Gilberthorpe Nicola J, Poole Robert K
Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, United Kingdom.
J Biol Chem. 2008 Apr 25;283(17):11146-54. doi: 10.1074/jbc.M708019200. Epub 2008 Feb 18.
Nitric oxide (NO) is generated in biological systems primarily via the activity of NO synthases and nitrate and nitrite reductases. Here we show that Salmonella enterica serovar Typhimurium (S. typhimurium) grown anaerobically with nitrate is capable of generating polarographically detectable NO after nitrite (NO(2)(-)) addition. NO accumulation is sensitive to the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Neither an fnr mutant nor an fnr hmp double mutant produces NO, indicating the involvement in NO evolution from NO(2)(-) of protein(s) positively regulated by FNR. Contrary to previous findings in Escherichia coli, we demonstrate that neither the periplasmic nitrite reductase (NrfA) nor the cytoplasmic nitrite reductase (NirB) is involved in NO production in S. typhimurium. However, mutant cells lacking the membrane-bound nitrate reductase, NarGHI, and membranes derived from these cells are unable to produce NO, demonstrating that, in wild-type S. typhimurium, this enzyme is responsible for NO production. Membrane terminal oxidases cannot account for the NO levels measured. The nitrate reductase inhibitor, azide, abrogates NO evolution by Salmonella, and production of NO occurs only in the absence from the assays of nitrate; both features reveal a marked similarity between the NO-generating activities of this bacterium and plants. Unlike the situation in E. coli, an S. typhimurium hmp mutant produces NO both aerobically and anaerobically. Under aerobic conditions, when a functional flavohemoglobin is present, no NO is detectable. We propose a homeostatic mechanism in S. typhimurium, in which NO produced from NO(2)(-) by nitrate reductase derepresses Hmp expression (via FNR and NsrR) and NorV expression (via NorR) and thus limits NO toxicity.
一氧化氮（NO）在生物系统中主要通过一氧化氮合酶以及硝酸盐和亚硝酸盐还原酶的活性产生。在此我们表明，在硝酸盐存在下厌氧生长的鼠伤寒沙门氏菌（Salmonella enterica serovar Typhimurium）在添加亚硝酸盐（NO₂⁻）后能够产生极谱可检测的NO。NO的积累对NO清除剂2-(4-羧基苯基)-4,4,5,5-四甲基咪唑啉-1-氧基-3-氧化物敏感。fnr突变体和fnr hmp双突变体均不产生NO，这表明由FNR正向调控的蛋白质参与了从NO₂⁻生成NO的过程。与先前在大肠杆菌中的发现相反，我们证明周质亚硝酸还原酶（NrfA）和细胞质亚硝酸还原酶（NirB）均不参与鼠伤寒沙门氏菌中NO的产生。然而，缺乏膜结合硝酸盐还原酶NarGHI的突变细胞以及源自这些细胞的膜无法产生NO，这表明在野生型鼠伤寒沙门氏菌中，该酶负责NO的产生。膜末端氧化酶无法解释所测得的NO水平。硝酸盐还原酶抑制剂叠氮化物可消除沙门氏菌产生的NO，并且仅在测定中没有硝酸盐时才会产生NO；这两个特征揭示了该细菌与植物产生NO的活性之间存在明显相似性。与大肠杆菌的情况不同，鼠伤寒沙门氏菌hmp突变体在需氧和厌氧条件下均产生NO。在有氧条件下，当存在功能性黄素血红蛋白时，无法检测到NO。我们提出了一种鼠伤寒沙门氏菌中的稳态机制，其中硝酸盐还原酶从NO₂⁻产生的NO通过FNR和NsrR抑制Hmp表达，并通过NorR抑制NorV表达，从而限制NO的毒性。
