Effect of ammonia on the synthesis and function of the N 2 -fixing enzyme system in Clostridium pasteurianum

The N(2)-fixing system of Clostridium pasteurianum operates under regulatory controls; no activity is found in cultures growing on excess NH(3). The conditions which are necessary for the synthesis and function of this system were studied in whole cells by using acetylene reduction as a sensitive assay for the presence of the N(2)-fixing system. Nitrogenase of N(2)-fixing cultures normally can fix twice as much N(2) as is needed to maintain the growth rate. When cultures that have grown for four or more generations on NH(3) exhaust NH(3) from the medium, a diauxic lag of about 90 min ensues before growth is resumed on N(2). Neither N(2)-fixing nor acetylene reduction activity can be detected before growth is resumed on N(2). N(2) is not a necessary requirement for this synthesis since under argon that contains less than 10(-8)m N(2), the N(2)-fixing system is made. If NH(3) is added to N(2)-dependent cultures, synthesis of the enzyme system is abruptly stopped, but the enzyme already present remains stable and functional for at least 6 hr (over three generations). Cultures grown under argon in a chemostat controlled by limiting ammonia have derepressed nitrogenase synthesis. If the argon is removed and replaced by N(2), partial repression of nitrogenase occurs.     

Feedback inhibition of nitrogenase.

No inhibition of nitrogenase activity by physiological levels of NH4+ or carbamyl phosphate was observed in extracts of Azotobacter vinelandii. All of the 15N2 reduced by cultures which received no NH4+ was found in the cells. By contrast, more than 95% of the 15N2 reduced by cultures which had been given NH4+ was found in the medium. Failure to examine the culture medium would lead to the erroneous conclusion that N2 fixation is inhibited by NH4+. Nitrogenase in a derepressed mutant strain of A. vinelandii was fully active in vivo in the presence of NH4+. The addition of NH4Cl to N2-fixing cultures resulted in no decrease in the N2-reducing activity of intact cells of Klebsiella pneumoniae or Clostridium pasteurianum and only a small (15%) decrease in A. vinelandii. Therefore, no significant inhibition of nitrogenase by NH4+ or metabolites derived from NH4+ exists in A. vinelandii, K. pneumoniae, or C. pasteurianum.     

Sucrose Catabolism in Clostridium pasteurianum and Its Relation to N2 Fixation

The growth constant and Y (sucrose) (grams of cells per mole of sucrose) for NH(3)-grown cultures of Clostridium pasteurianum were 1.7 times those of N(2)-grown cultures, whereas the rate of sucrose utilized per gram of cells per hour was similar for both conditions. The Y (sucrose) of chemostat cultures grown on limiting NH(3) under argon at generation times equal to those of N(2)-fixing cultures was less than that of cultures grown on excess NH(3), but cells of NH(3)-limited cultures contained the N(2)-fixing system in high concentration. The concentration of the N(2)-fixing system in whole cells, when measured with adenosine triphosphate (ATP) nonlimiting, was more than twofold greater than the amount needed for the N(2) actually fixed. Thus, energy production from sucrose, and not the concentration of the N(2)-fixing system nor the maximal rate at which N(2) could be fixed, was the limiting factor for growth of N(2)-fixing cells. Either NH(3) or some product of NH(3) metabolism partially regulated the rate of sucrose metabolism since, when cultures fixing N(2), growing on NH(3), or growing on limiting NH(3) in the absence of N(2) were deprived of their nitrogen source, the rate of sucrose catabplism decreased. Calculations showed that the rate of ATP production was the growth rate-limiting factor in cells grown on N(2), and that the increased sucrose requirement of N(2)-fixing cultures in part reflected the energy demand of N(2) fixation. Calculations indicated that whole cells require about 20 moles of ATP for the fixation of 1 mole of N(2) to 2 moles of NH(3).     

Photoproduction of ammonium ion from N2 in Rhodospirillum rubrum.

