Arginine Catabolism in Pseudomonas aeruginosa

铜绿假单胞菌中的精氨酸分解代谢

• 批准号: 9985660
• 负责人: Chung-Dar Lu
• 金额: $ 28.5万
• 依托单位: Georgia State University Research Foundation, Inc.
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9985660&HistoricalAwards=false
• 关键词: Arginine; Catabolism; Pseudomonas; aeruginosa

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.

精氨酸在铜绿假单胞菌生理学中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其作为该生物体的最强趋化性引诱剂之一的能力。本研究的长期目标是阐明精氨酸催化剂的调节机制。该项目的第一个目的是确定TCA循环的C4-二羧酸在反向分解代谢物阻遏中的作用。在铜绿假单胞菌中，乙酸盐和TCA循环中间体的存在抑制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，在外源精氨酸的存在下，ArgR的自诱导被废除的突变体的argSU基因编码的传感器激酶/响应调节的双组分调控系统。此外，利用各种化合物，包括葡萄糖，但不是琥珀酸，受到严重影响的argSU突变体。(i)将进行采用基因融合的研究，以检验TCA循环中C4-二羧酸盐作为argSU调控系统信号化合物的假设。(ii)将采用ArgS和ArgU的完整或His标记的融合蛋白的体外磷酸化测定来证明这两种蛋白中的信号转导，并且还将分析任何C4-二羧酸盐对这些生化反应的动力学的影响。该项目的第二个目的是确定在反向分解代谢物抑制中起作用的调控元件。ArgSU系统在这里提出了发挥作用，在反向分解代谢抑制。由于ArgU作为NtrC样转录调节因子出现，预期其仅对从s54启动子转录的操纵子具有直接作用。它对从s70启动子转录的操纵子的影响可以通过另外的调节子来介导。(i)为了鉴定ArgU的基因组靶标，将在选择策略中采用含有His标记的ArgU的硫酸镍亲和柱，以从粘粒基因组文库中分离其结合候选物。或者，可以通过分析argSU无效突变体的生长抑制因子并随后通过互补测试进行克隆来鉴定ArgU的基因组靶点。(ii)为了鉴定ArgU与其受影响的s70启动子之间缺失的连接，将采用转座子诱变来从aotJ：：lacZ融合体中分离具有改变的b-半乳糖苷酶表达水平的突变体，所述aotJ：：lacZ融合体已知在ArgSU的控制下。然后，在对含有转座子及其侧翼区的抗病基因的克隆进行序列分析后，可以鉴定受影响的基因。或者，将采用标准反向遗传学程序来鉴定该缺失元件。含有与aotJ调控区结合的ArgR以外的蛋白质的级分将通过迁移率变动测定法鉴定，并从DNA亲和柱进行进一步纯化。通过对铜绿假单胞菌全基因组序列进行N端氨基酸序列测定和BLAST搜索，可以确定其编码基因。铜绿假单胞菌及其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中的潜在致病性及其对植物的相关细菌通过显著的营养多样性而大大增强，所述营养多样性使该生物体能够在感染前在多样性和恶劣环境中存活，并且在感染后有效地调节其代谢活性以适应现有的环境限制。这些能力使得控制代谢活动的机制的进化成为必要，这些代谢活动与在肠道细菌和革兰氏阳性细菌中发现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，这对长期预防和控制疾病以及将这些微生物应用于生物技术至关重要。