• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Feb 2007; 189(3): 1138–1144.
Published online Nov 22, 2006. doi:  10.1128/JB.01368-06
PMCID: PMC1797324

Regulation of Denitrification Genes in Neisseria meningitidis by Nitric Oxide and the Repressor NsrR[down-pointing small open triangle]

Abstract

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 human pharynx is the only known natural habitat of Neisseria meningitidis (31). In the majority of cases, N. meningitidis colonization is asymptomatic, but occasionally invasive disease occurs following entry of meningococci into the bloodstream. N. meningitidis can grow aerobically, or, under oxygen limitation, growth is supported by denitrification using nitrite and nitric oxide as electron acceptors. Denitrification in this organism consists of the reduction of nitrite to nitric oxide via nitrite reductase AniA and reduction of nitric oxide to nitrous oxide via nitric oxide reductase NorB (1). The genes aniA and norB are divergently transcribed from one another and separated by an intergenic region of 370 base pairs (29). Nitric oxide (NO) in mammals is generated from arginine via the NO synthase enzymes (16). NO is present at high concentrations in the nasopharynx, as judged by its concentration in exhaled nasal breath (12). The ability of N. meningitidis to use NO is important for its lifestyle in several respects: (i) denitrification supports growth (23); (ii) NO is a toxic free radical gas, the toxicity of which is controlled by NO reductase in both pure culture (1) and tissue and organ culture models of N. meningitidis colonization (28); and (iii) bacterial NO reduction impacts upon host processes such as apoptosis (30).

Denitrification in N. meningitidis, and in its close relative Neisseria gonorrhoeae, is regulated by oxygen availability and also by the availability of nitrite and nitric oxide (10, 13, 23). The expression of the nitrite reductase gene aniA in N. meningitidis is controlled by oxygen via the transcriptional regulator FNR (fumarate and nitrate reduction regulator) and by nitrite via the two-component sensor-regulator NarQ/P (23). The expression of norB, on the other hand, appears to be consequent on the accumulation of NO in the culture medium. Here, we investigate the regulation of gene expression by NO and identify the NO-responsive regulator.

A number of different regulators have been implicated in the control of NO metabolism in bacteria. Regulators SoxR, FNR, and FUR, whose primary roles are considered to be the regulation of gene expression in response to superoxide stress, anaerobiosis, and iron limitation, respectively, have been shown to be capable of regulating gene expression in response to NO (5, 6, 19). Additionally, NO-specific regulators have been identified. NorR has been shown to regulate the expression of the flavorubredoxin NO detoxification system NorVW and other genes in Escherichia coli in direct response to NO (7). The regulator NsrR, which was originally identified as a nitrite-sensitive repressor in Nitrosomonas europaea (3), has homologues in E. coli and Bacillus subtilis which have been shown to be involved in control of gene expression in response to nitric oxide in these microorganisms (4, 17). The gene annotated as NMB0437 from the genome of N. meningitidis MC58 (29) was predicted by Rodionov et al. (24) to be an nsrR homologue. Here, we show that this gene from the pathogenic denitrifier N. meningitidis does indeed encode an nsrR homologue and that its gene product controls expression of denitrification genes.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

All N. meningitidis strains used in this study were derived from N. meningitidis MC58 (Table (Table1).1). N. meningitidis strains were routinely cultured at 37°C in 5% CO2 on Columbia horse blood agar plates or in liquid culture in Mueller-Hinton broth supplemented with 10 mM NaHCO3. Aerobic culture was carried out in 7.5 ml of broth in a 50-ml Falcon tube with shaking at 200 rpm. Microaerobic culture was carried out using 20 ml of broth in a 25-ml McCartney bottle with shaking at 90 rpm and, where appropriate, supplemented with 5 mM NaNO2. Antibiotics were used at the following concentrations: spectinomycin, 50 μg ml−1; erythromycin, 100 μg ml−1; and chloramphenicol, 2 μg ml−1.

TABLE 1.
Strains, plasmids and oligonucleotide primers

E. coli was cultured in LB liquid medium (5 ml in 25-ml McCartney bottles shaken at 37°C and 200 rpm) or on LB agar plates. Antibiotics were used at the following concentrations: spectinomycin, 50 μg ml−1; chloramphenicol, 25 μg ml−1.

Construction of strains.

