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

doi:10.21203/rs.2.9260/v1

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

https://doi.org/10.21203/rs.2.9260/v1

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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.

Microbiology

Nitrate reductase

assimilation

Mycobacterium

NarB

NarGHI

Nitrogen is an essential element for all life, being a vital component of proteins and nucleic acids in both eukaryotes and prokaryotes. The nitrogen cycle, shown in Figure 1A, is well characterized and involves the cycling of nitrogen from the atmosphere in various forms through the earth and back. The entire cycle involves different processes including nitrogen fixation, ammonification, nitrification and denitrification [1]. Bacteria are critical to this process as they are the only organisms capable of carrying out a number of the processes [1-3]. Although nitrogen is abundant in the atmosphere as a gas in a diatomic state (N2), the triple bond between the two nitrogen atoms requires a large amount of energy for hydrolysis and subsequent incorporation of nitrogen into other compounds. In addition to N2, other available sources of nitrogen include inorganic nitrate-containing compounds, urea, ammonia and proteins/amino acids. Nitrate reduction is considered the most important stage of nitrogen turnover in the nitrogen cycle and this reaction is catalyzed by nitrate reductase (NR) enzymes [4].

Prokaryotic NR’s are mononuclear molybdenum-containing enzymes and are assigned to one of three classes based on the ultimate purpose of the nitrate being reduced as well as their sub-cellular localization [2]. Assimilatory NR’s found in the cytoplasm are commonly referred to as Nas enzymes and catalyze the incorporation of nitrogen into cellular material thus directly contributing to growth [2, 5, 6]. Dissimilatory or periplasmic NR’s, known as Nap enzymes are generally involved in respiration through redox balancing via electron transfer. Nap proteins can also serve as respiratory enzymes in bacteria that lack the respiratory NR, Nar [2, 7, 8]. Nar enzymes are similar to their dissimilatory counterparts in that they are involved in electron transfer; however the electron flow through respiratory NR’s is coupled with proton translocation and thus the generation of a proton motive force (PMF) across the cytoplasmic membrane and energy production [5, 7]. Common to all three NRs is a catalytic site made up of the molybdenum cofactor (MoCo), 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.

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 ΔmoaD2 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 ΔmoaD2 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 ΔmoaD2 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). The ability of ΔnarB, ΔnarGHJI and ΔnarB ΔnarGHJI to grow in MPLN under aerobic and anaerobic conditions suggests that loss of NarB and NarGHI does not abrogate NR activity in M. smegmatis. To investigate this further, the expression pattern of narB and narGHJI was assessed for the different M. smegmatis strains grown in 7H9 and MPLN. When grown to late log phase in 7H9, no narB or narG transcript was detected in any of the strains, suggesting that these genes are not expressed in M. smegmatis under the conditions tested (data not shown). In these cases, robust levels of sigA were detected (Additional file 1: 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 ΔnarB 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.

Bioinformatics analyses of M. smegmatis NR proteins. M. smegmatis and M. tuberculosis both retain the necessary repertoire of genes encoding proteins required for each step of the nitrate assimilation pathway [18], shown in Figure 4A. The discrepancies observed between the two organisms include the increased number of nitrate/nitrite transporters present in M. tuberculosis and the presence of different additional putative NR-encoding genes in M. tuberculosis and M. smegmatis, namely the narX and narB respectively. A large degree of sequence homology is observed between the M. tuberculosis and M. smegmatis narG genes (76.8 %). In addition, the single nucleotide polymorphism (SNP) identified in the promoter region of narG in Mycobacterium bovis (-215 T →C), strongly associated with 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, including members of the Mycabacterium avium complex (MAC), Mycobacterium fortuitum, Mycobacterium yongonense, Mycobacterium goodii and Mycobacterium kansasii - all of which are capable of causing disease. However, a narB homologue was not identified in any of the organisms belonging to the M. tuberculosis complex. Analysis of the NarB protein sequence (A0QW69) revealed four domains, 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.

Table 1: M. smegmatis genes encoding proteins with the same domain architecture as NarB.

Gene	Identifier	EC classification	Gene length	Protein length	NarB % homology*
MSMEG_0161	Formate dehydrogenase	1.2.1.2	2820 bp	939 aa	21.6
MSMEG_2057 (nuoG)	NADH dehydrogenase subunit G	1.6.5.3	2385 bp	794 aa	17.6
MSMEG_3521	Formate dehydrogenase	1.2.1.2	2250 bp	749 aa	21.6
MSMEG_2837 (narB)	Assimilatory nitrate reductase	1.7.7.2	2388 bp	795 aa	-
MSMEG_5140 (narG)	Respiratory nitrate reductase	1.7.5.1/1.7.99.4	3675 bp	1224 aa	16.8
MSMEG_2237	Anaerobic dehydrogenase	Not assigned	2283 bp	760 aa	21.2
MSMEG_6816	Molybdopterin oxidoreductase	Not assigned	2163 bp	720 aa	24.4
MSMEI_4108	Putative assimilatory nitrate reductase/sulfite reductase	Not assigned	4056 bp	1351 aa	18.7

*Alignments were performed using EMBOSS Needle and protein sequences were obtained from KEGG.

