comparison of 61 sequenced escherichia coli genomes · comparison of 61 sequenced escherichia coli...

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Comparison of 61 Sequenced Escherichia coli Genomes

Oksana Lukjancenko & Trudy M. Wassenaar &

David W. Ussery

Received: 24 February 2010 /Accepted: 23 June 2010# The Author(s) 2010. This article is published with open access at Springerlink.com

Abstract Escherichia coli is an important component ofthe biosphere and is an ideal model for studies of processesinvolved in bacterial genome evolution. Sixty-one publicallyavailable E. coli and Shigella spp. sequenced genomes arecompared, using basic methods to produce phylogenetic andproteomics trees, and to identify the pan- and core genomesof this set of sequenced strains. A hierarchical clustering ofvariable genes allowed clear separation of the strains intoclusters, including known pathotypes; clinically relevantserotypes can also be resolved in this way. In contrast,when in silico MLST was performed, many of the variousstrains appear jumbled and less well resolved. The predictedpan-genome comprises 15,741 gene families, and only 993(6%) of the families are represented in every genome,comprising the core genome. The variable or ‘accessory’genes thus make up more than 90% of the pan-genomeand about 80% of a typical genome; some of thesevariable genes tend to be co-localized on genomicislands. The diversity within the species E. coli, and theoverlap in gene content between this and related species,suggests a continuum rather than sharp species borders inthis group of Enterobacteriaceae.

Introduction

The availability of complete genome sequences frommultiple isolates of a given species has opened up a wholenew range of research strategies. By far the best-studiedbacterial species is Escherichia coli, and the highestnumber of individual genome sequences is available forthis species, which has been the working horse ofbacteriology for as long as the specialization exists.Numerous basic molecular processes have been firstcharacterized and extensively studied in E. coli, leading toinsights that could subsequently be applied to other bacteria[47]. Despite the vast amount of knowledge alreadyavailable for E. coli, based on decades of experimentalresearch, genetic manipulation and, more recently, obser-vations based on single or multiple genome sequences,comparison of a large number of E. coli genome sequencescan still provide novel insights, such as the presence ofgenomic islands, present in some pathogenicity groups, butmissing in others. At the time of writing, there are morethan 100 E. coli genome sequence projects reported, manyof which have been deposited to GenBank. Here, wecompare 61 publically available genome sequences of E.coli and Shigella spp. isolates.

Escherichia spp. and Shigella spp. are Gram-negative,facultative anaerobic, intestinal bacteria belonging to theEnterobacteriaceae, which are taxonomically placed within thegamma subdivision of the Proteobacteria phylum. AlthoughShigella spp. isolates have been rewarded their own genus,which is divided into several species (representing differentsero-groups), its separation from Escherichia spp. is mainlyhistorical. For example, in Bergey’s Manual of SystematicBacteriology, the section on Shigella phylogeny begins withthe following sentence: “Scientific evidence accumulated todate strongly supports that view that Shigella species are

O. Lukjancenko : T. M. Wassenaar :D. W. Ussery (*)Center for Biological Sequence Analysis, Building 208,Department of Systems Biology,The Technical University of Denmark,2800 Kgs. Lyngby, Denmarke-mail: [email protected]

T. M. WassenaarMolecular Microbiology and Genomics Consultants,Tannenstrasse 7(55576 Zotzenheim, Germany

Microb EcolDOI 10.1007/s00248-010-9717-3

biotypes/pathotypes or clones of E. coli” [39]. More than50 years ago it was observed that Shigella spp. and E. colihave the same fertility system [25]; in 1972, Brenner et al. [3]found that based on DNA/DNA hybridization, that Shigellaspp. and E. coli are the same species. Experiments withmultilocus enzyme electrophoresis concluded that nearly allof the Shigella species are clones from within E. coli species[35]. Further, analysis of 16S rRNA sequence alignmentplaces Shigella spp. within E. coli [6]. Thus, all currentevidence indicates that Shigella spp. should be classified as E.coli [23, 36]. Both genera contain highly diverse species,although Shigella spp. are as related to E. coli as they are toeach other. E. coli is a ubiquitous component of the intestinalgut flora of animals including humans, and can survive andmultiply in abiotic environments as well. The speciescomprises both benign and pathogenic variants, whilstShigella spp. are all enteropathogens in mammals.

E. coli isolates have in the past been divided intosubgroups in various ways. Based on established patho-genicity towards the human host, pathogenic versuscommensal E. coli have been recognized, although it isacknowledged that 'pathogenic' E. coli strains maycolonize other animal species asymptomatically. Patho-genic E. coli have further been subdivided according totheir typical site of infection and clinical manifestations inhumans, for instance enteropathogenic, uropathogenic, orextra-intestinal pathogenic E. coli, or based on theirvirulence mechanisms, such as enterohemorragic (EHEC),enterotoxigenic, enteroinvasive, and enteroaggregative E.coli [1, 13]. Other divisions that are frequently used arebased on serology (e.g., serotypes O127:H7 or K12) or,mainly for population genetic purposes, on phylogeneticproperties of particular housekeeping genes, as establishedby MULTI-ENZYME electrophoresis and later by multi-locus sequence typing (MLST) [25]. Finally, some isolatesare described simply for their source of isolation, such asenvironmental isolates or avian pathogenic E. coli.

