A Nitrate Reductase-less Variant Isolated from Suspension Cultures of Datura innoxia (Mill.)

A comparative study has been carried out of the growth of two lines of Datura innoxia (Mill.) cells, designated DI-6 and NR1, their resistance to chlorate, and their ability to assimilate nitrate in sterile culture. The NR1 cell line was isolated from DI-6 cultures by first growing the latter in a nitrate-based medium for 5 days and then transferring the cells to a medium containing 2 grams liter⁻¹ of casein hydrolysate as the sole N source and 49 millimolar KClO₃ for a 6-week incubation period. Cells which survived the chlorate treatment then were transferred to casein hydrolysate medium and have been cultured in the absence of chlorate for more than 18 months (NR1).     

曼陀罗培养细胞的硝酸还原酶突变株

经紫外诱变氯酸钾筛选,得到一个低硝酸还原酶(NADH:硝酸氧化还原酶.EC1.6.6.1.,以下简写为NR)活力的细胞株。其主要特征:NR活力低,约为正常型的1/5;对氯酸钾具有较强的抗性;不适合在单纯以硝酸盐为氮源的培养基上生长,能在以(NH_4)_2SO_4为唯一氮源的培养基上生长。蛋白电泳表明,此细胞株与正常型有不同的蛋白带。这些特征在没有选择压力的培养基上培养二年后,仍保持不变,说明此细胞株是一个遗传型的变异株。     

Diverse effects of formate on the assimilatory metabolism of nitrate and nitrite inRhizobium

Two strains ofRhizobium, cowpeaRhizobium 32H1 andRhizobium japonicum CB 1809, showed a marked stimulation in growth on addition of formate to the minimal medium containing nitrate as the sole source of nitrogen. The amount of accumulated nitrite and specific nitrate reductase activity was much higher in cultures supplemented with formate than in the control medium. In contrast, growth, consumption of nitrite and specific nitrite reductase activity in minimal medium + nitrite was greatly reduced by the addition of formate. A chlorate resistant mutant (Chl-16) was isolated spontaneously which contained a nitrite reductase which was not inhibited by formate. The results suggest that formate serves as an electron donor for nitrate reductase and inhibits nitrite assimilation inRhizobium     

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.     

Effect of chlorate,molybdate, and shikimic acid on Salmonella enterica serovar Typhimurium in aerobic and anaerobic cultures

Experiments were conducted to determine factors that affect sensitivity of Salmonella enterica serovar Typhimurium to sodium chlorate (5 mM). In our first experiment, cultures grown without chlorate grew more rapidly than those with chlorate. An extended lag before logarithmic growth was observed in anaerobic but not aerobic cultures containing chlorate. Chlorate inhibition of growth during aerobic culture began later than that observed in anaerobic cultures but persisted once inhibition was apparent. Conversely, anaerobic cultures appeared to adapt to chlorate after approximately 10 h of incubation, exhibiting rapid compensatory growth. In anaerobic chlorate-containing cultures, 20% of total viable counts were resistant to chlorate by 6 h and had propagated to 100% resistance (>10⁹ CFU mL^?1) by 24 h. In the aerobic chlorate-containing cultures, 12.9% of colonies had detectable resistance to chlorate by 6 h, but only 1% retained detectable resistance at 24 h, likely because these cultures had opportunity to respire on oxygen and were thus not enriched via the selective pressure of chlorate. In another study, treatment with shikimic acid (0.34 mM), molybdate (1 mM) or their combination had little effect on aerobic or anaerobic growth of Salmonella in the absence of added chlorate. As observed in our earlier study, chlorate resistance was not detected in any cultures without added chlorate. Chlorate resistant Salmonella were recovered at equivalent numbers regardless of treatment after 8 h of aerobic or anaerobic culture with added chlorate; however, by 24 h incubation chlorate sensitivity was completely restored to aerobic but not anaerobic cultures treated with shikimic acid or molybdate but not their combination. Results indicate that anaerobic adaptation of S. Typhimurium to sodium chlorate during pure culture is likely due to the selective propagation of low numbers of cells exhibiting spontaneous resistance to chlorate and this resistance is not reversible by molybdenum supplementation.     

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.     

