH57 YP 0010471761 Methanoculleus marisnigri JR1
GH57 YP 0014309471 Roseiflexus castenholzii DSM 13941
GH57 YP 0029516361 Desulfovibrio magneticus RS-1
GH57 YP 0038064671 Desulfarculus baarsii DSM 2075
GH57 YP 0013789531 Anaeromyxobacter sp Fw109-5
GH57 ZP 063702071 Desulfovibrio sp FW1012B
GH57 YP 0035531061 Aminobacterium colombiense DSM 12261
GH57 ZP 064391271 Anaerobaculum hydrogeniformans ATCC BAA-1850
GH57 AKF06951 Sandaracinus amylolyticus DSM 53668
GH57 ZP 063915611 Dethiosulfovibrio peptidovorans DSM 11002
GH57 YP 0024634161 Chloroflexus aggregans DSM 9485
GH57 YP 0043692891 Desulfobacca acetoxidans DSM 11109
GH57 YP 8464661 Syntrophobacter fumaroxidans M
POB
GH57 CAJ738171 Candidatus Kuenenia stuttgartiensis
GH57 YP 0031969181 Desulfohalobium
retbaense DSM 5692
FIG 3mdashPhylogeny of Sandaracinus amylolyticus GH13 and GH57 a-amylases Sandaracinus amylolyticus a-amylases (GH13 and GH57) are colored in
pink along with their domain architecture displayed as Domain Color Shape [GH13 Red rectangle GH57 Black rectangle GH77 dark pink rectangle
CBM48 Dark blue circle CBM20 green circle CBM41 cyan circle] Myxobacterial homologs are colored fluorescent green Bootstrap values correspond-
ing to the tree nodes are provided
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2525
of the phyla Betaproteobacteria suggestive of a possible hor-
izontal transfer
AKF04017 and AKF03605 have close homologs in the ex-
tremophile Deinococcus desserti VCD115 and shares clade
with certain myxobacterial amylases belonging to
Myxococcus xanthus S cellulosum and Haliangium ocraceum
which further share clades with Deinococcus amylases
AKF04017 and AKF03605 share the highest sequence identity
with D desserti VCD115 plasmid protein (YP_002787899
533) and chromosomal protein (YP_002785382 456)
respectively that might point to its plasmid-borne horizontal
transfer across the clade (fig 3) The sequence conservation of
CSRs and the high percentage identity of these amylases to
Deinococcus support the inference for the putative horizontal
transfers among these members AKF09634 does not share its
sister branch with any member but the clade contains mem-
bers from diverse phyla that is Rhizobium from alphaproteo-
bacteria Opitutus from Chlamydae Chthonibacter from
Verrucomicrobia suggesting the multiple gain and loss of
this amylase among prokaryotes Similarly AKF07413 shares
the clade with members of Cyanobacteria AKF07406 shares
the clade with members of Deltaproteobacteria AKF03861
with members of Alphaproteobacteria and AKF03642 with
members of Bacteroidetes Taken together we can suggest
that many of the S amylolyticus amylases have been acquired
via horizontal gene transfer events from other bacterial species
belonging to the phylum Deinococcus-Thermus
Acidobacteria Cyanobacteria and Alphaproteobacteria
To further support these horizontal transfer events we
checked the sequence conservation between S amylolyticus
amylases and closely related members in the phylogenetic tree
and aligned using PROMALS-3D with Taka-Amylase A of A
oryzae (PDBid 2TAA) as a structural template We also per-
formed synteny studies analyzing proteins present in the vicin-
ity of all identified S amylolyticus GH13 and GH57 amylases
and their closely related homologs as revealed by phylogenetic
studies We could not trace any conserved syntenic regions in
these genomes whereas in the case of AKF03862 and
AKF09634 we identified glycogen debranching enzymes in
Stigmatella aurantiaca DW43-1 [56 identity and 98
query coverage of AKF03860 with WP_0133760911] and
Opitutus terrae PB90-1 [57 identity and 97 query cover-
age of AKF09635 YP_0018205421] which further support
their putative horizontal transfer in S amylolyticus
Similarly the phylogenetic inference was drawn from two
representative domains of g-amylases using the same meth-
odology as followed for a-amylases (fig 4) We found that
one of the g-amylases AKF10340 lies closer to M xanthus
amylase whereas the other AKF10487 shares its sister
branch with Nitrosococcus watsonii a Gammaproteobacteria
This suggests that while some of the a- and g-amylases
are present in many myxobacterial species a majority of the
a- and g-amylases are present only in S amylolyticus and not
in other myxobacterial genomes possibly due to the result of
horizontal transfer events from diverse bacterial phylum such
as Deinococcus-Thermus Cyanobacteria Alphaproteobac-
teria Gammaproteobacteria and Acidobacteria
Agar Degrading Enzymes
Some bacteria are known to degrade agar and use it as a
carbon source and possess agarase enzymes that help in hy-
drolyzing agar in oligosaccharides (Chi et al 2012) Depending
on the site of action they can be of two types a-agarase
(agarose 3-glycanohydrolase 321158 GH96) and
b-agarase (agarose 4-glycanohydrolase 32181 GH16
GH50 GH86 GH118) (table 1) Both families of agarases
GH15 EFV841771 Achrom
obacter xylosoxidans C54
GH
15 ZP 011267341 Nitrococcus m
obilis Nb-231
GH15 YP 4129921 Nitrosospira m
ultiformis ATCC 25196
GH15 YP 5716301 Nitrobacter ham
burgensis X14 plasmid
GH15 ZP 012260811 Aurantim
onas manganoxydans SI85-9A1
GH15 YP 0016032801 Gluconacetobacter diazotrophicus PA1 5
GH15 YP 7451371 Granulibacter bethesdensis CGDNIH1
GH15 AEA616211 Burkholderia gladioli BSR3
GH15 YP 0018344311 Beijerinckia indica subsp indica ATCC 9039
GH15 YP 0027560251 Acidobacterium capsulatum
ATCC 51196
GH15 CBJ387091 Ralstonia solanacearum CMR15
GH15 YP 3189151 Nitrobacter winogradskyi Nb-255
GH15 YP 0012344011 Acidiphilium cryptum JF-5
GH15 YP 0040115781 Rhodomicrobium vannielii ATCC 17100
GH15 YP 0016419391 Methylobacterium extorquens PA1GH15 YP 5252641 Rhodoferax ferrireducens T118
GH15 YP 0041811661 Terriglobus saanensis SP1PR4
GH15 ZP 031333281 Chthoniobacter flavus Ellin428
GH15 CCA537261 Streptomyces venezuelae ATCC 10712
GH15 YP 0015065621 Frankia sp EAN1pec
GH15 YP 0024892701 Arthrobacter chlorophenolicus A6
GH15 YP 0018202091 Opitutus terrae PB90-1
GH15 YP 8729011 Acidothermus cellulolyticus 11B
GH15 YP 0013795591 Anaeromyxobacter sp Fw109-5
GH15 YP 0024929091 Anaeromyxobacter dehalogenans 2CP-1
GH15 YP 6355121 Myxococcus xanthus DK 1622
GH15 YP 0011047051 Saccharopolyspora erythraea NRRL 2338
GH15 YP 0011092191 Saccharopolyspora erythraea NRRL 2338
GH15 AKF10340 Sandaracinus amylolyticus DSM 53668
GH15 YP 6342991 Myxococcus xanthus DK 1622
GH15 YP 0031091351 Acidimicrobium ferrooxidans DSM 10331
GH15 YP 0035703361 Salinibacter ruber M8
GH15 YP 0025241401 Thermomicrobium roseum DSM 5159 plasmid
GH15 YP 0033214441 Sphaerobacter thermophilus DSM 20745
GH15 NP 7336051 Streptomyces coelicolor A3-2
GH15 YP 0035282991 Nitrosococcus halophilus Nc 4
GH15 YP 0043676121 Marinithermus hydrothermalis DSM 14884
GH15 YP 0043687811 Marinithermus hydrothermalis DSM 14884
GH15 YP 0034042871 Haloterrigena turkmenica DSM 5511
GH15 ZP 080461001 Haladaptatus paucihalophilus DX253
GH1
5 YP
023
376
1 Pi
crop
hilu
s to
rrid
us D
SM 9
790
GH1
5 ZP
055
7131
11
Ferr
opla
sma
acid
arm
anus
fer1
GH1
5 BA
B604
801
The
rmop
lasm
a vo
lcan
ium
GSS
1
GH
15 Y
P 00
3323
761
1 Th
erm
obac
ulum
terr
enum
ATC
C B
AA
-798
GH
15 Y
P 00
3899
615
1 C
yano
thec
e sp
PC
C 7
822
plas
mid
GH
15 Y
P 00
3705
927
1 Tr
uepe
ra ra
diov
