Broad-host-range plasmid and M13 bacteriophage-derived vectors for promoter analysis in Escherichia coli and Pseudomonas aeruginosa.
A set of bacteriophage and plasmid vectors containing xylE as a reporter gene was constructed for the analysis of promoters functional in Escherichia coli and in other Gram-negative bacteria. Two M13 bacteriophage derivatives, M13mVDX18 and M13mMK010, were designed for rapid cloning, screening and sequencing of DNA fragments promoting transcription in E. coli. To demonstrate their utility, total cellular DNA from a variety of bacterial species including Pseudomonas aeruginosa strain PAO was shotgun cloned in M13 vectors and clones displaying promoter activity in E. coli were isolated. These randomly cloned promoters from P. aeruginosa, Borrelia burgdorferi, Streptococcus pneumoniae and other bacterial species were sequenced without a need for further subcloning manipulation. The promoter activity of P. aeruginosa clones was verified by subcloning inserts on a broad-host-range promoter probe vector pVDX18 and assaying the xylE transcription from these promoters in P. aeruginosa. The pVDX18 vector was also used for initial characterization of the algD promoter controlling mucoidy in P. aeruginosa. The activities of the wild-type and deletion clones of the algD promoter were compared. Results indicated that the region containing direct and inverted repeats at -55 to -110 bp upstream of the mRNA 5' end was important for the activation of the algD transcription in mucoid P. aeruginosa infecting cystic fibrosis patients.
Konyecsni
W M
WM
Department of Microbiology, University of Texas Health Science Center, San Antonio 78284.
Deretic
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D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D050557:Catechol 2,3-Dioxygenase; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D049308:Dioxygenases; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D014158:Transcription, Genetic
D004274:DNA, Recombinant; D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase