Elevated nitrate in the environment inhibits sulfate reduction by important microorganisms of sulfate-reducing bacteria (SRB). Several SRB may respire nitrate to survive under elevated nitrate, but how SRB that lack nitrate reductase survive to elevated nitrate remains elusive. To understand nitrate adaptation mechanisms, we evolved 12 populations of a model SRB (i.e., Desulfovibrio vulgaris Hildenborough, DvH) under elevated NaNO₃ for 1000 generations, analyzed growth and acquired mutations, and linked their genotypes with phenotypes. Nitrate-evolved (EN) populations significantly (p < 0.05) increased nitrate tolerance, and whole-genome resequencing identified 119 new mutations in 44 genes of 12 EN populations, among which six functional gene groups were discovered with high mutation frequencies at the population level. We observed a high frequency of nonsense or frameshift mutations in nitrosative stress response genes (NSR: DVU2543, DVU2547, and DVU2548), nitrogen regulatory protein C family genes (NRC: DVU2394-2396, DVU2402, and DVU2405), and nitrate cluster (DVU0246-0249 and DVU0251). Mutagenesis analysis confirmed that loss-of-functions of NRC and NSR increased nitrate tolerance. Also, functional gene groups involved in fatty acid synthesis, iron regulation, and two-component system (LytR/LytS) known to be responsive to multiple stresses, had a high frequency of missense mutations. Mutations in those gene groups could increase nitrate tolerance through regulating energy metabolism, barring entry of nitrate into cells, altering cell membrane characteristics, or conferring growth advantages at the stationary phase. This study advances our understanding of nitrate tolerance mechanisms and has important implications for linking genotypes with phenotypes in DvH.

环境中升高的硝酸盐会抑制硫酸盐还原菌 (SRB) 的重要微生物对硫酸盐的还原。几种 SRB 可能会呼吸硝酸盐以在硝酸盐升高的情况下存活，但缺乏硝酸还原酶的 SRB 如何在硝酸盐升高的情况下存活仍然难以捉摸。为了了解硝酸盐适应机制，我们在升高的 NaNO ₃下进化了 12 个模型 SRB（即Desulfovibrio vulgaris Hildenborough，DvH）群体1000 代，分析了生长和获得性突变，并将它们的基因型与表型联系起来。硝酸盐进化 (EN) 种群显着 ( p < 0.05) 增加硝酸盐耐受性，全基因组重测序在 12 个 EN 种群的 44 个基因中发现了 119 个新突变，其中在种群水平发现了 6 个具有高突变频率的功能基因组。我们观察到亚硝化应激反应基因（NSR：DVU2543、DVU2547 和 DVU2548）、氮调节蛋白 C 家族基因（NRC：DVU2394-2396、DVU2402 和 DVU2405）和硝酸盐簇（ -0249 和 DVU0251）。诱变分析证实 NRC 和 NSR 的功能丧失增加了硝酸盐耐受性。此外，参与脂肪酸合成、铁调节和双组分系统（LytR / LytS）的功能基因组) 已知对多种压力有反应，具有高频率的错义突变。这些基因组中的突变可以通过调节能量代谢、阻止硝酸盐进入细胞、改变细胞膜特性或在稳定期赋予生长优势来增加硝酸盐耐受性。这项研究增进了我们对硝酸盐耐受机制的理解，并对将基因型与 DvH 中的表型联系起来具有重要意义。