Functional evolution of bacterial genome

1
Functional evolution of bacterial genome

• The Escherichia coli case

2
Plan

• Presentation of E. coli
• Genomes sequenced
• Genomic island
• Definition
• Roles
• Examples
• Tools for functional analysis
• Conclusion

3
Escherichia coli

• Theodor Escherich (1857-1911), a German
  pediatrician and bacteriologist discovered
  Escherichia coli, which was named after him in
  1919
• Gram-negative bacilli, facultatively anaerobic,
  non motile or motile by peritrichous flagella

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E. coli the gram-negative model organism

• Catapulted to prominence by the discovery of
  strain K-12s ability to carry out genetic
  recombination by conjugation (Tatum, 1946) and by
  generalized transduction (Lennox, 1955)
• Easy to rear, small, cheap, rapid growth cycle,
  short lived, genetically manipulable

5
E. coli habitat

• Resident of animal intestinal tracts
• Well adapt to life in rivers, oceans and soils
• Can be found living at -2C in McMurdo Bay
  (Antarctica)
• Indicator of fecal pollution and water
  contamination

Number of E. coli/ml water
6
E. coli a versatile bacteria

• Commensal
• Use a probiotic
• Could be virulent

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E. coli as a commensal

• Intestinal tract of humans and many animal
  species
• Source of vitamin K and B-complex vitamins
• One of the first bacterial species to colonize
  the infants intestine

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E. coli as a pathogen

• Intra intestinal infections iPEC
• Extra intestinal infections ExPEC

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E. coli as a probiotic

• E. coli strain Nissle 1917 (O6K5H1) forms the
  basis of the probiotic preparation Mutaflor
• used for treatment of various intestinal
  disorders (Crohns disease)
• successful colonizer of the human gut

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iPEC

• ETEC - enterotoxigenic E. coli (Cholera-like)
• EIEC - enteroinvasive E. coli (Shigella-like)
• EHEC - enterohemorrhagic E. coli (Hamburger
  disease)
• EPEC - enteropathogenic E. coli (Neonatal
  diarrhea)
• EAEC - Enteroaggregative E. coli
• DAEC - Diffuse adhering E. coli

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ExPEC

• UPEC uropathogenic E. coli
• MENEC new born meningitis causing E. coli
• SEPEC septicemia-causing E. coli
• APEC avian pathogenic E. coli

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Serotyping

• Serotyping scheme based on three types of antigen
  
• the somatic (O) antigen
• the capsular (K) antigen
• the flagellar (H) antigen
• Over 700 antigenic types

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E. coli genomes sequences
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K-12 genome sequences

• Independent efforts by American and Japanese
  groups using two different strains of K-12
  MG1655 and W3110
• These strains were diverged from the same
  ancestral strain about 50 years ago
• Slight but significant differences including the
  large inversion involving the ribosomal RNA genes

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Genome sequencing

• First genome of E. coli that has been sequenced
  MG1655, a commensal strain in 1997(The complete
  genome sequence of Escherichia coli K-12, Science
  277 (5331), 1453-1474 (1997) )
• Determination of the complete E. coli sequence
  has required almost 6 years

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General features of MG1655 genome

• 4,639,221-base pair
• 4288 protein-coding genes annotated
• Protein-coding genes account for 87.8 of the
  genome

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Status of annotation of E. coli gene products
(2006)
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Genome of E. coli sequenced

• UPEC strain
• Welch et al., 2002, PNAS

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Genome of E. coli sequenced

• EHEC strains
• EDL933 (Perna et al., 2001, Nature)
• SaKai (Hayashi et al., 2001, DNA Research)

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Current E. coli genomes sequenced or in progress
Strains type Number of genes Length (bp) Numbers of contigs
B03 commensal 4387 4,629,810 1
MG1655 commensal 4,254 4,639,675 1
W3110 commensal 4,390 4,641,433 1
HS commensal 3,689 4,643,538 1
101NA1 EAEC 4,238 4,880,380 70
536 UPEC 4800 4,900,000 1
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Current E. coli genomes sequenced or in progress
Strains type Number of genes Length (bp) Numbers of contigs
E24377A ETEC 4,254 4,980,187 1
E2348 EPEC 4,594 5,072,200 4
RS218 NMEC 4,900 5,089,235 1
B7A ETEC 4,637 5,202,558 198
F11 UPEC 4,467 5,206,906 88
H10407 ETEC 5,000 5,208,000 225
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Current E. coli genomes sequenced or in progress
Strains type Number of genes Length (bp) Numbers of contigs
CFT073 UPEC 5,379 5,231,428 1
042 EAEC 4,899 5,241,977 2
53638 EIEC 4,783 5,289,471 119
B171 EPEC 4,467 5,299,753 159
E110019 Atypical EPEC 4,746 5,384,084 119
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Current E. coli genomes sequenced or in progress
Strains type Number of genes Length (bp) Numbers of contigs
SAKAI EHEC 5,361 5,498,450 1
E22 EPEC 4,788 5,516,160 109
EDL933 EHEC 5,349 5,289,471 1
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Undergoing project Coliscope

