The E. coli Extended Genome

1
The E. coli Extended Genome

• Fernando Baquero
• Dept. Microbiology, Ramón y Cajal University
  Hospital, and Laboratory for Microbial Evolution,
  CAB (INTA-CSIC)
• Madrid, Spain

2
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)

3
The Species E. coli
New way

• 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
4
Diversity at all hierarchical levels
Some strains are more mutable than others
Strain Mutation
Some populations tend to produce more clones?
Population Clonalization
Some bacterial groups tend to produce more
species?
Community Speciation
At any level, the origin of diversity is
probably stochastic
5
Adaptation Complexity MutationSingle adaptive
event
6
ClonalizationMultiple adaptive events
7
SpeciationVery complex adaptive events
8
Clonalization
Allopatric clonalization
Sympatric clonalization
9
Clonalization
Host Defenses
ExPEC
Non-ExPEC
Allopatric clonalization
Sympatric clonalization
From James R. Linneus Johnson
10
The elimination of intermediates
Impossibility of being a business man and a
little meermaid
11
Species-Environment Concerted Evolution
Phylogenetic groups
Core genome
Basic reproductive environment
species evolution
environmental evolution
12
Co-evolution Trees within Trees
Host
Bacteria or bacterial consortium
13
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

14
Not a single strain represents the whole species

• K12-MG1655 (4,289 ORFs)
• K12-W3110 (4,390 ORFs)
• O157H7 (Sakai) (5,361 ORFs)
• O157H7-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)

15
E. coli genomes
1,000 genes of difference!
http//colibase.bham.ac.uk
16
E. coli genomes
http//colibase.bham.ac.uk
17
Loops in a common core backbone
A-strain
B-strain
A-loop (A-island)
B-loops (B-islands)
18
Loops in a common core backbone
296 loops in E. coli Sakai
325 loops in E. coli K12
A-strain
B-strain
BB 3,730 kb
BB 3,730 kb
1,393 kb
537 kb
S-loops
K-loops
19
Loop sizes
Large loops arise from horizontal transfer events
Small loops may arise from replication errors
(small deletions or insertions), or correspond to
highly polymorphic regions
Chiapello et al., BMC Bioinformatics, 6171, 2005
20
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)

21
Gogarden et Townsend, Nature Rev. Mic. (2005)
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.
22
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...
23
Random-scale sub-network (loop)
ALIEN
nodes
Operative genes are more easily accepted
links
24
Scale free network (core)
Elaboration from Jain et al.
ALIEN
nodes
Informative genes less easily accepted
Number of links (log)
25
Scale free network (core)
Elaboration from Jain et al.
ALIEN Subnetwork
Informative genes less easily accepted except
alien replacement of an entire sub-network
nodes
Number of links (log)
26
3,256 E. coli genes are connected by 113,894 links
Predicted functional modules in E. coli (von
Mering et al., PNAS 10015428, 2003)
27
Loops as RD 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)
28
Gene flux
Excision Modification
Acquisition
Loss
More loss in sequences of recent
acquisition Insertions and deletions occur more
frequently in loops Overall less loss than
acquisition?
DuplicationModification
(Daubin et al., Genome Biol., 4R57, 2003 Ochman
and Jones, EMBO J., 196637, 2000)
29
Gene flux
Acquisition
Excision Modification
Loss
ConstantRandom Gene Influx?
DuplicationModification
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.
30
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 DaubinOchman, Genome Research, 2004)

