scholarly journals The roles of the nitrate reductase NarGHJI, the nitrite reductase NirBD and the response regulator GlnR in nitrate assimilation of Mycobacterium tuberculosis

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1332-1339 ◽  
Author(s):  
Sven Malm ◽  
Yvonne Tiffert ◽  
Julia Micklinghoff ◽  
Sonja Schultze ◽  
Insa Joost ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nitrate Reductase ◽  
Nitrite Reductase ◽  
Response Regulator ◽  
Specific Binding ◽  
Nitrate Assimilation ◽  
Cosmid Library ◽  
Nitrate Respiration ◽  
Mobility Shift ◽  
Nitrate And Nitrite

Mycobacterium tuberculosis can utilize various nutrients including nitrate as a source of nitrogen. Assimilation of nitrate requires the reduction of nitrate via nitrite to ammonium, which is then incorporated into metabolic pathways. This study was undertaken to define the molecular mechanism of nitrate assimilation in M. tuberculosis. Homologues to a narGHJI-encoded nitrate reductase and a nirBD-encoded nitrite reductase have been found on the chromosome of M. tuberculosis. Previous studies have implied a role for NarGHJI in nitrate respiration rather than nitrate assimilation. Here, we show that a narG mutant of M. tuberculosis failed to grow on nitrate. A nirB mutant of M. tuberculosis failed to grow on both nitrate and nitrite. Mutant strains of Mycobacterium smegmatis mc2155 that are unable to grow on nitrate were isolated. The mutants were rescued by screening a cosmid library from M. tuberculosis, and a gene with homology to the response regulator gene glnR of Streptomyces coelicolor was identified. A ΔglnR mutant of M. tuberculosis was generated, which also failed to grow on nitrate, but regained its ability to utilize nitrate when nirBD was expressed from a plasmid, suggesting a role of GlnR in regulating nirBD expression. A specific binding site for GlnR within the nirB promoter was identified and confirmed by electrophoretic mobility shift assay using purified recombinant GlnR. Semiquantitative reverse transcription PCR, as well as microarray analysis, demonstrated upregulation of nirBD expression in response to GlnR under nitrogen-limiting conditions. In summary, we conclude that NarGHJI and NirBD of M. tuberculosis mediate the assimilatory reduction of nitrate and nitrite, respectively, and that GlnR acts as a transcriptional activator of nirBD.

scholarly journals Nitrogen and Oxygen Regulation of Bacillus subtilis nasDEF Encoding NADH-Dependent Nitrite Reductase by TnrA and ResDE

1998 ◽  
Vol 180 (20) ◽  
pp. 5344-5350 ◽  
Author(s):  
Michiko M. Nakano ◽  
Tamara Hoffmann ◽  
Yi Zhu ◽  
Dieter Jahn
Keyword(s):  
Bacillus Subtilis ◽  
Nitrate Reductase ◽  
Nitrite Reductase ◽  
Nitrogen Limitation ◽  
Reductase Activity ◽  
Anaerobic Respiration ◽  
Regulatory System ◽  
Nitrate Respiration ◽  
Nitrite Reductase Activity ◽  
Nitrate And Nitrite

ABSTRACT The nitrate and nitrite reductases of Bacillus subtilishave two different physiological functions. Under conditions of nitrogen limitation, these enzymes catalyze the reduction of nitrate via nitrite to ammonia for the anabolic incorporation of nitrogen into biomolecules. They also function catabolically in anaerobic respiration, which involves the use of nitrate and nitrite as terminal electron acceptors. Two distinct nitrate reductases, encoded bynarGHI and nasBC, function in anabolic and catabolic nitrogen metabolism, respectively. However, as reported herein, a single NADH-dependent, soluble nitrite reductase encoded by the nasDE genes is required for both catabolic and anabolic processes. The nasDE genes, together with nasBC(encoding assimilatory nitrate reductase) and nasF(required for nitrite reductase siroheme cofactor formation), constitute the nas operon. Data presented show that transcription of nasDEF is driven not only by the previously characterized nas operon promoter but also from an internal promoter residing between the nasC andnasD genes. Transcription from both promoters is activated by nitrogen limitation during aerobic growth by the nitrogen regulator, TnrA. However, under conditions of oxygen limitation,nasDEF expression and nitrite reductase activity were significantly induced. Anaerobic induction of nasDEFrequired the ResDE two-component regulatory system and the presence of nitrite, indicating partial coregulation of NasDEF with the respiratory nitrate reductase NarGHI during nitrate respiration.