All these subdivisions have been applied more or lessfrequently to group isolates that share particular features.We were interested to see if any of these groupings wouldhold when isolates were compared based on their completegenome sequences, considering some or all of their genes.Isolates from some groups (based on whatever grouping)have been more frequently sequenced than from others, andcomplete information on all characteristics of interest(pathogenicity, source of isolation, serotype) is not avail-able for all sequenced isolates. Despite these recognizedshortcomings in sampling bias and recorded information,comparison of these 61 genome sequences revealed thatneither the 16S gene, nor gene fragments usually used forMLST, provides biologically meaningful information on therelatedness of the sequenced isolates. The best way toanalyze this is by taking into account all the genomic

content, rather than looking at one or a few individualgenes. The E. coli core genome has been previouslyreported to be less than half the genes [13], with morethan half the E. coli genes in any given genome beingfound in some strains, but missing in others. Many of thesevariable genes can be clustered to specific regions, locatedon genomic islands in an E. coli chromosome.

Materials and Methods

Bacterial Genomes and Gene Annotations

Sixty-one bacterial genomes of E. coli and Shigella spp.were used in this study (Table 1). Of these, 39 fullysequenced genomes and 19 genomes for which thesequence was still in progress at the time of extractionwere obtained from GenBank (1). Sequence from E. coliO103 Oslo was obtained from Norwegian VeterinaryInstitute and sequences from strains LANL ECA andLANL ECF were obtained from Los Alamos NationalLab. Genome sequences of Escherichia albertii, Escherichiafergusonii, and Salmonella enterica Typhimurium LT2 wereincluded for comparison (Table 1). The ‘quality score’ foreach genome is given in Table 1, based on the suggestedscale by Chain et al. [4]. A completely sequenced genomethat has been deposited to GenBank is given a score of ‘1’,with the only exception being E. coli O157:H7 isolateEDL933, which currently has more than 4,000 “N’s” in theDNA sequence of the GenBank file, representing unfilledgaps along the chromsomal sequence—hence, this genome isgiven a lower score of ‘2’. The higher scores represent lowerquality (and often more contigs, or pieces of the DNA,although sequence quality is not measured only by this, asdescribed in [4]).

16S Ribosomal RNA Analysis

The sequences encoding 16S ribosomal RNA wereextracted from the analyzed genomes using RNAmmer[22]; sequences with an RNAmmer score above 1,400 wereconsidered reliable and were kept for analysis. From everygenome, the gene with highest similarity to rrsH of E. coliK12 MG1655 was selected and these sequences werealigned using ClustalX [24]. A phylogenic tree wasgenerated by ClustalX using the Bootstrap neighborhood-joining method, showing the bootstrap values at branchpoints, visualized by NJPlot [34].

In Silico MLST

The alleles for seven housekeeping genes used for MLST ofvarious species (www.mlst.net) were analyzed. These were

O. Lukjancenko et al.

http://www.mlst.net

Tab

le1

Genom

esused

inthisstud

y

GPID

Strain

Size(bp)

Noof

genes

Genedensity

(genes/Kbp

)Noof

contigs

Accession

number

Quality

score

Patho

type,serotype,

othercharacteristics

Reference

225

E.coliK12

MG16

554,63

9,67

54,14

90.89

41

U00

096

1Com

mensal,K12

[2]

226

E.coli01

57:H7Sakai

5,59

4,47

75,23

00.93

43

BA00

0007

1EHEC,O15

7:H7

[15]

259

E.coli01

57:H7EDL93

35,62

0,52

25,31

20.94

52

AE00

5174

2EHEC,O15

7:H7

[33]

313

E.coliCFT07

35,23

1,42

85,33

91.02

01

AE01

4075

1UPEC,O6:K2:H1

[45]

1395

9E.coliHS

4,64

3,53

84,37

80.94

21

CP00

0802

1Com

mensal,O9

[37]

1396

0E.coliE24

377A

5,24

9,28

84,74

90.90

47

CP00

0800

1ETEC,O13

9:H28

[37]

1557

2E.coliB7A

5,30

0,24

24,64

80.87

628

9AAJT00

0000

004

ETEC,O14

8:H28

[37]

1557

6E.coliF11

5,21

5,96

14,70

40.90

1119

AAJU

0000

0000

3ExP

EC,O6:H31

[37]

1557

7E.coliE22

5,52

8,23

85,10

50.92

312

7AAJV

0000

0000

3EPEC,O10

3:H2

[37]

1557

8E.coliE1100

195,37

6,211

4,93

40.91

713

7AAJW

0000

0000

3EPEC,O111:H9

[37]

1563

9E.coli53

638

5,37

1,79

04,80

30.89

44

AAKB00

0000

002

EIEC,O14

4TIG

R

1619

3E.coli10

1-1

4,97

9,76

74,60

70.92

591

AAMK00

0000

003

EAEC,O-:H10

[18,

37]

1623

5E.coli53

64,93

8,92

04,62

00.93

51

CP00

0247

1UPEC,O6:K15

:H31

[7]

1625

9E.coliUTI89

5,17

9,97

15,02

10.96

92

CP00

0243

1UPEC

[5]

1635

1E.coliK12

W3110

4,64

6,33

24,22

60.90

91

AP00

9048

1Com

mensal,K12

[16]

1671

8E.coliAPECO1

5,49

7,65

34,42

80.80

13

CP00

0468

1APEC,O1:K1:H7

[21]

1808

3E.coliATCC87

394,74

6,21

84,20

00.88

41

CP00

0946

1K12

derivativ

eDOEJG

I-PGF

1805

7E.coliSE11

5,15

5,62

64,67

90.90

77

AP00

9240

1Com

mensal,O15

2:H28

[32]

