Data_Sheet_1_Stoichiometry of the Gene Products From the Tetrachloroethene Reductive Dehalogenase Operon pceABCT.PDF

Organohalide respiration (OHR) is a bacterial anaerobic process that uses halogenated compounds, e.g., tetrachloroethene (PCE), as terminal electron acceptors. Our model organisms are Dehalobacter restrictus strain PER-K23, an obligate OHR bacterium (OHRB), and Desulfitobacterium hafniense strain TCE1, a bacterium with a versatile metabolism. The key enzyme is the PCE reductive dehalogenase (PceA) that is encoded in the highly conserved gene cluster (pceABCT) in both above-mentioned strains, and in other Firmicutes OHRB. 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. We present a multilevel study aiming at deciphering the stoichiometry of 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 Pce proteins in cell-free extracts as well as in soluble and membrane fractions of both strains using heavy-labeled reference peptides. At RNA level, our results confirmed the co-transcription of all pce genes, while the quantitative analysis revealed a relative stoichiometry of the gene transcripts of pceA, pceB, pceC, and pceT at ~ 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, an apparent 2:1 stoichiometry of PceA and PceB was obtained 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. Despite challenges in the quantification of integral membrane proteins such as PceB and PceC, the similar abundance of PceA and PceB invites to consider them as forming a membrane-bound PceA2B protein complex, which, in contrast to the proposed model, seems to be devoid of PceC.

有机卤化物呼吸（Organohalide respiration, OHR）是一类以卤代化合物（如四氯乙烯（tetrachloroethene, PCE））为末端电子受体的细菌厌氧代谢过程。本研究选用的模式菌株为限制性脱卤杆菌（Dehalobacter restrictus）PER-K23——一种专性有机卤化物呼吸细菌（obligate OHR bacterium, OHRB），以及哈氏脱卤拟球菌（Desulfitobacterium hafniense）TCE1——一类代谢多样的细菌。该过程的关键酶为PCE还原脱卤酶（PCE reductive dehalogenase, PceA），其编码基因存在于上述两株菌及其他厚壁菌门（Firmicutes）有机卤化物呼吸细菌中高度保守的基因簇（pceABCT）内。
截至目前，PceA与负责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的具体作用仍未阐明。本研究开展多维度分析，旨在解析pceABCT各基因编码产物的化学计量比。在RNA层面，我们通过反转录及（定量）聚合酶链式反应进行检测；在蛋白质层面，则基于平行反应监测（parallel reaction monitoring）开展蛋白质组学分析，利用重标记参考肽段对两株菌的无细胞提取物、可溶性组分及膜组分中的Pce家族蛋白进行定量检测。
RNA层面的实验结果证实，所有pce基因均发生共转录；定量分析显示，在限制性脱卤杆菌PER-K23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。这一趋势在哈氏脱卤拟球菌TCE1中未被观测到，该菌株的四个基因转录水平无显著差异。
蛋白质层面的检测结果显示：膜组分中PceA与PceB呈现约2:1的化学计量比，且PceC的丰度显著低于另外两种蛋白；可溶性组分中则检测到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加以解释。尽管对PceB、PceC这类整合膜蛋白的定量分析仍存在挑战，但PceA与PceB的丰度相似，提示二者可能形成膜结合型PceA2B蛋白复合物——与已提出的模型不同，该复合物似乎不含PceC。