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
10
20
30
40
50
60
70
80
90
100
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
10
20
30
40
50
60
70
80
90
100
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
30
40
50
% 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
10
20
30
40
50
60
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
10
15
20
25
30
88 89 90 91 92 93 94 95 96 97 98 99 0
Year
No
. o
f p
atie
nts
/clo
ne
0
10
20
30
40
50
60
70
80
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