ictr
ix D
SM 1
7093
GH
15 Y
P 00
2478
443
1 A
rthr
obac
ter c
hlor
ophe
nolic
us A
6 pl
asm
idG
H15
AK
F104
87 S
anda
raci
nus
amyl
olyt
icus
DSM
536
68G
H15
YP
0037
6113
81
Nitr
osoc
occu
s w
atso
nii C
-113
GH1
5 ZP
069
7134
91
Kted
onob
acte
r rac
emife
r DSM
449
63
GH15
ZP
0697
6049
1 K
tedo
noba
cter
race
mife
r DSM
449
63
GH15
YP
0033
9335
81
Cone
xiba
cter
woe
sei D
SM 1
4684
GH15
YP
0042
3629
11 A
cido
vora
x av
enae
sub
sp a
vena
e ATC
C 19
860
GH15 Z
P 03
6273
271
Pedo
spha
era p
arvu
la El
lin51
4
GH15 Y
P 00
1618
7001
Sor
angi
um ce
llulo
sum
So
ce56
GH15 ZP
0145
9561
1 Stig
matella
aura
ntiac
a DW
4-3-1
GH15 ZP
0503
8177
1 Syn
echo
cocc
us sp
PCC 73
35
GH15 YP 64
4038
1 Rubrobac
ter xy
lanophilu
s DSM 99
41
GH15 ZP 064746651 Frankia symbiont of D
atisca glomerata
GH15 ZP 069744041 Ktedonobacter racemife
r DSM 44963
GH15 ZP 069653261 Ktedonobacter racemifer D
SM 44963
GH15 ZP 069661381 Ktedonobacter racemifer D
SM 44963
FIG 4mdashPhylogeny of Sandaracinus amylolyticus GH15 g-amylases
Sandaracinus amylolyticus g-amylases are colored dark pink and myxobac-
terial homologs are colored fluorescent green Bootstrap values corre-
sponding to the tree nodes are provided
Sharma et al GBE
2526 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
were absent in S amylolyticus genome which could be related
to its agar nondegrading property (Mohr et al 2012) as sup-
ported by colony morphology where agar was not penetrated
or liquefied by the bacteria (supplementary fig S5
Supplementary Material online)
Cellulose Degrading Enzymes
We identified only GH5 and GH74 in S amylolyticus proteome
in two and three proteins respectively GH5 domain was pre-
sent as a single unit in AKF04963 and AKF09194 whereas
GH74 was identified in AKF04859 AKF06963 and
AKF09775 associated with a GH74 domain CAZY domains
for cellobiohydrolaseexoglucanases (represented by GH6
GH7 GH9 and GH48) were not present in S amylolyticus
Cellobiohydrolaseexoglucanases plays a significant role in cel-
lulose degradation as they cut two to four sugar subunits from
the exposed ends formed by the endoglucanases which are
used as initial substrates for b-glucosidase (Mba Medie et al
2012) The absence of cellobiohydrolase or exoglucanases
could be a possible reason for the lack of cellulose degradation
in S amylolyticus Out of b-glucosidase representative CAZY
families GH1 GH3 GH5 GH9 GH30 and GH116 only GH1
GH3 and GH5 families were found in three two and two
proteins in S amylolyticus respectively AKF03843 AKF0
6028 and AKF10711 have a GH1 domain whereas
AKF09078 and AKF10875 have only the GH3 domain
Chitin Degrading Enzymes
Chitinases (EC32114) are involved in degradation of chitin
a component of the cell wall of fungi and exoskeleton of
arthropods GH18 GH19 GH23 and GH48 families have
been reported in the CAZY database as chitinases that hydro-
lyse b-14 linkages between N-acetylglucosamine units GH18
is a chitinase family with a characteristic ldquo[LIVM
FY][DN]G[LIVMF][DN][LIVMF][DN]Erdquo motif and is expressed
in archaea prokaryotes and eukaryotes (Funkhouser and
Aronson 2007) GH18 chitinases were not found in S amylo-
lyticus genome while members of GH19 (AKF03006 and
AKF10423) and GH23 (AKF04575 AKF05533 AKF06346
AKF08835 and AKF11377) could be identified GH19
family chitinases are mostly reported to be present in plants
with only a few members in prokaryotes (Kasprzewska 2003
Funkhouser and Aronson 2007) Among the two identified
GH19 homologs we found that AKF10423 is approximately
65 identical (~68 query coverage) with cyanobacteria
such as Nodosilinea nodulosa and Limnoraphis robusta
along with another myxobacterium Chondromyces crocatus
Besides these homologs we also found that AKF10423 shows
maximum similarity with eukaryotic GH19 chitinases counter-
parts as present in Acacia mangium Festuca arundinacea
Triticum aestivum and Leucaena leucocephala (55ndash59 iden-
tity with 70 query coverage) We identified that another
chitinase AKF06346 has GH23 domain along with three
copies of CBM50 which is generally present in the enzymes
cleaving either chitin or peptidoglycan
Conclusion
The complete 1033 Mb circular genome of S amylolyticus
type strain DSM 53668T was determined CAZy analysis re-
vealed the presence of 72 GT domains 72 CE domains 58 GH
domains 19 AA domains 4 PL domains and 26 CBM do-
mains Sixteen S amylolyticus proteins were identified of
which 15 are a-amylases of class GH13 and 1 GH57 Two
g-amylases of GH15 family were also identified Phylogeny
analysis revealed that ten GH13 a-amylases and one GH15
g-amylase have been aquired via horizontal gene transfer
from other bacterial phyla such as Deinococcus-Thermus
Cyanobacteria Alphaproteobacteria Gammaproteobacteria
and Acidobacteria
Table 1
Cellulose Starch Chitin and Agar Degrading Enzymes and Their Representative CAZY Domains Distribution
Substrate Enzyme Category Enzyme Name EC Number CAZY Families (protein repertoire in brackets)
Starch Amylase a-Amylase 3211 GH13 (15) GH57 (1) GH119 GH126
Amylase b-Amylase 3212 GH14
Amylase g-Amylase 3213 GH15 (2) GH97
Chitin Chitinase Chitinase 32114 GH18 GH19 (2) GH23 (5) GH48
Agar a-Agarase Agarose 3-glycanohydrolase 321158 GH96
b-Agarase Agarose 4-glycanohydrolase 32181 GH16 GH50 GH86 GH118
Cellulose Cellulase b-Glucosidase 32121 GH1 (3) GH3 (2) GH5 (2) GH9 GH30 GH116
Cellulase Cellobiohydrolase or Exoglucanase 321176 GH48 GH6 GH7 GH9
Cellulase Endoglucanase 3214 GH5 (2) GH6 GH7 GH8 GH9 GH12 GH44
GH45 GH48 GH51 GH74 (3) GH124
NOTEmdashEC number enzyme category and CAZY families for proteins involved in starch chitin agar and cellulose degradation are shown The number of Sandaracinusamylolyticus proteins identified having CAZY domains are represented in bold fonts in respective families
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2527
Supplementary Material
Supplementary figures S1ndashS5 and tables S1 and S2 are avail-
able at Genome Biology and Evolution online (httpwww
gbeoxfordjournalsorg)
Acknowledgments
This work is supported by a project ldquoExpansion and modern-
ization of Microbial Type Culture Collection and Gene Bank
(MTCC) jointly supported by Council of Scientific and
Industrial Research (CSIR) Grant No BSC0402 and
Department of Biotechnology (DBT) Govt of India Grant No
BTPR7368INF221772012rdquo The authors thank MTCC for
providing help in procuring the cultures from DSMZ They
acknowledge Dr Ramya TNC for providing workbench for cul-
turing myxobacteria GS and IK acknowledge the Council
of Scientific and Industrial Research (CSIR) and University
Grant Commission (UGC) for the research fellowship respec-
tively They also thank the Genome Quebec Innovation
Centre McGill University Canada and C-CAMP next-genera-
tion genomics facility Bangalore India for help in obtaining
PacBio and Illumina NGS data respectively
Literature CitedAlikhan NF Petty NK Ben Zakour NL Beatson SA 2011 BLAST Ring
Image Generator (BRIG) simple prokaryote genome comparisons
BMC Genomics 12402
Altschul SF Gish W Miller W Myers EW Lipman DJ 1990 Basic local
alignment search tool J Mol Biol 215403ndash410
Auch AF Von Jan M Klenk HP Goker M 2010 Digital DNA-DNA hybrid-
ization for microbial species delineation by means of genome-to-
