Molecular cloning and expression in Escherichia coli K-12 of the O101 rfb region from E. coli B41 (O101:K99/F41) and the genetic relationship to other O101 rfb loci.
The gene cluster (rfb region) which determines the synthesis of O101 lipopolysaccharide (LPS) O-antigen was cloned from the Escherichia coli O101:K99:F41 reference strain B41 to give plasmid pPM1301. The smallest subclones represented by pPM1305 and pPM1330 expressed O-antigen in E. coli K-12 similar to (but not identical to) B41, as judged by immunogold electron microscopy and silver staining of LPS separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). At least six proteins were detected by minicell analysis of proteins encoded by pPM1305, which suggests that O-antigen synthesis is genetically complex. Restriction and deletion analysis demonstrated that a minimum of 8.9 kb and a maximum of 11.8 kb are required for O101 O-antigen biosynthesis in E. coli K-12. Examination of LPS banding patterns of other O101 isolates by SDS-PAGE suggested heterogeneity of LPS structure. Southern DNA hybridization analysis using radiolabelled subclones of pPM1305 demonstrated that there was close relationship among the O101 ETEC isolates.
Heuzenroeder
M W
MW
Department of Microbiology and Immunology, University of Adelaide, South Australia.
Beger
D W
DW
Thomas
C J
CJ
Manning
P A
PA
D000942:Antigens, Bacterial / Q000235:genetics*; D015139:Blotting, Southern; D003001:Cloning, Molecular / Q000379:methods; D015342:DNA Probes; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000145:classification / Q000235:genetics*; D005798:Genes, Bacterial; D007150:Immunohistochemistry; D005810:Multigene Family; D019081:O Antigens; D010957:Plasmids; D015183:Restriction Mapping; D012703:Serotyping
D000942:Antigens, Bacterial; D015342:DNA Probes; D004269:DNA, Bacterial; D019081:O Antigens