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], suggesting that M. smegmatis is unable to reduce nitrate. However, several studies have shown that M. smegmatis is able to use nitrate as the sole nitrogen source [13-15, 17, 30]. Furthermore, it has been demonstrated that NR activity in a strain (ΔmoaD2 ΔmoaE2) unable to synthesise the bis-MGD cofactor was completely abrogated [13], providing further evidence that a functional NR is present in M. smegmatis which like the M. tuberculosis NR, is bis-MGD dependent. Using a modified Griess assay we confirmed that M. smegmatis does indeed reduce nitrate. We show that over time in culture, M. smegmatis is able to utilize all available nitrate in the medium for assimilation. The ability of M. smegmatis to reduce nitrate is not surprising as two putative NR-encoding genes are annotated in the genome i.e. narB and narGHJI. However, the contribution of each to NR activity was unknown. We sought to address this question.

NR activity is a phenotypic characteristic widely accepted to classify and differentiate between mycobacterial species, with M. tuberculosis displaying high NR activity while M. bovis and M. bovis BCG show no distinct NR activity [30]. The molecular basis for the difference in NR activity between M. tuberculosis and M. bovis BCG has been attributed to the lack of a functional narGHJI-encoded NR as a result of loss of narH expression in the latter [31]. The reduced NR activity in the pathogenic M. bovis has been attributed to a SNP in the promoter region of narG, although the authors couldn’t rule out that other SNPs in the coding region of the narGHJI operon also contributed to the differential activity between the two strains [19]. M. smegmatis retains a fully intact narGHJI operon and although there are sequence differences in narG between M. tuberculosis and M. smegmatis, the promoter mutation (-215 T>C) is not present in the latter, suggesting that the enzymes could have similar activities. It has recently been reported that M. smegmatis does harbour a T>C mutation in narG, however the authors refer to position -125 instead of -215 [32]. The different Griess assay results observed in M. tuberculosis and M. smegmatis could be due to a lack of extracellular nitrite as a result of either (i) more efficient nitrite reductase activity in M. smegmatis which leads to the complete reduction of nitrite to ammonia, (ii) reduced nitrite transporter activity in M. smegmatis or (iii) a combination of both. No intracellular nitrite was detected in M. smegmatis during this study, suggesting that it is able to completely assimilate nitrite.

NarGHI, encoded by the narGHJI operon has been well characterized in E. coli and is a respiratory enzyme which serves as the terminal electron acceptor during anaerobic respiration when nitrate is available. The second NR-encoding gene in M. smegmatis – narB, is a putative assimilatory NR-encoding gene [15]. NarB is a cytoplasmic enzyme that has been well characterized in Cyanobacteria where it facilitates growth on nitrate [26]. Based on the genome organisation, sequence similarities and predicted 3D structure, the M. smegmatis narB was expected to encode a functional NR. Although this cannot be ruled out, the inability to detect narB transcript in our study and the ability of strains lacking narB to assimilate nitrate suggests otherwise. Although narG transcript was detected in M. smegmatis, the strains lacking narGHJI were also able to assimilate nitrate, suggesting that NarGHI is not the only enzyme involved in nitrate assimilation in contrast to the M. tuberculosis counterpart [10]. Furthermore, NarB and NarGHI were dispensable for anaerobic survival in the presence of nitrate, suggesting that either these enzymes are not required for nitrate reduction or that in the absence of each, an alternate enzyme can compensate for the loss.

Herein, we demonstrate that narB and narGHJI are individually and collectively dispensable for nitrate reduction in M. smegmatis under the conditions tested. Our analysis identified MSMEG_4206 as another putative assimilatory NR in M. smegmatis. Further analysis of this locus may provide novel insight into nitrogen metabolism in mycobacteria.