Genetic mutation of gene nsrR (N. meningitidis MC58 gene NMB0437) was produced by allelic replacement with an insertionally inactivated cloned N. meningitidis MC58 gene. nsrR and its flanking region were amplified using primers NOregF1 and NOregR1 (Table (Table1),1), and the product was cloned into pGEM-T Easy (Promega Corp., Madison, WI). A partial deletion of the target gene was achieved by inverse PCR using primers (NoreginvF1 and NoreginvR1) that contain terminal HindIII sites. The resulting product was cut with HindIII, and the Ω cassette, encoding spectinomycin resistance from pHP45Ω (21), was ligated within the protein-coding region of nsrR. This plasmid (pJR113) was then transformed into N. meningitidis MC58 (26). Transformants were selected for by plating on Columbia horse blood agar containing spectinomycin, and correct chromosomal rearrangement was verified by PCR using primers NOregF1 and NOregR1. The norB-aniA promoter region (the intergenic region between NMB1622 and NMB1623 in N. meningitidis MC58) was amplified with MFA7 and MFA8 primers, and the product was cloned into pGEM-T Easy (Promega Corp., Madison, WI). The promoter region was extracted as a BamHI fragment and inserted upstream of the lacZ gene in pLES94 (27). Sequencing using primer MFA10, which complements the lacZ gene, was used to confirm orientation of the insert to yield an norB-lacZ fusion. Monoallelic promoter-lacZ fusions of the aniA and norB promoters were inserted into the proAB region of N. meningitidis MC58 as described previously (23). Chromosomal DNA from these strains was extracted and used to transform nsrR, fnr, and aniA strains of N. meningitidis MC58 to transfer the promoter aniA-lacZ or norB-lacZ reporter constructs. The double mutant nsrR fnr strains were constructed by using chromosomal DNA from the fnr mutant strain to transform the nsrR (PaniA-lacZ) and nsrR (PnorB-lacZ) strains. All strains were verified for correct chromosomal rearrangement by PCR.

Growth and activity assays.

Spermine NONOate (AG Scientific Inc., San Diego, CA) was resuspended in 0.1 M NaOH and was used to release NO into culture suspensions (half-life of 39 min at 37°C) at final concentrations of 10 to 50 μM. Spermine NONOate or nitrite was added to aerobic bacterial cultures in exponential phase, routinely with an optical density at 600 nm (OD600) of 0.3 to 0.6(approximately 3 to 4 h of growth), and 1-ml aliquots were harvested by centrifugation at hourly intervals after additions; cell pellets were stored at −20°C overnight for β-galactosidase activity assays. At 2 h after the addition of spermine NONOate (or nitrite), cell suspensions were assayed for their ability to utilize NO. The amount of protein in 1 ml of bacterial cells was measured by bicinchoninic acid assay (Pierce, Rockford, IL) or Bradford assay (Bio-Rad GmbH, Munich, Germany). β-Galactosidase activity assays were carried out on cell pellets (15). NO uptake assays were conducted by placing a maximum of 2 mg of whole cells in 5 ml of Mueller-Hinton broth in an electrode chamber. The respiration of the cells was used to make the chamber anaerobic, and once no oxygen was left, aliquots of aqueous solution containing saturated NO solution were added to the culture. Saturated NO solution was made by sparging NO gas (Sigma-Aldrich, United Kingdom) through 1 M NaOH and into 10 ml of 1 M Tris, pH 8, and a sample was analyzed to check that the pH had not become acidic. The presence of oxygen in culture was measured using a Clark-type oxygen electrode (Rank Bros, Bottisham, United Kingdom), and NO concentration was measured by an ISO-NOP Mark II 2-mm electrode (World Precision Instruments, Stevenage, United Kingdom). Nitrite concentration in culture was measured by colorimetric assay (18). All activity and nitrite assays were performed in triplicate on at least three different occasions. Data sets shown are from one representative occasion, and the error bars represent one standard deviation of the mean.

Western blotting.

Western blotting was carried out using antibodies raised in rabbits (Charles River Laboratories, France) against recombinant AniA protein overexpressed in E. coli and purified using standard biochemical techniques. Anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (Sigma) was used as a secondary antibody, and the horseradish peroxidase activity was detected on X-ray film (SLS, United Kingdom) using a Super Signal Western Dura (Pierce) chemiluminescence kit.

RESULTS

Nitric oxide reductase expression in N. meningitidis is induced in response to nitric oxide.

