Mukherjee Ashis K, Bhagowati Pabitra, Biswa Bhim Bahadur, Chanda Abhishek, Kalita Bhargab
ONGC-Center for Petroleum Biotechnology & Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur 784028, Assam, India.
ONGC-Center for Petroleum Biotechnology & Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur 784028, Assam, India.
J Proteomics. 2017 Sep 7;167:25-35. doi: 10.1016/j.jprot.2017.07.020. Epub 2017 Jul 31.
Pseudomonas aeruginosa strain ASP-53, isolated from a petroleum oil-contaminated soil sample, was found to be an efficient degrader of pyrene. PCR amplification of selected hydrocarbon catabolic genes (alkB gene, which encodes for monooxygenase, and the C12O, C23O, and PAH-RHDα genes encoding for the dioxygenase enzyme) from the genomic DNA of P. aeruginosa strain ASP-53 suggested its hydrocarbon degradation potential. The GC-MS analysis demonstrated 30.1% pyrene degradation by P. aeruginosa strain ASP-53 after 144h of incubation at pH6.5, 37°C. Expressions of 115 and 196 intracellular proteins were unambiguously identified and quantitated by shotgun proteomics analysis when the isolate was grown in medium containing pyrene and glucose, respectively. The pyrene-induced uniquely expressed and up-regulated proteins in P. aeruginosa strain ASP-53 in addition to substrate (pyrene) metabolism are also likely to be associated with different cellular functions for example-related to protein folding (molecular chaperone), stress response, metabolism of carbohydrate, proteins and amino acids, and fatty acids; transport of metabolites, energy generation such as ATP synthesis, electron transport and nitrate assimilation, and other oxidation-reduction reactions. Proteomic analyses identified some important enzymes involved in pyrene degradation by P. aeruginosa ASP-53 which shows that this bacterium follows the salicylate pathway of pyrene degradation.
This study is the first report on proteomic analysis of pyrene biodegradation pathway by Pseudomonas aeruginosa, isolated from a petroleum-oil contaminated soil sample. The pathway displays partial similarity with deduced pyrene degradation mechanisms of Mycobacterium vanbaalenii PYR-1. The GC-MS analysis as well as PCR amplification of hydrocarbon catabolic genes substantiated the potency of the bacterium under study to effectively degrade high molecular weight, toxic PAH such as pyrene for its filed scale bioremediation experiments. The proteomics approach (LC-MS/MS analysis) identified the differentially regulated intracellular proteins of the isolate P. aeruginosa ASP-53 when grown in pyrene medium. This study identified some important pyrene biodegradation enzymes in Pseudomonas aeruginosa ASP-53 and highlights that the bacterium follows salicylate pathway for pyrene degradation.
从石油污染土壤样本中分离出的铜绿假单胞菌ASP - 53菌株，被发现是芘的高效降解菌。从铜绿假单胞菌ASP - 53菌株的基因组DNA中对选定的碳氢化合物分解代谢基因（编码单加氧酶的alkB基因，以及编码双加氧酶的C12O、C23O和PAH - RHDα基因）进行PCR扩增，表明其具有碳氢化合物降解潜力。气相色谱 - 质谱分析表明，在pH6.5、37°C下培养144小时后，铜绿假单胞菌ASP - 53菌株对芘的降解率为30.1%。当该菌株分别在含有芘和葡萄糖的培养基中生长时，通过鸟枪法蛋白质组学分析明确鉴定并定量了115种和196种细胞内蛋白质的表达。除了底物（芘）代谢外，铜绿假单胞菌ASP - 53菌株中芘诱导的独特表达和上调的蛋白质也可能与不同的细胞功能相关，例如与蛋白质折叠（分子伴侣）、应激反应、碳水化合物、蛋白质和氨基酸以及脂肪酸的代谢；代谢物运输、能量产生（如ATP合成、电子传递和硝酸盐同化）以及其他氧化还原反应。蛋白质组学分析确定了铜绿假单胞菌ASP - 53降解芘过程中涉及的一些重要酶，这表明该细菌遵循芘降解的水杨酸途径。
本研究是关于从石油污染土壤样本中分离出的铜绿假单胞菌芘生物降解途径蛋白质组学分析的首次报道。该途径与范氏分枝杆菌PYR - 1推导的芘降解机制显示出部分相似性。气相色谱 - 质谱分析以及碳氢化合物分解代谢基因的PCR扩增证实了所研究细菌在现场规模生物修复实验中有效降解芘等高分子量有毒多环芳烃的能力。蛋白质组学方法（液相色谱 - 串联质谱分析）确定了铜绿假单胞菌ASP - 53菌株在芘培养基中生长时差异调节的细胞内蛋白质。本研究确定了铜绿假单胞菌ASP - 53中一些重要的芘生物降解酶，并强调该细菌遵循水杨酸途径进行芘降解。
