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fmicb-09-01033 May 18, 2018 Time: 15:56 # 1

ORIGINAL RESEARCHpublished: 18 May 2018

doi: 10.3389/fmicb.2018.01033

Edited by:Robert Czajkowski,

University of Gdansk, Poland

Reviewed by:Jessica Labonté,

Texas A&M University at Galveston,United States

Konstantin Anatolievich Miroshnikov,Institute of Bioorganic Chemistry

(RAS), Russia

*Correspondence:Francisco Rodriguez-Valera

[email protected]

†These authors have contributedequally to this work.

Specialty section:This article was submitted to

Virology,a section of the journal

Frontiers in Microbiology

Received: 13 March 2018Accepted: 01 May 2018Published: 18 May 2018

Citation:Fernández-Ruiz I, Coutinho FH and

Rodriguez-Valera F (2018) Thousandsof Novel Endolysins Discoveredin Uncultured Phage Genomes.

Front. Microbiol. 9:1033.doi: 10.3389/fmicb.2018.01033

Thousands of Novel EndolysinsDiscovered in Uncultured PhageGenomesIris Fernández-Ruiz†, Felipe H. Coutinho† and Francisco Rodriguez-Valera*

Evolutionary Genomics Group, Departamento de Producción Vegetal y Microbiología, Universidad Miguel Hernándezde Elche, San Juan de Alicante, Spain

Bacteriophages express endolysins toward the end of their replication cycle to degradethe microbial cell wall from within, allowing viral progeny to be released. Endolysins canalso degrade the prokaryotic cell wall from the outside, thus have potential to be usedfor biotechnological and medical purposes. Multiple endolysins have been identifiedwithin the genomes of isolated phages, but their diversity in uncultured phages hasbeen overlooked. We used a bioinformatics pipeline to identify novel endolysins fromnearly 200,000 uncultured viruses. We report the discovery of 2,628 putative endolysins,many of which displayed novel domain architectures. In addition, several of the identifiedproteins are predicted to be active against genera that include pathogenic bacteria.These discoveries enhance the diversity of known endolysins and are a stepping stonefor developing medical and biotechnological applications that rely on bacteriophages,the most diverse biological entities on Earth.

Keywords: bacteriophage, endolysins, metagenomics, biotechnology, protein discovery

INTRODUCTION

Bacteriophages (Phages) have evolved a multitude of endolysins for the purpose of degrading thecomplex cell wall structures of their bacterial hosts (Pimentel, 2014). Despite sharing a commonfunction, endolysins are a diverse class of enzymes with a multitude of action mechanisms andprotein architectures (Oliveira et al., 2013; Vidová et al., 2014). Protein domains within endolysinsgrant them their host specificity and efficiency (Hermoso et al., 2007). Multiple classes of endolysinshave been described and classified according to the specific enzymatic activities of their catalyticdomains, including glycosidases, amidases, and carboxy/endo-peptidase (Schmelcher et al., 2012).Endolysins of phages that infect Gram-positive bacteria often have a modular structure thatincludes one enzymatic catalytic domain (ECD) at the N-terminal and at least one cell wall bindingdomain (CBD) at the C-terminal portion of the protein connected by a flexible interdomain linker(Nelson et al., 2012). Meanwhile endolysins of phages that infect Gram-negative bacteria oftenexhibit a globular structure that contains a single catalytic domain (Pohane and Jain, 2015).

Some endolysins can degrade the cell wall from the outside, which grants them potentialto be used for biotechnological and medical applications (Nelson et al., 2012; Gutiérrez et al.,2018). The ongoing crisis of antibiotic based treatments calls for alternative strategies for fightingbacterial infections (Ventola, 2015) and endolysins have multiple advantages over antibiotics:they have higher target specificity and so far no forms of resistance have been reported (Nelsonet al., 2012). Furthermore, endolysins can be engineered to alter their host range and efficiency(Díez-Martínez et al., 2014; Blázquez et al., 2016). Several recombinant endolysins are nowin preliminary study phase for use in human and veterinary medicine, with promising results

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Fernández-Ruiz et al. Environmental Endolysins

for the treatment against both Gram-positive and Gram-negativebacteria (Briers et al., 2014; Cooper et al., 2016). Some endolysinscan even combat intracellular human pathogens (Shen et al.,2016), demonstrating that their potential applicabilities are widerthan originally conceived. Finally, these enzymes can be clonedinto expression vectors for large scale synthesis. Therefore thepotential applications of endolysins are not only of medical butalso of industrial (e.g., detecting food-borne pathogens) andagricultural relevance (e.g., treatment against phytopathogens)(Schmelcher and Loessner, 2016).

Despite the recognized diversity and potential of endolysins,our current understanding of these enzymes is limited. Analysisof sequence repositories suggests that less than 1,000 of theseproteins are currently known (Oliveira et al., 2013). Althoughprevious studies have characterized the diversity of endolysinsthrough bioinformatic approaches, they focused on referencegenomes of isolated phages and prophages, and overlookedenvironmental phages that have not yet been isolated or cultured(Oliveira et al., 2013; Vidová et al., 2014). Thus, currentlyavailable catalogs of endolysins do not cover the diversity ofenzymes encoded in the genomes of the many uncultured phagesspread across Earth’s many ecosystems. Culture independentapproaches have revolutionized our understanding of phagegenetic diversity, revealing thousands of phage genomes andentirely novel evolutionary lineages at an unprecedented scale(Mizuno et al., 2013; Roux et al., 2015; Paez-Espino et al.,2016; Yutin et al., 2017). These novel genomes are a richresource for the discovery of endolysins that could haveunique domain architectures and target hosts for which noendolysins are currently known. Thus, we sought to screenthe genomes of bacteriophages discovered through cultureindependent approaches to expand the known repertoire ofendolysins, asses their structural diversity, and determine howthis diversity changes across targeted hosts and ecosystems.

MATERIALS AND METHODS

A database of uncultured viral genomes was compiled frompublications aimed at large scale discovery of phages withoutculturing (Mizuno et al., 2013, 2016; Roux et al., 2015,2016; Paez-Espino et al., 2016; Coutinho et al., 2017). Thisdataset comprised 183,298 genomic sequences (SupplementaryFile S1) of uncultured viral genomes adding up to 2.9 Gbpof raw data (Supplementary Table S1). We also retrievedavailable metadata associated with those sequences regardingthe ecosystems from which they originated, and the predictedhosts reported in the original publications. These studies usedmultiple strategies to assign hosts to metagenomic contigswhich included: high genomic similarity to reference phagegenomes; homology matches between phage and prokaryoticgenomes; CRISPR spacers from prokaryotic genomes matchingmetagenomic contigs; similarity between phage and prokaryotictRNAs; and co-occurrence of phage genome pairs indicative ofa shared host. Prophages described by Roux et al. (2015) wereassigned hosts according to the prokaryotic genomes in whichthey were identified.

A dataset of bona fide endolysin sequences encoded ingenomes of double stranded DNA phages was compiled tobe used as a reference database. This database comprised 629proteins from NCBI RefSeq phages and was manually curatedso to exclude structural lysins (i.e., exolysins) (Oliveira et al.,2013). Prodigal (Hyatt et al., 2010) was run in metagenomicmode to identify protein encoding genes of uncultured phagegenomes. All predicted protein sequences were queried againstthe reference endolysin database using Diamond (Buchfinket al., 2015). Proteins that had hits to the reference databasewere classified as putative endolysins if matches were withinthe following thresholds: identity ≥ 50%, e-value ≤ 0.001,query coverage ≥ 30%, and alignment length ≥ 50 aminoacids. Protein domains of putative endolysins were identified byquerying sequences against the Pfam database using HMMERversion v3.1b2 (Finn et al., 2015) with default parameters.Additionally, putative endolysins were analyzed through SignalIP(Petersen et al., 2011) to detect signal peptide sequences. Finally,both the putative and reference endolysins were clustered intoorthologous groups (OGs) using OrthoMCL (Li et al., 2003)within the GET_HOMOLOGUES pipeline (Contreras-Moreiraand Vinuesa, 2013) by setting an inflation factor of 1 and all otherparameters set to default. Multiple sequence alignments wereconstructed through Clustal Omega (Sievers et al., 2014) for eachOG represented by at least 10 proteins. Alignments were usedto perform phylogenetic reconstructions through FastTree (Priceet al., 2010) using default parameters (Amino acid distancesBLOSUM45 and Jones-Taylor-Thorton model and support valuecalculation). An additional tree was built based on a multiplealignment of all proteins assigned to OGs of 10 or more proteins.

