The contribution of the NarB and NarGHI enzymes to nitrate reduction in Mycobacterium smegmatis

Background: Nitrate reduction in bacteria is an essential step in the nitrogen cycle. For this, the reduction of nitrate to nitrite is catalyzed by a variety of nitrate reductase enzymes. In the pathogen Mycobacterium tuberculosis, nitrate reduction is driven by the NarGHI respiratory and assimilatory nitrate reductase. In addition to this enzyme, Mycobacterium smegmatis carries a second putative narB-encoded nitrate reductase and the contribution of this enzyme to nitrate reduction remains unknown. Herein, we set out to investigate this. Results: To assess the relative contribution of NarGHI and NarB, the corresponding gene loci we deleted using two-step allelic replacement, individually and in combination, followed by investigation of nitrate reduction using the Griess assay. However, previous reports demonstrated that this assay was unable to report on nitrate reduction in M. smegmatis, as it yielded no detectable levels of the nitrite product. To address this, we modified the assay through the addition of zinc, which reduces nitrate remaining in the reaction to nitrite thus allowing for assessment of nitrate depletion. This then serves as a surrogate for nitrate reductase activity. The mutant strains lacking narB and/or narGHJI retained the ability to reduce nitrate at levels comparable to the wild type. We further investigated nitrate assimilation and all strains defective for these enzymes were able to grow on nitrate as the sole nitrogen source. Conclusions: Collectively, these data confirm that NarB and NarGHI are individually and collectively dispensable for both respiratory and assimilatory nitrate reduction in M. smegmatis. Furthermore, we identified MSMEG_4206 as a putative, previously unannotated, nitrate reductase in this organism.

specifically bis-molybdopterin guanine dinucleotide (bis-MGD) and iron-sulfur clusters, usually of the type [4Fe-4S] however, a distinct series of steps is followed for nitrate reduction by each enzyme [5].
The respiratory NR is a trimeric enzyme made up of the subunits NarG, NarH and NarI, and is encoded by the narGHJI operon. NarJ serves as a chaperone to facilitate the insertion MoCo [9]. In Escherichia coli during the catalytic reaction, electrons are donated to NarI from the electron carrier menaquinol (MQH2) and this is coupled with the transfer of two protons into the periplasm [5]. The two electrons are then transferred to bis-MGD for the reduction of nitrate to nitrite, in the process consuming two cytoplasmic protons and thus generating a PMF [5]. The causative agent of tuberculosis, Mycobacterium tuberculosis, has a similar respiratory NR, which has been shown to support both assimilatory and respiratory nitrate reduction [10]. Although a second "fused' respiratory NR, NarX, is annotated, no function has been assigned to this enzyme [11]. Nitrate reduction is important for M. tuberculosis with several lines of evidence implicating the activity of NarGHI in virulence and pathogenesis, reviewed in [12].
The model organism, Mycobacterium smegmatis, also carries a narGHJI-encoded NR and it has been demonstrated that strains lacking a functional bis-MGD biosynthetic pathway were unable to assimilate nitrate, presumably due to the lack of the catalytic MoCo cofactor required by the NarGHI [13,14]. However, in addition to this NR, M. smegmatis encodes for a second putative NR, narB, proposed to contribute to assimilatory nitrate reduction [15]. However, this has not been demonstrated. Furthermore, both NarB and NarGHI are annotated as MoCo-dependent enzymes, Figure 1B. Abrogation of bis-MGD biosynthesis would therefore abolish the function of both enzymes, thus making it difficult to determine relative contribution to nitrate reduction. Herein, we aimed to dissect the relative contribution of NarGHI and NarB to respiratory and assimilatory nitrate reduction in M. smegmatis using a gene-knockout approach and subsequent characterization of mutant strains.

