Phenotypic reversion of nitrogenase in pleiotropic mutants of Rhizobium meliloti.

In two out of three pleiotropic mutants of Rhizobium meliloti, defective in nitrate reductase induced by amino acid utilization in vegetative bacteria and in symbiotic nitrogen fixation, nitrogenase activity could be restored completely by purines and partially by the amino acids L-glutamate, L-aspartate, L-glutamine, and L-asparagine. The compounds restoring effectiveness in nitrogen fixation did not restore nitrate reductase activity in vegetative bacteria. The restoration of effectiveness supports our earlier conclusion that the mutation is not in the structural gene for a suggested common subunit of nitrogenase and nitrate reductase.     

Isolation and characterisation of nitrate reductase mutants and regulation of nitrate reductase and nitrogenase in the cyanobacterium Nostoc muscorum

Summary Chlorate resistant mutants of the cyanobacterium Nostoc muscorum isolated after N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis were found to be defective/blocked in nitrate reductase (NR).The parent strain possessed active NR in the presence of nitrogen as nitrate and only basal levels of activity in ammonia and N-free grown cultures. Addition of ammonia suppressed the NR activity in the parent strain whereas addition of L-methionine DL-sulphoximine (MSX) restored NR activity. A similar repression by ammonia, glutamine and derepression with MSX were also observed for nitrogenase synthesis.One class of mutants lacked NR activity (nar ^-) whereas the specific activity of NR was low in another class of mutants (nar ^def). Unlike the parent, the mutants synthesized nitrogenase and differentiated heterocysts in the presence of nitrate nitrogen. Uptake studies of nitrite and ammonia in mutants revealed that they possessed both nitrite reductase and glutamine synthetases (GS) at low levels, and the same level respectively in comparison with the parent.     

Physiological and agrochemical properties of different symbiotic genotypes of pea (Pisum sativum L.)

Physiological characters of symbiotic mutants of pea were studied: nodulation, activities of nitrogenase and nitrate reductase, chlorophyll content in leaves and their water-holding capacity, biomass accumulation, and nitrogen forms. The parameters reflecting the genotype state of the macrosymbiont under soil conditions considerably varied. Supernodulation mutants stood out against symbiotic pea genotypes by high contents of chlorophyll and nonprotein nitrogen compounds, high nitrogenase activity, and low nitrate reductase activity. The efficiency of the legume-rhizobium symbiosis was largely mediated by the macrosymbiont genotype. The highest atmospheric nitrogen fixation (50–80%) was observed in the parental pea varieties. Despite the highest nitrogenase activity in the nodules, the supernodulation mutants were inferior to the parental varieties by the nitrogen fixation capacity (40–60%), which was due to their low productivity.     

Expression of Klebsiella pneumoniae nitrogen fixation genes in nitrate reductase mutants of Escherichia coli.

Nitrate reductase (nar) A, B and E mutants of Escherichia coli with plasmids carrying Klebsiella pneumoniae nitrogen fixation (nif) genes reduced acetylene independently of added molybdate, but nar D mutants showed pleiotropic dependence on the concentration of added molybdate for expression of both nar and nif. No complementation of nar mutations by nif occurred; nitrite but not nitrate repressed nif in nar hosts. Derepression of nif occurred in molybdenum-deficient nar D (nif) strains since nitrogenase peptides were present. nifB mutants, thought to have a lesion in the pathway of molybdenum to nitrogenase, as well as nif deletion mutants, had normal nitrate reductase activity.     

Nitrogen fixation in nitrate reductase-deficient mutants of cultured rhizobia.

Forty-eight mutants unable to reduce nitrate were isolated from "cowpea" Rhizobium sp. strain 32Hl and examined for nitrogenase activity in culture. All but two of the mutants had nitrogenase activity comparable with the parental sttain and two nitrogenase-defective strains showed alterations in their symbiotic properties. One strain was unable to nodulate either Macroptilium atropurpureum or Vigna uguiculata and, with the other, nodules appeared promptly, but effective nitrogen fixation was delayed. These results, and the relatively low proportion of nitrate reductase mutants with impaired nitrogenase activity, do not support the proposed commanality between nitrogenase and nitrate reductase in cowpea rhizobia. Inhibition studies of the effect of nitrate and its reduction products on the nitrogenase activity in cultured strains 32Hl and the nitrate reductase-deficient, Nif+ strains, indicated that nitrogenase activity was sensitive to nitrite rather than to nitrate.     