RESULTS AND DISCUSSION

A total of 2,628 putative endolysins were identified(Supplementary File S2). Homolog identification clusteredthese proteins into 297 OGs. We focused downstream analysison 46 OGs represented by 10 or more proteins. Each ofthese OGs was manually validated as true endolysins byinspecting for: presence of bona fide endolysins from thereference database; prevalence of typical endolysins domains(e.g., Lysozyme, Amidase, and Glycosyl Hydrolase); and closephylogenetic relationship between putative and bona fideendolysins as assessed by inspecting phylogenetic trees generatedfor each OG. Considering the degree of diversity among phagegenomes and the rate in which their genes evolve, we usedvery conservative thresholds to identify putative endolysins(identity ≥ 50%, e-value ≤ 0.001, query coverage ≥ 30% andalignment length ≥ 50 amino acids). Yet, due to high levels ofstructural similarities between structural lysins and endolysins,it is possible that some of the putative endolysins identified areactually structural lysins (enzymes used by phages to breach thecell wall at the beginning of the infection process). Yet, using acurated database of bona fide endolysins from reference phagegenomes, conservative thresholds, and manual validation of theOGs should minimize the occurrence of false positives in ourdataset.





fmicb-09-01033 May 18, 2018 Time: 15:56 # 3


Proteins assigned to the same OG often displayed identicaldomain architectures, although some exceptions were observed(Figure 1 and Supplementary File S3). A total of 62 domainswere identified across 46 OGs (Table 1), including 26 types ofECDs, 12 CBDs, and 24 domains of unknown function. For 27OGs only a single catalytic domain was observed. Meanwhile, 19OGs harbored at least one protein with both cell wall bindingand ECDs. Signal peptide sequences were detected only in sixOGs, suggesting that most of the discovered endolysins rely onother mechanisms for membrane translocation such as holin-dependent transportation. The most frequent domain observedin the putative endolysins was phage_lysozyme (PF00959) aGlycoside Hydrolase, followed by Peptidase_M15 (PF08291), andAmidase_2 (PF01510).

We investigated associations between OGs and predictedhosts of uncultured phages. A total of 32 OGs had at leastone endolysin derived from a phage genome for which hostprediction was available. Endolysins from OGs predicted totarget Gram-negatives were often composed of a single ECD,while those derived from OGs predicted to target Gram-positive bacteria were often composed of both a CBD andan ECD. Often a single bacterial genus could be targeted

by multiple OGs (Figure 2). For example, enzymes targetingStreptococcus were present in nine distinct OGs and thosetargeting Bacillus were found in four OGs. Most OGs arepredicted to be active against multiple bacterial genera withonly two exceptions: OG44 and OG26 which respectively targetStreptococcus and Bacillus. In general, OGs predicted to targetmultiple taxa were restricted to organisms with the same gramstaining patterns. Interestingly endolysins from phages predictedto infect Mycobacterium were all assigned to a single group(OG6), likely due to the unique cell wall composition of theseorganisms.

A total of 34 OGs harbor at least one endolysin predictedto be active against genera that include potentially pathogenicbacteria (e.g., Staphylococcus, Clostridium, and Klebsiella). Tenof those OGs have at least one protein predicted to targetbacteria currently classified by the World Health Organization ascritical priority for the development of alternative therapies. Inaddition, five OGs include proteins predicted to be active againstphytopathogens (e.g., Xylella, Erwinina, and Burkholderia)(Mansfield et al., 2012; Blomme et al., 2017). In vivo experimentsare necessary to confirm the efficiency of these proteins forfighting pathogens, since different species or strains of bacteria

