Nitrate Nonutilizing Mutants and Vegetative Compatibility Groups in Fusarium poae

Nitrate nonutilizing (nit) mutants were recovered from 24 isolates of Fusarium poae and used to force heterokaryons between these isolates and to determine vegetative compatibility. Between 30 and 90% of the mycelial blocks, cultured on medium containing chlorate, produced nit mutants. The amount of chlorate in the medium altered the frequency and spectrum of nit mutants recovered. Most of the mutants (63%) had lesions at a nitrate reductase structural locus (nit1). Another 30% were mutants at one or more loci that control the production of a molybdenum-containing cofactor necessary for nitrate reductase activity (NitM). A few (6%) of the mutations occurred in a regulatory gene specific for the nitrate reduction pathway (nit3). Pairings between nit1 and NitM mutants were made on minimal medium containing nitrate as the sole nitrogen source. A mutant grows thinly unless it forms a complementary heterokaryon upon contact with another mutant. Heterokaryon formation was indicated by dense growth where the two mutant colonies touched. The 24 isolates could be divided into 13 nonoverlapping vegetative compatibility groups, suggesting that asexual exchange of genetic information within F. poae is subject to significant limitations.     

The isolation and survival characterization of radiation and chemical-mutagen sensitive mutants of Pseudomonas aeruginosa

Chlorate-resistant mutants of Arabidopsis thaliana were isolated in order to find nitrate reductase-less mutants. It appeared that chlorate resistance in higher plants can arise by mutations concerning two different mechanisms: (1) a lower reduction rate of chlorate due to a lower level of nitrate reductase activity; (2) a lower increase in content of chlorate and/or chlorite and of chloride after chlorate treatment. One mutant of the first type and two mutants of the second type are described. The nitrate reductase-less mutant grows poorly on a medium with nitrate as the only nitrogen source but is not blocked in the uptake of nitrate. Both the other mutants exhibit a nitrate reductase activity equal to or higher than that of the wild type, but probably have a much lowered uptake of chlorate. The latter two mutants belong to the same complementation group, whereas the nitrate reductase-less mutant belongs to a different group.     

Isolation and characterization of nitrate reductase deficient mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum

To clarify the role of the fungal nitrate assimilation pathway in nitrate reduction by mycorrhizal plants, nitrate reductase (NR)-deficient (NR⁻) mutants of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum Romagnesi have been selected. These mutants were produced by u.v. mutagenesis on protoplasts originating from homokaryotic mycelia belonging to complementary mating types of this heterothallic tetrapolar species. Chlorate-resistant mutants were first selected in the presence of different nitrogen (N) sources in the culture medium. Among 1495 chlorate resistant mycelia, 30 failed to grow on nitrate and lacked a detectable NR activity. Growth tests on different N sources suggested that the NR activity of all the different mutants is specifically impaired as a result of mutations in either the gene coding for NR apoprotein or genes controlling the synthesis of the molybdenum cofactor. Furthermore, restoration of NR activity in some of the dikaryons obtained after crosses between the different mutant mycelia suggested that not all the selected mutations mapped in the same gene. Utilization of N on a NH₄¹⁵NO₃ medium was studied for two mutant strains and their corresponding wild-type homokaryons. None of the mutants could use nitrate whereas ¹⁵N enrichment values indicated that 13–27% of N present in 13-d-old wild-type mycelia originated from nitrate. Apparently, the mutant mycelia do not compensate their inability to use nitrate by a more efficient use of ammonium. These different NR mutants still form mycorrhizas with the habitual host plant, Pinus pinaster (Ait.), making them suitable for study of the contribution of the fungal nitrate assimilation pathway to nitrate assimilation by mycorrhizal plants.     