NH+4 excretion was undetectable in N2-fixing cultures of Rhodospirillum rubrum (S-1) and nitrogenase activity in these cultures was repressed by the addition of 10 mM NH+4 to the medium. The glutamate analog, L-methionine-DL-sulfoximine (MSX), derepressed N2 fixation even in the presence of 10 mM extracellular NH+4. When 10 mg MSX/ml was added to cultures just prior to nitrogenase induction they developed nitrogenase activity (20% of the control activities) and excreted most of their fixed N2 as NH+4. Nitrogenase activities and NH+4 production from fixed N2 were increased considerably when a combined nitrogen source, NH+4 (greater than 40 mumoles NH+4/mg cell protein in 6 days) or L-glutamate (greater than 60 mumoles NH+4/ mg cell protein in 6 days) was added to the cultures together with MSX. Biochemical analysis revealed that R. rubrum produced glutamine synthetase and glutamate synthase (NADP-dependent) but no detectable NADP-dependent glutamate dehydrogenase. The specific activity of glutamine synthetase was observed to be maximal when nitrogenase activity was also maximal. Nitrogenase and glutamine synthetase activities were repressed by NH+4 as well as by glutamate. The results demonstrate that utilization of solar energy to photoproduce large quantities of NH+4 from N2 is possible with photosynthetic bacteria by interfering with their regulatory control of N2 fixation.     

Regulation of molybdate transport by Clostridium pasteurianum.

The regulation of the molybdate (MoO42-) transport activity of Clostridium pasteurianum has been studied by observing the effects of NH3, carbamyl phosphate, MoO42-, and chloramphenicol on the ability of cells to take up MoO42-. Compared with cells fixing N2, cells grown in the presence of 1 mM NH3 are greater than 95% repressed for MoO42- transport. Uptake activity begins to increase just before NH exhaustion (under Ar or N2) and continues to increase throughout the lag period as cells shift from NH3-growing to N2-fixing conditions. When cells are shifted from N2-fixing to NH3-growing conditions the transport activity per fixed number of cells decreases by increase of bells in absence of transport synthesis. Carbamyl phosphate (greater than or equal to 15 mM) but not NH3 inhibits 58% of the in vitro uptake activity. When 1 mM carbamyl phosphate is added just before the exhaustion of NH3, the transport activity, measured 2 h later, is 100% repressed. Cells grown in the presence of high MoO42- (1mM) are 80% repressed for MoO42- transport. Synthesis of the MoO42- transport system is also completely stopped when chloramphenicol (300 mug/ml) is added just before the exhaustion oNH 3 from the medium. These findings demonstrate that the ability of cells to transport MoO42- is dependent upon new protein synthesis and can be repressed by high levels of substrate. The regulation of MoO42- uptake by NH3 or carbamyl phosphate closely parallels the regulation of nitrogenase activity. Activity of neither nitrogenase component (Fe protein or MoFe protein) was detected even 3 h after the exhaustion of the NH3 if either MoO42- was absent or if WO42- was present in place of MoO42-. The duration of the diauxic lag increases with decreasing concentration of MoO42- in the medium. If no MoO42- is present the lag continues indefinitely. If MoO42- is added late in the lag period, growth under N2-fixing conditions resumes but only after a normal induction period.     

Role of molybdenum in dinitrogen fixation by Clostridium pasteurianum.

The role of Mo in the activity and synthesis of the nitrogenase components of Clostridium pasteurianum has been studied by observing the competition of Mo with its structural analogue W. Clostridial cells when fixing N2 appeared strictly dependent upon the available Mo, showing maximal N2-fixing activity at molybdate concentrations in the media of 10 muM. Cells grown in media with 3 times 10(-6) muM Mo, although showing good growth, had only 15% as much N2-fixing activity. In the presence of W the synthesis of both nitrogenase components, molybdoferredoxin and azoferredoxin, was affected. Attempts to produce nitrogenase in W-grown cells by addition of high molybdenum to the media in the presence of inhibitors of protein synthesis showed that Mo incorporation into a possible inactive preformed apoenzyme did not occur. Unlike other molybdoenzyme-containing cells, in which W either is incorporated in place of Mo to yield inactive protein or initiates the production of apoprotein, C. pasteurianum forms neither a tungsten substituted molybdoferredoxin nor an apoprotein. It is concluded that in C. pasteurianum molybdenum is an essential requirement for both the biosynthesis and activity of its nitrogenase.     

