Stoichiometry of the gene products from the tetrachloroethene reductive dehalogenase operon pceABCT

Organohalide respiration (OHR) is a bacterial anaerobic process in which halogenated compounds, e.g. tetrachloroethene (PCE), are used as terminal electron acceptors. Our model organisms are Dehalobacter restrictus strain PER-K23, an obligate OHR bacterium, and Desulfitobacterium hafniense strain TCE1, a bacterium with a versatile energy metabolism. The key catalytic enzyme is the tetrachloroethene reductive dehalogenase (PceA) that is, at genomic level, encoded in a highly conserved gene cluster (pceABCT) in both D. restrictus and D. hafniense strain TCE1. To date, the functions of PceA and PceT, a dedicated molecular chaperone for the maturation of PceA, are well defined. However, the role of PceB and PceC are still not elucidated. Here, we present a multilevel study aiming at deciphering the stoichiometry of the pceABCT individual gene products. The investigation was assessed at RNA level by reverse transcription and (quantitative) polymerase chain reaction, while at protein level, proteomic analyses based on parallel reaction monitoring were performed to quantify the PceA, B, C and T proteins in cell-free extracts as well as in soluble and membrane fractions of both strains using heavy-labelled reference peptides. At RNA level, our results confirmed the co-transcription of all pce gene products, while the quantitative analysis revealed a relative stoichiometry of the gene transcripts of pceA, pceB, pceC and pceT at approximately 1.0:3.0:0.1:0.1 in D. restrictus. This trend was not observed in D. hafniense strain TCE1, where no substantial difference was measured for the four genes. At proteomic level, a 2:1 stoichiometry of PceA and PceB was observed in the membrane fraction, and a low abundance of PceC in comparison to the other two proteins. In the soluble fraction, a 1:1 stoichiometry of PceA and PceT was identified. In summary, we show that the pce gene cluster is transcribed as an operon with, however, a level of transcription that differs for individual genes, an observation that could be explained by post-transcriptional events, such as RNA processing and differential RNA stability. Results at protein level suggest that PceA and PceB form a membrane-bound PceA2B protein complex, which, in contrast to the proposed model, seems to be devoid of PceC.

卤化呼吸（Organohalide respiration，OHR）是一类细菌厌氧代谢途径，该途径以卤代化合物（例如四氯乙烯（tetrachloroethene，PCE））作为末端电子受体。本研究选用的模式生物为专性卤化呼吸细菌——限制脱卤杆菌（Dehalobacter restrictus）PER-K23菌株，以及具备多样化能量代谢能力的哈芬尼脱硫杆菌（Desulfitobacterium hafniense）TCE1菌株。该过程的关键催化酶为四氯乙烯还原脱卤酶（tetrachloroethene reductive dehalogenase，PceA），在基因组层面，限制脱卤杆菌与哈芬尼脱硫杆菌TCE1菌株的pceABCT基因簇中均存在高度保守的该酶编码基因。截至目前，PceA以及负责PceA成熟的专属分子伴侣PceT的功能已得到明确阐释，但PceB与PceC的功能仍未阐明。本研究开展了一项多维度研究，旨在解析pceABCT基因簇各编码产物的化学计量比。在RNA层面，研究通过反转录（及定量）聚合酶链式反应开展分析；在蛋白质层面，则基于平行反应监测开展蛋白质组学分析，利用重标参考肽对两种菌株的无细胞提取物、可溶性组分与膜组分中的PceA、PceB、PceC及PceT蛋白进行定量检测。RNA层面的实验结果证实，所有pce基因产物均发生共转录；定量分析则显示，限制脱卤杆菌中pceA、pceB、pceC与pceT的转录本相对化学计量比约为1.0:3.0:0.1:0.1。这一趋势在哈芬尼脱硫杆菌TCE1菌株中并未出现，该菌株的四个基因转录水平未检测到显著差异。蛋白质组层面，膜组分中观测到PceA与PceB的化学计量比为2:1，且PceC的丰度远低于另外两种蛋白；在可溶性组分中，则鉴定出PceA与PceT的化学计量比为1:1。综上，本研究证实pce基因簇以操纵子形式进行转录，但各基因的转录水平存在差异，这一现象可通过转录后事件（如RNA加工与RNA稳定性差异）加以解释。蛋白质层面的结果表明，PceA与PceB可形成膜结合型PceA₂B蛋白复合物，与此前提出的模型不同，该复合物似乎并不包含PceC。