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Peptidase_M15

VanY

Peptidase_M15

Pha

ge_l

ysoz

yme

Peptidase_M15

Peptidase_M15

Glyco_hydro

Glyco_hydro

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Glyco_hydro

Peptidase_M15

Peptidase_M15

Glyco_hydroPG_binding

Glyco_hydro

Peptidase_M15

Glyco_hydro

Peptidase_M15

Amidase

Phag

e_ly

sozy

me

Peptidase_M15

Amidase

Peptidase_M15

Amidase

Amidase

Peptidase_M15

Peptidase_M15

Peptidase_M15

Phage_lysozyme

Peptidase_M15

Peptidase_M15

SLT

Phage_lysozyme

SLT

Phage_lysozyme

Glyco_hydro

Mur

aida

sePG

_bin

ding

PG_b

indi

ng

PG_b

indi

ng

Glyco_hydro

Peptidase_M15

Peptidase_M15

Peptidase_M15

Phage_lysozyme

Peptidase_M15

TcpQ

Glyco_hydro

Mur

aida

sePG

_bin

ding

Phage_lysozyme

Phage_lysozyme

DU

F4691

Amidase

Amidase

Phag

e_ly

sozy

me

Amidase

Peptidase_M15

Peptidase_M15

Phag

e_ly

sozy

me

Amidase

Phag

e_lys

ozym

e

Amidase

Pept

idas

e_M

15Va

nY


Glyco_hydro

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Amidase

Peptidase_M15

GlucosaminidasePeptidase_M15

Phag

e_ly

sozy

me

Peptidase_M15

Peptidase_M15

Peptidase_M15

Phag

e_ly

sozy

me


Amidase

Phag

e_ly

sozy

me

Peptidase_M15

Peptidase_M15

Peptidase_M15

SLT

Phag

e_ly

sozy

me

Amidase

Amidase

Peptidase_M15

Amidase

Amidase

Peptidase_M15

Peptidase_M15

Amidase

Peptidase_M15

Peptidase_M15

Phag

e_ly

sozy

me

Phag

e_ly

sozy

me

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Phag

e_lys

ozym

e

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Amidase

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Amidase

Peptidase_M15

Phag

e_ly

sozy

me

Peptidase_M15

Phag

e_ly

sozy

me

Peptidase_M15

Peptidase_M15

Amidase

Peptidase_M15

Phage_lysozyme

Amidase

Amidase

Phag

e_ly

sozy

me

Phag

e_ly

sozy

me

Phag

e_ly

sozy

me

Peptidase_M15

Peptidase_M15

Peptidase_M15

Phag

e_ly

sozy

me

Glyco_

hydro

Glyco_hydro

Phag

eMin

_Tai

l

SLT

Pept

idas

e_M

23

Peptidase_M23

Phage_lysozyme

Amidase

SH3

Am

idaseS

H3

SH

3S

H3

SH

3

Amid

ase0

2_CA

mid

ase

esad

im

A

AmidasePG_bindingPG_bindingPG_binding

esad

im

Aes

adi

mA

esad

im

Aes

adi

mA

Peptidase_M15

Phage_lysozyme

Phage_lysozym

e

Phage_lysozyme

Mur

aida

sePG

_bin

ding

PG_b

indi

ng

PG_b

indi

ng

Phage_lysozyme

Glyco_hydroPeptidase_M15 Glyco_hydroPeptidase_M15

Glyco_hydro

PG_bindingGlyco_hydroPG_bindingGlyco_hydroPG_binding

Glyco_hydro

PG_bindingGlyco_hydroPG_bindingPG_binding


Glyco_hydro

Peptidase_M15

Phage_lysozyme

Glyco_hydro

Phage_lysozyme

Glyco_hydro


Glucos

amini

dase

Glyco_hydro

Glyco_hydro

Glyco_hydro


Glyco_hydro

PG_bindingGlyco_hydroPG_bindingPG_bindingGlyco_hydro

Glyco_hydro

Peptidase_M15

Glyco_hydro

Phage_lysozyme

Glyco_hydro

Glyco_hydro

Phage_lysozyme

Glyco_hydro

Glyco_hydro

Pep

tidas

e_M

15Va

nY

Peptidase_M15

VanY

Peptidase_M15

Mur

aida

sePG

_bin

ding

PG_b

indi

ngPG

_bin

ding

Peptidase_M15

Pep

tidas

e_M

15Va

nY

Peptidase_M15

Phage_lysozyme


Phag

e_ly

sozy

me

emyzosyl_egah

P

Phage_lysozyme

LysM

LysM

LysM

Glyc

o_hy

dro

esad

im

APG

_bindingP

G_binding

PG

_bindingP

G_binding

PG

_binding

Phag

e_ly

sozy

me

Mur

aida

sePG

_bin

ding

PG_b

indi

ng

Amidase

SH3

Amidase

SH3

Amidase

SH3

SH3

Glyco_hydro

Mur

aida

sePG

_bin

ding

PG_b

indi

ng

Mur

aida

sePG

_bin

ding

PG_b

indi

ng

Peptidase_M15


15

Amidase

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Glyco_hydro

51M_esaditpe

Pgnidnib_

GP

VanY

gnidnib_G

Pgnidnib_

GP

Peptidase_M15

Amidase

Glyco_hydro

emy

zosy

l_eg

ahP

Glyco_hydro

Glyco_hydro

Glyco_hydro

51M_esaditpe

PVa

nY

emyzosyl_egah

Pe

myzosyl_egahP

Glyco_hydro

Glyco_hydro


Glyco_hydro

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Phage_lysozyme


Peptidase_M15

Peptidase_M15VanY

Peptidase_M15

Peptidase_M15

Glyco_hydroPeptidase_M15 Glyco_hydroPeptidase_M15

Peptidase_M15

GlucosaminidasePeptidase_M15

Glucosaminidase

Phage_lysozyme

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

emy

zosy

l_eg

ahP

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Amidase

Peptidase_M15

Peptidase_M15

Amidase

Amidase

Peptidase_M15

Peptidase_M15

emy

zosy

l_eg

ahP

Mur

aida

sePG

_bin

ding

Mur

aida

sePG

_bin

ding

Mur

aida

sePG

_bin

ding

Glyco_hydro

emyzosyl_egah

P Pha

ge_l

ysoz

yme

Glyco_hydro

Glyco_hydro

51M_esaditpe

PVa

nY

emy

zosy

l_eg

ahP

emyzosyl_egah

Pe

myzosyl_egahP

Glyco_hydro

Peptidase_M15

Peptidase_M15

Glyco_hydro

Phage_lysozyme

Phage_lysozyme

Mur

aida

sePG

_bin

ding

gnidnib_G

Pgnidnib_

GP

51M_esaditpe

PYnaV

gnidnib_G

Pgnidnib_

GP

gnidnib_G

Pgnidnib_

GP

Amidase

AmidaseAmidase02_C

Amidase

SH3

SH3

SH3

SH3

esadim

Agnidnib_G

P

Phage_lysozyme e

myzo

syl_

egah

P

Glyco_hydro

Phage_lysozyme

Phage_lysozyme

Pha

ge_l

ysoz

ymePhage_lysozym

e

Peptid

ase_

C39

Glyco_hydro

Glyco_hydro_cc

SH3CHAP

SH3

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

Kelch

Amidase

ZoocinA_TRDCHAP

SH3CHAP

SH3

Amidase

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ingNLP

C_P60

CHAP

CHAP

AmidaseSH3 NLPC_P60

CHAP

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingCHAP

Glyc

o_hy

dro

CHAP

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

SLT

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

Phag

e_ly

sozy

me

PG_b

indi

ng

Peptidase_M15

Peptidase_M15

Glyco_hydro

CW_binding

CW_binding

CW_binding

CW_bindingCHAP

Glyco_

hydr

o

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

ZoocinA_TRDAmidase

NLPC_P60

Amidase

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ingNLP

C_P60

CHAPZoocin

A_TRDAmidase

NLPC_P60

Amid

ase

Peptidase_M15

AmidaseSH3 CHAP

CHAP

SH3

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingNLPC_P60

SLT

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingCHAP

Amid

ase

Zooc

inA_

TRD

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingCHAP

Amida

se

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

NLPC_P

60

CHAP

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

Glyco_

hydr

o

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amid

ase

Zooc

inA_

TRD

Hin

t

SH3CHAP

SH3

CHAP

Peptidase_M15

Peptidase_M15

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingNLPC_P60

CHAP

Amidase

ZoocinA_TRDCHAPNLPC_P60

CHAP

SH3Peptidase_M15


Peptidase_M15

ZoocinA_TRDAmidase

NLPC_P60

SH3CHAP

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingCHAP

Amida

se

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

NLPC_P

60

CHAP

Zooc

inA_

TRDA

mid

ase

Glyc

o_hy

dro

CHAP

ZoocinA_TRDAmidase

NLPC_P60

ZoocinA_TRDAmidase

ZoocinA_TRDAmidase

NLPC_P60Peptidase_M15

Peptidase_M15

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CHAP

NLPC_P60

Phag

e_ly

sozy

me

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

HH_signal

Amidase


Glyc

o_hy

dro

CHAP

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingNLPC_P60

CHAP

AmidaseNa_Ca_ex_C

Peptidase_M15

Peptidase_M15

HH_signal

Glyco_

hydr

o

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amid

ase

Peptidase_M15

Peptidase_M15

HH_signal

CW

CW

esad

im

AS

H3

Amidase

AmidaseNa_Ca_ex_C

Peptidase_M15

Peptidase_M15

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingNLPC_P60

CHAP

Peptidase_M15

Extensin-like_C

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingCHAP

Glyco_hydro

Amidase


CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

Peptidase_M15

Peptidase_M15

ZoocinA_TRDAmidase

NLPC_P60

Amidase

ZoocinA_TRDCHAP

SH3CHAP

CHAPSH3

Amidase

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ingNLP

C_P60

CHAP

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

Glyc

o_hy

dro

CHAP

Zooc

inA_

TRDA

mid

ase

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingNLPC_P60

Glyc

o_hy

dro

CHAP

ZoocinA_TRDAmidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingCHAP

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingCHAPCW_binding

CW_binding

CW_binding

CW_binding

CW_binding

Glyc

o_hy

dro

CHAP

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CHAP

NLPC_P60

CHAPSH3

Glyco_hydro

SH3CHAP Peptidase_M15

Peptidase_M15

Extensin-like_CPeptidase_M15

Peptidase_M15

HH_signal

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ingAmida

se

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

SH3CHAP

SH3

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

Glyco_hydro

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

Peptidase_M15

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingCHAP

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amida

se

Peptidase_M15

Peptidase_M15

Glyco_hydro

CHAP

SH3

SH3CHAP

SH3

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingNLPC_P60

Peptidase_M15

Peptidase_M15

Phag

e_ly

sozy

me

PG_b

indi

ng

Amidase

ZoocinA_TRDCHAP

esad

im

A

Peptidase_M15

Extensin-like_C

SH3 CHAP

SH3

Glyc

o_hy

dro

CHAP

Peptidase_M15

Glyco_

hydr

o

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Glyco_hydro

Glyco_

hydr

o

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

SH3CHAP

SH3

Gly

co_h

ydro

