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 The YNT1 gene encoding the nitrate transporter in the yeast Hansenula polymorpha is clustered with genes YNI1 and YNR1 encoding nitrite reductase and nitrate reductase, and its disruption causes inability to grow in nitrate

1997 ◽  
Vol 321 (2) ◽  
pp. 397-403 ◽  
Author(s):  
M. Dolores PÉREZ ◽  
Celedonio GONZÁLEZ ◽  
Julio ÁVILA ◽  
Nélida BRITO ◽  
José M. SIVERIO
Keyword(s):  
Nitrate Reductase ◽  
Nitrite Reductase ◽  
Northern Blot Analysis ◽  
Hansenula Polymorpha ◽  
Gene Encoding ◽  
Reading Frame ◽  
Dna Library ◽  
Nitrate And Nitrite ◽  
Encoding Genes ◽  
Nitrate Transporters

DNA sequencing in the phage λJA13isolated from a λEMBL3 Hansenula polymorphagenomic DNA library containing the nitrate reductase- (YNR1) and nitrite reductase- (YNI1) encoding genes revealed an open reading frame (YNT1) of 1524 nucleotides encoding a putative protein of 508 amino acids with great similarity to the nitrate transporters from Aspergillus nidulansand Chlamydomonas reinhardtii. Disruption of the chromosomal YNT1copy resulted in incapacity to grow in nitrate and a significant reduction in rate of nitrate uptake. The disrupted strain is still sensitive to chlorate, and, in the presence of 0.1 mM nitrate, the expression of YNR1 and YNI1 and the activity of nitrate reductase and nitrite reductase are significantly reduced compared with the wild-type. Northern-blot analysis showed that YNT1 is expressed when the yeast is grown in nitrate and nitrite but not in ammonium solution.

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 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.

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 Involvement of NarK1 and NarK2 Proteins in Transport of Nitrate and Nitrite in the Denitrifying Bacterium Pseudomonas aeruginosa PAO1

2006 ◽  
Vol 72 (1) ◽  
pp. 695-701 ◽  
Author(s):  
Vandana Sharma ◽  
Chris E. Noriega ◽  
John J. Rowe
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Nitrate Reductase ◽  
Nitrate Uptake ◽  
Reductase Activity ◽  
Physiological Role ◽  
Genome Project ◽  
Anaerobic Growth ◽  
Wild Type ◽  
Uptake Rates ◽  
Nitrate And Nitrite

ABSTRACT Two transmembrane proteins were tentatively classified as NarK1 and NarK2 in the Pseudomonas genome project and hypothesized to play an important physiological role in nitrate/nitrite transport in Pseudomonas aeruginosa. The narK1 and narK2 genes are located in a cluster along with the structural genes for the nitrate reductase complex. Our studies indicate that the transcription of all these genes is initiated from a single promoter and that the gene complex narK1K2GHJI constitutes an operon. Utilizing an isogenic narK1 mutant, a narK2 mutant, and a narK1K2 double mutant, we explored their effect on growth under denitrifying conditions. While the ΔnarK1::Gm mutant was only slightly affected in its ability to grow under denitrification conditions, both the ΔnarK2::Gm and ΔnarK1K2::Gm mutants were found to be severely restricted in nitrate-dependent, anaerobic growth. All three strains demonstrated wild-type levels of nitrate reductase activity. Nitrate uptake by whole-cell suspensions demonstrated both the ΔnarK2::Gm and ΔnarK1K2::Gm mutants to have very low yet different nitrate uptake rates, while the ΔnarK1::Gm mutant exhibited wild-type levels of nitrate uptake. Finally, Escherichia coli narK rescued both the ΔnarK2::Gm and ΔnarK1K2::Gm mutants with respect to anaerobic respiratory growth. Our results indicate that only the NarK2 protein is required as a nitrate/nitrite transporter by Pseudomonas aeruginosa under denitrifying conditions.

scholarly journals Effect of tungsten on nitrate and nitrite reductases in Azospirillum brasilense Sp7

