Mapping of a gene causing resistance to chlorate inSalmonella typhimurium

Chlorate-resistant mutants ofS. typhimurium LT2 and LT7 and ofS. abony have been isolated, which are deficient in the biosynthesis of nicotinic acid and thiamin and in the fermentation of inositol. These mutants could be divided into 5 groups. The most likely gene order isnicB-chlG-thiB-inlB. This segment is transferred early in conjugation experiments with Hfr H2 and Hfr B2 as donors. In time-of-entry experiments with Hfr B2 as donor the segment entered about 3 minutes afterpur C. Consequently this segment maps in the 79- to 82-minutes region of the genetic map. From recombinant analysis of nic⁺ recombinants obtained in a four-point cross between Hfr B2 and ahis carBpur C del (nic chl G) acceptor the incorporation frequency of the transferred donor fragment was calculated to be about 0.41. The number of crossing-over events per minute length of the chromosome was about the same as in similar crosses betweenE. coli Hfr and F^–. However, between thenic and thepur C markers it was much higher; it may therefore be inferred that there is a higher probability for a crossing-over event in the regions adjacent to the region that is deleted in the recipient.In crosses betweenS. abony Hfr H2 del (nic thi inl chl) and F^– strains no recombinants were observed which have obtained the deletion from the donor. Nearly all auxotrophic or nic⁺ recombinants obtained in a cross between Hfr B2 and a F^– del (nicBthiBchlGinlB) strain have inherited all markers of the donor, which are present in the deletion of the recipient.     

Involvement of a gene of the chl E locus in the regulation of the nitrate reductase operon

Summary A strain of E. coli carrying a Mudl insertion leading to chlorate resistance was found to lack nitrate reductase and formate dehydrogenase activities, but to synthesize b-type cytochrome constitutively. Introduction of this insertion mutation into a strain bearing a fusion between the nitrate reductase operon (chl C, chl I) and the lac structural genes resulted in the constitutive expression of the lac genes of this last fusion. Identical results were found when the Mudl was eliminated promoting a deletion in the original insertion site. This mutation was located midway between gal and aro A, at the chl E locus. Study of a chl E strain already described revealed similar behaviour. Absence of nitrate reductase activity in these strains which constitutively express the structural genes of the nitrate reductase operon was tentatively attributed to the simultaneous lack of a cofactor of the nitrate reductase terminal enzyme, possibly cofactor Mo-X, and of a repressor of the operon.     

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.     

A genetic assay for transversion mutations in bacteriophage T4

Summary Seventy-two mutants deficient in formate-nitrate reductase activity were selected in Escherichia coli strain PK 27, by two different procedures. Forty-five strains were selected on the basis of chlorate resistance and 27 strains were selected by their inability to reduce nitrate with formate as an electron donor. Genetic analysis of these strains showed that the two techniques yield distinctly different distributions of mutants among the various controlling genetic loci. Chlorate resistance appears to select for severe alterations in the nitrate reductase system; 98% of these mutants fell into the pleiotropic chl A, B, D and E classes and are deficient in all the activities of the formate-hydrogenlyase pathway as well as formate-nitrate reductase pathway. In contrast, 48% of the mutants selected for their inability to reduce nitrate with formate as the electron donor were of the chl C class and two new classes were identified among mutants selected by this procedure. Chl F mutants are linked to tryptophan and the chl C locus. Chl G mutants map at zero minutes on the E. coli genetic map.     

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.     

Mapping of chlorate-resistant mutants of Citrobacter freundii by deletion and complementation analysis

Summary From Citrobacter freundii mutants have been isolated, with deletions extending chl genes. 53% of these mutants mapped in the gal-bio region of the chromosome. Genetic mapping by three methods—deletion analysis, linkage analysis in crosses with C. freundii Hfr donors and complementation with Escherichia coli F factors—establishes the gene order: chl H-gal-chl D-hut-bio-uvr B-chl A-chl I-chl E In an other segment a gene order ilv-chl-pdx was found. Furthermore chl loci were found adjacent to 7 different chromosomal markers. C. freundii can form nitrate reductase A, thiosulfate reductase, tetrathionate reductase and formic dehydrogenase. These enzymes are not formed in most of the chlorate-resistant mutants. In some of these mutants the enzyme activities can be restored by complementation with F factors of E. coli.     

