[Construction and identification of rpoS gene inserted and fused in frame with the promoterless lacZ in Pseudomonas sp. M18].
With hybridization in situ and Southern blots, an Eco RI- Xho I DNA fragment of 3.1 kb in length containing an rpoS gene and its flanking sequence was first cloned into pBluescript SK to generate pBLS by screening the genomic DNA library of Pseudomonas sp. M18. In order to identify the potential factors involved in rpoS gene expression and the regulatory mechanism of RpoS in strain M18, the rpoS gene was inserted and fused in frame with a promoterless and truncated lacZ gene, and a mutant named as M18SZ was then constructed through homologous recombination. Growth curves in KMB medium indicated that loss of RpoS made the mutant strain M18SZ more sensitive to alteration of some environmental factors. With detection and comparison of beta-galactosidase activities from both the wild type strain M18 and its derivative M18SZ cultivated in KMB medium respectively, it was found that the expression level of beta-galactosidase activities in the mutant M18SZ was high and could come to 480U. Expression of beta-galactosidase activities of the wild type strain M18 in KMB medium was not almost detected during its whole growth phase. With these results, it was confirmed that the rpoS gene did be fused in frame with the truncated lacZ gene in chromosome of the mutant M18SZ. Meanwhile, it is suggested that construction of a mutation, which is made with fusion in frame with the truncated lacZ gene, may be verified by detecting its beta-galactosidase activity, not using Southern blot or PCR.
Ge
Yi-He
YH
School of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai 200240, China. geyihe@126.com
Xu
Yu-Quan
YQ
D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000235:genetics*; D015139:Blotting, Southern; D003001:Cloning, Molecular; D007763:Lac Operon; D016254:Mutagenesis, Insertional; D011549:Pseudomonas / Q000235:genetics* / Q000254:growth & development; D012808:Sigma Factor / Q000235:genetics*
D001426:Bacterial Proteins; D012808:Sigma Factor; C069567:sigma factor KatF protein, Bacteria