Altered Utilization of N-Acetyl-D-Galactosamine by Escherichia coli O157:H7 from the 2006 Spinach Outbreak

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

^* To whom correspondence should be addressed. Email: Thomas.Cebula{at}fda.hhs.gov.

	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 N-acetyl-D-galactosamine phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) and other catabolic enzymes responsible for transport and catabolism of N-acetyl-D-galactosamine. As the complete coding sequences for Enzyme IIA (EIIA)^Aga/Gam, EIIB^Aga, EIIC^Aga, and EIID^Aga of the N-acetyl-D-galactosamine PTS are present, E. coli O157:H7 strains normally are able to utilize N-acetyl-D-galactosamine as a sole carbon source. The D-galactosamine PTS complex, in contrast, lacks EIIC^Gam, and, consequently, E. coli O157:H7 strains cannot utilize D-galactosamine. 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:CA:T) in the agaF gene in the spinach-associated isolates. The base substitution in agaF, which encodes EIIA^Aga/Gam of the PTS system, 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:CA: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 N-acetyl-D-galactosamine, thus demonstrating that the Gly91Ser substitution underlies the Aga^- phenotype in these isolates.