Results
Deletion of the narB and narGHJI loci individually and collectively does not affect nitrate utilization. We set out to investigate the role of NarB and NarGHJI in nitrate reduction by generating deletion mutant strains lacking each locus as well as a strain lacking both loci.
The strains ΔnarB, ΔnarGHJI and ΔnarB ΔnarGHJI were generated by homologous recombination and genotypically confirmed by Southern blot analyses (Additional File 1: Figures S1 and S2). Following this, nitrate utilization assays were carried out. M. smegmatis has been reported as NR negative in the Griess assay, due to the inability to detect nitrite under anaerobic conditions when nitrate was provided as a substrate [16]. Hence, to investigate NR activity in M. smegmatis, we modified the Griess assay to measure nitrate utilization as described in Figure 2A. As a negative control, a Δ moaD2 ΔmoaE2 double mutant, which was previously shown to be defective for bis-MGD production and as a result, nitrate reduction [13,14], was included as it is unable to assimilate nitrate. When the Griess reagents were added to the culture samples taken at Day 0, no colour change was observed ( Figure 2B) which was in contrast to the immediate change to pink observed for the nitrite standards. Once zinc was added to the samples, a dark pink colour was produced, confirming the presence of nitrate at the beginning of the experiment. This colour started fading after 1 day's incubation at 37 ˚C ( Figure 2B). At the end of the experiment, the nitrate was completely consumed for all strains, with the exception of the Δ moaD2 ΔmoaE2 negative control. These effects were quantified ( Figure 2C) and further confirmed that loss of both narB and narGHJI does not affect nitrate utilization.
To confirm that the depletion of nitrate from the medium was because of utilization and not merely transport of nitrate into the cell, intracellular nitrate levels were also quantified. The intracellular levels of nitrate were approximately 10-fold lower than the extracellular levels on Day 1 and were further depleted by Day 6, confirming that the nitrate was being assimilated and not accumulating intracellularly ( Figure 2C). No statistically significant differences in the amount of intracellular nitrate were detected between the wild type, ΔnarB, ΔnarGHJI and ΔnarB ΔnarGHJI strains. Increased intracellular levels of nitrate were detected in ΔmoaD2 ΔmoaE2, again confirming the inability of this strain to reduce nitrate.
NarB and NarGHI are both dispensable for nitrate assimilation in minimal media. After confirming that no change in nitrate utilization occurred upon deletion of both narB and narGHJI, we next assessed the ability of mutant strains to assimilate nitrate as previously described [14]. The growth curves for both the single mutants and the double deletion mutant in MPLN, were indistinguishable from the wild type ( Figure 3A). The Δ moaD2 ΔmoaE2 strain was included as a negative control and as expected was unable to assimilate nitrate in order to grow under these conditions. In addition, no differences in growth were observed between the strains when grown in 7H9 medium (Additional file 1: Figure S3), providing evidence that NarB and NarGHI are dispensable for the growth of M. smegmatis under carbon and nitrogen replete conditions.
M. smegmatis NarGHI is dispensable under anaerobic conditions. In M. tuberculosis, NarGHI is a respiratory enzyme which serves as the terminal electron acceptor under anaerobic conditions when nitrate is available, thus facilitating growth and/or survival [11]. It has been shown that M. smegmatis is able to survive under anaerobic conditions in the presence of nitrate [17]. We sought to assess if this effect is mediated by NarGHI. For this, we scored the proportionate increase in CFUs under anaerobic conditions after 7 days' incubation and normalized this with the initial inoculum. We observed between 30-45 fold increases in bacterial biomass under aerobic conditions ( Figure 3B). Under anaerobic conditions, less growth was observed with no significant difference in bacterial yield between the wild type and mutant strains ( Figure 3B).  Figure S4). Next, we assessed the expression of narB and narG in MPLN to determine if growth in nitrate leads to an induction of these genes. We could not detect any narB transcript under these conditions (data not shown). For narG, transcript was detected in wild type and Δ narB in equal amounts when grown in MPLN (Additional file 1: Figure S4). Despite the low amount of narG transcript in samples grown in MPLN, no transcript was detected when grown in 7H9 (data not shown).
These data thus provide evidence that narGHJI expression in M. smegmatis is induced in the presence of nitrate, albeit to a low level. differences in NR activity [19], is not present in M. smegmatis (Additional file 1: Figure S5), suggesting that the expression profile of narG in M. smegmatis and M. tuberculosis could be similar. Furthermore, pairwise protein sequence alignments of NarG performed using the EMBOSS Needle tool [20], revealed high sequence homology (~80%) between different mycobacterial organisms and when compared to the E. coli protein, all the catalytic residues are conserved (Additional file 1: Figure S6). These bioinformatics analyses suggest that M. smegmatis encodes a functional narGHJI-encoded NR.
BLAST [21] analysis revealed that several mycobacterial species harbour a copy of narB,  Figure 4B. This domain architecture is most commonly found in the catalytic subunit of periplasmic nitrate reductases and interestingly, a prediction of the 3D structure of M. smegmatis NarB models best to the Desulfovibrio desulfuricans periplasmic nitrate reductase (Pdb 2V45) [22,23], Figure 4C. However, this is likely attributable to the lack of a resolved NarB crystal structure. A signal sequence was not identified using SignalP [24], for M. smegmatis NarB, suggesting that it is most likely located in the cytoplasm. The protein shares homology (31.1%) with the Synechococcus sp.
PCC 7942 NarB and according to STRING [25] analysis, it could possibly interact with MSMEG_1847, a ferrodoxin which is the predicted physiological electron donor to NarB in Cyanobacteria [26].
Considering that nitrate reduction continued to occur after deletion of both narB and narGHJI, it is possible that an alternate MoCo-dependent NR reductase is present in M.
smegmatis which is solely responsible for nitrate assimilation or works in combination with other MoCo-dependent NR's for the reduction of nitrate to nitrite. Using the domains shown in Figure 4B, the genome of M. smegmatis was examined in KEGG [27] to identify enzymes with the same architecture which could possibly catalyze the reaction. Seven genes encoding enzymes with the same architecture were identified and are listed in Table 1, two of which were narB and narG. Three of the seven genes encoded the MoCo-dependent oxidoreductases formate dehydrogenase and NADH dehydrogenase. The two remaining hits were MSMEG_2237 and MSMEG_6816 which are annotated as an anaerobic dehydrogenase and molybdopterin oxidoreductase respectively [28] and could possibly reduce nitrate to nitrite. In addition to the seven genes identified in the M. smegmatis genome with the MSMEG annotations, one gene was identified in the M. smegmatis genome with the newer MSMEI gene annotations curated by EcoGene-RefSeq [29], MSMEI_4108. MSMEI_4108 is annotated as a putative assimilatory nitrate/sulfite reductase. In order to identify this gene in SmegmaList, a BLAST search was performed using the nucleotide sequence and 100 % homology was detected for an un-annotated region [28]. The region was between coordinates 4288072-4292127 and the surrounding gene organisation for both databases was exactly the same (Additional File 1: Figure S7). No signal sequence or transmembrane domains were detected for MSMEI_4108, suggesting that it is cytoplasmic. In addition, it possesses binding domains for different cytoplasmic electron donors, Figure S7. We thus propose to annotate MSMEG_4206 as a putative assimilatory nitrate/sulfite reductase.
Further investigation into this gene and its role in nitrate assimilation is currently being conducted.