Deletion-mapping of resistance against chlorate in Klebsiella aerogenes

Summary Chlorate-resistant mutants with deletions have been isolated from Klebsiella aerogenes. Mutants with deletions in the gal-bio region of the genetic map can be divided into 9 groups. From the properties of the various groups of mutants the gene order nic A-aro G-gal-chl D-hut-bio-uvr B-chl A is deduced. Furthermore deletions have been observed in a segment of the chromosome containing nic B, thi B, inl B, and chl G. Thi B and chl G are adjacent but no more information about the gene order in this segment can be given. In both segments a great homology is observed with the corresponding regions of the genetic maps of E. coli and S. typhimurium. Deletions of chl A, chl D or chl G have a pleiotropic effect. Mutants with a deletion of one of these genes do not produce nitrite from nitrate or gas from glucose.     

Membrane reconstitution in chl-r mutants of Escherichia Coli K 12: VIII. Purification and properties of the FA factor,the product of the chl B gene

The isolation and purification of the product of the chl B gene of Escherichia coli K 12 from the chl A mutant have been attempted. The purified protein gives a single band in 10 % sodium dodecylsulfate/polyacrylamide gel electrophoresis. The molecular weight is estimated to be 35 000. This protein, that we have named “F_A factor”, does not contain any lipid, has a strong tendency to lose its activity by polymerizing but can be kept in an active state when stored in buffer containing NaCl. The addition of purified F_A protein to a soluble extract from the chl B mutant strain grown under anaerobiosis in the presence of nitrate initiates the “complementation reaction”, i.e. the reconstitution of the nitrate reductase activity and the formation of particulate material similar to the native membrane with respect to the structure and enzymatic function. F_A protein acts both on the rate of reconstitution and on the total amount of reconstituted enzyme. The complementation leads to the reconstitution of nonsedimentable nitrate reductase and to the formation of three types of particles of different buoyant densities (1.10, 1.18 and 1.23) the two lightest of which contain nitrate reductase. It is shown that F_A factor is incorporated only into the particles of intermediate density. In vivo, this factor is located in the native membranes of chl A, chl C, chl D and wild-type strains, whatever the growth conditions, aerobiosis or anaerobiosis, and in the presence or absence of nitrate. Protein F_A can be released from either of these membranes (native or reconstituted) by removing Mg²⁺ or by subjecting Kaback's vesicles to mechanical treatments; in the case of 1.18-reconstituted particles and wild-type membranes, the release of F_A protein does not exert any effect on the level of the nitrate reductase activity.     

Polar mutations in lac, gal and phage lambda consist of a few IS-DNA sequences inserted with either orientation

Summary Twenty nine nitrate reductase-less mutants ofEscherichia coli K12 were isolated and characterised asnar(chl)A ^-,B ^- orE ^-. Data from phageP1-mediated transduction studies permitted the construction of fine structure maps fornarA andnarE and suggested that both loci may be functionally divisible into two contiguous genes.NarB was shown to comprise at least three complementation groups.     

Overexpression of Rice Genes OsNRT1.1A and OsNRT1.1B Restores Chlorate Uptake and NRT2.1/NAR2.1 Expression in Arabidopsis thaliana chl1-5 Mutant

Nitrogen uptake by plants is a key step for efficient nitrogen use, which affects plant growth and yield. Arabidopsis thaliana gene NRT1.1 was identified as a transporter related to nitrate (NO₃⁻) signaling and uptake. In rice, three orthologs of NRT1.1, named OsNRT1.1A, OsNRT1.1B, and OsNRT1.1C, have been identified. This study evaluated the potential of OsNRT1.1A, OsNRT1.1B, and OsNRT1.1C in NO₃⁻ signaling and uptake through overexpression in the Arabidopsis chl1-5 mutant. The expression of OsNRT1.1A, OsNRT1.1B, and OsNRT1.1C was evaluated in the roots and shoots of rice cultivated with NO₃⁻ or NH₄⁺. OsNRT1.1A was expressed in the roots and shoots cultivated with NO₃⁻ and NH₄⁺. OsNRT1.1B was expressed predominantly in roots of rice cultivated with NO₃⁻, while the expression of OsNRT1.1C was low in roots and shoots. Arabidopsis chl1-5 plants were transformed by the floral dip method using Agrobacterium tumefaciens to overexpress OsNRT1.1A and the alternative splicing product named OsNRT1.1As, OsNRT1.1B, and OsNRT1.1C. The chlorate test showed the ability of OsNRT1.1A, OsNRT1.1B or OsNRT1.1C to take up chlorate, as evidenced by the decrease in fresh weight. The OsNRT1.1B lineages presented higher toxicity to chlorate. Gene expression analyses showed that the insertion of OsNRT1.1A and OsNRT1.1B into Arabidopsis chl1-5 induced the expression of NRT2.1 and NAR2.1. OsNRT1.1As overexpression did not significantly affect the expression of NRT2.1 and NAR2.1. The results show the differential ability of NRT1.1 orthologs in rice to take up chlorate and signal the expression of other nitrate transporters, which may affect the efficiency of nitrogen utilization and its uptake.

     

Membrane reconstitution in chl-r mutants of Escherichia coli K 12 VI. Morphological study of membrane assembly during complementation between extracts of chl-r mutants

The particles formed during the complementation between supernatant extracts of mutants chl A⁻ and chl B⁻ of Escherichia coli K 12 have been examined under the electron microscope. These particles look like membranous formations with a vesicular structure limited by a triple-layered membrane. The observations made on these particles at various incubation times during complementation show that the aggregation progresses through several stages of organization. When the phenomenon becomes steady, the population of reaggregated particles shows a large structural heterogeneity: it consists of small aggregates, filaments and particulary well-organized closed vesicles. As we had previously shown for the reconstitution of nitrate reductase, the rate of formation and the structure of newly formed aggregates depend on various parameters (temperature, dialysis).     

Identification of two tungstate-sensitive molybdenum cofactor mutants, chl2 and chl7, of Arabidopsis thaliana.

Summary The characterization of mutants that are resistant to the herbicide chlorate has greatly increased our understanding of the structure and function of the genes required for the assimilation of nitrate. Hundreds of chlorate-resistant mutants have been identified in plants, and almost all have been found to be defective in nitrate reduction due to mutations in either nitrate reductase (NR) structural genes or genes required for the synthesis of the NR cofactor molybdenum-pterin (MoCo). The chlorate-resistant mutant ofArabidopsis thaliana, ch12, is also impaired in nitrate reduction, but the defect responsible for this phenotype has yet to be explained.chl2 plants have low levels of NR activity, yet the map position of thechl2 mutation is clearly distinct from that of the two NR structural genes that have been identified inArabidopsis. In addition,chl2 plants are not thought to be defective in MoCo, as they have near wild-type levels of xanthine dehydrogenase activity, which has been used as a measure of MoCo in other organisms. These results suggest thatchl2 may be a NR regulatory mutant. We have examinedchl2 plants and have found that they have as much NR (NIA2) mRNA as wild type a variable but often reduced level of NR protein, and one-eighth the NR activity of wild-type plants. It is difficult to explain these results by a simple regulatory model; therefore, we reexamined the MoCo levels inchl2 plants using a sensitive, specific assay for MoCo: complementation ofNeurospora MoCo mutant extracts. We found thatchl2 has low levels of MoCo — about one-eighth the wild-type level and less than the level in anotherArabidopsis MoCo mutantchl6 (B73). To confirm this result we developed a new diagnostic assay for MoCo mutants, growth inhibition by tungstate. Bothchl2 andchl6 are sensitive to tungstate at concentrations that have no effect on wildtype plants. The tungstate sensitivity as well as the chlorate resistance, low NR activity and low MoCo levels all cosegregate, indicating that all are due to a single mutation that maps to thechl2 locus, 10 centimorgans fromerecta on chromosome 2. We also report on the isolation of a new chlorate-resistant mutant ofArabidopsis, ch17, which is a MoCo mutant with the same phenotypes aschl2 andchl6.     

Isolation from Salmonella typhimurium LT2 of mutants lacking specifically nitrate reductase activity and mapping of the chl-C gene

Summary Addition of tetrathionate and nitrate to EMB medium allows the isolation from Salmonella typhimurium LT2 of mutants lacking specifically nitrate reductase A activity. Genetic localization of such chl-C mutations leads to the conclusion that the affected gene is close to tdk but outside the trp-tdk segment and that chl-C is an early marker in Hfr B2.     