genome sequence comparison Stand Genomic Sci 2117ndash134
Aziz RK et al 2008 The RAST Server rapid annotations using subsystems
technology BMC Genomics 975
Blesak K Janecek S 2012 Sequence fingerprints of enzyme specificities
from the glycoside hydrolase family GH57 Extremophiles 16497ndash
506
Chi WJ Chang YK Hong SK 2012 Agar degradation by microorganisms
and agar-degrading enzymes Appl Microbiol Biotechnol 94917ndash
930
Chin CS et al 2013 Nonhybrid finished microbial genome assemblies
from long-read SMRT sequencing data Nat Methods 10563ndash569
Eddy SR 2011 Accelerated profile HMM searches PLoS Comput Biol
7e1002195
Edgar RC 2004 MUSCLE multiple sequence alignment with high accu-
racy and high throughput Nucleic Acids Res 321792ndash1797
Funkhouser JD Aronson NN Jr 2007 Chitinase family GH18 evolutionary
insights from the genomic history of a diverse protein family BMC Evol
Biol 796
Garcia R Muller R 2014 The family Polyangiaceae In Rosenberg E
Delong E Lory S Stackebrandt E Thompson F editors The prokary-
otes Berlin Heidelberg Springer p 247ndash279
Grissa I Vergnaud G Pourcel C 2007 CRISPRFinder a web tool to identify
clustered regularly interspaced short palindromic repeats Nucleic
Acids Res 35W52ndashW57
Hall TA 1999 BioEdit a user-friendly biological sequence alignment editor
and analysis program for Windows 9598NT Nucleic Acids Symp Ser
4195ndash98
Han K et al 2013 Extraordinary expansion of a Sorangium cellulosum
genome from an alkaline milieu Sci Rep 32101
Janecek S Kuchtova A 2012 In silico identification of catalytic residues
and domain fold of the family GH119 sharing the catalytic machinery
with the alpha-amylase family GH57 FEBS Lett 5863360ndash3366
Janecek S Svensson B Macgregor EA 2014 alpha-Amylase an enzyme
specificity found in various families of glycoside hydrolases Cell Mol
Life Sci 711149ndash1170
Kasprzewska A 2003 Plant chitinasesndashregulation and function Cell Mol
Biol Lett 8809ndash824
Lagesen K et al 2007 RNAmmer consistent and rapid annotation of
ribosomal RNA genes Nucleic Acids Res 353100ndash3108
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B
2014 The carbohydrate-active enzymes database (CAZy) in 2013
Nucleic Acids Res 42D490ndashD495
Lowe TM Eddy SR 1997 tRNAscan-SE a program for improved detection
of transfer RNA genes in genomic sequence Nucleic Acids Res
25955ndash964
Matsuura Y Kusunoki M Harada W Kakudo M 1984 Structure
and possible catalytic residues of Taka-amylase A J Biochem
95697ndash702
Mba Medie F Davies GJ Drancourt M Henrissat B 2012 Genome anal-
yses highlight the different biological roles of cellulases Nat Rev
Microbiol 10227ndash234
Mohr KI Garcia RO Gerth K Irschik H Muller R 2012
Sandaracinus amylolyticus gen nov sp nov a starch-degrading
soil myxobacterium and description of Sandaracinaceae fam
nov Int J Syst Evol Microbiol 621191ndash1198
Noguchi J et al 2011 Crystal structure of the branching enzyme I
(BEI) from Oryza sativa L with implications for catalysis and sub-
strate binding Glycobiology 211108ndash1116
Patel RK Jain M 2012 NGS QC Toolkit a toolkit for quality control of next
generation sequencing data PLoS One 7e30619
Pei J Kim BH Grishin NV PROMALS3D a tool for multiple protein se-
quence and structure alignments Nucleic Acids Res 36 2295ndash2300
Reichenbach H Dworkin M 1992 The myxobacteria In Balows A
Truper HG Dworkin M Harder W Schleifer KH editors The pro-
karyotes a handbook on the biology of bacteria ecophysiology
isolation identification applications New York Springer New
York p 3416ndash3487
Reichenbach H 2005 ldquoOrder VIII Myxococcalesrdquo Bergeyrsquos Manual of
Systematic Bacteriology In Brenner DJ Krieg NR Staley JT Garrity
GM editors Vol 2 2nd ed part C New York Springer-Verlag New
York Inc pp 1074ndash1079
Richter M Rossello-Mora R 2009 Shifting the genomic gold standard for
the prokaryotic species definition Proc Natl Acad Sci U S A 106
19126ndash19131
Schneiker S et al 2007 Complete genome sequence of the myxobacter-
ium Sorangium cellulosum Nat Biotechnol 25 1281ndash1289
Shimkets LJ 1990 Social and developmental biology of the myxobacteria
Microbiol Rev 54 473ndash501
Siguier P Perochon J Lestrade L Mahillon J Chandler M 2006 ISfinder
the reference centre for bacterial insertion sequences Nucleic Acids
Res 34 D32ndashD36
Steinmetz H et al 2012 Indiacens A and B prenyl indoles from the
myxobacterium Sandaracinus amylolyticus J Nat Prod 75 1803ndash
1805
Tamura K Stecher G Peterson D Filipski A Kumar S 2013 MEGA6
molecular evolutionary genetics analysis version 60 Mol Biol Evol
30 2725ndash2729
Van Der Maarel MJ Van Der Veen B Uitdehaag JC Leemhuis H Dijkhuizen
L 2002 Properties and applications of starch-converting enzymes of
the alpha-amylase family J Biotechnol 94 137ndash155
Sharma et al GBE
2528 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
Weissman KJ Muller R 2010 Myxobacterial secondary metabolites
bioactivities and modes-of-action Nat Prod Rep 27 1276ndash1295
Whitworth DE 2008 Myxobacteria multicellularity and differentiation
Washington DC ASM Press American Society for Microbiology
Yin Y et al 2012 dbCAN a web resource for automated carbohydrate-
active enzyme annotation Nucleic Acids Res 40 W445ndashW451
Zhao JY et al 2008 Discovery of the autonomously replicating plasmid
pMF1 from Myxococcus fulvus and development of a gene cloning
system in Myxococcus xanthus Appl Environ Microbiol 74 1980ndash
1987
Associate editor Howard Ochman
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2529
of the phyla Betaproteobacteria suggestive of a possible hor-
izontal transfer
AKF04017 and AKF03605 have close homologs in the ex-
tremophile Deinococcus desserti VCD115 and shares clade
with certain myxobacterial amylases belonging to
Myxococcus xanthus S cellulosum and Haliangium ocraceum
which further share clades with Deinococcus amylases
AKF04017 and AKF03605 share the highest sequence identity
with D desserti VCD115 plasmid protein (YP_002787899
533) and chromosomal protein (YP_002785382 456)
respectively that might point to its plasmid-borne horizontal
transfer across the clade (fig 3) The sequence conservation of
CSRs and the high percentage identity of these amylases to
Deinococcus support the inference for the putative horizontal
transfers among these members AKF09634 does not share its
sister branch with any member but the clade contains mem-
bers from diverse phyla that is Rhizobium from alphaproteo-
bacteria Opitutus from Chlamydae Chthonibacter from
Verrucomicrobia suggesting the multiple gain and loss of
this amylase among prokaryotes Similarly AKF07413 shares
the clade with members of Cyanobacteria AKF07406 shares
the clade with members of Deltaproteobacteria AKF03861
with members of Alphaproteobacteria and AKF03642 with
members of Bacteroidetes Taken together we can suggest
that many of the S amylolyticus amylases have been acquired
via horizontal gene transfer events from other bacterial species
belonging to the phylum Deinococcus-Thermus
Acidobacteria Cyanobacteria and Alphaproteobacteria
To further support these horizontal transfer events we
checked the sequence conservation between S amylolyticus
amylases and closely related members in the phylogenetic tree
