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

2019 ◽  
Author(s):  
Nicole Collette Cardoso ◽  
Bavesh Davandra Kana
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reduction ◽  
Mycobacterium Smegmatis ◽  
Reductase Activity ◽  
Nitrate Assimilation ◽  
Wild Type ◽  
Relative Contribution ◽  
Allelic Replacement ◽  
Mutant Strains ◽  
Assimilatory Nitrate Reduction

Abstract 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.

scholarly journals Mycobacterium smegmatis does not display functional redundancy in nitrate reductase enzymes

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245745
Author(s):  
Nicole C. Cardoso ◽  
Andrea O. Papadopoulos ◽  
Bavesh D. Kana
Keyword(s):  
Nitrate Reductase ◽  
Mycobacterium Smegmatis ◽  
Reductase Activity ◽  
Nitrate Assimilation ◽  
Anaerobic Conditions ◽  
Relative Contribution ◽  
Allelic Replacement ◽  
Encoding Genes ◽  
Aerobic And Anaerobic ◽  
Aerobic And Anaerobic Conditions

Reduction of nitrate to nitrite in bacteria is an essential step in the nitrogen cycle, catalysed by a variety of nitrate reductase (NR) enzymes. The soil dweller, Mycobacterium smegmatis is able to assimilate nitrate and herein we set out to confirm the genetic basis for this by probing NR activity in mutants defective for putative nitrate reductase (NR) encoding genes. In addition to the annotated narB and narGHJI, bioinformatics identified three other putative NR-encoding genes: MSMEG_4206, MSMEG_2237 and MSMEG_6816. To assess the relative contribution of each, the corresponding gene loci were deleted using two-step allelic replacement, individually and in combination. The resulting strains were tested for their ability to assimilate nitrate and reduce nitrate under aerobic and anaerobic conditions, using nitrate assimilation and modified Griess assays. We demonstrated that narB, narGHJI, MSMEG_2237 and MSMEG_6816 were individually dispensable for nitrate assimilation and for nitrate reductase activity under aerobic and anaerobic conditions. Only deletion of MSMEG_4206 resulted in significant reduction in nitrate assimilation under aerobic conditions. These data confirm that in M. smegmatis, narB, narGHJI, MSMEG_2237 and MSMEG_6816 are not required for nitrate reduction as MSMEG_4206 serves as the sole assimilatory NR.

scholarly journals Expression of Mycobacterium smegmatisPyrazinamidase in Mycobacterium tuberculosis Confers Hypersensitivity to Pyrazinamide and Related Amides

2000 ◽  
Vol 182 (19) ◽  
pp. 5479-5485 ◽  
Author(s):  
Helena I. M. Boshoff ◽  
Valerie Mizrahi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Intracellular Localization ◽  
Wild Type ◽  
Gene Encoding ◽  
Allelic Exchange ◽  
Mutant Strains ◽  
Established Role ◽  
Amidase Gene

ABSTRACT A pyrazinamidase (PZase)-deficient pncA mutant ofMycobacterium tuberculosis, constructed by allelic exchange, was used to investigate the effects of heterologous amidase gene expression on the susceptibility of this organism to pyrazinamide (PZA) and related amides. The mutant was highly resistant to PZA (MIC, >2,000 μg/ml), in accordance with the well-established role ofpncA in the PZA susceptibility of M. tuberculosis (A. Scorpio and Y. Zhang, Nat. Med. 2:662–667, 1996). Integration of the pzaA gene encoding the major PZase/nicotinamidase from Mycobacterium smegmatis (H. I. M. Boshoff and V. Mizrahi, J. Bacteriol. 180:5809–5814, 1998) or the M. tuberculosis pncA gene into the pncAmutant complemented its PZase/nicotinamidase defect. In bothpzaA- and pncA-complemented mutant strains, the PZase activity was detected exclusively in the cytoplasm, suggesting an intracellular localization for PzaA and PncA. ThepzaA-complemented strain was hypersensitive to PZA (MIC, ≤10 μg/ml) and nicotinamide (MIC, ≥20 μg/ml) and was also sensitive to benzamide (MIC, 20 μg/ml), unlike the wild-type andpncA-complemented mutant strains, which were highly resistant to this amide (MIC, >500 μg/ml). This finding was consistent with the observation that benzamide is hydrolyzed by PzaA but not by PncA. Overexpression of PzaA also conferred sensitivity to PZA, nicotinamide, and benzamide on M. smegmatis (MIC, 150 μg/ml in all cases) and rendered Escherichia colihypersensitive for growth at low pH.