1828

1E.coliB

str.REL60

64,62

9,81

24,20

50.90

81

CP00

0819

1Com

mensal,strain

B[19]

1905

3E.coliSE15

4,83

9,68

34,48

80.92

72

AP00

9378

1Com

mensal,O15

0:H5

[42]

1946

9E.coliSMS-3-5

5,21

5,37

74,74

30.90

95

CP00

0970

1Env

iron

mentalisolate

[11]

2007

9E.coliK12

DH10

B4,68

6,13

74,12

60.88

01

CP00

0948

1K12

derivativ

e[8]

2071

3E.coliBL21

(DE3)

4,55

7,50

84,15

70.91

21

CP00

1509

1Com

mensal,strain

Bph

ylog

roup

Korea

Research

Institu

reof

Bioscienceand

Biotechno

logy

2773

7E.coli01

57:H7EC40

425,61

7,72

85,23

20.93

14

ABHM00

0000

002

EHEC,O15

7:H7

J.Craig

VenterInstitu

te

2773

3E.coli01

57:H7EC40

455,66

0,95

85,34

30.94

38

ABHL00

0000

002

EHEC,O15

7:H7

J.Craig

VenterInstitu

te

2774

5E.coli01

57:H7EC40

765,70

5,64

55,34

20.93

613

5ABHQ00

0000

002

EHEC,O15

7:H7

J.Craig

VenterInstitu

te

2774

3E.coli01

57:H7EC4113

5,65

5,84

75,00

50.88

423

1ABHP00

0000

003

EHEC,O15

7:H7

J.Craig

VenterInstitu

te

2773

9E.coli01

57:H7EC4115

5,70

4,17

15,31

50.93

11

CP00

1164

1EHEC,O15

7:H7

J.Craig

VenterInstitu

te

2774

1E.coli01

57:H7EC41

965,62

0,60

65,07

20.90

218

6ABHO00

0000

003

EHEC,O15

7:H7

J.Craig

VenterInstitu

te

2773

5E.coli01

57:H7EC42

065,62

9,93

25,20

20.92

37

ABHK00

0000

002

EHEC,O15

7:H7

J.Craig

VenterInstitu

te

2774

9E.coli01

57:H7EC44

015,73

3,13

35,18

50.90

418

6ABHR00

0000

003

EHEC,O15

7:H7

J.Craig

VenterInstitu

te

2775

1E.coli01

57:H7EC44

865,93

3,16

65,42

90.91

516

5ABHS00

0000

003

EHEC,O15

7:H7

J.Craig

VenterInstitu

te

2775

3E.coli01

57:H7EC45

015,67

7,18

15,12

40.90

225

0ABHT00

0000

004

EHEC,O15

7:H7

J.Craig

VenterInstitu

te

2775

5E.coli01

57:H7EC50

85,65

6,66

65,01

90.88

727

2ABHW00

0000

004

EHEC,O15

7:H7

J.Craig

VenterInstitu

te


Tab

le1

(con

tinued)

GPID

Strain

Size(bp)

Noof

genes

Genedensity

(genes/Kbp

)Noof

contigs

Accession

number

Quality

score

Patho

type,serotype,

othercharacteristics

Reference

2775

7E.coli01

57:H7EC86

95,73

1,06

55,22

00.91

014

7ABHU00

0000

003

EHEC,O15

7:H7

J.Craig

VenterInstitu

te

2884

7E.coli01

57:H7TW14

588

5,67

0,29

75,80

31.02

310

ABKY00

0000

002

EHEC,O15

7:H7

J.Craig

VenterInstitu

te

2896

5E.coliBL21

(DE3)

4,55

7,04

14,08

70.89

61

AM94

6981

1Com

mensal,strain

BAustralianCenter

forBioph

armaceutical

Techno

logy

3003

1E.coliDH1

4,63

0,70

74,16

01

CP00

1637

1K12

derivativ

eDOEJG

I-PGF

3068

1E.coliBL21

(DE3)

4,57

0,93

84,22

80.94

91

CP00

1665

1Com

mensal,strain

BDOEJG

I-PGF

3257

1E.coli01

27:H6E23

48/69

5,06

9,67

84,55

40.89

83

FM18

0568

1EPEC,O12

7:H6

[17]

3341

3E.coli55

989

5,15

4,86

24,76

30.92

31

CU92

8145

1EAEC

[43]

3337

3E.coliIA

I14,70

0,56

04,35

30.92

61

CU92

8160

1Com

mensal,O8

[43]

3337

5E.coliS88

5,03

2,26

84,69

60.93

32

CU92

8161

1ExP

EC,O45

:K1:H7

[43]

3340

9E.coliED1a

5,20

9,54

84,91

50.94

31

CU92

8162

1Com

mensal,O81

[43]

33411

E.coliIA

I39

5,13

2,06

84,73

20.92

21

CU92

8164

1UPEC,O7:K1

[43]

3341

5E.coliUMN02

65,32

4,39

14,82

60.90

63

CU92

8163

1UPEC,O7:K1

[43]

3377

5E.coliBW29

524,57

8,15

94,08

40.89

21

CP00

1396

1K12

derivativ

e[10]

4101

3E.coli01

03:H212

009

5,52

4,86

05,12

10.92

62

AP01

0958

1EHEC,O10

3:H2

[31]

4102

1E.coliO26

:H11

1136

85,85

1,45

85,51

60.94

24

AP01

0958

1EHEC,O26

:H11

[31]

4102

3E.coli0111:H-1112

85,76

6,08

15,40

70.93

76

AP01

0960

1EHEC,O111:H-

[31]

