The E. coli Extended Genome

Fernando BaqueroDept. Microbiology, Ramón y Cajal University

Hospital, and Laboratory for Microbial Evolution, CAB (INTA-CSIC)

Madrid, Spain

The Species E. coli

Roles of the concept of “species”• Units of taxonomic classification: Units in the

general reference system that microbiologists use to order the isolates

• Units of generalization: Kinds of microorganisms over which explanatory-predictive generalizations can be made

• Units of evolution: Bacterial entities that participate in evolutionary processes and undergo evolutionary change

(Modified from T.A.C. Reydon, Ph.D. Dissertation, Leiden University, 2005)

The Species E. coli

• Units of taxonomic classification: Units in the general reference system that microbiologists use to order the isolates

• Units of generalization: Kinds of microorganisms over which explanatory-predictive generalizations can be made

• Units of evolution: Bacterial entities that participate in evolutionary processes and undergo evolutionary change

Classic way

New way

Diversity at all hierarchical levels

Strain Mutation

Population Clonalization

Community Speciation

Some strains are more mutable than others

Some populations tend to produce more

clones?

Some bacterial groups tend to produce more

species? At any level, the origin of diversity is probably stochastic

Adaptation Complexity: MutationSingle adaptive event

ClonalizationMultiple adaptive events

SpeciationVery complex adaptive events

Clonalization

Allopatric clonalization

Sympatric clonalization

Clonalization

Allopatric clonalization

Sympatric clonalization

Host Defenses

ExPEC*

Non-ExPEC

* From James R. “Linneus” Johnson

Impossibility of being a business man andand a little meermaid

The elimination of intermediates

Species-Environment Concerted Evolution

species evolution environmental evolution

Basic reproductive environment

Phylogenetic groups Core

genome

Co-evolution: Trees within Trees

Host

Bacteria or bacterial consortium

The clues of E. coli genetic diversity

• Errors in DNA replication and repair

• Horizontal genetic transfer from other organisms

• Creation of mosaic genes from parts of other genes

• Duplication and divergence of pre-existing genes

• De novo invention of genes from DNA that had previously a non-coding sequence

Modified from Wolfe and Li, Nat. Genet. 33, 2003

Not a single strain represents the whole species

• K12-MG1655 (4,289 ORFs)

• K12-W3110 (4,390 ORFs)

• O157:H7 (Sakai) (5,361 ORFs)

• O157:H7-EDL933 (5,349 ORFs)

• E2348/69

• CFT073 (UPEC) (5,379 ORFs)

• O42 (EAEC), HS, E24377A (ETEC), Nissle (PBEC)

• Shigella floxneri SF-301 and 2457T (4,084)

E. coli genomes

http://colibase.bham.ac.uk 1,000 genes of difference!

E. coli genomes

http://colibase.bham.ac.uk

Loops in a common core backbone

A-strain B-strain

A-loop (A-island) B-loops (B-islands)

Loops in a common core backbone

A-strain B-strain

S-loops K-loops

296 loops in E. coli Sakai

325 loops in E. coli K12

BB: 3,730 kb BB: 3,730 kb

1,393 kb 537 kb

Loop sizes

Chiapello et al., BMC Bioinformatics, 6:171, 2005

Small loops may arise from replication errors (small deletions or insertions), or correspond to highly polymorphic regions

Large loops arise from horizontal transfer events

The core backbone is not the minimal genome

• The “core backbone” is not the “minimal E. coli genome”, because of high level of gene redundancy.

• A high number of genes are members of gene families (2-30 copies), similar enough to be assigned similar functions (paralogs)

• Such redundancy involves 20-40 % of the E. coli coding sequences (more in the largest genomes)

• “In-silico metabolic phenotype” including all basic functions, predict about 700 genes in minimal genome (Blattner at al., Science

1997, Edwards and Palsson, PNAS 2000)

The blue gene, unexpected in the species “C”, might have arisen: i) by horizontal gene transfer; or ii) by an ancient gene duplication followed by differential gene loss.

Gogarden et Townsend, Nature Rev. Mic. (2005)

The loops

• The backbone evolves by vertical transfer.

• Large loops are probably acquired by horizontal gene transfer, but also evolve by vertical transfer.

• Loops tend to have a different

codon usage and higher AT % than the backbone.

• Loops tend to contain more frequently

operational genes (actions) than informative

genes (complex regulation) (R. Jain, 1999)

PAIs, islets, phages, plasmids, transposable, repetitive elements...