LAG

LID

ADG

LAG

LID

ADG

CH

AP

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingCHAP

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

ZoocinA_TRDAmidase

NLPC_P60

SH3 CHAP

SH3

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

Peptidase_M15

Peptidase_M15

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

Amida

se

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

NLPC_P

60

CHAPGlyc

o_hy

dro

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase


Glyc

o_hy

dro

Glyco_

hydr

o

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

ZoocinA_TRDAmidase

NLPC_P60

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingCHAP

CW

_bin

ding

CHAP

CW_binding

CW_binding

CW_binding

CW_binding

SH3CHAP

SH3

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

SH3CHAP

SH3

CHAPSH3

Amid

ase

Zooc

inA_

TRD

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingCHAP

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

CHAP

SH3

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

Peptidase_M15

Peptidase_M15

Extensin-like_C

Zooc

inA_

TRDA

mid

ase

CHAP

SH3CHAP

SH3

CHAPSH3

Glyc

o_hy

dro

CHAP

Glyc

o_hy

dro

CHAP

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingNLPC_P60

Glyco_hydro

Amid

ase

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingNLPC_P60

Glyco_hydro

CHAP

SH3

ZoocinA_TRDAmidase

NLPC_P60

ZoocinA_TRDAmidase

NLPC_P60

Amida

se

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

NLPC_P

60

CHAP

Amidase

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ingNLP

C_P60

CHAP

SH3CHAP

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

Peptidase_M15

SH3 CHAP

SH3

Amidase

ZoocinA_TRDCHAP

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingNLPC_P60

Amidase

ZoocinA_TRDCHAP

Amid

ase

Phag

e_ly

sozy

me

Glyc

o_hy

dro

CHAP

CHAP

SH3

Amida

se

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

NLPC_P

60

CHAP

esad

im

A

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingCHAP

ZoocinA_TRDAmidase

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingNLPC_P60

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingNLPC_P60

CHAP

Amida

se

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amida

se

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingNLPC_P60

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingCHAP

NLPC_P60

Glyco_

hydr

o

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding Gl

yco_

hydr

o

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

Zooc

inA_

TRDA

mid

ase

Peptidase_M15

CHAP

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

Peptidase_M15

Peptidase_M15

Extensin-like_C

Glyco_

hydr

o

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Glyco_

hydr

o

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ingAmida

se

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ingAmida

se

Peptidase_M15

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

SH3 CHAP

Glyco_

hydr

o

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding Glyc

o_hy

dro

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Peptidase_M15

Extensin-like_C

SH3CHAP

Peptidase_M15

Amidase

Phag

e_ly

sozy

me

PG_b

indi

ng

Peptidase_M15

Peptidase_M15CHAP

SH3CHAP

SH3

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingCHAP

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

Glyco_hydro

Amida

se

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

CW_binding

CW_binding

CW_binding

CW_bindingNLPC_P60

Mur

aida

sePG

_bin

ding

PG_b

indi

ng

PG_b

indi

ngPG

_bin

ding

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingNLPC_P60

CHAP

Peptidase_M15

Peptidase_M15

Peptidase_M15

Amidase

Amid

ase

Zooc

inA_

TRD

Amidase


SH3CHAP

SH3

ZoocinA_TRDAmidase

NLPC_P60

SH3 CHAP

SH3

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

Amidase

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CHAP

NLPC_P

60

Peptidase_M15

Peptidase_M15

Glyc

o_hy

dro

CHAP

Glyc

o_hy

dro

CHAP

SLT

Glyco_

hydr

o

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

SH3CHAP

SH3

Amidase

ZoocinA_TRDNLPC_P60CHAP

Glyco_

hydr

o

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amida

se

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

NLPC_P

60

CHAP

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

Glyco_hydro

Amidase

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ingNLP

C_P60

CHAP

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

Glyco_

hydr

o

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amida

se

Glyc

o_hy

dro

Inte

in_s

plici

ng

LAG

LIDA

DG

LAG

LIDA

DG

CHAP

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

Peptidase_M15


Peptidase_M15

Glyco_

hydr

o

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amid

ase

Zooc

inA_

TRD

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

CHAP

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

Kelch

Amidase

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ingNLP

C_P60

CHAP

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingCHAP

NLPC_P60

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

ZoocinA_TRDAmidase

NLPC_P60

Amid

ase

AmidaseNa_Ca_ex_C

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingCHAP

CHAPSH3

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingNLPC_P60

SH3CHAP

SH3

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ingAmida

se

Peptidase_M15

Phag

e_ly

sozy

me

Phag

e_lys

ozym

e

Phag

e_lys

ozym

e

Phag

e_lys

ozym

e

Amidase

Peptidase_M15

Peptidase_M15

Phag

e_ly

sozy

me

AmidaseMelibiaseMelibiase

Peptidase_M15

Peptidase_M15

Phag

e_lys

ozym

e

Amidase

Phag

e_lys

ozym

e

Peptidase_M15

Peptidase_M15

Phag

e_ly

sozy

me

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Amidase

Phag

e_ly

sozy

me

Peptid

ase_

C70

Peptid

ase_

C39

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Amidase

Peptid

ase_

C70

Peptid

ase_

C39

Phag

e_ly

sozy

me

Phag

e_lys

ozym

e

Amidase

Phag

e_lys

ozym

e

Peptidase_M15

Phag

e_lys

ozym

e

AmidaseAmidase

Glucos

amini

dase

Amidase

Phag

e_lys

ozym

e

Phag

e_ly

sozy

me

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Amidase

Amidase

Peptidase_M15

Peptidase_M15

Peptidase_M15

Amidase

Phag

e_ly

sozy

me

Peptidase_M15

Amidase

Peptidase_M15

Phag

e_ly

sozy

me

Phag

e_ly

sozy

me

Peptidase_M15

Peptidase_M15

Amidase

Peptidase_M15

Peptidase_M15

Amidase

Phag

e_lys

ozym

e

Phage_lysozym

e

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15 Glyco_hydro

PG_binding

Peptid

ase_

C70

Peptid

ase_

C39

Peptid

ase_

C70

Amidase

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Phag

e_ly

sozy

me

Peptidase_M15

Peptidase_M15

Amidase

Phag

e_ly

sozy

me

Amidase

Peptidase_M15

Peptidase_M15

PG_binding

Glyco_hydroPG_bindingPG_binding

Peptidase_M15

Amidase

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Phag

e_lys

ozym

e

Peptidase_M15

Glucosaminidase

Peptid

ase_

C39

Peptid

ase_

C70

Phag

e_lys

ozym

e

Peptid

ase_

C70

Peptid

ase_

C39

Peptidase_M15

Peptidase_M15

Amidase

Peptidase_M15

Phage_lysozyme

Peptidase_M15

Amidase

Amidase

Peptidase_M15

Peptidase_M15

Peptid

ase_

C70

Peptid

ase_

C39

Glyco_hydro

Amidase

Phag

e_ly

sozy

me

Amidase

Peptidase_M15

Peptidase_M15

Peptidase_M15

Amidase

Amidase

Peptidase_M15

Phage_lysozyme

Pha

ge_l

ysoz

yme

Phage_lysozyme

Peptidase_M15

Amidase

Peptidase_M15

Peptidase_M15

Peptidase_M15

Amidase

Phag

e_lys

ozym

e

Phag

e_ly

sozy

me

Peptidase_M15

Peptidase_M15

Phag

e_ly

sozy

me

Phag

e_ly

sozy

me

Amidase

Phag

e_ly

sozy

me

Amidase

Peptidase_M15

Phage_lysozymePeptidase_C

39Peptidase_C

70

Phag

e_ly

sozy

me

Peptidase_M15

Peptidase_M15

Phag

e_lys

ozym

e

Glyco_hydro

PG_binding

Amidase

Amidase

Phag

e_ly

sozy

me

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

SLT

Lyso

zym

e_lik

eG

luco

sam

inid

ase

SLT

Peptid

ase_

C70

Peptid

ase_

C39

Phage_lysozym

e

Peptidase_M15

Peptidase_M15

Phag

e_lys

ozym

e

Peptidase_M15

Peptidase_M15

Amidase

Phag

e_lys

ozym

e

Phag

e_ly

sozy

me

Phag

e_lys

ozym

e

Amidase

Phag

e_ly

sozy

me

Phag

e_ly

sozy

me

Phag

e_lys

ozym

e

Phag

e_lys

ozym

e

Peptidase_M15

Peptidase_M15

Peptid

ase_

C39

Peptid

ase_

C70

BtrH_N

ERAP1_C

Peptidase_M15

Peptidase_M15

Amidase

Peptidase_M15

Phag

e_lys

ozym

e

Phag

e_lys

ozym

e

Peptidase_M15

Amidase

Amidase

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Amidase

Phag

e_ly

sozy

me

Phag

e_lys

ozym

e

Amidase

Peptidase_M15

Glyco_hydro

Peptidase_M15

Peptidase_M15

Phag

e_lys

ozym

e

Amidase

Peptidase_M15

Amidase

Peptidase_M15

Peptidase_M15

Phag

e_ly

sozy

me

Amidase

emy

zosy

l_eg

ahP

Amidase

Peptidase_M15

Peptidase_M15

Phag

e_ly

sozy

me

Phag

e_ly

sozy

me

Amidase

Peptidase_M15

Amidase

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Phage_lysozyme

Peptidase_M15

Peptidase_M15

Phag

e_ly

sozy

me

Phag

e_ly

sozy

me

Peptidase_M15

Amidase

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Phag

e_lys

ozym

e

Peptidase_M15

Peptidase_M15

Peptidase_M15

