Transcription and mass-spectroscopic proteomic studies of electron transport oxidoreductases in Dehalococcoides ethenogenes
Transcription and mass-spectroscopic proteomic studies of electron transport oxidoreductases in Dehalococcoides ethenogenes
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DOI：
10.1111/j.1462-2920.2006.01090.x
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发表时间：
2006-09-01
影响因子：
5.1
通讯作者：
Richardson, R.
中科院分区：
文献类型：
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作者：
Morris, R. M.;Sowell, S.;Richardson, R.
除了 19 个潜在的还原性脱卤酶基因外，Dehalococcoides ethenogenes 菌株 195 的基因组还包含 60 多个注释为编码氧化还原酶的基因，包括 5 个氢化酶复合物和一个甲酸脱氢酶 (Fdh)。使用定量逆转录酶聚合酶链式反应，我们发现编码周质Hup氢化酶和Fdh的基因在分批培养的纯培养物中表达最高，其中H-2分压> 0.1 atm，并且在丁酸盐/四氯乙烯混合培养物中表达最高，其中H-2分压为10(-4)-10(-5) atm。使用鸟枪电喷雾电离 (ESI) 和基质辅助激光解吸/电离 (MALDI) 串联质谱法来鉴定纯培养物膜富集级分中的多种肽，这些肽与几种高表达的呼吸酶相匹配，包括三种氢化酶、两种还原脱卤酶、Fdh 和 DET1407，一种我们认为是 S 层细胞壁一部分的 105.5 kDa 蛋白质。转录本和质谱方法均表明假定的 Fdh 是这些细胞中重要的氧化还原酶；然而，D.ethenogenes培养物不能使用甲酸作为还原脱氯的电子供体。对编码Fdh大亚基的基因的分析表明，虽然它与其他Fdh蛋白相关，但其序列在关键位置编码丝氨酸而不是半胱氨酸或硒代半胱氨酸，这让人对其功能产生怀疑。总体而言，基因组学和蛋白质组学方法为这种难以培养的生物体的新陈代谢提供了新的见解。
Besides 19 potential reductive dehalogenase genes, the genome of Dehalococcoides ethenogenes strain 195 contains over 60 genes annotated as encoding oxidoreductases, including five hydrogenase complexes and a formate dehydrogenase (Fdh). Using quantitative reverse transcriptase polymerase chain reaction, we found that genes encoding a periplasmic Hup hydrogenase and the Fdh were the most highly expressed in batch-grown pure cultures, in which the H-2 partial pressure was > 0.1 atm, and in butyrate/tetrachloroethene-mixed cultures, in which H-2 partial pressures were 10(-4)-10(-5) atm. Shotgun electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry were used to identify multiple peptides in pure culture membrane-enriched fractions matching several highly expressed respiratory enzymes, including three hydrogenases, two reductive dehalogenases, Fdh and DET1407, a 105.5-kDa protein we propose to be part of an S-layer cell wall. Both transcript and mass spectrometric approaches indicated that the putative Fdh was an important oxidoreductase in these cells; nevertheless, D. ethenogenes cultures could not use formate as an electron donor for reductive dechlorination. Analysis of the gene encoding the large subunit of Fdh indicated that while it was related to other Fdh proteins, its sequence encodes serine rather than cysteine or selenocysteine at a critical position, casting doubt on its function. Overall, genomic and proteomic approaches have provided novel insights into the metabolism of this difficult to culture organism.