On transition to anaerobic conditions, NO reduction proceeds following the up-regulation and activity of the nitrite reductase aniA and appears to be a consequence of nitric oxide accumulation (23). In order to test the hypothesis that NO induces NO reductase expression, we incubated aerobically grown N. meningitidis with the NO releaser compound spermine NONOate. Spermine NONOate breaks down spontaneously at neutral pH to yield 1.5 molecules of NO per molecule of the NONOate with a half-life of 39 min at 37°C (AG Scientific Inc., San Diego, CA). Incubation with 50 μM spermine NONOate is sufficient to bring about induction of expression of a nitric oxide reductase activity (Table (Table2).2). The slow rate of NO removal in untreated cells is equivalent to the rate of NO disappearance from the electrode chamber in the absence of cells or with a cell suspension of a norB-deficient strain of N. meningitidis MC58 (Table (Table2).2). Under aerobic conditions NO breaks down into nitrite and nitrate, but neither of these anions was capable of inducing NO reductase at a 50 μM concentration (data not shown).

TABLE 2.
Nitric oxide uptake rates in N. meningitidis in response to nitric oxide

Mutagenesis of nsrR from N. meningitidis.

NMB0437 (nsrR) was mutated by insertion of a spectinomycin resistance gene. Spectinomycin-resistant meningococcal transformants were checked for correct insertion of the antibiotic resistance gene by PCR. Chromosomal DNA from this original mutant strain was isolated and transformed into wild-type N. meningitidis MC58. The resultant spectinomycin-resistant colonies were screened for disruption of nsrR by PCR. A number of nsrR mutant strains independently derived by this procedure were analyzed and found to have identical phenotypes, indicating that the likelihood that the phenotype resulted from a second site mutation was extremely low.

NMB0437 is monocistronic: the adjacent genes NMB0436 and NMB0438 are both transcribed from the opposite strand of the N. meningitidis DNA. Quantitative real-time PCR studies confirm that the expression of these flanking genes was not altered in nsrR mutant strains compared to the N. meningitidis MC58 wild type (data not shown).

Growth of N. meningitidis nsrR versus wild type.

To test whether the mutation in nsrR has an impact on growth by denitrification, N. meningitidis strains were grown under oxygen-limited conditions in the presence of 5 mM nitrite. N. meningitidis nsrR grew more rapidly than the wild type and consumed nitrite at a more rapid rate (Fig. (Fig.1).1). Under aerobic conditions, the nsrR mutant strain grew somewhat more slowly than the wild type and reached a lower final optical density (Fig. (Fig.1).1). These results suggest that in the nsrR mutant, the denitrification apparatus is expressed constitutively such that under oxygen limitation there is no lag in growth to allow for up-regulation of denitrification enzymes and no lag due to NO accumulation. Furthermore, under aerobic conditions, there may be a metabolic burden on the cell due to the expression of these enzymes that are not necessary for aerobic growth.

FIG. 1.
Growth of wild-type and nsrR mutant strains of N. meningitidis. (A) Growth of strains was monitored (by OD600 values) during incubation of cultures under oxygen-limited conditions in the presence of 5 mM nitrite. (B) Disappearance of nitrite during growth ...

Denitrification enzymes are expressed aerobically and under oxygen limitation in an nsrR mutant.

During aerobic growth of wild-type N. meningitidis and the nsrR mutant, nitrite is not utilized (data not shown). We have previously shown that oxygen is the preferred electron acceptor for respiration under these conditions (23), and therefore the lack of NO2 reduction does not necessarily imply that AniA is not being synthesized. Aerobically grown N. meningitidis wild type and the nsrR mutant were assayed for nitrite reductase activity under anaerobic conditions. While there was no nitrite reduction within ~1 h for the wild type, nitrite reduction occurred instantaneously for the nsrR mutant. Treatment with chloramphenicol to inhibit protein synthesis confirmed that nitrite reduction in the nsrR mutant was independent of de novo protein synthesis under anaerobic conditions (data not shown). Similarly, nitric oxide reduction was observed following aerobic growth of the N. meningitidis nsrR mutant strain but not the wild type (Table (Table2).2). Exogenous NO did not lead to further induction of NO reductase activity in the nsrR mutant, unlike the wild type, in which NO reductase expression is regulated by NO (Table (Table22).

In order to clarify the regulation of aniA and norB expression by NsrR and other regulatory proteins, we introduced aniA and norB promoter lacZ fusions into N. meningitidis MC58 and derivative strains with mutations in nsrR, fnr, and aniA.

norB expression is elevated in an nsrR mutant, and NsrR-dependent repression is lifted by NO.

