Boes Nelli, Schreiber Kerstin, Härtig Elisabeth, Jaensch Lothar, Schobert Max
Institute of Microbiology, Technical University Braunschweig, Spielmannstr. 7D-38106, Braunschweig, Germany.
J Bacteriol. 2006 Sep;188(18):6529-38. doi: 10.1128/JB.00308-06.
During infection of the cystic fibrosis (CF) lung, Pseudomonas aeruginosa microcolonies are embedded in the anaerobic CF mucus. This anaerobic environment seems to contribute to the formation of more robust P. aeruginosa biofilms and to an increased antibiotic tolerance and therefore promotes persistent infection. This study characterizes the P. aeruginosa protein PA4352, which is important for survival under anaerobic energy stress conditions. PA4352 belongs to the universal stress protein (Usp) superfamily and harbors two Usp domains in tandem. In Escherichia coli, Usp-type stress proteins are involved in survival during aerobic growth arrest and under various other stresses. A P. aeruginosa PA4352 knockout mutant was tested for survival under several stress conditions. We found a decrease in viability of this mutant compared to the P. aeruginosa wild type during anaerobic energy starvation caused by the missing electron acceptors oxygen and nitrate. Consistent with this phenotype under anaerobic conditions, the PA4352 knockout mutant was also highly sensitive to carbonyl cyanide m-chlorophenylhydrazone, the chemical uncoupler of the electron transport chain. Primer extension experiments identified two promoters upstream of the PA4352 gene. One promoter is activated in response to oxygen limitation by the oxygen-sensing regulatory protein Anr. The center of a putative Anr binding site was identified 41.5 bp upstream of the transcriptional start site. The second promoter is active only in the stationary phase, however, independently of RpoS, RelA, or quorum sensing. This is the second P. aeruginosa Usp-type stress protein that we have identified as important for survival under anaerobic conditions, which resembles the environment during persistent infection.
在囊性纤维化（CF）肺部感染期间，铜绿假单胞菌微菌落嵌入厌氧的CF黏液中。这种厌氧环境似乎有助于形成更坚固的铜绿假单胞菌生物膜，并增加抗生素耐受性，从而促进持续性感染。本研究对铜绿假单胞菌蛋白PA4352进行了表征，该蛋白在厌氧能量应激条件下的生存中起重要作用。PA4352属于通用应激蛋白（Usp）超家族，串联有两个Usp结构域。在大肠杆菌中，Usp型应激蛋白参与有氧生长停滞期间及其他各种应激条件下的生存。对铜绿假单胞菌PA4352基因敲除突变体在几种应激条件下的生存情况进行了测试。我们发现，在因缺乏电子受体氧气和硝酸盐导致的厌氧能量饥饿期间，与铜绿假单胞菌野生型相比，该突变体的活力下降。与这种厌氧条件下的表型一致，PA4352基因敲除突变体对电子传递链的化学解偶联剂羰基氰化物间氯苯腙也高度敏感。引物延伸实验确定了PA4352基因上游的两个启动子。一个启动子在氧气感应调节蛋白Anr的作用下响应氧气限制而被激活。在转录起始位点上游41.5 bp处确定了一个假定的Anr结合位点的中心。第二个启动子仅在稳定期有活性，然而，独立于RpoS、RelA或群体感应。这是我们鉴定出的第二种对厌氧条件下生存很重要的铜绿假单胞菌Usp型应激蛋白，这种条件类似于持续性感染期间的环境。
