Regulation of denitrification genes in Neisseria meningitidis by nitric oxide and the repressor NsrR

The human pathogen Neisseria meningitidis is capable of growth using the denitrification of nitrite to nitrous oxide under microaerobic conditions. This process is catalyzed by two reductases: nitrite reductase (encoded by aniA) and nitric oxide (NO) reductase (encoded by norB). Here, we show that in N. meningitidis MC58 norB is regulated by nitric oxide via the product of gene NMB0437 which encodes NsrR. NsrR is a repressor in the absence of NO, but norB expression is derepressed by NO in an NsrR-dependent manner. nsrR-deficient mutants grow by denitrification more rapidly than wild-type N. meningitidis, and this is coincident with the upregulation of both NO reductase and nitrite reductase even under aerobic conditions in the absence of nitrite or NO. The NsrR-dependent repression of aniA (unlike that of norB) is not lifted in the presence of NO. The role of NsrR in the control of expression of aniA is linked to the function of the anaerobic activator protein FNR: analysis of nsrR and fnr single and nsrR fnr double mutants carrying an aniA promoter lacZ fusion indicates that the role of NsrR is to prevent FNR-dependent aniA expression under aerobic conditions, indicating that FNR in N. meningitidis retains considerable activity aerobically.     

The nitric oxide (NO)-sensing repressor NsrR of Neisseria meningitidis has a compact regulon of genes involved in NO synthesis and detoxification

We have analyzed the extent of regulation by the nitric oxide (NO)-sensitive repressor NsrR from Neisseria meningitidis MC58, using microarray analysis. Target genes that appeared to be regulated by NsrR, based on a comparison between an nsrR mutant and a wild-type strain, were further investigated by quantitative real-time PCR, revealing a very compact set of genes, as follows: norB (encoding NO reductase), dnrN (encoding a protein putatively involved in the repair of nitrosative damage to iron-sulfur clusters), aniA (encoding nitrite reductase), nirV (a putative nitrite reductase assembly protein), and mobA (a gene associated with molybdenum metabolism in other species but with a frame shift in N. meningitidis). In all cases, NsrR acts as a repressor. The NO protection systems norB and dnrN are regulated by NO in an NsrR-dependent manner, whereas the NO protection system cytochrome c' (encoded by cycP) is not controlled by NO or NsrR, indicating that N. meningitidis expresses both constitutive and inducible NO protection systems. In addition, we present evidence to show that the anaerobic response regulator FNR is also sensitive to NO but less so than NsrR, resulting in complex regulation of promoters such as aniA, which is controlled by both FNR and NsrR: aniA was found to be maximally induced by intermediate NO concentrations, consistent with a regulatory system that allows expression during denitrification (in which NO accumulates) but is down-regulated as NO approaches toxic concentrations.     

Nitric oxide metabolism in Neisseria meningitidis

Neisseria meningitidis, the causative agent of meningococcal disease in humans, is likely to be exposed to nitrosative stress during natural colonization and disease. The genome of N. meningitidis includes the genes aniA and norB, predicted to encode nitrite reductase and nitric oxide (NO) reductase, respectively. These gene products should allow the bacterium to denitrify nitrite to nitrous oxide. We show that N. meningitidis can support growth microaerobically by the denitrification of nitrite via NO and that norB is required for anaerobic growth with nitrite. NorB and, to a lesser extent, the cycP gene product cytochrome c' are able to counteract toxicity due to exogenously added NO. Expression of these genes by N. meningitidis during colonization and disease may confer protection against exogenous or endogenous nitrosative stress.     

Phylogenetic analysis of nitrite, nitric oxide, and nitrous oxide respiratory enzymes reveal a complex evolutionary history for denitrification

Denitrification is a facultative respiratory pathway in which nitrite (NO2(-)), nitric oxide (NO), and nitrous oxide (N2O) are successively reduced to nitrogen gas (N(2)), effectively closing the nitrogen cycle. The ability to denitrify is widely dispersed among prokaryotes, and this polyphyletic distribution has raised the possibility of horizontal gene transfer (HGT) having a substantial role in the evolution of denitrification. Comparisons of 16S rRNA and denitrification gene phylogenies in recent studies support this possibility; however, these results remain speculative as they are based on visual comparisons of phylogenies from partial sequences. We reanalyzed publicly available nirS, nirK, norB, and nosZ partial sequences using Bayesian and maximum likelihood phylogenetic inference. Concomitant analysis of denitrification genes with 16S rRNA sequences from the same organisms showed substantial differences between the trees, which were supported by examining the posterior probability of monophyletic constraints at different taxonomic levels. Although these differences suggest HGT of denitrification genes, the presence of structural variants for nirK, norB, and nosZ makes it difficult to determine HGT from other evolutionary events. Additional analysis using phylogenetic networks and likelihood ratio tests of phylogenies based on full-length sequences retrieved from genomes also revealed significant differences in tree topologies among denitrification and 16S rRNA gene phylogenies, with the exception of the nosZ gene phylogeny within the data set of the nirK-harboring genomes. However, inspection of codon usage and G + C content plots from complete genomes gave no evidence for recent HGT. Instead, the close proximity of denitrification gene copies in the genomes of several denitrifying bacteria suggests duplication. Although HGT cannot be ruled out as a factor in the evolution of denitrification genes, our analysis suggests that other phenomena, such gene duplication/divergence and lineage sorting, may have differently influenced the evolution of each denitrification gene.     

Determinants of nitric oxide steady-state levels during anaerobic respiration by Neisseria gonorrhoeae

Nitric oxide (NO) is an important host defence molecule that varies its immune stimulatory effects depending on the concentrations at which it is produced, with low concentrations (< 1 microM) promoting an anti-inflammatory host response while higher concentrations (>1 microM) lead to inflammatory responses. Neisseria gonorrhoeae grows anaerobically by anaerobic respiration using nitrite reductase (Nir) to convert nitrite to NO and nitric oxide reductase (Nor) to convert NO to nitrous oxide. As N. gonorrhoeae can both produce and degrade NO, we have begun a study of NO metabolism in this bacterium to understand how gonococcal manipulation of NO concentration may influence the inflammatory response during infection. N. gonorrhoeae has an apparent Nir Km of 33 microM nitrite and an apparent Nor Km of 1.2 microM NO. The maximum specific activities for Nir and Nor were 135 nmoles nitrite reduced per minute per OD600 (pH 6.7) and 270 nmoles NO reduced per minute per OD600 (pH 7.5) respectively. N. gonorrhoeae established a steady-state concentration of NO after nitrite addition that was dependent on the nitrite concentration until saturation at 1 mM nitrite. The NO steady-state level decreased as pH increased, and the ratio of activities of Nir and Nor correlated to the NO steady-state level. When the NO donor DETA/NO was used to simulate host NO production, N. gonorrhoeae also established a NO steady-state level. The concentration of NO at steady state was found to be a function of the concentration of NO generated by DETA/NO, with N. gonorrhoeae reducing the NO from proinflammatory (>1 microM) to anti-inflammatory (approximately 100 nM) concentrations. The implications of the ability of N. gonorrhoeae to maintain an anti-inflammatory NO concentration is discussed in relation to asymptomatic infection in women.