Voltammetric characterization of the aerobic energy-dissipating nitrate reductase of Paracoccus pantotrophus: exploring the activity of a redox-balancing enzyme as a function of electrochemical potential

2007 ◽  
Vol 409 (1) ◽  
pp. 159-168 ◽  
Author(s):  
Andrew J. Gates ◽  
David J. Richardson ◽  
Julea N. Butt
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reduction ◽  
Reductase Activity ◽  
Electrochemical Potential ◽  
Intact Cells ◽  
Protein Film ◽  
Protein Film Voltammetry ◽  
Voltammetric Studies ◽  
Paracoccus Pantotrophus

Paracoccus pantotrophus expresses two nitrate reductases associated with respiratory electron transport, termed NapABC and NarGHI. Both enzymes derive electrons from ubiquinol to reduce nitrate to nitrite. However, while NarGHI harnesses the energy of the quinol/nitrate couple to generate a transmembrane proton gradient, NapABC dissipates the energy associated with these reducing equivalents. In the present paper we explore the nitrate reductase activity of purified NapAB as a function of electrochemical potential, substrate concentration and pH using protein film voltammetry. Nitrate reduction by NapAB is shown to occur at potentials below approx. 0.1 V at pH 7. These are lower potentials than required for NarGH nitrate reduction. The potentials required for Nap nitrate reduction are also likely to require ubiquinol/ubiquinone ratios higher than are needed to activate the H+-pumping oxidases expressed during aerobic growth where Nap levels are maximal. Thus the operational potentials of P. pantotrophus NapAB are consistent with a productive role in redox balancing. A Michaelis constant (KM) of approx. 45 μM was determined for NapAB nitrate reduction at pH 7. This is in line with studies on intact cells where nitrate reduction by Nap was described by a Monod constant (KS) of less than 15 μM. The voltammetric studies also disclosed maximal NapAB activity in a narrow window of potential. This behaviour is resistant to change of pH, nitrate concentration and inhibitor concentration and its possible mechanistic origins are discussed.

Nitrate Reductase from a Mutant Strain of Chlamydomonas reinhardii Incapable of Nitrate Assimilation

1983 ◽  
Vol 38 (5-6) ◽  
pp. 439-445 ◽  
Author(s):  
Emilio Fernández ◽  
Jacobo Cárdenas
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Wild Strain ◽  
Nitrate Assimilation ◽  
Pyridine Nucleotide ◽  
Spectral Studies ◽  
Binding Region ◽  
Chlamydomonas Reinhardii ◽  
Blue Sepharose

Nitrate reductase from mutant 305 of Chlamydomonas reinhardii has been purified about 90-fold and biochemically characterized. The enzyme can use reduced flavins and viologens as electron donors to reduce nitrate but, unlike the nitrate reductase complex from its parental wild strain, lacks NAD(P)H-nitrate reductase and NAD(P)H-cytochrome c reductase activities, does not bind to Blue-Agarose or Blue-Sepharose and exhibits a significantly lower molecular weight (177.000 vs. 241.000), whereas its kinetic characteristics and its sensitivity against several inhibitors and treatments are very similar to those of the terminal nitrate reductase activity of the wild strain complex. Spectral studies and antagonistic experiments with tungstate show the presence of cytochrome b557 and molybdenum. These facts lead us to propose that nitrate reductase from mutant 305 has a protein deletion which affects the pyridine nucleotide binding region of the diaphorase protein but without any effect on the terminal nitrate reductase activity.

