oriC environ speciates staphylococciWhat can be found by comparative

genomics of related bacterial species

2005.12.16 @JuntendoFumihiko Takeuchi

J. Bacteriology (2005) vol.187, p.7292

Background:We compare the genomes of

• Staphylococcus aureus• Major pathogen for hospital- and community-

acquired infection• Notorious for its virulence and antibiotic resistance

as in Methicillin-resistant S. aureus (MRSA)• Staphylococcus epidermidis

• Colonizes the skin of almost all humans• Benign but causes foreign-body infection

• Staphylococcus haemolyticus• Also among the normal skin flora• Remarkable for its historically early acquisition of

antibiotic resistance

A fundamental concept:homologs

• open reading frames (orfs)• Genes encoding proteins

• homologs• orfs grouped by their nucleotide sequence similarity• Definition: two orfs with quality ratio ≥70% by alignment belong to the same

homolog• e.g. the homolog of intercellular adhesion A (icaA) genes comprises

• COL, #2565, icaA, intercellular adhesion protein A• MRSA252, #2608, icaA, glucosaminyltransferase• MSSA476, #2533, -, glucosaminyltransferase• Mu50, #2666, icaA, intercellular adhesion protein A• N315, #2458, icaA, intercellular adhesion protein A• NCTC8325, #2761• MW2, #2586, icaA, intercellular adhesion protein A• RP62A, #2243, icaA, intercellular adhesion protein A

• Two orfs that are homologous/orthologous/paralogous should belong to thesame homolog

26782494241926322810(draft)

25942714257926562615

JCSC1435RP62aATCC12228

MW2NCTC8325

N315Mu50MSSA476

MRSA252

COL

S. haemolyticusS. epidermidisS. aureus

Two basic profiles ofhomologs

• Phylogenetic profile• Which species/strains share a homolog

• Location profile• Where on chromosome are the orfs (of a homolog) located

• e.g. icaA homolog

• Viewpoint from comparative genomics:association of these profiles might tell us something…

#2243#2586#2761#2458#2666#2533#2608#25652763320..2764558

Locationprofile

Not in S.haemolyticus

Partially in S.epidermidis

Common to S. aureus

Phylogeneticprofile

-+-+++++++

JCSC1435RP62aATCC12228

MW2NCTC8325

N315Mu50MSSA476

MRSA252

COL

S. haemolyticusS. epidermidisS. aureus

Vertical and horizontal transferof DNA

cell division

vertical transfer

vertical transferwithin "recipient"

horizontal transferfrom "doner"e.g. phage

Horizontal transfer

The phylogenetic profile &location profile of homologsidentify horizontal transfer.

1. homologs are classifiedand color-coded accordingto their phylogeneticprofile; homologs commonto three species arerepresented by blacksegmented bars

2. Each orf of a genome iscolored according to thephylogenetic profile (of itsbelonging homolog)

3. Chromosomal regionslacking the commonhomologs are suggested tobe introduced by horizontaltransfer

• Genomic islands(prophages, integratedplasmids, SCC, etc.)

• oriC environ

2127

1 7

96

336

73

1158

491983

666

28

155

780106

common to 7

S. aureus strains

only in someS. aureus strains

in S. haemolyticus(JCSC1435)

common to 2

S. epidermidis strains

only in oneS. epidermidis strain

!Sh1 "Sh2 #Sh1

!Sh2

"Sh1 "Sh3

#Sh2

SCC

oriC environ

SCC

oriC environ

"Sa$ "Sa% !Sa3

"Sa4

SCCoriC environ

!Se1"Se4"Se3 "Se6SE1128

ebhA,B

SH1471

"Se5

Row 1: transposase, integrase

Row 2: pathogenic orfs

Row 3: (see above)

Row 4: GC3 in height

(highly expressed,

putative alien,

possible alien, others)

Row 5: genomic islandsS. haemolyticus JCSC1435

0.5 1.0 1.5 2.0 2.5 (Mbp)

