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.     

Effect of an ntrC mutation on amino acid or urea utilization and on nitrogenase switch-off in Herbaspirillum seropedicae

Herbaspirillum seropedicae is a nitrogen-fixing bacterium that grows well with ammonium chloride or sodium nitrate as alternative single nitrogen sources but that grows more slowly with L-alanine, L-serine, L-proline, or urea. The ntrC mutant strain DCP286A was able to utilize only ammonium or urea of these nitrogen sources. The addition of 1 mmol.L-1 ammonium chloride to the nitrogen-fixing wild-type strain inhibited nitrogenase activity rapidly and completely. Urea was a less effective inhibitor; approximately 20% of nitrogenase activity remained 40 min after the addition of 1 mmol x L-1 urea. The effect of the ntrC mutation on nitrogenase inhibition (switch-off) was studied in strain DCP286A containing the constitutively expressed gene nifA of H. seropedicae. In this strain, nitrogenase inhibition by ammonium was completely abolished, but the addition of urea produced a reduction in nitrogenase activity similar to that of the wild-type strain. The results suggest that the NtrC protein is required for assimilation of nitrate and the tested amino acids by H. seropedicae. Furthermore, NtrC is also necessary for ammonium-induced switch-off of nitrogenase but is not involved in the mechanism of nitrogenase switch-off by urea.     

Nitrogenase in synchronized Azotobacter vinelandii OP.

Azotobacter vinelandii OP was synchronized by the continuous phased culture technique. The nitrogenase (nitrogen:(acceptor)oxidoreductase)(EC 1.7.99.2) activity of the culture was determined continuously within the fermentor by acetylene reduction. Addition of NH4+ in excess of 5 x 10(-3)M to the culture lowered nitrogenase activity immediately. Other sources of fixed nitrogen had no immediate effect on nitrogenase activity, but nitrogenase synthesis decreased in the cell cycle following the one in which the fixed nitrogen was added.     

Isolation, sequencing, and mutagenesis of the nifF gene encoding flavodoxin from Azotobacter vinelandii

The nifF gene encoding flavodoxin from Azotobacter vinelandii OP was cloned and its DNA sequence determined. It is located adjacent to, or possibly within, the major nif cluster and it is preceded by nif-specific regulatory elements. Southern hybridization analysis revealed that there is only a single copy of the nifF gene on the A. vinelandii OP genome. Mutant strains were constructed which have an insertion mutation or an insertion and a deletion mutation within the nifF gene coding sequence. These mutant strains are capable of diazotrophic growth, indicating that flavodoxin is not the unique physiological electron donor to nitrogenase. The results of nifF-lacZYA gene fusion experiments and Northern hybridization analyses indicated that the nifF gene is both transcribed and translated under nitrogen fixing and non-nitrogen fixing conditions. However, under nitrogen fixing conditions a substantial increase in both nifF synthesis and in accumulation of an approximately 800-base pair nifF-encoding mRNA species was observed. Furthermore, strains mutated within the nifF gene have only 70% of the wild type in vivo nitrogenase activity as determined by whole cell acetylene reduction assays. These data demonstrate that the nifF-encoded flavodoxin of A. vinelandii OP, although not essential for nitrogen fixation, is required for maximum in vivo nitrogenase activity.     

Control of Nitrogenase mRNA Levels by Products of Nitrate Assimilation in the Cyanobacterium Anabaena sp. Strain PCC 7120

Nitrate inhibited nitrogenase synthesis and heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120. Inhibition of dinitrogen fixation by nitrate did not take place, however, in nitrate reductase-deficient derivatives of this strain. Hybridization of total RNA isolated from cells grown on different nitrogen sources with an internal fragment of the nifD gene showed that regulation of nitrogenase activity by nitrate is exerted through a negative control of the nitrogenase mRNA levels.     

Oxygen inactivation and recovery of nitrogenase activity in cyanobacteria.