Differential effects of ozone on in vivo nitrate reduction in soybean cultivars. I. Response to exogenous sugars

1978 ◽  
Vol 56 (13) ◽  
pp. 1540-1544 ◽  
Author(s):  
Albert C. Purvis
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reduction ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Foliar Injury ◽  
Glycolytic Intermediates ◽  
Glycine Max L ◽  
Growth Chambers ◽  
Visible Foliar Injury

Two cultivars of soybeans (Glycine max (L.) Merr.) differing widely in their resistance to ozone were exposed to 0.5 μl/ℓ ozone for 2 h in growth chambers. In vivo nitrate reduction was depressed by more than 50% in the primary leaves of Dare, the ozone-sensitive cultivar, but was not significantly altered in Hood, the ozone-resistant cultivar. Sucrose, up to 1.5% (w/v), added to excised seedlings of the Dare cultivar during exposure to ozone eliminated the ozone depression of in vivo nitrate reductase activity and also reduced foliar injury. Addition of two glycolytic intermediates, glyceraldehyde-3-phosphate and fructose-1,6-diphosphate, to the infiltration medium recovered some in vivo nitrate reduction in treated Dare leaves. The levels of extractable nitrate reductase and glyceraldehyde-3-phosphate dehydrogenase in the primary leaves of both cultivars were unaltered by ozone fumigations. These observations led to the conclusion that ozone depression of in vivo nitrate reduction is not due to ozone inactivation of nitrate reductase or of the enzymes coupling nitrate reduction to glycolysis, but may be caused by an inadequate supply of photosynthetic sugars. It was also noted that ozone depression of in vivo nitrate reduction only occurred with treatments which subsequently caused the development of visible foliar injury.

Seasonal variation in nitrate reductase activity in needles of high-elevation red spruce trees

1992 ◽  
Vol 22 (3) ◽  
pp. 375-380 ◽  
Author(s):  
M.G. Tjoelker ◽  
S.B. McLaughlin ◽  
R.J. DiCosty ◽  
S.E. Lindberg ◽  
R.J. Norby
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Nitrate Assimilation ◽  
High Elevation ◽  
Preliminary Evidence ◽  
Air Pollutant ◽  
Red Spruce ◽  
Acid Vapor ◽  
Assimilation Capacity

To assess seasonal and site variation in foliar nitrate reductase activity and its utility as a biochemical marker for the uptake of nitrogen oxide pollutants in high-elevation forests, we measured nitrate reductase activity in current-year needles of red spruce (Picearubens Sarg.) saplings at two high-elevation stands (1935 and 1720 m) in the Great Smoky Mountains, North Carolina. Measurements spanned two growing seasons between September 1987 and September 1988. Nitrate reductase activity peaked near 60 nmol•g−1•h−1 at both sites in September and October 1987 and August 1988 and declined 80% in November 1987 and 65% in September 1988. Although nitrate reductase activity was 30% greater in saplings at the higher site relative to the lower site in September and October 1987, activity dropped to approximately 10 nmol•g−1•h−1 at both sites in November 1987. No differences among sites were evident the following year. Comparing deposition of nitric acid vapor at a nearby site to nitrate reductase activity suggests that needle nitrate reductase activity is not an unequivocal marker for foliar uptake of nitrogen oxides during air pollutant episodes. The changes in soil nitrate levels in this system provide preliminary evidence that foliar nitrate assimilation may, in part, include nitrate taken up from the soil, as the highest activity occurred during periods of higher A-horizon nitrate concentrations in 1988. These measurements of nitrate reductase activity suggest that red spruce are capable of assimilating nitrate in foliage in the field and that the nitrate assimilation capacity varies throughout the year.

scholarly journals Respiratory Nitrate Ammonification by Shewanella oneidensis MR-1

2006 ◽  
Vol 189 (2) ◽  
pp. 656-662 ◽  
Author(s):  
Claribel Cruz-García ◽  
Alison E. Murray ◽  
Joel A. Klappenbach ◽  
Valley Stewart ◽  
James M. Tiedje
Keyword(s):  
Nitrous Oxide ◽  
Nitrate Reductase ◽  
Electron Acceptor ◽  
Nitrate Reduction ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Shewanella Oneidensis ◽  
Gene Encoding ◽  
Nitrate Ammonification ◽  
Sequential Reduction