1991 ◽  
Vol 37 (10) ◽  
pp. 744-750 ◽  
Author(s):  
Christian Chauret ◽  
Roger Knowles
Keyword(s):  
Nitrate Reductase ◽  
Nitrite Reductase ◽  
Azospirillum Brasilense ◽  
Nitrate Reductase Activity ◽  
Paracoccus Denitrificans ◽  
Reductase Activity ◽  
Whole Cells ◽  
Azospirillum Brasilense Sp7 ◽  
Nitrite Reductase Activity ◽  
Nitrate And Nitrite

Tungstate, at concentrations that completely suppressed nitrate reductase activity in Paracoccus denitrificans, caused only partial inhibition of nitrate reductase in Azospirillum brasilense Sp7. Nitrate reductase activity in cell-free extracts was much more sensitive than whole cells to tungstate, suggesting that there may be a barrier to its transport. Nitrite reductase activity was partially inhibited by tungstate in both whole cells and cell-free extracts. Azospirillum brasilense apparently scavenged enough contaminating molybdenum from molybdenum-limited medium to allow maximum nitrate reductase activity, which was not stimulated by added molybdate. Cells grown in molybdenum-depleted medium could not reduce nitrate. Nitrate concentrations less than 0.25 mM inhibited activity, but not synthesis, of nitrite reductase and caused significant accumulation of nitrite during reduction of nitrate. Key words: Azospirillum brasilense, Paracoccus denitrificans, nitrate reductase, nitrite reductase, tungsten, molybdenum, denitrification.

scholarly journals Isolation and characterization of PEP5, a gene essential for vacuolar biogenesis in Saccharomyces cerevisiae.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 739-752 ◽  
Author(s):  
C A Woolford ◽  
C K Dixon ◽  
M F Manolson ◽  
R Wright ◽  
E W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mass ◽  
Open Reading Frame ◽  
Relative Molecular Mass ◽  
Cell Fractionation ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Dna Library ◽  
Isolation And Characterization ◽  
Genomic Dna Library

Abstract pep5 mutants of Saccharomyces cerevisiae accumulate inactive precursors to the vacuolar hydrolases. The PEP5 gene was isolated from a genomic DNA library by complementation of the pep5-8 mutation. Deletion analysis localized the complementing activity to a 3.3-kb DNA fragment. DNA sequence analysis of the PEP5 gene revealed an open reading frame of 1029 codons with a calculated molecular mass for the encoded protein of 117,403 D. Deletion/disruption of the PEP5 gene did not kill the cells. The resulting strains grow very slowly at 37 degrees. The disruption mutant showed greatly decreased activities of all vacuolar hydrolases examined, including PrA, PrB, CpY, and the repressible alkaline phosphatase. Apparently normal precursors forms of the proteases accumulated in pep5 mutants, as did novel forms of PrB antigen. Antibodies raised to a fusion protein that contained almost half of the PEP5 open reading frame allowed detection by immunoblot of a protein of relative molecular mass 107 kD in extracts prepared from wild-type cells. Cell fractionation showed the PEP5 gene product is enriched in the vacuolar fraction and appears to be a peripheral vacuolar membrane protein.

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.

Effects of oxygen on each step of denitrification on Pseudomonas nautica

1989 ◽  
Vol 35 (11) ◽  
pp. 1061-1064 ◽  
Author(s):  
P. Bonin ◽  
M. Gilewicz ◽  
J. C. Bertrand
Keyword(s):  
Nitrous Oxide ◽  
Nitrate Reductase ◽  
Oxygen Concentration ◽  
Nitrite Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
A Value ◽  
Nitrite Reductase Activity ◽  
Effect Of Oxygen

Studies on the effect of oxygen on denitrification have shown that denitrification on Pseudomonas nautica 617 can take place in the presence of oxygen. The enzymes associated with denitrification are affected differently with respect to oxygen concentration. Nitrate reductase was less sensitive toward oxygen than nitrite and nitrous oxide reductases. Nitrate reductase activity was completely blocked at an oxygen concentration greater than 4.05 mg/L, compared with 2.15 and 0.25 mg/L for nitrite and nitrous oxide reductases, respectively. After an aerobic–anaerobic shift, nitrate reductase activity remained unchanged whereas the rate of nitrite reductase activity rose to a value only 20% that of the original rate.Key words: denitrification, oxygen, Pseudomonas.

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