Isolation and characterization of biochemical and morphological mutants in chlamydomonas smithii

Mutants of Chlamydomanas smithii resistant to chlorate were induced after UV irradiation. Some were unable to grow on nitrate and suspected to be defective in nitrate reductase. One of them (nit-a) was analyzed and shown to complement nit-1 nit-2 of C. reinhardtii.Mutants resistant to streptomycin, spectinomycin or erythromycin selected from nit-a pre-grown in the presence of 5-fluorodeoxyridine. Two spectinomycin resistant strains were genetically analyzed from crosses with C. reinhardtii carrying different chloroplast mutations. The transmission of resistance was shown to be non-Mendelian and similar to that obtained in intraspecific crosses involving C. reinhardtii chloroplast mutants.Mutants lacking a cell wall (CW⁻) were selected after UV treatment on the basis of the particular flat amoeboid shape of the colonies. Seven of them were tested for complementation with CW15 and CW_d mutants of C. reinhardtii: none was found to be allelic with these two mutations.     

Nitrate reductase activity and growth in Paul's Scarlet rose suspension cultures in relation to nitrogen source and molybdenum

Growth and nitrate reductase activity were measured in Paul's Scarlet rose cell suspensions, cultured in media purified from molybdenum and containing nitrate or urea as sole nitrogen source with or without added Mo. Urea could replace nitrate to yield 80% of the fresh weight in nitrate medium. Nitrate reductase activities were compared by in vivo and in vitro assays. The latter varied due to inactivation during extraction. Compared with activities in cells in complete NO₃ ^- medium, activity in NO₃ ^--Mo cells was reduced to 30% and, in urea-grown cells, to trace amounts. Increases in nitrate reductase activity were found when NO₃ ^- alone was added to NO₃ ^- or urea+Mo cultures. In NO₃ ^--Mo cultures, Mo alone or with NO₃ ^- caused a similar increase in activity, whereas urea-Mo cultures required both NO₃ ^- and Mo for enzyme induction.Abbreviations FAD flavin adenine dinucleotide - Mo molybdenum - NADH reduced nicotinamide adenine dinucleotide - NO₃ ^-+Mo standard MX₁ culture medium - NO₃ ^--Mo MX₁ medium purified of Mo and used for continuous subculture with nitrate - NR nitrate reductase - PSR Paul's Scarlet rose - PVP polyvinylpyrrolidone - U urea - U+Mo MX₁ medium containing urea instead of nitrate - U-Mo MX₁ medium containing urea instead of nitrate and also purified of Mo     

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.     

Chlamydomonas reinhardtii strains expressing nitrate reductase under control of the cabII-1 promoter: isolation of chlorate resistant mutants and identification of new loci for nitrate assimilation

The Chlamydomonas reinhardtii strain Tx11-8 is a transgenic alga that bears the nitrate reductase gene (Nia1) under control of the CabII-1 gene promoter (CabII-1-Nia1). Approximately nine copies of the chimeric CabII-1-Nia1 gene were found to be integrated in this strain and to confer a phenotype of chlorate sensitivity in the presence of ammonium. We have used this strain for the isolation of spontaneous chlorate resistant mutants in the presence of ammonium that were found to be defective at loci involved in MoCo metabolism and light-dependent growth in nitrate media. Of a total of 45 mutant strains analyzed first, 44 were affected in the MoCo activity (16 Nit⁻, unable to grow in nitrate, and 28 Nit⁺, able to grow in nitrate). All the Nit⁻ strains lacked MoCo activity. Diploid complementation of Nit⁻, MoCo⁻ strains with C. reinhardtii MoCo mutants and genetic analysis indicated that some strains were defective at known loci for MoCo biosynthesis, while three strains were defective at two new loci, hereafter named Nit10 and Nit11. The other 28 Nit⁺ strains showed almost undetectable MoCo activity or activity was below 20% of the parental strain. Second, only one strain (named 23c⁺) showed MoCo and NR activities comparable to those in the parental strain. Strain 23c⁺ seems to be affected in a locus, Nit12, required for growth in nitrate under continuous light. It is proposed that this locus is required for nitrate/chlorate transport activity. In this work, mechanisms of chlorate toxicity are reviewed in the light of our results.     

Isolation of a nitrate reductase deficient mutant of Pisum sativum by means of selection for chlorate resistance

Summary After EMS treatment of seeds of the Pisum variety Rondo a chlorate resistant mutant was isolated which showed a decrease in the in vitro activity of the enzyme nitrate reductase of roughly 95%. The mutation is monogenic and recessive. The mutant shows a decrease in protein content, and an increase in the amount of nitrate accumulated and in the activity of the enzyme nitrite reductase. On a liquid nutrient medium containing nitrate as the sole nitrogen source and in soil, the mutant grows very poorly due to necrosis of the leaves. On liquid medium containing ammonium, either with or without nitrate, growth is as good as that of the parent variety.     