Identification of molybdoproteins in Clostridium pasteurianum.

Cells of Clostridium pasteurianum whose N source is switched from NH3 to N2 accumulate large amounts of molybdenum beginning 1.5 h before the detection of nitrogenase activity. Anaerobic multiphasic gel electrophoresis and anion-exchange chromatography were used to identify the molybdoproteins and molybdenum-containing components present in N2-fixing cells. In addition to molybdate, six distinct 99Mo-labeled species were detected, i.e., a membrane fragment, the MoFe protein of nitrogenase, formate dehydrogenase, a Mo "binding-storage" protein, a 30-kilodalton molybdoprotein, and a low-molecular-weight molybdenum species. Of these, the MoFe protein, formate dehydrogenase, and the Mo binding-storage protein were present in more than one zone because of complex formation with other proteins, partial denaturation, and variation in the amount of Mo bound to the protein, respectively. In addition to the six proteins, a soluble "free" Mo cofactor in the cytosol was detected by showing that it reconstituted nitrate reductase activity in crude extracts of the Neurospora crassa mutant nit-1.     

Glutamine synthetase and nitrogen cycling in colonies of the marine diazotrophic cyanobacteria Trichodesmium spp.

We examined freshly collected samples of the colonial planktonic cyanobacterium Trichodesmium thiebautii to determine the pathways of recently fixed N within and among trichomes. High concentrations of glutamate and glutamine were found in colonies. Glutamate and glutamine uptake rates and concentrations in cells were low in the early morning and increased in the late morning to reach maxima near midday; then uptake and concentration again fell to low values. This pattern followed that previously observed for T. thiebautii nitrogenase activity. Our results suggest that recently fixed nitrogen is incorporated into glutamine in the N2-fixing trichomes and may be passed as glutamate to non-N2-fixing trichomes. The high transport rates and concentrations of glutamate may explain the previously observed absence of appreciable uptake of NH4+, NO3-, or urea by Trichodesmium spp. Immunolocalization, Western blots (immunoblots), and enzymatic assays indicated that glutamine synthetase (GS) was present in all cells during both day and night. GS appeared to be primarily contained in cells of T. thiebautii rather than in associated bacteria or cyanobacteria. Double immunolabeling showed that cells with nitrogenase (Fe protein) contained levels of the GS protein that were twofold higher than those in cells with little or no nitrogenase. GS activity and the uptake of glutamine and glutamate dramatically decreased in the presence of the GS inhibitor methionine sulfoximine. Since no glutamate dehydrogenase activity was detected in this species, GS appears to be the primary enzyme responsible for NH3 incorporation.     

Glutamine synthetase and nitrogen cycling in colonies of the marine diazotrophic cyanobacteria Trichodesmium spp.

We examined freshly collected samples of the colonial planktonic cyanobacterium Trichodesmium thiebautii to determine the pathways of recently fixed N within and among trichomes. High concentrations of glutamate and glutamine were found in colonies. Glutamate and glutamine uptake rates and concentrations in cells were low in the early morning and increased in the late morning to reach maxima near midday; then uptake and concentration again fell to low values. This pattern followed that previously observed for T. thiebautii nitrogenase activity. Our results suggest that recently fixed nitrogen is incorporated into glutamine in the N2-fixing trichomes and may be passed as glutamate to non-N2-fixing trichomes. The high transport rates and concentrations of glutamate may explain the previously observed absence of appreciable uptake of NH4+, NO3-, or urea by Trichodesmium spp. Immunolocalization, Western blots (immunoblots), and enzymatic assays indicated that glutamine synthetase (GS) was present in all cells during both day and night. GS appeared to be primarily contained in cells of T. thiebautii rather than in associated bacteria or cyanobacteria. Double immunolabeling showed that cells with nitrogenase (Fe protein) contained levels of the GS protein that were twofold higher than those in cells with little or no nitrogenase. GS activity and the uptake of glutamine and glutamate dramatically decreased in the presence of the GS inhibitor methionine sulfoximine. Since no glutamate dehydrogenase activity was detected in this species, GS appears to be the primary enzyme responsible for NH3 incorporation.     

