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Metabolic and Transcriptional Analysis of Acid Stress in Lactococcus lactis, with a Focus on the Kinetics of Lactic Acid Pools

机译:乳酸乳球菌酸胁迫的代谢和转录分析,重点是乳酸池的动力学

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摘要

The effect of pH on the glucose metabolism of non-growing cells of L. lactis MG1363 was studied by in vivo NMR in the range 4.8 to 6.5. Immediate pH effects on glucose transporters and/or enzyme activities were distinguished from transcriptional/translational effects by using cells grown at the optimal pH of 6.5 or pre-adjusted to low pH by growth at 5.1. In cells grown at pH 5.1, glucose metabolism proceeds at a rate 35% higher than in non-adjusted cells at the same pH. Besides the upregulation of stress-related genes (such as dnaK and groEL), cells adjusted to low pH overexpressed H+-ATPase subunits as well as glycolytic genes. At sub-optimal pHs, the total intracellular pool of lactic acid reached approximately 500 mM in cells grown at optimal pH and about 700 mM in cells grown at pH 5.1. These high levels, together with good pH homeostasis (internal pH always above 6), imply intracellular accumulation of the ionized form of lactic acid (lactate anion), and the concomitant export of the equivalent protons. The average number, n, of protons exported with each lactate anion was determined directly from the kinetics of accumulation of intra-and extracellular lactic acid as monitored online by C-13-NMR. In cells non-adjusted to low pH, n varies between 2 and 1 during glucose consumption, suggesting an inhibitory effect of intracellular lactate on proton export. We confirmed that extracellular lactate did not affect the lactate: proton stoichiometry. In adjusted cells, n was lower and varied less, indicating a different mix of lactic acid exporters less affected by the high level of intracellular lactate. A qualitative model for pH effects and acid stress adaptation is proposed on the basis of these results.
机译:通过体内NMR在4.8至6.5范围内研究了pH对乳酸乳球菌MG1363非生长细胞的葡萄糖代谢的影响。通过使用在6.5的最佳pH下生长或通过在5.1的生长下预先调节至低pH值的细胞,可以将即时的pH对葡萄糖转运蛋白和/或酶活性的影响与转录/翻译作用区分开。在pH为5.1的细胞中,在相同pH下,葡萄糖的代谢速率比未调整的细胞高35%。除了上调与压力相关的基因(例如dnaK和groEL)外,将细胞调节至低pH值也可以过表达H + -ATPase亚基以及糖酵解基因。在最佳pH值下,在最佳pH下生长的细胞中乳酸的总细胞内池达到约500 mM,在pH 5.1下生长的细胞中约700 mM。这些高水平,加上良好的pH稳态(内部pH始终高于6),暗示了离子化形式的乳酸(乳酸阴离子)在细胞内的蓄积,并伴随着等价质子的输出。与每种乳酸根阴离子一起输出的质子平均数n,直接由C-13-NMR在线监测的细胞内和细胞外乳酸积累的动力学确定。在未调节至低pH的细胞中,葡萄糖消耗期间n在2到1之间变化,表明细胞内乳酸对质子输出的抑制作用。我们证实细胞外乳酸不影响乳酸:质子化学计量。在调整后的细胞中,n较低且变化较小,表明乳酸输出蛋白的不同混合物较少受到高水平细胞内乳酸的影响。在这些结果的基础上,提出了pH效应和酸适应性的定性模型。

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