Exposure of nitrogen-fixing cultures of Anabaena spp. to 100% oxygen resulted in the rapid decline of nitrogenase activity. When oxygen-treated cells were transferred to 100% argon, nitrogenase activity was quickly restored in a process that required protein synthesis. Anaerobiosis was not essential for the recovery process; in fact, cells of Anabaena sp. strains CA and 1F will recover nitrogenase activity after prolonged incubation in 100% oxygen. Oxygen treatment acted directly on the intracellular nitrogenase and did not affect other metabolic processes. Examination of crude extracts of oxygen-treated Anabaena sp. strain CA indicated that both components of nitrogenase are inactivated. However, several lines of evidence suggest that oxygen treatment does not result in irreversible denaturation of nitrogenase, but rather results in a reversible inactivation which may serve as a protection mechanism. Nitrogenase present in crude extracts from cells of Anabaena sp. strain 1F which had been incubated for a prolonged period in 100% oxygen was less sensitive to oxygen in vitro than was nitrogenase of a crude extract of untreated cells.     

Growth physiology of a marine nitrogen-fixing cyanobacterium (Nodularia harveyana) in outdoor culture

The performance ofNodularia harveyana, a N₂-fixing cyanobacterium isolated from seawater, has been studied outdoors in two different culture systems: open pond (OP) and tubular photobioreactor (TPR). The productivity in both devices was influenced by areal density. The maximum yield obtained was 12.0 g (d.wt) m^–2 day^–1 in OP and 14.0 g (d.wt) m^–2 day^–1 in TPR in August, corresponding to the highest solar radiation received. In a month-long experiment with the cyanobacterium cultivated in TPR at high circulation speed, a net increase in productivity was obtained over that at low circulation speed. The influence of temperature on the productivity of the cultures grown in open ponds and tubular photobioreactors has been investigated. The higher productivity obtained in TPR compared to OP was attributed to its better controlled temperature conditions. In outdoor culture the maximum nitrogenase activity did not coincide with the maximum light intensity, but occurred in early afternoon. The amount of carbohydrate accumulated during the day probably influenced the rate of dark nitrogenase activity and its duration in the night.     

Oxydation of substrates in organic acids utilization negative mutants and the wild typeRhizobium meliloti strain S'14

Two mutants defective in succinate utilization were isolated by NTG mutagenesis of the effective wild typeRhizobium meliloti strain S₁₄. The mutants used carbon sources in a fashion similar to strain S₁₄, but they were not able to grow on succinate, fumarate or malate. The mutants nodulated alfalfa plants but did not exhibit any nitrogenase activity. The mutants oxidized glucose and fructose, but were not able to oxidize organic acids. Cultured free-living bacteria of strain S₁₄ appeared to have an inducible C₄-dicarboxylic acid uptake system and a constitutive glucose uptake system. When S₁₄ cells were grown on glucose in the presence of 5mM or more succinate or malate, the rate of glucose-dependent O₂ consumption significantly decreased suggesting the presence of a catabolite repression like phenomenom. Contribution no. 301, Station de Recherches, Agriculture Canada.     

In vitro andin vivo nitrogenase activity ofRhizobium mutants and their symbiotic effectivity

A slow growing nitrogen-fixing strain ofVigna radiata var.aureus (mung bean)Rhizobium which expressed nitrogenase activity in a synthetic medium was isolated from its native population. Mutants with decreased and increased nitrogenase activity were derived from this strain by treatment with acridine orange and ethidium bromide. These mutants were tested for symbiotic effectivity invivo. The effectivity of mutants with decreased nitrogenase activity in the culture medium was lower than the parent strain; however, the effectivity of mutants with higher nitrogenase activity did not increase above that of the parent. This suggests that the plant is perhaps a limiting factor in the full expression of rhizobial nitrogenase in the nodules.     

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.     

Presence of a second mechanism for the posttranslational regulation of nitrogenase activity in Azospirillum brasilense in response to ammonium.

Although ADP-ribosylation of dinitrogenase reductase plays a significant role in the regulation of nitrogenase activity in Azospirillum brasilense, it is not the only mechanism of that regulation. The replacement of an arginine residue at position 101 in the dinitrogenase reductase eliminated this ADP-ribosylation and revealed another regulatory system. While the constructed mutants had a low nitrogenase activity, NH4+ still partially inhibited their nitrogenase activity, independent of the dinitrogenase reductase ADP-ribosyltransferase/dinitrogenase reductase activating glycohydrolase (DRAT/DRAG) system. These mutated dinitrogenase reductases also were expressed in a Rhodospirillum rubrum strain that lacked its endogenous dinitrogenase reductase, and they supported high nitrogenase activity. These strains neither lost nitrogenase activity nor modified dinitrogenase reductase in response to darkness and NH4+, suggesting that the ADP-ribosylation of dinitrogenase reductase is probably the only mechanism for posttranslational regulation of nitrogenase activity in R. rubrum under these conditions.     