Nitrogenase activity and nitrate respiration inAzospirillum spp.

The interaction between nitrate respiration and nitrogen fixation inAzospirillum lipoferum andA. brasilense was studied. All strains examined were capable of nitrogen fixation (acetylene reduction) under conditions of severe oxygen limitation in the presence of nitrate. A lag phase of about 1 h was observed for both nitrate reduction and nitrogenase activity corresponding to the period of induction of the dissimilatory nitrate reductase. Nitrogenase activity ceased when nitrate was exhausted suggesting that the reduction of nitrate to nitrite, rather than denitrification (the further reduction of nitrite to gas) is coupled to nitrogen fixation. The addition of nitrate to nitrate reductase negative mutants (nr^-) ofAzospirillum did not stimulate nitrogenase activity. Under oxygen-limited conditionsA. brasilense andA. lipoferum were also shown to reduce nitrate to ammonia, which accumulated in the medium. Both species, including strains ofA. brasilense which do not possess a dissimilatory nitrite reductase (nir^-) were also capable of reducing nitrous oxide to N₂.     

Genetic relationship between nitrogen fixation and nitrate utilization in Cylindrospermum fertilissimum

Summary A series of nitrate reductaseless mutants of the blue-green alga (cyanobacterium), Cylindrospermum fertilissimum, have been isolated by selecting clones resistant to chlorate. Chlorate resistant mutants obtained spontaneously showed partial block in nitrate utilization and nitrogen fixation. Resistant derivatives were also obtained after NG mutagenesis. Some of these mutants were found to be double mutants, i.e., blocked in assimilation of nitrate and dinitrogen, simultaneously showing loss of heterocyst diffentiation. All the chlorate resistant/nitrate reductaseless mutants were either partially or completely blocked in utilization of dinitrogen supporting the proposed commonality between nitrogenase and nitrate reductase in the blue-green alga, Cylindrospermum fertilissimum.     

Regulation of nitrogen fixation in Azotobacter vinelandii OP: the role of nitrate reductase

A number of chlorate-resistant mutants were selected, and one of these, clr68-5, was studied in detail. This mutant cannot utilize nitrate in vivo to overcome the effect of nonmetabolizable repressors of nitrogenase. The reason for this inability was that strain clr68-5 lacked nitrate reductase. Nitrate inhibited the activity of nitrogenase but did not act as a corepressor of nitrogenase in strain clr68-5 as it does in the wild type. Ammonia seemed to act as corepressor of nitrogenase in both strains.     

Isolation of ntrA-like mutants of Azotobacter vinelandii.

A number of chlorate-resistant mutants of Azotobacter vinelandii affected in a general control of nitrogen metabolism were isolated. These mutants could not utilize dinitrogen, nitrate, or nitrite as a nitrogen source. The reason for this inability is that they were simultaneously deficient in nitrogenase and nitrate and nitrite reductase activities. They were complemented by a cosmid carrying a DNA fragment of A. vinelandii able to complement ntrA mutants of Escherichia coli, so they seemed to be ntrA-like mutants.     

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.     

Pathways of energy metabolism required for phenotypic expression of nif+Kp genes in Escherichia coli.

In E. coli K12 (F'nif+Kp) hybrids, electron-transport-dependent phosphorylation is not necessary for anaerobic nitrogen fixation, and substrate level phosphorylation can provide sufficient ATP from glucose for nitrogenase activity. The fumarate-reduction system, however, is essential in these hybrids for the transfer of electrons to nitrogenase. This system is probably also involved in maintaining the membrane in the energized state, thereby allowing nitrogen fixation to occur. The nitrate-reduction system, which can energize the membrane like the fumarate-reduction system, is not necessary for nitrogenase activity in the E. coli K12(F'nif+Kp) hybrids. However, two nitrate reductase genes, chlA, and chlB, are essential for inhibition of nitrogen fixation by nitrate. Moreover, nitrate inhibits nitrogenase activity and this inhibition is most probably effected through a regulator factor coded by chlA and chlB.     