E.coli01

03Oslo

4,96

0,07

64,57

10.92

115

3Unp

ublished

4EHEC,O10

3NorwegianVet

Institu

te

E.coliLANLECA

5,48

9,84

54,75

00.86

564

Unp

ublished

3EHEC,O15

7:H7

Los

Alamos

National

Lab

E.coliLANLECF

5,55

6,39

34,81

30.86

663

Unp

ublished

3EHEC,O15

7:H7

Los

Alamos

National

Lab

310

S.flexneri2a

301

4,82

8,82

14,17

70.86

51

AE00

5674

1Dysentery,Serog

roup

2a[20]

408

S.flexneri2a

2457

T4,59

9,35

44,06

10.88

21

AE01

4073

1Dysentery,Serog

roup

2a[44]

1637

5S.

flexneri584

014,57

4,28

44,115

0.89

91

CP00

0266

1Dysentery,Serog

roup

5[29]

1563

7S.

boydiiCDC

3083

-94

4,87

4,65

94,24

60.87

11

CP00

1063

1Dysentery,Serog

roup

1[35]

1314

6S.

boydiiSb2

274,64

6,52

04,13

40.88

91

CP00

0036

1Dysentery,Serog

roup

4[41]

1314

5S.

dysenteriaeSd1

974,56

0,911

4,27

10.93

61

CP00

0034

1Dysentery,Serog

roup

1[41]

1619

4S.

dysenteriae10

125,23

5,53

54,41

90.84

618

9AAMJ000

0000

03

Dysentery,Serotyp

e4

TIG

R,[34]

1315

1S.

sonn

eiSs046

5,05

5,31

64,21

90.83

41

CP00

0038

1Dysentery

[47]

2885

1E.albertiiTW07

627

4,69

8,53

34,38

60.93

364

ABKX01

0000

003

Enterop

atho

genic

J.Craig

VenterInstitu

te

3336

9E.ferguson

iiATCC35

469

4,64

3,86

14,31

90.93

02

CU92

8158

1Com

mensal

[43]

241

S.enterica

serovarTyp

himurium

LT2

4,95

1,37

14,52

50.91

32

AE00

6468

1Enterop

atho

genic

[27]

GPID

geno

meprojectidentifierat

NCBI(http

://www.ncbi.n

lm.nih.gov

/genom

es/lp

roks.cgi).

EPECenteropathog

enic,UPECurop

atho

genic,

ExP

ECextra-intestinal

pathog

enic,ETECenterotoxigenic,

EIECenteroinvasive,EAECenteroaggregative,

APECavianpathog

enic

E.coli


http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi

fragments of adk, fumC, icd, gyrB, mdh, purA, and recA.The obtained DNA sequences were extracted from thegenome sequence, concatenated and phylogenically analyzedas described above. Alignments were not manually adjusted toavoid subjective interpretation of the outcome.

Predicted Proteome Analysis

The predicted proteomes comprising all protein-codinggenes were extracted from the GenBank files for thepublished genomes. For unpublished genomes, they werepredicted using EasyGene [30]. All predicted proteomeswere compared by BLASTP reciprocal pairwise compari-son. Two genes were attributed to a single gene family andconsidered 'conserved' when they shared at least 50%amino acid identity over at least 50% of the length of thelongest gene.

A hierarchical clustering was performed for the completepan-genome as described by Snipen et al. [38]. Briefly, apan-genome matrix was constructed consisting of 1 s and0 s where each row corresponds to a gene family, asdescribed above, and each column to a genome. Cell (i,j) inthe matrix is 1 if gene family i is present in genome j, or 0if it is absent. Manhattan distances were calculated andused for hierarchical clustering to generate the tree. Theplotted distance between two genomes shows the proportionof gene families where their present/absent status differs.Thus, pan-genome hierarchical clustering analyses genes thatare not conserved, but vary in their presence or absencebetween genomes. Shorter distances represent genomes withmore gene families in common. Genes only occurring in asingle genome (singletons) were not included in the analysis.Bootstrap values (per mil) were computed for each inner nodeby re-sampling the rows of the matrix.

A pan- and core genome plot was constructed accordingto [12]. The order of genomes was chosen based on thepan-genome tree, starting with the largest E. coli O157genome. For the pan-genome curve, all cumulative BLASThits found in the genomes were plotted as a running total,which increases as more genomes are added. The numberof gene families with at least one representative in everygenome was plotted for the core genome and this slowlydecreases with the addition of more genomes, as thesegenomes may lack genes from gene families that had beenconserved in the previously plotted genomes.

A BLAST atlas was constructed as described by Hallinet al. [14].

Results and Discussion

A number of characteristics of each of the 61 genomes aresummarized in Table 1, such as their size, their number of

recognized protein genes, and their gene density. Their GCcontent varies around 50% for all genomes (not shown), buttheir size and number of genes varies extensively. Thesmallest E. coli genome included is that of strain BL21(DE3) sequenced by the Korean consortium, which is only4.56 Mbp, and the smallest Shigella genome is that ofShigella dysenteriae Sd197, with 4.56 Mbp. The longestgenome of the completed genomes so far belongs to E. coliO157:H7 strain EC4115, with 5.70 Mbp. Longer genomesare listed in Table 1, but since those sequences are still inmultiple contigs, it is possible that their stated length isoverestimated. These size differences mean that around onemillion nucleotides (approximately 20% of a genome) canbe absent in one E. coli or Shigella isolate and present inanother. These 'extra' sequences are not void, as indicatedby the variation in number of genes: the longest E coligenome has 1,158 more predicted genes than the shortest E.coli genome (5,315 genes for strain EC4115 and 4157genes for BL21). Further, the observed gene density isrelatively constant, at 0.911±0.04 genes per 1,000 basepairs. It should be noted that published proteomes havebeen defined using different gene prediction programs anddefinitions, so that the observed slight variation in genedensity might be explained by non-standardized geneidentification.

