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Abstract

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 pharaonisNOS (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 H2O2-supported reaction. Additionally, thenosgene was successfully targeted and disrupted to create aNmn. pharaonis nosmutant by adapting an establishedNatrialba magadiitransformation 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 thenosmutant. Inactivation ofNmn. pharaonis noswas also associated with several phenotypes congruent with bacterialnosmutants (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 aNmn. pharaonisgene and provides enhanced tools for probing its physiology.