Polypeptide synthesis by Rhizobium bacteroids and bacteria.

When Rhizobium bacteroids (strain NZP 2257) from lupin nodules were isolated and incubated aerobically at high osmolarity, they incorporated [35S]-methionine into a characteristic set of polypeptides; many of these polypeptides coelectrophoresed on SDS-polyacrylamide gels with the bacteroid polypeptide bands stained by Coomassie blue. The labelled polypeptides were stable for several hours in pulse-chase experiments. Changes in the concentration of H+, K+ and Mg2+ in the incubation mixture affected overall incorporation of label, but not the relative incorporation into different polypeptides. A similar set of bacteroid polypeptides was labelled in situ when detached nodules were fed [35S]methionine. Distinctive labelling patterns were observed with bacteroid suspensions from mature and immature nodules, with a transitional pattern at the time when nitrogenase activity appeared. Two of the major labelled components in mature bacteroids had estimated molecular weights of 60- and 34-kilodaltons similar to values reported by others for the constituent polypeptides of nitrogenase. Bacteroids of the same Rhizobium strain grown in different plant hosts gave similar polypeptide labelling patterns in purified suspensions, but bacteroids of different Rhizobium strains gave different patterns. The polypeptide labelling patterns obtained using broth-cultured Rhizobium bacteria from various growth stages and growth media differed from those obtained using bacteroids of the same strain.     

The role of formate metabolism in nitrogen fixation in Rhizobium Spp.

Formate metabolism supported nitrogen-fixation activity in free-living cultures of Rhizobium japonicum. However, formate0dependent nitrogense activity was observed only in the presence of carbon sources such as glutamate, ribose or aspartate which by themselves were unable to support nitrogenase activity. Formate-dependent nitrogenase activity was not detected in the presence of carbon sources such as malate, gluconate or glycerol which by themselves supported nitrogenase activity. A mutant strain of R. japonicum was isolated that was unable to utilise formate and was shown to lack formate dehydrogenase activity. This mutant strain exhibited no formate-dependent nitrogenase activity. Both the wild-type and mutant strains nodulated soybean plants effectively and there were no significant differences in the plant dry weight or total nitrogen content of the respective plants. Furthermore pea bacteroids lacked formate dehydrogenase activity and exogenously added formate had no stimulatory effect on the endogenous oxygen uptake rate. The role of formate metabolism in symbiotic nitrogen fixation is discussed.Abbreviation FDH formate dehydrogenase     

The effect of waterlogging on the synthesis of the nitrogenase components in bacteroids of Rhizobium leguminosarum in root nodules of Pisum sativum

The effect of waterlogging of root nodules on nitrogenase activity and synthesis was studied in Pisum sativum inoculated with Rhizobium leguminosarum (strain PRE). It was shown that: 1. nitrogenase activity of intact pea plants was decreased by waterlogging, 2. this decrease was paralleled by a decline of the amount of active nitrogenase determined in toluene EDTA treated bacteroids, 3. SDS-polyacrylamide gel electrophoresis revealed that the amount of nitrogenase component II (CII) decreased by waterlogging while the amount of component I (CI) was not markedly affected, and 4. analysis of bacteroid proteins after ³⁵SO₄ labeling of pea plants showed that CII synthesis was repressed while CI synthesis continued indicating that the synthesis of CI and CII is regulated by independent mechanisms.     

Nitrogenase activity in the non-heterocystous cyanobacterium Oscillatoria sp. grown under alternating light-dark cycles

