Analysis of the genes responsible for the O-antigen synthesis in enterohaemorrhagic Escherichia coli O157.

The genes responsible for O157 O-antigen synthesis were cloned from Shiga toxin 1 (Stx1)-producing enterohaemorrhagic Escherichia coli O157:H-. Based on the published sequence of the rfbE(EcoO157:H7) gene, the rfbE gene 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, comprising orf2 to orf16 and orf17 to orf24 regions, which were aligned in opposite directions. The GC contents of orf1, orf12 and orf14 to orf26 were similar to the overall GC content of the E. coli chromosome (51%). However, orf2 to orf11 and orf13 showed a much lower GC content of 30.0 to 39.4%. The minimum region essential for O157 O-antigen expression in E. coli K-12 lay between orf2 and orf13, which corresponded to the region of low GC content in E. coli O157. A colony hybridization test was carried out against reference strains of E. coli representing serogroups O1 to O173 using O157 O-antigen synthesis genes (orf2-orf13) of E. coli O157 as probes. With the exception of orf12, all probes from E. coli O157 O-antigen synthesis gene cluster did not hybridize with DNA from E. coli O1 to O173, except O157 reference strains, under high stringency conditions. These data suggest that the region from orf2 to orf13 has originated from a non-E. coli species with a low GC content. Copyright 1999 Academic Press.