The expression of norB was quantified by introducing a single copy of a norB promoter-lacZ fusion into the chromosome of N. meningitidis MC58 and an nsrR mutant. Following aerobic growth in the absence of nitric oxide, the β-galactosidase activity derived from this fusion construct was negligible in the wild type, but a significant activity was measured for the nsrR mutant strain bearing the fusion (Fig. (Fig.2),2), consistent with NsrR's being a repressor. Culturing N. meningitidis MC58 (PnorB-lacZ) under conditions that allow denitrification to occur (i.e., oxygen limitation plus nitrite) or incubation of the same strain aerobically cultured in the presence of spermine NONOate led to elevated expression of norB, whereas these culture conditions had no effect on the levels of expression of the norB-lacZ reporter in the nsrR mutant strain (Fig. (Fig.2).2). Clearly, NsrR is functioning as a repressor, but the presence of NO is able to relieve the repression and allow NO reductase to be expressed. Under aerobic conditions, nitrite has no impact on PnorB-lacZ expression in N. meningitidis wild type or the nsrR mutant strain (data not shown), whereas under microaerobic conditions nitrite increases expression in the wild type but not the nsrR mutant (Fig. (Fig.2),2), presumably due to NO accumulation as an intermediate in denitrification.

FIG. 2.
Expression of norB monitored using a norB promoter lacZ fusion. β-Galactosidase activities were measured following aerobic growth or microaerobic growth. Aerobic samples were treated with 50 μM spermine NONOate when the OD600 was ≈0.3. ...

aniA expression is elevated in an nsrR mutant strain but is not strongly induced in response to NO.

The activity of an aniA promoter lacZ fusion was analyzed in N. meningitidis MC58 and in an nsrR mutant background. The promoter activity was judged to be increased approximately 5- to 10-fold in the absence of the nsrR gene (Fig. (Fig.3).3). The activity of the PaniA-lacZ fusion is increased in the nsrR mutant strain over the wild type when N. meningitidis is cultured aerobically or under oxygen limitation plus or minus nitrite. Clearly, aniA expression is still regulated by other environmental variables (oxygen availability and nitrite) in the absence of nsrR, whereas these variables do not affect expression of norB in an nsrR mutant strain.

FIG. 3.
Expression of aniA monitored using an aniA promoter lacZ fusion. β-Galactosidase activities were measured following aerobic growth or microaerobic growth. Aerobic samples were treated with 50 μM spermine NONOate when the OD600 was ≈0.3. ...

Treatment of N. meningitidis MC58 (PaniA-lacZ) (or indeed the nsrR mutant strain bearing this fusion construct) with spermine NONOate has little effect on the activity of β-galactosidase (Fig. (Fig.3),3), indicating that nitric oxide itself has little impact on expression of the aniA gene, even though the aniA promoter is strongly repressible by NsrR. The PaniA-lacZ fusion was introduced into an aniA-deficient strain of N. meningitidis, and this was used to confirm that aniA expression is controlled by both oxygen limitation and the presence of nitrite but is not reliant on the generation of NO (i.e., via nitrite reductase) (Fig. (Fig.3C).3C). Western blotting of total cell extracts with antibodies raised against AniA confirmed that aniA expression is derepressed in an nsrR mutant strain but that NO has little effect on aniA expression (Fig. (Fig.3D).3D). In the N. meningitidis nsrR mutant strain, aniA expression remains inducible by nitrite (Fig. (Fig.3B),3B), whereas norB is not inducible by nitrite under denitrifying conditions (Fig. (Fig.2B),2B), consistent with the idea that NarQP controls aniA in a nitrite-dependent manner. Rodionov (24) predicted that NsrR in Neisseria might control narQP expression. However, we have found, using real-time PCR, that narQP expression is unaffected by mutation of nsrR (data not shown). It is evident that the norB promoter is strongly regulated by NO in an NsrR-dependent manner but that the aniA promoter activity is barely affected by NO regardless of the presence or otherwise of an intact nsrR gene.

The role of NsrR in control of aniA expression involves prevention of FNR-dependent expression under aerobic conditions.