Modeling the dynamic regulation of nitrogen fixation in the cyanobacterium Trichodesmium sp

A physiological, unbalanced model is presented that explicitly describes growth of the marine cyanobacterium Trichodesmium sp. at the expense of N(2) (diazotrophy). The model involves the dynamics of intracellular reserves of carbon and nitrogen and allows the uncoupling of the metabolism of these elements. The results show the transient dynamics of N(2) fixation when combined nitrogen (NO(3)(-), NH(4)(+)) is available and the increased rate of N(2) fixation when combined nitrogen is insufficient to cover the demand. The daily N(2) fixation pattern that emerges from the model agrees with measurements of rates of nitrogenase activity in laboratory cultures of Trichodesmium sp. Model simulations explored the influence of irradiance levels and the length of the light period on fixation activity and cellular carbon and nitrogen stoichiometry. Changes in the cellular C/N ratio resulted from allocations of carbon to different cell compartments as demanded by the growth of the organism. The model shows that carbon availability is a simple and efficient mechanism to regulate the balance of carbon and nitrogen fixed (C/N ratio) in filaments of cells. The lowest C/N ratios were obtained when the light regime closely matched nitrogenase dynamics.     

Ammonium inhibition of nitrogenase activity in Herbaspirillum seropedicae.

The effect of oxygen, ammonium ion, and amino acids on nitrogenase activity in the root-associated N2-fixing bacterium Herbaspirillum seropedicae was investigated in comparison with Azospirillum spp. and Rhodospirillum rubrum. H. seropedicae is microaerophilic, and its optimal dissolved oxygen level is from 0.04 to 0.2 kPa for dinitrogen fixation but higher when it is supplied with fixed nitrogen. No nitrogenase activity was detected when the dissolved O2 level corresponded to 4.0 kPa. Ammonium, a product of the nitrogenase reaction, reversibly inhibited nitrogenase activity when added to derepressed cell cultures. However, the inhibition of nitrogenase activity was only partial even with concentrations of ammonium chloride as high as 20 mM. Amides such as glutamine and asparagine partially inhibited nitrogenase activity, but glutamate did not. Nitrogenase in crude extracts prepared from ammonium-inhibited cells showed activity as high as in extracts from N2-fixing cells. The pattern of the dinitrogenase and the dinitrogenase reductase revealed by the immunoblotting technique did not change upon ammonium chloride treatment of cells in vivo. No homologous sequences were detected with the draT-draG probe from Azospirillum lipoferum. There is no clear evidence that ADP-ribosylation of the dinitrogenase reductase is involved in the ammonium inhibition of H. seropedicae. The uncoupler carbonyl cyanide m-chlorophenylhydrazone decreased the intracellular ATP concentration and inhibited the nitrogenase activity of whole cells. The ATP pool was not significantly disturbed when cultures were treated with ammonium in vivo. Possible mechanisms for inhibition by ammonium of whole-cell nitrogenase activity in H. seropedicae are discussed.     

Effect of amino acids on the nitrogenase system of Klebsiella pneumoniae

Yoch, D. C. (South Dakota State University, Brookings), and R. M. Pengra. Effect of amino acids on the nitrogenase system of Klebsiella pneumoniae. J. Bacteriol. 92:618-622. 1966.-The effect of exogenous amino acids and the free amino acid pool on the synthesis of the nitrogenase system of Klebsiella pneumoniae M5al (formerly Aerobacter aerogenes M5al) was investigated. When an actively N(2)-fixing culture was used to inoculate a medium containing a limiting concentration of NH(4) (+), an induction lag period was observed. When either a single amino acid or a mixture of amino acids was substituted at the same nitrogen concentration, growth was uninterrupted by the induction period. It appears that a step or steps in the formation of the nitrogenase system are repressed by NH(4) (+) and are not affected by amino acid N. The amino acids, far from repressing formation of nitrogenase as does NH(4) (+), actually stimulate its formation. It appears that both free and amino nitrogen are used simultaneously. The amino acids that served concomitantly with N(2) as a source of nitrogen were: aspartic acid, serine, threonine, leucine, and histidine. Of these amino acids, it was shown that aspartic acid is readily taken up by the cells. Of the amino acids not serving as an immediate nitrogen source, isoleucine is not taken up by the cells. The free amino acid pool of the cells was measured at the onset and termination of the induction period. Ninhydrin-positive material in the amino acid pool was depleted by 35% during the induction period.     