新月弯孢菌Curvularia lunata营养体亲和性的进一步研究

来自全国39个地区的70株新月弯孢菌Curvularialunata在WAC培养基上诱导培养后,随机挑取10197个抗KClO3突变体,经CDA鉴定获得2207株nit突变体,nit突变体频率为21.64%。在这些nit突变体中,1397个为nit1,占63.30%;734个为NitM,占33.26%;76个为nit3,占3.44%。70个菌株全部获得了稳定的nit突变体,其中52个菌株获得了NitM突变体。结果表明WAC比先前报道的KPS更适合用于C.lunatanit突变体的筛选。通过不同菌株间互补nit突变体配对测试,将其中的65个C.lunata菌株划分为22个营养体亲和群(VCGS),而另外5个菌株因未获得NitM突变体暂时无法确定其VCG。划分出的22个VCGs中,有11个VCGs是由多菌株组成的,VCG3为优势类群,含18个菌株,其地理来源最复杂,主要为致病性中等以上的菌株;其余11个VCGs内均仅有1个自身亲和的菌株。以上结果初步表明,在C.lunata群体内存在丰富的VCG多样性,VCG3可能是与致病性相关的优势VCG,但营养体亲和性与菌株地理来源没有明显的直接关系。     

Genetic and molecular characterization of pathogenic isolates of Pyricularia grisea from wheat (Triticum aestivum Lam.) and triticale (x Triticosecale Wittmack) in the state of Paraná, Brazil

Isolates of Pyricularia grisea from wheat (Triticum aestivum Lam.) and triticale (x Triticosecale Wittmack) spikes with blast symptoms were analyzed by classical (VCG) and molecular (RAPD) techniques. P. grisea mutants, unable to use sodium nitrate (nit) as nitrogen source, were obtained with potassium chlorate. For vegetative compatibility (VCG) tests, genetically complementary nit mutant pairs were inoculated in a medium with sodium nitrate as a single nitrogen source. P. grisea isolates were divided into two vegetative compatibility groups and two RAPD groups. Since vegetative compatible strains may mutually exchange genetic and cytoplasmatic material, the contribution of the parasexual cycle in the genetic variability of Brazilian P. grisea isolates is discussed.     

Isolation and characterization of two new negative regulatory mutants for nitrate assimilation inChlamydomonas reinhardtii obtained by insertional mutagenesis

Plasmid DNA carrying either the nitrate reductase (NR) gene or the argininosuccinate lyase gene as selectable markers and the correspondingChlamydomonas reinhardtii mutants as recipient strains have been used to isolate regulatory mutants for nitrate assimilation by insertional mutagenesis. Identification of putative regulatory mutants was based on their chlorate sensitivity in the presence of ammonium. Among 8975 transformants, two mutants, N1 and T1, were obtained. Genetic characterization of these mutants indicated that they carry recessive mutations at two different loci, namedNrg1 andNrg2. The mutation in N1 was shown to be linked to the plasmid insertion. Two copies of the nitrate reductase plasmid, one of them truncated, were inserted in the N1 genome in inverse orientation. In addition to the chlorate sensitivity phenotype in the presence of ammonium, these mutants expressed NR, nitrite reductase and nitrate transport activities in ammonium-nitrate media. Kinetic constants for ammonium (¹⁴C-methylammonium) transport, as well as enzymatic activities related to the ammonium-regulated metabolic pathway for xanthine utilization, were not affected in these strains. The data strongly suggest thatNrg1 andNrg2 are regulatory genes which specifically mediate the negative control exerted by ammonium on the nitrate assimilation pathway inC. reinhardtii.     

Isolation and characterization of two new negative regulatory mutants for nitrate assimilation inChlamydomonas reinhardtii obtained by insertional mutagenesis

