narGHJI Operon Research Articles

Inflammation-associated nitrate facilitates ectopic colonization of oral bacterium Veillonella parvula in the intestine.

Colonization of the intestine by oral microbes has been linked to multiple diseases such as inflammatory bowel disease and colon cancer, yet mechanisms allowing expansion in this niche remain largely unknown. Veillonella parvula, an asaccharolytic, anaerobic, oral microbe that derives energy from organic acids, increases in abundance in the intestine of patients with inflammatory bowel disease. Here we show that nitrate, a signature metabolite of inflammation, allows V. parvula to transition from fermentation to anaerobic respiration. Nitrate respiration, through the narGHJI operon, boosted Veillonella growth on organic acids and also modulated its metabolic repertoire, allowing it to use amino acids and peptides as carbon sources. This metabolic shift was accompanied by changes in carbon metabolism and ATP production pathways. Nitrate respiration was fundamental for ectopic colonization in a mouse model of colitis, because a V. parvula narG deletion mutant colonized significantly less than a wild-type strain during inflammation. These results suggest that V. parvula harness conditions present during inflammation to colonize in the intestine.

Proteomic analysis of drug-susceptible and multidrug-resistant nonreplicating Beijing strains of Mycobacterium tuberculosis cultured in vitro

The existence of latent tuberculosis infection (LTBI) is one of the main obstacles hindering eradication of tuberculosis (TB). To better understand molecular mechanisms and explore biomarkers for the pathogen during LTBI, we cultured strains of Mycobacterium tuberculosis (Mtb) under stress conditions, mimicking those in the host granuloma intracellular environment, to induce entry into the non-replicating persistence stage. The stresses included hypoxia, low pH (5.0), iron deprivation (100 μM of 2, 2’˗dipyridyl) and nutrient starvation (10% M7H9 medium). Three Mtb strains were studied: two clinical isolates (drug-susceptible Beijing (BJ) and multidrug-resistant Beijing (MDR-BJ) strains) and the reference laboratory strain, H37Rv. We investigated the proteomics profiles of these strains cultured in stressful conditions and then validated the findings by transcriptional analysis. NarJ (respiratory nitrate reductase delta chain) was significantly up-regulated at the protein level and the mRNA level in all three Mtb strains. The narJ gene is a member of the narGHJI operon encoding all nitrate reductase subunits, which play a role in nitrate metabolism during the adaptation of Mtb to stressful intracellular environments and the subsequent establishment of latent TB. The identification of up-regulated mRNAs and proteins of Mtb under stress conditions could assist development of biomarkers, drug targets and vaccine antigens.

Phylogenomics of Mycobacterium Nitrate Reductase Operon

NarGHJI operon encodes a nitrate reductase that can reduce nitrate to nitrite. This process enhances bacterial survival by nitrate respiration under anaerobic conditions. NarGHJI operon exists in many bacteria, especially saprophytic bacteria living in soil which play a key role in the nitrogen cycle. Most actinomycetes, including Mycobacterium tuberculosis, possess NarGHJI operons. M. tuberculosis is a facultative intracellular pathogen that expands in macrophages and has the ability to persist in a non-replicative form in granuloma lifelong. Nitrogen and nitrogen compounds play crucial roles in the struggle between M. tuberculosis and host. M. tuberculosis can use nitrate as a final electron acceptor under anaerobic conditions to enhance its survival. In this article, we reviewed the mechanisms regulating nitrate reductase expression and affecting its activity. Potential genes involved in regulating the nitrate reductase expression in M. tuberculosis were identified. The conserved NarG might be an alternative mycobacterium taxonomic marker.

Differences in nitrate reduction between Mycobacterium tuberculosis and Mycobacterium bovis are due to differential expression of both narGHJI and narK2

Both Mycobacterium tuberculosis and Mycobacterium bovis can produce tuberculosis in humans. Mycobacterium tuberculosis has low nitrate reductase activity during aerobic growth (AG), but shows strong hypoxic induction. Virulent M. bovis has weak activity during AG with no hypoxic induction. Bacille Calmette-Guerin (BCG) lacks activity in both stages. Transcription of narG of the nitrate reductase enzyme operon was higher in M. tuberculosis than in M. bovis or BCG. Transcription of narK2 encoding the nitrate transporter was induced by hypoxia in M. tuberculosis but not M. bovis or BCG. Insertion of the M. tuberculosis narGHJI operon into M. bovis resulted in increased activity only during AG. Regulation of both the nitrate reductase enzyme and transporter are regulated differently in the two species.