ABSTRACT Anaerobic cultures of Shewanella oneidensis MR-1 grown with nitrate as the sole electron acceptor exhibited sequential reduction of nitrate to nitrite and then to ammonium. Little dinitrogen and nitrous oxide were detected, and no growth occurred on nitrous oxide. A mutant with the napA gene encoding periplasmic nitrate reductase deleted could not respire or assimilate nitrate and did not express nitrate reductase activity, confirming that the NapA enzyme is the sole nitrate reductase. Hence, S. oneidensis MR-1 conducts respiratory nitrate ammonification, also termed dissimilatory nitrate reduction to ammonium, but not respiratory denitrification.

scholarly journals The genes YNI1 and YNR1, encoding nitrite reductase and nitrate reductase respectively in the yeast Hansenula polymorpha, are clustered and co-ordinately regulated

1996 ◽  
Vol 317 (1) ◽  
pp. 89-95 ◽  
Author(s):  
Nélida BRITO ◽  
Julio AVILA ◽  
M. Dolores PEREZ ◽  
Celedonio GONZALEZ ◽  
José M. SIVERIO
Keyword(s):  
Nitrate Reductase ◽  
Nitrite Reductase ◽  
Reductase Activity ◽  
Hansenula Polymorpha ◽  
Enzymic Activity ◽  
Wild Type ◽  
Reading Frame ◽  
Dna Library ◽  
Genomic Dna Library ◽  
Nitrate And Nitrite

The nitrite reductase-encoding gene (YNI1) from the yeast Hansenula polymorpha was isolated from a lambda EMBL3 H. polymorpha genomic DNA library, using as a probe a 481 bp DNA fragment from the gene of Aspergillus nidulans encoding nitrite reductase (niiA). An open reading frame of 3132 bp, encoding a putative protein of 1044 amino acids with high similarity with nitrite reductases from fungi, was located by DNA sequencing in the phages λNB5 and λJA13. Genes YNI1 and YNR1 (encoding nitrate reductase) are clustered, separated by 1700 bp. Northern blot analysis showed that expression of YNI1 and YNR1 is co-ordinately regulated; induced by nitrate and nitrite and repressed by sources of reduced nitrogen, even in the presence of nitrate. A mutant lacking nitrite reductase activity was obtained by deletion of the chromosomal copy of YNI1. The mutant does not grow in nitrate or in nitrite; it exhibits a similar level of transcription of YNR1 to the wild type, but the nitrate reductase enzymic activity is only about 50% of the wild type. In the presence of nitrate the Δyni1::URA3 mutant extrudes approx. 24 nmol of nitrite/h per mg of yeast (wet weight), about five times more than the wild type.

Nitrate reduction and nitrogenase activity in Spirillum lipoferum

1977 ◽  
Vol 23 (3) ◽  
pp. 306-310 ◽  
Author(s):  
Carlos A. Neyra ◽  
Peter Van Berkum
Keyword(s):  
Nitrate Reduction ◽  
Time Course ◽  
Nitrogenase Activity ◽  
Reductase Activity ◽  
Nitrite Reduction ◽  
Lag Phase ◽  
Nitrite Accumulation ◽  
Assimilatory Nitrate Reduction ◽  
Nitrate And Nitrite ◽  
Anaerobic Pretreatment

Nitrate and nitrite reduction under aerobic, microaerophillic, and anaerobic conditions was demonstrated in Spirillum lipoferum (ATCC 29145). Nitrite did not accumulate during assimilatory nitrate reduction in air. The nitrite produced during dissimilatory nitrate reduction accumulated in the medium but not in the cells. On exposure of the bacteria to nitrate and anaerobiosis, a low initial rate (lag) was followed by accelerated rates of nitrite accumulation. A 3-h anaerobic pretreatment, in the absence of nitrate, did not avoid the lag phase. No nitrate reductase activity (NRA) developed in the presence of chloramphenicol. The data suggest that induction of anaerobic NRA in S. lipoferum required nitrate and protein synthesis.Anaerobic N2ase activity by S. lipoferum was greatly stimulated in the presence of nitrate. The time course of nitrate reduction was coincidental with the pattern of nitrate-stimulated N2ase activity indicating that a relationship exists between these two processes.

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