Random-scale sub-network (loop)

ALIEN

nodes

links

Operative genes are more easily accepted

Scale free network (core)

Informative genes less easily accepted

Elaboration from Jain et al. ALIEN

Number of links (log)

nodes

Scale free network (core)

Informative genes less easily accepted

except alien replacement of an

entire sub-network

Elaboration from Jain et al.

ALIEN Subnetwork

Number of links (log)

nodes

Predicted functional modules in E. coli

(von Mering et al., PNAS 100:15428, 2003)

3,256 E. coli genes are connected by 113,894 links

Loops as R&D E. coli laboratories

• Proteins expressed

(bars in red)

Positions of K-loops (bars in blue)

The genes in the loops express proteins in only 10% of the cases

M. Taoka et al., Mol & Cell. Proteomics (2004)

Gene flux

Acquisition Loss

DuplicationModification

Excision Modification

More loss in sequences of recent

acquisition*

Insertions and deletions occur more frequently in loops

Overall less loss than acquisition?(Daubin et al., Genome Biol., 4:R57, 2003;

Ochman and Jones, EMBO J., 19:6637, 2000)

Gene fluxAcquisition

Loss

DuplicationModification

Excision Modification

ConstantRandom

Gene Influx?

As in the case of random mutation, there might be a blind, random uptake and loss of available foreign genetic sequences;

environmental selection and random drift determines the fate of these constructions.

E. coli - where alien genes come from?

• Enterobacteriaceae (56 %) (Klebsiella, Salmonella, Serratia, Yersinia); Aeromonas, Xylella, Ralstonia, Caulobacter, Agrobacterium

• Plasmids (28 %) - about 250 plasmids identified in E. coli. • Phages (10%) + many ORFan genes (64 MG1655-specific)

(Modified from Duphraigne et al., NAR 33, 2005, and Daubin&Ochman, Genome Research, 2004)

The E. coli “Gene Exchange Community” should be better identified!

E. coli Recipient Barriers for Horizontal Gene Transfer

• Ecological separation from donor• DNA sequence divergence• Low numbers• Inadequate phage receptors• Inadequate pilus specificity for mating• Contact-killing or inhibition• Surface exclusion• Restriction*; no anti-restriction mechanisms, gene inactivation• Absence of replication of foreign gene, incompatibility• Absence of integration of foreign gene in specific sites• No recombination with host genome (AT/CG), MMR system• Decrease in fitness of recipient after DNA acquisition • No more room for new DNA: Headroom (Maximal Genome?)

*200 enzymes!

Sequence divergence reduces

acquisition of foreign DNA

If the acquisition produce neutral events the tolerance increases

Deleterious events are frequent with high

divergence, but eventual beneficial

events are rare with low divergence rates

Modified from Gogarten and Towsend, Nature RM, 2005

Species-Environment Concerted Evolution

species evolution environmental evolution

Basic reproductive environment

Phylogenetic groups Core

genome

Genome Size in E. coli strains ECOR Phylogenetic Groups

4

4,2

4,4

4,6

4,8

5

5,2

5,4

A B1 B2 D

K12 level

kb

Data: Bergthorsson and Ochman, Microb. Biol. Evol. 15:6-16, 1998

Phylogenetic groups: clinical associations

0

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60

70

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90

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A B1 B2 D

Clinical Cystitis Febrile UTI

Rectal (FUTI) Faecal HV-Fr Faecal HV-Sp

Clinical: Johnson et al., EID 11:141, 2005; Cystitis: Johnson et al., AAC 49:26, 2005; FUTI and rectal FUTI: Johnson et al., JCM 43:3895, 2005; Faecal Fr/Cr/Ma, Duriez et al., Microbiology 147:1671, 2001; Faecal HV Spain, Machado et al., AAC 49, 2005

Phylogenetic groups: clinical associations

0

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40

50

60

70

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A B1 B2 D

Clinical Cystitis Febrile UTI

Rectal (FUTI) Faecal HV-Fr Faecal HV-Sp

Clinical: Johnson et al., EID 11:141, 2005; Cystitis: Johnson et al., AAC 49:26, 2005; FUTI and rectal FUTI: Johnson et al., JCM 43:3895, 2005; Faecal Fr/Cr/Ma, Duriez et al., Microbiology 147:1671, 2001; Faecal HV Spain, Machado et al., AAC 49, 2005

But: “Epidemic extraintestinal strains”, many SxT-R in UTI

in US, Israel, France (Johnson et al.,EID 11:141, 2005)

Groups B2 and D are the more frequently found in E. coli

bacteremia (Hilali et al., Inf.Imm 68:3983, 2000; Johnson et al.,

JID15:2121, 2004, Bingen, yesterday)

