Biomedical subjects

D Jahn

Publications and source records attributed to D Jahn.

At least 19 recordsLinked to original sources

PubMed2003

[Laser-induced thermotherapy (LITT) of lung metastases: description of a miniaturized applicator, optimization, and initial treatment of patients].

PURPOSE: A thin-caliber applicator system was developed for introducing a laser fiber under CT guidance into lung metastases with only minimal complications. MATERIALS AND METHODS: A space-saving 5.5 French Teflon cannula with a titanium trocar and connectors for a laser light guide (2 or 3 cm Dornier Diffusor-Tip H-6111-T2 or H-6111-T3 coupled to a Dornier Medilas Fibertom 5100 laser, wavelength of 1064 nm) and a perfusion line for physiologic saline solution were developed. After puncture the laser Diffusor-Tip remains in the cannula and is cooled during its tissue passage by slowly flowing saline solution. The miniaturized applicator system (Monocath) was calibrated in nonperfused bovine liver for maximum energy supply and necessary flow of the cooling saline solution in reference to a commercially available 9 French laser catheter with an 11.5 French inducer sheath (Power-Applicator). The new applicator system was used for treating lung metastases in 10 patients over a period of 21 months. RESULTS: The size of heat coagulation in bovine liver was 24 +/- 2 ml using the miniaturized system with application of 15 W for 20 min and a saline flow of 0.75 ml/min, in comparison to a size of 29 +/- 7 ml for the commercial applicator (30 W, 20 min, 60 ml/min). All metastases could be safely approached with the miniaturized applicator, except for two metastatic lesions at the lung base in two patients. A minor pneumothorax developed in three patients and intrapulmonary bleeding in two. Contrast-enhanced CT demonstrated necrosis of the treated metastatic areas in 6 patients. Follow-up of three patients after 5, 6, and 8 months showed complete tumor regression with minimal scarring in one patient. CONCLUSION: The miniaturized applicator system enables the introduction of a laser fiber into pulmonary metastases with only minor complications. Complete ablation seems to be achievable in suitable patients with the applied laser energy and a slow cooling fluid flow rate.

Adult↗

PubMed2002

The Pseudomonas aeruginosa hemA promoter is regulated by Anr, Dnr, NarL and Integration Host Factor.

Pseudomonas aeruginosa forms most of its heme under anaerobic denitrifying conditions. To study the regulation of the hemA gene, which codes for the first enzyme of heme biosynthesis in P. aeruginosa, a lacZ reporter gene fusion was constructed. Expression of lacZ under the control of the hemA promoter was found to be increased by 2.8-fold under anaerobic conditions in the presence of the alternative electron acceptor nitrate, relative to the level observed under aerobic growth conditions. Anaerobic fermentative growth or the presence of nitrite did not affect the lacZ expression. The genes encoding the oxygen sensor protein Anr, the redox regulator Dnr, the nitrate regulator NarL and the DNA-bending Integration Host Factor (IHF) are all required for the cooperative anaerobic induction of the hemA promoter hemAp (1). Potential binding sites for these regulatory proteins were identified by site-directed mutagenesis of the promoter fused to the reporter gene. The mode of regulation of P. aeruginosa hemA differs significantly from that described for the hemA gene of Escherichia coli K-12.

Aldehyde Oxidoreductases↗

PubMed2002

Structure and function of glutamyl-tRNA reductase involved in 5-aminolaevulinic acid formation.

In most bacteria, in archaea and in plants, the general precursor of all tetrapyrroles, 5-aminolaevulinic acid, is formed by two enzymes. The initial substrate, glutamyl-tRNA, is reduced by NADPH-dependent glutamyl-tRNA reductase to form glutamate 1-semialdehyde. The aldehyde is subsequently transaminated by glutamate-1-semialdehyde 2,1-aminomutase to yield 5-aminolaevulinic acid. The enzymic mechanism and the solved crystal structure of Methanopyrrus kandleri glutamyl-tRNA reductase are described. A pathway for metabolic channelling of the reactive aldehyde between glutamyl-tRNA reductase and the aminomutase is proposed.

Aldehyde Oxidoreductases↗

PubMed2001

Microbial production of vitamin B12.