The E. coli Gene Exchange Community should be
better identified!
31
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!
32
Sequence divergence reduces acquisition of
foreign DNA
Modified from Gogarten and Towsend, Nature RM,
2005
Deleterious events are frequent with high
divergence, but eventual beneficial events are
rare with low divergence rates
If the acquisition produce neutral events the
tolerance increases
33
Species-Environment Concerted Evolution
Phylogenetic groups
Core genome
Basic reproductive environment
species evolution
environmental evolution
34
Genome Size in E. coli strains ECOR Phylogenetic
Groups
kb
K12 level
Data Bergthorsson and Ochman, Microb. Biol.
Evol. 156-16, 1998
35
Phylogenetic groups clinical associations
Clinical Johnson et al., EID 11141, 2005
Cystitis Johnson et al., AAC 4926, 2005 FUTI
and rectal FUTI Johnson et al., JCM 433895,
2005 Faecal Fr/Cr/Ma, Duriez et al.,
Microbiology 1471671, 2001 Faecal HV Spain,
Machado et al., AAC 49, 2005
36
Phylogenetic groups clinical associations
Groups B2 and D are the more frequently found in
E. coli bacteremia (Hilali et al., Inf.Imm
683983, 2000 Johnson et al., JID152121, 2004,
Bingen, yesterday)
But Epidemic extraintestinal strains, many
SxT-R in UTI in US, Israel, France (Johnson et
al.,EID 11141, 2005)
Clinical Johnson et al., EID 11141, 2005
Cystitis Johnson et al., AAC 4926, 2005 FUTI
and rectal FUTI Johnson et al., JCM 433895,
2005 Faecal Fr/Cr/Ma, Duriez et al.,
Microbiology 1471671, 2001 Faecal HV Spain,
Machado et al., AAC 49, 2005
37
Distribution of E. coli isolates from
hospitalized patients and from healthy volunteers
among the four phylogenetic groups
Machado, Cantón, Baquero et al., AAC 49 (2005)
ESBLs (red) predominates among strains of group D
Pathogenic strains, non ESBL, predominates
among group B2 Commensal strains, non ESBL,
predominates among group A
38
Antimicrobial-R in phylogenetic groups
SxT-R and Cipro-R(1) Johnson et al, AAC 4926,
2005 ESBL Machado et al., AAC 49, 2005
Cipro-R(2) Kuntaman et al., EID 111363, 2005
(Indonesia).
The phylogenetic group B2, the more pathogenic
one, tends to be the less resistant?
39
Species-Environment Concerted Evolution
Ecotypes
Core genome
Basic reproductive environment
species evolution
environmental evolution
40
Models for Multiple Ecotypes (Gevers
et al., Nature MR 3733, 2005)
Clonalization
41
Patients with different ESBL clonesRamón y Cajal
Hospital, Madrid (Baquero, Coque Cantón,
Lancet I.D. 2591, 2002)
42
Mutation Intra-Clonal Diversity
E. coli Faecal - Urine - Blood - ESBLs
Baquero et al, AAC 2004 and Nov.
2005
43
Clonal Ensembles Metastability through
Intermittent Fixation
Line of best fit clones
time
Different clones peak in frequency at different
times, accordingly to the best-fit clone in each
epoch of a changing environment
The maintenance of clonal ensembles is favored by
the assymetry of fitness abilities in different
clones in different epochs
Clonal ensemble
epochal evolution
44
Shared Environments and Maintenance of
DiversityA regional polyclonal community
structure
1
1
2
Alternative stable equilibria and the coexistence
of variant organisms
On this topic Geographic mosaic theory of
coevolution, Forde et al, Nature, 2004
45
Maintenance of diversityA regional polyclonal
community structure
1
1
2
Local Migration
Local Gene Flow
46
Diversity Collapse and Resurrection
Kin effects in open systems
SELECTION
47
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
48
Maintenance of diversityA regional polyclonal
community structure
Gradients and concentration-dependent selection
(F. Baquero and C. Negri, Bioessays, 1997)
49
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 418171, 2002
50
Rock, Paper, Scissors Model
2. Scissors increase its power against paper...
3. And less paper means more stones...
1. If the stones reduces its attack again
scissors....
51
Rock, Paper, Scissors Model
B. Kerr et al., Local dispersal promotes
biodiversity in a real-life game of
rock-paper-scissors. Nature 418171, 2002
52
Rock, Paper, Scissors Model
B. Kerr et al., Local dispersal promotes
biodiversity in a real-life game of
rock-paper-scissors. Nature 418171, 2002
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In60-like integrons
Kindly provided by Teresa Coque et al., 2005
CTX-M-9
CTX-M-2
55
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
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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 50468, 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
  693687, 2003)

57
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 SantamaríaToranzos,
  Int.Microbiol. 65-9, 2003)

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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)

59
Temperature fitness profiles
Absolute fitness
E. coli
K. pneumoniae
5 0 -5 -10 -15 -20
10 20 30 40 50
10 20 30 40 50
Temperature (ºC)
Modified from Okada and Gordon, Mol. Ecol.
102499, 2001
60
CTX-M-10 linked to Kluyvera and phage sequences
Oliver, Coque, Alonso, Valverde, Baquero, Cantón.
AAC 2005 1567-1571

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

61
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

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Extended Genome a Genetic Space
Core Loops Peripheral
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Extended Genome Core Gravity
Foreign sequences of different base composition
tends to ameliorate to resemble the features of
the resident genome
Core Loops Peripheral
Ochman and Jones, EMBO J., 196637, 2000
64
Extended Genome a Genetic Space
Filling the Carrying Capacity of the Environment
for the Species
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Genetic Space
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Complex Genetic Space
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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

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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)

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Gerdes SY et al, JB 2003
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