Plasmid DNA carrying either the nitrate reductase (NR) gene or the argininosuccinate lyase gene as selectable markers and the correspondingChlamydomonas reinhardtii mutants as recipient strains have been used to isolate regulatory mutants for nitrate assimilation by insertional mutagenesis. Identification of putative regulatory mutants was based on their chlorate sensitivity in the presence of ammonium. Among 8975 transformants, two mutants, N1 and T1, were obtained. Genetic characterization of these mutants indicated that they carry recessive mutations at two different loci, namedNrg1 andNrg2. The mutation in N1 was shown to be linked to the plasmid insertion. Two copies of the nitrate reductase plasmid, one of them truncated, were inserted in the N1 genome in inverse orientation. In addition to the chlorate sensitivity phenotype in the presence of ammonium, these mutants expressed NR, nitrite reductase and nitrate transport activities in ammonium-nitrate media. Kinetic constants for ammonium (¹⁴C-methylammonium) transport, as well as enzymatic activities related to the ammonium-regulated metabolic pathway for xanthine utilization, were not affected in these strains. The data strongly suggest thatNrg1 andNrg2 are regulatory genes which specifically mediate the negative control exerted by ammonium on the nitrate assimilation pathway inC. reinhardtii.     

Isolation and characterization of transposon Tn5 mutants of Rhodobacter sphaeroides deficient in both nitrate and chlorate reduction.

Abstract The wild-type strain Rhodobacter sphaeroides DSM 158 is a nitrate-reducing bacterium with a periplasmic nitrate reductase. Addition of chlorate to the culture medium causes a stimulation of the phototrophic growth, indicating that this strain is able to use chlorate as an ancillary oxidant. Several mutant strains of R. sphaeroides deficient in nitrate reductase activity were obtained by transposon Tn5 mutagenesis. Mutant strain NR45 exhibited high constitutive nitrate and chlorate reductase activities and phototrophic growth was also increased by the presence of chlorate. In contrast, the stimulation of growth by chlorate was not observed in mutant strains NR8 and NR13, in which transposon Tn5 insertion causes the simultaneous loss of both nitrate and chlorate reductase activities. Tn5 insertion probably does not affect molybdenum metabolism since NR8 and NR13 mutants exhibit both xanthine dehydrogenase and nitrogenase activities. These results that a single enzyme could reduce both nitrate and chlorate in R. sphaeroides DSM 158.     

Nitrate Utilization by Nitrate Reductase-deficient Barley Mutants

Two nitrate reductase-deficient barley mutants were studied for growth on nitrate and ammonium sources of nitrogen and for resistance to chlorate. Although nitrate reductase-deficient mutants in some species are chlorate-resistant (unable to reduce chlorate to chlorite), the barley mutants used in these studies when grown on nitrate and treated with chlorate were only slightly more resistant to chlorate than the control. When grown to maturity on vermiculite supplemented with either nitrate or ammonium nutrient solutions, the mutants produced as much dry weight and reduced nitrogen per plant as the control. The in vivo and in vitro nitrate reductase activities in the roots and shoots of the mutants grown on nitrate were consistently less than 10% of the control. To avoid the possibility that the mutants received reduced nitrogen from microbial sources, excised embryos were cultured under sterile conditions. Again the mutants were capable of growth and reduced nitrogen accumulation with nitrate as the sole source of nitrogen. In spite of the low apparent nitrate reductase activity, the nitrate reductase-deficient mutants are capable of substantial nitrate reduction.     

Induction and Characterization of Chlorate-resistant Strains of Rosa damascena Cultured Cells

The sensitivity of Rosa damascena cultured cells to chlorate was measured by plating samples of suspensions in agar containing NaClO₃. This sensitivity depended on the age of the cultures that were plated. Chlorate-resistant colonies isolated from 5- to 7-day cultures retained their resistance through many generations of growth in medium lacking NaClO₃; they also retained resistance when mixed with sensitive cells. Treating cell aggregates with ultraviolet (UV) light (254 nanometers), or UV light (360 nanometers) in the presence of 4′-methoxymethyltrioxsalen, increased the proportion that was resistant to NaClO₃. However, the amount of increase was low (three times) and required very specific doses of UV light. The UV treatments did not select for chlorate-resistant cells over chlorate-sensitive cells. The data suggested that UV had induced mutations leading to chlorate resistance. Approximately 15% of the resistant strains did not grow on medium containing nitrate as the sole nitrogen source. These strains lacked ability to reduce chlorate to chlorite. This observation supports the current idea that chlorate toxicity depends on the activity of nitrate reductase. Approximately 85% of the resistant strains grew on medium containing nitrate as the sole nitrogen source. These strains lost catalase activity following chlorate treatment, indicating that they took up and reduced chlorate. These strains have a mechanism for tolerating chlorate and its reduction products, rather than avoiding them.     