Amidase

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptid

ase_

C39

Peptid

ase_

C70

Peptidase_M15

Peptid

ase_

C70

Peptid

ase_

C39

Amidase

Phage_lysozyme

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Amidase

Peptidase_M15

Peptidase_M15

Peptid

ase_

C39

Peptid

ase_

C70

Gmx_pa

ra_CXXCG

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Amidase

Peptidase_M15

Peptidase_M15

Peptid

ase_

C70

Peptid

ase_

C39

Peptidase_M15

Peptid

ase_

C70

Peptid

ase_

C39

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

DUF2099

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Amidase

Peptidase_M15

Peptid

ase_

C39

Peptid

ase_

C70

Peptidase_M15

Peptidase_M15

Peptidase_M15

Phag

e_ly

sozy

me

Peptidase_M15

Peptidase_M15

Amidase

Peptid

ase_

C70

Peptid

ase_

C39

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Phag

e_lys

ozym

e

Amidase

Phag

e_ly

sozy

me

Peptidase_M15

Peptidase_M15

Phag

e_ly

sozy

me

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Amidase

Peptidase_M15

Amidase

Peptidase_M15

Peptidase_M15

Peptidase_M15

Peptidase_M15

Phag

e_ly

sozy

me

Met

hyltr

ansf

Peptid

ase_

C70

Peptid

ase_

C39

Peptidase_M15

Phag

e_lys

ozym

e

Peptidase_M15

Peptidase_M15

SH3CHAP

SH3

Amidase

CH

APAm

idaseC

HAP

SH3

Amid

ase0

2_CA

mid

ase

Amidase

SH3

esad

im

AS

H3

SH

3S

H3

SH

3

Amid

ase

Amidase

SH3

AmidaseAmidase02_C

Amidase

SH3

SH3

Amidase

SH3

SH3

SH3

esad

im

AS

H3

SH

3S

H3

Amidase02_C

Amidase

Amidase

SH3

AmidaseAmidase02_C

Amidase

SH3

AmidaseAmidase02_C

Amid

ase0

2_CA

mid

ase

Amidase

SH3

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

SH3 CHAPSH3

SLT

Pept

idas

e_M

23

Phag

eMin

_Tai

l

MsyLesadi

mA

gnidnib_G

P

Glyc

o_hy

dro

LysM

LysM

Amidase

Glyc

o_hy

dro

LysM

LysM

Glyc

o_hy

dro

LysM

LysM

Amidase

SH3

SH3

Amidase

SH3

SH3

SH3

Amidase

SH3

Amidase

SH3

SH3

Amidase

SH3

SH3

Amidase

SH3

SH3

Amidase

SH3

SH3

Amid

ase

Amid

ase0

2_C

Amidase02_C

Amidase

Am

idaseS

H3

SH

3S

H3

Amidase

SH3

SH3

Glucosaminidase

CHAP

SH3

AmidaseCWCW

Glucosaminidase NLPC_P60DUF4407

AmidaseCW

Glucosaminidase

Glyc

o_hy

dro

LysM es

adi

mA

SH

3S

H3

SH

3

Glucosaminidase

CHAP

SH3

Am

idaseS

H3

SH

3S

H3

SH

3

LysM

LysM

Glyc

o_hy

dro

AmidaseCWCW

Glucosaminidase NLPC_P60

esad

im

AS

H3

SH

3S

H3gnidnib_

GP

esadim

AMsyL

Glucosaminidase

CHAP

SH3

CW

CW

Glu

cosa

min

idas

eD

UF4

407

Amid

ase

SH3

Glyc

o_hy

dro

LysM

LysM

Amidase

CH

AP

Glyc

o_hy

dro

SH3

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ingAmida

se

LysM

LysM

Glyc

o_hy

dro

Glucosaminidase

CHAP

Glyco_hydro

SLT

Phage_lysozyme

Glyco_hydro

esad

im

A

Amid

ase

SLTSLT

SLT

SLT

SLT

SLT

Phage_lysozyme

SLTSLT

Phage_lysozyme

Glyco_hydro

Phage_lysozyme

Phage_lysozyme

Phag

e_ly

sozy

me

Phag

e_ly

sozy

me

Glyco_hydro

Glyco_hydro

Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

Phage_lysozyme Ph

age_

lyso

zym

e

Phage_lysozyme

Phag

e_ly

sozy

me

Glyco_hydro

Mur

aida

sePG

_bin

ding

PG_b

indi

ng

PG_b

indi

ng


SLT

Phage_lysozyme

Phage_lysozyme

SLT

Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

SLT

Phage_lysozyme

Glyco_hydro

Phage_lysozyme


Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

Glyco_hydro

Phage_lysozyme

Peptidase_M15

Peptidase_M15

Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

Mur

aida

se

Phage_lysozyme

Glyco_hydro

Glyco_hydro

Peptidase_M23Amidase

Amidase

emy

zosy

l_eg

ahP

Amidase

Amidase02_C

Amidase

Amidase

SH3

SH3

Glyc

o_hy

dro

LysM

LysM

esadim

AMsyL

gnidnib_G

Pesadi

mA

MsyL

Glucosaminidase

CHAP

SH3

Amida

se

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

NLPC_P

60

CHAP

SH3

Amidase

CH

AP

CHAP

CW_binding

CW_binding

CW_binding

Amidase

CH

AP

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ingAmida

se

esadim

AMsyLgnidnib_

GP

Amidase

SH3

CH

AP

SH3

Amidase

CH

AP

Glyc

o_hy

dro

SH3

SH3

Glucosaminidase

CHAP

SH3

AmidaseCWCW


Glyc

o_hy

dro

SH3

Phag

eMin

_Tai

l

SLT

Pept

idas

e_M

23G

luco

sam

inid

ase

esadim

Agnidnib_

GP

gnidnib_G

Pgnidnib_

GP

esadim

AMsyL

Amidase

SH3

SH3

SH3

SH3

AmidaseCW

GlucosaminidaseNLPC_P60CHAPLCCL

Glucosaminidase

SH3CHAP

SH3

Glyc

o_hy

dro

SH3

Glyc

o_hy

dro

SH3

Glyc

o_hy

dro

SH3

esadim

Agnidnib_

GP

SLT

Phag

eMin

_Tai

l

Phag

eMin

_Tai

l

Pept

idas

e_M

23

AmidaseCWCW

Glucosaminidase NLPC_P60CHAP

Glyco_

hydr

o

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

esadim

AMsyL

AmidaseCW


Glucosaminidase

CHAP

Glucosaminidase

CHAP

SH3

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ingAmida

se

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ingAmida

se

Amidase

SH3

Phage_lysozyme

Mur

aida

sePG

_bin

ding

PG_b

indi

ng

PG_b

indi

ng

Glyco_hydro

Phage_lysozyme

Mur

aida

sePG

_bin

ding

PG_b

indi

ng

Amid

ase

Amid

ase

Amid

ase

Amid

ase

DU

F116

1 Amid

ase

DU

F116

1Amid

ase

Amid

ase

Amid

ase

SLT

Phag

e_ly

sozy

me

Phag

e_ly

sozy

me

SLT

Glyco_hydro

Phage_lysozyme Ph

age_

lyso

zym

e

Mur

aida

se

Peptidase_M15

VanY

Phage_lysozyme

Phage_lysozyme

Glyco_hydro

Phag

e_ly

sozy

me

SLT

Phage_lysozyme

SLT

Glyco_hydro

PG_binding

Glyco_hydro

Phage_lysozyme

Glyco_hydro

DUF2514DUF2514

Glyco_hydro

Mur

aida

sePG

_bin

ding

PG_b

indi

ng

PG_b

indi

ng

Phage_lysozyme

Mur

aida

sePG

_bin

ding

Glyco_hydroDUF2514

Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

Glyco_hydro

SLT

Phage_lysozyme

Phage_lysozyme

Glyco_hydro

Glyco_hydro

Phag

e_ly

sozy

me

Mur

aida

sePG

_bin

ding

Phage_lysozymePeptidase_M

15

VanY

Peptidase_M15

Phage_lysozyme Ph

age_

lyso

zym

ePh

age_

lyso

zym

ePh

age_

lyso

zym

e

Phag

e_ly

sozy

me

Phage_lysozyme


Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

Glyco_hydro

Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

Phage_lysozymePeptidase_M

15

Peptidase_M15

Phag

e_ly

sozy

me

Phag

e_ly

sozy

me

Phag

e_ly

sozy

me

Phag

e_ly

sozy

me

Phag

e_ly

sozy

me

Phag

e_ly

sozy

me

Glyco_hydro

Amid

ase

SH3

SH3

Amidase

SH3

Peptidase_M15

Amid

ase

Amid

ase

Glyco_hydro

Phag

e_ly

sozy

me

AmidaseAmidase

Phag

e_ly

sozy

me

Phage_lysozyme

Amidase

Phag

e_ly

sozy

me

Phage_lysozyme

Phag

e_ly

sozy

me

Amidase

SLT

SLT

SLT

SLTSL

TSL

TSL

TSL

T

Phag

e_ly

sozy

me

Phag

e_ly

sozy

me

Phag

e_ly

sozy

me

Phag

e_ly

sozy

me

Amidase

Phag

e_ly

sozy

me

Glyc

o_hy

dro

LysM

LysM

Glyc

o_hy

dro

LysM

LysM

Glyc

o_hy

dro

LysM

LysM

Glyc

o_hy

dro

LysM

LysM

Glyc

o_hy

dro

LysM

LysM Ph

age_

lyso

zym

e

Phage_lysozyme

AmidaseCSD

Phag

e_ly

sozy

me

Phag

e_ly

sozy

me

Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

Glyco_hydro

Glyco_hydro

AmidaseAmidase

Amidase

Amid

ase

LysM

LysM

Glyc

o_hy

dro

LysM

LysM

Glyc

o_hy

dro

Amid

aseG

lyco_

hydr

o

LysM

LysM

Glyc

o_hy

dro

LysM

LysM

Amid

ase

Amid

ase

Amid

ase

Amid

ase

Amid

ase

Amid

ase

Amid

ase

Amid

ase

Zooc

inA_

TRD

Zooc

inA_

TRD

Glyco_hydro

Peptidase_M23Amidase PG_bindingPG_bindingPG_binding

Peptidase_C39PG_binding

Amidase PG_bindingPG_binding

Amidase

Amidase


Peptid

ase_

M15Glyc

o_hy

dro

Peptid

ase_

M15Glyc

o_hy

dro

Amidase


Peptid

ase_

M15

Peptidase_M15

D123

Peptidase

_M15

PG_bindingPeptidase_M15PG_bindingPG_binding

Peptid

ase_

M15

Trans

glyco

sylas

SLT

Amidase

PG_binding

Peptidase_M15

Peptidase_M15

D123

Amidase

Amidase

Amidase

AmidaseGlyco_hydro

Amidase

Peptidase_M23Amidase PG_bindingPG_binding

PG_binding

Amidase

Peptidase

_M15

Amidase

Amidase

Transg

lycos

ylas

Peptid

ase_

M15SLT

Peptidase_M15

AmidaseAmidase

Peptidase_M15

D123

Amidase

Amidase

Amidase

Peptidase

_M15

Amidase

Glyco_

hydr

o

Amidase

Peptidase_M15


Amidase

Amidase

Amidase

Amidase

Transg

lycos

ylas

Peptid

ase_

M15SLT

Amidase


Peptid

ase_

M15

Trans

glyco

sylas

SLT

Peptidase_M23Amidase PG_binding

Peptid

ase_

M15Glyc

o_hy

dro


PG_binding


Amidase


PG_binding

Peptidase_M15

Peptidase_M15

D123

AmidaseAmidase

Peptidase_M15



PG_binding

PG_bindingPeptidase_M15

Peptidase_M15

Peptidase

_M15Peptid

ase_

M15


Amidase

Amidase

Peptidase_M15

Amidase

Amidase

Peptidase

_M15


Amidase


Peptidase

_M15

Amidase

AmidasePeptidase_M15PG_binding


PG_binding


PG_binding

Amidase

PG_binding


Peptid

ase_

M15

Peptidase_M15

D123

Amidase


Amidase

Amidase

Amidase

Peptidase_M15PG_bindingPG_bindingPeptidase_M15PG_bindingPG_binding


PG_binding

Peptid

ase_

M15

Amidase

Peptidase_M15

Amidase

Amidase

Peptid

ase_

M15Glyc

o_hy

dro

Amidase


AmidaseAmidase

Peptidase

_M15Glyco_hyd

ro


PG_binding

Amidase

Peptidase

_M15

Amidase

Amidase

Peptidase_M15PG_binding

Peptidase_M15

Peptidase_M15

Amidase

Amidase

Peptidase

_M15


PG_binding

Peptid

ase_

M15

Peptidase

_M15Transg

lycos

ylas

SLT

Peptidase_M15PG_binding

Trans

glyco

sylas

SLT

Peptidase

_M15


Amidase

Peptidase_M23Amidase PG_bindingPG_bindingPG_bindingPeptidase_M23Amidase PG_bindingPG_binding