scholarly journals Induction of the Nitrate AssimilationnirAOperon and Protein-Protein Interactions in the Maturation of Nitrate and Nitrite Reductases in the Cyanobacterium Anabaena sp. Strain PCC 7120

2015 ◽  
Vol 197 (14) ◽  
pp. 2442-2452 ◽  
Author(s):  
José E. Frías ◽  
Enrique Flores
Keyword(s):  
Nitrate Reductase ◽  
Protein Interactions ◽  
Nitrite Reductase ◽  
Nitrate Assimilation ◽  
Protein Protein Interactions ◽  
Content Type ◽  
Nitrite Reductases ◽  
Nitrate And Nitrite ◽  
Pcc 7120 ◽  
Assimilation System

ABSTRACTNitrate is widely used as a nitrogen source by cyanobacteria, in which the nitrate assimilation structural genes frequently constitute the so-callednirAoperon. This operon contains the genes encoding nitrite reductase (nirA), a nitrate/nitrite transporter (frequently an ABC-type transporter;nrtABCD), and nitrate reductase (narB). In the model filamentous cyanobacteriumAnabaenasp. strain PCC 7120, which can fix N2in specialized cells termed heterocysts, thenirAoperon is expressed at high levels only in media containing nitrate or nitrite and lacking ammonium, a preferred nitrogen source. Here we examined the genes downstream of thenirAoperon inAnabaenaand found that a small open reading frame of unknown function,alr0613, can be cotranscribed with the operon. The next gene in the genome,alr0614(narM), showed an expression pattern similar to that of thenirAoperon, implying correlated expression ofnarMand the operon. A mutant ofnarMwith an insertion mutation failed to produce nitrate reductase activity, consistent with the idea that NarM is required for the maturation of NarB. BothnarMandnarBmutants were impaired in the nitrate-dependent induction of thenirAoperon, suggesting that nitrite is an inducer of the operon inAnabaena. It has previously been shown that the nitrite reductase protein NirA requires NirB, a protein likely involved in protein-protein interactions, to attain maximum activity. Bacterial two-hybrid analysis confirmed possible NirA-NirB and NarB-NarM interactions, suggesting that the development of both nitrite reductase and nitrate reductase activities in cyanobacteria involves physical interaction of the corresponding enzymes with their cognate partners, NirB and NarM, respectively.IMPORTANCENitrate is an important source of nitrogen for many microorganisms that is utilized through the nitrate assimilation system, which includes nitrate/nitrite membrane transporters and the nitrate and nitrite reductases. Many cyanobacteria assimilate nitrate, but regulation of the nitrate assimilation system varies in different cyanobacterial groups. In the N2-fixing, heterocyst-forming cyanobacteria, thenirAoperon, which includes the structural genes for the nitrate assimilation system, is expressed in the presence of nitrate or nitrite if ammonium is not available to the cells. Here we studied the genes required for production of an active nitrate reductase, providing information on the nitrate-dependent induction of the operon, and found evidence for possible protein-protein interactions in the maturation of nitrate reductase and nitrite reductase.

scholarly journals Two Nitrate/Nitrite Transporters Are Encoded within the Mobilizable Plasmid for Nitrate Respiration of Thermus thermophilus HB8

2000 ◽  
Vol 182 (8) ◽  
pp. 2179-2183 ◽  
Author(s):  
Sandra Ramírez ◽  
Renata Moreno ◽  
Olga Zafra ◽  
Pablo Castán ◽  
Cristina Vallés ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Average Distance ◽  
Thermus Thermophilus ◽  
Major Facilitator Superfamily ◽  
Nitrate Respiration ◽  
Mobilizable Plasmid ◽  
Excretion Rates ◽  
Major Facilitator ◽  
Nitrate And Nitrite