and aligned using PROMALS-3D with Taka-Amylase A of A
oryzae (PDBid 2TAA) as a structural template We also per-
formed synteny studies analyzing proteins present in the vicin-
ity of all identified S amylolyticus GH13 and GH57 amylases
and their closely related homologs as revealed by phylogenetic
studies We could not trace any conserved syntenic regions in
these genomes whereas in the case of AKF03862 and
AKF09634 we identified glycogen debranching enzymes in
Stigmatella aurantiaca DW43-1 [56 identity and 98
query coverage of AKF03860 with WP_0133760911] and
Opitutus terrae PB90-1 [57 identity and 97 query cover-
age of AKF09635 YP_0018205421] which further support
their putative horizontal transfer in S amylolyticus
Similarly the phylogenetic inference was drawn from two
representative domains of g-amylases using the same meth-
odology as followed for a-amylases (fig 4) We found that
one of the g-amylases AKF10340 lies closer to M xanthus
amylase whereas the other AKF10487 shares its sister
branch with Nitrosococcus watsonii a Gammaproteobacteria
This suggests that while some of the a- and g-amylases
are present in many myxobacterial species a majority of the
a- and g-amylases are present only in S amylolyticus and not
in other myxobacterial genomes possibly due to the result of
horizontal transfer events from diverse bacterial phylum such
as Deinococcus-Thermus Cyanobacteria Alphaproteobac-
teria Gammaproteobacteria and Acidobacteria
Agar Degrading Enzymes
Some bacteria are known to degrade agar and use it as a
carbon source and possess agarase enzymes that help in hy-
drolyzing agar in oligosaccharides (Chi et al 2012) Depending
on the site of action they can be of two types a-agarase
(agarose 3-glycanohydrolase 321158 GH96) and
b-agarase (agarose 4-glycanohydrolase 32181 GH16
GH50 GH86 GH118) (table 1) Both families of agarases
GH15 EFV841771 Achrom
obacter xylosoxidans C54
GH
15 ZP 011267341 Nitrococcus m
obilis Nb-231
GH15 YP 4129921 Nitrosospira m
ultiformis ATCC 25196
GH15 YP 5716301 Nitrobacter ham
burgensis X14 plasmid
GH15 ZP 012260811 Aurantim
onas manganoxydans SI85-9A1
GH15 YP 0016032801 Gluconacetobacter diazotrophicus PA1 5
GH15 YP 7451371 Granulibacter bethesdensis CGDNIH1
GH15 AEA616211 Burkholderia gladioli BSR3
GH15 YP 0018344311 Beijerinckia indica subsp indica ATCC 9039
GH15 YP 0027560251 Acidobacterium capsulatum
ATCC 51196
GH15 CBJ387091 Ralstonia solanacearum CMR15
GH15 YP 3189151 Nitrobacter winogradskyi Nb-255
GH15 YP 0012344011 Acidiphilium cryptum JF-5
GH15 YP 0040115781 Rhodomicrobium vannielii ATCC 17100
GH15 YP 0016419391 Methylobacterium extorquens PA1GH15 YP 5252641 Rhodoferax ferrireducens T118
GH15 YP 0041811661 Terriglobus saanensis SP1PR4
GH15 ZP 031333281 Chthoniobacter flavus Ellin428
GH15 CCA537261 Streptomyces venezuelae ATCC 10712
GH15 YP 0015065621 Frankia sp EAN1pec
GH15 YP 0024892701 Arthrobacter chlorophenolicus A6
GH15 YP 0018202091 Opitutus terrae PB90-1
GH15 YP 8729011 Acidothermus cellulolyticus 11B
GH15 YP 0013795591 Anaeromyxobacter sp Fw109-5
GH15 YP 0024929091 Anaeromyxobacter dehalogenans 2CP-1
GH15 YP 6355121 Myxococcus xanthus DK 1622
GH15 YP 0011047051 Saccharopolyspora erythraea NRRL 2338
GH15 YP 0011092191 Saccharopolyspora erythraea NRRL 2338
GH15 AKF10340 Sandaracinus amylolyticus DSM 53668
GH15 YP 6342991 Myxococcus xanthus DK 1622
GH15 YP 0031091351 Acidimicrobium ferrooxidans DSM 10331
GH15 YP 0035703361 Salinibacter ruber M8
GH15 YP 0025241401 Thermomicrobium roseum DSM 5159 plasmid
GH15 YP 0033214441 Sphaerobacter thermophilus DSM 20745
GH15 NP 7336051 Streptomyces coelicolor A3-2
GH15 YP 0035282991 Nitrosococcus halophilus Nc 4
GH15 YP 0043676121 Marinithermus hydrothermalis DSM 14884
GH15 YP 0043687811 Marinithermus hydrothermalis DSM 14884
GH15 YP 0034042871 Haloterrigena turkmenica DSM 5511
GH15 ZP 080461001 Haladaptatus paucihalophilus DX253
GH1
5 YP
023
376
1 Pi
crop
hilu
s to
rrid
us D
SM 9
790
GH1
5 ZP
055
7131
11
Ferr
opla
sma
acid
arm
anus
fer1
GH1
5 BA
B604
801
The
rmop
lasm
a vo
lcan
ium
GSS
1
GH
15 Y
P 00
3323
761
1 Th
erm
obac
ulum
terr
enum
ATC
C B
AA
-798
GH
15 Y
P 00
3899
615
1 C
yano
thec
e sp
PC
C 7
822
plas
mid
GH
15 Y
P 00
3705
927
1 Tr
uepe
ra ra
diov
ictr
ix D
SM 1
7093
GH
15 Y
P 00
2478
443
1 A
rthr
obac
ter c
hlor
ophe
nolic
us A
6 pl
asm
idG
H15
AK
F104
87 S
anda
raci
nus
amyl
olyt
icus
DSM
536
68G
H15
YP
0037
6113
81
Nitr
osoc
occu
s w
atso
nii C
-113
GH1
5 ZP
069
7134
91
Kted
onob
acte
r rac
emife
r DSM
449
63
GH15
ZP
0697
6049
1 K
tedo
noba
cter
race
mife
r DSM
449
63
GH15
YP
0033
9335
81
Cone
xiba
cter
woe
sei D
SM 1
4684
GH15
YP
0042
3629
11 A
cido
vora
x av
enae
sub
sp a
vena
e ATC
C 19
860
GH15 Z
P 03
6273
271
Pedo
spha
era p
arvu
la El
lin51
4
GH15 Y
P 00
1618
7001
Sor
angi
um ce
llulo
sum
So
ce56
GH15 ZP
0145
9561
1 Stig
matella
aura
ntiac
a DW
4-3-1
GH15 ZP
0503
8177
1 Syn
echo
cocc
us sp
PCC 73
35
GH15 YP 64
4038
1 Rubrobac
ter xy
lanophilu
s DSM 99
41
GH15 ZP 064746651 Frankia symbiont of D
atisca glomerata
GH15 ZP 069744041 Ktedonobacter racemife
r DSM 44963
GH15 ZP 069653261 Ktedonobacter racemifer D
SM 44963
GH15 ZP 069661381 Ktedonobacter racemifer D
SM 44963
FIG 4mdashPhylogeny of Sandaracinus amylolyticus GH15 g-amylases
Sandaracinus amylolyticus g-amylases are colored dark pink and myxobac-
terial homologs are colored fluorescent green Bootstrap values corre-
sponding to the tree nodes are provided
Sharma et al GBE
2526 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
were absent in S amylolyticus genome which could be related
to its agar nondegrading property (Mohr et al 2012) as sup-
ported by colony morphology where agar was not penetrated
or liquefied by the bacteria (supplementary fig S5
Supplementary Material online)
Cellulose Degrading Enzymes
We identified only GH5 and GH74 in S amylolyticus proteome
in two and three proteins respectively GH5 domain was pre-
sent as a single unit in AKF04963 and AKF09194 whereas
GH74 was identified in AKF04859 AKF06963 and
AKF09775 associated with a GH74 domain CAZY domains
for cellobiohydrolaseexoglucanases (represented by GH6
GH7 GH9 and GH48) were not present in S amylolyticus
Cellobiohydrolaseexoglucanases plays a significant role in cel-
lulose degradation as they cut two to four sugar subunits from
the exposed ends formed by the endoglucanases which are
used as initial substrates for b-glucosidase (Mba Medie et al
2012) The absence of cellobiohydrolase or exoglucanases
could be a possible reason for the lack of cellulose degradation
in S amylolyticus Out of b-glucosidase representative CAZY
families GH1 GH3 GH5 GH9 GH30 and GH116 only GH1
GH3 and GH5 families were found in three two and two
proteins in S amylolyticus respectively AKF03843 AKF0
6028 and AKF10711 have a GH1 domain whereas
AKF09078 and AKF10875 have only the GH3 domain
Chitin Degrading Enzymes
Chitinases (EC32114) are involved in degradation of chitin
a component of the cell wall of fungi and exoskeleton of
arthropods GH18 GH19 GH23 and GH48 families have
been reported in the CAZY database as chitinases that hydro-
lyse b-14 linkages between N-acetylglucosamine units GH18
is a chitinase family with a characteristic ldquo[LIVM
FY][DN]G[LIVMF][DN][LIVMF][DN]Erdquo motif and is expressed
