A rapid screen for functional mutants of TraM, an autoregulatory protein required for F conjugation.
TraM is an autoregulatory protein required for conjugative transfer of the F plasmid. A rapid screening procedure was developed to select for traM mutants constructed by random PCR mutagenesis. The mutated traM gene was cloned into pT7-5, without the traM promoters (collectively called P( traM)), such that these mutants were expressed from the downstream traJ promoter, resulting in constitutive, low-level, transcription of traM by polymerases that had circumnavigated the plasmid. P( traM) was cloned into pPR9tt as a translational fusion in which a DNA fragment containing P( traM), the ribosome binding site and first 24 codons of traM was fused to the 5' end of lacZ. To downregulate beta-galactosidase expression, a -1 frameshift mutation was introduced at the junction between traM and lacZ in the fusion. Selected TraM mutants were further characterized for their intracellular levels, electrophoretic mobility on nondenaturing gels, and activity in F conjugation. Point mutations throughout TraM were found to affect both autoregulation and conjugative function.
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Department of Biological Sciences, CW405 Biological Sciences Bldg., University of Alberta, 11455 Saskatchewan Drive, Edmonton, T6G 2E9, Canada.
Fekete
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Frost
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D001426:Bacterial Proteins / Q000235:genetics*; D003062:Codon; D004247:DNA / Q000235:genetics; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D016368:Frameshift Mutation; D015151:Immunoblotting; D008957:Models, Genetic; D016296:Mutagenesis; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D001616:beta-Galactosidase / Q000378:metabolism
D001426:Bacterial Proteins; D003062:Codon; D011993:Recombinant Fusion Proteins; C074281:TraM protein, bacterial; D004247:DNA; D001616:beta-Galactosidase