Phylogeny of 16S Ribosomal RNA and MLST Genes

A phylogenetic tree based on the 16S ribosomal RNAsequences extracted from a representative set of 20 Enter-obacteriacea genomes is shown in panel a of Fig. 1, whichis in agreement with the known phylogeny of the family.The tree for the full set of the 61 E. coli and Shigellastrains, including two additional species of Escherichia andone from S. enterica is shown in Fig. 1b. From this figure,it is obvious that phylogeny of the 16S rRNA gene does notresolve well within the genus level, as is known, becausethe rRNA operons are so similar. Although some of the treenodes are predicted with uncertainty, clearly the generaShigella and Escherichia are not separated, nor are E. coligenes separated from those of E. fergusonii or E. albertii.This finding was expected, considering the close related-ness between Escherichia spp. and Shigella spp. In general,16S sequences are not suitable to analyze inter-strainrelationships within a species or between closely relatedspecies, as illustrated with this set of Enterobacteriaceaegenes. This questions the reliability to use 16S as anindicator for the species to which unknown sequencedDNA belongs [45].

Next, it was investigated if conserved housekeepinggenes, frequently assessed for MLST, provide a betterrepresentation of the relatedness of the investigatedgenomes. Various MLST schemes are in use for E. coli


[9, 28] or Shigella spp. [35] but these are not standardizedand the genes assessed in these schemes are not conservedin all genomes. We used the combination of sevenhousekeeping genes that has been applied to a number ofbacterial species [26] (www.mlst.net). Since S. entericalacks fumC (an observation that somewhat weakens the

general applicability of this MLST gene set), that genomewas not included in the analysis. The resulting tree, shownin Fig. 2, still mixes E. coli with Shigella species, and doesnot separate all pathogenic strains from commensal strains.Some of the phylogroups previously defined by multilocusenzyme electrophoresis are clearly separated, such as the E

Buchnera aphidicola str 5A (Acyrthosiphon pisum)

Candidatus Hamiltonella defensa 5AT (Acyrthosiphon pisum)

Sodalis glossinidius 'morsitans'

Escherichia coli K-12 MG1655

Shigella boydii CDC 3083-94

Citrobacter koseri ATCC BAA-895

1000

Salmonella enterica serovar Typhimurium LT2

609

Citrobacter sakazakii ATCC BAA-894

954

Erwinia amylovora ATCC 49946

648

Eneterobacter species 638

Klebsiella pneumoniae 342

846

668

Pectobacterium atrosepticum SCRI1043

Serratia proteamaculans 568

Dickeya dadantii Ech586

586

921

737

Edwardsiella ictaluri 93-146

586

420

Photorhabdus asymbiotica ATCC 43949

Xenorhabdus bovienii SS-2004

Proteus mirabilis HI4320

356

428

876

Yersinia pestis Angola

261

926

999

Wigglesworthia glossinidia endosymbiont of Glossina brevipalpis

0.02

Enterobacteriaceae genera 16S rRNA treeaFigure 1 Phylogenetic treebased on extracted 16S rRNAsequences. a Comparison of 20different Enterobacteriaceae,based on extracted 16S rRNAsequences from the GenBanksequence files. E. coli andShigella are shown in green. bTree of 61 sequenced E. coli(black) and related species(colored), based on the alignmentof the 16S rRNA gene sequence.Apart from Shigella spp., thegenes from E. albertii and E.fergusonii are also included(arrows). The 16S rRNA gene ofS. enterica Typhimurium LT2was used as the root. Bootstrapvalues, indicated in red, showthat most nodes are predictedwith uncertainty; nevertheless,the genera Escherichia spp.and Shigella spp. are notseparated in this tree, and thethree Escherichia species are alsomixed


http://www.mlst.net

cluster containing all O157 strains, the A/B cluster ofcommensal K12 and B strains, and the B2 clustercontaining some of the uropathogenic strains, in accordanceto comparisons carried out by others [40]. Other authorsconcluded that the O157 serotype of EHEC probablyevolved in successive evolutionary events [9]; however,that conclusion is not supported by the MLST tree. Andalthough the B phylogroup is known for its commensal

isolates, one of which being used by Delbrück and Luria fortheir famous phage work, this branch also contains theenteroaggregative strain 101-1 (Fig. 2). Moreover, the twoS. dysenteriae strains are widely separated from each other.Pupo et al. [36], who used a different set of MLST genes,also found that isolates of the three species Shigellaflexneri, Shigella boydii, and S. dysenteriae, could notalways be grouped together nor separated from E. coli.