in archaea prokaryotes and eukaryotes (Funkhouser and
Aronson 2007) GH18 chitinases were not found in S amylo-
lyticus genome while members of GH19 (AKF03006 and
AKF10423) and GH23 (AKF04575 AKF05533 AKF06346
AKF08835 and AKF11377) could be identified GH19
family chitinases are mostly reported to be present in plants
with only a few members in prokaryotes (Kasprzewska 2003
Funkhouser and Aronson 2007) Among the two identified
GH19 homologs we found that AKF10423 is approximately
65 identical (~68 query coverage) with cyanobacteria
such as Nodosilinea nodulosa and Limnoraphis robusta
along with another myxobacterium Chondromyces crocatus
Besides these homologs we also found that AKF10423 shows
maximum similarity with eukaryotic GH19 chitinases counter-
parts as present in Acacia mangium Festuca arundinacea
Triticum aestivum and Leucaena leucocephala (55ndash59 iden-
tity with 70 query coverage) We identified that another
chitinase AKF06346 has GH23 domain along with three
copies of CBM50 which is generally present in the enzymes
cleaving either chitin or peptidoglycan
Conclusion
The complete 1033 Mb circular genome of S amylolyticus
type strain DSM 53668T was determined CAZy analysis re-
vealed the presence of 72 GT domains 72 CE domains 58 GH
domains 19 AA domains 4 PL domains and 26 CBM do-
mains Sixteen S amylolyticus proteins were identified of
which 15 are a-amylases of class GH13 and 1 GH57 Two
g-amylases of GH15 family were also identified Phylogeny
analysis revealed that ten GH13 a-amylases and one GH15
g-amylase have been aquired via horizontal gene transfer
from other bacterial phyla such as Deinococcus-Thermus
Cyanobacteria Alphaproteobacteria Gammaproteobacteria
and Acidobacteria
Table 1
Cellulose Starch Chitin and Agar Degrading Enzymes and Their Representative CAZY Domains Distribution
Substrate Enzyme Category Enzyme Name EC Number CAZY Families (protein repertoire in brackets)
Starch Amylase a-Amylase 3211 GH13 (15) GH57 (1) GH119 GH126
Amylase b-Amylase 3212 GH14
Amylase g-Amylase 3213 GH15 (2) GH97
Chitin Chitinase Chitinase 32114 GH18 GH19 (2) GH23 (5) GH48
Agar a-Agarase Agarose 3-glycanohydrolase 321158 GH96
b-Agarase Agarose 4-glycanohydrolase 32181 GH16 GH50 GH86 GH118
Cellulose Cellulase b-Glucosidase 32121 GH1 (3) GH3 (2) GH5 (2) GH9 GH30 GH116
Cellulase Cellobiohydrolase or Exoglucanase 321176 GH48 GH6 GH7 GH9
Cellulase Endoglucanase 3214 GH5 (2) GH6 GH7 GH8 GH9 GH12 GH44
GH45 GH48 GH51 GH74 (3) GH124
NOTEmdashEC number enzyme category and CAZY families for proteins involved in starch chitin agar and cellulose degradation are shown The number of Sandaracinusamylolyticus proteins identified having CAZY domains are represented in bold fonts in respective families
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2527
Supplementary Material
Supplementary figures S1ndashS5 and tables S1 and S2 are avail-
able at Genome Biology and Evolution online (httpwww
gbeoxfordjournalsorg)
Acknowledgments
This work is supported by a project ldquoExpansion and modern-
ization of Microbial Type Culture Collection and Gene Bank
(MTCC) jointly supported by Council of Scientific and
Industrial Research (CSIR) Grant No BSC0402 and
Department of Biotechnology (DBT) Govt of India Grant No
BTPR7368INF221772012rdquo The authors thank MTCC for
providing help in procuring the cultures from DSMZ They
acknowledge Dr Ramya TNC for providing workbench for cul-
turing myxobacteria GS and IK acknowledge the Council
of Scientific and Industrial Research (CSIR) and University
Grant Commission (UGC) for the research fellowship respec-
tively They also thank the Genome Quebec Innovation
Centre McGill University Canada and C-CAMP next-genera-
tion genomics facility Bangalore India for help in obtaining
PacBio and Illumina NGS data respectively
Literature CitedAlikhan NF Petty NK Ben Zakour NL Beatson SA 2011 BLAST Ring
Image Generator (BRIG) simple prokaryote genome comparisons
BMC Genomics 12402
Altschul SF Gish W Miller W Myers EW Lipman DJ 1990 Basic local
alignment search tool J Mol Biol 215403ndash410
Auch AF Von Jan M Klenk HP Goker M 2010 Digital DNA-DNA hybrid-
ization for microbial species delineation by means of genome-to-
genome sequence comparison Stand Genomic Sci 2117ndash134
Aziz RK et al 2008 The RAST Server rapid annotations using subsystems
technology BMC Genomics 975
Blesak K Janecek S 2012 Sequence fingerprints of enzyme specificities
from the glycoside hydrolase family GH57 Extremophiles 16497ndash
506
Chi WJ Chang YK Hong SK 2012 Agar degradation by microorganisms
and agar-degrading enzymes Appl Microbiol Biotechnol 94917ndash
930
Chin CS et al 2013 Nonhybrid finished microbial genome assemblies
from long-read SMRT sequencing data Nat Methods 10563ndash569
Eddy SR 2011 Accelerated profile HMM searches PLoS Comput Biol
7e1002195
Edgar RC 2004 MUSCLE multiple sequence alignment with high accu-
racy and high throughput Nucleic Acids Res 321792ndash1797
Funkhouser JD Aronson NN Jr 2007 Chitinase family GH18 evolutionary
insights from the genomic history of a diverse protein family BMC Evol
Biol 796
Garcia R Muller R 2014 The family Polyangiaceae In Rosenberg E
Delong E Lory S Stackebrandt E Thompson F editors The prokary-
otes Berlin Heidelberg Springer p 247ndash279
Grissa I Vergnaud G Pourcel C 2007 CRISPRFinder a web tool to identify
clustered regularly interspaced short palindromic repeats Nucleic
Acids Res 35W52ndashW57
Hall TA 1999 BioEdit a user-friendly biological sequence alignment editor
and analysis program for Windows 9598NT Nucleic Acids Symp Ser
4195ndash98
Han K et al 2013 Extraordinary expansion of a Sorangium cellulosum
genome from an alkaline milieu Sci Rep 32101
Janecek S Kuchtova A 2012 In silico identification of catalytic residues
and domain fold of the family GH119 sharing the catalytic machinery
with the alpha-amylase family GH57 FEBS Lett 5863360ndash3366
Janecek S Svensson B Macgregor EA 2014 alpha-Amylase an enzyme
specificity found in various families of glycoside hydrolases Cell Mol
Life Sci 711149ndash1170
Kasprzewska A 2003 Plant chitinasesndashregulation and function Cell Mol
Biol Lett 8809ndash824
Lagesen K et al 2007 RNAmmer consistent and rapid annotation of
ribosomal RNA genes Nucleic Acids Res 353100ndash3108
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B
2014 The carbohydrate-active enzymes database (CAZy) in 2013
Nucleic Acids Res 42D490ndashD495
Lowe TM Eddy SR 1997 tRNAscan-SE a program for improved detection
of transfer RNA genes in genomic sequence Nucleic Acids Res
25955ndash964
Matsuura Y Kusunoki M Harada W Kakudo M 1984 Structure
and possible catalytic residues of Taka-amylase A J Biochem
95697ndash702
Mba Medie F Davies GJ Drancourt M Henrissat B 2012 Genome anal-
yses highlight the different biological roles of cellulases Nat Rev
Microbiol 10227ndash234
Mohr KI Garcia RO Gerth K Irschik H Muller R 2012
Sandaracinus amylolyticus gen nov sp nov a starch-degrading
soil myxobacterium and description of Sandaracinaceae fam
nov Int J Syst Evol Microbiol 621191ndash1198
Noguchi J et al 2011 Crystal structure of the branching enzyme I
(BEI) from Oryza sativa L with implications for catalysis and sub-