S. epidermidis ATCC12228

S. aureus N315

Genomic islands

• Chromosomalregions (>10 kbp)putatively introducedby horizontal transfer

• Anomalous codonusage, biased GC3,and existence ofintegrase andtransposase supporttheir alien origin

• Differ betweenstrains (even within aspecies)

!Sh1 "Sh2 #Sh1

!Sh2

"Sh1 "Sh3

#Sh2

SCC

oriC environ

SCC

oriC environ

"Sa$ "Sa% !Sa3

"Sa4

SCCoriC environ

!Se1"Se4"Se3 "Se6SE1128

ebhA,B

SH1471

"Se5

Row 1: transposase, integrase

Row 2: pathogenic orfs

Row 3: (see above)

Row 4: GC3 in height

(highly expressed,

putative alien,

possible alien, others)

Row 5: genomic islandsS. haemolyticus JCSC1435

0.5 1.0 1.5 2.0 2.5 (Mbp)

S. epidermidis ATCC12228

S. aureus N315

oriC environ• Located in the downstream of

the origin of DNA replication(oriC), and defined asstretches of 20-kbp windowswith <45% commonhomologs

• 34,153–400,000 bp in S.aureus N315

• 32,492–280,000 bp in S.epidermidis ATCC12228

• 29,763–660,000 bp in S.haemolyticus JCSC1435

• Staphylococcus CassetteChromosome (SCC) occupiesthe left part

• The remaining right part isspecific to species (andconserved among strainswithin a species)

• Codon usage and GC3 issimilar to the totalchromosome

• The rest of the talk focuseson oriC environ

!Sh1 "Sh2 #Sh1

!Sh2

"Sh1 "Sh3

#Sh2

SCC

oriC environ

SCC

oriC environ

"Sa$ "Sa% !Sa3

"Sa4

SCCoriC environ

!Se1"Se4"Se3 "Se6SE1128

ebhA,B

SH1471

"Se5

Row 1: transposase, integrase

Row 2: pathogenic orfs

Row 3: (see above)

Row 4: GC3 in height

(highly expressed,

putative alien,

possible alien, others)

Row 5: genomic islandsS. haemolyticus JCSC1435

0.5 1.0 1.5 2.0 2.5 (Mbp)

S. epidermidis ATCC12228

S. aureus N315

oriC environ (other than SCC):functional profile

• In oriC environ (other than SCC)of any species, higher proportionwas observed for

• Transport/binding proteins andlipoproteins (I-2)

• Specific pathways (II-1-1)• In S. aureus N315 90–400 kbp

• protein A (spa), coagulase(coa), capsular polyscaccharidebiosynthesis operon(cap5[8]A–P)

• In S. epidermidis ATCC12228130–280 kbp

• Detoxification (IV-2)• Antibiotic production (IV-3) such

as iturin A and surfactin• In S. haemolyticus JSCS1435

120–660 kbp• Capsule operon (capA–M)

belonging to adaptation toatypical condition (IV-1)

• Mannitol-specificphosphotransferase system(PTS)

oriC environ (other than SCC):BLASTP top-hit species profile• BLASTP top-hit species of and orf suggests the

previous (or next) hosting species of the orf• oriC environ (other than SCC) had fewer top-hit in the

staphylococci’s most related Bacillus species,implying their acquisition by horizontal transfer.

12695145Streptococcus

151110111411Oceanobacillus

7111011613Listeria

254437472646Bacillus

S. haemolyticusJCSC1435120–520 kbp

S. haemolyticusJCSC1435

S. epidermidisATCC12228134–280 kbp

S. epidermidisATCC12228

S. aureusN31587–400 kbp

S. aureusN315

%

Chromosomal rearrangementsThe phylogenetic profile and location

profile of homologs also indicatechromosomal rearrangements duringvertical transfer

• For the common homolog that hasonly one orf in each genome, weconnected the orfs by lines. Thisindicates the history of chromosomalrearrangements (inversion in red)

• Probable ancestor type was S.haemolyticus and S. epidermidisRP62a

• Their ica operons were in oriC environ

• S. aureus and S. epidermidisATCC12228 were inverted aroundoriC, and remnats of oriC environ werefound transolocated (pink highlight).

