Application of an inducible system to engineer unmarked conditional mutants of essential genes of Pseudomonas aeruginosa

作者：Morita Yuji; Narita Shin Ichiro; Tomida Junko; Tokuda Hajime; Kawamura Yoshiaki^*

来源：Journal of Microbiological Methods, 2010, 82(3): 205-213.

DOI：10.1016/j.mimet.2010.06.001

• Essential genes
• Pseudomonas aeruginosa
• Unmarked gene disruption

The phi CTX-based integration vector pYM101 harboring a tightly controlled modified phage T7 early gene promoter/Lacl(q) repressor (T7/Lacl) system was constructed for the generation of unmarked conditional mutants in Pseudomonas aeruginosa. Promoter activity of the T7/Lacl system was demonstrated to be dependent on the presence of the inducer isopropyl -beta-D-1-thiogalactopyranoside (IPTG), as evaluated by measuring beta-galactosidase activity. In the absence of the inducer, the promoter was silent as its activity was lower than those of a promoter-less lacZ control. Unmarked conditional mutants of four predicted essential genes (lolCDE (PA2988-86), lpxC (PA4406), rho (PA5239), and def (PA0019)) were successfully constructed using this recombination system. In the absence of IPTG, the growth of all mutants was repressed: however, the addition of either 0.1 or 1 mM IFTG restored growth rates to levels nearly identical to wild-type cells. It was therefore demonstrated that the inducible integration vector pYM101 is suitable for the creation of unmarked conditional mutants of P. aeruginosa, and is particularly useful for examining the function of essential genes.

• 出版日期2010-9