0

10

20

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% of

stra

ins

A B1 B2 D

Distribution of Distribution of E. coliE. coli isolates from hospitalized isolates from hospitalized

patients and frompatients and from healthy volunteers among the healthy volunteers among the

four phylogenetic groupsfour phylogenetic groups

ESBLs (red) predominates among strains of group D Pathogenic strains, non ESBL, predominates among group B2 Commensal strains, non ESBL, predominates among group A

Machado, Cantón, Baquero et al., AAC 49

(2005)

Antimicrobial-R in phylogenetic groups

SxT-R and Cipro-R(1): Johnson et al, AAC 49:26, 2005; ESBL: Machado et al., AAC 49, 2005; Cipro-R(2): Kuntaman et al., EID 11:1363, 2005 (Indonesia).

0

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70

80

A B1 B2 D

SxT-R ESBLs Cipro-R(1) Cipro-R(2)

The phylogenetic group B2, the more pathogenic one, tends to be the less resistant?

Species-Environment Concerted Evolution

species evolution environmental evolution

Basic reproductive environment

Ecotypes

Core genome

Models for Multiple Ecotypes (Gevers et al., Nature MR 3:733, 2005)

Clonalization

Patients with different ESBL clonesRamón y Cajal Hospital, Madrid

(Baquero, Coque & Cantón, Lancet I.D. 2:591, 2002)

0

5

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25

30

88 89 90 91 92 93 94 95 96 97 98 99 0

Year

No

. o

f p

atie

nts

/clo

ne

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10

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30

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50

60

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Hypo Normo Weak Strong

Mutation frequency

% o

f str

ain

sE. coli : Faecal - Urine - Blood - ESBLs

Baquero et al, AAC 2004 and Nov. 2005

Mutation: Intra-Clonal Diversity

Clonal Ensembles: Metastability through Intermittent Fixation

Different clones peak in frequency at different times, accordingly to the best-fit clone in each epoch* of a changing environment

Clonal ensemble*epochal evolution

Line of best fit clones

time

The maintenance of clonal ensembles is favored by the assymetry of fitness abilities in different clones in different epochs

Shared Environments and Maintenance of Diversity

A regional polyclonal community structure

Alternative stable equilibria and the coexistence of variant organisms

1 12

On this topic: Geographic mosaic theory of coevolution, Forde et al, Nature, 2004

Maintenance of diversityA regional polyclonal community structure

1 12

Local Migration

Local Gene Flow

Diversity: Collapse and Resurrection

SELECTION

Kin effects in open systems

Maintenance of diversityA regional polyclonal community structure

1

Environmental gradients are composed by a multiplicity of patches

that may act as discrete selective points for bacterial variants

Maintenance of diversityA regional polyclonal community structure

Gradients and concentration-dependent selection

(F. Baquero and C. Negri, Bioessays, 1997)

Maintenance of Diversity by Scissors, Rock, Paper Model

B. Kerr et al., Local dispersal promotes biodiversity in a real-life game of rock-paper-scissors. Nature 418:171, 2002

Rock, Paper, Scissors Model

1. If the stones reduces its attack again scissors....

2. Scissors increase its power against paper...

3. And less paper means more stones...

B. Kerr et al., Local dispersal promotes biodiversity in a real-life game of rock-paper-scissors. Nature 418:171, 2002

Rock, Paper, Scissors Model

B. Kerr et al., Local dispersal promotes biodiversity in a real-life game of rock-paper-scissors. Nature 418:171, 2002

Rock, Paper, Scissors Model

Int1 aacA4 blaOXA-2 orfD qacE1sul1 orf513 blaCTXM-2 orf3:: qacE1 sul1

Int1 aacA4 aadA2 qacE1sul1 orf513 catA2 qacE1 sul1 orf5

Int1 aadB qacE1sul1 orf513 dfrA10 qacE1 sul1 orf5

Int1 aadA2 qacE1sul1 orf513 ampC ampR qacE1 sul1 orf5

Int1 dfrA16 aadA22 qacE1sul1 orf513 blaCTXM-9 orf3-like IS3000 qacE1 sul1

Int1 dfrA16 aadA22 qacE1sul1 orf513 qnr ampR qacE1 sul1 orf5 orf6 IS6100

Int1 aac(6) blaoxA30 catB3 aar-3 qacE1sul1 orf513 qnr ampR qacE1 sul1 orf5 orf6 IS6100

qacE1sul1 orf513 dfrA18 int1 oxa1 aadA1 qacE1 sul1

qacE1sul1 orf513 blaDHA ampR qacE1 sul1

qacE1sul1 orf513 orf1 blaDHA ampR qacE1 sul1

In60-like integrons Kindly provided by Teresa Coque et al., 2005

CTX-M-9

CTX-M-2

Extensive “McFarlane-Burnett” Model and Evolution of Bacterial Pathogenicity

• Every evolutionary element (clones, chromosomal sequences, plasmids, transposons, islands, recombinases, insertion sequences...) is independently submitted to apparently random spontaneous variation.

