The E. coli Extended Genome
Fernando Baquero
Dept. 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
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
Diversity at all hierarchical levels
Strain
Population
Mutation
Clonalization
Some strains are more
mutable than others
Some populations tend
to produce more
clones?
Some bacterial
Community Speciation
groups tend to
produce more
species?
At any level, the origin of diversity is probably stochastic
Adaptation Complexity: Mutation
Single adaptive event
Clonalization
Multiple adaptive events
Speciation
Very complex adaptive events
Clonalization
Allopatric clonalization
Sympatric clonalization
Host
Defenses
Clonalization
ExPEC*
Allopatric clonalization
Sympatric clonalization
NonExPEC
* From James R.
“Linneus” Johnson
The elimination of intermediates
Impossibility of being a business man and a little meermaid
Species-Environment Concerted Evolution
Phylogenetic
groups
Core
genome
Basic
reproductive
environment
species evolution
environmental evolution
Co-evolution: Trees within Trees
Host
Bacteria or bacterial consortium
The clues of E. coli
genetic diversity
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•
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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
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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
1,000 genes of difference!
http://colibase.bham.ac.uk
E. coli genomes
http://colibase.bham.ac.uk
Loops in a common core backbone
A-strain
A-loop (A-island)
B-strain
B-loops (B-islands)
Loops in a common core backbone
1,393 kb
296 loops in
E.A-strain
coli Sakai
325 loops in
E.B-strain
coli K12
BB: 3,730 kb
BB: 3,730 kb
S-loops
K-loops
537 kb
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, 6:171, 2005
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)
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.
The loops
• The backbone evolves by vertical transfer.
• Large loops are probably acquired by horizontal
gene transfer, but also evolve by vertical transfer.
PAIs, islets, phages, plasmids, transposable, repetitive elements...
• 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)
Random-scale sub-network (loop)
ALIEN
nodes
Operative genes are
more easily accepted
links
Elaboration from
Jain et al.
ALIEN
Scale free network (core)
nodes
Informative genes
less easily accepted
Number of links (log)
Elaboration from
Jain et al.
Informative genes
less easily accepted
except alien
replacement of an
entire sub-network
ALIEN
Subnetwork
Scale free network (core)
nodes
Number of links (log)
3,256 E. coli genes are
connected by 113,894 links
Predicted functional
modules in E. coli
(von Mering et al., PNAS
100:15428, 2003)
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
Excision
Modification
Acquisition
Loss
Duplication
Modification
(Daubin et al., Genome Biol., 4:R57, 2003;
Ochman and Jones, EMBO J., 19:6637, 2000)
More loss in
sequences of recent
acquisition*
Insertions and
deletions occur more
frequently in loops
Overall less loss than
acquisition?
Gene flux
Acquisition
Excision
Modification
Constant
Random
Gene
Influx?
Loss
Duplication
Modification
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 MG1655specific)
(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
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Ecological separation from donor
DNA sequence divergence
Low numbers
Inadequate phage receptors
Inadequate pilus specificity for mating
Contact-killing or inhibition
Surface exclusion
*200 enzymes!
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?)
Sequence
divergence reduces
acquisition of
foreign DNA
If the acquisition
produce neutral events
the tolerance increases
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
Species-Environment Concerted Evolution
Phylogenetic
groups
Core
genome
Basic
reproductive
environment
species evolution
environmental evolution
Genome Size in E. coli strains
ECOR Phylogenetic Groups
kb
5,4
5,2
5
4,8
K12
level
4,6
4,4
4,2
4
A
B1
B2
D
Data: Bergthorsson and Ochman, Microb. Biol. Evol. 15:6-16, 1998
Phylogenetic groups: clinical associations
100
90
80
70
60
50
40
30
20
10
0
A
B1
B2
Clinical
Cystitis
Febrile UTI
Rectal (FUTI)
Faecal HV-Fr
Faecal HV-Sp
D
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
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)
But: “Epidemic extraintestinal
100 many SxT-R in UTI
strains”,
90Israel, France (Johnson
in US,
et 80
al.,EID 11:141, 2005)
70
60
50
40
30
20
10
0
A
B1
B2
Clinical
Cystitis
Febrile UTI
Rectal (FUTI)
Faecal HV-Fr
Faecal HV-Sp
D
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
Distribution of E. coli isolates from hospitalized
patients and from healthy volunteers among the
four phylogenetic groups
50
% of strai n s
40
30
20
10
0
A
B1
B2
D
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
Antimicrobial-R in phylogenetic groups
80
70
60
50
40
30
20
10
0
A
B1
SxT-R
ESBLs
B2
D
Cipro-R(1)
Cipro-R(2)
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).