Constitutive nitrate reductase: a dominant conditional marker for plant genetics

We describe the development of a counter-selection system based on the use of an engineered nitrate reductase (NR) gene. The engineered gene consists of an NR cDNA placed under the control of the CaMV 35S promoter (35S-NR). Seedlings and cells derived from transgenic Nicotiana plumbaginifolia plants transformed with 35S-NR are efficiently killed by the selective agent chlorate on medium containing ammonium as the sole nitrogen source. Under these nitrate-free conditions wild-type plants are not affected by chlorate because the endogenous wild-type Niagene is not expressed.     

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.     

Isolation of molybdenum cofactor defective cell lines of Nicotiana tabacum

Summary Thirty-nine chlorate resistant cell lines were isolated after plating ethylmethane sulphonate treated allodihaploid cells of Nicotiana tabacum cv. Xanthi on agar medium containing 20 mM chlorate. Thirty-two of these cell lines grew as well on nitrate medium as on amino acid medium and three other cell lines grew well on amino acid medium but poorly on nitrate medium. Four other cell lines, 042, P12, P31 and P47 which could grow on amino acid medium, but not on nitrate medium, were examined further. They lacked in vitro nitrate reductase activity but were able to accumulate nitrate. All lines possessed nitrite reductase activity. Lines 042, P12, and P31 had a cytochrome c reductase species which was the same size as the wild type nitrate reductase associated cytochrome c reductase species, whilst the cytochrome c reductase species in line P47 was slightly smaller. All four lines lacked xanthine dehydrogenase activity and neither nitrate reductase nor xanthine dehydrogenase activity was restored by subculture of the four lines into either nitrate medium or glutamine medium supplemented with 1 mM sodium molybdate. These four lines are different from other molybdenum cofactor defective cell lines so far described in N. tabacum and possess similar properties to certain other cnx mutants described in Aspergillus nidulans.     

Isolation and characterization of five genotypic mutants of chlorate-resistant cyanobacteria unable to utilize nitrate

Spontaneous mutants of the cyanobacteriumSynechococcus PCC 7002 resistant to chlorate were isolated. Either 40mM or 400mM Na₂ClO₃ was used as the selective agent. Putative Chl^r colonies were picked onto medium containing ammonia as the sole N source, then replicaplated to media containing either NH₄ ⁺, NO₂ ^– as N sources. Of 252 putative mutants, 106 were able to use either NH₄Cl or NaNO₂ but not NaNO₃ as their sole source of nitrogen. All of the mutant isolates had generation times similar to wild-type 7002 when grown on either ammonium (3.8–4.1 h/generation) or nitrite (4.5–4.7 h/generation). None had detectable methyl viologensupported nitrate reductase activity and are thus phenotypically NRase^–. The Chl^r mutants had photomediated O₂ production and dark O₂ uptake rates similar to the wild type and responded similarly to selected metabolic inhibitors. They expressed increased levels of phycocyanin (PC) synthesis under normal, nitrogen-replete growth conditions, but rapidly lapsed into a chlorotic state upon a shift to either medium containing nitrate or to N-free medium. Genetic analysis of the Chl⁴ mutants indicated that each could be rescued by direct transformation with chromosomally derived DNA from the wild-type strain. Frequencies of transformation for the mutants were characteristic for single genetic lesions in this cyanobacterium. On the basis of marker rescue by a cosmid library of wild-type DNA, the NRase^– mutants could be grouped into five distinctive genotypic families.     