One of the most alluring and fascinating molecules in the world of science and medicine is vitamin B12 (cobalamin), which was originally discovered as the anti pernicious anemia factor and whose enigmatic complex structure is matched only by the beguiling chemistry that it mediates. The biosynthesis of this essential nutrient is intricate, involved and, remarkably, confined to certain members of the prokaryotic world, seemingly never have to have made the eukaryotic transition. In humans, the vitamin is required in trace amounts (approximately 1 microg/day) to assist the actions of only two enzymes, methionine synthase and (R)-methylmalonyl-CoA mutase; yet commercially more than 10 t of B12 are produced each year from a number of bacterial species. The rich scientific history of vitamin B12 research, its biological functions and the pathways employed by bacteria for its de novo synthesis are described. Current strategies for the improvement of vitamin B12 production using modern biotechnological techniques are outlined.

Archaea↗

PubMed2001

V-shaped structure of glutamyl-tRNA reductase, the first enzyme of tRNA-dependent tetrapyrrole biosynthesis.

Processes vital to life such as respiration and photosynthesis critically depend on the availability of tetrapyrroles including hemes and chlorophylls. tRNA-dependent catalysis generally is associated with protein biosynthesis. An exception is the reduction of glutamyl-tRNA to glutamate-1-semialdehyde by the enzyme glutamyl-tRNA reductase. This reaction is the indispensable initiating step of tetrapyrrole biosynthesis in plants and most prokaryotes. The crystal structure of glutamyl-tRNA reductase from the archaeon Methanopyrus kandleri in complex with the substrate-like inhibitor glutamycin at 1.9 A resolution reveals an extended yet planar V-shaped dimer. The well defined interactions of the inhibitor with the active site support a thioester-mediated reduction process. Modeling the glutamyl-tRNA onto each monomer reveals an extensive protein-tRNA interface. We furthermore propose a model whereby the large void of glutamyl-tRNA reductase is occupied by glutamate-1-semialdehyde-1,2-mutase, the subsequent enzyme of this pathway, allowing for the efficient synthesis of 5-aminolevulinic acid, the common precursor of all tetrapyrroles.

Aldehyde Oxidoreductases↗

PubMed2001

Modulation of anaerobic energy metabolism of Bacillus subtilis by arfM (ywiD).

Bacillus subtilis grows under anaerobic conditions utilizing nitrate ammonification and various fermentative processes. The two-component regulatory system ResDE and the redox regulator Fnr are the currently known parts of the regulatory system for anaerobic adaptation. Mutation of the open reading frame ywiD located upstream of the respiratory nitrate reductase operon narGHJI resulted in elimination of the contribution of nitrite dissimilation to anaerobic nitrate respiratory growth. Significantly reduced nitrite reductase (NasDE) activity was detected, while respiratory nitrate reductase activity was unchanged. Anaerobic induction of nasDE expression was found to be significantly dependent on intact ywiD, while anaerobic narGHJI expression was ywiD independent. Anaerobic transcription of hmp, encoding a flavohemoglobin-like protein, and of the fermentative operons lctEP and alsSD, responsible for lactate and acetoin formation, was partially dependent on ywiD. Expression of pta, encoding phosphotransacetylase involved in fermentative acetate formation, was not influenced by ywiD. Transcription of the ywiD gene was anaerobically induced by the redox regulator Fnr via the conserved Fnr-box (TGTGA-6N-TCACT) centered 40.5 bp upstream of the transcriptional start site. Anaerobic induction of ywiD by resDE was found to be indirect via resDE-dependent activation of fnr. The ywiD gene is subject to autorepression and nitrite repression. These results suggest a ResDE --> Fnr --> YwiD regulatory cascade for the modulation of genes involved in the anaerobic metabolism of B. subtilis. Therefore, ywiD was renamed arfM for anaerobic respiration and fermentation modulator.

Anaerobiosis↗

PubMed2000

Fermentative metabolism of Bacillus subtilis: physiology and regulation of gene expression.