PG_binding

Amidase

AmidaseGlyco_hydro

Peptid

ase_

M15


PG_binding

Amidase


PG_binding

Peptidase_M15

Mur

aida

sePG

_bin

ding

PG_b

indi

ng

PG_b

indi

ng

Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

AmidaseAmidase

SLT

SLT

Phage_lysozyme

Phag

e_ly

sozy

me

Phage_lysozyme

SH3

Amidase

CH

APSH

3Am

idaseC

HAP

SH3

Amidase

CH

AP

Amidase

CH

APSH

3Am

idaseC

HAP

SH3

SH3

SH3

Amidase

CH

APSH

3Am

idaseC

HAP

Amidase

CH

APSH

3SH

3Am

idaseC

HAP

SH3

SH3

SH3

Amidase

CH

AP

SH3

Amidase

CH

AP

SH3

Amidase

CH

APSH

3Am

idaseC

HAP

Amidase

CH

APSH

3

SH3

Amidase

CH

AP

SH3

Amidase

CH

AP

Amidase

CH

APSH

3SH

3

Amidase

CH

APSH

3Am

idaseC

HAP

SH3

Amidase

CH

APSH

3Am

idaseC

HAP

SH3

SH3

Amidase

CH

APSH

3

Amidase

CH

APSH

3

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ing

CW_bind

ingAmida

se

Glucosaminidase

SH3CHAP

AmidaseCW

Glucosaminidase

Glucosaminidase

CHAP

SH3

CHAP

SH3

ZoocinA_TRDAmidase

NLPC_P60

ZoocinA_TRDAmidase

NLPC_P60

ZoocinA_TRDAmidase

NLPC_P60

Glyco_

hydr

o

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_bindingNLPC_P60

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

Amidase

ZoocinA_TRDAmidase

NLPC_P60

Glucosaminidase

CHAP

SH3

Glyco_

hydr

o

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

CW_b

inding

ZoocinA_TRDAmidase

NLPC_P60

ZoocinA_TRDAmidase

NLPC_P60

ZoocinA_TRDAmidase

NLPC_P60

Amidase

CW_binding

CW_binding

CW_binding

CW_binding

CW_binding

AmidaseCWCW

GlucosaminidaseCHAPNLPC_P60

Glucosaminidase

CHAP

SH3

LGFPLGFPLGFP

AmidaseAmidaseAmidaseAmidase

Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

Phage_lysozyme

Amidase

AmidaseAmidase

AmidaseAmidase

Colored ranges

OG0OG1

OG2OG3

OG4

Multiple

OG5OG6OG7OG8

OG9

OG10OG11OG12OG13OG14OG15OG17

OG16OG18OG19

OG21

OG20

OG22

OG23

OG27

OG25

OG24

OG26

OG29OG30

OG28

OG37

OG41OG40

OG32

OG33

OG34OG35OG36

OG42OG43

OG45

OG44

OG39OG38

Tree scale: 1

PEPTIDASE

LYSO

SLTPEPTIDASE

GH

AM

IDA

SE

AM

IDA

SE

AMIDASE +CW

_1GH

MURAIDASEAMIDASE SH3

PEPTIDASE

VanY GLUCO

SLT

PEPTIDASE

PG

PG

PG

LysM

NPLC_P60CHAP

PEPTIDASE

GLUCOLYSO

LYSOCW_1

PG

SH3CHAP

Zoo PEPTIDASE

LYSO

LysM

CHAP

CHA

PN

PLC_P60Zoo

Zoo

LYSO

+ES

ADITPEP

VanY

GLUCO

LYSO

AMIDASE

Zoo

GLUCO

CW-7

AMIDASE

PEPTIDASEPG

SH3

GH

FIGURE 1 | Novel diversity of endolysins discovered in uncultured phage genomes. Cladogram displaying the phylogenetic reconstruction of endolysins assigned tothe 46 OGs represented by 10 or more proteins. Branches are colored according to OG assignments. Protein architecture is displayed adjacent to each leaf,identical colors represent identical Pfam domains.





fmicb-09-01033 May 18, 2018 Time: 15:56 # 4


TAB

LE1

|Pre

vale

nce

ofdo

mai

ns,s

igna

lpep

tide

and

infe

rred

cell

wal

ltar

geto

fend

olys

inor

thol

ogou

sgr

oups

.H

oja

1

OG

_#

OG

0O

G1

OG

2O

G3

OG

4O

G5

OG

6O

G7

OG

8O

G9

OG

10

OG

11

OG

12

OG

13

OG

14

OG

15

OG

16

OG

17

OG

18

OG

19

OG

20

OG

21

OG

22

OG

23

OG

24

OG

25

OG

26

OG

27

OG

28

OG

29

OG

30

OG

31

OG

32

OG

33

OG

34

OG

35

OG

36

OG

37

OG

38

OG

39

OG

40

OG

41

OG

42

OG

43

OG

44

OG

45

Nu

mb

er

of

pro

tein

s2

30

21

01

96

16

31

57

58

47

43

40

39

36

34

33

31

30

27

25

24

23

23

22

22

20

19

17

17

16

16

15

15

15

15

15

14

14

12

11

11

11

11

11

11

11

10

10

10

Sig

na

l p

ep

tid

eN

oN

oN

oN

oN

oN

oN

oN

oN

oN

oN

oN

oY

es

No

No

No

Ye

sY

es

No

No

No

No

No

Ye

sN

oN

oY

es

No

No

No

No

No

No

No

No

No

Ye

sN

oN

oN

oN

oN

oN

oN

oN

oN

o

Do

main

Fu

ncti

on

Ce

ll W

all

Ta

rge

t ty

pe

UU

UU

UU

UG

+U

UU

UG

-U

UU

UG

-U

UU

UU

UG

-U

G+

UU

UU

G+

UG

+U

UG

-U

UU

UU

UU

G+

G+

Am

ida

se

PF

12

12

3 |

Am

ida

se

02

_C

(N

-ac

ety

lmu

ram

oyl-

l-a

lan

ine

am

ida

se

)0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

78.6

00

00

00

00

00

00

Am

ida

se

PF

01

51

0 |

Am

ida

se

_2

(N

-ac

ety

lmu

ram

oyl-

L-a

lan

ine

am

ida

se

)0

11

93.2

0.6

089.4

93

90

2.6

00

00

90

00

00

04.5

00

05.9

093.8

93.8

06.7

93.3

093.3

00

100

00

00

0100

00

00

Am

ida

se

PF

01

52

0 |

Am

ida

se

_3

(N

-ac

ety

lmu

ram

oyl-

L-a

lan

ine

am

ida

se

)0

00.5

00

00

00

00

00

00

00

00

00

00

00

94.1

00

00

06.7

092.9

00

00

00

00

00

0100

Am

ida

se

PF

05

38

2 |

Am

ida

se

_5

(B

ac

teri

op

ha

ge

pe

pti

do

gly

ca

n h

yd

rola

se

)0

00

00

00

00

94.9

00

00

00

00

095.7

00

00

00

00

93.3

00

00

00

00

00

00

00

090

0

Am

ida

se

/Pe

pti

da

se

PF

05

25

7 |

C

HA

P d

om

ain

00

00

00

00

059

94.4

00

77.4

00

00

60.9

00

00

00

94.1

00

86.7

00

93.3

93.3

00

00

00

00

9.1

00

30

0

Ca

rbo

xy/E

nd

o-p

ep

tid

as

eP

F1

18

14

| D

UF

33

35

(P

ep

tid

as

e_

C3

9 l

ike

fa

mil

y)

00.5

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Ca

rbo

xy/E

nd

o-p

ep

tid

as

eP

F0

08

77

| N

LP

C_

P6

0 (

Nlp

C/P

60

fa

mil

y)

00

00

00

00

087.2

00

00

00

00

082.6

00

00

00

00

46.7

00

00

00

00

00

00

00

070

0

Ca

rbo

xy/E

nd

o-p

ep

tid

as

eP

F1

35

29

| P

ep

tid

as

e_

C3

9_

2 (

Pe

pti

da

se

_C

39

lik

e f

am

ily)0.9

0.5

0.5

0.6

01.7

00

00

00

00

6.7

092

00

00

00

00

00

00

00

00

00

00

00

36.4

00

00

00

Ca

rbo

xy/E

nd

o-p

ep

tid

as

eP

F1

23

85

| P

ep

tid

as

e_

C7

0 (

Pa

pa

in-l

ike

cys

tein

e p

rote

as

e A

vrR

pt2

)0.9

0.5

0.5

0.6

01.7

00

00

00

00

3.3

096

00

00

00

00

00

00

00

00

00

00

00

18.2

00

00

00

Ca

rbo

xy/E

nd

o-p

ep

tid

as

eP

F0

14

27

| P

ep

tid

as

e_

M1

5 (

D-a

la-D

-ala

dip

ep

tid

as

e)

00

00

00

00

00

02.9

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Ca

rbo

xy/E

nd

o-p

ep

tid

as

eP

F0

59

51

| P

ep

tid

as

e_

M1

5_

2 (

Ba

cte

ria

l p

rote

in o

f u

nk

no

wn

fu

nc

tio

n (

DU

F8

8))

0.9

26.9

52.8

1.9

6.5

00

2.3

2.5

02.8

61.8

06.5

00

40

00

95.5

00

00

00

00

00

00

07.1

00

00

9.1

00

00

00

Ca

rbo

xy/E

nd

o-p

ep

tid

as

eP

F0

82

91

| P

ep

tid

as

e_

M1

5_

3 (

Pe

pti

da

se

M1

5)2.2

2.4

93.8

1.9

90

2.1

4.7

50

2.8

97.1

06.5

00

40

4.3

095.5

05

00

00

00

00

00

7.1

00

00

036.4

00

00

00

Ca

rbo

xy/E

nd

o-p

ep

tid

as

eP

F1

35

39

| P

ep

tid

as

e_

M1

5_

4 (

D-a

lan

yl-

D-a

lan

ine

ca

rbo

xyp

ep

tid

as

e)1.3

92.8

1.5

2.5

0.6

82.8

02.3

2.5

00

2.9

30

03.7

04.2

00

4.5

00

00

5.9

6.3

00

00

00

07.1

00

100

00

00

00

00

Ca

rbo

xy/E

nd

o-p

ep

tid

as

eP

F0

15

51

| P

ep

tid

as

e_

M2

3 (

Pe

pti

da

se

fa

mil

y M

23

)0

00

00

02.1

00

00

00

090

00

00

00

4.5

00

00

00

00

00

00

00

00

00

00

040

00

Ca

rbo

xy/E

nd

o-p

ep

tid

as

eP

F0

25

57

| V

an

Y (

D-a

lan

yl-

D-a

lan

ine

ca

rbo

xyp

ep

tid

as

e)