The non-heterocystous cyanobacterium Oscillatoria sp. strain 23 fixes nitrogen under aerobic conditions. If nitrate-grown cultures were transferred to a medium free of combined nitrogen, nitrogenase was induced within about 1 day. The acetylene reduction showed a diurnal variation under conditions of continuous light. Maximum rates of acetylene reduction steadily increased during 8 successive days. When grown under alternating light-dark cycles, Oscillatoria sp. fixes nitrogen preferably in the dark period. For dark periods longer than 8 h, nitrogenase activity is only present during the dark period. For dark periods of 8 h and less, however, nitrogenase activity appears before the beginning of the dark period. This is most pronounced in cultures grown in a 20 h light – 4 h dark cycle. In that case, nitrogenase activity appears 3–4 h before the beginning of the dark period. According to the light-dark regime applied, nitrogenase activity was observed during 8–11 h. Oscillatoria sp. grown under 16 h light and 8 h dark cycle, also induced nitrogenase at the usual point of time, when suddenly transferred to conditions of continuous light. The activity appeared exactly at the point of time where the dark period used to begin. No nitrogenase activity was observed when chloramphenicol was added to the cultures 3 h before the onset of the dark period. This observation indicated that for each cycle, de novo nitrogenase synthesis is necessary.     

The role of O2-limitation in control of nitrogenase in continuous cultures of Rhizobrium sp.

Oxygen-limited continuous cultures of the cowpea $\text{Rhizobium}$ sp. strain CB756, had high levels of nitrogenase activity, which were not significantly affected by excess ammonium ions or glutamine. When the growth-restricting O₂-limitation was partially relieved, nitrogenase was repressed and this was accompanied by increased adenylylation of glutamine synthetase. It is suggested that the restricted supply of ATP interferes with adenylylation of glutamine synthetase during O₂-limited growth, thus preventing repression of nitrogenase in the presence of excess ammonium ions.     

Role of bacterial polysaccharides in the derepression of ex-planta nitrogenase activity with rhizobia

Abstract Since bacterial polysaccharides may limit the availability of oxygen to the cells, we have investigated the role of rhizobial extracellular polysaccharides (EPS) and the non-rhizobial polyscharide, xanthan, in the depression of ex-planta nitrogenase activity with rhizobia in liquid medium. Two rhizobial strains known to exhibit ex-planta nitrogenase activity on solid media were used; the slow-growing Bradyrhizobium japonicum USDA 110 and the arctic Rhizobium strain N31, both being prolific EPS producers. In low nitrogen mannitol (LNM) liquid medium strain N31 exhibited nitrogenase activity only after 15 days, when sufficient EPS had accumulated in the medium, and activity was correlated with EPS production. When rhizobial EPS from an old culture was added to the LNM medium, nitrogenase activity was detected after 48 h incubation, indicating that EPS of the medium decreased oxygen diffusion to cells to a level that depressed nitrogenase activity. In modified LNM medium with xanthan nitrogenase activity was readily depressed. In both strains activity increased with increased xanthan concentration, but decreased sharply at higher concentrations. Strain N31 exhibited a narrower range of polysaccharide concentration for nitrogenase activity than the slow strain USDA 110. Thus, the condition for derepression of nitrogenase might be a careful balancing of the oxygen concentration surrounding the cells, and this condition is met when a balancing of polsaccharide, either synthesized by the rhizobia or added to the medium, can permit oxygen diffusion to within the narrow range required for the depression and expression of nitrogenase.     

Soluble di- and aminopeptidases in Escherichia K-12. Dispensible enzymes.

As part of a study of the peptidase content of Escherichia coli K-12, two peptidase-deficient amino acid auxotrophs isolated and characterized by Miller as pepD- (strain CM17) and pepD- pepN- pepA- pepB- pepQ- (strain CM89) were examined for the presence of several peptidases previously obtained from strain K-12 in this laboratory. The soluble fraction of each mutant was found to lack the broad-specificity strain K-12 dipeptidase DP and the strain CM89 fraction also lacked activity characteristic of the strain K-12 aminopeptidases AP, L, and OP; like strain CM17, strain CM89 contained the tripeptide-specific aminopeptidase TP. Strain CM89 (but not CM17) appeared to contain little if any activity attributable to the ribosome-bound aminopeptidase I of strain K-12. Whereas loss of DP, AP, OP, and aminopeptidase I activity may be attributed to the pepD-, pepB-, pepN-, and pepA- mutations, respectively, the reason for the loss of L activity remains uncertain. Grown responses of strain CM89 in liquid media containing di- or tripeptides were in accord with absence of enzymes catalyzing rapid hydrolysis of dipeptides. In synthetic liquid media supplemented with the required amino acids per se or with peptone, cultures of both CM strains grew more slowly than strain K-12 and produced smaller cell-yields than those produced by strain K-12.