ABSTRACT Thermus thermophilus HB8 can grow anaerobically by using a membrane-bound nitrate reductase to catalyze the reduction of nitrate as a final electron acceptor in respiration. In contrast to other denitrifiers, the nitrite produced does not continue the reduction pathway but accumulates in the growth medium after its active extrusion from the cell. We describe the presence of two genes,narK1 and narK2, downstream of the nitrate reductase-encoding gene cluster (nar) that code for two homologues to the major facilitator superfamily of transporters. The sequences of NarK1 and NarK2 are 30% identical to each other, but whereas NarK1 clusters in an average-distance tree with putative nitrate transporters, NarK2 does so with putative nitrite exporters. To analyze whether this differential clustering was actually related to functional differences, we isolated derivatives with mutations of one or both genes. Analysis revealed that single mutations had minor effects on growth by nitrate respiration, whereas a double narK1 narK2 mutation abolished this capability. Further analysis allowed us to confirm that the double mutant is completely unable to excrete nitrite, while single mutants have a limitation in the excretion rates compared with the wild type. These data allow us to propose that both proteins are implicated in the transport of nitrate and nitrite, probably acting as nitrate/nitrite antiporters. The possible differential roles of these proteins in vivo are discussed.

A composite biochemical system for bacterial nitrate and nitrite assimilation as exemplified by Paracoccus denitrificans

2011 ◽  
Vol 435 (3) ◽  
pp. 743-753 ◽  
Author(s):  
Andrew J. Gates ◽  
Victor M. Luque-Almagro ◽  
Alan D. Goddard ◽  
Stuart J. Ferguson ◽  
M. Dolores Roldán ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Nitrogen Source ◽  
Nitrite Reductase ◽  
Paracoccus Denitrificans ◽  
Gene Clusters ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Nitrite Reduction ◽  
Anaerobic Growth ◽  
Nitrate And Nitrite

The denitrifying bacterium Paracoccus denitrificans can grow aerobically or anaerobically using nitrate or nitrite as the sole nitrogen source. The biochemical pathway responsible is expressed from a gene cluster comprising a nitrate/nitrite transporter (NasA), nitrite transporter (NasH), nitrite reductase (NasB), ferredoxin (NasG) and nitrate reductase (NasC). NasB and NasG are essential for growth with nitrate or nitrite as the nitrogen source. NADH serves as the electron donor for nitrate and nitrite reduction, but only NasB has a NADH-oxidizing domain. Nitrate and nitrite reductase activities show the same Km for NADH and can be separated by anion-exchange chromatography, but only fractions containing NasB retain the ability to oxidize NADH. This implies that NasG mediates electron flux from the NADH-oxidizing site in NasB to the sites of nitrate and nitrite reduction in NasC and NasB respectively. Delivery of extracellular nitrate to NasBGC is mediated by NasA, but both NasA and NasH contribute to nitrite uptake. The roles of NasA and NasC can be substituted during anaerobic growth by the biochemically distinct membrane-bound respiratory nitrate reductase (Nar), demonstrating functional overlap. nasG is highly conserved in nitrate/nitrite assimilation gene clusters, which is consistent with a key role for the NasG ferredoxin, as part of a phylogenetically widespread composite nitrate and nitrite reductase system.

scholarly journals The bZIP transcription factor MdHY5 regulates anthocyanin accumulation and nitrate assimilation in apple

2017 ◽  
Vol 4 (1) ◽  
Author(s):  
Jian-Ping An ◽  
Feng-Jia Qu ◽  
Ji-Fang Yao ◽  
Xiao-Na Wang ◽  
Chun-Xiang You ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Nitrate Reductase ◽  
Leucine Zipper ◽  
Anthocyanin Biosynthesis ◽  
Nitrate Assimilation ◽  
Bzip Transcription Factor ◽  
Anthocyanin Accumulation ◽  
Mobility Shift ◽  
Basic Leucine Zipper ◽  
Transgenic Apple