Enzymes of ammonia assimilation in hyphae and vesicles of Frankia sp. strain CpI1.

Frankia spp. are filamentous actinomycetes that fix N2 in culture and in actinorhizal root nodules. In combined nitrogen-depleted aerobic environments, nitrogenase is restricted to thick-walled spherical structures, Frankia vesicles, that are formed on short stalks along the vegetative hyphae. The activities of the NH4(+)-assimilating enzymes (glutamine synthetase [GS], glutamate synthase, glutamate dehydrogenase, and alanine dehydrogenase) were determined in cells grown on NH4+ and N2 and in vesicles and hyphae from N2-fixing cultures separated on sucrose gradients. The two frankial GSs, GSI and GSII, were present in vesicles at levels similar to those detected in vegetative hyphae from N2-fixing cultures as shown by enzyme assay and two-dimensional polyacrylamide gel electrophoresis. Glutamate synthase, glutamate dehydrogenase, and alanine dehydrogenase activities were restricted to the vegetative hyphae. Vesicles apparently lack a complete pathway for assimilating ammonia beyond the glutamine stage.     

Effect of nitrogen starvation on ammonium-inhibition of nitrogenase activity in Azotobacter chroococcum

When Azotobacter chroococcum cells grown in batch culture under N₂-fixing conditions were transferred to a medium lacking a nitrogen source, the cellular C/N ratio, the amount of alginic acid released into the external medium and the rate of endogenous respiration increased appreciably after 6 h to the exclusion of dinitrogen, whereas nitrogenase activity did not undergo any significant change. Nitrogen deficiency caused a decrease in the ammonium inhibition of nitrogenase activity from 95% inhibition at zero time to 14% after 6 h incubation under dinitrogen starvation, with no difference in the rate of ammonium utilization by N₂-fixing and N₂-starved cells being observed. This suggests that a balance of nitrogen and carbon assimilation is necessary for the ammonium inhibition of nitrogenase activity in A. chroococcum to take place.     

Regulation of nitrogen fixation in Rhizobium sp.

Regulation of nitrogen fixation by ammonium and glutamate was examined in Rhizobium sp. 32H1 growing in defined liquid media. Whereas nitrogenase synthesis in Klebsiella pneunoniae is normally completely repressed during growth on NH4+, nitrogenase activity was detected in cultures of Rhizobium sp. grown with excess NH4+. However, an "ammonium effect" on activity was invariably observed in cultures grown on NH4+ as sole nitrogen source; the nitrogenase activity was, depending on conditions, 14 to 36% of that of comparable glutamate-grown cultures. Glutamate inhibited utilization of exogenous NH4+ and, in one of two procedures described, glutamate partially alleviated the ammonium effect on nitrogenase activity. NH4+, apparently produced from N2, was excreted into the culture medium when growth was initiated on glutamate, but not when NH4+ was thesole source of fixed nitrogen for growth. These findings are discussed in relation to nitrogen fixation by Rhizobium bacteroids.     

Growth of Spirillum lipoferum at constant partial pressures of oxygen, and the properties of its nitrogenase in cell-free extracts.

Spirillum lipoferum, an N2-fixing organism, was grown at constant concentrations of dissolved O2. When supplied with NH4+ aerobically, its doubling time was 1 h; when it fixed N2 microaerophilically, its doubling time was 5-5 to 7 h and the optimal PO2 for growth was 0-005 to 0-007 atm. At its optimal PO2 for growth on N2, S. lipoferum assimilated 8 to 10 mg nitrogen/g carbon substrate used; its efficiency was less at higher PO2 levels. Nitrogenase in cell-free extracts required Mg2+ and Mn2+, and the Fe-protein was activated by Rhodospirillum rubrum activating factor. The nitrogenase had an optimal pH of 7-1 to 7-4 and an apparent Km for acetylene of 0-0036 atm. Extracts of S. lipoferum lost their nitrogenase activity on storage at -18 degrees C, and activity was restored by adding purified Fe-protein from other N2-fixing bacteria.     