NsrR represses aniA expression, but under no conditions that we have found does NO lift this repression. What, therefore, is the functional significance of NsrR in the control of AniA expression? To address this question we examined the regulation of aniA expression in response to oxygen availability and nitrite in N. meningitidis MC58, an nsrR mutant, an fnr mutant, and an nsrR fnr double mutant. High levels of expression from the aniA promoter observed in the nsrR mutant strain (both aerobically and under oxygen limitation), in contrast with much lower levels of aniA expression in the nsrR fnr double mutant (Fig. (Fig.4),4), demonstrate that aniA expression in the nsrR mutant strain is FNR dependent. Oxygen limitation does elevate the level of expression of aniA in the nsrR mutant strain, yet there is clearly a very high background activity aerobically in the absence of NsrR. To confirm that the aniA promoter was active aerobically, the activity of PaniA-lacZ was monitored in cell suspensions of N. meningitidis MC58 and nsrR maintained at 60 to 80% air saturation in the chamber of a Clark oxygen electrode. This showed that the PaniA-lacZ fusion was active aerobically in the nsrR mutant but not the wild type (Fig. (Fig.4).4). This is consistent with the observation that FNR from N. meningitidis retains considerable activity as a transcriptional activator under aerobic culture conditions.

FIG. 4.
β-Galactosidase activity of the aniA promoter in N. meningitidis MC58 and nsrR, fnr, and nsrR fnr mutant strains under aerobic and oxygen-limiting (with or without nitrite) conditions. The activity of PaniA-lacZ was measured following growth of ...

DISCUSSION

We have defined a new regulator of denitrification in the pathogenic denitrifying bacterium N. meningitidis. Analysis of nsrR mutant strains indicates that the nsrR gene encodes a repressor protein that prevents expression of both genes required for denitrification in this organism, namely, the nitrite reductase aniA and the nitric oxide reductase norB. We also report that the elevated expression of norB is achieved in response to NO (confirming prior indirect observations indicating that norB appeared be regulated by NO [23]) and that this NO responsiveness is dependent upon the presence of a functional nsrR gene. This is in keeping with findings for NsrR homologues in Nitrosomonas europaea, E. coli, and B. subtilis (3, 4, 17). Since the initial submission of this paper, NsrR has been shown to regulate denitrification in N. gonorrhoeae (20). In that paper, mutation of nsrR rendered N. gonorrhoeae insensitive to nitrite; however, we find that the N. meningitidis nsrR strain retains sensitivity to regulation of gene expression by nitrite (Fig. (Fig.3B),3B), indicating that there are key differences in the regulatory networks of these two bacteria.

NsrR was first identified as a repressor of the nitrite reductase nirK in the nitrifying bacterium N. europaea (3). In the presence of nitrite and a low pH, the repression of nirK via NsrR was lifted, leading these authors to conclude that NsrR is a nitrite-sensing transcriptional repressor. The physiological function of nitrite reduction in N. europaea was argued to be a defense mechanism against the toxic accumulation of nitrite, which is the product of the nitrification process in this organism (3). Although NsrR from N. europaea may respond to nitrite, an alternative interpretation, consistent with the pH dependence of the nitrite sensitivity, is that NsrR in fact senses nitric oxide, which can be generated chemically from nitrite under acidic conditions. Although this remains to be determined, the weight of evidence suggests that NsrR homologues from other microorganisms (E. coli, B. subtilis, and, here, N. meningitidis) are sensitive to nitric oxide rather than nitrite. E. coli nsrR (previously yjeB) was identified as a negative regulator of a number of genes that have been shown to be regulated by nitrosative stress (4). Focusing on the regulation of ytfE, the authors showed that the concentrations of NO necessary to bring about derepression of this NsrR-dependent promoter were 100 times lower than the concentration of nitrite required, supporting their conclusion that NsrR senses NO. In B. subtilis expression of flavohemoglobin (Hmp), a gene product that acts to detoxify nitric oxide, is activated by nitric oxide (100 μM) and nitrite (5 mM), and the nitric oxide scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide ablates this activation. In nsrR mutants hmp expression was derepressed and no longer sensitive to nitric oxide (17). Here, we have shown that the NO-dependent up-regulation of nitric oxide reductase (norB) in N. meningitidis is controlled by NsrR. Nitrite fails to activate norB, except under conditions when NO is generated as an intermediate of denitrification, providing further evidence that NsrR is responding to nitric oxide, not nitrite. The product of norB has a dual physiological function in N. meningitidis: NO detoxification (1) and supporting conservation of energy through respiration (20). As such, the role of regulator NsrR is, as in N. europaea and E. coli, to protect against toxicity, but it additionally acts to ensure optimal synthesis of key metabolic enzymes that allow N. meningitidis to exploit the variable availability of electron acceptors that support growth.

