Molecular cloning into Tn5 and integration in the Pseudomonas aeruginosa chromosome: a tool for heterologous gene expression.
The DNA primase gene of the promiscuous IncP-1 conjugative plasmid RP1, encoding two polypeptides of 118 and 80 kDa, was inserted into the transposon Tn5 in Escherichia coli. The derivative transposon, Tn2523, was then transposed to a temperature-sensitive replication mutant of the promiscuous IncP-1 conjugative plasmid R68 at permissive temperature and the plasmid transferred to Pseudomonas aeruginosa strain PAO. The latter strain was then grown at non-permissive temperature to identify transposition of Tn2523 into the P. aeruginosa chromosome. Immunological and enzymic analysis showed the expression of functional primase polypeptides in the constructed P. aeruginosa strain. This strain also restored wild-type conjugational transfer proficiency, by complementation, to mutants of the IncP-1 plasmid R18 affected in transfer from P. aeruginosa to P. stutzeri or to Acinetobacter calcoaceticus due to transposon Tn7 insertion mutations in the primase gene. This strategy of cloning into a transposon and integration into the bacterial chromosome should facilitate genetic manipulation and studies of gene expression in a range of Gram-negative bacteria.
D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D019915:DNA Primase; D004251:DNA Transposable Elements; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D007118:Immunoassay; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D012316:RNA Nucleotidyltransferases / Q000235:genetics
D004251:DNA Transposable Elements; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases