Molybdenum and chlorate resistant mutants in Escherichiacoli K12

Radioactive molybdenum is used to detect the existence of molybdo compounds in $\text{E.}\text{coli}$ K12. Three membrane bound Mo-proteins are found, using sodium dodecyl sulfate. One of them is the nitrate reductase. The nature of the other two is discussed. The soluble fraction of the cellular extract contains a small Mo binding molecule which could be peptidic in nature (MW is about 1,500). Different chlorate resistant mutants are analyzed on the basis of these molybdo-compounds. None of the mutants is found to contain radioactivity bound to nitrate reductase protein. Defects in the biosynthesis of a molybdenum coenzyme is deduced for chlorate resistant pleiotropic mutants.     

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.     

The role of molybdenum in formation of the NADPH-nitrate reductase by Aspergillus nidulans

Non-nitrate reducing mutants of $\text{Aspergillus}$$\text{nidulans}$ have been noted to produce either a nitrate inducible or constitutive NADPH-cytochrome $\text{c}$ reductase which resides in either a 4.5s or a 7.8s protein. The latter closely resembles the nitrate inducible, FAD dependent NADPH-nitrate reductase from the wild type. Measurement of flavin adenine dinucleotide (FAD) and molybdenum (Mo) in these two proteins revealed significant differences particularly in Mo. The concepts that a nitrate inducible $\text{nia}$ gene product constitutes the major flavin bearing component of the enzyme and that a constitutively produced $\text{cnx}$ gene product is implicated in formation of the larger Mo bearing multimer are further supported.     

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.     

Regulation of reductase formation in Proteus mirabilis

Summary Prteus mirabilis can form four reductases after anaerobic growth: nitrate reductase A, chlorate reductase C, thiosulfate reductase and tetrathionate reductase. The last three enzymes are formed constitutively. Nitrate reductase is formed only after growth in the presence of nitrate, which causes repression of the formation of thiosulfate reductase, chlorate reductase C, tetrathionate reductase and hydrogenase. Formic dehydrogenase assayed with methylene blue as hydrogen acceptor is formed under all conditions.Two groups of chlorate resistant mutants were obtained. One group does not form the reductases and formic dehydrogenase. The second group does not form nitrate reductase, chlorate reductase and hydrogenase, but forms formic dehydrogenase and small amounts of formic hydrogenlyase after growth without hydrogen acceptor or after growth in the presence of thiosulfate or tetrathionate. Nitrate prevents the formation of formic dehydrogenase, thiosulfate reductase and tetrathionate reductase in this group of mutants. Only after growth with thiosulfate or tetrathionate the reductases for these compounds are formed. Anaerobic growth of the wild type in complex medium without a fermentable carbon source is strongly stimulated by the presence of nitrate. Tetrathionate and thiosulfate have no effect at all or only a small effect. The results show that in the presence of tetrathionate or thiosulfate the bacterial metabolism is fully anaerobic, as these cells also contain formic hydrogenlyase.     

Evidence for a Second Nitrate Reductase Activity That Is Distinct from the Respiratory Enzyme in Salmonella typhimurium

Significant nitrate reductase activity was detected in mutants of Salmonella typhimurium which mapped at or near chlC and which were incapable of growth with nitrate as electron acceptor. The same mutants were sensitive to chlorate and performed sufficient nitrate reduction to permit anaerobic growth with nitrate as the sole nitrogen source in media containing glucose. The mutant nitrate-reducing protein did not migrate with the wild-type nitrate reductase in polyacrylamide electrophoretic gels. Studies of the electrophoretic mobility in gels of different polyacrylamide concentration revealed that the wild-type and mutant nitrate reductases differed significantly in both size and charge. The second enzyme also differed from the wild-type major enzyme in its response to repression by low pH and its lack of response to repression by glucose. The same mutants were found to be derepressed for nitrite reductase and for a cytochrome with a maximal reduced absorbance at 555 nm at 25°C. This cytochrome was not detected in preparations of the wild type grown under the same conditions. Extracts of these mutants contained normal amounts of the b-type cytochromes which, in the wild type, were associated with nitrate reductase and formate dehydrogenase, respectively, although they could not mediate the oxidation of these cytochromes with nitrate. They were capable of oxidizing the derepressed 555-nm peak cytochrome with nitrate. It is suggested that these mutants synthesize a nitrate-reducing enzyme which is distinct from the chlC gene product and which is repressed in the wild type during anaerobic growth with nitrate.     

The correlation between the protein composition of cytoplasmic membranes and the formation of nitrate reductase A,chlorate reductase C and tetrathionate reductase in Proteus mirabilis wild type and some chlorate resistant mutants

Three genotypically different chlorate resistant mutants, chl I, chl II and chl III, appeared to lack completely nitrate reductase A, chlorate reductase C and tetrathionate reductase activity. Fumarate reductase is only partially affected in chl I and chl III and unaffected in chl II. Formate dehydrogenase is only partially diminished in chl II, hydrogenase is diminished in chl I and chl II and completely absent in chl III.Subunits of nitrate reductase A, chlorate reductase C and tetrathionate reductase have been identified in protein profiles of purified cytoplasmic membranes from the wild type and the three mutant strains, grown under various conditions. Only the presence and absence of the largest subunits of these enzymes appeared to be correlated with their repression and derepression in the wild type membranes. On the cytoplasmic membranes of the chl I and chl III mutants these subunits lack for the greater part. In the chl II mutant, however, these subunits are inserted in the membrane all together after anaerobic growth with or without nitrate.A model for the repression/derepression mechanism for the reductases has been proposed. It includes repression by cytochrome b components, whereas the redox-state of the nitrate reductase A molecule itself is also involved in its derepression under anaerobic conditions.     

Characterization of a membrane-bound nitrate reductase from Azotobacter chroococcum.

Nitrate reductase from the aerobic bacterium $\text{Azotobacter chroococcum}$ is a soluble enzyme with the characteristic features of Pichinoty's type B nitrate reductase. When cell suspensions of $\text{A. chroococcum}$ are repeatedly subcultured in liquid medium with nitrate as the nitrogen source, most of the nitrate-reducing activity is incorporated into the cytoplasmic membrane. The properties of the particulate nitrate reductase closely resemble Pichinoty's type A enzyme.     

Energy-dependence of nitrate reductase induction in Candidautilis

The requirement of a suitable energy source during the induced synthesis of nitrate reductase in $\text{Candida}\text{utilis}$ was investigated. The levels of nitrate reductase induced were shown to be energy-dependent, and to vary in response to the type of carbon source provided. Glycerol, fructose, ethanol, glucose, and sucrose served as efficient energy sources. Growth rate of the yeast and the induced level of nitrate reductase were dependent on the ratio of carbon to nitrogen in the induction medium, and ratio of 2 being optimal. Induction of nitrate reductase was inhibited by uncouplers, 2,4-dinitrophenol (DNP), dicumarol and carbonyl cyanide $\text{p}$-trifluoromethoxy phenyl hydrazone (CCCP), and by cyanide and azide, indicating an absolute energy-dependency. The facilitation of induction of a high level of nitrate reductase by exogenously added ATP as sole source of energy confirmed the obligate requirement of ATP for the synthesis of nitrate reductase in $\text{Candida}$$\text{utilis}$.     

Isolation and characterization of chlorate resistant mutants from nitrate-nonutilizing fungusPhycomyces blakesleeanus

Chlorate resistant mutants, which were first isolated in the zygomycetous fungusPhycomyces blakesleeanus, were found to be resistant up to a concentration of at least 300 mM of potassium chlorate. The dose-response relationship showed that although the mutants could be divided into two groups based on chlorate resistance in the mycelial elongation assay on the solid minimal medium, this was not observed in the assay using liquid culture. Genetic analysis of heterokaryons revealed the mutant alleles to be dominant. Enzymatic activities of three nitrate reductases and chlorate reductase were deficient in both the parent strain and the mutants. Intracellular incorporation of chlorate ion varied from strain to strain; however, the variation could not explain the mechanism of chlorate resistance. One unexpected characteristic of the mutants was that the intracellular sulfate ion concentration was 3.5 to 5.5 times higher than in the parent strain. We designated this mutant genotypecrw, chlorate resistant mutant from nitrate-nonutilizing wild type.     