Chlorate and nitrate reduction in the phototrophic bacteriaRhodobacter capsulatus andRhodobacter sphaeroides

Chlorate or trimethylamine-N-oxide (TMAO) added to phototrophic cultures ofRhodobacter sphaeroides DSM 158 increased both the growth rate and the growth yield although this stimulation was not observed in the presence of tungstate. This strain, exhibited basal activities of nitrate, chlorate, and TMAO reductases independently of the presence of these substrates in the culture medium, and nitrate reductase (NR) activity was competitively inhibited by chlorate. Phototrophic growth ofRhodobacter capsulatus B10, a strain devoid of NR activity, was inhibited only by 100 mM chlorate. However, growth of the nitrate-assimilatingR. capsulatus strains E1F1 and AD2 was sensitive to 10mm chlorate, and their NR activities were not inhibited by chlorate. Both NR and chlorate reductase (CR) activities of strain E1F1 were induced in the presence of nitrate or chlorate respectively, whereas strain AD2 showed basal levels of these activities in the absence of the substrates. A basal TMAO reductase (TR) activity was also observed when these strains ofR. capsulatus were cultured in the absence of this electron acceptor. These results suggest that chlorate and TMAO can be used as ancillary oxidants byRhodobacter strains and that a single enzyme could be responsible for nitrate and chlorate reduction inR. sphaeroides DSM 158, whereas these reactions are catalyzed by two different enzymes inR. capsulatus E1F1 and AD2.     

Isolation of Cobalt-Resistant Strains of Propionic Acid Bacteria,Potent Producers of Vitamin B12

A method of adaptation to cobalt nitrate at high concentrations allowed us to isolate 46 strains of propionic acid bacteria Propionibacterium acidipropionici, resistant to excessive amounts of Co²⁺ in the medium. Studies of these strains revealed cultures that were most potent in synthesizing vitamin B₁₂. The yield of vitamin B₁₂ was increased 3 times, compared to parent strains.     

Nitrate reductase deficient cell lines from haploid protoplast cultures ofNicotiana plumbaginifolia

Summary Nitrate reductase deficient (NR^-) cell lines were selected indirectly by their resistance to 40 mM chlorate in protoplast cultures of haploidNicotiana plumbaginifolia. Frequency of the chlorate resistant clones was 5.8×10^-5 in non-mutagenized cultures, which could be increased up to 25 times by treatment with N-ethyl-N-nitrosourea (NEU) or gamma irradiation.Out of 136 chlorate resistant clones 29 were fully deficient in nitrate reductase. The rest of the clones contained decreased or normal levels of NR activity (91 and 16 clones, respectively).Further characterization was carried out in 9 clones which were fully deficient in NR and in 2 clones containing resisdual (0–5%) NR activity. The clones were tentatively classified as defective in the apoenzyme (7 clones including the 2 with residual NR activity) or the cofactor (4 clones) of NR by the xanthine dehydrogenase activity and in vitro enzyme complementation. The cofactor defectives could be further classified into two groups. In one of these (2 clones) the NR activity could be partially restored by unphysiologically high (0.2–1 mM) molybdate in the culture medium. The other two are new types which have not been described in flowering plants.Plant regeneration was obtained only in the clones which contained residual NR activity.     

Nitrate reductase activity of free-living and symbiotic uptake hydrogenase-positive and uptake hydrogenase-negative strains of Bradyrhizobium japonicum

The nitrate reductase activity (NR) of selected uptake hydrogenase-positive (hup ⁺) and uptake hydrogenase-negative (hup ^-) strains of Bradyrhizobium japonicum were examined both in free-living cells and in symbioses with Glycine max L. (Marr.) cv. Williams. Bacteria were cultured in a defined medium containing either 10 mM glutamate or nitrate as the sole nitrogen source. Nodules and bacteriods were isolated from plants that were only N₂-dependent or grown in the presence of 2 mM KNO₃. Rates of activity in nodules were determined by an in vivo assay, and those of cultured cells and bacteriods were assayed after permeabilization of the cells with alkyltrimethyl ammonium bromide. All seven strains examined expressed NR activity as free-living cells and as symbiotic forms, regardless of the hup genotype of the strain used for inoculation. Although the presence of nitrate increased nitrate reduction by cultures cells and nodules, no differences in NR activity were observed between bacteroids isolated from nodules of plants fed with nitrate or grown on N₂-fixation exclusively. Cultured cells, nodules and bacteriods of strains with hup ^- genotype (USDA 138, L-236, 3. 15B3 and PJ17) had higher rates of NR activity than those with hup ⁺ genotype (USDA 110, USDA 122 DES and CB1003). These results suggest that NR activity is reduced in the presence of a genetic determinant associated with the hup region of B. japonicum.Abbreviations EDTA ethylene-diamine tetraacetic acid - Hup hydrogen uptake - MOPS 3-(N-morpholino)-propane sulfonic acid - NR nitrate reductase - PVP polyvinyl-polypyrrolidone - Tris Tris(hydroxymethyl)-aminomethane