NO induces NO reductase activity and PnorB-lacZ activity in the wild type but not in an nsrR mutant, whereas nitrite reductase expression is repressed by NsrR but is not affected by NO per se. Thus, it appears that NsrR can act as both an NO-responsive regulator (with norB) and a NO-independent regulator (with aniA). This difference in NO sensitivity for expression of norB and aniA may relate to different binding affinities of NsrR to the two predicted sites for NsrR binding in the aniA/norB promoter region that have been suggested from bioinformatic analysis (24). Modulation of gene expression at the aniA promoter relies on binding of FNR. Presumably, NsrR and FNR compete for binding at the promoter, and only when FNR is bound is the promoter active. This is in keeping with our finding that the derepression of PaniA in an nsrR mutant is quashed by introducing a mutation in fnr (i.e., expression of aniA relies on FNR).

In the absence of NsrR, N. meningitidis FNR retains considerable activity at the aniA promoter under aerobic conditions, and further FNR-dependent activation is achieved under oxygen limitation. FNR has been most extensively studied in E. coli where it acts a global regulator of gene expression in response to anaerobiosis (11). A recent transcriptomic study of N. meningitidis FNR indicated that it regulates a smaller set of genes than in E. coli (2). Our work here indicates that an additional distinction between FNR in these organisms is that the N. meningitidis protein retains transcriptional activation activity with oxygen concentrations of 60 to 80% of air saturation such that a corepressor protein is required to prevent excessive expression aerobically. An explanation of the apparent oxygen tolerance of N. meningitidis FNR may lie in the physiology of this bacterium. We have shown previously (23) that N. meningitidis is unable to grow under fully anaerobic conditions, due to the absence of an anaerobic ribonucleotide reductase, and thus has to tolerate a supply of molecular oxygen even under conditions when the bacterium is oxygen limited and using denitrification to grow. N. meningitidis FNR may thus have adapted to be more tolerant to molecular oxygen per se than its homologues in facultative anaerobes such as E. coli.

FNR is principally regarded as an oxygen-sensitive transcriptional activator, but it has been reported that FNR from E. coli can respond to NO, such that NO can cause down-regulation of FNR-activated genes. Similarly, the iron responsive regulator FUR, which has also been implicated in regulation of aniA and norB in N. meningitidis (8), may also be regulated by NO directly. However, since mutating nsrR ablates NO-dependent regulation, there is no evidence that FNR or FUR play significant roles in NO-dependent regulation of denitrification genes in N. meningitidis.

NsrR is a member of the Rrf2 family of transcriptional repressor proteins. The structure of MarA, which is a member of this family, has been solved bound to DNA, revealing that the protein has two DNA-binding helix-turn-helix domains (22). The NsrR proteins are most closely related to another repressor, the protein IscR, which has been characterized from E. coli. This protein senses the iron-sulfur cluster status of the cell, probably by reversible binding of an iron sulfur cluster (25). IscR and NsrR proteins contain three conserved cysteine residues that may be involved in the binding of an iron-sulfur cluster in both of these classes of protein. Nakano and colleagues indicated that NsrR from Bacillus may, indeed, contain a labile iron-sulfur cluster. Comparing the sequences of IscR/NsrR with MarA indicates that the sequence region containing the key predicted sensory cysteine residues is found between the two DNA-binding helix-turn-helix domains, suggesting that iron-sulfur cluster binding (IscR) or NO interaction with an iron-sulfur cluster (NsrR) could perturb the structure of the repressor proteins appropriately to impact upon DNA binding. Both NsrR and IscR appear to be dual functional. While some target genes of IscR are repressed specifically under anaerobic conditions, others are only repressed aerobically (9). Likewise, we have shown here that NsrR acts as a repressor in both an NO-dependent and an NO-independent manner at different target genes.

Acknowledgments

This work was supported by a Wellcome Trust grant (070268/Z/03/Z) awarded to J.W.B.M. and R.C.R. and by a BBSRC quota studentship awarded to M.J.T.

Footnotes

[down-pointing small open triangle]Published ahead of print on 22 November 2006.