Toxicity of and mutagenesis by chlorate are independent of nitrate reductase activity in Chlamydomonas reinhardtii

Summary Spontaneous chlorate-resistant (CR) mutants have been isolated from Chlamydomonas reinhardtii wildtype strains. Most of them, 244, were able to grow on nitrate minimal medium, but 23 were not. Genetic and in vivo complementation analyses of this latter group of mutants indicated that they were defective either at the regulatory locus nit-2, or at the nitrate reductase (NR) locus nit-1, or at very closely linked loci. Some of these nit-1 or nit-2 mutants were also defective in pathways not directly related to nitrate assimilation, such as those of amino acids and purines. Chlorate treatment of wild-type cells resulted in both a decrease in cell survival and an increase in mutant cells resistant to a number of different chemicals (chlorate, methylammonium, sulphanilamide, arsenate, and streptomycin). The toxic and mutagenic effects of chlorate in minimal medium were not found when cells were grown either in darkness or in the presence of ammonium, conditions under which nitrate uptake is drastically inhibited. Chlorate was also able to induce reversion of nit ^– mutants of C. reinhardtii, but failed to produce His ⁺ revertants or Ara^r mutants in the BA-13 strain of Salmonella typhimurium. In contrast, chlorate treatment induced mutagenesis in strain E1F1 of the phototrophic bacterium Rhodobacter capsulatus. Genetic analyses of nitrate reductase-deficient CR mutants of C. reinhardtii revealed two types of CR, to low (1.5 mM) and high (15 mM) chlorate concentrations. These two traits were recessive in heterozygous diploids and segregated in genetic crosses independently of each other and of the nit-1 and nit-2 loci. Three her loci and four lcr loci mediating resistance to high (HC) and low (LC) concentrations of chlorate were identified. Mutations at the nit-2 locus, and deletions of a putative locus for nitrate transport were always epistatic to mutations responsible for resistance to either LC or HC. In both nit ⁺ and nit ^– chlorate-sensitive (CS) strains, nitrate and nitrite gave protection from the toxic effect of chlorate. Our data indicate that in C. reinhardtii chlorate toxicity is primarily dependent on the nitrate transport system and independent of the existence of an active NR enzyme. At least seven loci unrelated to the nitrate assimilation pathway and mediating CR are thought to control indirectly the efficiency of the nitrate transporter for chlorate transport. In addition, chlorate appears to be a mutagen capable of inducing a wide range of mutations unrelated to the nitrate assimilation pathway.     

Chlorate toxicity in Aspergillus nidulans. Studies of mutants altered in nitrate assimilation.

Summary It had previously been held that chlorate is not itself toxic, but is rendered toxic as a result of nitrate reductase-catalysed conversion to chlorite. This however cannot be the explanation of chlorate toxicity in Aspergillus nidulans, even though nitrate reductase is known to have chlorate reductase activity. Among other evidence against the classical theory for the mechanism of chlorate toxicity, is the finding that not all mutants lacking nitrate reductase are clorate resistant. Both chlorate-sensitive and resistant mutants lacking nitrate reductase, also lack chlorate reductase. Data is presented which implicates not only nitrate reductase but also the product of the nirA gene, a positive regulator gene for nitrate assimilation, in the mediation of chlorate toxicity. Alternative mechanisms for chlorate toxicity are considered. It is unlikely that chlorate toxicity results from the involvement of nitrate reductase and the nirA gene product in the regulation either of nitrite reductase, or of the pentose phosphate pathway. Although low pH has an effect similar to chlorate, chlorate is not likely to be toxic because it lowers the pH; low pH and chlorate may instead have similar effects. A possible explanation for chlorate toxicity is that it mimics nitrate in mediating, via nitrate reductase and the nirA gene product, a shut-down of nitrogen catabolism. As chlorate cannot act as a nitrogen source, nitrogen starvation ensures.     

