title: 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.
authors.affiliation: Division of Molecular Biology, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Laurel, Maryland 20708, USA.
authors.forename: Amit
authors.initials: A
authors.lastname: Mukherjee
authors.forename: Mark K
authors.initials: MK
authors.lastname: Mammel
authors.forename: J Eugene
authors.initials: JE
authors.lastname: LeClerc
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Cebula
mesh_terms: D000116:Acetylgalactosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D019453:Escherichia coli O157 / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005947:Glucose / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000378:metabolism; D010935:Plant Diseases / Q000382:microbiology; D017354:Point Mutation; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000382:microbiology*
chemical_list: D029968:Escherichia coli Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D005947:Glucose; D000116:Acetylgalactosamine