• ica operon of S. aureus was in thetranslocated region (2,550 kbp inN315).

• Loci flanking ica in S. epidermidisRP62a was found in 280 and 2,330kbp of S. epidermidis ATCC12228 (=breakpoints of rearrangements) andthe ica operon was missing inATCC12228.

S. aureusN315

inverted(Fig. 1a)

S. haemolyticusJCSC1435

S. epidermidisRP62A

(Fig. 1b)

S. epidermidisATCC12228

inverted(Fig. 1d)

replichore 1 replichore 2

S. haemolyticusJCSC1435

inverted(Fig. 1c)

0.5 1.0 1.5 2.0 2.5 (Mbp)

replichore 1 replichore 2

replichore 1 replichore 2

replichore 1 replichore 2

replichore 1 replichore 2

oriC environ

Summary

Phylogenetic profile and location profile ofhomologs could extract

• Chromosomal regions transferredhorizontally––genomic islands and oriCenviron

• Genomic islands were introduced in specificstrains

• oriC environ included SCC (varies by strains) in itsleft part, and a species-specific region in the rightpart

• Chromosomal rearrangements during verticaltransfer

• The inverted genomes had a remnant of oriCenviron translocated in the upstream of oriC

Epilogue 1: in vitro chromosomalrearrangements in oriC environ [Watanabe]

• Method: prolonged (11 or 18days) culture of S.haemolyiticus JCSC1435,followed by isolation ofsusceptible mutants

• Deletions (pink boxes)exclusively in oriC environmediated by the numerousIS’s

• Changes in biochemicalproperties

• --> oriC environ is notessential for cell viability butbiochemically characterizesthe species

S.haemolyticus JCSC1435

2,685,015 bp

0.0(Mbp)

0.5

1.0

1.5

2.0

2.5

WP12

3DC1

8HT4

8GC1

4IA1

!Sh1

Tn4001

"Sh1

SCC

Tn552

#Sh1

"Sh2

Tn552-like element mecA

ccrC

aacA-aphD

blaZ

msrSAmphBM

mtlA (MAN)

mtlF (MAN)argF (ARG)

ptsG (MAL)

arcA (ARG)

SH0462 (VP)

fmhA (NIT)

scrA (SAC)

SH0741 (SAC)

lacF (LAC)

lacE (LAC)

glpD (GLY)

treP (TRE)

fruA (FRU)

SH2645 (GLN)

SH0326

#Sh2

!Sh2

!Sh3ISS

Epilogue 2: oriC environ in S.saprophyticus

• Does oriC environ exist in the recently sequencedStaphylococcus saprophyticus genome?

• --> Yes

5000001·10

61.5·10

62·10

62.5·10

6N315

500000

1·106

1.5·106

2·106

2.5·106Ssap

N315 v.s. Ssap

5000001·10

61.5·10

62·10

62.5·10

6RP62Ashifted

500000

1·106

1.5·106

2·106

2.5·106Ssap RP62Ashifted v.s. Ssap

5000001·10

61.5·10

62·10

62.5·10

6haem

500000

1·106

1.5·106

2·106

2.5·106Ssap

haem v.s. Ssap

S. aureus N315 S. epidermidis RP62A S. haemolyticus JCSC1435

Epilogue 3: a region similar tooriC environ in Bacillus

• Does oriC environ exist in other species?• --> Species-specific region around oriC exists in

Bacillus species. However, orfX (where the SCC isintegrated in staphylococci) is not located in thisregion (4,134,123..4,133,647 in B. subtilis)

1·106

2·106

3·106

4·106

Bsub

1·106

2·106

3·106

4·106

5·106

BantAmesBsub v.s. BantAmes

1·106

2·106

3·106

4·106

Bsub

1·106

2·106

3·106

4·106

BhalBsub v.s. Bhal

B. subtilis B. subtilisorfX orfX