Arginine Catabolism in Pseudomonas aeruginosa

精氨酸在P. aeruginosa生理上的重要性反映在其作为碳、能量和氮的来源的四种分解代谢途径的存在，以及它作为该生物最强的趋化引诱剂之一的能力。本研究的长期目标是阐明精氨酸分解代谢的调控机制。这个项目的第一个目的是确定c4 -二羧酸的TCA循环在反分解代谢抑制中的作用。在P. aeruginosa中，乙酸和TCA循环中间体的存在抑制了糖以及包括精氨酸在内的其他化合物的利用。本实验室的研究已经确定精氨酸特异性调节蛋白ArgR对于诱导精氨酸分解代谢操纵子是必不可少的。初步数据还表明，在编码一对双组分调节系统的激酶/反应调节因子的argSU基因突变体中，外源精氨酸存在时的ArgR自诱导被消除。此外，argSU突变体对包括葡萄糖在内的多种化合物的利用受到严重影响，但琥珀酸盐不受影响。(i)将进行基因融合研究，以检验TCA循环中c4 -二羧酸盐作为argSU调节系统信号化合物的假设。（ii）使用完整的或his标记的ArgS和ArgU融合蛋白进行体外磷酸化实验，以证明这两种蛋白的信号转导，并分析任何c4 -二羧化物对这些生化反应动力学的影响。该项目的第二个目的是确定在反向分解代谢抑制中起作用的调控元件。本文提出ArgSU系统在反向分解代谢抑制中发挥作用。由于ArgU作为ntrc样转录调控因子出现，预计它只对s54启动子转录的操纵子有直接作用。它对从70个启动子转录而来的操纵子的作用可能由一个额外的调节因子介导。(i)为了确定ArgU的基因组靶点，将采用含有his标记ArgU的硫酸镍亲和柱作为选择策略，从cosmid基因组文库中分离其结合候选者。另外，ArgU的基因组靶点可以通过分析ArgU零突变体的生长抑制因子和随后的互补试验克隆来确定。（ii）为了确定ArgU与其受影响的s70启动子之间缺失的连接，将采用转座子诱变技术从aotJ::lacZ融合中分离出b-半乳糖苷酶表达水平改变的突变体，已知该融合是由ArgSU控制的。然后，在对含有转座子及其侧翼区域耐药基因的克隆进行序列分析后，可以鉴定受影响的基因。或者，将采用标准的反向遗传学程序来识别这种缺失的元素。含有与aotJ调控区结合的ArgR以外的蛋白质的组分将通过迁移位移测定来鉴定，并从DNA亲和柱中进一步纯化。对铜绿假单胞菌完整的基因组序列进行n端氨基酸序列的确定和BLAST搜索，即可实现编码基因的鉴定。铜绿假单胞菌和其他相关细菌的巨大分解代谢能力使其在工业和环境生物技术中的潜在意义得到广泛认识。P. aeruginosa对人类及其相关细菌对植物的潜在致病性因其显著的营养多样性而大大增强，这使得该生物在感染前能够在多样化和恶劣的环境中生存，并在感染后有效地调整其代谢活性以适应现有的环境限制。这些能力使得代谢活动控制机制的进化成为必要，这与肠道细菌和革兰氏阳性细菌的代谢活动控制机制明显不同。这项研究的完成将有助于我们了解各种生物化学反应和调节机制，这对于预防和控制疾病的长期努力以及将这些微生物应用于生物技术至关重要。

The significance of arginine in the physiology of P. aeruginosa is reflected in the presence of four catabolic pathways for its utilization as a source of carbon, energy, and nitrogen as well as its ability to serve as one of the strongest chemotactic attractants for this organism. The long-term objective of this research is to elucidate the regulatory mechanisms for arginine catabolism. The first aim of this project is to determine the role of C4-dicarboxylate of the TCA cycle in reverse catabolite repression. In P. aeruginosa, the presence of acetate and the TCA cycle intermediates inhibits the utilization of sugars as well as other compounds including arginine. Studies in this laboratory have established that arginine-specific regulatory protein, ArgR, is essential for the induction of arginine catabolic operons. Preliminary data also indicated that auto-induction of ArgR in the presence of exogenous arginine was abolished in the mutants of argSU genes encoding a pair of sensor kinase/response regulator of a two-component regulatory system. In addition, the utilization of a variety of compounds including glucose, but not succinate, was severely affected in the argSU mutant. (i) Studies employing gene fusions will be conducted to test the hypothesis of C4-dicarboxylates in the TCA cycle as the signal compound of the argSU regulatory system. (ii) In vitro phosphorylation assays with either intact or His-tagged fusion proteins of ArgS and ArgU will be employed to demonstrate signal transduction in these two proteins, and effects of any of the C4-dicarboxylates on the kinetics of these biochemical reactions will also be analyzed. The second aim of this project is to identify the regulatory elements that function in reverse catabolite repression. The ArgSU system is proposed here to play a role in reverse catabolite repression. Since ArgU appears as an NtrC-like transcriptional regulator, it is expected that it could only have a direct effect on operons transcribed from s54 promoters. Its effects on operons that are transcribed from s70 promoters could be mediated by an additional regulator. (i) To identify the genomic targets of ArgU, a nickel sulfate affinity column containing His-tagged ArgU will be employed in a selection strategy to isolate its binding candidates from a cosmid genomic library. Alternatively, genomic targets of ArgU can be identified by analyses of growth suppressors of the argSU null mutant and the subsequent cloning by complementation tests. (ii) To identify the missing linking between ArgU and its affected s70 promoters, transposon mutagenesis will be employed to isolate mutants with altered expression level of b-galactosidase from an aotJ::lacZ fusion, which is known to be under the control of ArgSU. The affected gene can then be identified after sequence analysis of clones containing antibiotic-resistant genes of the transposon and its flanking regions. Alternatively, standard reverse genetics procedures will be adapted to identify this missing element. Fractions containing proteins other than ArgR that bind to the aotJ regulatory region will be identified by mobility shift assays, and subject to further purification from a DNA affinity column. Identification of the encoding gene can then be achieved after determination of the N-terminal amino acid sequence and BLAST search on the completed genomic sequence of P. aeruginosa.The enormous catabolic capability of P. aeruginosa and other related bacteria have led to a wide recognition of its potential significance in industrial and environmental biotechnology. The potential pathogenicity of P. aeruginosa in human and its related bacteria to plants is greatly enhanced by a remarkable nutritional versatility that enables this organism to survive in diverse and in harsh environments prior to infection and to efficiently adjust its metabolic activity to the existing environmental constraints following infection. These capacities have necessitated the evolution of mechanisms in control of metabolic activities that are significantly different from those found in enteric bacteria and gram-positive bacteria. Completion of this research will contribute to our knowledge of the diverse biochemical reactions and the regulatory mechanisms that is critical for long term efforts to prevent and control diseases, and to apply these microorganisms in biotechnology.