Role of glycoside hydrolase genes in sinigrin degradation by E. coli O157:H7

This work examined Escherichia colt 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 beta-glucosidase families in E. coli O157:H7. Specifically, 6-phospho-beta-glucosidase encoded by the genes bglA and ascB (family 1), and chbF (family 4) present in E. colt 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 0157: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. colt 0157:H7 was also assessed by comparing gene expression and sinigrin degradation rates among the E. colt 0157: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 0157:H7 strain 02-0304. (C) 2015 Elsevier B.V. All rights reserved.