Construction and complementation of mutant strains. The upstream and downstream regions flanking the narB gene and narGHJI operon were amplified from wild type genomic DNA using the primer sets listed in Additional file 1: Table S1. The amplicons were digested with the appropriate restriction enzymes and a three-way cloning strategy was employed to generate intermediate constructs carrying the upstream and downstream regions of the narB gene and the narGHJI operon. The selectable – counter-selectable marker cassette was released from the pGOAL19 plasmid with PacI, and was subsequently ligated to the PacI-linearized p2nil intermediate constructs to generate the suicide vectors pΔnarB and pΔnarGHJI. M. smegmatis mutants were constructed by two-step allelic exchange mutagenesis as previously described [33]. The strains used and generated during this study are listed in Additional file 1: Table S2.

Bacterial growth. E. coli strains were grown in Luria Bertani liquid medium (LB) or solid medium (LA) supplemented with the appropriate antibiotics at concentrations of 100 µg/ml Ampicillin (Amp), 200 µg/ml Hygromycin (Hyg) or 50 µg/ml Kanamycin (Kan). E. coli strains used for the propagation of suicide vectors were grown at 30 ºC to prevent DNA re-arrangements, while all other strains were grown at 37 ºC. M. smegmatis strains were grown in Middlebrook 7H9 liquid medium (Difco) supplemented with Middlebrook oleic acid-albumin-dextrose-catalase (OADC) enrichment (Difco) or 0.085 % NaCl, 0.2 % glucose, 0.2 % glycerol and 0.05 % Tween80 and on Middlebrook 7H10 solid medium (Difco) 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 negative Griess result does not necessarily represent a lack of NR activity. Zinc rapidly reduces nitrate to nitrite [34]. A colour change upon the addition of zinc to a sample containing the Griess reagents measures the remaining nitrate, which allows for a measurement of nitrate depletion, thus confirming NR activity in an indirect way. This method can be exploited to quantify the amount of nitrite present in a sample by generating a nitrite standard curve with absorbance values recorded at λmax530nm. In addition, the rate of nitrate utilization can be determined. To investigate nitrate utilization M. smegmatis cultures were inoculated into MPLN and incubated until stationary phase. The modified 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.

Ethics approval and consent to participate

This study did not involve human subjects and consequently, an ethics waiver was obtained from the Human Research Ethics Committee of the University of the Witwatersrand (Reference number: W-CJ-160208-2)

Consent for publication

Not applicable

Abbreviations

Amp: Ampicillin, bis-MGD: bis-molybdopterin guanine dinucleotide, CFU: colony forming units, Hyg: Hygromycin, Kan: Kanamycin, MAC: Mycobacterium avium complex, MoCo: molybdenum cofactor, MPLN: modified M. phlei minimal medium, MQH2: menaquinol, NEDD: N-(1-naphthyl) ethylenediamine dihydrochloride, NR: Nitrate reductase, PMF: proton motive force, qRT PCR: quantitative reverse transcriptase polymerase chain reaction, SNP: single nucleotide polymorphism.

Acknowledgments

The authors thank Valerie Mizrahi (Molecular Mycobacteriology Research Unit) for guidance and insightful comment and Christopher Ealand for critical review the manuscript.

Funding

This work was supported by funding from an International Early Career Scientist Award from the Howard Hughes Medical Institute (to BDK), the South African National Research Foundation (to BDK), the South African Medical Research Council (to BDK and NCC). NCC was supported by a PhD internship from South African Medical Research Council and a BTC Fellowship sponsored by the Belgian Embassy in South Africa.

Availability of data and materials

Data supporting the conclusions in this article are included within the article and additional supporting information. All datasets analyzed in this article are available from the corresponding author.

Authors’ contributions

NCC and BDK conceived and designed experiments in this study. NCC performed all experimental procedures. NCC and BDK wrote and approved the manuscript.

Competing interests

The authors declare that they have no competing interests.

1. Thamdrup, B., New Pathways and Processes in the Global Nitrogen Cycle. Annual Review of Ecology, Evolution, and Systematics, 2012. 43(1): p. 407-428.

2. Moreno-Vivian, C., et al., Prokaryotic nitrate reduction: molecular properties and functional distinction among bacterial nitrate reductases. J Bacteriol, 1999. 181(21): p. 6573-84.

3. Richardson, D.J. and N.J. Watmough, Inorganic nitrogen metabolism in bacteria. Curr Opin Chem Biol, 1999. 3(2): p. 207-19.

4. Morozkina, E.V. and R.A. Zvyagilskaya, Nitrate reductases: structure, functions, and effect of stress factors. Biochemistry (Mosc), 2007. 72(10): p. 1151-60.

5. Gonzalez, P.J., et al., Bacterial nitrate reductases: Molecular and biological aspects of nitrate reduction. J Inorg Biochem, 2006. 100(5-6): p. 1015-23.