REFERENCES

1. Anjum, M. F., T. M. Stevanin, R. C. Read, and J. W. Moir. 2002. Nitric oxide metabolism in Neisseria meningitidis. J. Bacteriol. 184:2987-2993. [PMC free article] [PubMed]
2. Bartolini, E., E. Frigimelica, S. Giovinazzi, G. Galli, Y. Shaik, C. Genco, J. A. Welsch, D. M. Granoff, G. Grandi, and R. Grifantini. 2006. Role of FNR and FNR-regulated, sugar fermentation genes in Neisseria meningitidis infection. Mol. Microbiol. 60:963-972. [PMC free article] [PubMed]
3. Beaumont, H. J., S. I. Lens, W. N. Reijnders, H. V. Westerhoff, and R. J. van Spanning. 2004. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Mol. Microbiol. 54:148-158. [PubMed]
4. Bodenmiller, D. M., and S. Spiro. 2006. The yjeB (nsrR) gene of Escherichia coli encodes a nitric oxide-sensitive transcriptional regulator. J. Bacteriol. 188:874-881. [PMC free article] [PubMed]
5. Cruz-Ramos, H., J. Crack, G. Wu, M. N. Hughes, C. Scott, A. J. Thomson, J. Green, and R. K. Poole. 2002. NO sensing by FNR: regulation of the Escherichia coli NO-detoxifying flavohaemoglobin, Hmp. EMBO J. 21:3235-3244. [PMC free article] [PubMed]
6. D'Autreaux, B., D. Touati, B. Bersch, J. M. Latour, and I. Michaud-Soret. 2002. Direct inhibition by nitric oxide of the transcriptional ferric uptake regulation protein via nitrosylation of the iron. Proc. Natl. Acad. Sci. USA 99:16619-16624. [PMC free article] [PubMed]
7. D'Autreaux, B., N. P. Tucker, R. Dixon, and S. Spiro. 2005. A non-haem iron centre in the transcription factor NorR senses nitric oxide. Nature 437:769-772. [PubMed]
8. Delany, I., R. Rappuoli, and V. Scarlato. 2004. Fur functions as an activator and as a repressor of putative virulence genes in Neisseria meningitidis. Mol. Microbiol. 52:1081-1090. [PubMed]
9. Giel, J. L., D. Rodionov, M. Liu, F. R. Blattner, and P. J. Kiley. 2006. IscR-dependent gene expression links iron-sulphur cluster assembly to the control of O-regulated genes in Escherichia coli. Mol. Microbiol. 60:1058-1075. [PubMed]
10. Householder, T. C., E. M. Fozo, J. A. Cardinale, and V. L. Clark. 2000. Gonococcal nitric oxide reductase is encoded by a single gene, norB, which is required for anaerobic growth and is induced by nitric oxide. Infect. Immun. 68:5241-5246. [PMC free article] [PubMed]
11. Kiley, P. J., and H. Beinert. 1998. Oxygen sensing by the global regulator, FNR: the role of the iron-sulfur cluster. FEMS Microbiol. Rev. 22:341-352. [PubMed]
12. Kimberly, B., B. Nejadnik, G. D. Giraud, and W. E. Holden. 1996. Nasal contribution to exhaled nitric oxide at rest and during breath-holding in humans. Am. J. Respir. Crit. Care Med. 153:829-836. [PubMed]
13. Lissenden, S., S. Mohan, T. Overton, T. Regan, H. Crooke, J. A. Cardinale, T. C. Householder, P. Adams, C. D. O'Conner, V. L. Clark, H. Smith, and J. A. Cole. 2000. Identification of transcription activators that regulate gonococcal adaptation from aerobic to anaerobic or oxygen-limited growth. Mol. Microbiol. 37:839-855. [PubMed]
14. McGuinness, B. T., I. N. Clarke, P. R. Lambden, A. K. Barlow, J. T. Poolman, D. M. Jones, and J. E. Heckels. 1991. Point mutation in meningococcal porA gene associated with increased endemic disease. Lancet 337:514-517. [PubMed]
15. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
16. Moncada, S., R. M. Palmer, and E. A. Higgs. 1988. The discovery of nitric oxide as the endogenous nitrovasodilator. Hypertension 12:365-372. [PubMed]
17. Nakano, M. M., H. Geng, S. Nakano, and K. Kobayashi. 2006. The nitric oxide-responsive regulator NsrR controls ResDE-dependent gene expression. J. Bacteriol. 188:5878-5887. [PMC free article] [PubMed]
