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.     

Nitric oxide homeostasis in Salmonella typhimurium: roles of respiratory nitrate reductase and flavohemoglobin

Nitric oxide (NO) is generated in biological systems primarily via the activity of NO synthases and nitrate and nitrite reductases. Here we show that Salmonella enterica serovar Typhimurium (S. typhimurium) grown anaerobically with nitrate is capable of generating polarographically detectable NO after nitrite (NO(2)(-)) addition. NO accumulation is sensitive to the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Neither an fnr mutant nor an fnr hmp double mutant produces NO, indicating the involvement in NO evolution from NO(2)(-) of protein(s) positively regulated by FNR. Contrary to previous findings in Escherichia coli, we demonstrate that neither the periplasmic nitrite reductase (NrfA) nor the cytoplasmic nitrite reductase (NirB) is involved in NO production in S. typhimurium. However, mutant cells lacking the membrane-bound nitrate reductase, NarGHI, and membranes derived from these cells are unable to produce NO, demonstrating that, in wild-type S. typhimurium, this enzyme is responsible for NO production. Membrane terminal oxidases cannot account for the NO levels measured. The nitrate reductase inhibitor, azide, abrogates NO evolution by Salmonella, and production of NO occurs only in the absence from the assays of nitrate; both features reveal a marked similarity between the NO-generating activities of this bacterium and plants. Unlike the situation in E. coli, an S. typhimurium hmp mutant produces NO both aerobically and anaerobically. Under aerobic conditions, when a functional flavohemoglobin is present, no NO is detectable. We propose a homeostatic mechanism in S. typhimurium, in which NO produced from NO(2)(-) by nitrate reductase derepresses Hmp expression (via FNR and NsrR) and NorV expression (via NorR) and thus limits NO toxicity.     

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.     

Substrate binding site for nitrate reductase of Escherichia coli is on the inner aspect of the membrane.

Escherichia coli grown anaerobically on nitrate exhibited the same transport barrier to reduction of chlorate, relative to nitrate, as that exhibited by Paracoccus denitrificans. This establishes that the nitrate binding site of nitrate reductase (EC 1.7.99.4) in E. coli must also lie on the cell side of the nitrate transporter which is associated with the plasma membrane. Because nitrate reductase is membrane bound, the nitrate binding site is thus located on the inner aspect of the membrane. Nitrate pulse studies on E. coli in the absence of valinomycin showed a small transient alkalinization (leads to H+/NO3- congruent to --0.07) which did not occur with oxygen pulses. By analogy with P. denitrificans, the alkaline transient is interpreted to arise from proton-linked nitrate uptake which is closely followed by nitrite efflux. The result is consistent with internal reduction of nitrate, whereas external reduction would be expected to give leads to H+/NO3-ratios approaching --2.     

Reactions of nitrite with hemoglobin measured by membrane inlet mass spectrometry

Membrane inlet mass spectrometry was used to observe nitric oxide in the well-studied reaction of nitrite with hemoglobin. The membrane inlet was submerged in the reaction solutions and measured NO in solution via its flux across a semipermeable membrane leading to the mass spectrometer detecting the mass-to-charge ratio m/z 30. This method measures NO directly in solution and is an alternate approach compared with methods that purge solutions to measure NO. Addition to deoxy-Hb(Fe(II)) (near 38 microM heme concentration) of nitrite in a range of 80 microM to 16 mM showed no accumulation of either NO or N(2)O(3) on a physiologically relevant time scale with a sensitivity near 1 nM. The addition of nitrite to oxy-Hb(Fe(II)) and met-Hb(Fe(III)) did not accumulate free NO to appreciable extents. These observations show that for several minutes after mixing nitrite with hemoglogin, free NO does not accumulate to levels exceeding the equilibrium level of NO. The presence of cyanide ions did not alter the appearance of the data; however, the presence of 2 mM mercuric ions at the beginning of the experiment with deoxy-Hb(Fe(II)) shortened the initial phase of NO accumulation and increased the maximal level of free, unbound NO by about twofold. These experiments appear consistent with no role of met-Hb(Fe(III)) in the generation of NO and an increase in nitrite reductase activity caused by the presumed binding of mercuric to cysteine residues. These results raise questions about the ability of reduction of nitrite mediated by deoxy-Hb(Fe(II)) to play a role in vasodilation.