6. Richardson, D.J., et al., Functional, biochemical and genetic diversity of prokaryotic nitrate reductases. Cell Mol Life Sci, 2001. 58(2): p. 165-78.

7. Hille, R., J. Hall, and P. Basu, The mononuclear molybdenum enzymes. Chem Rev, 2014. 114(7): p. 3963-4038.

8. Moreno-Vivian, C. and S.J. Ferguson, Definition and distinction between assimilatory, dissimilatory and respiratory pathways. Mol Microbiol, 1998. 29(2): p. 664-6.

9. Blasco, F., et al., NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli. Mol Microbiol, 1998. 28(3): p. 435-47.

10. Malm, S., et al., The roles of the nitrate reductase NarGHJI, the nitrite reductase NirBD and the response regulator GlnR in nitrate assimilation of Mycobacterium tuberculosis. Microbiology, 2009. 155(Pt 4): p. 1332-9.

11. Sohaskey, C.D. and L.G. Wayne, Role of narK2X and narGHJI in hypoxic upregulation of nitrate reduction by Mycobacterium tuberculosis. J Bacteriol, 2003. 185(24): p. 7247-56.

12. Gouzy, A., Y. Poquet, and O. Neyrolles, Nitrogen metabolism in Mycobacterium tuberculosis physiology and virulence. Nat Rev Microbiol, 2014. 12(11): p. 729-37.

13. Williams, M.J., B.D. Kana, and V. Mizrahi, Functional analysis of molybdopterin biosynthesis in mycobacteria identifies a fused molybdopterin synthase in Mycobacterium tuberculosis. J Bacteriol, 2011. 193(1): p. 98-106.

14. Narrandes, N.C., et al., Cleavage of the moaX-encoded fused molybdopterin synthase from Mycobacterium tuberculosis is necessary for activity. BMC Microbiol, 2015. 15: p. 22.

15. Khan, A., et al., Presence of functional nitrate assimilation pathway in Mycobacterium smegmatis. Microbial Pathogenesis, 2008. 44: p. 71-77.

16. Weber, I., et al., Anaerobic nitrate reductase (narGHJI) activity of Mycobacterium bovis BCG in vitro and its contribution to virulence in immunodeficient mice. Mol Microbiol, 2000. 35(5): p. 1017-25.

17. Khan, A. and D. Sarkar, Identification of a respiratory-type nitrate reductase and its role for survival of Mycobacterium smegmatis in Wayne model. Microbial Pathogenesis, 2006. 41: p. 90-95.

18. Amon, J., F. Titgemeyer, and A. Burkovski, A genomic view on nitrogen metabolism and nitrogen control in mycobacteria. J Mol Microbiol Biotechnol, 2009. 17(1): p. 20-9.

19. Stermann, M., et al., A promoter mutation causes differential nitrate reductase activity of Mycobacterium tuberculosis and Mycobacterium bovis. J Bacteriol, 2004. 186(9): p. 2856-61.

20. Rice, P., I. Longden, and A. Bleasby, EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet, 2000. 16(6): p. 276-7.

21. Altschul, S.F., et al., Basic local alignment search tool. J Mol Biol, 1990. 215(3): p. 403-10.

22. Zhang, Y., I-TASSER server for protein 3D structure prediction. BMC Bioinformatics, 2008. 9: p. 40.

23. Najmudin, S., et al., Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic molybdenum. J Biol Inorg Chem, 2008. 13(5): p. 737-53.

24. Petersen, T.N., et al., SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods, 2011. 8(10): p. 785-6.

25. Szklarczyk, D., et al., The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. 2017. 45(D1): p. D362-d368.

26. Rubio, L.M., A. Herrero, and E. Flores, A cyanobacterial narB gene encodes a ferredoxin-dependent nitrate reductase. Plant Mol Biol, 1996. 30(4): p. 845-50.

27. Kanehisa, M., et al., KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res, 2016. 44(D1): p. D457-62.

28. Kapopoulou, A., J.M. Lew, and S.T. Cole, The MycoBrowser portal: a comprehensive annotated resource for mycobacterial genomes. Tuberculosis (Edinb), 2011. 91(1): p. 8-13.

29. Zhou, J., A.J. Richardson, and K.E. Rudd, EcoGene-RefSeq:EcoGene tools applied to the RefSeq Prokaryote genomes. 2013.

30. Khan, A. and D. Sarkar, Nitrate reduction pathways in mycobacteria and their implications during latency. Microbiology, 2012. 158(Pt 2): p. 301-7.

31. Hutter, B. and T. Dick, Up-regulation of narX, encoding a putative 'fused nitrate reductase' in anaerobic dormant Mycobacterium bovis BCG. FEMS Microbiol Lett, 1999. 178(1): p. 63-9.

