Toya Yoshihiro, Nakahigashi Kenji, Tomita Masaru, Shimizu Kazuyuki
Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan.
Mol Biosyst. 2012 Oct;8(10):2593-604. doi: 10.1039/c2mb25069a.
It is of practical interest to investigate the effect of nitrates on bacterial metabolic regulation of both fermentation and energy generation, as compared to aerobic and anaerobic growth without nitrates. Although gene level regulation has previously been studied for nitrate assimilation, it is important to understand this metabolic regulation in terms of global regulators. In the present study, therefore, we measured gene expression using DNA microarrays, intracellular metabolite concentrations using CE-TOFMS, and metabolic fluxes using the (13)C-labeling technique for wild-type E. coli and the ΔarcA (a global regulatory gene for anoxic response control, ArcA) mutant to compare the metabolic state under nitrate conditions to that under aerobic and anaerobic conditions without nitrates in continuous culture conditions at a dilution rate of 0.2 h(-1). In wild-type, although the measured metabolite concentrations changed very little among the three culture conditions, the TCA cycle and the pentose phosphate pathway fluxes were significantly different under each condition. These results suggested that the ATP production rate was 29% higher under nitrate conditions than that under anaerobic conditions, whereas the ATP production rate was 10% lower than that under aerobic conditions. The flux changes in the TCA cycle were caused by changes in control at the gene expression level. In ΔarcA mutant, the TCA cycle flux was significantly increased (4.4 times higher than that of the wild type) under nitrate conditions. Similarly, the intracellular ATP/ADP ratio increased approximately two-fold compared to that of the wild-type strain.
与无硝酸盐的有氧和厌氧生长相比，研究硝酸盐对细菌发酵和能量产生的代谢调节作用具有实际意义。尽管之前已经对硝酸盐同化的基因水平调节进行了研究，但从全局调节因子的角度理解这种代谢调节很重要。因此，在本研究中，我们使用DNA微阵列测量基因表达，使用CE-TOFMS测量细胞内代谢物浓度，并使用(13)C标记技术测量野生型大肠杆菌和ΔarcA（缺氧反应控制的全局调节基因，ArcA）突变体的代谢通量，以比较在0.2 h(-1)的稀释率下连续培养条件下硝酸盐条件下的代谢状态与无硝酸盐的有氧和厌氧条件下的代谢状态。在野生型中，尽管在三种培养条件下测得的代谢物浓度变化很小，但三羧酸循环和戊糖磷酸途径通量在每种条件下都有显著差异。这些结果表明，硝酸盐条件下的ATP产生率比厌氧条件下高29%，而比有氧条件下低10%。三羧酸循环中的通量变化是由基因表达水平的控制变化引起的。在ΔarcA突变体中，硝酸盐条件下三羧酸循环通量显著增加（比野生型高4.4倍）。同样，细胞内ATP/ADP比值与野生型菌株相比增加了约两倍。
