School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan
School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan.
Appl Environ Microbiol. 2019 Jan 23;85(3). doi: 10.1128/AEM.02668-18. Print 2019 Feb 1.
MR-1 is a facultative anaerobe that respires using a variety of electron acceptors. Although this organism is incapable of fermentative growth in the absence of electron acceptors, its genome encodes LdhA (a putative fermentative NADH-dependent d-lactate dehydrogenase [d-LDH]) and Dld (a respiratory quinone-dependent d-LDH). However, the physiological roles of LdhA in MR-1 are unclear. Here, we examined the activity, transcriptional regulation, and traits of deletion mutants to gain insight into the roles of LdhA in the anaerobic growth of MR-1. Analyses of d-LDH activity in MR-1 and the deletion mutant confirmed that LdhA functions as an NADH-dependent d-LDH that catalyzes the reduction of pyruvate to d-lactate. and assays revealed that expression was positively regulated by the cyclic-AMP receptor protein, a global transcription factor that regulates anaerobic respiratory pathways in MR-1, suggesting that LdhA functions in coordination with anaerobic respiration. Notably, we found that a deletion mutant of all four NADH dehydrogenases (NDHs) in MR-1 (ΔNDH mutant) retained the ability to grow on -acetylglucosamine under fumarate-respiring conditions, while an additional deletion of or deprived the ΔNDH mutant of this growth ability. These results indicate that LdhA-Dld serves as a bypass of NDH in electron transfer from NADH to quinones. Our findings suggest that the LdhA-Dld system manages intracellular redox balance by utilizing d-lactate as a temporal electron sink under electron acceptor-limited conditions. NADH-dependent LDHs are conserved among diverse organisms and contribute to NAD regeneration in lactic acid fermentation. However, this type of LDH is also present in nonfermentative bacteria, including members of the genus , while their physiological roles in these bacteria remain unknown. Here, we show that LdhA (an NADH-dependent d-LDH) works in concert with Dld (a quinone-dependent d-LDH) to transfer electrons from NADH to quinones during sugar catabolism in MR-1. Our results indicate that d-lactate acts as an intracellular electron mediator to transfer electrons from NADH to membrane quinones. In addition, d-lactate serves as a temporal electron sink when respiratory electron acceptors are not available. Our study suggests novel physiological roles for d-LDHs in providing nonfermentative bacteria with catabolic flexibility under electron acceptor-limited conditions.
MR-1 是一种兼性厌氧菌，可利用多种电子受体进行呼吸。尽管该生物体在没有电子受体的情况下无法进行发酵生长，但它的基因组编码了 LdhA（一种假定的发酵 NADH 依赖性 d-乳酸脱氢酶[d-LDH]）和 Dld（一种呼吸醌依赖性 d-乳酸脱氢酶）。然而，LdhA 在 MR-1 中的生理作用尚不清楚。在这里，我们研究了 LdhA 缺失突变体的活性、转录调控和特性，以深入了解 LdhA 在 MR-1 无氧生长中的作用。在 MR-1 和缺失突变体中对 d-LDH 活性的分析证实，LdhA 作为 NADH 依赖性 d-LDH 发挥作用，可催化丙酮酸还原为 d-乳酸。实时荧光定量 PCR 和 LacZ 报告基因测定表明，LdhA 的表达受环 AMP 受体蛋白的正调控，该蛋白是一种全局转录因子，可调节 MR-1 中的无氧呼吸途径，表明 LdhA 与无氧呼吸协调发挥作用。值得注意的是，我们发现 MR-1 中所有四个 NADH 脱氢酶（NDH）的缺失突变体（ΔNDH 突变体）在富马酸盐呼吸条件下仍能利用乙酰葡萄糖胺生长，而额外缺失或剥夺了ΔNDH 突变体的这种生长能力。这些结果表明，LdhA-Dld 作为电子从 NADH 向醌传递的旁路，在电子受体有限的条件下，将 d-乳酸作为临时电子汇来管理细胞内的氧化还原平衡。NADH 依赖性 LDH 在不同生物体中保守，并有助于乳酸发酵中的 NAD 再生。然而，这种类型的 LDH 也存在于非发酵细菌中，包括属的成员，而它们在这些细菌中的生理作用仍然未知。在这里，我们表明 LdhA（一种 NADH 依赖性 d-LDH）在 MR-1 中的糖分解代谢过程中与 Dld（一种醌依赖性 d-LDH）协同作用，将电子从 NADH 传递到醌上。我们的结果表明，d-乳酸作为一种细胞内电子介体，将电子从 NADH 传递到膜醌上。此外，当呼吸电子受体不可用时，d-乳酸作为临时电子汇。我们的研究表明，d-LDH 在电子受体有限的条件下为非发酵细菌提供了代谢灵活性的新的生理作用。
