Initial studies on aBacillus subtilis mutant lacking the dnaK-homologue protein |
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Authors: | Robin R Staples Brian S Miller Marie L Hoover Qun Chou Dr Uldis N Streips |
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Institution: | (1) Department of Microbiology and Immunology, School of Medicine, University of Louisville, 40292 Louisville, Kentucky, USA;(2) Cetus Corporation, 40292 Emeryville, California, USA;(3) Present address: Department of Bicohemistry, University of Arizona, 85721 Tucson, Arizona, USA |
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Abstract: | A sequence of 412bp, spanning the terminal half of thegrpE and the proximal portion of thednaK-homologues inBacillus subtilis, was amplified with PCR technology. This fragment was cloned into pJH101, anEscherichia coli plasmid, and transformed intoB. subtilis strain YB886. Several chloramphenicol-resistant colonies were obtained from this transformation. The integration of the plasmid into theB. subtilis chromosome was verified by restriction endonuclease analysis and Southern hybridization. Strain BUL101, a chloramphenicol-resistant transformant, lacked the DnaK-homologue as demonstrated by two-dimensional polyacrylamide gel electrophoresis and Western blot analysis. BUL101 grew at slower rates than parental cells at both 37°C and 48°C, produced abnormal cell shapes at 48°C, and was unable to grow at 51°C. The 412bp fragment did not exhibit detectable promoter activity. |
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