strate binding Glycobiology 211108ndash1116
Patel RK Jain M 2012 NGS QC Toolkit a toolkit for quality control of next
generation sequencing data PLoS One 7e30619
Pei J Kim BH Grishin NV PROMALS3D a tool for multiple protein se-
quence and structure alignments Nucleic Acids Res 36 2295ndash2300
Reichenbach H Dworkin M 1992 The myxobacteria In Balows A
Truper HG Dworkin M Harder W Schleifer KH editors The pro-
karyotes a handbook on the biology of bacteria ecophysiology
isolation identification applications New York Springer New
York p 3416ndash3487
Reichenbach H 2005 ldquoOrder VIII Myxococcalesrdquo Bergeyrsquos Manual of
Systematic Bacteriology In Brenner DJ Krieg NR Staley JT Garrity
GM editors Vol 2 2nd ed part C New York Springer-Verlag New
York Inc pp 1074ndash1079
Richter M Rossello-Mora R 2009 Shifting the genomic gold standard for
the prokaryotic species definition Proc Natl Acad Sci U S A 106
19126ndash19131
Schneiker S et al 2007 Complete genome sequence of the myxobacter-
ium Sorangium cellulosum Nat Biotechnol 25 1281ndash1289
Shimkets LJ 1990 Social and developmental biology of the myxobacteria
Microbiol Rev 54 473ndash501
Siguier P Perochon J Lestrade L Mahillon J Chandler M 2006 ISfinder
the reference centre for bacterial insertion sequences Nucleic Acids
Res 34 D32ndashD36
Steinmetz H et al 2012 Indiacens A and B prenyl indoles from the
myxobacterium Sandaracinus amylolyticus J Nat Prod 75 1803ndash
1805
Tamura K Stecher G Peterson D Filipski A Kumar S 2013 MEGA6
molecular evolutionary genetics analysis version 60 Mol Biol Evol
30 2725ndash2729
Van Der Maarel MJ Van Der Veen B Uitdehaag JC Leemhuis H Dijkhuizen
L 2002 Properties and applications of starch-converting enzymes of
the alpha-amylase family J Biotechnol 94 137ndash155
Sharma et al GBE
2528 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
Weissman KJ Muller R 2010 Myxobacterial secondary metabolites
bioactivities and modes-of-action Nat Prod Rep 27 1276ndash1295
Whitworth DE 2008 Myxobacteria multicellularity and differentiation
Washington DC ASM Press American Society for Microbiology
Yin Y et al 2012 dbCAN a web resource for automated carbohydrate-
active enzyme annotation Nucleic Acids Res 40 W445ndashW451
Zhao JY et al 2008 Discovery of the autonomously replicating plasmid
pMF1 from Myxococcus fulvus and development of a gene cloning
system in Myxococcus xanthus Appl Environ Microbiol 74 1980ndash
1987
Associate editor Howard Ochman
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2529
were absent in S amylolyticus genome which could be related
to its agar nondegrading property (Mohr et al 2012) as sup-
ported by colony morphology where agar was not penetrated
or liquefied by the bacteria (supplementary fig S5
Supplementary Material online)
Cellulose Degrading Enzymes
We identified only GH5 and GH74 in S amylolyticus proteome
in two and three proteins respectively GH5 domain was pre-
sent as a single unit in AKF04963 and AKF09194 whereas
GH74 was identified in AKF04859 AKF06963 and
AKF09775 associated with a GH74 domain CAZY domains
for cellobiohydrolaseexoglucanases (represented by GH6
GH7 GH9 and GH48) were not present in S amylolyticus
Cellobiohydrolaseexoglucanases plays a significant role in cel-
lulose degradation as they cut two to four sugar subunits from
the exposed ends formed by the endoglucanases which are
used as initial substrates for b-glucosidase (Mba Medie et al
2012) The absence of cellobiohydrolase or exoglucanases
could be a possible reason for the lack of cellulose degradation
in S amylolyticus Out of b-glucosidase representative CAZY
families GH1 GH3 GH5 GH9 GH30 and GH116 only GH1
GH3 and GH5 families were found in three two and two
proteins in S amylolyticus respectively AKF03843 AKF0
6028 and AKF10711 have a GH1 domain whereas
AKF09078 and AKF10875 have only the GH3 domain
Chitin Degrading Enzymes
Chitinases (EC32114) are involved in degradation of chitin
a component of the cell wall of fungi and exoskeleton of
arthropods GH18 GH19 GH23 and GH48 families have
been reported in the CAZY database as chitinases that hydro-
lyse b-14 linkages between N-acetylglucosamine units GH18
is a chitinase family with a characteristic ldquo[LIVM
FY][DN]G[LIVMF][DN][LIVMF][DN]Erdquo motif and is expressed
in archaea prokaryotes and eukaryotes (Funkhouser and
Aronson 2007) GH18 chitinases were not found in S amylo-
lyticus genome while members of GH19 (AKF03006 and
AKF10423) and GH23 (AKF04575 AKF05533 AKF06346
AKF08835 and AKF11377) could be identified GH19
family chitinases are mostly reported to be present in plants
with only a few members in prokaryotes (Kasprzewska 2003
Funkhouser and Aronson 2007) Among the two identified
GH19 homologs we found that AKF10423 is approximately
65 identical (~68 query coverage) with cyanobacteria
such as Nodosilinea nodulosa and Limnoraphis robusta
along with another myxobacterium Chondromyces crocatus
Besides these homologs we also found that AKF10423 shows
maximum similarity with eukaryotic GH19 chitinases counter-
parts as present in Acacia mangium Festuca arundinacea
Triticum aestivum and Leucaena leucocephala (55ndash59 iden-
tity with 70 query coverage) We identified that another
chitinase AKF06346 has GH23 domain along with three
copies of CBM50 which is generally present in the enzymes
cleaving either chitin or peptidoglycan
Conclusion
The complete 1033 Mb circular genome of S amylolyticus
type strain DSM 53668T was determined CAZy analysis re-
vealed the presence of 72 GT domains 72 CE domains 58 GH
domains 19 AA domains 4 PL domains and 26 CBM do-
mains Sixteen S amylolyticus proteins were identified of
which 15 are a-amylases of class GH13 and 1 GH57 Two
g-amylases of GH15 family were also identified Phylogeny
analysis revealed that ten GH13 a-amylases and one GH15
g-amylase have been aquired via horizontal gene transfer
from other bacterial phyla such as Deinococcus-Thermus
Cyanobacteria Alphaproteobacteria Gammaproteobacteria
and Acidobacteria
Table 1
Cellulose Starch Chitin and Agar Degrading Enzymes and Their Representative CAZY Domains Distribution
Substrate Enzyme Category Enzyme Name EC Number CAZY Families (protein repertoire in brackets)
Starch Amylase a-Amylase 3211 GH13 (15) GH57 (1) GH119 GH126
Amylase b-Amylase 3212 GH14
Amylase g-Amylase 3213 GH15 (2) GH97
Chitin Chitinase Chitinase 32114 GH18 GH19 (2) GH23 (5) GH48
Agar a-Agarase Agarose 3-glycanohydrolase 321158 GH96
b-Agarase Agarose 4-glycanohydrolase 32181 GH16 GH50 GH86 GH118
Cellulose Cellulase b-Glucosidase 32121 GH1 (3) GH3 (2) GH5 (2) GH9 GH30 GH116
Cellulase Cellobiohydrolase or Exoglucanase 321176 GH48 GH6 GH7 GH9
Cellulase Endoglucanase 3214 GH5 (2) GH6 GH7 GH8 GH9 GH12 GH44
GH45 GH48 GH51 GH74 (3) GH124
NOTEmdashEC number enzyme category and CAZY families for proteins involved in starch chitin agar and cellulose degradation are shown The number of Sandaracinusamylolyticus proteins identified having CAZY domains are represented in bold fonts in respective families
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2527
Supplementary Material
Supplementary figures S1ndashS5 and tables S1 and S2 are avail-
able at Genome Biology and Evolution online (httpwww
gbeoxfordjournalsorg)