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.     

Genetical and biochemical evidence that a novel dinucleoside polyphosphate coordinates salvage and de novo nucleotide biosynthetic pathways in mammalian cells

Studies with ‘wild type’ Chinese hamster ovary cells and mutant derivatives defective in purine salvage and $\text{de novo}$ nucleotide biosynthesis pathways have brought to light the possibility that an unusual dinucleoside polyphosphate, HS-3 (see appendix) is a crucial regulator of these two pathways. Three antitumor drugs, methotrexate, 5-fluorouracil and azaserine as well as L-glutamine, purines and pyrimidines were used to define the loci of HS-3 metabolism. Wild type and salvage pathways mutants accumulated HS-3 in the absence of glutamine. $\text{De novo}$ pathways mutant accumulated HS-3 only when purine was absent. Depletion of HS-3 was induced in wild type and $\text{de novo}$ mutant cell lines by purine compounds. Salvage pathways mutants did not cause depletion of HS-3 when supplied with purines or pyrimidines, except 5-fluorouracil. Data indicate that HS-3 is probably synthesised when an early step in purine nucleotide synthesis is blocked and depleted when the salvage pathways are operative. HS-3 may be an important factor in certain diseases involving nucleotide metabolism.     

A genetical and biochemical study of chlorate-resistant mutants ofSalmonella typhimurium

S.typhimurium can form nitrate reductase A, chlorate reductase C, thiosulfate reductase, tetrathionate reductase and formic dehydrogenase. None of these enzymes are formed in chlorate-resistant mutants. Conjugation experiments showed the presence of a strong linkage between thechl andgal markers of the bacterial chromosome. By deletion mapping the gene ordernic A aro G gal bio chl D uvr B chl A was found. Strains with deletions terminating betweenbio anduvr B or betweenuvr B andchl A have a number of aberrant properties. Though resistant against chlorate they reduce nitrate and form gas. After growth with nitrate they form less nitrate reductase than the wild type which may explain the resistance against chlorate. After growth with thiosulfate they form small amounts of thiosulfate reductase and chlorate reductase C. In crosses between anE.coli Hfrchl ⁺ strain and aS.typhimurium chl A strain recombinants were obtained, forming nitrate reductase A and chlorate reductase C. These recombinants do not form gas, which indicates that thechl ⁺ gene fromE.coli does not function normally inS.typhimurium.The author is very gratefull to Miss C. W. Bettenhaussen, Miss W. M. C. Kapteijn and Mr. K. Pietersma for technical assistance. Helpfull suggestions of Dr. P. van de Putte (Medical Biological Laboratory of the National Defence Organization TNO, Rijswijk) are gratefully acknowledged.     

Interaction between the nitrate respiratory system of Escherichia coli K12 and the nitrogen fixation genes of Klebsiella pneumoniae.

Hybrids were constructed between $\text{E. coli}$ K12 $\text{chl}$^? mutants defective in nitrate respiration and an F′ plasmid carrying nitrogen fixation genes from $\text{K. pneumoniae}$. Examination of these hybrids showed that expression of $\text{nif}$_Kp⁺ genes does not require a functional nitrate respiratory system, but that nitrate reductase and nitrogenase do share some Mo-processing functions. For nitrate repression of nitrogenase activity, reduction of nitrate to nitrite is not necessary, but the Mo-X cofactor encoded by $\text{chl}$ genes is essential. Nitrate probably inhibits nitrogen fixation by affecting the membrane relationship of the nitrate and fumarate reduction systems such that the membrane cannot be energized for nitrogenase activity.     

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.     

