Dataset Information

Altered utilization of N-acetyl-D-galactosamine by Escherichia coli O157:H7 from the 2006 spinach outbreak.

ABSTRACT: In silico analyses of previously sequenced strains of Escherichia coli O157:H7, EDL933 and Sakai, localized the gene cluster for the utilization of N-acetyl-D-galactosamine (Aga) and D-galactosamine (Gam). This gene cluster encodes the Aga phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) and other catabolic enzymes responsible for transport and catabolism of Aga. As the complete coding sequences for enzyme IIA (EIIA)(Aga/Gam), EIIB(Aga), EIIC(Aga), and EIID(Aga) of the Aga PTS are present, E. coli O157:H7 strains normally are able to utilize Aga as a sole carbon source. The Gam PTS complex, in contrast, lacks EIIC(Gam), and consequently, E. coli O157:H7 strains cannot utilize Gam. Phenotypic analyses of 120 independent isolates of E. coli O157:H7 from our culture collection revealed that the overwhelming majority (118/120) displayed the expected Aga+ Gam- phenotype. Yet, when 194 individual isolates, derived from a 2006 spinach-associated E. coli O157:H7 outbreak, were analyzed, all (194/194) displayed an Aga- Gam- phenotype. Comparison of aga/gam sequences from two spinach isolates with those of EDL933 and Sakai revealed a single nucleotide change (G:C-->A:T) in the agaF gene in the spinach-associated isolates. The base substitution in agaF, which encodes EIIA(Aga/Gam) of the PTS, changes a conserved glycine residue to serine (Gly91Ser). Pyrosequencing of this region showed that all spinach-associated E. coli O157:H7 isolates harbored this same G:C-->A:T substitution. Notably, when agaF+ was cloned into an expression vector and transformed into six spinach isolates, all (6/6) were able to grow on Aga, thus demonstrating that the Gly91Ser substitution underlies the Aga- phenotype in these isolates.

SUBMITTER: Mukherjee A

PROVIDER: S-EPMC2258687 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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Altered utilization of N-acetyl-D-galactosamine by Escherichia coli O157:H7 from the 2006 spinach outbreak.

Mukherjee Amit A Mammel Mark K MK LeClerc J Eugene JE Cebula Thomas A TA

Journal of bacteriology 20071221 5

In silico analyses of previously sequenced strains of Escherichia coli O157:H7, EDL933 and Sakai, localized the gene cluster for the utilization of N-acetyl-D-galactosamine (Aga) and D-galactosamine (Gam). This gene cluster encodes the Aga phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) and other catabolic enzymes responsible for transport and catabolism of Aga. As the complete coding sequences for enzyme IIA (EIIA)(Aga/Gam), EIIB(Aga), EIIC(Aga), and EIID(Aga) of the Aga PTS ar ...[more]

PMID: 18156259

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Project description:The bovine gastrointestinal (GI) tract is the main reservoir for enterohaemorrhagic Escherichia coli (EHEC) responsible for food-borne infections. Characterization of nutrients preferentially used by EHEC in the bovine intestine would help to develop ecological strategies to reduce EHEC carriage. However, the carbon sources that support the growth of EHEC in the bovine intestine are poorly documented. In this study, a very low concentration of glucose, the most abundant monomer included in the cattle dietary polysaccharides, was detected in bovine small intestine contents (BSIC) collected from healthy cows at the slaughterhouse. Six carbohydrates reported to be included in the mucus layer covering the enterocytes [galactose, N-acetyl-glucosamine (GlcNAc), N-acetyl- galactosamine (GalNAc), fucose, mannose and N-acetyl neuraminic acid (Neu5Ac)] have been quantified for the first time in BSIC and accounted for a total concentration of 4.2?mM carbohydrates. The genes required for enzymatic degradation of the six mucus-derived carbohydrates are highly expressed during the exponential growth of the EHEC strain O157:H7 EDL933 in BSIC and are more strongly induced in EHEC than in bovine commensal E.?coli. In addition, EDL933 consumed the free monosaccharides present in the BSIC more rapidly than the resident microbiota and commensal E.?coli, indicating a competitive ability of EHEC to catabolize mucus-derived carbohydrates in the bovine gut. Mutations of EDL933 genes required for the catabolism of each of these sugars have been constructed, and growth competitions of the mutants with the wild-type strain clearly demonstrated that mannose, GlcNAc, Neu5Ac and galactose catabolism confers a high competitive growth advantage to EHEC in BSIC and probably represents an ecological niche for EHEC strains in the bovine small intestine. The utilization of these mucus-derived monosaccharides by EDL933 is apparently required for rapid growth of EHEC in BSIC, and for maintaining a competitive growth rate as compared with that of commensal E.?coli. The results suggest a strategy for O157:H7 E.?coli survival in the bovine intestine, whereby EHEC rapidly consumes mucus-derived carbohydrates that are poorly consumed by bacteria belonging to the resident intestinal microbiota, including commensal E.?coli.

Project description:Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL933 harbors multiple prophage-associated open reading frames (ORFs) in its genome which are highly homologous to the chromosomal nanS gene. The latter is part of the nanCMS operon, which is present in most E. coli strains and encodes an esterase which is responsible for the monodeacetylation of 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac2). Whereas one prophage-borne ORF (z1466) has been characterized in previous studies, the functions of the other nanS-homologous ORFs are unknown. In the current study, the nanS-homologous ORFs of EDL933 were initially studied in silico Due to their homology to the chromosomal nanS gene and their location in prophage genomes, we designated them nanS-p and numbered the different nanS-p alleles consecutively from 1 to 10. The two alleles nanS-p2 and nanS-p4 were selected for production of recombinant proteins, their enzymatic activities were investigated, and differences in their temperature optima were found. Furthermore, a function of these enzymes in substrate utilization could be demonstrated using an E. coli C600?nanS mutant in a growth medium with Neu5,9Ac2 as the carbon source and supplementation with the different recombinant NanS-p proteins. Moreover, generation of sequential deletions of all nanS-p alleles in strain EDL933 and subsequent growth experiments demonstrated a gene dose effect on the utilization of Neu5,9Ac2 Since Neu5,9Ac2 is an important component of human and animal gut mucus and since the nutrient availability in the large intestine is limited, we hypothesize that the presence of multiple Neu5,9Ac2 esterases provides them a nutrient supply under certain conditions in the large intestine, even if particular prophages are lost.In this study, a group of homologous prophage-borne nanS-p alleles and two of the corresponding enzymes of enterohemorrhagic E. coli (EHEC) O157:H7 strain EDL933 that may be important to provide alternative genes for substrate utilization were characterized.

Dataset Information

Altered utilization of N-acetyl-D-galactosamine by Escherichia coli O157:H7 from the 2006 spinach outbreak.

Publications

Altered utilization of N-acetyl-D-galactosamine by Escherichia coli O157:H7 from the 2006 spinach outbreak.

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