Bacillus subtilis grows in the absence of oxygen using nitrate ammonification and various fermentation processes. Lactate, acetate, and 2,3-butanediol were identified in the growth medium as the major anaerobic fermentation products by using high-performance liquid chromatography. Lactate formation was found to be dependent on the lctEP locus, encoding lactate dehydrogenase and a putative lactate permease. Mutation of lctE results in drastically reduced anaerobic growth independent of the presence of alternative electron acceptors, indicating the importance of NADH reoxidation by lactate dehydrogenase for the overall anaerobic energy metabolism. Anaerobic formation of 2,3-butanediol via acetoin involves acetolactate synthase and decarboxylase encoded by the alsSD operon. Mutation of alsSD has no significant effect on anaerobic growth. Anaerobic acetate synthesis from acetyl coenzyme A requires phosphotransacetylase encoded by pta. Similar to the case for lctEP, mutation of pta significantly reduces anaerobic fermentative and respiratory growth. The expression of both lctEP and alsSD is strongly induced under anaerobic conditions. Anaerobic lctEP and alsSD induction was found to be partially dependent on the gene encoding the redox regulator Fnr. The observed fnr dependence might be the result of Fnr-induced arfM (ywiD) transcription and subsequent lctEP and alsSD activation by the regulator ArfM (YwiD). The two-component regulatory system encoded by resDE is also involved in anaerobic lctEP induction. No direct resDE influence on the redox regulation of alsSD was observed. The alternative electron acceptor nitrate represses anaerobic lctEP and alsSD transcription. Nitrate repression requires resDE- and fnr-dependent expression of narGHJI, encoding respiratory nitrate reductase. The gene alsR, encoding a regulator potentially responding to changes of the intracellular pH and to acetate, is essential for anaerobic lctEP and alsSD expression. In agreement with its known aerobic function, no obvious oxygen- or nitrate-dependent pta regulation was observed. A model for the regulation of the anaerobic fermentation genes in B. subtilis is proposed.

Acetates↗

PubMed1999

Methanopyrus kandleri glutamyl-tRNA reductase.

The initial reaction of tetrapyrrole formation in archaea is catalyzed by a NADPH-dependent glutamyl-tRNA reductase (GluTR). The hemA gene encoding GluTR was cloned from the extremely thermophilic archaeon Methanopyrus kandleri and overexpressed in Escherichia coli. Purified recombinant GluTR is a tetrameric enzyme with a native M(r) = 190,000 +/- 10,000. Using a newly established enzyme assay, a specific activity of 0.75 nmol h(-1) mg(-1) at 56 degrees C with E. coli glutamyl-tRNA as substrate was measured. A temperature optimum of 90 degrees C and a pH optimum of 8.1 were determined. Neither heme cofactor, nor flavin, nor metal ions were required for GluTR catalysis. Heavy metal compounds, Zn(2+), and heme inhibited the enzyme. GluTR inhibition by the newly synthesized inhibitor glutamycin, whose structure is similar to the 3' end of the glutamyl-tRNA substrate, revealed the importance of an intact chemical bond between glutamate and tRNA(Glu) for substrate recognition. The absolute requirement for NADPH in the reaction of GluTR was demonstrated using four NADPH analogues. Chemical modification and site-directed mutagenesis studies indicated that a single cysteinyl residue and a single histidinyl residue were important for catalysis. It was concluded that during GluTR catalysis the highly reactive sulfhydryl group of Cys-48 acts as a nucleophile attacking the alpha-carbonyl group of tRNA-bound glutamate with the formation of an enzyme-localized thioester intermediate and the concomitant release of tRNA(Glu). In the presence of NADPH, direct hydride transfer to enzyme-bound glutamate, possibly facilitated by His-84, leads to glutamate-1-semialdehyde formation. In the absence of NADPH, a newly discovered esterase activity of GluTR hydrolyzes the highly reactive thioester of tRNA(Glu) to release glutamate.

Amino Acid Substitution↗

PubMed1999

Production, purification, and characterization of a Mg2+-responsive porphobilinogen synthase from Pseudomonas aeruginosa.