Acknowledgments
This work is supported by a project ldquoExpansion and modern-
ization of Microbial Type Culture Collection and Gene Bank
(MTCC) jointly supported by Council of Scientific and
Industrial Research (CSIR) Grant No BSC0402 and
Department of Biotechnology (DBT) Govt of India Grant No
BTPR7368INF221772012rdquo The authors thank MTCC for
providing help in procuring the cultures from DSMZ They
acknowledge Dr Ramya TNC for providing workbench for cul-
turing myxobacteria GS and IK acknowledge the Council
of Scientific and Industrial Research (CSIR) and University
Grant Commission (UGC) for the research fellowship respec-
tively They also thank the Genome Quebec Innovation
Centre McGill University Canada and C-CAMP next-genera-
tion genomics facility Bangalore India for help in obtaining
PacBio and Illumina NGS data respectively
Literature CitedAlikhan NF Petty NK Ben Zakour NL Beatson SA 2011 BLAST Ring
Image Generator (BRIG) simple prokaryote genome comparisons
BMC Genomics 12402
Altschul SF Gish W Miller W Myers EW Lipman DJ 1990 Basic local
alignment search tool J Mol Biol 215403ndash410
Auch AF Von Jan M Klenk HP Goker M 2010 Digital DNA-DNA hybrid-
ization for microbial species delineation by means of genome-to-
genome sequence comparison Stand Genomic Sci 2117ndash134
Aziz RK et al 2008 The RAST Server rapid annotations using subsystems
technology BMC Genomics 975
Blesak K Janecek S 2012 Sequence fingerprints of enzyme specificities
from the glycoside hydrolase family GH57 Extremophiles 16497ndash
506
Chi WJ Chang YK Hong SK 2012 Agar degradation by microorganisms
and agar-degrading enzymes Appl Microbiol Biotechnol 94917ndash
930
Chin CS et al 2013 Nonhybrid finished microbial genome assemblies
from long-read SMRT sequencing data Nat Methods 10563ndash569
Eddy SR 2011 Accelerated profile HMM searches PLoS Comput Biol
7e1002195
Edgar RC 2004 MUSCLE multiple sequence alignment with high accu-
racy and high throughput Nucleic Acids Res 321792ndash1797
Funkhouser JD Aronson NN Jr 2007 Chitinase family GH18 evolutionary
insights from the genomic history of a diverse protein family BMC Evol
Biol 796
Garcia R Muller R 2014 The family Polyangiaceae In Rosenberg E
Delong E Lory S Stackebrandt E Thompson F editors The prokary-
otes Berlin Heidelberg Springer p 247ndash279
Grissa I Vergnaud G Pourcel C 2007 CRISPRFinder a web tool to identify
clustered regularly interspaced short palindromic repeats Nucleic
Acids Res 35W52ndashW57
Hall TA 1999 BioEdit a user-friendly biological sequence alignment editor
and analysis program for Windows 9598NT Nucleic Acids Symp Ser
4195ndash98
Han K et al 2013 Extraordinary expansion of a Sorangium cellulosum
genome from an alkaline milieu Sci Rep 32101
Janecek S Kuchtova A 2012 In silico identification of catalytic residues
and domain fold of the family GH119 sharing the catalytic machinery
with the alpha-amylase family GH57 FEBS Lett 5863360ndash3366
Janecek S Svensson B Macgregor EA 2014 alpha-Amylase an enzyme
specificity found in various families of glycoside hydrolases Cell Mol
Life Sci 711149ndash1170
Kasprzewska A 2003 Plant chitinasesndashregulation and function Cell Mol
Biol Lett 8809ndash824
Lagesen K et al 2007 RNAmmer consistent and rapid annotation of
ribosomal RNA genes Nucleic Acids Res 353100ndash3108
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B
2014 The carbohydrate-active enzymes database (CAZy) in 2013
Nucleic Acids Res 42D490ndashD495
Lowe TM Eddy SR 1997 tRNAscan-SE a program for improved detection
of transfer RNA genes in genomic sequence Nucleic Acids Res
25955ndash964
Matsuura Y Kusunoki M Harada W Kakudo M 1984 Structure
and possible catalytic residues of Taka-amylase A J Biochem
95697ndash702
Mba Medie F Davies GJ Drancourt M Henrissat B 2012 Genome anal-
yses highlight the different biological roles of cellulases Nat Rev
Microbiol 10227ndash234
Mohr KI Garcia RO Gerth K Irschik H Muller R 2012
Sandaracinus amylolyticus gen nov sp nov a starch-degrading
soil myxobacterium and description of Sandaracinaceae fam
nov Int J Syst Evol Microbiol 621191ndash1198
Noguchi J et al 2011 Crystal structure of the branching enzyme I
(BEI) from Oryza sativa L with implications for catalysis and sub-
strate binding Glycobiology 211108ndash1116
Patel RK Jain M 2012 NGS QC Toolkit a toolkit for quality control of next
generation sequencing data PLoS One 7e30619
Pei J Kim BH Grishin NV PROMALS3D a tool for multiple protein se-
quence and structure alignments Nucleic Acids Res 36 2295ndash2300
Reichenbach H Dworkin M 1992 The myxobacteria In Balows A
Truper HG Dworkin M Harder W Schleifer KH editors The pro-
karyotes a handbook on the biology of bacteria ecophysiology
isolation identification applications New York Springer New
York p 3416ndash3487
Reichenbach H 2005 ldquoOrder VIII Myxococcalesrdquo Bergeyrsquos Manual of
Systematic Bacteriology In Brenner DJ Krieg NR Staley JT Garrity
GM editors Vol 2 2nd ed part C New York Springer-Verlag New
York Inc pp 1074ndash1079
Richter M Rossello-Mora R 2009 Shifting the genomic gold standard for
the prokaryotic species definition Proc Natl Acad Sci U S A 106
19126ndash19131
Schneiker S et al 2007 Complete genome sequence of the myxobacter-
ium Sorangium cellulosum Nat Biotechnol 25 1281ndash1289
Shimkets LJ 1990 Social and developmental biology of the myxobacteria
Microbiol Rev 54 473ndash501
Siguier P Perochon J Lestrade L Mahillon J Chandler M 2006 ISfinder
the reference centre for bacterial insertion sequences Nucleic Acids
Res 34 D32ndashD36
Steinmetz H et al 2012 Indiacens A and B prenyl indoles from the
myxobacterium Sandaracinus amylolyticus J Nat Prod 75 1803ndash
1805
Tamura K Stecher G Peterson D Filipski A Kumar S 2013 MEGA6
molecular evolutionary genetics analysis version 60 Mol Biol Evol
30 2725ndash2729
Van Der Maarel MJ Van Der Veen B Uitdehaag JC Leemhuis H Dijkhuizen
L 2002 Properties and applications of starch-converting enzymes of
the alpha-amylase family J Biotechnol 94 137ndash155
Sharma et al GBE
2528 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
Weissman KJ Muller R 2010 Myxobacterial secondary metabolites
bioactivities and modes-of-action Nat Prod Rep 27 1276ndash1295
Whitworth DE 2008 Myxobacteria multicellularity and differentiation
Washington DC ASM Press American Society for Microbiology
Yin Y et al 2012 dbCAN a web resource for automated carbohydrate-
active enzyme annotation Nucleic Acids Res 40 W445ndashW451
Zhao JY et al 2008 Discovery of the autonomously replicating plasmid
pMF1 from Myxococcus fulvus and development of a gene cloning
system in Myxococcus xanthus Appl Environ Microbiol 74 1980ndash
1987
Associate editor Howard Ochman
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2529
Supplementary Material
Supplementary figures S1ndashS5 and tables S1 and S2 are avail-
able at Genome Biology and Evolution online (httpwww
gbeoxfordjournalsorg)
Acknowledgments
This work is supported by a project ldquoExpansion and modern-
ization of Microbial Type Culture Collection and Gene Bank
(MTCC) jointly supported by Council of Scientific and
Industrial Research (CSIR) Grant No BSC0402 and
Department of Biotechnology (DBT) Govt of India Grant No