Abstract The basic leucine zipper (bZIP) transcription factor HY5 plays a multifaceted role in plant growth and development. Here the apple MdHY5 gene was cloned based on its homology with Arabidopsis HY5. Expression analysis demonstrated that MdHY5 transcription was induced by light and abscisic acid treatments. Electrophoretic mobility shift assays and transient expression assays subsequently showed that MdHY5 positively regulated both its own transcription and that of MdMYB10 by binding to E-box and G-box motifs, respectively. Furthermore, we obtained transgenic apple calli that overexpressed the MdHY5 gene, and apple calli coloration assays showed that MdHY5 promoted anthocyanin accumulation by regulating expression of the MdMYB10 gene and downstream anthocyanin biosynthesis genes. In addition, the transcript levels of a series of nitrate reductase genes and nitrate uptake genes in both wild-type and transgenic apple calli were detected. In association with increased nitrate reductase activities and nitrate contents, the results indicated that MdHY5 might be an important regulator in nutrient assimilation. Taken together, these results indicate that MdHY5 plays a vital role in anthocyanin accumulation and nitrate assimilation in apple.

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.

scholarly journals Molecular Components of Nitrate and Nitrite Efflux in Yeast

2013 ◽  
Vol 13 (2) ◽  
pp. 267-278 ◽  
Author(s):  
Elisa Cabrera ◽  
Rafaela González-Montelongo ◽  
Teresa Giraldez ◽  
Diego Alvarez de la Rosa ◽  
José M. Siverio
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Uptake ◽  
Reductase Activity ◽  
Hansenula Polymorpha ◽  
Nitrate Assimilation ◽  
Nitrate Reductase Gene ◽  
Content Type ◽  
Nitrite Toxicity ◽  
Essential Components ◽  
Nitrate And Nitrite

ABSTRACTSome eukaryotes, such as plant and fungi, are capable of utilizing nitrate as the sole nitrogen source. Once transported into the cell, nitrate is reduced to ammonium by the consecutive action of nitrate and nitrite reductase. How nitrate assimilation is balanced with nitrate and nitrite efflux is unknown, as are the proteins involved. The nitrate assimilatory yeastHansenula polymorphawas used as a model to dissect these efflux systems. We identified the sulfite transporters Ssu1 and Ssu2 as effective nitrate exporters, Ssu2 being quantitatively more important, and we characterize the Nar1 protein as a nitrate/nitrite exporter. The use of strains lacking eitherSSU2orNAR1along with the nitrate reductase geneYNR1showed that nitrate reductase activity is not required for net nitrate uptake. Growth test experiments indicated that Ssu2 and Nar1 exporters allow yeast to cope with nitrite toxicity. We also have shown that the well-knownSaccharomyces cerevisiaesulfite efflux permease Ssu1 is also able to excrete nitrite and nitrate. These results characterize for the first time essential components of the nitrate/nitrite efflux system and their impact on net nitrate uptake and its regulation.

scholarly journals Kinetics of nirS Expression (Cytochromecd1 Nitrite Reductase) in Pseudomonas stutzeri during the Transition from Aerobic Respiration to Denitrification: Evidence for a Denitrification-Specific Nitrate- and Nitrite-Responsive Regulatory System

1999 ◽  
Vol 181 (1) ◽  
pp. 161-166 ◽  
Author(s):  
Elisabeth Härtig ◽  
Walter G. Zumft
Keyword(s):  
Nitrite Reductase ◽  
Response Regulator ◽  
Gene Activation ◽  
Regulatory System ◽  
Pseudomonas Stutzeri ◽  
Low Oxygen ◽  
Gene Encoding ◽  
Regulatory Circuit ◽  
Nitrate And Nitrite ◽  
Denitrification Genes

ABSTRACT After shifting an oxygen-respiring culture of Pseudomonas stutzeri to nitrate or nitrite respiration, we directly monitored the expression of the nirS gene by mRNA analysis.nirS encodes the 62-kDa subunit of the homodimeric cytochrome cd 1 nitrite reductase involved in denitrification. Information was sought about the requirements for gene activation, potential regulators of such activation, and signal transduction pathways triggered by the alternative respiratory substrates. We found that nirS, together withnirT and nirB (which encode tetra- and diheme cytochromes, respectively), is part of a 3.4-kb operon. In addition, we found a 2-kb monocistronic transcript. The half-life of each of these messages was approximately 13 min in denitrifying cells with a doubling time of around 2.5 h. When the culture was subjected to a low oxygen tension, we observed a transient expression of nirSlasting for about 30 min. The continued transcription of thenirS operon required the presence of nitrate or nitrite. This anaerobically manifested N-oxide response was maintained in nitrate sensor (NarX) and response regulator (NarL) knockout strains. Similar mRNA stability and transition kinetics were observed for the norCB operon, encoding the NO reductase complex, and the nosZ gene, encoding nitrous oxide reductase. Our results suggest that a nitrate- and nitrite-responsive regulatory circuit independent of NarXL is necessary for the activation of denitrification genes.