Anaerobic growth of Corynebacterium glutamicum using nitrate as a terminal electron acceptor

Corynebacterium glutamicum, a gram-positive soil bacterium, has been regarded as an aerobe because its growth by fermentative catabolism or by anaerobic respiration has, to this date, not been demonstrated. In this study, we report on the anaerobic growth of C. glutamicum in the presence of nitrate as a terminal electron acceptor. C. glutamicum strains R and ATCC13032 consumed nitrate and excreted nitrite during growth under anaerobic, but not aerobic, conditions. This was attributed to the presence of a narKGHJI gene cluster with high similarity to the Escherichia coli narK gene and narGHJI operon. The gene encodes a nitrate/nitrite transporter, whereas the operon encodes a respiratory nitrate reductase. Transposonal inactivation of C. glutamicum narG or narH resulted in mutants with impaired anaerobic growth on nitrate because of their inability to convert nitrate to nitrite. Further analysis revealed that in C. glutamicum, narK and narGHJI are cotranscribed as a single narKGHJI operon, the expression of which is activated under anaerobic conditions in the presence of nitrate. C. glutamicum is therefore a facultative anaerobe.

Role of the Escherichia coli nitrate transport protein, NarU, in survival during severe nutrient starvation and slow growth

Escherichia coli K-12 strains expressing either NarU or NarK as the only nitrate transport protein are both able to support nitrate-dependent anaerobic growth. The narK gene is highly expressed during anaerobic growth in the presence of nitrate, consistent with a role for NarK in nitrate transport coupled to nitrate reduction by the most active nitrate reductase encoded by the adjacent narGHJI operon. The physiological role of NarU is unknown. Reverse transcriptase PCR experiments established that, unlike the monocistronic narK gene, narU is co-transcribed with narZ as the first gene of a five-gene narUZYWV operon. The narK and narU genes were fused in-frame to a myc tag: the encoded fusion proteins complemented the nitrate-dependent growth defect of chromosomal narK and narU mutations. A commercial anti-Myc antibody was used to detect NarK and NarU in membrane fractions. During anaerobic growth in the presence of nitrate, the quantity of NarU-Myc accumulated during exponential growth was far less than that of NarK-Myc, but NarU was more abundant than NarK in stationary-phase cultures in the absence of nitrate. Although the concentration of NarU-Myc increased considerably during the post-exponential phase of growth, NarK-Myc was still more abundant than NarU-Myc in stationary-phase bacteria in the presence of nitrate. In chemostat competition experiments, a strain expressing only narU had a selective advantage relative to a strain expressing only narK during nutrient starvation or very slow growth, but NarK(+) bacteria had a much greater selective advantage during rapid growth. The data suggest that NarU confers a selective advantage during severe nutrient starvation or slow growth, conditions similar to those encountered in vivo.

A membrane-bound nitrate reductase encoded by the narGHJI operon is responsible for anaerobic respiration in Halomonas maura

The halophilic bacterium Halomonas maura is capable of anaerobic respiration on nitrates. By insertional mutagenesis with the minitransposon Tn-5 we obtained the mutant Tc62, which was incapable of anaerobic respiration on nitrates. An analysis of the regions adjacent to the transposon allowed us to characterize the membrane-bound anaerobic-respiratory nitrate reductase narGHJI gene cluster in H. maura. We identified consensus sequences for fumarate and nitrate reductase regulator (FNR)-like protein-binding sites in the promoter regions of the nar genes and consensus sequences corresponding to the NarL binding sites upstream of the nar genes. RT-PCR analysis showed that the narGHJI operon was expressed in response to anaerobic conditions when nitrate was available as electron acceptor. This membrane-bound nitrate reductase is the only enzyme responsible for anaerobic respiration on nitrate in H. maura. In this article we discuss the possible relationship between this enzyme and a dissimilatory nitrate-reduction-to-ammonia process (DNRA) in H. maura and its role in the colonization of the rhizosphere.

