The expression profile of the <Emphasis Type="Italic">Tuber borchii</Emphasis> nitrite reductase suggests its positive contribution to host plant nitrogen nutrition

Ectomycorrhizal symbiosis is a ubiquitous association between plant roots and numerous fungal species. One of the main aspects of the ectomycorrhizal association are the regulation mechanisms of fungal genes involved in nitrogen acquisition. We report on the genomic organisation of the nitrate gene cluster and functional regulation of tbnir1, the nitrite reductase gene of the ectomycorrhizal ascomycete Tuber borchii. The sequence data demonstrate that clustering also occurs in this ectomycorrhizal fungus. Within the TBNIR1 protein sequence, we identified three functional domains at conserved positions: the FAD box, the NADPH box and the two (Fe/S)-siroheme binding site signatures. We demonstrated that tbnir1 presents an expression pattern comparable to that of nitrate transporter. In fact, we found a strong down-regulation in the presence of primary nitrogen sources and a marked tbnir1 mRNA accumulation following transfer to either nitrate or nitrogen limited conditions. The real-time PCR assays of tbnir1 and nitrate transporter revealed that both nitrate transporter and nitrite reductase expression levels are about 15-fold and 10-fold higher in ectomycorrhizal tissues than in control mycelia, respectively. The results reported herein suggest that the symbiotic fungus Tuber borchii contributes to improving the host plant’s ability to make use of nitrate/nitrite in its nitrogen nutrition.     

Cloning of the nitrate reductase gene of Stagonospora (Septoria) nodorum and its use as a selectable marker for targeted transformation

The nitrate reductase gene (NIA1) of the phytopathogenic fungus Stagonospora (Septoria) nodorum has been cloned from a cosmid library by homologous hybridisation with a PCR-generated probe. A 6.7-kb fragment carrying the NIA1 gene was subcloned and partially characterised by restriction mapping. Sequencing of the gene indicated a high degree of homology, both at the nucleotide and amino-acid levels, with nitrate reductase genes of other filamentous fungi. Furthermore, consensus regulatory signals thought to be involved in the control of nitrogen metabolism are present in the 5′ flanking region. The cloned NIA1 gene has been used to develop a gene-transfer system based on nitrate assimilation. Stable nia1 mutants of S. nodorum defective in nitrate reductase were isolated by virtue of their resistance to chlorate. These were transformed back to nitrate utilisation with the wild-type S. nodorum NIA1 gene. Southern analyses revealed that transformation occurred as a result of the integration of transforming DNA into the fungal genome; in all cases examined, integration was targeted to the homologous sequence. Received: 30 March / 9 June 1998     

Assimilatory nitrate reductase in a chlorate-resistant mutant of Escherichia coli

Nitrate reductase was investigated in extracts from cells of a chlorate-resistant mutant strain of E. coli which grew anaerobically on nitrate as the sole source of nitrogen. The nitrate reductase was of particulate nature and reduced chlorate like the nitrate reductase from the wild strain, but in contrast was inhibited only weakly by azide or cyanide. Nitrate reductase activity was found in extracts from the mutant cells grown on nitrate as the sole source of nitrogen, but not in extracts from cells grown in complex nutrient medium. Addition of ammonia also caused a decrease in activity. Accordingly, the nitrate reductase in the chlorateresistant mutant is of the assimilatory type.     

Vanadium requirement for growth on N2 or nitrate as nitrogen source in a tungsten-resistant mutant of the cyanobacterium Nostoc muscorum

A tungstate-resistant mutant of Nostoc muscorum was isolated with severely defective molybdate transport activity. It did not show nitrogenase activity or nitrate reductase activity in the presence or absence of Mo, but expressed both activities in the presence of vanadyl sulphate. V-dependent nitrate assimilation was accompanied by V-dependent nitrate reductase mediated nitrate repression of heterocyst formation and nitrogenase in a way Mo-nitrate reductase did in the parent.     

Characterisation of the nitrite reductase gene (NII1) and the nitrate-assimilation gene cluster of Stagonospora (Septoria) nodorum

By sequencing downstream of the cloned nitrate reductase gene (NIA1) in the phytopathogenic fungus Stagonospora (Septoria) nodorum, a second open reading frame was found. Further analysis revealed this to be the nitrite reductase gene (NII1). Both genes are transcribed in the same direction, and are separated by an intergenic region of 829-bp. The coding sequence of NII1 is interrupted by three small introns and corresponds to a predicted protein of 1141 amino acids in length. Consensus binding sites for regulatory proteins are present in the promoter region of NII1. There is no indication, however, from hybridisation or sequence analysis that the nitrate transporter gene is closely associated with the NIA1-NII1 cluster, as has been found for a number of fungi. Received: 23 November 1998 / 26 April 1999     

Aerobic inhibition of nitrate assimilation in Escherichia coli

Growth of Escherichia coli, strain K-10, in synthetic medium with nitrate as a sole source of nitrogen was not found under aerobic conditions, but found in the medium under anaerobic conditions as well as in synthetic medium with ammonium or nitrite under both aerobic and anaerobic conditions. The presence of nitrate reductase activity in cells indicates that the growth inhibition is due to the inhibition of the enzyme by oxygen and not due to its absence. Conditions of preculture, inoculation size, pH, and composition of the nitratesynthetic medium did not affect the inhibition. All the 37 strains of E. coli tested were grown in the nitrate medium only anaerobically, while 18 strains of Klebsiella-Aerobacter among 22 strains tested were grown in the nitrate medium both aerobically and anaerobically.     