E. coli 55989S. sonnei Ss046

E. coli BL21 (DE3)E. coli BL21 (DE3)E. coli B str. REL606E. coli O103:H2 str. 12009E. coli E22

E. coli CFT073S. dysenteriae Sd197

E. coli 101-1E. albertii TW07627

E. coli B7AE. coli O26:H11 str. 11368

S. boydii CDC 3083-94S. flexneri 2a str. 301

790

707

S. boydii Sb227S. flexneri 2a str. 2457T

S. flexneri 5 str. 8401E. coli 536

510

221

E. coli E110019

196

E. coli APEC O1E. coli BL21 (DE3)

87

E. coli ED1aE. coli S88

S. dysenteriae 1012

500

E. coli O127:H6 str. E2348/69E. coli SE15

E. coli UTI89E. fergusonii ATCC35469

493

124

E. coli F11

292

E. coli Oslo O103

383

494

377

E. coli K12 str. ATCC8739E. coli HS

E. coli IAI39E. coli E24377AE. coli O111:H- str. 11128

E. coli UMN026E. coli SE11E. coli SMS-3-5E. coli O157:H7 str. TW14588E. coli O157:H7 str. SakaiE. coli O157:H7 str. EC4401E. coli O157:H7 str. EC4196E. coli O157:H7 str. EC4115E. coli O157:H7 str. EC4113E. coli O157:H7 str. EC4076E. coli O157:H7 str. EC4045E. coli O157:H7 str. EC4042E. coli IAI1E. coli 53638

E. coli K12 str. BW2952E. coli K12 str. DH1

E. coli K12 str. DH10BE. coli K12 str. MG1655

272

E. coli K12 str. W3110E. coli O157:H7 str. LANL ECA

E. coli O157:H7 str. EC508E. coli O157:H7 str. LANL ECFE. coli O157:H7 str. EC4206E. coli O157:H7 str. EC4486E. coli O157:H7 str. EC4501E. coli O157:H7 str. EC869

639

604

E. coli O157:H7 str. EDL933

225

951

774

404

423

101

56

161

108

343

S. enterica Typhimurium str. LT20.005

bFigure 1 (continued)


Various enteroinvasive E. coli serotypes have been sug-gested as ancestral to the different Shigella serogroups [23],which could explain the lack of differentiation power ofMLST in this case. Apparently, neither MLST gene sets aresuitable to group these Enterobacteriaceae organisms in a

meaningful way. The performance of MLST could in theorybe improved by selecting different genes, for instance usinga set of genes specifically chosen to produce the desiredgrouping. However, the strength of MLST analysis shouldbe that a conserved set of genes is able to identify

E. coli BL21(DE3)E. coli BL21(DE3)E. coli BL21(DE3)E. coli B str. REL606E. coli 101-1

E. coli K12 str. MG1655E. coli K12 str. DH1E. coli K12 str. DH10B

E. coli K12 str. W3110E. coli BW2952

1000

1000

Shigella dysenteriae 1012

830

E. coli 536E. coli F11E. coli S88E. coli UTI89E. coli APEC O1E. coli CFT073

1000

1000

669

E. coli ED1aE. coli O127:H6 str. E2348/69

E. coli SE15E. coli IAI39E. coli SMS-3-5

1000

998

939

E. coli UMN026E. coli K12 str. ATCC 8739

E. coli HSE. coli 53638

741

E. coli IAI1

1000

E. coli 55989E. coli Oslo O103

E. coli E22E. coli O103:H2 str. 12009

708

1000

E. coli B7AE. coli SE11

E. coli O111:H- str. 11128E. coli O26:H11 str. 11368

E. coli E1100191000

Shigella sonnei Ss046

792

Shigella boydii CDC 3083-94Shigella boydii Sb227E. coli E24377A1000

702

Shigella dysenteriae Sd197Shigella flexneri 2a str. 2457TShigella flexneri 5 str. 8401

Shigella flexneri 2a str. 301E. coli O157:H7 str. EC4401E. coli O157:H7 str. EC4206E. coli O157:H7 str. SakaiE. coli O157:H7 str. EC4115E. coli O157:H7 str. EC4076E. coli O157:H7 str. EC4045E. coli O157:H7 str. EDL933E. coli O157:H7 str. EC4501E. coli O157:H7 str. LANL ECFE. coli O157:H7 str. EC4042E. coli O157:H7 str. EC508E. coli O157:H7 str. EC4486E. coli O157:H7 str. EC869E. coli O157:H7 str. EC4196E. coli O157:H7 str. TW14588E. coli O157:H7 str. EC4113E. coli O157:H7 str. LANL ECA

1000

1000

E. fergusonii ATCC354

1000

E. albertii TW07627

0.01

Figure 2 Phylogenetic tree ofconcatenated MLST gene alleles(adk, fumC, icd, gyrB, mdh,purA, recA), extracted from thegenome sequences. Color use isthe same as in Fig. 1


phylogenetic relationships in any collection of isolates fromone species. If one has to select a 'standard' gene setspecifically for the species under investigation, it weakensthe general application of MLST considerably.

Pan-Genome Comparisons

MLST analyzes allelic differences in genes whosepresence has to be conserved in all genomes. However,we hypothesized that genes that are variably present couldprovide useful information as to the true relatedness of theanalyzed genomes. Since the variable fraction contain genesthat are present in some, absent in other genomes, aphylogenetic analysis cannot be performed to capture allinformation. Figure 3 displays a pan-genome clustering tree,based on the gene families that are variably present in theanalyzed genomes (gene families comprising singletons wereexcluded). The hierarchical clustering obtained by thisanalysis correctly separates the Shigella spp. and S.Typhimurium from Escherichia spp. and, within the latter

genus, separates E. coli from the other Escherichia spp(Fig. 3). Moreover, all E. coli O157:H7 genomes now clustertogether, as do the K12 derivatives (W3110, MG1655, DH1,BW2952, DH10B, and ATCC8739). The strains belongingto phylogenic group B are also positioned in one cluster,to which the non-pathogenic commensal strain HS alsoseems to belong. All these are avirulent isolates, and it isquite impressive that all these are positioned closetogether in the tree. We conclude that this analysis ofvariable genes identifies inter-strain relationships that canbe correlated to the lifestyle of the organisms.

