The Multifaceted Proteins MvaT and MvaU, Members of the H-NS Family, Control Arginine Metabolism, Pyocyanin Synthesis, and Prophage Activation in Pseudomonas aeruginosa PAO1
The Multifaceted Proteins MvaT and MvaU, Members of the H-NS Family, Control Arginine Metabolism, Pyocyanin Synthesis, and Prophage Activation in Pseudomonas aeruginosa PAO1
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DOI：
10.1128/jb.00888-09
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发表时间：
2009-10-15
影响因子：
3.2
通讯作者：
Lu, Chung-Dar
中科院分区：
文献类型：
--
作者：
Li, Congran;Wally, Hassan;Lu, Chung-Dar
属于H-NS家族的MvaT和MvaU蛋白被鉴定为dna结合蛋白，它们与aotJQMOP-argR操纵子的调控区域相互作用，以进行精氨酸摄取和调控。重组MvaT和MvaU蛋白被纯化，这些纯化蛋白结合到aotJ调控区，通过电迁移转移实验证明。制备了针对纯化MvaT和MvaU的多克隆抗体，并用于超移实验来支持这些观察结果。利用双亲本偶联技术构建了mvaT或mvaU中单个病变的敲除突变，以及导致双病变的敲除突变，并通过免疫印迹分析证实了mvaT和mvaU在所产生的突变体中缺失。通过测量aotJ::lacZ在突变体和亲本菌株中融合的β -半乳糖苷酶活性，发现MvaT和MvaU是控制aotJ表达的抑制因子。分析了MvaT和MvaU对这些突变体花青素合成和CupA毛层表达的影响。单突变体诱导了花青素的合成，而双突变体则完全取消了花青素的合成，这表明花青素的合成有一个复杂的调控机制，其中MvaT和MvaU是必不可少的元素。相比之下，MvaT对cupA表达的抑制作用比MvaU更为深刻；然而，MvaT和MvaU消耗的组合对cupA有很强的协同效应。此外，整合到铜绿假单胞菌PAO1染色体上的噬菌体Pf4在mvaT mvaU双突变体中被激活，而在mvaT mvaU单突变体中没有被激活。这些结果得到了复制形式DNA的纯化和核苷酸测序以及斑块试验中噬菌体颗粒释放的支持。综上所述，mvaT和mvaU双突变体是有活力的，mvaT和mvaU的耗尽对铜绿假单胞菌的多种生理功能产生了严重影响。
The MvaT and MvaU proteins belonging to the H-NS family were identified as DNA-binding proteins that interact with the regulatory region of the aotJQMOP-argR operon for arginine uptake and regulation. Recombinant MvaT and MvaU proteins were purified, and binding of these purified proteins to the aotJ regulatory region was demonstrated using electromobility shift assays. Polyclonal antibodies against purified MvaT and MvaU were prepared and employed in supershift assays to support these observations. Knockout mutations resulting in a single lesion in mvaT or mvaU, as well as knockout mutations resulting in double lesions, were constructed using biparental conjugation, and the absence of MvaT and MvaU in the resulting mutants was confirmed by immunoblot analysis. Using measurements of the beta-galactosidase activities from aotJ::lacZ fusions in the mutants and the parental strain, it was found that MvaT and MvaU serve as repressors in control of aotJ expression. The effects of MvaT and MvaU on pyocyanin synthesis and CupA fimbrial expression in these mutants were also analyzed. Pyocyanin synthesis was induced in the single mutants but was completely abolished in the double mutant, suggesting that there is a complicated regulatory scheme in which MvaT and MvaU are essential elements. In comparison, MvaT had a more profound role than MvaU as a repressor of cupA expression; however, a combination of MvaT depletion and MvaU depletion had a strong synergistic effect on cupA. Moreover, prophage Pf4 integrated into the chromosome of Pseudomonas aeruginosa PAO1 was activated in an mvaT mvaU double mutant but not in a single mutant. These results were supported by purification and nucleotide sequencing of replicative-form DNA and by the release of phage particles in plaque assays. In summary, the mvaT mvaU double mutant was viable, and depletion of MvaT and MvaU had serious effects on a variety of physiological functions in P. aeruginosa.