NIT2, the nitrogen regulatory protein of Neurospora crassa,binds upstream of nia,the tomato nitrate reductase gene,in vitro

Summary The nit-2 gene of Neurospora crassa encodes a trans-acting regulatory protein that activates the expression of a number of structural genes which code for nitrogen catabolic enzymes, including nitrate reductase. The NIT2 protein contains a Cys2/Cys2-type zinc-finger DNA-binding domain that recognizes promoter regions of the Neurospora nitrogen-related genes. The NIT2 zincfinger domain/-Gal fusion protein was shown to recognize and bind in a specific manner to two upstream fragments of the nia gene of Lycopersicon esculentum (tomato) in vitro, whereas two mutant NIT2 proteins failed to bind to the same fragments. The dissociation kinetics of the complexes formed between the NIT2 protein and the Neurospora nit-3 and the tomato nia gene promoters were examined; NIT2 binds more strongly to the nit-3 promoter DNA fragment than it does to fragments derived from the plant nitrate reductase gene itself. The observed specificity of the binding suggests the existence of a NIT2-like homolog which regulates the expression of the nitrate assimilation pathway of higher plants.     

The effects of salinity and other factors on nitrite reduction by Ochrobactrum anthropi 49187

The nitrite reductase (NIR) gene was cloned from Ochrobactrum anthropi 49187 and found to contain an open reading frame of 1131 nucleotides, encoding a polypeptide of 376 amino acids. The O. anthropi NIR gene encodes a copper-type dissimilatory reductase based on sequence homology with other genes. The polypeptide product is predicted to form a trimeric holoenzyme of 37 kDa subunits based on molecular weight estimates of extracts in activity gels. Expression of the enzyme is up-regulated by nitrate, presumably through the intermediate nitrite, and its activity is influenced by inhibitors. Salinity enhances the activity of existing NIR enzyme, but appears to decrease the expression of new enzyme.     

Assimilatory reduction of nitrate in Rhizobium meliloti

Nitrate and nitrite reductases in the crude extract of aerobically grown Rhizobium meliloti were determined with methylviologen as electron donor at pH 7. Nitrate reductase was detected in the cells grown in the medium that did not contain nitrate, and in the presence of nitrate the specific activity increased about 2-fold. Nitrite reductase was induced by nitrate and produced ammonia from nitrite. In nitrate reducing cells, two kinds of O₂ labile nitrate reductase were found. One enzyme had optimal pH at 7 and was stabilized to O₂ by treating with DEAE-Toyopearl 650M. The other had optimal pH at 9 and was stabilized by the addition of dithiothreitol and EDTA. Nitrate reductase stabilized by DEAE-Toyopearl 650M treatment was purified 3,360-fold from crude extract. The purified enzyme showed a single protein band in polyacrylamide gel electrophoresis, and there was no absorption peak in the visible region. It had a molecular weight of 64,000 in SDS PAGE and 58,000 on Sephadex G-100 gel filtration. K_m for nitrate was 0.9 mM. It was inhibited by p-chloromercuribenzoate, cyanide, and α,α'-dipyridyl.     

Presence of a functional nitrate assimilation pathway in Mycobacterium smegmatis

Ability of Mycobacterium smegmatis to assimilate nitrate was evaluated in its active and dormant phase. Nitrate (10 mM), nitrite (0.5 mM) and ammonia (10mM) allowed growth of M. smegmatis concomitant with their complete depletion from the culture in 144, 120 and 96 h, respectively, when used as sole nitrogen source. Azide (50 microM) stopped the growth of M. smegmatis when nitrate was used as sole nitrogen source. l-methionine-S-sulfoximine (l-MSO), which is a well-known inhibitor of glutamine synthetase, an enzyme also involved in nitrogen metabolic pathway, when applied at 10 microg/ml concentration, completely inhibited the growth of the organism when nitrate or nitrite was used as sole nitrogen source. There was no effect of either azide or l-MSO at above concentrations on the growth of the organism when asparagine or ammonia was used as sole nitrogen source. More significantly, utilization of nitrate, nitrite and ammonia continued even in oxygen depletion induced dormant culture at the rates of 289, 25 and 354 microM/day, respectively. These rates were 5-8 times slower than the rates of 1966, 127 and 2890 microM/day, respectively, in active replicating phase. In the presence of azide (50 microM) and l-MSO (10 microg/ml), 2.1 and 1.51 logs reduction in viability of dormant M. smegmatis was observed using nitrate and nitrite, respectively, as sole nitrogen source. Altogether, the results indicated the presence of nitrate assimilation pathway operating in both active and dormant stage of M. smegmatis.     

