Krishnapillai V, Pühler A, Lanka E
J Gen Microbiol. 1986 Mar;132(3):707-15. doi: 10.1099/00221287-132-3-707.
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.
编码118 kDa和80 kDa两种多肽的滥交型IncP - 1接合质粒RP1的DNA引发酶基因，被插入到大肠杆菌的转座子Tn5中。然后，将衍生转座子Tn2523在允许温度下转座到滥交型IncP - 1接合质粒R68的温度敏感复制突变体上，并将该质粒转移到铜绿假单胞菌PAO菌株中。随后，使后一种菌株在非允许温度下生长，以鉴定Tn2523转座到铜绿假单胞菌染色体中。免疫和酶分析表明，在构建的铜绿假单胞菌菌株中功能性引发酶多肽得以表达。该菌株还通过互补作用，将野生型接合转移能力恢复到因引发酶基因中的转座子Tn7插入突变而在从铜绿假单胞菌转移到施氏假单胞菌或乙酸钙不动杆菌过程中受到影响的IncP - 1质粒R18突变体。这种克隆到转座子并整合到细菌染色体中的策略，应有助于对一系列革兰氏阴性菌进行基因操作和基因表达研究。
