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Comparative Study
. 2010 Jan 20;5(1):e8801.
doi: 10.1371/journal.pone.0008801.

Complete Genome Sequence and Comparative Metabolic Profiling of the Prototypical Enteroaggregative Escherichia Coli Strain 042

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Free PMC article
Comparative Study

Complete Genome Sequence and Comparative Metabolic Profiling of the Prototypical Enteroaggregative Escherichia Coli Strain 042

Roy R Chaudhuri et al. PLoS One. .
Free PMC article

Abstract

Background: Escherichia coli can experience a multifaceted life, in some cases acting as a commensal while in other cases causing intestinal and/or extraintestinal disease. Several studies suggest enteroaggregative E. coli are the predominant cause of E. coli-mediated diarrhea in the developed world and are second only to Campylobacter sp. as a cause of bacterial-mediated diarrhea. Furthermore, enteroaggregative E. coli are a predominant cause of persistent diarrhea in the developing world where infection has been associated with malnourishment and growth retardation.

Methods: In this study we determined the complete genomic sequence of E. coli 042, the prototypical member of the enteroaggregative E. coli, which has been shown to cause disease in volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains revealing previously uncharacterised virulence factors including a variety of secreted proteins and a capsular polysaccharide biosynthetic locus. In addition, by using Biolog Phenotype Microarrays we have provided a full metabolic profiling of E. coli 042 and the non-pathogenic lab strain E. coli K-12. We have highlighted the genetic basis for many of the metabolic differences between E. coli 042 and E. coli K-12.

Conclusion: This study provides a genetic context for the vast amount of experimental and epidemiological data published thus far and provides a template for future diagnostic and intervention strategies.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Circular representation of the E. coli O42 chromosome.
From the outside in, the outer circle 1 marks the position of regions of difference (mentioned in the text) including prophage (light pink) fimbrial operons (Dark green) as well as regions differentially present in other E. coli strains: blue (Present in 0157:H7 & absent/divergent in UPEC CFT073) Light Green (Present in 0157:H7 absent/divergent in UPEC CFT073). Circle 2 shows the size in bps. Circles 3 and 4 show the position of CDSs transcribed in a clockwise and anticlockwise direction, respectively (for colour codes see below); circle 4 to 13 show the position of E. coli O42 genes which have orthologues (by reciprocal FASTA analysis) in other E. coli strains (see methods): Sakai (0157:H7; red), UT189 (UPEC; dark blue), CFT073 (UPEC; light blue), 536 (UPEC; orange), APEC 01 (APEC; dark pink), E2348/69 (EPEC; black), H10407 (ETEC; salmon pink), E24377A (ETEC; pale pink), HS (grey), and K-12 MG1655 (green). Circle 14 sows the position of genes unique to E. coli 042 unique (red). Circle 15 shows a plot of G+C content (in a 10 Kb window). Circle 16 shows a plot of GC skew ([G−C]/[G+C]; in a 10 Kb window). Genes in circles 3 and 4 are colour coded according to the function of their gene products: dark green = membrane or surface structures, yellow = central or intermediary metabolism, cyan = degradation of macromolecules, red = information transfer/cell division, cerise  = degradation of small molecules, pale blue  = regulators, Salmon pink = pathogenicity or adaptation, black = energy metabolism, orange = conserved hypothetical, pale green = unknown, brown = pseudogenes.
Figure 2
Figure 2. Gene organisation of the Tn21 element containing loci encoding antibiotic resistance.
The Tn21 element is inserted between genes lpfA and glmS and constitutes ROD 66. The presence of this locus is consistent with the phenotypic information garned from the BioLog assays.
Figure 3
Figure 3. Measurement of small molecule uptake.
Hoescht 33342 is a substrate of the major AcrAB-TolC efflux systems. Accumulation by E. coli MG1655 and EAEC 042 was measured fluorometrically in the presence or absence of the efflux pump inhibitor PAβN and the proton-motive force inhibitor CCCP. Efflux of Hoescht 33342 is inhibited in the presence of both PAβN and CCCP in EAEC 042 and E. coli MG1655. However, Hoescht 33342 accumulates to higher levels in EAEC 042 than E. coli MG1655 suggesting EAEC 042 possesses a more permeable membrane.
Figure 4
Figure 4. Phylogenetic analyses of the porin CDS from EAEC 042 and E. coli K-12.
The CDS encoding ompN, ompC, ompF and phoE demonstrated little divergence. By comparison that encoding ompD(nmpC) demonstrates greater divergence and Ec042-2121 is present on an evolutionary distinct lineage.
Figure 5
Figure 5. Comparison of the genetic content of the three genome sequenced EAEC isolates (042, 55989 and 101) with the commensal strain E. coli HS.
The four strains share a large proportion of common genes. Only 210 EAEC specific genes were found (see text for details).
Figure 6
Figure 6. Functional characteristics of the EAEC 042 agn43 alleles.
The three Antigen 43 encoding genes (Ec042-2242, Ec042-4511 and Ec042-4803) were cloned into a high copy number vector (pCRII-TOPO) and expressed in E. coli TOP10. (A) Western immunoblot of E. coli outer membrane fractions probed with anti-α43 antisera demonstrating all three alleles produced cross reacting species. As expected from nucleotide sequence analysis Ec042-4803 produces a smaller passenger domain than Ec042-2242, Ec042-4511 and Antigen 43 from E. coli K-12. Autoaggregation assays (B) and biofilm assays (C) of E. coli expressing each agn43 allele demonstrated that each gene product was capable of promoting autoaggregation or biofilm in a manner similar to that previously described. There was no appreciable difference between the proteins in their capacity to induce autotaggregation or biofilm formation. A strain lacking a functional agn43 allele (E. coli deltaflu) failed to autoaggregate and failed to form a biofilm.
Figure 7
Figure 7. Structural predictions of YtfM.
(A) The conserved TpsB homologue YtfM was modelled using SWISS-MODEL and is predicted to form an outer membrane β-barrel structure with two POTRA domains extending into the periplasm. The model was based on the crystal structure of FhaC, the well-characterised TpsB translocator of B. pertussis filamentous haemagglutinin; the first POTRA domain could not be modelled. (B) Secondary structure predictions of the N-terminal domain of YtfM using PsiPred predicts the presence of three POTRA domains based on the structural motif β–α–α–β–β; the POTRA domains are highlighted by red, green and cyan lines. The amino acid sequence corresponding to the β-barrel is truncated. Arrows corresponds to β-strands whereas α-helices are depicted by cylinders.

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