Department of Biological Sciences, Michigan Technological University, Houghton, MI 49931, USA.
FEMS Microbiol Lett. 2012 Oct;335(2):140-8. doi: 10.1111/j.1574-6968.2012.02646.x. Epub 2012 Aug 21.
Pseudomonas sp. TLC6-6.5-4 is a multiple metal resistant plant growth-promoting bacteria isolated from copper-contaminated lake sediments. In this study, a comprehensive analysis of genes involved in copper resistance was performed by generating a library of transposon (Tn5) mutants. Two copper-sensitive mutants with significant reduction in copper resistance were identified: CSM1, a mutant disrupted in trpA gene (tryptophan synthase alpha subunit), and CSM2, a mutant disrupted in clpA gene (ATP-dependent Clp protease). Proteomic and metabolomic analyses were performed to identify biochemical and molecular mechanisms involved in copper resistance using CSM2 due to its lower minimum inhibitory concentration compared with CSM1 and the wild type. Proteomic analysis revealed that disruption of Clp protease gene up-regulated molecular chaperones and down-regulated the expression of enzymes related to tRNA modification, whereas metabolomic analysis showed that amino acid and oligosaccharide transporters that are part of ATP-binding cassette (ABC) transporters pathways were down-regulated. Further, copper stress altered metabolic pathways including the tricarboxylic acid cycle, protein absorption and glyoxylate metabolism.
假单胞菌 TLC6-6.5-4 是一种从受铜污染的湖底沉积物中分离出来的具有多种金属抗性的植物促生菌。在这项研究中，通过生成转座子 (Tn5) 突变体文库，对涉及铜抗性的基因进行了全面分析。鉴定出两个铜敏感性突变体，其铜抗性显著降低：CSM1，trpA 基因（色氨酸合酶 α 亚基）缺失突变体，以及 CSM2，clpA 基因（ATP 依赖性 Clp 蛋白酶）缺失突变体。由于 CSM2 的最小抑菌浓度 (MIC) 低于 CSM1 和野生型，因此使用 CSM2 进行了蛋白质组学和代谢组学分析，以确定涉及铜抗性的生化和分子机制。蛋白质组学分析表明，Clp 蛋白酶基因的缺失上调了分子伴侣，下调了与 tRNA 修饰相关的酶的表达，而代谢组学分析表明，参与 ABC 转运体途径的氨基酸和寡糖转运体下调。此外，铜胁迫改变了代谢途径，包括三羧酸循环、蛋白质吸收和乙醛酸代谢。