Transformants of Neurospora crassa with the nit-4 nitrogen regulatory gene: copy number,growth rate and enzyme activity

Summary nit-4 is a pathway-specific regulatory gene which controls nitrate assimilation in Neurospora crassa, and appears to mediate nitrate induction of nitrate and nitrite reductase. The NIT4 protein consists of 1090 amino-acid residues and possesses a single GAL4-like putative DNA-binding domain plus acidic, glutaminerich, and polyglutamine regions. Several mutants with amino-acid substitutions in the putative DNA-binding domain and a nit-4 deletion mutant, which encodes a truncated NIT4 protein lacking the polyglutamine region, are functional, i.e., they are capable of transforming a nit-4 mutant strain. However, transformants obtained with most of these nit-4 mutant genes possess a markedly reduced level of nitrate reductase and grow only slowly on nitrate, emphasizing the need to examine quantitatively the affects of in vitro-manipulated genes. The possibility that some mutant genes could yield transformants only if multiple copies were integrated was examined. The presence of multiple copies of wild-type or mutant nit-4 genes did not generally lead to increased enzyme activity or growth rate, but instead frequently appeared to be detrimental to nit-4 function. A hybrid nit-4-nirA gene transforms nit-4 mutants but only allows slow growth on nitrate and has a very low level of nitrate reductase.     

Development of biocatalysts carrying naphthalene dioxygenase and dihydrodiol dehydrogenase genes inducible in aerobic and anaerobic conditions

We developed biocatalysts carrying naphthalene dioxygenase and dihydrodiol dehydrogenase genes cloned from plasmid pN3 of Pseudomonas fluoresceins N3 involved in naphthalene degradation, as an alternative approach to the production of hydroxylated compounds by chemical synthesis. Naphthalene dioxygenase is responsible for hydroxylation of the hydrocarbon into the corresponding 1,2-dihydro-1,2-dihydroxy derivative and dihydrodiol dehydrogenase is involved in the subsequent transformation into the 1,2-dihydroxy derivative. The first reaction strictly requires the presence of oxygen, essential for the dioxygenation reaction, while the second one can also be performed in anaerobic conditions that are optimal to avoid the easy oxidation of bioconversion products. Consequently, we constructed biocatalysts carrying the genes responsible for the biotransformation of hydrocarbons, inducible under aerobic and anaerobic conditions. We cloned the dioxygenase gene under its promoter, inducible by salicylic acid and the dihydrodiol dehydrogenase under the Pnar promoter of Escherichia coli, inducible by nitrate, in a nitrogen atmosphere, in order to develop biological systems with the possibility of controlling the expression of the cloned genes by the shift from aerobic to anaerobic conditions. Bioconversion experiments performed in aerobic conditions showed dihydrodiol production and dehydrogenase repression; as soon as cultures were switched to nitrogen, dihydrodiol dehydrogenation with an efficient production of 1,2-dihydroxyderivatives was observed.     

Transformation of Botrytis cinerea with the nitrate reductase gene (niaD) shows a high frequency of homologous recombination

The nitrate reductase (niaD) gene was isolated from the phytopathogenic ascomycete Botrytis cinerea using a probe obtained by a polymerase chain reaction (PCR) with degenerate oligonucleotides corresponding to domains conserved among three fungal nitrate reductases. The B. cinerea niaD gene encodes a predicted protein of 907 amino acids and contains no intron. Nitrate reductase-deficient mutants of B. cinerea have been isolated. One of them was transformed with the niaD genes of Fusarium oxysporum f.sp. melonis and B. cinerea. The transformation was always ectopic when the donor DNA originated from F. oxysporum, but there was 80% gene replacement when the donor DNA originated from B. cinerea. Received: 19 February / 8 April 1997     

Effect of cyanate on nitrate reduction by Rhizobium meliloti SU47

Effect of cyanate on nitrate reduction by Rhizobium meliloti SU47 was studied. In the presence of cyanate assimilatory nitrate reduction activity appeared in the culture after a long lag and a conspicuously low level of nitrite was accumulated. Dissimilatory nitrate reduction activity was low in the intact cells precultured in KCNO of KNO₃ as compared to that in the control. Dissimilatory nitrate reductase activity in the extracts of cells precultured in KCNO was also compared to be low. In the extracts, dissimilatory nitrate reductase activity assayed with increasing concentrations of KCNO confirmed that KCNO acts as an inhibitor of the enzyme.