02.4

00.6

00

00

00

02.9

00

00

00

00

00

00

00

00

00

00

00

00

0100

00

00

00

00

Gly

co

sid

as

eP

F0

18

32

| G

luc

os

am

inid

as

e (

Ma

nn

os

yl-

gly

co

pro

tein

en

do

-be

ta-N

-ac

ety

lglu

c)0.4

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

86.7

00

00

00

081.8

00

020

100

0

Gly

co

sid

as

eP

F0

01

82

| G

lyc

o_

hyd

ro_

19

(C

hit

ina

se

cla

ss

I)0.9

0.5

0.5

0.6

83.2

12.1

6.4

00

00

00

00

00

00

00

00

5.3

00

06.3

093.3

00

00

00

00

00

00

00

00

Gly

co

sid

as

eP

F0

11

83

| G

lyc

o_

hyd

ro_

25

(G

lyc

os

yl

hyd

rola

se

s f

am

ily 2

5)

00

00

00

00

00

00

00

00

00

95.7

00

095

94.7

00

00

00

00

00

00

00

00

00

00

00

Gly

co

sid

as

eP

F1

17

90

| G

lyc

o_

hyd

ro_

cc

(G

lyc

os

yl

hyd

rola

se

ca

taly

tic

co

re)

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

7.1

00

00

00

00

00

0

Gly

co

sid

as

eP

F1

37

02

| L

ys

ozym

e_

lik

e (

Lys

ozym

e-l

ike

)0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

10

00

Gly

co

sid

as

eP

F1

64

99

| M

eli

bia

se

_2

(G

lyc

os

yl

hyd

rola

se

s f

am

ily 2

7)

00

00.6

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Gly

co

sid

as

eP

F1

18

60

| M

ura

ida

se

(N

-ac

ety

lmu

ram

ida

se

)0

01

00

00

00

00

00

00

96.3

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Gly

co

sid

as

eP

F0

09

59

| P

ha

ge

_ly

so

zym

e (

Gly

co

syl

hyd

rola

se

s f

am

ily 2

4)95.2

2.4

11.9

2.6

1.7

6.4

00

2.6

2.8

097

03.3

04

00

4.3

095.5

00

94.1

00

06.7

06.7

06.7

00

0100

0100

0100

0100

00

0

Gly

co

sid

as

eP

F0

14

64

| S

LT

(S

olu

ble

Lyti

c T

ran

sg

lyc

os

yla

se

do

ma

in)

00

00

010.3

00

00

00

00

00

095.8

00

00

00

00

00

00

00

00

00

00

00

00

0100

00

Gly

co

sid

as

eP

F1

34

06

| S

LT

_2

(S

olu

ble

Lyti

c T

ran

sg

lyc

os

yla

se

do

ma

in)

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

060

00

Gly

co

sid

as

eP

F0

67

37

| T

ran

sg

lyc

os

yla

se

(T

ran

sg

lyc

os

yla

se

-lik

e d

om

ain

)0

00

00

10.3

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Ce

ll W

all

Bin

din

gP

F0

82

30

| C

W_

7 (

CW

_7

re

pe

at)

00

00

00

04.7

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

100

0

Ce

ll W

all

Bin

din

gP

F0

14

73

| C

W_

bin

din

g_

1 (

Pu

tati

ve

ce

ll w

all

bin

din

g r

ep

ea

t)0

00

00

00

090

94.9

00

074.2

00

00

00

00

95

00

00

00

00

00

00

100

00

00

00

00

00

Ce

ll W

all

Bin

din

gP

F0

38

15

| L

CC

L (

LC

CL

do

ma

in)

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

10

0

Ce

ll W

all

Bin

din

gP

F0

83

10

| L

GF

P (

LG

FP

re

pe

at)

00

00

00

2.1

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

0

Ce

ll W

all

Bin

din

gP

F0

14

76

| L

ys

M (

Lys

M d

om

ain

)0

00

00

00

18.6

00

00

00

00

00

00

00

094.7

00

00

00

00

00

00

00

00

00

00

00

Ce

ll W

all

Bin

din

gP

F0

14

71

| P

G_

bin

din

g_

1 (

Pu

tati

ve

pe

pti

do

gly

ca

n b

ind

ing

do

ma

in)1.8

00.5

1.9

7.7

19

020.9

00

00

00

90

85.2

00

00

00

00

00

00

00

00

00

00

018.2

00

00

00

00

Ce

ll W

all

Bin

din

gP

F0

88

23

| P

G_

bin

din

g_

2 (

Pu

tati

ve

pe

pti

do

gly

ca

n b

ind

ing

do

ma

in)

00

00

00

00

00

00

00

03.7

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Ce

ll W

all

Bin

din

gP

F0

93

74

| P

G_

bin

din

g_

3 (

Pre

dic

ted

Pe

pti

do

gly

ca

n d

om

ain

)0

00.5

1.2

7.1

6.9

07

00

00

00

80

59.3

00

00

00

00

00

00

00

00

00

00

018.2

00

00

00

00

Ce

ll W

all

Bin

din

gP

F0

82

39

| S

H3

_3

(B

ac

teri

al

SH

3 d

om

ain

)0

00

00

00

41.9

00

50

00

3.2

00

00

26.1

00

00

00

29.4

87.5

00

00

6.7

07.1

00

00

00

00

00

00

Ce

ll W

all

Bin

din

gP

F0

63

47

| S

H3

_4

(B

ac

teri

al

SH

3 d

om

ain

)0

00

00

00

25.6

00

00

00

00

00

4.3

00

00

00

031.3

00

00

00

00

00

00

00

00

00

0

Ce

ll W

all

Bin

din

gP

F0

84

60

| S

H3

_5

(B

ac

teri

al

SH

3 d

om

ain

)0

00

00

00

00

094.4

00

6.5

00

00

00

00

00

082.4

00

13.3

06.7

80

86.7

7.1

00

00

00

00

00

00

Ce

ll W

all

Bin

din

gP

F1

67

75

| Z

oo

cin

A_

TR

D (

Ta

rge

t re

co

gn

itio

n d

om

ain

of

lyti

c e

xo

en

zym

e)

00

00

00

00

00

00

00

00

00

095.7

00

00

00

00

73.3

00

00

00

00

00

00

90.9

00

00

Un

kn

ow

nP

F1

43

99

| B

trH

_N

(B

uti

ros

in b

ios

yn

the

sis

pro

tein

H,

N-t

erm

ina

l)0

00

00

00

00

00

00

00

08

00

00

00

00

00

00

00

00

00

00

00

00

00

00

0

Un

kn

ow

nP

F0

03

13

| C

SD

(C

old

-sh

oc

k' D

NA

-bin

din

g d

om

ain

)0

00

0.6

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Un

kn

ow

nP

F0

70

65

| D

12

30

00

00

8.6

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Un

kn

ow

nP

F0

66

49

| D

UF

11

61

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

020

Un

kn

ow

nP

F0

98

72

| D

UF

20

99

(U

nc

ha

rac

teri

ze

d p

rote

in c

on

se

rve

d i

n a

rch

ae

a)

00.5

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Un

kn

ow

nP

F1

07

21

| D

UF

25

14

00

00

1.3

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

0

Un

kn

ow

nP

F1

43

62

| D

UF

44

07

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

20

0

Un

kn

ow

nP

F1

57

62

| D

UF

46

91

0.4

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

0

Un

kn

ow

nP

F1

18

38

| E

RA

P1

_C

(E

RA

P1

-lik

e C

-te

rmin

al

do

ma

in)

00

00

00

00

00

00

00

00

40

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Un

kn

ow

nP

F0

69

04

| E

xte

ns

in-l

ike

_C

(E

xte

ns

in-l

ike

pro

tein

C-t

erm

inu

s)

00

00

00

00

00

017.6

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Un

kn

ow

nP

F0

95

35

| G

mx

_p

ara

_C

XX

CG

00

00

00

00

00

00

00

00

40

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Un

kn

ow

nP

F0

10

85

| H

H_

sig

na

l (H

ed

ge

ho

g a

min

o-t

erm

ina

l s

ign

all

ing

do

ma

in)

00

00

00

00

00

011.8

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Un

kn

ow

nP

F1

27

59

| H

TH

_T

np

_IS

1 (

Ins

A C

-te

rmin

al

do

ma

in)

00

0.5

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

0

Un

kn

ow

nP

F1

34

03

| H

int_

2 (

Hin

t d

om

ain

)0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

9.1

00

00

Un

kn

ow

nP

F0

09

77

| H

is_

bio

syn

th (

His

tid

ine

bio

syn

the

sis

pro

tein

)0

0.5

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Un

kn

ow

nP

F1

48

90

| In

tein

_s

pli

cin

g (

Inte

in s

pli

cin

g d

om

ain

)0

00

00

00

00

00

00

00

00

04.3

00

00

00

00

00

00

00

00

00

00

00

00

00

0

Un

kn

ow

nP

F1

34

18

| K

elc

h_

4 (

Ga

lac

tos

e o

xid

as

e,

ce

ntr

al

do

ma

in)

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

016.7

00

00

00

00

00

Un

kn

ow

nP

F1

45

28

| L

AG

LID

AD

G_

3 (

LA

GL

IDA

DG

-lik

e d

om

ain

)0

00

00

00

00

00

00

00

00

08.7

00

00

00

00

00

00

00

00

00

00

00

00

00

0

Un

kn

ow

nP

F1

34

72

| L

ipa

se

_G

DS

L_

2 (

GD

SL

-lik

e L

ipa

se

/Ac

ylh

yd

rola

se

fa

mil

y)