Complementation analysis of a nitrate reductase deficient Hyoscyamus muticus cell line by somatic hybridisation

Summary Complementation of a nitrate reductase deficient variant of Hyoscyamus muticus (MA-2) and nitrate reductase apoenzyme (nia-115) and cofactor mutants (cnx-68) of Nicotiana tabacum was studied by protoplast fusion. Selection of prototrophic intergeneric somatic hybrids was achieved in combination of MA-2 with the apoenzyme mutant nia-115 of N. tabacum. The H. muticus MA-2 line was therefore classified to be a cnx type variant possessing an altered molybdenum cofactor of the nitrate reductase enzyme complex but unaffected in the apoprotein of nitrate reductase. The nitrate reductase deficient and chlorate resistant characters of MA-2 were functionally coupled recessive traits. Nitrate reductase activity accompanied by chlorate sensitivity could be detected only under inductive conditions in the somatic hybrids. The inductive expression of nitrate reductase in the somatic hybrids arising from the combination of cells harbouring either the inductive or constitutive type nitrate reductase is discussed.Abbreviations DTT 1,4-Dithio-DL-threitol - Mo-co molybdenum containing cofactor - PEG polyethylene glycol     

Mutants of Azotobacter vinelandii altered in the regulation of nitrate assimilation

The two enzymes involved in the assimilatory pathway of nitrate in Azotobacter vinelandii are corregulated. Nitrate reductase and nitrite reductase are inducible by nitrate and nitrite. Ammonium represses induction by nitrate of both reductases. Repression by ammonium is higher in media containing 2-oxo-glutarate as carbon source than in media containing sucrose. Mutants in the gene ntrC lost nitrate and nitrite reductase simultaneously. Ten chlorate-resistant mutants with a new phenotype were isolated. In media without ammonium they had a normal phenotype, being sensitive to the toxic effect of chlorate. In media containing low ammonium concentrations they were resistant to chlorate. These mutants seem to be affected in the repression of nitrate and nitrite reductases by ammonium.     

Repair of deaminated cytosine residues of DNA: biological significance of the absence of uracil from DNA.

Uracil-DNA glycosylase of $\text{Bacillus}\text{subtilis}$ is involved in repair of deaminated cytosine residues of DNA. Survivals of SPO2 phage after treatment with bisulfite and weak alkali are considerably higher in wild type strains than in $\text{urg}$ mutants, which are deficient in the enzyme activity, whereas survivals of bisulfite/alkali-treated PBS1 phage in the two types of cells are essentially the same. The spontaneous mutation frequency of a $\text{urg}$ mutant is three fold higher than is that of a wild type strain.     

Isolation and characterization of Nicotiana plumbaginifolia nitrate reductase-deficient mutants: genetic and biochemical analysis of the NIA complementation group

Summary Two hundred and eleven nitrate reductase-deficient mutants (NR^–) were isolated from mutagenized Nicotiana plumbaginifolia protoplast cultures by chlorate selection and regenerated into plant. More than 40% of these clones were classified as cnx and presumed to be affected in the biosynthesis of the molybdenum cofactor, the remaining clones being classified as nia mutants. A genetic analysis of the regenerated plants confirmed this proportion of nia and cnx clones. All mutants regenerated were found to carry monogenic recessive mutations that impaired growth on nitrate as sole nitrogen source. Mutants propagated by grafting on N. tabacum systematically displayed a chlorotic leaf phenotype. This chlorosis was therefore related to the NR deficiency. The observation of leaves with NR^– chlorotic sectors surrounded by NR⁺ wild-type tissues suggeests that an NR deficiency is not corrected by diffusible factors. Periclinal chimeras between wild-type tobacco and the NR^– graft were also observed. In this type of chimeric tissue chlorosis was no longer detectable when NR⁺ cells were in the secondmost (L2) layer, but was still detectable when NR^– cells were in the secondmost layer. The genetic analysis of nia mutants revealed that they belong to a single complementation group. However three nia mutants were found to complement some of the other nia mutants. The apoenzyme of nitrate reductase was immunologically detected in several nia mutants but not in other members of this complementation group. Some of the nia mutants, although they were NR^–, still displayed methylviologenitrate reductase activity at a high level. These data show that the nia complementation group corresponds to the structural gene of nitrate reductase. Some of the mutations affecting this structural gene result in the overproduction of an inactive nitrate reductase, suggesting a feedback regulation of the level of the apoenzyme in the wild type.