BTPR7368INF221772012rdquo The authors thank MTCC for
providing help in procuring the cultures from DSMZ They
acknowledge Dr Ramya TNC for providing workbench for cul-
turing myxobacteria GS and IK acknowledge the Council
of Scientific and Industrial Research (CSIR) and University
Grant Commission (UGC) for the research fellowship respec-
tively They also thank the Genome Quebec Innovation
Centre McGill University Canada and C-CAMP next-genera-
tion genomics facility Bangalore India for help in obtaining
PacBio and Illumina NGS data respectively
Literature CitedAlikhan NF Petty NK Ben Zakour NL Beatson SA 2011 BLAST Ring
Image Generator (BRIG) simple prokaryote genome comparisons
BMC Genomics 12402
Altschul SF Gish W Miller W Myers EW Lipman DJ 1990 Basic local
alignment search tool J Mol Biol 215403ndash410
Auch AF Von Jan M Klenk HP Goker M 2010 Digital DNA-DNA hybrid-
ization for microbial species delineation by means of genome-to-
genome sequence comparison Stand Genomic Sci 2117ndash134
Aziz RK et al 2008 The RAST Server rapid annotations using subsystems
technology BMC Genomics 975
Blesak K Janecek S 2012 Sequence fingerprints of enzyme specificities
from the glycoside hydrolase family GH57 Extremophiles 16497ndash
506
Chi WJ Chang YK Hong SK 2012 Agar degradation by microorganisms
and agar-degrading enzymes Appl Microbiol Biotechnol 94917ndash
930
Chin CS et al 2013 Nonhybrid finished microbial genome assemblies
from long-read SMRT sequencing data Nat Methods 10563ndash569
Eddy SR 2011 Accelerated profile HMM searches PLoS Comput Biol
7e1002195
Edgar RC 2004 MUSCLE multiple sequence alignment with high accu-
racy and high throughput Nucleic Acids Res 321792ndash1797
Funkhouser JD Aronson NN Jr 2007 Chitinase family GH18 evolutionary
insights from the genomic history of a diverse protein family BMC Evol
Biol 796
Garcia R Muller R 2014 The family Polyangiaceae In Rosenberg E
Delong E Lory S Stackebrandt E Thompson F editors The prokary-
otes Berlin Heidelberg Springer p 247ndash279
Grissa I Vergnaud G Pourcel C 2007 CRISPRFinder a web tool to identify
clustered regularly interspaced short palindromic repeats Nucleic
Acids Res 35W52ndashW57
Hall TA 1999 BioEdit a user-friendly biological sequence alignment editor
and analysis program for Windows 9598NT Nucleic Acids Symp Ser
4195ndash98
Han K et al 2013 Extraordinary expansion of a Sorangium cellulosum
genome from an alkaline milieu Sci Rep 32101
Janecek S Kuchtova A 2012 In silico identification of catalytic residues
and domain fold of the family GH119 sharing the catalytic machinery
with the alpha-amylase family GH57 FEBS Lett 5863360ndash3366
Janecek S Svensson B Macgregor EA 2014 alpha-Amylase an enzyme
specificity found in various families of glycoside hydrolases Cell Mol
Life Sci 711149ndash1170
Kasprzewska A 2003 Plant chitinasesndashregulation and function Cell Mol
Biol Lett 8809ndash824
Lagesen K et al 2007 RNAmmer consistent and rapid annotation of
ribosomal RNA genes Nucleic Acids Res 353100ndash3108
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B
2014 The carbohydrate-active enzymes database (CAZy) in 2013
Nucleic Acids Res 42D490ndashD495
Lowe TM Eddy SR 1997 tRNAscan-SE a program for improved detection
of transfer RNA genes in genomic sequence Nucleic Acids Res
25955ndash964
Matsuura Y Kusunoki M Harada W Kakudo M 1984 Structure
and possible catalytic residues of Taka-amylase A J Biochem
95697ndash702
Mba Medie F Davies GJ Drancourt M Henrissat B 2012 Genome anal-
yses highlight the different biological roles of cellulases Nat Rev
Microbiol 10227ndash234
Mohr KI Garcia RO Gerth K Irschik H Muller R 2012
Sandaracinus amylolyticus gen nov sp nov a starch-degrading
soil myxobacterium and description of Sandaracinaceae fam
nov Int J Syst Evol Microbiol 621191ndash1198
Noguchi J et al 2011 Crystal structure of the branching enzyme I
(BEI) from Oryza sativa L with implications for catalysis and sub-
strate binding Glycobiology 211108ndash1116
Patel RK Jain M 2012 NGS QC Toolkit a toolkit for quality control of next
generation sequencing data PLoS One 7e30619
Pei J Kim BH Grishin NV PROMALS3D a tool for multiple protein se-
quence and structure alignments Nucleic Acids Res 36 2295ndash2300
Reichenbach H Dworkin M 1992 The myxobacteria In Balows A
Truper HG Dworkin M Harder W Schleifer KH editors The pro-
karyotes a handbook on the biology of bacteria ecophysiology
isolation identification applications New York Springer New
York p 3416ndash3487
Reichenbach H 2005 ldquoOrder VIII Myxococcalesrdquo Bergeyrsquos Manual of
Systematic Bacteriology In Brenner DJ Krieg NR Staley JT Garrity
GM editors Vol 2 2nd ed part C New York Springer-Verlag New
York Inc pp 1074ndash1079
Richter M Rossello-Mora R 2009 Shifting the genomic gold standard for
the prokaryotic species definition Proc Natl Acad Sci U S A 106
19126ndash19131
Schneiker S et al 2007 Complete genome sequence of the myxobacter-
ium Sorangium cellulosum Nat Biotechnol 25 1281ndash1289
Shimkets LJ 1990 Social and developmental biology of the myxobacteria
Microbiol Rev 54 473ndash501
Siguier P Perochon J Lestrade L Mahillon J Chandler M 2006 ISfinder
the reference centre for bacterial insertion sequences Nucleic Acids
Res 34 D32ndashD36
Steinmetz H et al 2012 Indiacens A and B prenyl indoles from the
myxobacterium Sandaracinus amylolyticus J Nat Prod 75 1803ndash
1805
Tamura K Stecher G Peterson D Filipski A Kumar S 2013 MEGA6
molecular evolutionary genetics analysis version 60 Mol Biol Evol
30 2725ndash2729
Van Der Maarel MJ Van Der Veen B Uitdehaag JC Leemhuis H Dijkhuizen
L 2002 Properties and applications of starch-converting enzymes of
the alpha-amylase family J Biotechnol 94 137ndash155
Sharma et al GBE
2528 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016
Weissman KJ Muller R 2010 Myxobacterial secondary metabolites
bioactivities and modes-of-action Nat Prod Rep 27 1276ndash1295
Whitworth DE 2008 Myxobacteria multicellularity and differentiation
Washington DC ASM Press American Society for Microbiology
Yin Y et al 2012 dbCAN a web resource for automated carbohydrate-
active enzyme annotation Nucleic Acids Res 40 W445ndashW451
Zhao JY et al 2008 Discovery of the autonomously replicating plasmid
pMF1 from Myxococcus fulvus and development of a gene cloning
system in Myxococcus xanthus Appl Environ Microbiol 74 1980ndash
1987
Associate editor Howard Ochman
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2529
Weissman KJ Muller R 2010 Myxobacterial secondary metabolites
bioactivities and modes-of-action Nat Prod Rep 27 1276ndash1295
Whitworth DE 2008 Myxobacteria multicellularity and differentiation
Washington DC ASM Press American Society for Microbiology
Yin Y et al 2012 dbCAN a web resource for automated carbohydrate-
active enzyme annotation Nucleic Acids Res 40 W445ndashW451
Zhao JY et al 2008 Discovery of the autonomously replicating plasmid
pMF1 from Myxococcus fulvus and development of a gene cloning
system in Myxococcus xanthus Appl Environ Microbiol 74 1980ndash
1987
Associate editor Howard Ochman
Complete Genome of a Starch-Degrading Myxobacteria GBE
Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2529
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