Identification of the Arabidopsis CHL3 gene as the nitrate reductase structural gene NIA2.

Chlorate, the chlorine analog of nitrate, is a herbicide that has been used to select mutants impaired in the process of nitrate assimilation. In Arabidopsis thaliana, mutations at any one of eight distinct loci confer resistance to chlorate. The molecular identities of the genes at these loci are not known; however, one of these loci--chl3--maps very near the nitrate reductase structural gene NIA2. Through the isolation, characterization, and genetic analysis of new chlorate-resistant mutants generated by gamma irradiation, we have been able to demonstrate that the CHL3 gene and the NIA2 gene are identical. Three new chlorate-resistant mutants were identified that had deletions of the entire NIA2 gene. These nia2 null mutants were viable and still retained 10% of wild-type nitrate reductase activity in the leaves of the plants. All three deletion mutations were found to be new alleles of chl3. Introduction of the NIA2 gene back into these chl3 mutants by Agrobacterium-mediated transformation partially complemented their mutant phenotype. From these data, we conclude that Arabidopsis has at least two functional nitrate reductase genes and that the NIA2 gene product accounts for the majority of the leaf nitrate reductase activity and chlorate sensitivity of Arabidopsis plants.     

Nitrate and nitrite reductase negative mutants of N2-fixingAzospirillum spp.

Chlorate resistant spontaneous mutants ofAzospirillum spp. (syn.Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants fromA. brasilense and 13 fromA. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr^–). Most of the mutants were also nitrite reductase negative (nir^–), only 3 remaining nir⁺. Two mutants from nr⁺ nir⁺ parent strains lost only nir and became like the nr⁺ nir^– parent strain ofA. brasilense. No parent strain or nr⁺ mutant showed any nitrogenase activity with 10 mM NO ₃ ^– . In all nr^– mutants, nitrogenase was unaffected by 10 mM NO ₃ ^– . Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir^–. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr^– nir^–) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.     

Biochemical analysis of mutants defective in nitrate assimilation in Neurospora crassa: evidence for autogenous control by nitrate reductase

Summary A biochemical analysis of mutants altered for nitrate assimilation in Neurospora crassa is described. Mutant alleles at each of the nine nit (nitrate-nonutilizing) loci were assayed for nitrate reductase activity, for three partial activities of nitrate reductase, and for nitrite reductase activity. In each case, the enzyme deficiency was consistent with data obtained from growth tests and complementation tests in previous studies. The mutant strains at these nit loci were also examined for altered regulation of enzyme synthesis. Such exeriments revealed that mutations which affect the structural integrity of the native nitrate reductase molecule can result in constitutive synthesis of this enzyme protein and of nitrite reductase. These results provide very strong evidence that, as in Aspergillus nidulans, nitrate reductase autogenously regulates the pathway of nitrate assimilation. However, only mutants at the nit-2 locus affect the regulation of this pathway by nitrogen metabolite repression.     

Characterization of Tn5 mutants deficient in dissimilatory nitrite reduction in Pseudomonas sp. strain G-179, which contains a copper nitrite reductase.

Tn5 was used to generate mutants that were deficient in the dissimilatory reduction of nitrite for Pseudomonas sp. strain G-179, which contains a copper nitrite reductase. Three types of mutants were isolated. The first type showed a lack of growth on nitrate, nitrite, and nitrous oxide. The second type grew on nitrate and nitrous oxide but not on nitrite (Nir-). The two mutants of this type accumulated nitrite, showed no nitrite reductase activity, and had no detectable nitrite reductase protein bands in a Western blot (immunoblot). Tn5 insertions in these two mutants were clustered in the same region and were within the structural gene for nitrite reductase. The third type of mutant grew on nitrate but not on nitrite or nitrous oxide (N2O). The mutant of this type accumulated significant amounts of nitrite, NO, and N2O during anaerobic growth on nitrate and showed a slower growth rate than the wild type. Diethyldithiocarbamic acid, which inhibited nitrite reductase activity in the wild type, did not affect NO reductase activity, indicating that nitrite reductase did not participate in NO reduction. NO reductase activity in Nir- mutants was lower than that in the wild type when the strains were grown on nitrate but was the same as that in the wild type when the strains were grown on nitrous oxide. These results suggest that the reduction of NO and N2O was carried out by two distinct processes and that mutations affecting nitrite reduction resulted in reduced NO reductase activity following anaerobic growth with nitrate.     