Adaptation of Bacillus subtilis to oxygen limitation

Bacillus subtilis grows anaerobically by at least two different pathways, respiration using nitrate as an electron acceptor and fermentation in the absence of electron acceptors. Regulatory mechanisms have evolved allowing cells to shift to these metabolic capabilities in response to changes in oxygen availability. These include transcriptional activation of fnr upon oxygen limitation, a process requiring the ResD-ResE two-component signal transduction system that also regulates aerobic respiration. FNR then activates transcription of other anaerobically induced genes including the narGHJI operon which encodes a respiratory nitrate reductase. Genes involved in fermentative growth are controlled by an unidentified FNR-independent regulatory pathway.

Nitrate respiration in the actinomycete Streptomyces coelicolor

Streptomyces coelicolor is an obligate aerobic, filamentous soil-dwelling bacterium. Remarkably, the genome of S. coelicolor has three copies of the narGHJI operon that encodes respiratory nitrate reductase. This review summarizes our current views on the requirements for multiple nitrate reductases in S. coelicolor.

Construction of a whole-cell gene reporter for the fluorescent bioassay of nitrate

The development of a whole-cell fluorescence-based biosensor for nitrate is reported. The sensor is Escherichia coli transformed with a plasmid (pPNARGFP) in which the promoter and regulatory regions of the membrane-bound nitrate reductase narGHJI operon ( Pnar) are fused to a gfp gene encoding green fluorescent protein (GFP). Pnar-gfp activity was measured at a range of nitrate concentrations using whole-cell GFP fluorescence. The bioassay conditions have been optimized so that the fluorescence intensity is proportional to the extracellular nitrate concentration. The developed bioassay has established that E. coli (pPNARGFP) can be used for the quantitative determination of nitrate in environmental waters without interference from other electron acceptors, e.g., nitrite, dimethyl sulfoxide, trimethylamine- N-oxide and fumerate, and azide, an inhibitor of redox-active proteins.

Genetic characterization of the nitrate reducing community based on narG nucleotide sequence analysis.

The ability of facultative anerobes to respire nitrate has been ascribed mainly to the activity of a membrane-bound nitrate reductase encoded by the narGHJI operon. Respiratory nitrate reduction is the first step of the denitrification pathway, which is considered as an important soil process since it contributes to the global cycling of nitrogen. In this study, we employed direct PCR, cloning, and sequencing of narG gene fragments to determine the diversity of nitrate-reducing bacteria occurring in soil and in the maize rhizosphere. Libraries containing 727 clones in total were screened by restriction fragment analysis. Phylogenetic analysis of 128 narG sequences separated the clone families into two main groups that represent the Gram-positive and Gram-negative nitrate-reducing bacteria. Novel narG lineages that branch distinctly from all currently known membrane bound nitrate-reductase encoding genes were detected within the Gram-negative branch. All together, our results revealed a more complex nitrate-reducing community than did previous culture-based studies. A significant and consistent shift in the relative abundance of the nitrate-reducing groups within this functional community was detected in the maize rhizosphere. Thus a substantially higher abundance of the dominant clone family and a lower diversity index were observed in the rhizosphere compared to the unplanted soil, suggesting that a bacterial group has been specifically selected within the nitrate-reducing community. Furthermore, restriction fragment length polymorphism analysis of cloned narG gene fragments proved to be a powerful tool in evaluating the structure and the diversity of the nitrate-reducing community and community shifts therein.

Nitric oxide signaling and transcriptional control of denitrification genes in Pseudomonas stutzeri.