• Commensal IAI1 (serogroup O8)
• EAEC 55989
• UPEC IAI39 (serogroup 07)
• UPEC UMN026 (serogroup 017)
• Commensal ED1a (serogroup O81)
• MNEC S88 (serogroup O45)

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Average genome size

• From 4.62 Mb (Commensal isolates)
• To 5.28 Mb (Virulent isolates)
• Much of this diversity comes from bacteriophages
  BUT
• Presence of foreign DNA concept of genomic
  island (GEIs)

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Genomic islands
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Definition of genomic island

• Concept of PAI (pathogenicity island) was founded
  in the late 1980s by Jörg Hacker and colleagues
  in Werner Goebel's group at the University of
  Würzburg, Würzburg, Germany

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Common features of genomic island

• presence of virulence genes
• Specific presence in pathogens, absence in benign
  relatives
• Large distinct chromosomal regions (10 to 200 kb)
• Characteristic base composition different from
  core genome
• Insertion of PAI adjacent to tRNA genes

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Common features of genomic island

• Frequent association with mobile genetic
  elements, i.e., presence of    
• DR   
• Cryptic or functional integrase or
  transposase    
• IS elements    
• Chromosomally integrated conjugative transposons,
  plasmids, and phages
• Genetic instability (if functional mobility
  elements are present)
• Mosaic structures of several acquisitions

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GEIs can be involved in

• Pathogenicity
• Symbiosis
• Fitness
• Metabolism
• Resistance to xenobiotics

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Comparison between EHEC and K-12

• Common linear backbone of 4.1 Mb
• 1.34 Mb specific to EHEC (O-islands)
• 0.53 Mb specific to K-12 (K-islands)

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O-islands larger than 15 kb encoding factors

• LEE (locus of enterocyte effacement)
• Macrophage toxin and ClpB-like chaperone (IAHP
  island)
• RTX-like exoprotein and a transport system

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Contribution of mobile genetic elements to the
evolution of pathogenic E. coli
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One of the best studied PAI The LEE

• LEE locus of enterocyte effacement
• Present in enteropathogenic and
  enterohaemmoragic strains
• Encode a type III secretory system that injects
  proteins into host cells to modulate function.

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A/E lesions caused by EPEC
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A/E lesions caused by EPEC
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A/E lesions caused by EPEC
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The TTSS

• Secretion and translocation of bacterial effector
  proteins into and through the host cell membrane

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Identification and characterization of a genomic
island from an avian ExPEC strain

• tRNA target screening

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Avian ExPEC

• Localized infection
• Respiratory diseases
• Some virulence factors identified
• Pathogenicity not fully understood

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tRNA target screening

• Design of primers after comparison of 4
  completely sequenced strains (MG1655, CFT073,
  EDL933 and Sakai)
• PCR analysis
• Focus on some loci to identify the EcDNA inserted
  (arbitrary PCR followed by a screening of a
  genomic library)

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selC an integration hot spot
Strain PAI Length (kb) function
E.coli 536 (UPEC) PAI-I536 70 hemolysin
E.coli CFT073 (UPEC) GEI 68
E.coli E2348/69 (EPEC) LEE 35 Type III secretion, invasion
E.coli O157H7 (EHEC) LEE 43 Type III secretion, invasion
E.coli (ETEC) Tia PAI 46 invasion
S.flexneri SHI-2 ND Aerobactin synthesis, transport
S.enterica SPI-3 17 Invasion, survival in monocytes
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AGI-3

• 49600pb inserted at selC. Mosaic structure 5
  modules of genes
• Presence of mobile genetic elements such as IS
  elements, integrase gene and direct repeats

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Features of aec35 to 37
DR2
DR1
DR2
DR1
36
yicM
selC
33
37
81
nlpA
34
35
yicL

• aec35 360 aa putative transcriptional regulator
  of the LacI regulator family (99 CFT073 C4494,
  74 Yersinia pestis C092, Yersinia pestis Kim,
  Yersinia pseudotuberculosis)
• aec36 452 aa putative MFS superfamily
  hexuronate transporter (100 CFT073 C4495, 85
  Yersinia pseudotuberculosis)
• aec37 795 aa putative a-glucosidase family 31
  (100 CFT073 C4496 and C4497, 83 Yersinia
  pseudotuberculosis)

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Prevalence in other bacteria
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Role in virulence for chicken

• Deletion of 3 orfs aec35, aec36 and aec37 (5kb)
  (Datsenko and Wanner, 2000)
• Experimental reproduction of colibacillosis
• 2 inocula preparations
• cullture at 37C with shaking since OD600nm 0.6,
  frozen at -70C
• culture at 37C without neither shaking nor
  freezing

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Experimental procedure

• Inoculation of 5.106 CFU (shaken inoculum) or 107
  CFU (non shaken inoculum) to 3 weeks old SPF
  chickens (12/set randomly constituted) into the
  right thoracic air sac
• Blood sampling at 24h and 48h p.i. and bacterial
  numeration
• 48h p.i., euthanasia, necropsy
• Lesion score determination
• Liver sampling and bacterial numeration
• Statistical analysis Wilcoxon-Mann-Withney (non
  parametric test)

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Bacteraemia
The mutant derivative is less bacteraemic than
the wt strain
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Liver colonization
The mutant derivative less colonized the liver in
comparison to the wt strain
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Lesions scores
No lesion score difference
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Conclusion (1)

• The ?aec35 to aec37 mutant induces same lesions
  as the wt but is less bacteriaemic and is less
  able to colonize liver
• Involvement of these three orfs in the
  pathogenicity of BEN2908 for chickens
• Functional analysis of these three orfs
  (determination of the sugar involved....)

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Phenotypic analysis of aec35, aec36 and aec37

• Reminder
• Protein identity
• aec35 putative transcriptional regulator LacI
  family
• aec36 putative MFS superfamily hexuronate
  transporter
• aec37 putative glucosidase family 31 of
  glycosyl hydrolases
• Phenotype Microarrays (Biolog Inc.)
• minimal media
• 190 carbon sources
• aerobic conditions 37C
• OD590 mesures (ELISA reader)

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Phenotypic micro arrays results
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Growth in minimal media

• M9 Supplemented by trehalose or galacturonate
• Growth followed during 24h in a Bioscreen
  apparatus
• The mutant has a two-fold lower growth rate
  compared to the wt strain

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Conclusion (2)

• AGI-3 has all the features of a pathogenicity
  island
• Is involved in virulence
• Played a role in carbohydrate metabolism

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Tools for E. coli functional analysis
57

• E. coli K-12 one of the best-characterized
  organisms in molecular biology
• Many key ressources for functional genomics and
  systems biology of E. coli are still lacking

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Tools available for K-12 strains

• DNA microarrays transcriptome (available for
  K-12, UPEC and development of patho-arrays)
• The Keio collection mutants 3985 deletions (in
  duplicate) of the K-12 strain strain BW25113
• The ASKA library (A complete Set of E. coli K-12
  ORF Archive)
• Metabolome

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Transcriptome

• Transcriptome of K-12 in various growth
  conditions
• Transcriptome of uropathogenic Escherichia coli
  during urinary tract infection
• Transcriptome of carbon utilisation in the mouse
  intestine

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Proteome

• Many in vitro studies for K-12 strains
• Proteomic analysis of extracellular proteins from
  E. coli W3110
• carbon source variation
• Response to stress

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Metabolome

• Definition the full complement of metabolites
  of an organism
• The E. coli metabolome has been characterized
  using the two-dimensional structures of 745
  metabolites, obtained from the EcoCyc and KEGG
  databases.

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Conclusion

• E. coli is a versatile bacteria
• Many genomes of several E. coli strains are
  available
• Tools for functional analysis are developed
• A lot of data are available
• Three worlds exist in the field of E. coli
  research (non pathogenic, intestinal pathogenic
  and extra intestinal pathogenic strains)