Application of an inducible system to engineer unmarked conditional mutants of essential genes of Pseudomonas aeruginosa
Application of an inducible system to engineer unmarked conditional mutants of essential genes of Pseudomonas aeruginosa
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DOI：
10.1016/j.mimet.2010.06.001
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发表时间：
2010-09-01
影响因子：
2.2
通讯作者：
Kawamura, Yoshiaki
中科院分区：
文献类型：
--
作者：
Morita, Yuji;Narita, Shin-Ichiro;Kawamura, Yoshiaki
构建了基于phi CTX的整合载体pYM 101，该载体含有严格控制的修饰的噬菌体T7早期基因启动子/Lacl（q）阻遏子（T7/Lacl）系统，用于铜绿假单胞菌无标记条件突变体的产生。通过测量β-半乳糖苷酶活性来评估，证明T7/Lacl系统的启动子活性取决于诱导剂异丙基-β-D-1-硫代半乳糖苷（IPTG）的存在。在不存在诱导物的情况下，启动子是沉默的，因为其活性低于无启动子的lacZ对照。利用该重组系统成功地构建了四个预测的必需基因（lolCDE（PA 2988 -86）、lpxC（PA 4406）、rho（PA 5239）和def（PA 0019））的未标记的条件突变体。在不存在IPTG的情况下，所有突变体的生长被抑制：然而，添加0.1或1 mM IFTG将生长速率恢复到与野生型细胞几乎相同的水平。因此，证明了诱导型整合载体pYM 101适合于产生铜绿假单胞菌的未标记条件突变体，并且对于检查必需基因的功能特别有用。(C)2010 Elsevier B. V.保留所有权利。
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. (C) 2010 Elsevier B.V. All rights reserved.