• Combinations of the variant elements are constantly constructed apparently at random.

• Eventually a given combination is selected and enriched by an unexpected advantage (colonization-pathogenicity) or fixed by drift.

Pre-pathogens are probably constantly constructed; many of them eliminated by immunity and normal microbiota

The opportunity of meeting interesting people: E. coli in the environment

• It has been suggested that one-half of E. coli population resides in primary habitats (warm-blooded hosts) and one-half in soil or water.

• Tropical waters harbor natural populations of E. coli (Carrillo et al., AEM 50:468, 1985)

• In nutrient-rich soils, particularly with cyclic periods of wet and dry weather, E. coli is member of normal microflora (Winfield and Groisman, AEM 69:3687, 2003)

E. coli in the environment

• Land disposal practices of sewage and sewage sludges that result from wastewater treatment.

• More than 3 million gallons of sewage effluent from more than 3,000 land treatment sites and 15 million septic tanks were applied to land every day in 1984 (Keswick, BH. 1984)

• More than 7 million dry tons of sewage sludge are produced anually and 54 % of this is applied to soil

(Environmental Protection Agency, http:// www.epa.gov./oigearth; 2002;

Santamaría&Toranzos, Int.Microbiol. 6:5-9, 2003)

E. coli in the environment

• EPA Class A Biosolids

Less than 103 thermotolerant coliforms/g, for lawns, home gardens, as commercial fertilizer.

• EPA Class B Biosolids

Less than 106 thermotolerant coliforms/g, for land application, forest lands, reclamation sites. During a period, access is limited to public and livestock.

(Environmental Protection Agency)

Temperature fitness profiles

5

0

-5

-10

-15

-20

10 20 30 40 50 10 20 30 40 50

Temperature (ºC)

E. coli K. pneumoniae

Absolute fitness

Modified from: Okada and Gordon, Mol. Ecol. 10:2499, 2001

Oliver, Coque, Alonso, Valverde, Baquero, Cantón. AAC 2005; 1567-1571

EcoRI

Tn1000-likeTransposase(fragment) ORF2

ORF3 DNA invertase

CTX-M-10ORF7

ORF8 Transposase IS432 ORF10

Transposase IS5

Invertibleregion

K. cryocrescenshomol. region (90%)

Tn 5708 fragment

IS4321 IS5

Phage related region

ORF4 ORF11

EcoRI EcoRIEcoRIBamHI

BamHI BamHI

CTX-M-10 linked to Kluyvera and phage sequences

Present in different clones at Ramón y Cajal Hospital Variability in the sequence among different clones Probably linked to the same plasmid structure

The Extended Genome

A genetic space composed by the sum of:• The sequences corresponding to the maximal

core genome of all clones (ortologs-paralogs), plus • The sequences of all loops that have been

inserted in such a core in the different natural (successful at one time) clones or lineages: ecotypes, geotypes, pathotypes.., plus

• The sequences of all extra-chromosomal elements stably associated with any clone

Extended Genome: a Genetic Space

Core

Loops

Peripheral

Extended Genome: Core Gravity

Core

Loops

Peripheral

Foreign sequences of different base composition tends to “ameliorate” to resemble the features of the resident genome*

*Ochman and Jones, EMBO J., 19:6637, 2000

Extended Genome: a Genetic Space

Filling the Carrying Capacity of the Environment for the Species

Genetic Space

Complex Genetic Space

The Extended E. coli Genome

• Research to increase our interpretative, predictive and preventive capability about Escherichia coli evolutionary biology.

• Catalog of sequences of all evolutionary relevant pieces* in E. coli.

• Network of all interactions between pieces.

• Modelization of combinations that might emerge under particular environmental or clinical conditions.

*F.Baquero, From Pieces to Patterns, Nature Reviews 2004

A lot of work, a lot of fun.

Particular thanks to some of my friends in the lab...

• Rafael Cantón

• Teresa Coque

• Juan-Carlos Galán

• José-Luis Martínez (CNB, CSIC)

Gerdes SY et al, JB 2003

http://www.pnas.org/content/vol99/issue26/images/large/pq2525297001.jpeg