The contribution of every genome to the completepan-genome of E. coli and related organisms is demon-strated in Fig. 4, where the pan-genome and core genome,as defined by other authors [40] of the analyzed sequencesis plotted. The number of novel gene families for everyadded genome is also shown. As can be seen, all genomescontribute to the increase of the pan-genome. Thisincrease is less strong when similar genomes are added(for instance all four K12 genomes, or the B strains). The

E. coli 101 1E. coli B7AE. coli E22E. coli E110019E. coli APECO1E. coli F11E. coli ED1aE. coli O127:H6 str. E2348/69E. coli 536E. coli SE15E. coli CFT073E. coli S88E. coli UTI89E. coli IAI39E. coli 53638E. coli UMN026E. coli SMS 3 5E. coli O103 OsloE. coli O111:H str.11128E. coli O103:H2 str. 12009E. coli O26:H11 str. 11368 E. coli E24377AE. coli 55989E. coli IAI1E. coli SE11E. coli HSE. coli BL21 (DE3 DOE)E. coli BL21 (DE3 AU)E. coli BL21 (DE3Korea)E. coli B str. REL606E. coli K12ATCC8739 E. coli K12 DH10BE. coli K12 str. BW2952E. coli K12 str. DH1E. coli K12 str. MG1655E. coli K12 str. W3110E. coli O157:H7 str. EC4045E. coli O157:H7 str. LANL ECAE. coli O157:H7 str. LANL ECFE. coli O157:H7 str. TW14588E. coli O157:H7 str. SakaiE. coli O157:H7 str. EDL933E. coli O157:H7 str. EC4401E. coli O157:H7 str. EC4206E. coli O157:H7 str. EC869E. coli O157:H7 str. EC4486E. coli O157:H7 str. EC4042E. coli O157:H7 str. EC4115E. coli O157:H7 str. EC4076E. coli O157:H7 str. EC4501E. coli O157:H7 str. EC508E. coli O157:H7 str. EC4113E. coli O157:H7 str. EC4196

100

100

100

42

61

51

61

86

83

89

40

79

38

65

96

56

86

50

85

62

60

60

97

83

58

69

35

97

42

16

99

36

67

35

82

59

82

84

100

95

S. flexneri 5 str. 8401S. flexneri 2a str. 301S. flexneri 2a str. 2457TS. sonnei Ss046S. boydii Sb227S. boydii CDC 3083 94

100

6060

0.000.15 0.10 0.050.20

Relati ve manhattan distance

S. dysenteriae Sd197S. dysenteriae 1012E. albertii TW07627 E. fergusonii ATCC 35469 Salmonella enterica Typhimurium str. LT2

99

58

69

70

70

Figure 3 Pan-genome clustering of E. coli (black) and related species(colored), based on the alignment of their variable gene content. Thegenomes now cluster according to species and a relatedness between

E. coli K12 derivatives (green block) and group B isolates (orangeblock) is visible


addition of Shigella spp. genomes does not alter the shapeof the pan-genome curve, but addition of the otherEscherichia genomes causes a sharp increase. Thecontribution of E. fergusonii to the pan-genome has beennoted before [42].

The core genome reduces in size as more genomes areadded, with an expected significant drop when theshorter genomes are assessed (starting with E. coli K12DH10B, at position 18 in Fig. 4). The core genomereaches 1,472 gene families conserved in 53 E. coligenomes, which is further reduced to 993 gene familiesif Shigella spp. are considered as well. The bars show howmany novel gene families each genome contributes to thegrowing pan-genome. It should be noted that the order inwhich genomes are analyzed influences the number ofthese reported novel gene families other than for single-tons. When novel genes are considered, instead of novelgene families, the findings can be even more dramatic. Forinstance, six novel E. coli genome sequences identifiedapproximately 10,000 novel genes [42]. Previous work has

estimated a core genome of 1,976 genes for 20 E. coligenomes and a pan-genome of 17,831 genes. Our analysisof 53 E. coli genomes identified 1,472 conserved genefamilies and 13,296 gene families comprising the pan-genome. We prefer to report these findings as gene families,instead of individual genes, using clearly defined criteria forinclusion of genes into a gene family (described in the“Materials and Methods” section).

Where are all these variable genes located in agenome? Gene order is not strongly conserved betweenthe analyzed genomes, so that gene location dependswhich genome is considered. Nevertheless, by visualizingwhere a gene, whose presence can vary, is located on asingle reference genome provides further information,and this can be visualized in a BLAST atlas [14]. In theBLAST atlas of Fig. 5, it becomes apparent that the variablegene content is not evenly distributed over the referencegenome, but appears to be distributed over various islands.The reference chromosome of E. coli O157:H7 EC4115was chosen, as it is the largest chromosome for which a