Discussion
It has long been accepted that M. smegmatis is NR negative, based solely on the results of the Griess assay [16]. When performed following the conventional protocol, this assay cannot be used to assess NR activity in M. smegmatis unless the strain carries a constitutively expressed copy of the M. tuberculosis narGHJI operon [13,16] supplemented with 0.085 % NaCl, 0.2 % glucose and 0.5 % glycerol. For nitrate assimilation and utilization assays, strains were grown in modified M. phlei minimal media (MPLN) made up of 5 g KH2PO4, 2 g sodium citrate, 0.6 g MgSO4, 0.85 g NaNO3, 20 ml glycerol and 5 ml 10 % tyloxapol in 1 L as previously described [14]. Media for M. smegmatis growth was supplemented with antibiotics at concentrations of 50 µg/ml Hyg and/or 25 µg/ml Kan where appropriate. Nitrate assimilation assays were carried out as previously described [14]. All liquid E. coli and M. smegmatis cultures were grown with shaking at 115 rpm.
Gene expression. RNA extractions and quantitative reverse transcriptase PCR (qRT PCR) were carried out as previously described [14]. Primers used for qRT PCR are listed in Additional file 1: Table S3. Expression of each gene was normalized against the sigA expression level.
Nitrate utilization. Nitrate utilization was measured using the Griess assay, which is based on the production of a red diazonium dye from the reaction of nitrite with naphthylamide under acidic conditions. This assay therefore relied on the availability of nitrite in the sample being tested. Nitrate reduction leads to the export of nitrite outside the cell, in which case a positive Griess result is observed. Alternatively, nitrite is further assimilated into ammonia or reduced to nitric oxide which leads to a negative Griess result. Therefore, a Griess assay was performed at the time of inoculation and once cultures had reached the stationary phase, as described by Weber, Fritz [16] with minor modifications, Figure 2A.
Briefly, 100 μl of 1 % sulfinilic acid and 100 μl of 1 % N-(1-Naphthyl) ethylenediamine dihydrochloride (NEDD) were added to 1 ml of culture and mixed thoroughly. In those samples wherein no colour change was observed, a few grains of zinc powder was added, mixed thoroughly and incubated for 5 min at room temperature. The samples were then clarified by centrifugation at 12 470 x g and the absorbance of the supernatants were measured at 530 nm and compared to known standards of nitrite (0.1 nM-1000 nM).
Anaerobic growth. Cultures were grown in MPLN overnight to early-log phase. These cultures were used to inoculate, in duplicate, 2 ml MPLN in 10 ml culture tubes to OD 0.05.
An aliquot was taken from each tube to determine the starting colony forming units (CFU/ml) and one set of tubes was incubated at 37 ºC with no shaking. The other set of tubes were placed in the Oxoid AnaeroGen chamber which creates an anaerobic environment.
Once the indicator showed that the environment was anaerobic, the chamber was incubated at 37 ºC with no shaking. After 7 days an aliquot from each tube was plated to determine CFU.

Declarations
Ethics approval and consent to participate        Expression was normalized against the house-keeping sigA and data from three independent experiments is depicted with standard error bars. Figure S5: Sequence alignments of narG promoter regions. Grey text shows the narG upstream promoter region. Black text depicts nucleotides of the narG gene sequence.
Green text represents the start codon of each homologue while the red uppercase text depicts the promoter mutation site with which differing NR activity is associated. Ecoli:    The Students t-test was used to compare between strains. *P=0.01  with the best fit template, the periplasmic NR from Desulfovibrio desulfuricans (Red). The crystal structure prediction was done using i-TASSER and the NarB amino acid sequence obtained from SmegmaList.

Supplementary Files
This is a list of supplementary files associated with the primary manuscript. Click to download. Cardoso_et_al_Additional_File_AV4.docx