Pyruvate fermentation vs Lactate-Sulfate. Nitratidesulfovibrio vulgaris str. Hildenborough

In order to obtain a global view of energy metabolism pathways of the sulfate-reducer Desulfovibrio vulgaris Hildenborough and the proteins involved therein whole-genome microarrays were used to compare the transcriptional response of cells grown with hydrogen/sulfate, pyruvate/sulfate, lactate/thiosulfate, and pyruvate with limiting sulfate, relative to growth in standard lactate/sulfate condition. Growth with hydrogen/sulfate showed the largest number of differently expressed genes and the largest changes in expression levels. The most up-regulated energy metabolism genes were those coding for the periplasmic [NiFeSe] hydrogenase, followed by the Ech hydrogenase, and the most down-regulated were genes coding for the Coo hydrogenase. The results point to the involvement of formate cycling and the ethanol pathway during growth on hydrogen, whereas there is evidence for CO cycling during growth on lactate and pyruvate, but not on H2. Growth with thiosulfate showed the hallmarks of a reduced energy status of the cells with down regulation of the ATP synthase and the Qmo and Dsr complexes., whereas growth with pyruvate showed the smallest differences but an increased role for the Ech hydrogenase.that in this case functions in reverse from the case of growth with H2. The multiple periplasmic hydrogenases and formate dehydrogenases, do not display the same regulation pattern showing that their metabolic roles are not totally interchangeable. This result together with the observation that several genes coding for proteins that have not been biochemically characterised were considerably affected in this study, reveals a more complex energy metabolism than previously considered and provides guidance for further studies. Keywords: Growth protocol Overall design: Desulfovibrio vulgaris from our laboratory culture collection was cultured at 37°C with pyruvate as the only substrate (without sulfate as the electron acceptor) to mid-log phase. Cultures were also cultivated similarly in Lactate-Sulfate medium to mid-log phase. Gene expression profiles of cultures grown under pyruvate fermentation were compared with those of the cultures grown via sulfate reduction. Total RNA was harvested from four replicate cultures for microarray analysis. RNA extraction, purification, and labeling were performed independently on each cell sample.Two samples of each total RNA preparation were labeled, one with Cy3-dUTP and another with Cy5-dUTP for microarray hybridization.

为全面解析脱硫弧菌（Desulfovibrio vulgaris）Hildenborough菌株的能量代谢通路及其相关蛋白的转录调控特征，本研究借助全基因组微阵列（whole-genome microarrays）技术，以标准乳酸盐-硫酸盐培养条件为对照，对比分析该菌株在氢/硫酸盐、丙酮酸/硫酸盐、乳酸盐/硫代硫酸盐，以及硫酸盐限制型丙酮酸培养条件下的细胞转录响应（transcriptional response）差异。其中，氢/硫酸盐培养组的差异表达基因（differently expressed genes）数量最多，表达水平的变化幅度也最为显著。上调幅度最高的能量代谢基因为编码周质[NiFeSe]氢化酶（periplasmic [NiFeSe] hydrogenase）的基因，其次为Ech氢化酶（Ech hydrogenase）编码基因；而下调最为显著的则为编码Coo氢化酶（Coo hydrogenase）的基因。研究结果显示，菌株以氢气为底物生长时，甲酸循环（formate cycling）与乙醇代谢通路（ethanol pathway）参与其中；而在乳酸盐与丙酮酸培养条件下，则存在CO循环（CO cycling）的相关证据，但氢培养条件下未观测到此现象。硫代硫酸盐培养组中，细胞呈现能量状态降低的特征：ATP合酶（ATP synthase）、Qmo复合物（Qmo complex）与Dsr复合物（Dsr complex）均出现下调表达。丙酮酸培养组的转录差异最小，但Ech氢化酶的作用显著增强，此时其功能与氢培养组中恰好相反。多种周质氢化酶与甲酸脱氢酶的调控模式并不统一，提示其代谢功能并非完全可相互替代。此外，本研究中多个尚未经生化表征的蛋白编码基因的表达水平发生显著改变，这一结果与前述发现共同表明，该菌株的能量代谢过程较此前认知更为复杂，可为后续相关研究提供重要指引。关键词：培养方案 整体实验设计：本研究从实验室菌种保藏库中获取脱硫弧菌（Desulfovibrio vulgaris）菌株，以丙酮酸为唯一底物（无硫酸盐作为电子受体）于37℃培养至对数中期；同时以乳酸盐-硫酸盐培养基进行相同条件的培养至对数中期。将丙酮酸发酵培养的菌体基因表达谱与硫酸盐还原培养的菌体表达谱进行对比分析。从4个重复培养样本中提取总RNA（total RNA）用于微阵列分析（microarray analysis）。每个细胞样本独立完成RNA提取、纯化与标记流程。每份总RNA制备物制备两个标记样本：分别以Cy3-dUTP与Cy5-dUTP进行标记，用于微阵列杂交（microarray hybridization）。