The phylogenetic group B2, the more pathogenic one, tends to
be the less resistant?
Species-Environment Concerted Evolution
Ecotypes
Core
genome
Basic
reproductive
environment
species evolution
environmental evolution
Models for Multiple Ecotypes
(Gevers et al., Nature MR 3:733, 2005)
Clonalization
Patients with different ESBL clones
Ramón y Cajal Hospital, Madrid
(Baquero, Coque & Cantón, Lancet I.D. 2:591, 2002)
30
No. of patients/clone
25
20
15
10
5
0
88 89 90 91 92 93 94 95 96 97 98 99
Ye ar
0
Mutation: Intra-Clonal Diversity
80
E. coli : Faecal - Urine - Blood - ESBLs
70
% of strains
60
50
40
30
20
10
0
Hypo
Normo
Weak
Mutation frequency
Baquero et al, AAC 2004 and Nov. 2005
Strong
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
*epochal evolution
The maintenance
of clonal
ensembles is
favored by the
assymetry of
fitness abilities
in different
clones in
different epochs
Clonal ensemble
Shared Environments and Maintenance of
Diversity
A regional polyclonal community structure
1
2
1
Alternative stable equilibria and the
coexistence of variant organisms
On this topic: Geographic mosaic theory of coevolution, Forde et al, Nature, 2004
Maintenance of diversity
A regional polyclonal community structure
1
2
Local
Migration
1
Local
Gene Flow
Diversity: Collapse and Resurrection
Kin effects in
open systems
SELECTION
Maintenance of diversity
A 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 diversity
A regional polyclonal community structure
Gradients and concentrationdependent 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 reallife game of rock-paper-scissors. Nature 418:171, 2002
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....
Rock, Paper, Scissors Model
B. Kerr et al., Local dispersal promotes biodiversity in a reallife game of rock-paper-scissors. Nature 418:171, 2002
Rock, Paper, Scissors Model
B. Kerr et al., Local dispersal promotes biodiversity in a reallife game of rock-paper-scissors. Nature 418:171, 2002
Kindly provided by
Teresa Coque et al., 2005
In60-like integrons
Int1
Int1
aadB
qacED1sul1
orf513
dfrA10
aadA2
qacED1sul1
orf513
ampC
Int1
aac(6)
aadA2 2
qacED1sul1
blaOXA-2 orfD qacED1sul1
aacA4
Int1
Int1
dfrA16
dfrA16
blaoxA30
aadA22
catB3
qacED1sul1
aar-3 qacED1sul1
qacED1sul1
orf513
qacED1 sul1
aadA2
Int1
Int1
qacED1sul1
aacA4
catA2
qacE D1 sul1
orf5
orf5
qacE D1 sul1
ampR
orf5
CTX-M-9
orf513
bla CTXM-9
orf3-like
CTX-M-2
orf513
orf513
orf513
orf513
qacED1sul1
orf513
qacED1sul1
orf513
blaCTXM-2
qnr
qnr
bla DHA
orf3:: qacED1 sul1
ampR
int1
ampR
orf1
qacED1 sul1 orf5 orf6
qacED1 sul1 orf5 orf6
ampR
dfrA18
qacED1 sul1
IS3000
oxa1 aadA1
IS6100
IS6100
qacED1 sul1
qacED1 sul1
blaDHA
ampR
qacED1 sul1
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 (colonizationpathogenicity) 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 (warmblooded 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
Absolute
fitness
E. coli
K. pneumoniae
5
0
-5
-10
-15
-20
10
20
30
40 50
10
Temperature (ºC)
20
30
40
Modified from: Okada and Gordon, Mol. Ecol. 10:2499, 2001
50
CTX-M-10 linked to Kluyvera and phage sequences
Tn1000-like
Transposase
ORF3
(fragment) ORF2
ORF4
BamHI
EcoRI BamHI
DNA CTX-M-10
Transposase
ORF8 Transposase
ORF11
invertase
ORF7
IS432 ORF10
IS5
EcoRI
Invertible
region
Phage related region
EcoRI
EcoRI
BamHI
Tn 5708
fragment
IS4321
IS5
K. cryocrescens
homol. region (90%)
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
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
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., 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...
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Rafael Cantón
Teresa Coque
Juan-Carlos Galán
José-Luis Martínez (CNB, CSIC)
Gerdes SY et al, JB 2003