The expression of denitrification by a facultatively anaerobic bacterium requires as exogenous signals a low oxygen tension concomitant with an N oxide. We have studied the role of nitric oxide (NO), nitrous oxide (N2O), and nitrite as signal molecules for the expression of the denitrification apparatus of Pseudomonas stutzeri. Transcriptional kinetics of structural genes were monitored by Northern blot analysis in a 60-min time frame after cells were exposed to an N oxide signal. To differentiate the inducer role of NO from that of nitrite, mRNA kinetics were monitored under anoxic conditions in a nirF strain, where NO generation from nitrite is prevented because of a defect in heme D(1) biosynthesis. NO-triggered responses were monitored from the nirSTB operon (encoding cytochrome cd(1) nitrite reductase), the norCB operon (encoding NO reductase), nosZ (encoding nitrous oxide reductase), and nosR (encoding a putative regulator). Transcription of nirSTB and norCB was activated by 5 to 50 nM NO, whereas the nosZ promoter required about 250 nM. Nitrite at 5 to 50 nM elicited no response. At a threshold concentration of 650 nM N2O, we observed in the anoxic cell the transient appearance of nosZ and nosR transcripts. Constant levels of transcripts of both genes were observed in an anoxic cell sparged with N2O. NO at 250 nM stimulated in this cell type the expression of nos genes severalfold. The transcription factor DnrD, a member of the FNR-CRP family, was found to be part of the NO-triggered signal transduction pathway. However, overexpression of dnrD in an engineered strain did not result in NirS synthesis, indicating a need for activation of DnrD. NO modified the transcriptional pattern of the dnrD operon by inducing the transcription of dnrN and dnrO, located upstream of dnrD. Insertional mutagenesis of dnrN altered the kinetic response of the nirSTB operon towards nitrite. Our data establish NO and DnrD as key elements in the regulatory network of denitrification in P. stutzeri. The NO response adds to the previously identified nitrate-nitrite response mediated by the NarXL two-component system for the expression of respiratory nitrate reductase encoded by the narGHJI operon.

Cloning and sequencing of the genes encoding the periplasmic-cytochrome B-containing selenate reductase of Thauera selenatis.

The periplasmic selenate reductase (Ser) of Thauera selennatis is a component of the electron transport chain catalyzing selenate reduction with acetate as the electron donor (i.e., selenate respiration). The purified enzyme consists of three subunits (SerA, SerB and SerC). Using transposon (i.e., Tn5) mutagenesis selenate reductase mutants were isolated. Junction fragments of DNA adjacent to the integrated Tn5 were used, together with oligonucleotides derived from the N-termini of SerA and SerB, to clone from a gene bank a DNA fragment that contained the corresponding genes. After sequencing, serA, serB and serC were identified by sequence comparison with the N-termini of the three subunits. The genes are arranged in the order serA, serB, serC; a fourth open reading frame (serD) in between, but overlapping serB and serC, is also present. The serA gene product contains an apparent leader peptide with a twin-arginine motif. The remainder of the translated amino acid sequence is similar to that of a number of prokaryotic molybdenum-containing enzymes (e.g., nitrate reductases and formate dehydrogenases of Escherichia coli). The serB gene product contains four cysteine clusters and is similar to various iron-sulfur protein subunits. The serC gene product contains a putative Sec-dependent leader peptide, but there are no similarities between the remainder of the translated protein and other protein subunits. The SerC contains two histidine and four methionine residues, and these may noncovalently bind heme b--which is a component of the active selenate reductase. The serD gene product encodes a putative protein that shows no significant sequence similarities to other proteins. However, the location of the serD within the other ser genes is similar to that of narJ within the E. coli narGHJI operon (nitrate reductase A); thus suggesting that the role of SerD may be similar to that of NarJ, which is a system-specific chaperone protein.

Molybdate-dependent transcription of hyc and nar operons of Escherichia coli requires MoeA protein and ModE-molybdate.

In Escherichia coli, ModE-molybdate, a repressor of modABCD operon (molybdate transport), was previously shown to be an additional transcriptional activator of hyc operon (formate hydrogenlyase) and narGHJI operon (respiratory nitrate reductase). However, in a modE mutant, both operons were expressed at about 50% of the wild-type level in a molybdate-dependent manner. This ModE-independent, molybdate-dependent, expression of hyc, narG and narK operons required MoeA protein. An E. coli modE, moeA double mutant failed to produce formate hydrogenlyase or respiratory nitrate reductase activity irrespective of the growth medium. Tungstate substituted for molybdate in the activation of transcription of hyc and nar operons by ModE could not replace molybdate for MoeA-dependent expression. It is proposed that the MoeA-catalyzed product, an activated form of molybdate, interacts with a transcriptional activator/regulator other than ModE and regulates hyc and nar operons.

