Department of Microbiology and Cell Science, IFAS, University of Florida, Gainesville, FL, USA.
Department Oesterhelt, Max Planck Institut für Biochemie, Martinsried, Germany.
Microbiologyopen. 2020 Nov;9(11):e1124. doi: 10.1002/mbo3.1124. Epub 2020 Oct 14.
Bacterial nitric oxide (NO) synthases (bNOS) play diverse and important roles in microbial physiology, stress resistance, and virulence. Although bacterial and mammalian NOS enzymes have been well-characterized, comparatively little is known about the prevalence and function of NOS enzymes in Archaea. Analysis of archaeal genomes revealed that highly conserved bNOS homologs were restricted to members of the Halobacteria. Of these, Natronomonas pharaonis NOS (npNOS) was chosen for further characterization. NO production was confirmed in heterologously expressed His-tagged npNOS by coupling nitrite production from N-hydroxy-L-arginine in an HO-supported reaction. Additionally, the nos gene was successfully targeted and disrupted to create a Nmn. pharaonis nos mutant by adapting an established Natrialba magadii transformation protocol. Genome re-sequencing of this mutant revealed an additional frameshift in a putative cation-acetate symporter gene, which could contribute to altered acetate metabolism in the nos mutant. Inactivation of Nmn. pharaonis nos was also associated with several phenotypes congruent with bacterial nos mutants (altered growth, increased oxygen consumption, increased pigment, increased UV susceptibility), suggesting that NOS function may be conserved between bacteria and archaea. These studies are the first to describe genetic inactivation and characterization of a Nmn. pharaonis gene and provides enhanced tools for probing its physiology.
细菌一氧化氮 (NO) 合酶 (bNOS) 在微生物生理学、抗应激和毒力方面发挥着多样化且重要的作用。尽管细菌和哺乳动物的 NOS 酶已得到充分研究，但关于古菌中 NOS 酶的普遍性和功能却知之甚少。对古菌基因组的分析表明，高度保守的 bNOS 同源物仅限于盐杆菌属成员。在这些同源物中，选择嗜盐碱杆菌 NOS (npNOS) 进行进一步表征。通过在 HO 支持的反应中耦合 N-羟基-L-精氨酸产生的亚硝酸盐，在异源表达的 His 标记 npNOS 中确认了 NO 的产生。此外，成功地靶向并破坏了 nos 基因，通过适应已建立的盐生盐杆菌转化方案创建了 Nmn。嗜盐碱杆菌 nos 突变体。对该突变体的基因组重测序揭示了一个假定阳离子-乙酸盐转运蛋白基因中的额外移码，这可能导致 nos 突变体中乙酸盐代谢的改变。Nmn 的失活。嗜盐碱杆菌 nos 也与几种与细菌 nos 突变体一致的表型相关（生长改变、耗氧量增加、色素增加、对 UV 的敏感性增加），这表明 NOS 功能在细菌和古菌之间可能是保守的。这些研究首次描述了 Nmn 的遗传失活和嗜盐碱杆菌基因的表征，并为探究其生理学提供了增强的工具。
