Abstract
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 β-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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Acknowledgements
We thank Jan Manchak and Troy Locke for technical assistance. pPR9tt was kindly provided by Svein Valla (Dept. of Biotechnology, Norwegian University of Science and Technology, Trondheim). This work is supported by the Canadian Institutes for Health Research. J. L. is supported by a studentship from the Alberta Ingenuity Fund. R.A.F is supported by a studentship from the Alberta Heritage Foundation for Medical Research. This work was done in compliance with the laws of Canada
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Lu, J., Fekete, R.A. & Frost, L.S. A rapid screen for functional mutants of TraM, an autoregulatory protein required for F conjugation. Mol Gen Genomics 269, 227–233 (2003). https://doi.org/10.1007/s00438-003-0826-2
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DOI: https://doi.org/10.1007/s00438-003-0826-2