Arginine Catabolism in Pseudomonas aeruginosa

精氨酸在铜绿假单胞菌生理中的重要性体现在它作为碳源、能量源和氮源的四条分解代谢途径的存在，以及它作为最强趋化诱导剂的能力。本研究的长期目标是阐明精氨酸分解代谢的调节机制。这个项目的第一个目标是确定TCA循环中的C4-二羧酸盐在反向分解代谢抑制中的作用。在铜绿假单胞菌中，醋酸盐和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证实，精氨酸特异性调节蛋白ArgR对于精氨酸分解代谢操纵子的诱导是必不可少的。初步数据还表明，在一个双组分的调控系统中，编码一对传感器激酶/反应调节因子的argSU基因突变取消了在外源精氨酸存在下对ArgR的自动诱导。此外，包括葡萄糖在内的多种化合物的利用在argSU突变体中受到严重影响，但不包括琥珀酸。(I)将进行利用基因融合的研究，以检验C4-二羧酸盐在TCA循环中作为argSU调节系统的信号化合物的假设。(Ii)用完整的或His标记的Args和Argu融合蛋白的体外磷酸化分析来展示这两种蛋白的信号转导，并分析任何C4-二羧酸盐对这些生化反应动力学的影响。该项目的第二个目标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的作用。由于Argu似乎是一个类似NTRC的转录调控因子，预计它只能对从s54启动子转录的操纵子产生直接影响。它对转录自s70启动子的操纵子的影响可以通过另一个调控因子来调节。(I)为了确定精氨酸的基因组靶点，将使用含有组氨酸标记的精氨酸的硫酸镍亲和层析柱从粘粒基因组文库中分离其结合候选者。或者，可以通过分析argSU缺失突变体的生长抑制因子和随后通过互补试验进行克隆来确定argu的基因组靶标。(Ii)为了确定argu与其受影响的s70启动子之间缺失的连接，将利用转座子突变从已知受ArgSU控制的aotJ：：lacZ融合中分离出b-半乳糖苷酶表达水平发生变化的突变株。然后，在对包含转座子及其侧翼区域的抗生素耐药基因的克隆进行序列分析后，可以识别受影响的基因。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ArgR以外的蛋白质与aotJ调节区结合的组份将通过迁移率变化分析进行鉴定，并需要从DNA亲和层析中进一步纯化。通过对铜绿假单胞菌N端氨基酸序列的测定和BLAST搜索，可以确定编码基因。铜绿假单胞菌和其他相关细菌巨大的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了广泛的认识。铜绿假单胞菌在人类及其相关细菌中对植物的潜在致病性因其显著的营养多样性而大大增强，使其能够在感染前在不同的恶劣环境中生存，并在感染后有效地调整其代谢活动以适应现有的环境限制。这些能力要求进化出控制代谢活动的机制，这些机制与肠道细菌和革兰氏阳性细菌的代谢活动显著不同。这项研究的完成将有助于我们了解各种生化反应和调控机制，这对长期努力预防和控制疾病至关重要，并将这些微生物应用于生物技术。

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.