炭疽菌不利用硝酸盐突变体的筛选及鉴定

将从杉木和大叶黄杨上分离获得的８个胶孢炭疽菌（Colletoctrichum gloeosporioides）分离物培养在含氯酸钾的平板上,得到快速生长抗氯酸钾的不利用硝酸盐的突变体(Nit)。所有的突变体经鉴定分属于３种表现型,即硝酸还原酶结构位点（nit１）,硝酸盐同化途径的专化调节位点（nit３）,和钼辅因位点（nitM）。分离物发生突变的频率随氯酸钾浓度的增加而提高,并且不同的氮源在一定程度上会影响突变表型种类。除ＣＣ３外,所有的分离物都是自身亲和的,即不同表型的突变体能遗传互补,其     

Targeted disruption of the NIT8 gene in Chlamydomonas reinhardtii.

We have used homologous recombination to disrupt the nuclear gene NIT8 in Chlamydomonas reinhardtii. This is the first report of targeted gene disruption of an endogenous locus in C. reinhardtii and only the second for a photosynthetic eukaryote. NIT8 encodes a protein necessary for nitrate and nitrite assimilation by C. reinhardtii. A disruption vector was constructed by placing the CRY1-1 selectable marker gene, which confers emetine resistance, within the NIT8 coding region. nit8 mutants are unable to grow on nitrate as their sole nitrogen source (Nit-) and are resistant to killing by chlorate. One of 2,000 transformants obtained after selection on emetine-chlorate medium contained a homologous insertion of five copies of the disruption plasmid into the NIT8 gene, producing an emetine-resistant, chlorate-resistant Nit- phenotype. The mutant phenotype was rescued by the wild-type NIT8 gene upon transformation. Seven other mutations at the nit8 locus, presumably resulting from homologous recombination with the disruption plasmid, were identified but were shown to be accompanied by deletions of the surrounding genomic region.     

Isolation and characterization of cell lines of Nicotiana tabacum lacking nitrate reductase

Summary Chlorate-resistant cell lines were established from survivors after plating allodihaploid cells of Nicotiana tabacum into solid medium containing 20 mM chlorate and amino acids as sole nitrogen source. Data characterizing 9 of the most resistant lines are presented. The mutational origin of these lines was inferred on the basis of the enhancement of the variant frequency by mutagen treatment, and of the persistance of the variant phenotype in cell progeny during growth in the absence of selection for more than 3 years and in plants regenerated from two of the lines.Seven lines completely lacked in vivo nitrate reductase (NR) activity and two lines exhibited low (less than 5% of the wild type) NR activity. The abolition of NR activity was found to be not due to an impaired induction by nitrate. Data reported elsewhere show that one of the NR-negative mutants simultaneously lacks xanthine dehydrogenase activity. This pleiotropic mutation is interpreted to affect the synthesis of a molybdenum-containing cofactor, whereas the 8 other lines carry mutations specifically affecting the synthesis of the NR. Both types of NR-negative mutants were unable to grow on minimal medium containing nitrate as sole nitrogen source, but grew well on amino acids. They proved extremely sensitive to the standard medium containing nitrate and ammonium. Differences between the NR-negative mutants with respect to chlorate resistance suggest that chlorate inhibits cultured N tabacum cells not only via its NR-catalysed conversion to chlorite, but also by NR-independent mechanisms.