Isotopic Fractionation During Reduction of Nitrate and Nitrite by Extracts of Spinach Leaves

1985 ◽  
Vol 12 (6) ◽  
pp. 631 ◽  
Author(s):  
SF Ledgard ◽  
KC Woo ◽  
FJ Bergersen
Keyword(s):  
Nitrate Reductase ◽  
Nitrite Reductase ◽  
Spinacia Oleracea ◽  
Reductase Activity ◽  
Isotopic Fractionation ◽  
Cytosolic Extract ◽  
Nitrite Reductase Activity ◽  
Reconstituted System ◽  
No2 Reduction ◽  
Nitrate And Nitrite

The isotopic fractionations of nitrogen during the reduction of NO3- and NO2- in a cytosolic fraction and in a chloroplast preparation from spinach (Spinacia oleracea L.) leaves were determined. The reduction of NO3- to NH3 was studied using a reconstituted system containing cytosolic extract and intact chloroplasts, while a chloroplast system was used for NO2- reduction. In the reconstituted systems the ratio of nitrate reductase activity to nitrite reductase activity had a large effect on the relative amounts of NO2- and NH3 formed. Ammonia predominated when the nitrate reductase to nitrite reductase activity ratio was 1 : 5 and this ratio was used in the isotopic fractionation studies. Significant isotopic fractionation of N was observed in the reconstituted system but not in the chloroplast system. This indicates that the observed isotopic fractionation was associated with the reduction of NO3- to NO2- by nitrate reductase. The isotopic fractionation (i.e. δ15Nproduct - δ15Nsubstrate) for this reaction was - 15‰.

scholarly journals THE ISOLATION AND CHARACTERIZATION OF MUTANTS DEFECTIVE IN NITRATE ASSIMILATION IN NEUROSPORA CRASSA

Genetics ◽  
1980 ◽  
Vol 95 (3) ◽  
pp. 649-660
Author(s):  
A Brian Tomsett ◽  
Reginald H Garrett
Keyword(s):  
Nitrate Reductase ◽  
Neurospora Crassa ◽  
Nitrate Assimilation ◽  
Xanthine Dehydrogenase ◽  
Growth Patterns ◽  
Isolation And Characterization ◽  
Complex Locus ◽  
Complementation Groups ◽  
Nitrate And Nitrite

ABSTRACT The isolation and characterization of mutants altered for nitrate assimilation in Neurospora crassa is described, The mutants isolated can be subdivided into five classes on the basis of growth tests that correspond to the growth patterns of existing mutants at six distinct loci. Mutants with growth characteristics like those of nit-2, nit-3 and nit-6 are assigned to those loci on the basis of noncomplementation and lack of recombination. Mutants that, from their growth patterns, appear to lack the molybdenum-containing cofactor for both nitrate reductase and xanthine dehydrogenase subdivide into three loci (nit-7, nit4 and nit-9), all of which are genetically distinct from nit-1. nit-9 is a complex locus consisting of three complementation groups and thus appears similar to the cnxABC locus of Asperillus nidulans. Extensive complementational and recombinational analyses reveal that nit-4 and nit-5 are alleles of the same locus, and two new alleles of that locus have been isolated. The results indicate that, as in A. nidulans, nitrate assimilation in N. crassa requires at least four loci (nit-1,7,8 and 9) to produce the molybdenum co-factor for nitrate reductase (and xanthine dehydrogenase), one locus (nit-3) to code for the nitrate reductase apoprotein, one locus (nit-6) to code for the nitrite reductase approtein and only one locus (nit-4/5) for the regulation of induction of the pathway by nitrate and nitrite.

Sign in / Sign up

Export Citation Format

Share Document