title: Role of glycoside hydrolase genes in sinigrin degradation by E. coli O157:H7.
abstract: This work examined Escherichia coli O157:H7 strain 02-0304 for putative genes responsible for sinigrin hydrolysis. Sinigrin is a glucosinolate present in Oriental mustard (Brassica juncea), and its hydrolysis is mediated in plants by the enzyme myrosinase. Sinigrin hydrolysis by plant or bacterial myrosinase yields allyl isothiocyanate (AITC) which is bactericidal. In silico analysis using public databases found sequence similarity between plant myrosinase and enzymes encoded by genes from β-glucosidase families in E. coli O157:H7. Specifically, 6-phospho-β-glucosidase encoded by the genes bglA and ascB (family 1), and chbF (family 4) present in E. coli O157:H7 showed the highest similarity. Polymerase chain reaction (PCR) confirmed the presence of bglA, ascB, and chbF in the clinical E. coli strain tested. Disruption of these genes in wild-type E. coli O157:H7 strain 02-0304 using lambda-red replacement created single and double mutants. The relative importance of each gene in the hydrolysis of sinigrin by E. coli O157:H7 was also assessed by comparing gene expression and sinigrin degradation rates among the E. coli O157:H7 wild-type strain and its mutants. The results suggested that the genes bglA and ascB play a substantial role in the degradation of sinigrin by E. coli O157:H7 strain 02-0304.
authors.affiliation: Department of Food Science, Faculty of Agriculture and Food Sciences, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.
authors.forename: Roniele P
authors.initials: RP
authors.lastname: Cordeiro
authors.affiliation: Department of Animal Science, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.
authors.forename: Juan H
authors.initials: JH
authors.lastname: Doria
authors.affiliation: Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, MB R3E 3P5, Canada.
authors.forename: George G
authors.initials: GG
authors.lastname: Zhanel
authors.affiliation: Department of Microbiology, Faculty of Science, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.
authors.forename: Richard
authors.initials: R
authors.lastname: Sparling
authors.affiliation: Department of Food Science, Faculty of Agriculture and Food Sciences, University of Manitoba, Winnipeg, MB R3T 2N2, Canada. Electronic address: rick_holley@umanitoba.ca.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Holley
mesh_terms: D019453:Escherichia coli O157 / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005959:Glucosidases / Q000235:genetics* / Q000378:metabolism; D005961:Glucosinolates / Q000378:metabolism*; D006868:Hydrolysis; D009149:Mustard Plant / Q000737:chemistry / Q000382:microbiology; D009154:Mutation
chemical_list: D005961:Glucosinolates; C010330:sinigrin; D005959:Glucosidases; C066586:6-phospho-beta-glucosidase
keywords: E. coli O157:H7; Glucosinolates; Glycoside hydrolase; Mustard; Myrosinase; Sinigrin