Azide-resistant mutants ofAzorhizobium caulinodans with enhanced symbiotic effectiveness

Azide-resistant mutants ofAzorhizobium caulinodans strains Sb3, S78, SrR13 and SrS8 were isolated and screened for nitrate reductase activity. Selected nitrate reductase negative mutants were inoculated onSesbania bispinosa andS. rostrata under sterile conditions in chillum jars to study their symbiotic behavior. Azide-resistant mutants exhibited either similar or higher symbiotic effectiveness than the parent strain after 30 d of plant growth. Nodule mass, nitrogenase activity and uptake hydrogenase activity of the mutants varied depending on the host as well as on the plant growth stage. In comparison to wild-type parent strains, four azide-resistant mutants, Sb3Az18, S78Az21, SrR13Az17 and SrS8Az6 showed significant increase in nodulation and nitrogen fixation as well as shoot dry mass of the inoculated plants.     

Biosynthesis of the iron-molybdenum cofactor and the molybdenum cofactor in Klebsiella pneumoniae: effect of sulfur source.

NifQ- and Mol- mutants of Klebsiella pneumoniae show an elevated molybdenum requirement for nitrogen fixation. Substitution of cystine for sulfate as the sulfur source in the medium reduced the molybdenum requirement of these mutants to levels required by the wild type. Cystine also increased the intracellular molybdenum accumulation of NifQ- and Mol- mutants. Cystine did not affect the molybdenum requirement or accumulation in wild-type K. pneumoniae. Sulfate transport and metabolism in K. pneumoniae were repressed by cystine. However, the effect of cystine on the molybdenum requirement could not be explained by an interaction between sulfate and molybdate at the transport level. Cystine increased the molybdenum requirement of Mol- mutants for nitrate reductase activity by at least 100-fold. Cystine had the same effect on the molybdenum requirement for nitrate reductase activity in Escherichia coli ChlD- mutants. This shows that cystine does not have a generalized effect on molybdenum metabolism. Millimolar concentrations of molybdate inhibited nitrogenase and nitrate reductase derepression with sulfate as the sulfur source, but not with cystine. The inhibition was the result of a specific antagonism of sulfate metabolism by molybdate. The effects of nifQ and mol mutations on nitrogenase could be suppressed either by the addition of cystine or by high concentrations of molybdate. This suggests that a sulfur donor and molybdenum interact at an early step in the biosynthesis of the iron-molybdenum cofactor. This interaction might occur nonenzymatically when the levels of the reactants are high.     

Temperature sensitive nitrogen fixation mutants of Azotobacter vinelandii

Summary Temperature-sensitive nitrogen fixation mutants of Azotobacter vinelandii were obtained by nitrosoguanidine mutagenesis and penicillin selection. The mutants were unable to grow on N₂ at 39° but grew normally at 30° on N₂ and at both temperatures in the presence of metabolizable nitrogen compounds. Growth experiments and assays of whole cells for nitrogenase activity separated the mutants into two classes: 1. mutants in which the nitrogenase activity present in cells grown at 30° was unaffected by a shift to 39°, and 2. mutants which lost their nitrogen fixation activity after such a temperature shift. Assays of cell-free extracts of the second class of mutants showed that in all cases tested the enzymatic activity of the nitrogenase complex itself was not affected by the mutation. These mutants might therefore contain some other temperature-sensitive proteins specifically involved in nitrogen fixation.     

Nitrate reductase from bacteroides of Rhizobium japonicum: enzyme characteristics and possible interaction with nitrogen fixation.

The soluble nitrate reductase of Rhizobium japonicum bacteroids has been purified and its properties compared to those of aerobically grown cells. The enzymes from both sources are similar with molecular weights of about 70 000 suggesting no close relationship with the molybdo-protein component of nitrogenase. Nitrite, the product of nitrate reductase, strongly inhibited the nitrogenase activity from bacteroids, at concentrations less than 100 muM. Thus, an interference in the rate of nitrogen fixation is possible as a result of nitrate reductase activity. A study of the distribution of nitrate reductase in bacteroids indicates that a proportion of the total activity is membrane-bound but that this activity is similar to that in the soluble fraction. Purified nitrate reductase required reduced viologen dyes for activity. Neither NADPH or NADH or FAD could substitute as electron donors. Dithionite is a strong inhibitor and inactivated nitrate reductase from all sources examined. This inactivation is prevented by methyl viologen. Purified nitrate reductase from bacteroids and bacteria Rhizobium japonicum is practically unaffected by exposure to oxygen.     