Effects of nitrate on intracellular nitrite and growth of Microcystis aeruginosa

Although nitrate is a macronutrient and can serve as good nitrogen source for many species of phytoplankton, high nitrate concentrations do not benefit the growth of phytoplankton. We hypothesise that algae cultured under high nitrate concentrations can accumulate intracellular nitrite, which is produced by nitrate reductase (NR) and can inhibit the growth of algae. To assess the validity of this hypothesis, Microcystis aeruginosa was grown under different nitrate concentrations from 3.57 to 21.43 mM in low CO₂ and high CO₂ conditions for 15 days. We observed that, with increasing nitrate concentrations, the intracellular nitrite concentrations of the alga increased and the growth rates and photosynthesis declined. When grown under high CO₂ conditions, M. aeruginosa showed lower intracellular nitrite concentrations and higher growth rates and \textP_\textm^\textchla {\text{P}}_{\text{m}}^{{\text{chl}}a} , \textR_\textd^\textchla {\text{R}}_{\text{d}}^{{\text{chl}}a} , α^chla than under low CO₂ conditions. These results suggest that the accumulation of intracellular nitrite could be the cause of inhibition of algal growth under high nitrate concentrations.     

Multiple Forms of Inorganic Pyrophosphatase from the Leaf of Amarantus blitum L.

The existence of a carrier-bound pathway for inorganic sulfate assimilation has been proposed in Chlorella and Escherichia coli. The possibility that the sulfonyl group of active sulfate is transferred to a specific organic acceptor to form thiosulfate ester was examined with Salmonella typhimurium LT-2. Some 11% of the radioactive products from [³⁵S]-3′-phosphoadenosine 5′-phosphosulfate were transferred to high molecular weight compounds, and the remainder of the product is identified as free inorganic sulfite. Apparent thiosulfonate reductase activity was detected in the reaction mixtures containing S-sulfoglutathione and NADPH as conceivable substrates, but not with partially purified sulfite reductase. The former activity was attributable to the nonenzymatic reaction, sulfitolysis. Through these in vitro experiments the existence of the carrier-bound pathway was disproved.     

Comparison between the chromosomal maps of Escherichia coli and Salmonella typhimurium. Length of the inverted segment in the trp region

Summary Genetic analysis of nitrate and tetrathionate reductase-less mutants of Salmonella typhimurium and the comparison of the trp region of this organism with that of Escherichia coli lead to the conclusion that the inversion involves the whole segment between ttr and chl C genes, i.e. approximately 10 percent of the genome.     

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.     

Catabolite repression of thiosulfate reduction bySalmonella typhimurium

The effect of glucose and other carbon sources on thiosulfate reduction and on the expression ofphs bySalmonella typhimurium was examined. Glucose repressed both H₂S production from thiosulfate and methyl viologen-linked thiosulfate reductase activity. Cyclic AMP (2 mM) in the growth medium restored both activities. Cyclic AMP was essential for both activities in acya mutant. Glucose and many other sugars repressedphs expression in both Cya⁺ and Cya^– phs::Mu d1(Ap^r lac) operon fusion mutants. Increasing cyclic AMP to 10 mM increasedphs expression in the presence of some, but not all, sugars. It appears that catabolite repression of thiosulfate reduction inS. typhimurium involves more than a simple requirement for cyclic AMP.     

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.     

Properties of a temperature sensitive plasmid in Citrobacter freundii

Summary The properties of a strain of Citrobacter freundii with a deletion of the gal, chl D, bio, uvr B, chl A region of the chromosome, harbouring a temperature sensitive plasmid F _{ts 114} ¹ aro G⁺ gal ⁺ chl D⁺ bio ⁺ uvr B⁺, originating from Escherichia coli, are described.The isolation of strains with an integrated plasmid was tried by incubation of the partial diploid strain at the restrictive temperature and selection for retained plasmid properties. However C. freundii Hfr strains were not obtained, since only fragments of the plasmid were integrated. Integration occurred at seven different sites of the chromosome and resulted in an inactivation of the gene in which the fragment was integrated. Mutants with deficiencies for arginine, isoleucine and valine, tryptophan, guanine and either tryptophan or tyrosine were obtained. In another type the deficiency, resulting from integration, could not be identified, whereas in the seventh type integration had occurred in one or more non-essential genes, because no deficiency was present. Release of the integrated fragment occurred in such a way that gene activity was restored. The released fragment was lost or was integrated again at one of the other six integration sites, resulting in another mutant type.