Characterization of Molecular Mechanisms Controlling fabAB Transcription in Pseudomonas aeruginosa

BackgroundThe FabAB pathway is one of the unsaturated fatty acid (UFA) synthesis pathways for Pseudomonas aeruginosa. It was previously noted that this operon was upregulated in biofilms and repressed by exogenous UFAs. Deletion of a 30 nt fabA upstream sequence, which is conserved in P. aeruginosa, P. putida, and P. syringae, led to a significant decrease in fabA transcription, suggesting positive regulation by an unknown positive regulatory mechanism. Methods/Principal FindingsHere, genetic and biochemical approaches were employed to identify a potential fabAB activator. Deletion of candidate genes such as PA1611 or PA1627 was performed to determine if any of these gene products act as a fabAB activator. However, none of these genes were involved in the regulation of fabAB transcription. Use of mariner-based random mutagenesis to screen for fabA activator(s) showed that several genes encoding unknown functions, rpoN and DesA may be involved in fabA regulation, but probably via indirect mechanisms. Biochemical attempts performed did fail to isolate an activator of fabAB operon. Conclusion/SignificanceThe data suggest that fabA expression might not be regulated by protein-binding, but by a distinct mechanism such as a regulatory RNA-based mechanism.背景：FabAB通路是铜绿假单胞菌（Pseudomonas aeruginosa）的不饱和脂肪酸（unsaturated fatty acid, UFA）合成途径之一。此前已有研究发现，该操纵子在生物被膜中表达上调，且可被外源不饱和脂肪酸抑制。对一段在铜绿假单胞菌、恶臭假单胞菌（P. putida）及丁香假单胞菌（P. syringae）中保守的fabA上游30 nt序列进行敲除，会导致fabA转录水平显著下降，提示该基因的正向转录调控由一种未知的调控机制介导。 方法与主要发现：本研究采用遗传与生化实验手段，旨在鉴定fabAB操纵子的潜在激活因子。我们对PA1611、PA1627等候选基因开展敲除实验，以验证其编码产物是否为fabAB的激活因子，但结果显示上述基因均未参与fabAB的转录调控。利用基于mariner转座子的随机诱变技术筛选fabA激活因子时发现，若干功能未知基因、rpoN及DesA可能参与fabA的调控，但该调控大概率通过间接机制实现。生化分离尝试未能成功分离得到fabAB操纵子的激活因子。 结论与意义：本研究数据表明，fabA的表达可能并非通过蛋白质结合的方式进行调控，而是依赖于一类独特的调控机制，例如基于调控RNA的调控途径。