1887

Abstract

Over the past decade, the flavohaemoglobin Hmp has emerged as the most significant nitric oxide (NO)-detoxifying protein in many diverse micro-organisms, particularly pathogenic bacteria. Its expression in enterobacteria is dramatically increased on exposure to NO and other agents of nitrosative stress as a result of transcriptional regulation of gene expression, mediated by (at least) four regulators. One such regulator, NsrR, has recently been shown to be responsible for repression of transcription in the absence of NO in and , but the roles of other members of this regulon in , particularly in surviving nitrosative stresses and , have not been elucidated. This paper demonstrates that an mutant of Serovar Typhimurium expresses high levels of Hmp both aerobically and anaerobically, exceeding those that can be elicited by supplementing media with -nitrosoglutathione (GSNO). Elevated transcription of , , and is also observed, but no evidence was obtained for upregulation. The hyper-resistance to GSNO of an mutant is attributable solely to Hmp, since an double mutant has a wild-type phenotype. However, overexpression of NsrR-regulated genes other than confers some resistance of respiratory oxygen consumption to NO. The ability to enhance, by mutating NsrR, Hmp levels without recourse to exposure to nitrosative stress was used to test the hypothesis that control of Hmp levels is required to avoid oxidative stress, Hmp being a potent generator of superoxide. Within IFN--stimulated J774.2 macrophages, in which high levels of nitrite accumulated (indicative of NO production) an mutant was severely compromised in survival. Surprisingly, under these conditions, an mutant (as well as an double mutant) was also disadvantaged relative to the wild-type bacteria, attributable to the combined oxidative effect of the macrophage oxidative burst and Hmp-generated superoxide. This explanation is supported by the sensitivity of an mutant to superoxide and peroxide. Fur has recently been confirmed as a weak repressor of transcription, and a mutant was also compromised for survival within macrophages even in the absence of elevated NO levels in non-stimulated macrophages. The results indicate the critical role of Hmp in protection of from nitrosative stress within and outside macrophages, but also the key role of transcriptional regulation in tuning Hmp levels to prevent exacerbation of the oxidative stress encountered in macrophages.

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2007-06-01
2019-10-23
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