Inorganic nitrogen regulation of glutamate uptake in the cyanobacterium Nostoc muscorum

In Nostoc muscorum (Anabaena ATCC 27893) glutamate was not metabolised as a fixed nitrogen source, rather it functioned as an inhibitor of growth. The latter effect was nitrogen source specific and occurred in N₂-fixing cultures but not in cultures assimilating nitrate or ammonium. NO₃^--grown cultures lacked heterocysts and nitrogenase activity and showed a nearly 50% reduction in glutamate uptake rates, as well as in the final extent of glutamate taken up, compared to N₂-fixing or nitrogen-limited control cultures. NH₄⁺-grown cultures showed a similar response, except that the reduction in glutamate uptake rates and the final exten of glutamate taken up was over 80%. The present results suggest a relation between nitrate/ammounium nitrogen-dependent inhibition of glutamate uptake, probably via repression of the glutamate transport system, and glutamate toxicity.     

Nitrogen fixation in transposon mutants from Bradyrhizobium japonicum USDA 110 impaired in nitrate reductase

Tn5 transposon mutagenesis was carried out in Bradyrhizobium japonicum strain USDA 110 to produce defective mutants. From over one thousand clones expressing low levels of nitrate reductase activity as free-living bacteria, approximately five percent had significantly different ratios of nodulation, N2 fixation or nitrate reductase activity compared to the wild strain when determined in bacteroids from soybean nodules. Tn5 insertions were checked previously and mutants were arranged into four different groups. Only one of these groups, designated AN, was less effective at N2 fixation than the wild strain, suggesting a mutation in a domain shared by nitrogenase and NR. The remaining groups of insertions successfully nodulated and were as effective at N2 fixation as the wild strain, but showed diminished ability to reduce nitrate both in nodules and in the isolated bacteroids when assayed in vitro with NADH or methyl viologen as electron donors. PCR amplification demonstrated that Tn5 insertions took place in different genes on each mutant group and the type of mutant (CC) expressing almost no nitrate reductase activity under all treatments seemed to possess transposable elements in two genes. Induction of nitrate reductase activity by nitrate was observed only in those clones expressing a low constitutive activity (AN and AE). Nitrate reductase activity in bacteroids along nodule growth decreased in all groups including the ineffective AN group, whose nodulation was highly inhibited by nitrate at 5 mmol/L N. Host-cultivar interaction seemed to influence the regulation of nitrate reductase activity in bacteroids. Total or partial repression of nitrate reductase activity in bacteroids unaffected by N2 fixation (CC, AJ and AE groups) improved nodule resistance to nitrate and N yields of shoots over those of the wild strain. These observations may suggest that some of the energy supplied to bacteroids was wasted by its constitutive NRA.     

Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium

The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (mol C₂H₄g^–1 nodule fr. wt. h^–1), were both affected by the added nitrate.     

Regulation of nitrogen fixation. Nitrogenase-derepressed mutants of Klebsiella pneumoniae.

1. A new procedure is described for selecting nitrogenase-derepressed mutants based on the method of Brenchley et al. (Brenchley, J.E., Prival, M.J. and Magasanik, B. (1973) J. Biol. Chem. 248, 6122-6128) for isolating histidase-constitutive mutants of a non-N2-fixing bacterium. 2. Nitrogenase levels of the new mutants in the presence of NH4+ were as high as 100% of the nitrogenase activity detected in the absence of NH4+. 3. Biochemical characterization of these nitrogen fixation (nif) derepressed mutants reveals that they fall into three classes. Three mutants (strains SK-24, 28 and 29), requiring glutamate for growth, synthesize nitrogenase and glutamine synthetase constitutively (in the presence of NH4+). A second class of mutants (strains SK-27 and 37) requiring glutamine for growth produces derepressed levels of nitrogenase activity and synthesized catalytically inactive glutamine synthetase protein, as determined immunologically. A third class of glutamine-requiring, nitrogenase-derepressed mutants (strain SK-25 and 26) synthesizes neither a catalytically active glutamine synthetase enzyme nor an immunologically cross-reactive glutamine synthetase protein. 4. F-prime complementation analysis reveals that the mutant strains SK-25, 26, 27, 37 map in a segment of the Klebsiella chromosome corresponding to the region coding for glutamine synthetase. Since the mutant strains SK-27 and SK-37 produce inactive glutamine synthetase protein, it is concluded that these mutations map within the glutamine synthetase structural gene.