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The E. coli Extended Genome Fernando Baquero Dept. Microbiology, Ramón y Cajal University Hospital, and Laboratory for Microbial Evolution, CAB (INTA-CSIC)

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Presentation on theme: "The E. coli Extended Genome Fernando Baquero Dept. Microbiology, Ramón y Cajal University Hospital, and Laboratory for Microbial Evolution, CAB (INTA-CSIC)"— Presentation transcript:

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 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

4 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

5 Adaptation Complexity: Mutation Single adaptive event

6 Clonalization Multiple adaptive events

7 Speciation Very complex adaptive events

8 Clonalization Allopatric clonalization Sympatric clonalization

9 Clonalization Allopatric clonalization Sympatric clonalization Host Defenses ExPEC* Non- ExPEC * From James R. “Linneus” Johnson

10 and Impossibility of being a business man and a little meermaid The elimination of intermediates

11 Species-Environment Concerted Evolution species evolution environmental evolution Basic reproductive environment Phylogenetic groups Core genome

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) 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)

15 E. coli genomes 1,000 genes of difference!

16 E. coli genomes

17 Loops in a common core backbone A-strainB-strain A-loop (A-island) B-loops (B-islands)

18 Loops in a common core backbone A-strainB-strain S-loopsK-loops 296 loops in E. coli Sakai 325 loops in E. coli K12 BB: 3,730 kb 1,393 kb537 kb

19 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

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 % 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 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)

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 links Operative genes are more easily accepted

24 Scale free network (core) Informative genes less easily accepted Elaboration from Jain et al. ALIEN Number of links (log) nodes

25 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

26 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

27 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)

28 Gene flux AcquisitionLoss Duplication Modification 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)

29 Gene flux Acquisition Loss Duplication Modification Excision Modification Constant Random 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.

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 Daubin&Ochman, 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 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

33 Species-Environment Concerted Evolution species evolution environmental evolution Basic reproductive environment Phylogenetic groups Core genome

34 Genome Size in E. coli strains ECOR Phylogenetic Groups K12 level kb Data: Bergthorsson and Ochman, Microb. Biol. Evol. 15:6-16, 1998

35 Phylogenetic groups: clinical associations 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

36 Phylogenetic groups: clinical associations 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)

37 Distribution of E. coli isolates from hospitalized patients and from healthy volunteers among the four 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)

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

39 Species-Environment Concerted Evolution species evolution environmental evolution Basic reproductive environment Ecotypes Core genome

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

41 Patients with different ESBL clones Ramón y Cajal Hospital, Madrid (Baquero, Coque & Cantón, Lancet I.D. 2:591, 2002)

42 E. coli : Faecal - Urine - Blood - ESBLs Baquero et al, AAC 2004 and Nov Mutation: Intra-Clonal Diversity

43 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

44 Shared Environments and Maintenance of Diversity A regional polyclonal community structure Alternative stable equilibria and the coexistence of variant organisms 112 On this topic: Geographic mosaic theory of coevolution, Forde et al, Nature, 2004

45 Maintenance of diversity A regional polyclonal community structure 112 Local Migration Local Gene Flow

46 Diversity: Collapse and Resurrection SELECTION Kin effects in open systems

47 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

48 Maintenance of diversity A 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 418:171, 2002

50 Rock, Paper, Scissors Model 1. If the stones reduces its attack again scissors Scissors increase its power against paper And less paper means more stones...

51 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

52 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

53

54 Int1 aacA4 bla OXA-2 orfD qacE  1sul1 orf513 bla CTXM-2 orf3:: qacE  1 sul1 Int1 aacA4 aadA2 qacE  1sul1 orf513 catA2 qacE  1 sul1 orf5 Int1 aadB qacE  1sul1 orf513 dfrA10 qacE  1 sul1 orf5 Int1 aadA2 qacE  1sul1 orf513 ampC ampR qacE  1 sul1 orf5 Int1 dfrA16 aadA2 2 qacE  1sul1 orf513 bla CTXM-9 orf3-like IS3000 qacE  1 sul1 Int1 dfrA16 aadA2 2 qacE  1sul1 orf513 qnr ampR qacE  1 sul1 orf5 orf6 IS6100 Int1 aac(6) bla oxA30 catB3 aar-3 qacE  1sul1 orf513 qnr ampR qacE  1 sul1 orf5 orf6 IS6100 qacE  1sul1 orf513 dfrA18 int1 oxa1 aadA1 qacE  1 sul1 qacE  1sul1 orf513 bla DHA ampR qacE  1 sul1 qacE  1sul1 orf513 orf1 bla DHA ampR qacE  1 sul1 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

56 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)

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, 2002; Santamaría&Toranzos, Int.Microbiol. 6:5-9, 2003)

58 E. coli in the environment EPA Class A Biosolids Less than 10 3 thermotolerant coliforms/g, for lawns, home gardens, as commercial fertilizer. EPA Class B Biosolids Less than 10 6 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 Temperature (ºC) E. coliK. pneumoniae Absolute fitness Modified from: Okada and Gordon, Mol. Ecol. 10:2499, 2001

60 Oliver, Coque, Alonso, Valverde, Baquero, Cantón. AAC 2005; EcoRI Tn1000-like Transposase (fragment) ORF2 ORF3 DNA invertase CTX-M-10 ORF7 ORF8 Transposase IS432 ORF10 Transposase IS5 Invertible region K. cryocrescens homol. region (90%) Tn 5708 fragment IS4321 IS5 Phage related region ORF4ORF11 EcoRI 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

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

62 Extended Genome: a Genetic Space Core Loops Peripheral

63 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

64 Extended Genome: a Genetic Space Filling the Carrying Capacity of the Environment for the Species

65 Genetic Space

66 Complex Genetic Space

67 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

68 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)

69 Gerdes SY et al, JB 2003

70


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