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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
Author(s) -
Heuzenroeder M. W.,
Beger D. W.,
Thomas C. J.,
Manning P. A.
Publication year - 1989
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1989.tb00174.x
Subject(s) - biology , escherichia coli , plasmid , microbiology and biotechnology , antigen , gel electrophoresis , molecular cloning , cloning (programming) , restriction fragment , enterobacteriaceae , polyacrylamide gel electrophoresis , restriction map , gene , genetics , gene expression , biochemistry , enzyme , computer science , programming language
Summary 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 pPM 1330expressed 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 a close relationship among the 0101 ETEC isolates.