00

00.6

00

00

02.6

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Un

kn

ow

nP

F1

02

94

| M

eth

ylt

ran

sf_

16

(L

ys

ine

me

thylt

ran

sfe

ras

e)0.4

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

0

Un

kn

ow

nP

F0

22

11

| N

Ha

se

_b

eta

(N

itri

le h

yd

rata

se

be

ta s

ub

un

it)

00

00

00

02.3

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Un

kn

ow

nP

F1

64

94

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a_

Ca

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x_

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C-t

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xte

ns

ion

of

so

diu

m/c

alc

ium

ex

ch

an

ge

r)0

00

1.9

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Un

kn

ow

nP

F1

01

45

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ha

ge

Min

_T

ail

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ha

ge

-re

late

d m

ino

r ta

il p

rote

in)

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

040

00

Un

kn

ow

nP

F1

06

71

| T

cp

Q (

To

xin

co

-re

gu

late

d p

ilu

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ios

yn

the

sis

pro

tein

)0

0.5

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Pág

ina

1





fmicb-09-01033 May 18, 2018 Time: 15:56 # 5


Act

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FIGURE 2 | Heatmap depicting prevalence of targeted host genus across endolysin orthologous groups (OGs). Numbers within cells represent the amount ofproteins targeting each host genus (columns) among the 46 OGs (rows) represented by 10 proteins or more. Cell colors depict the percentage frequency of eachhost among OGs. Calculated percentages do not include proteins derived from genomic sequences without host assignments.

that belong to the same genus may differ in their pathogenicpotential.

Next we investigated the frequency of associations betweenendolysin domains and targeted bacteria (Figure 3). On the onehand, some genera were targeted by few domain categories. Forexample, most of the enzymes that targeted Bacillus have amidase(PF12123/PF01510/PF01520) activity in their catalytic domainand SH3 (PF08239/PF06347) on their CBDs, suggesting that thismight be the most efficient mechanism to degrade the cell wallof the members of this clade. On the other hand, genera such asStreptococcus were targeted by endolysins that rely on multipletypes of catalytic activities such as Amidase (PF01510/PF05382),Cysteine Histidine-dependent Amidohydrolases/Peptidases

(CHAP) domain (PF05257), Glucosaminidase (PF01832),Glycosyl hydrolases family 25 (PF01183), as well as multipleCBDs such as CW1 (PF01473), CW7 (PF08230), and SH3(PF08239/PF06347). This pattern suggests that multiple domainarchitectures might be efficient for targeting the cell wallsof Streptococcus. Finally, the Phage_lysozyme (PF00959)and Soluble Lytic Transglycosylase (SLT, PF01464/PF13406)domains were often detected in endolysins predicted to targetProteobacteria, demonstrating that these domains are tuned todegrade the cell walls of this diverse phylum of Gram-negatives.

Host assignments for phage genomes derived frommetagenomic datasets are based on bioinformatic predictionswhich vary on their degree of precision and recall. These





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Amidase_3 (N−acetylmuramoyl−L−alanine amidase)

Amidase_2 (N−acetylmuramoyl−L−alanine amidase)

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FIGURE 3 | Heatmap depicting associations between targeted host genus and endolysin domains. Numbers and colors within cells represent the frequency ofprotein domains (rows) among endolysins targeting each host genera (columns).

approaches have high accuracy for higher taxonomic rankssuch as phylum or class but sometimes lead to incorrectpredictions at the lower ranks such as genus or species (Edwardset al., 2015; Coutinho et al., 2017). Thus, the associationsbetween OGs/domains and targeted hosts inferred frommetagenomic data should be interpreted as putative and inneed of experimental validation (which is currently out of ourscope). Yet the validity of our findings is corroborated by: (1)Use of conservative thresholds to maximize accuracy during thehost prediction analysis described in the original publicationsthat first reported phage genomes obtained from metagenomicsamples. (2) Endolysin domains identified in uncultured phagesmatch those previously described for isolated and culturedbacteriophages (Oliveira et al., 2013). (3) Approximately, 74%of host assignments obtained for the 46 OGs analyzed indepth were derived from prophage sequences integrated intohost genomes that were obtained from pure cultures (insteadof metagenomic data). Thus the bulk of the host/OG andhost/domain associations are based on high confidence hostassignments.

Several of the identified protein domains are not typicallypresent in endolysins from reference phage genomes. Thoseinclude domains that are likely associated with peptidoglycanlysis such as Melibiase_2 (PF16499; Glycosyl hydrolases family27) and Glyco_hydro_cc (PF11790; Glycosyl hydrolase catalyticcore), but also domains with no obvious role in this process,such as: Methyltransf_16 (PF10294; Lysine methyltransferase),

Lipase_GDSL_2 (PF13472; GDSL-like Lipase/Acylhydrolasefamily), and PhageMin_Tail (PF10145; Phage-related minor tailprotein). Presence of the latter could be either the result of proteinfusions or represent novel functions or action mechanisms ofendolysins. Since computational domain identification maybe subjected to errors, experimental validation is necessary tocorroborate the presence of these domains and to determine theirmolecular functions. Nevertheless, our results demonstrate thatthe molecular versatility of endolysins is still poorly understoodconsidering the lack of information available for so many of theidentified domains.

Finally, we explored associations between endolysinarchitecture and their ecosystem of origin. Most of theputative endolysins were derived from aquatic ecosystemsor human microbiome samples, which are the habitats mostoften sampled by metagenomic studies (SupplementaryFigure S1). This pattern highlights the potential for endolysindiscovery in these sites but also the need for investigationsof other ecosystems such as plant associated and terrestrialhabitats, that also yielded novel endolysins. Aquatic phagesappear to be a rich resource for endolysins containing amidase,peptidase and glycosidase domains, which points to them asa source of endolysins active against both Gram-positive andGram-negative bacteria. Meanwhile, endolysins derived fromphages of the human microbiome most often have amidase andpeptidase domains, typical of enzymes acting on Gram-positivehosts.





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CONCLUSION

We were able to notably expand the known diversity of endol-ysins and describe proteins with novel and often complex domainarchitectures, thus challenging the current understanding ofthe diversity of those enzymes. Our findings show thatenvironmental phage genomes, specially those from aquatic andhuman associated microbiomes, are a rich resource for endolysinsdiscovery. Since several of the identified endolysins are predictedto be effective against plant and animal pathogens, those are idealcandidates for purification and further characterization, speciallythose predicted to act on Gram-positive bacteria, against whichendolysin therapy has showed the most promising results sofar (Gerstmans et al., 2016). Our findings regarding associationsbetween bacterial targets and domains provide insights for theengineering of recombinant proteins that have higher efficiencyor an extended host spectrum. Further experimental researchwill be necessary to corroborate our findings regarding theendolytic activity, domain architecture, and target spectrumof these proteins and to evaluate their potential applicationsas antimicrobial agents. Culture independent approaches willcontinue to expand the genetic diversity of phages, and ourstrategy represents a simple, fast, and scalable approach fordiscovering endolysins encoded in their genomes.

AUTHOR CONTRIBUTIONS

IF-R, FC, and FR-V conceived the study and designed theexperiments. IF-R and FC performed the experiments andanalyzed the data. All authors contributed to writing themanuscript.

FUNDING

FC and FR-V were supported by grants “VIREVO” CGL2016-76273-P (AEI/FEDER, EU) (co-funded with FEDER funds);Acciones de Dinamización “REDES DE EXCELENCIA”CONSOLIDER- CGL2015-71523-REDC from the SpanishMinistério de Economía, Industria y Competitividad andPROMETEO II/2014/012 “AQUAMET” from GeneralitatValenciana.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be foundonline at: https://www.frontiersin.org/articles/10.3389/fmicb.2018.01033/full#supplementary-material

FIGURE S1 | Heatmap depicting associations between ecosystem sources andendolysin domains. Numbers and colors within cells represent the frequency ofprotein domains (rows) among endolysins identified in phage genomes from eachecosystem category (columns).

TABLE S1 | Detailed metadata of the genomic sequences of uncultured phagesanalyzed in this study: including sequence identifier, original dataset, sequencelength, number of identified protein encoding genes, ecosystem source and hosttaxonomic classification.

FILE S1 | Multifasta file containing the 183,6298 genomic sequences ofuncultured viruses analyzed in this study.File can be downloaded here: https://drive.google.com/open?id=15rRJMfguPGhfiuQor7puIrlIr2J02tJQ

FILE S2 | Multifasta file containing the amino acid sequences of 2,628 putativeendolysins identified in the genomes of uncultured viruses.

FILE S3 | Newick format file of the phylogenetic reconstruction of endolysinproteins displayed in Figure 1.

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Conflict of Interest Statement: The authors declare that the research wasconducted in the absence of any commercial or financial relationships that couldbe construed as a potential conflict of interest.

Copyright © 2018 Fernández-Ruiz, Coutinho and Rodriguez-Valera. This is an open-access article distributed under the terms of the Creative Commons AttributionLicense (CC BY). The use, distribution or reproduction in other forums is permitted,provided the original author(s) and the copyright owner are credited and that theoriginal publication in this journal is cited, in accordance with accepted academicpractice. No use, distribution or reproduction is permitted which does not complywith these terms.


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https://doi.org/10.2478/s11756-014-0358-8

https://doi.org/10.1038/s41564-017-0053-y









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