32. Huang, Q., A.E. Abdalla, and J. Xie, Phylogenomics of Mycobacterium Nitrate Reductase Operon. Curr Microbiol, 2015. 71(1): p. 121-8.

33. Parish, T. and N.G. Stoker, Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology, 2000. 146 ( Pt 8): p. 1969-75.

34. Angeby, K.A., L. Klintz, and S.E. Hoffner, Rapid and inexpensive drug susceptibility testing of Mycobacterium tuberculosis with a nitrate reductase assay. J Clin Microbiol, 2002. 40(2): p. 553-5.

35. Morozkina, E.V. and A.V. Kurakov, [Dissimilatory nitrate reduction in fungi under conditions of hypoxia and anoxia: a review]. Prikl Biokhim Mikrobiol, 2007. 43(5): p. 607-13.

The supplemental file (downloadable below) contains the following items:

Supplementary tables.

Table S1: Primers used for the construction of suicide vectors

Table S2: Plasmids and strains used and generated during this study

Table S3: Primers used for qRT PCR

Figure S1: Genotypic confirmation of ΔnarB. (A) Schematic representation of genomic maps of wild type and mutant narB regions. Restriction enzymes, probes and expected fragment sizes for southern blot confirmation are depicted. (B) Southern blot with upstream probe (US). Lane 1: Marker λIV, Lane 2: Empty, Lane 3: NotI digested wild type DNA, Lane 4: NotI digested ΔnarB DNA, Lane 5: Empty, Lane 6: SacI digested wild type DNA, Lane 7: SacI digested ΔnarB DNA. (C) Southern blot with downstream probe (DS). Lane 1: Marker λIV, Lane 2: Empty, Lane 3: NotI digested wild type DNA, Lane 4: NotI digested ΔnarB DNA, Lane 5: Empty, Lane 6: NcoI digested wild type DNA, Lane 7: NcoI digested ΔnarB DNA.

Figure S2: Genotypic confirmation of ΔnarGHJI and ΔnarB ΔnarGHJI. (A) Schematic representation of genomic maps of wild type and mutant narGHJI regions. Restriction enzymes, probes and expected fragment sizes for southern blot confirmation are depicted. (B) Southern blot with upstream probe (US). Lane 1: Marker λIV, Lane 2: BamHI digested wild type DNA, Lane 3: BamHI digested ΔnarGHJI DNA, Lane 4: BamHI digested ΔnarB ΔnarGHJI DNA. (C) Southern blot with downstream probe (DS). Lane 1: Marker λIV, Lane 2: PstI digested wild type DNA, Lane 3: PstI digested ΔnarGHJI DNA, Lane 4: PstI digested ΔnarB ΔnarGHJI DNA, Lane 5: Empty, Lane 6: MuI digested wild type DNA, Lane 7: MluI digested ΔnarGHJI DNA, Lane 8: MluI digested ΔnarB ΔnarGHJI DNA.

Figure S3: Growth kinetics of M. smegmatis strains grown in rich 7H9 medium. Cultures were grown in 7H9 supplemented with OADC and tyloxapol with absorbance readings recorded daily for five days. A representative of a single biological replicate is shown.

Figure S4: The expression analysis of strains grown in 7H9 or MPLN. (A) Transcript abundance of sigA in 7H9. (B) Transcript abundance of sigA in MPLN. (C) The normalized expression of narG in MPLN. Strains were grown to OD600 0,8-1 in MPLN liquid media. 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: Escherichia coli, Mleprae: Mycobacterium leprae, Msmeg: Mycobacterium smegmatis, M. tuberculosis: Mycobacterium tuberculosis, BCG: Mycobacterium bovis BCG.

Figure S6: Sequence alignments of NarG from different organisms. The amino acids shaded in different colours represent different secondary structures and ligand binding sites. Ecoli: Escherichia coli, MSMEG: Mycobacterium smegmatis, M.tuberculosis: Mycobacterium tuberculosis, M. africanum: Mycobacterium africanum, MTBC: Mycobacterium tuberculosis complex, M. canetti: Mycobacterium canettii, M. bovis: Mycobacterium bovis BCG.

Figure S7: The genome organization of MSMEI_4108, an additional putative assimilatory NR in M. smegmatis. (A) Shown is the organization of a region of the M. smegmatis chromosome as annotated on two different databases, namely Mycobrowser and EcoGene-RefSeq. (B) Shown is a table comparing the gene coordinates and annotations from the two databases. (C) The protein domain architecture of MSMEI_4108 according to KEGG is shown.

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

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