18. Nicholas, D. J. D., and A. Nason. 1957. Determination of nitrite and nitrate. Methods Enzymol. 3:981-984.
19. Nunoshiba, T., T. DeRojas-Walker, J. S. Wishnok, S. R. Tannenbaum, and B. Demple. 1993. Activation by nitric oxide of an oxidative-stress response that defends Escherichia coli against activated macrophages. Proc. Natl. Acad. Sci. USA 90:9993-9997. [PMC free article] [PubMed]
20. Overton, T. W., R. Whitehead, Y. Li, L. A. Snyder, N. J. Saunders, H. Smith, and J. A. Cole. 2006. Coordinated regulation of the Neisseria gonorrhoeae-truncated denitrification pathway by the nitric oxide-sensitive repressor, NsrR, and nitrite-insensitive NarQ-NarP. J. Biol. Chem. 281:33115-33126. [PubMed]
21. Prentki, P., and H. M. Krisch. 1984. In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29:303-313. [PubMed]
22. Rhee, S., R. G. Martin, J. L. Rosner, and D. R. Davies. 1998. A novel DNA-binding motif in MarA: the first structure for an AraC family transcriptional activator. Proc. Natl. Acad. Sci. USA 95:10413-10418. [PMC free article] [PubMed]
23. Rock, J. D., M. R. Mahnane, M. F. Anjum, J. G. Shaw, R. C. Reid, and J. W. B. Moir. 2005. The pathogen Neisseria meningitidis requires oxygen, but supplements growth by denitrification. Nitrite, nitric oxide and oxygen control respiratory flux at genetic and metabolic levels. Mol. Microbiol. 58:800-809. [PubMed]
24. Rodionov, D. A., I. L. Dubchak, A. P. Arkin, E. J. Alm, and M. S. Gelfand. 2005. Dissimilatory metabolism of nitrogen oxides in bacteria: comparative reconstruction of transcriptional networks. PLOS Comput. Biol. 1:e55. [PMC free article] [PubMed]
25. Schwartz, C. J., J. Giel, T. Patschkowski, C. Luther, F. J. Ruzicka, H. Beinert, and P. J. Kiley. 2001. IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins. Proc. Natl. Acad. Sci. USA 98:14895-14900. [PMC free article] [PubMed]
26. Seifert, H. S., R. S. Ajioka, D. Paruchuri, F. Heffron, and M. So. 1990. Shuttle mutagenesis of Neisseria gonorrhoeae: pilin null mutations lower DNA transformation competence. J. Bacteriol. 172:40-46. [PMC free article] [PubMed]
27. Silver, L. E., and V. L. Clark. 1995. Construction of a translational lacZ fusion system to study gene regulation in Neisseria gonorrhoeae. Gene 166:101-104. [PubMed]
28. Stevanin, T. M., J. W. Moir, and R. C. Read. 2005. Nitric oxide detoxification systems enhance survival of Neisseria meningitidis in human macrophages and in nasopharyngeal mucosa. Infect. Immun. 73:3322-3329. [PMC free article] [PubMed]
29. Tettelin, H., N. J. Saunders, J. Heidelberg, A. C. Jeffries, K. E. Nelson, J. A. Eisen, K. A. Ketchum, D. W. Hood, J. F. Peden, R. J. Dodson, W. C. Nelson, M. L. Gwinn, R. DeBoy, J. D. Peterson, E. K. Hickey, Haft, D. H., S. L. Salzberg, O. White, R. D. Fleischmann, B. A. Dougherty, T. Mason, A. Ciecko, D. S. Parksey, E. Blair, H. Cittone, E. B. Clark, M. D. Cotton, T. R. Utterback, H. Khouri, H. Qin, J. Vamathevan, J. Gill, V. Scarlato, V. Masignani, M. Pizza, G. Grandi, L. Sun, H. O. Smith, C. M. Fraser, Moxon, E. R., R. Rappuoli, and J. C. Venter. 2000. Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 287:1809-1815. [PubMed]
30. Tunbridge, A. J., T. M. Stevanin, M. Lee, t. H. M. Marriot, J. W. Moir, R. C. Read, and D. H. Dockrell. 2006. Inhibition of macrophage apoptosis by Neisseria meningitidis requires nitric oxide detoxification mechanisms. Infect. Immun. 74:729-733. [PMC free article] [PubMed]
31. van Deuren, M., P. Brandtzaeg, and J. van der Meer. 2000. Update on meningococcal disease with emphasis on pathogenesis and clinical management. Clin. Microbiol. Rev. 13:144-166. [PMC free article] [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...