Interaction between hydrogenase, nitrogenase, and respiratory activities in a Frankia isolate from Alnus rubra

H2 uptake and H2-supported O2 uptake were measured in N2-fixing cultures of Frankia strain ArI3 isolated from root nodules of Alnus rubra. H2 uptake by intact cells was O2 dependent and maximum rates were observed at ambient O2 concentrations. No hydrogenase activity could be detected in NH4+-grown, undifferentiated filaments cultured aerobically indicating that uptake hydrogenase activity was associated with the vesicles, the cellular site of nitrogen fixation in Frankia. Hydrogenase activity was inhibited by acetylene but inhibition could be alleviated by pretreatment with H2. H2 stimulated acetylene reduction at supraoptimal but not suboptimal O2 concentrations. These results suggest that uptake hydrogenase activity in ArI3 may play a role in O2 protection of nitrogenase, especially under conditions of carbon limitation.     

Nodulated N2-fixing Casuarina cunninghamiana is the sink for net N transfer from non-N2-fixing Eucalyptus maculata via an ectomycorrhizal fungus Pisolithus sp. using 15NH4+ or 15NO3- supplied as ammonium nitrate

To determine the effects of nitrogen source on rates of net N transfer between plants connected by a common mycorrhizal network, we measured transfer of N supplied as 15NH4 14NO3 or 14NH4 15NO3 in three Casuarina/Eucalyptus treatments interconnected by a Pisolithus sp. The treatments were nonnodulated nonmycorrhizal/nonmycorrhizal; nonnodulated mycorrhizal/mycorrhizal; and nodulated mycorrhizal/mycorrhizal. Mycorrhization was 67% in Eucalyptus and 36% in Casuarina. N2 fixation supplied 38% of the N in Casuarina. Biomass, N and 15N contents were lowest in nonmycorrhizal plants and greatest in plants in the nodulated/mycorrhizal treatment. Nitrogen transfer was enhanced by mycorrhization and by nodulation, and was greater when N was supplied as 15NH4+ than 15NO3-. Nitrogen transfer rates were lowest in the nonmycorrhizal treatment for either 15N source, and greatest in the nodulated, mycorrhizal treatment. Transfer was greater to Casuarina than to Eucalyptus and where ammonium rather than nitrate was the N source. Irrespective of 15N source and of whether Casuarina or Eucalyptus was the N sink, net N transfer was low and was similar in both nonnodulated treatments. However, when Casuarina was the N sink in the nodulated, mycorrhizal treatment, net N transfer was much greater with 15NH4+ than with 15NO3-. High N demand by Casuarina resulted in greater net N transfer from the less N-demanding Eucalyptus. Net transfer of N from a non-N2-fixing to an N2-fixing plant may reflect the very high N demand of N2-fixing species.     

Interactions between nitrogen fixation and osmoregulation in the methanogenic archaeon methanosarcina barkeri 227

The nitrogenase enzyme complex of Methanosarcina barkeri 227 was found to be more sensitive to NaCl than previously studied molybdenum nitrogenases are, with total inhibition of activity occurring at 190 mM NaCl, compared with >600 mM NaCl for Azotobacter vinelandii and Clostridium pasteurianum nitrogenases. Na+ and K+ had equivalent effects, whereas Mg2+ was more inhibitory than either monovalent cation, even on a per-charge basis. The anion Cl- was more inhibitory than acetate was. Because M. barkeri 227 is a facultative halophile, we examined the effects of external salt on growth and diazotrophy and found that inhibition of growth was not greater with N2 than with NH4+. Cells grown with N2 and cells grown with NH4+ produced equal concentrations of alpha-glutamate at low salt concentrations and equal concentrations of Nepsilon-acetyl-beta-lysine at NaCl concentrations greater than 500 mM. Despite the high energetic cost of fixing nitrogen for these osmolytes, we obtained no evidence that there is a shift towards nonnitrogenous osmolytes during diazotrophic growth. In vitro nitrogenase enzyme assays showed that at a low concentration (approximately 100 mM) potassium glutamate enhanced activity but at higher concentrations this compound inhibited activity; 50% inhibition occurred at a potassium glutamate concentration of approximately 400 mM.