During tetrapyrrole biosynthesis the metalloenzyme porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of 5-aminolevulinic acid to form the pyrrole porphobilinogen. Pseudomonas aeruginosa PBGS was synthesized in Escherichia coli, and the enzyme was purified as a fusion protein with glutathione S-transferase (GST). After removal of GST, a molecular mass of 280 000 +/- 10 000 with a Stokes radius of 57 A was determined for native PBGS, indicating a homooctameric structure of the enzyme. Mg2+ stabilized the oligomeric state but was not essential for octamer formation. Alteration of N-terminal amino acids changed the oligomeric state and reduced the activity of the enzyme, revealing the importance of this region for oligomerization and activity. EDTA treatment severely inhibited enzymatic activity which could be completely restored by the addition of Mg2+ or Mn2+. At concentrations in the micromolar range Co2+, Zn2+, and Ni2+ partially restored EDTA-inhibited enzymatic activity while higher concentrations of Zn2+ inhibited the enzyme. Pb2+, Cd2+, and Hg2+ did not restore activity. A stimulatory effect of monovalent ions was observed. A Km of 0.33 mM for ALA and a maximal specific activity of 60 micromol h-1 mg-1 at the pH optimum of 8.6 in the presence of Mg2+ and K+ were found. pH-dependent kinetic studies were combined with protein modifications to determine the structural basis of two observed pKa values of approximately 7.9 (pKa1) and 9.5 (pKa2). These are postulated respectively as ionization of an active site lysine residue and of free substrate during catalysis. Some PBGS inhibitors were characterized. Finally, we succeeded in obtaining well-ordered crystals of P. aeruginosa PBGS complexed with the substrate analogue levulinic acid.

Amino Acid Sequence↗

PubMed1999

Pseudomonas aeruginosa contains a novel type V porphobilinogen synthase with no required catalytic metal ions.

Porphobilinogen synthases (PBGS) are metalloenzymes that catalyze the first common step in tetrapyrrole biosynthesis. The PBGS enzymes have previously been categorized into four types (I-IV) by the number of Zn(2+) and/or Mg(2+) utilized at three different metal binding sites termed A, B, and C. In this study Pseudomonas aeruginosa PBGS is found to bind only four Mg(2+) per octamer as determined by atomic absorption spectroscopy, in the presence or absence of substrate/product. This is the lowest number of bound metal ions yet found for PBGS where other enzymes bind 8-16 divalent ions. These four Mg(2+) allosterically stimulate a metal ion independent catalytic activity, in a fashion dependent upon both pH and K(+). The allosteric Mg(2+) of PBGS is located in metal binding site C, which is outside the active site. No evidence is found for metal binding to the potential high-affinity active site metal binding sites A and/or B. P. aeruginosa PBGS was investigated using Mn(2+) as an EPR probe for Mg(2+), and the active site was investigated using [3,5-(13)C]porphobilinogen as an NMR probe. The magnetic resonance data exclude the direct involvement of Mg(2+) in substrate binding and product formation. The combined data suggest that P. aeruginosa PBGS represents a new type V enzyme. Type V PBGS has the remarkable ability to synthesize porphobilinogen in a metal ion independent fashion. The total metal ion stoichiometry of only 4 per octamer suggests half-sites reactivity.

Allosteric Regulation↗

PubMed1999

High resolution crystal structure of a Mg2+-dependent porphobilinogen synthase.

Common to the biosynthesis of all known tetrapyrroles is the condensation of two molecules of 5-aminolevulinic acid to the pyrrole porphobilinogen catalyzed by the enzyme porphobilinogen synthase (PBGS). Two major classes of PBGS are known. Zn2+-dependent PBGSs are found in mammals, yeast and some bacteria including Escherichia coli, while Mg2+-dependent PBGSs are present mainly in plants and other bacteria. The crystal structure of the Mg2+-dependent PBGS from the human pathogen Pseudomonas aeruginosa in complex with the competitive inhibitor levulinic acid (LA) solved at 1.67 A resolution shows a homooctameric enzyme that consists of four asymmetric dimers. The monomers in each dimer differ from each other by having a "closed" and an "open" active site pocket. In the closed subunit, the active site is completely shielded from solvent by a well-defined lid that is partially disordered in the open subunit. A single molecule of LA binds to a mainly hydrophobic pocket in each monomer where it is covalently attached via a Schiff base to an active site lysine residue. Whereas no metal ions are found in the active site of both monomers, a single well-defined and highly hydrated Mg2+is present only in the closed form about 14 A away from the Schiff base forming nitrogen atom of the active site lysine. We conclude that the observed differences in the active sites of both monomers might be induced by Mg2+-binding to this remote site and propose a structure-based mechanism for this allosteric Mg2+in rate enhancement.

Allosteric Site↗

PubMed1999

Transcriptional control of Bacillus subtilis hemN and hemZ.

