| The ability of Neisseria gonorrhoeae to reduce nitric oxide (NO) may have important immunomodulary effects on the host during infection. Therefore, a comprehensive understanding of the regulatory mechanism of the nitric oxide reductase gene (norB) needs to be elucidated. To accomplish this, the functional regions of the norB upstream region were analyzed. The promoter contains an extended -10 motif (TGNTACAAT) that is required for high level expression. Deletion and substitution analysis of the norB upstream region revealed that no sequence upstream of the -10 motif is involved in norB regulation under anaerobic conditions or in the presence of NO. However, replacement of a 29 base pair inverted repeat sequence immediately downstream of the extended -10 motif gave high level aerobic expression of a norB::lacZ fusion. Insertional inactivation of gonococcal nsrR, an Rrf2-type transcriptional repressor, resulted in the loss of norB repression and eliminated NO induction capacity. Single-copy complementation of nsrR in trans restored regulation of both norB transcription and NorB activity by NO. In E. coli, expression of a gonococcal nsrR gene repressed gonococcal norB; induction of norB occurred in the presence of exogenously added NO but not nitrite. In addition, NsrR was found to regulate expression of aniA, dnrN, and nnrS. It was also determined that Fur regulates norB by a novel indirect activation method, by preventing the binding of a gonococcal ArsR homolog, a second repressor whose putative binding site overlaps that of the Fur binding site.;A highly pure extract of epitope tagged NsrR was isolated and mass spectroscopic analysis demonstrated that the protein contained a [2Fe-2S] cluster. NsrR/DNA interactions were thoroughly analyzed in vitro. Using EMSA analysis, NsrR::FLAG was shown to interact with predicted operators in the norB, aniA, and nnrS upstream regions with a K d of 7 nM, 19 nM, and 35 nM respectively. DNase I footprint analysis was performed on the upstream regions of norB and nnrS, where NsrR was shown to protect the predicted 29 bp binding sites. The presence of exogenously added NO inhibited DNA binding by NsrR. Alanine substitution of C90, C97, or C103 in NsrR abrogated repression of norB::lacZ and inhibited DNA binding. Mass spectroscopic analysis of NsrR C90A demonstrated the absence of a [2Fe-2S] cluster, consistent with the presumed role of these cysteine residues in the coordination of a NO-sensitive Fe-S center required for high affinity DNA binding.;Due to the fact that N. gonorrhoeae is an obligate human pathogen, it has been a challenge to establish an animal model that would allow direct testing of the involvement of denitrification as it relates to virulence. Comparative genomics may be a useful method to evaluate the role of specific proteins in neisserial pathogenesis, as only two neisserial species are capable of causing disease in healthy individuals. Recent completion of genome sequencing projects for the commensal Neisseriaceae allowed a comparison of the cis- and trans-acting elements involved in the regulation of denitrification in Neisseria spp. From this analysis it was shown that, in contrast to the pathogenic Neisseria spp., many commensal species may have decreased regulation of the norB gene in response to NO, and some species may have lost the ability to regulate norB in response to iron. This analysis also suggested that, unlike N. gonorrhoeae, other Neisseria spp. may be capable of sensing and responding to nitrite, which may reflect the different ecological niche in which these organisms reside. |