Molybdate-dependent transcription of hyc and nar operons of Escherichia coli requires MoeA protein and ModE-molybdate

In Escherichia coli, ModE-molybdate, a repressor of modABCD operon (molybdate transport), was previously shown to be an additional transcriptional activator of hyc operon (formate hydrogenlyase) and narGHJI operon (respiratory nitrate reductase). However, in a modE mutant, both operons were expressed at about 50% of the wild-type level in a molybdate-dependent manner. This ModE-independent, molybdate-dependent, expression of hyc, narG and narK operons required MoeA protein. An E. coli modE, moeA double mutant failed to produce formate hydrogenlyase or respiratory nitrate reductase activity irrespective of the growth medium. Tungstate substituted for molybdate in the activation of transcription of hyc and nar operons by ModE could not replace molybdate for MoeA-dependent expression. It is proposed that the MoeA-catalyzed product, an activated form of molybdate, interacts with a transcriptional activator/regulator other than ModE and regulates hyc and nar operons.

Characterization of anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end products and genes required for growth.

Bacillus subtilis can grow anaerobically by respiration with nitrate as a terminal electron acceptor. In the absence of external electron acceptors, it grows by fermentation. Identification of fermentation products by using in vivo nuclear magnetic resonance scans of whole cultures indicated that B. subtilis grows by mixed acid-butanediol fermentation but that no formate is produced. An ace mutant that lacks pyruvate dehydrogenase (PDH) activity was unable to grow anaerobically and produced hardly any fermentation product. These results suggest that PDH is involved in most or all acetyl coenzyme A production in B. subtilis under anaerobic conditions, unlike Escherichia coli, which uses pyruvate formate lyase. Nitrate respiration was previously shown to require the ResDE two-component signal transduction system and an anaerobic gene regulator, FNR. Also required are respiratory nitrate reductase, encoded by the narGHJI operon, and moaA, involved in biosynthesis of a molybdopterin cofactor of nitrate reductase. The resD and resDE mutations were shown to moderately affect fermentation, but nitrate reductase activity and fnr are dispensable for fermentative growth. A search for genes involved in fermentation indicated that ftsH is required, and is also needed to a lesser extent for nitrate respiration. These results show that nitrate respiration and fermentation of B. subtilis are governed by divergent regulatory pathways.

Characterization of NarJ, a System-specific Chaperone Required for Nitrate Reductase Biogenesis in Escherichia coli

The narGHJI operon encodes the three subunits, alpha, beta, and gamma, of the respiratory nitrate reductase complex in Escherichia coli. A fourth open reading frame of the operon encodes a putative protein, NarJ, which is not present in purified nitrate reductase, but is required for biogenesis of the membrane-bound complex. NarJ was identified with a T7 expression system and was produced at significantly less than stoichiometric levels relative to the three enzyme subunits. A functional His-tagged NarJ fusion protein was overexpressed from a multicopy plasmid, purified by Ni2+ affinity chromatography, and characterized. Western blot analysis with antibodies raised against the fusion protein demonstrated that NarJ remained in the cytosol after assembly of the active membrane complex. The cytosolic alphabeta complex accumulated in a narJ insertion mutant was rapidly degraded after induction, but was stabilized by NarJ expressed from a multicopy plasmid. Overproduction of the His-tagged NarJ fusion protein in the same mutant led to the formation of an alphabeta.NarJ complex, which was resolved by Ni2+ affinity chromatography. The NarJ protein therefore has the properties of a system-specific (private) chaperone that reacts directly with and modifies the properties of the cytosolic alphabeta subunit complex, but remains in the cytoplasm after the assembly of the active alphabetagamma complex in the membrane.

A reassessment of the genetic determinants, the effect of growth conditions and the availability of an electron donor on the nitrosating activity of Escherichia coli K-12.