Previous characterization of Bacillus subtilis hemN, encoding a protein involved in oxygen-independent coproporphyrinogen III decarboxylation, indicated the presence of a second hemN-like gene (B. Hippler, G. Homuth, T. Hoffmann, C. Hungerer, W. Schumann, and D. Jahn, J. Bacteriol. 179:7181-7185, 1997). The corresponding hemZ gene was found to be split into the two potential open reading frames yhaV and yhaW by a sequencing error of the genome sequencing project. The hemZ gene, encoding a 501-amino-acid protein with a calculated molecular mass of 57,533 Da, complemented a Salmonella typhimurium hemF hemN double mutant under aerobic and anaerobic growth conditions. A B. subtilis hemZ mutant accumulated coproporphyrinogen III under anaerobic growth conditions. A hemN hemZ double mutant exhibited normal aerobic and anaerobic growth, indicating the presence of a third alternative oxygen-independent enzymatic system for coproporphyrinogen III oxidation. The hemY gene, encoding oxygen-dependent protoporphyrinogen IX oxidase with coproporphyrinogen III oxidase side activity, did not significantly contribute to this newly identified system. Growth behavior of hemY mutants revealed the presence of an oxygen-independent protoporphyrinogen IX oxidase in B. subtilis. A monocistronic hemZ mRNA, starting 31 bp upstream of the translational start codon, was detected. Reporter gene fusions of hemZ and hemN demonstrated a fivefold anaerobic induction of both genes under nitrate ammonifying growth conditions. No anaerobic induction was observed for fermentatively growing B. subtilis. The B. subtilis redox regulatory systems encoded by resDE, fnr, and ywiD were indispensable for the observed transcriptional induction. A redox regulation cascade proceeding from an unknown sensor via resDE, through fnr and ywiD to hemN/hemZ, is suggested for the observed coregulation of heme biosynthesis and the anaerobic respiratory energy metabolism. Finally, only hemZ was found to be fivefold induced by the presence of H(2)O(2), indicating further coregulation of heme biosynthesis with the formation of the tetrapyrrole enzyme catalase.

Amino Acid Sequence↗

PubMed1998

The Alcaligenes eutrophus hemN gene encoding the oxygen-independent coproporphyrinogen III oxidase, is required for heme biosynthesis during anaerobic growth.

The insertion mutant HF231 of Alcaligenes eutrophus H16 failed to grow anaerobically on nitrate and nitrite. When grown under oxygen limitation, mutant HF231 specifically excreted coproporphyrin III, an intermediate of heme biosynthesis. With the help of a Tn5-labeled fragment, we identified and cloned the corresponding wild-type fragment. Sequence analysis of the mutant locus revealed an open reading frame consisting of 1,473 bp, predicting a protein of 491 amino acids that corresponds to a size of 54.2 kDa. In the non-coding upstream region, consensus elements that are indicative for binding sites of the anaerobic transcriptional regulator Fnr were identified. The deduced polypeptide showed extensive sequence similarity with various bacterial oxygen-independent coproporphyrinogen III oxidases designated HemN. HemN catalyzes the oxidative decarboxylation of coproporphyrinogen III to yield protoporphyrinogen IX. Anaerobic growth on nitrate and nitrite of mutant HF231 was restored by introducing the hemN gene of A. eutrophus or of Pseudomonas aeruginosa on a broad-host-range vector. Likewise, the A. eutrophus hemN complemented heme biosynthesis of a Salmonella typhimurium hemF/hemN double mutant during anaerobic and aerobic growth. Analysis of a transcriptional lacZ gene fusion showed that expression of hemN in A. eutrophus is nitrate-independent and repressed by oxygen.

Alcaligenes↗

PubMed1998

Changes in protein synthesis as a consequence of heme depletion in Escherichia coli.

Two-dimensional gel electrophoresis and N-terminal amino acid sequence determination were used to compare the protein synthesis of exponentially growing Escherichia coli with heme-deficient cells. Mutation of the E. coli hemA gene encoding glutamyl-tRNA reductase resulted in the absence of detectable amounts of heme. As a consequence of heme deficiency, the induction of tryptophanase (trpA), citrate synthase (gltA), and aldehyde dehydrogenase (aldA) and the repression of enolase (eno) and phosphoglycerate kinase (pgk) were observed. All induced genes are under the control of the catabolite repressor protein Crp. The observed changes in gene expression as a consequence of heme depletion are discussed.