1 : Escherichia coli 0157:H7 str. EC4196 2 : Escherichia coli 0157:H7 str. EC4113 3 : Escherichia coli 0157:H7 str. EC508 4 : Escherichia coli 0157:H7 str. EC4501 5 : Escherichia coli 0157:H7 str. EC4076 6 : Escherichia coli 0157:H7 str. EC4115 7 : Escherichia coli 0157:H7 str. EC4042 8 : Escherichia coli 0157:H7 str. EC4486 9 : Escherichia coli 0157:H7 str. EC86910 : Escherichia coli 0157:H7 str. EC420611 : Escherichia coli 0157:H7 str. EC440112 : Escherichia coli 0157:H7 str. EDL93313 : Escherichia coli 0157:H7 str. TW1458814 : Escherichia coli 0157:H7 str. Sakai15 : Escherichia coli 0157:H7 EC404516 : Escherichia coli 0157:H7 str. LANL ECF17 : Escherichia coli 0157:H7 str. LANL ECA18 : Escherichia coli K12 str. DH10B19 : Escherichia coli K12 str. MG165520 : Escherichia coli K12 str. W311021 : Escherichia coli K12 str. DH122 : Escherichia coli BW295223 : Escherichia coli ATCC873924 : Escherichia coli B REL60625 : Escherichia coli BL21 (DE3 Korea)26 : Escherichia coli BL21 (DE3 AU)27 : Escherichia coli BL21 (DE3 DOE)28 : Escherichia coli HS29 : Escherichia coli SE1130 : Escherichia coli IAI131 : Escherichia coli 5598932 : Escherichia coli E24377A33 : Escherichia coli O26:H11 str. 1136834 : Escherichia coli 0127:H6 str. E2348/6935 : Escherichia coli O103:H2 str. 1200936 : Escherichia coli O111:H- str. 1112837 : Escherichia coli 0103 Oslo38 : Escherichia coli SMS 3 539 : Escherichia coli UMN02640 : Escherichia coli 5363841 : Escherichia coli IAI3942 : Escherichia coli UTI8943 : Escherichia coli S8844 : Escherichia coli CFT07345 : Escherichia coli SE1546 : Escherichia coli 53647 : Escherichia coli ED1a48 : Escherichia coli F1149 : Escherichia coli APECO150 : Escherichia coli E11001951 : Escherichia coli E2252 : Escherichia coli B7A53 : Escherichia coli 101 154 : Shigella flexneri 2a 2457T55 : Shigella flexneri 2a 30156 : Shigella flexneri 5 840157 : Shigella boydii CDC 3083 9458 : Shigella boydii Sb22759 : Shigella sonnei Ss04660 : Escherichia fergusonii ATCC 3546961 : Escherichia albertii TW0762762 : Salmonella enterica Typhimurium LT263 : Shigella dysenteriae Sd19764 : Shigella dysenteriae 1012

New gene families

Core genome

Pan genome

5000

10000

15000

1 3 5 7 9 11 13 15 17 19 21 23 61 6325 27 33 3729 31 35 39 41 43 45 47 53 5749 51 55 59

Numberof genefamilies

Figure 4 Pan- and core genome plot of the analyzed genomes. Theblue pan-genome curve connects the cumulative number of genefamilies present in the analyzed genomes. The red core genome curve

connects the conserved number of gene families. The gray bars showthe numbers of novel gene families identified in each genome


complete sequence is currently available. Around this,all other genomes are plotted, whereby lack of colorindicates that particular gene from EC4115 is missing inthe shown genome. The strong conservation of genepresence within the O157 serotype (in green) contrasts withthe multiple 'gaps' seen in the other lanes. Every gaprepresents multiple genes in strain EC4115, illustrating thatgene variation is not evenly distributed along the genome,but located in islands.

Concluding Remarks

“This gene is not found in E. coli”, is an expression oftenheard in discussions about novel genes in variousorganisms, and when people are looking for functionalmatches in databases. It is a sobering thought to realizethat any given E. coli genome sequenced will have onlyroughly 20% of its genes part of the E. coli core, and theremaining 80% are not found in all other E. coli genomes.After a comparison of the diversity with many sequenced

E. coli genomes, it has become clear such a statement canonly be valid when it is specified which E. coli genomesequence has been searched. Of the predicted pan-genomecomprising about 16,000 gene families, the core (slightlyless than a thousand genes) is found to be only about afifth of a typical E. coli genome which contains around5,000 genes. Many of the accessible or variable genes,making up more than 90% of the pan-genome and roughlyfour fifth of a typical genome, are often found co-localizedon genomic islands. The diversity within the species E.coli, and the overlap in gene content between this andrelated species is far greater than many had anticipated,and represents a broad set of functions for adapting tomany different environments. The comparative methodsused here are generally applicable to genomes of relatedspecies, and are considered a valuable tool to evaluatecurrent insights of species' relatedness and evolutionaryhistory.

Acknowledgments We would like to thank the Danish ResearchCouncils and the DTU Globalization funds for financial support.

0M 0.5M1M

1.5

M2M

2.5M3M3.5

M

4M

4.5

M

5M

Terminus

Origin

Escherichia coli O157:H7 strain EC4115

Figure 5 BLAST atlas. In themiddle, a genome atlas of E. coliO157:H7 strain EC4115 isshown, around which BLASTlanes are shown. Every lanecorresponds to a genome, withthe following colors (goingoutwards): green E. coli O157:H7 (15 lanes); light blue E. coliLANL strains (two lanes); darkblue Shigella spp. (eight lanes);red E. coli K12 and derivatives(six lanes); orange E. coli strainB phylogroup (four lanes);followed by all other E. coligenomes in different colors.The outermost three lanesrepresent E. fergusonii, E.albertii, and S. entericaTyphimurium LT2. Lack ofcolor indicates that the genes atthat position in strain EC4115were not found in the genomeof that lane. The position ofreplication origin and terminusis indicated


Open Access This article is distributed under the terms of theCreative Commons Attribution Noncommercial License which per-mits any noncommercial use, distribution, and reproduction in anymedium, provided the original author(s) and source are credited.

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