Analysis of the genes responsible for the O-antigen synthesis in enterohaemorrhagicEscherichia coliO157

Abstract

The genes responsible for O157 O-antigen synthesis were cloned from Shiga toxin 1 (Stx1)-producing enterohaemorrhagicEscherichia coliO157:H-. Based on the published sequence of therfbEEcoO157:H7gene, therfbEgene was amplified by PCR and used as the probe. One clone (1-4-1) out of ten agglutinated using the slide agglutination test with O157 antiserum. The DNA sequence of a 31.5 kb fragment in p1-4-1 was determined and found to contain 26 open reading frames (ORFs).These ORFs were organized as two clusters of genes, comprisingorf2toorf16andorf17toorf24regions, which were aligned in opposite directions. The GC contents oforf1,orf12andorf14toorf26were similar to the overall GC content of theE. colichromosome (51%). However,orf2toorf11andorf13showed a much lower GC content of 30.0 to 39.4%. The minimum region essential for O157 O-antigen expression inE.coliK-12 lay betweenorf2andorf13, which corresponded to the region of low GC content inE.coliO157. A colony hybridization test was carried out against reference strains ofE. colirepresenting serogroups O1 to O173 using O157 O-antigen synthesis genes (orf2–orf13) ofE. coliO157 as probes. With the exception oforf12, all probes fromE. coliO157 O-antigen synthesis gene cluster did not hybridize with DNA fromE. coliO1 to O173, except O157 reference strains, under high stringency conditions. These data suggest that the region fromorf2toorf13has originated from a non-E. colispecies with a low GC content.