Aldehyde Oxidoreductases↗

PubMed1998

Cloning, mapping and functional characterization of the hemB gene of Pseudomonas aeruginosa, which encodes a magnesium-dependent 5-aminolevulinic acid dehydratase.

During tetrapyrrole biosynthesis 5-aminolevulinic acid dehydratase (ALAD) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA) to form one molecule of the pyrrole derivative porphobilinogen. In Escherichia coli, the enzyme is encoded by the gene hemB. The hemB gene was cloned from Pseudomonas aeruginosa by functional complementation of an E. coli hemB mutant. An open reading frame of 1011 bp encoding a protein of 336 amino acids (M(r) 37,008) was identified. The gene was mapped to SpeI fragment G and DpnI fragment G of the P. aeruginosa chromosome, corresponding to the 10 to 12 min region of the new map or 19 to 22 min interval of the old map. The 5' end of the hemB mRNA was determined and the -10 and -35 regions of a potential sigma 70-dependent promoter were localized. No obvious regulation of the hemB gene by oxygen, nitrate, heme or iron was detected. Alignment of the amino acid sequences deduced from hemB revealed a potential metal-binding site and indicated that the enzyme is Mg(2+)-dependent. P. aeruginosa hemB was overexpressed in an E. coli hemB mutant using the phage T7 RNA polymerase system and its Mg(2+)-dependent activity was directly demonstrated.

Amino Acid Sequence↗

PubMed1998

Regulation of Pseudomonas aeruginosa hemF and hemN by the dual action of the redox response regulators Anr and Dnr.

The oxidative decarboxylation of coproporphyrinogen III catalysed by an oxygen-dependent oxidase (HemF) and an oxygen-independent dehydrogenase (HemN) is one of the key regulatory points of haem biosynthesis in Pseudomonas aeruginosa. To investigate the oxygen-dependent regulation of hemF and hemN, the corresponding genes were cloned from the P. aeruginosa chromosome. Recognition sequences for the Fnr-type transcriptional regulator Anr were detected -44.5 bp from the 5' end of the hemF mRNA transcript and at an optimal distance of -41.5 bp with respect to the transcriptional start of hemN. An approximately 10-fold anaerobic induction of hemN gene expression was mediated by the dual action of Anr and a second Fnr-type regulator, Dnr. Regulation by both proteins required the Anr recognition sequence. Surprisingly, aerobic expression of hemN was dependent only on Anr. An anr mutant did not contain detectable amounts of hemN mRNA and accumulated coproporphyrin III both aerobically and anaerobically, indicating the importance of HemN for aerobic and anaerobic haem formation. Mutation of hemN and hemF did not abolish aerobic or anaerobic growth, indicating the existence of an additional HemN-type enzyme, which was termed HemZ. Expression of hemF was induced approximately 20-fold during anaerobic growth and, as was found for hemN, both Anr and Dnr were required for anaerobic induction. Paradoxically, oxygen is necessary for HemF catalysis, suggesting the existence of an additional physiological function for the P. aeruginosa HemF protein.

Aerobiosis↗

PubMed1998

Ammonification in Bacillus subtilis utilizing dissimilatory nitrite reductase is dependent on resDE.

During anaerobic nitrate respiration Bacillus subtilis reduces nitrate via nitrite to ammonia. No denitrification products were observed. B. subtilis wild-type cells and a nitrate reductase mutant grew anaerobically with nitrite as an electron acceptor. Oxygen-sensitive dissimilatory nitrite reductase activity was demonstrated in cell extracts prepared from both strains with benzyl viologen as an electron donor and nitrite as an electron acceptor. The anaerobic expression of the discovered nitrite reductase activity was dependent on the regulatory system encoded by resDE. Mutation of the gene encoding the regulatory Fnr had no negative effect on dissimilatory nitrite reductase formation.

Ammonia↗

PubMed1998

Nitrogen and oxygen regulation of Bacillus subtilis nasDEF encoding NADH-dependent nitrite reductase by TnrA and ResDE.

The nitrate and nitrite reductases of Bacillus subtilis have 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 by narGHI 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 and nasD 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 nasDEF required 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.

Aerobiosis↗