Anaerobic, but not aerobic, cultures of Escherichia coli K-12 catalysed the rapid nitrosation of the model substrate 2,3-diaminonaphthalene when incubated with nitrite. Formate and lactate were effective electron donors for the nitrosation reaction, which was inhibited by nitrate. Optimal growth conditions for the expression of nitrosation activity by various strains and mutants were determined. Highest activities were found with bacteria that had been grown anaerobically in a minimal medium rather than in Lennox broth, with glycerol and fumarate rather than glucose as the main carbon and energy source, and in the presence of a low concentration of nitrate. Bacteria harvested in the early exponential phase were more active than those harvested in later stages of growth. Well-characterized mutants defective in the synthesis of one or more anaerobically induced electron transfer chains were screened for nitrosation activity under these optimal growth conditions: only the respiratory nitrate reductase encoded by the narGHJI operon was implicated as a major contributor to nitrosation activity. Due to the limited sensitivity of the assays currently available, a minor contribution from the two alternative nitrate reductases or even other molybdoproteins could not be excluded. The role of formate in nitrosation was complex and was clearly not limited simply to that of an electron donor in the bacterial reduction of nitrite to nitric oxide: at least two further, chemical roles were inferred. This extensive study of more than 400 independent cultures of E. coli K-12 and its derivatives resolved some, but not all, of the apparently conflicting data in the literature concerning nitrosation catalysed by enteric bacteria.

Analysis of nitrate regulatory protein NarL-binding sites in the fdnG and narG operon control regions of Escherichia coli K-12.

During anaerobic growth, expression of the fdnGHI and narGHJI operons of Escherichia coli is induced by the NarL protein in response to nitrate. The fdnG operon control region contains four NarL-binding sites (termed NarL heptamers) between positions -70 and -130. The two central NarL heptamers of fdnG are arranged as an inverted repeat and are essential for regulation by NarL. We used mutational analysis of these central heptamers to investigate the precise sequence requirements for NarL-dependent induction. Mutations were examined for their effects on NarL-dependent expression in vivo. Substitutions at position 1 of either heptamer had the strongest effect whereas substitutions at position 7 had the weakest effect. For some positions, alterations in both heptamers had a stronger effect than either of the single changes. The 2 bp spacing between these NarL heptamers was also important for normal nitrate induction. The narG operon control region has at least eight NarL heptamers arranged in two groups. Previous work has shown that nucleotide substitutions in two of these heptamers, centred at positions -195 and -89, severely reduce nitrate induction of narG operon expression in vivo and significantly interfere with NarL-DNA interactions in vitro. Substitutions in heptamers -185 and -101 affected narG operon induction only when the concentration of phospho-NarL was low (during growth in the presence of nitrite). Changes in each of the other four NarL heptamers studied had little or no effect on nitrate or nitrite induction of narG operon expression or on NarL-DNA interactions in vitro.

Anaerobic transcription activation in Bacillus subtilis: identification of distinct FNR-dependent and -independent regulatory mechanisms.

Bacillus subtilis is able to grow anaerobically using alternative electron acceptors, including nitrate or fumarate. We characterized an operon encoding the dissimilatory nitrate reductase subunits homologous to the Escherichia coli narGHJI operon and the narK gene encoding a protein with nitrite extrusion activity. Downstream from narK and co-transcribed with it a gene (fnr) encoding a protein homologous to E.coli FNR was found. Disruption of fnr abolished both nitrate and fumarate utilization as electron acceptors and anaerobic induction of narK. Four putative FNR binding sites were found in B.subtilis sequences. The consensus sequence, centred at position -41.5, is identical to the consensus for the DNA site for E.coli CAP. Bs-FNR contained a four cysteine residue cluster at its C-terminal end. This is in contrast to Ec-FNR, where a similar cluster is present at the N-terminal end. It is possible that oxygen modulates the activity of both activators by a similar mechanism involving iron. Unlike in E.coli, where fnr expression is weakly repressed by anaerobiosis, fnr gene expression in B.subtilis is strongly activated by anaerobiosis. We have identified in the narK-fnr intergenic region a promotor activated by anaerobiosis independently of FNR. Thus induction of genes involved in anaerobic respiration requires in B.subtilis at least two levels of regulation: activation of fnr transcription and activation of FNR to induce transcription of FNR-dependent promoters.