GENETIC-ANALYSIS OF UIDA EXPRESSION IN ENTEROHEMORRHAGIC ESCHERICHIA-COLI SEROTYPE O157-H7

Isolates of enterohaemorrhagic Escherichia coli serotype O157:H7 do not exhibit beta-glucuronidase (GUD) activity; however, they carry nucleotide sequences for the uidA gene that encodes the GUD enzyme. Polymerase chain reaction analysis using uidA-specific primers confirmed that a genetic region homologous in size to the E. coli uidA structural gene, including the regulatory region, was present in E. coli O157:H7 isolates. DNA sequencing analysis of the regulatory region and the 5' terminus of the uidA gene of E. coli O157:H7 showed a base substitution in the putative -10 promotor region and at 93 bases downstream from the initiation codon. Neither base alteration, however, appeared to affect uidA allele gene expression in the O157:H7 isolates. Immunoblotting of cell extracts with an anti-GUD antibody showed that E. coli O157:H7 isolates produced an antibody-reactive protein that was homologous in site to E. coli CUD, but no CUD activity was observed in cell-free extracts of these isolates. These results suggest that the antibody-reactive protein produced by E. coli O157:H7 may be an inactive GUD enzyme. Sequencing of the uidA structural gene in O157:H7 showed the presence of 18 additional nucleotide base substitutions. but only six altered the amino acid sequence. Also, there were two frame shift mutations, 18 bases apart, that altered the sequence of six consecutive amino acids. These genetic alterations in the uidA structural gene of O157:H7 may account for the absence of CUD activity in this serotype.