Duhl Kody L, TerAvest Michaela A
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, United States.
Front Energy Res. 2019 Oct;7. doi: 10.3389/fenrg.2019.00116. Epub 2019 Oct 24.
MR-1 is a dissimilatory metal reducing bacterium with a highly branched respiratory electron transport chain. The MR-1 genome encodes four NADH dehydrogenases, any of which may be used during respiration. We previously determined that a double-knockout of two NADH dehydrogenases, Nuo and Nqr1, eliminated aerobic growth in minimal medium. However, the double-knockout strain was able to grow aerobically in rich medium. Here, we determined that amino acid supplementation rescued growth of the mutant strain in oxic minimal medium. To determine the mechanism of the growth defect, we monitored growth, metabolism, and total NAD(H) pools in MR-1 and the NADH dehydrogenase knockout strain. We also used a genetically encoded redox sensing system and determined that NADH/NAD was higher in the mutant strain than in the wild-type. We observed that the double-knockout strain was able to metabolize d,l-lactate and N-acetylglucosamine when supplemented with tryptone, but excreted high concentrations of pyruvate and acetate. The requirement for amino acid supplementation, combined with an apparent inability of the mutant strain to oxidize pyruvate or acetate suggests that TCA cycle activity was inhibited in the mutant strain by a high NADH/NAD.
MR-1是一种异化金属还原菌，具有高度分支的呼吸电子传递链。MR-1基因组编码四种NADH脱氢酶，在呼吸过程中可使用其中任何一种。我们之前确定，两种NADH脱氢酶Nuo和Nqr1的双敲除消除了基本培养基中的有氧生长。然而，双敲除菌能够在丰富培养基中进行有氧生长。在这里，我们确定补充氨基酸可挽救突变菌株在有氧基本培养基中的生长。为了确定生长缺陷的机制，我们监测了MR-1和NADH脱氢酶敲除菌的生长、代谢和总NAD(H)库。我们还使用了一种基因编码的氧化还原传感系统，确定突变菌株中的NADH/NAD高于野生型。我们观察到，当补充胰蛋白胨时，双敲除菌能够代谢d，l-乳酸和N-乙酰葡糖胺，但会分泌高浓度的丙酮酸和乙酸。对氨基酸补充的需求，以及突变菌株明显无法氧化丙酮酸或乙酸，表明突变菌株中的三羧酸循环活性因高NADH/NAD而受到抑制。
