PURIFICATION AND REGULATION OF THE F PLASMID TRAJ PROTEIN OF ESCHERICHIA COLI K-12 (CONJUGATION)

Abstract

The TraJ protein encoded by the conjugative plasmid F is required for transcription of the other F plasmid transfer (tra) genes, and hence for conjugal DNA donor activity and surface exclusion of F' and Hfr strains of E. coli. In addition, TraJ protein synthesis is itself subject to plasmid as well as host regulation.;To investigate its role in donor activity, the TraJ protein was purified using a (lamda)traJ lysogen that overproduces the protein after heat induction of the prophage. Sufficient TraJ was synthesized after induction to follow its purification by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The identity of the purified protein was confirmed by analysis of its amino-terminal amino acid sequence.;A polyclonal antiserum was prepared using purified TraJ protein. Anti-TraJ antibodies were isolated from the antiserum by affinity chromatography. This antibody was used in Western blots as an assay for TraJ protein, which is present in F('+) cells in otherwise undetectable amounts.;Donor strains were found to contain 2000-4000 molecules of TraJ protein per cell. Moreover, this level was reduced in chromosomal mutants in genes cpxA/B and sfrA which are phenotypically F('-), even though they contain a normal F plasmid. Hence, cellular gene products contribute to the maintenance of F plasmid-encoded TraJ protein levels.;Previous studies of TraJ localization in minicells or under conditions of gross overproduction suggested an outer membrane location for the TraJ protein. Studies of the sedimentation properties of overproduced TraJ protein revealed that the protein is insoluble, but not membrane associated. The antibody was used to assay the TraJ protein during fractionation of normal F('+) cell extracts. These fractionations indicated that the TraJ protein is cytoplasmic, which is compatible with a direct genetic regulatory role.;As a first step to characterize the native protein, the sedimentation coefficient of TraJ protein as it occurs in normal donor strains was determined. This value will be of further use in studies of native TraJ molecular weight and subunit analysis.