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.     

Mechanism of Carbamyl Phosphate Inhibition of Nitrogenase of Clostridium pasteurianum

Carbamyl phosphate caused a maximal inhibition of 50% of the in vitro nitrogenase activity measured by acetylene reduction and dinitrogen reduction. The addition of 1 mM carbamyl phosphate to a N(2)-fixing culture caused a rapid decrease of 30% of the acetylene reduction activity and also repression of nitrogenase biosynthesis. However, carbamyl phosphate had no effect on the reductant-dependent adenosine triphosphate hydrolysis and H(2) evolution reactions catalyzed by nitrogenase. Studies on the binding of carbamyl phosphate to nitrogenase and each of its two components (azoferredoxin and molybdoferredoxin) indicated that optimal binding was obtained only in the presence of an operating nitrogenase system. Moreover, the binding seemed to be on the molybdoferredoxin component rather than azoferredoxin. From a Scatchard plot and a reciprocal plot of the data, the values of n = 2 and dissociation constant (K) of approximately 5 x 10(-5) M were obtained. The value for the dissociation constant was of the same order of magnitude as the endogenous level of carbamyl phosphate in a N(2)-fixing cell. The carbamyl phosphate pool in NH(3)-grown cells was twice that of N(2)-fixing cells.     

Regulation and order of involvement of molybdoproteins during synthesis of molybdoenzymes in Clostridium pasteurianum.

The accumulation of 99Mo (from 99MoO4(2-) into molybdenum-containing species in Clostridium pasteurianum was investigated to identify the molybdoprotein(s) involved in Mo metabolism. Mo accumulation by clostridial cells during the derepression of the nitrogenase system increased substantially beginning 1.5 h before nitrogenase activity was detected. The increase in Mo accumulation by the cells is a result of the incorporation of Mo into a high-molecular-weight molybdenum species (suspected membrane fragments), a low-molecular-weight molybdenum species, a Mo binding-storage protein, a 30-kilodalton molybdoprotein, and formate dehydrogenase. Mo incorporation into the MoFe protein was detected 1 h after the onset of metal uptake. Kinetics of Mo accumulation into the molybdoproteins during the derepression of nitrogenase suggests that Mo incorporation or uptake or both occur in the following sequence: (i) membranes and MoO4(2-), (ii) a low-molecular-weight molybdenum species, (iii) Mo binding-storage protein and a 30-kilodalton molybdoprotein, (iv) formate dehydrogenase, and (v) the MoFe protein. The intracellular level of all molybdenum components except the MoFe protein appears to be influenced by the availability of Mo. Clostridial cells grown in the presence of a limiting amount of Mo became Mo deficient as a result of growth and a MoO4(2-) supplement added to such cells rapidly accumulated within the cells to levels five times that found in steady-state nitrogen-fixing cells. The Mo accumulated by the Mo-deficient cells was rapidly incorporated into preformed demolybdoproteins in the absence of de novo protein synthesis. The increase in Mo accumulation by Mo-deficient cells was a result of an increase in all molybdoproteins except the MoFe protein.     

In vivo energetics and control of nitrogen fixation: changes in the adenylate energy charge and adenosine 5'-diphosphate/adenosine 5'-triphosphate ratio of cells during growth on dinitrogen versus growth on ammonia.

The effects of the intracellular energy balance and adenylate pool composition on N2 fixation were examined by determining changes in the energy charge (EC) and the ADP/ATP (D/T) ratio of cells in chemostat and batch cultures of Clostridium pasteurianum, Klebsiella pneumoniae, and Azotobacter vinelandii. When cells of C. pasteurianum, K. pneumoniae, and A. vinelandii in sucrose-limited chemostats were examined, in all cases the EC increased greater than or equal to 15% when the nitrogen source was switched from N2 to NH3 and decreased greater than or equal to 15% when the nitrogen source was switched from NH3 to N2. The D/T ratio of the same cultures decreased greater than or equal to 70% when they were switched from N2 to NH3. In such cultures the adenylate pools remained constant when the cells were grown on either NH3 or N2. In nitrogen (NH3)-limited cultures, the adenylate pool was two- to threefold higher than the adenylate pool in sucrose-limited cultures, and the nitrogenase content of such cells was two- to threefold greater than the nitrogenase content of sucrose-limited N2-fixing cells. The EC and D/T ratio of cells from batch cultures of C. pasteurianum growing on NH3 in the presence of N2 were 0.82 and 0.83, respectively, but when the NH3 was consumed and the cells were switched to a nitrogen-fixing metabolism, the EC and D/T ratio changed to 0.70 and 0.90, respectively. Conversely, when NH3 was added to N2-fixing cultures the EC and D/T ratio changed within 1.5 h the EC and D/T ratio of NH3-grown cells. The nitrogen content of N2-fixing cells to which NH3 was added decreased at a rate greater could be accounted for by cell growth in the absence of further synthesis. This decay of nitrogenase activity (with a half-life about 1.2 to 1.4 h) suggests that some type of inactivation of nitrogenase occurs during repression. The nitrogenase of whole cells was estimated to be operating at about 32% of its theoretical maximum activity during steady-state N2-fixing conditions. Similarities in the data from chemostat and batch cultures of both aerobic and anaerobic N2-fixing organisms suggest that low EC and high D/T ratio are normal manifestations of an N2-fixing physiology.     

Molybdenum independence of nitrogenase component synthesis in the non-heterocystous cyanobacterium Plectonema.

The cyanobacterium Plectonema boryanum (IU 594-UTEX 594) fixes N2 only in the absence of combined N and of O2. We induced nitrogenase by transfer to anaerobic N-free medium and studied the effect of Mo starvation on nitrogenase activity and synthesis. Activity was first detected within 3 h after transfer by the acetylene reduction assay in controls, increasing for at least 25 h. Cells grown on nitrate and Mo and then transferred to N-free, Mo-free medium produced 8% of the control nitrogenase activity. Addition of W to the Mo-free medium reduced the activity to 0.5%. Under both Mo starvation conditions, nitrogenase protein components were synthesized. Component II of the cyanobacterial enzyme was detected by in vitro complementation with Mo-containing component I from Klebsiella pneumoniae or Azotobacter vinelandii but not Clostridium pasteurianum. Component I activity was restored by addition of Mo to cultures in which new enzyme synthesis was blocked by chloramphenicol. Acidified extracts of Plectonema induced in Mo-containing medium contained the Fe-Mo cofactor required to activate extracts of the Azotobacter mutant UW45 in vitro, but they did not activate extracts of Mo-starved Plectonema. Analysis of 35SO4(2-)-labeled proteins by polyacrylamide gel electrophoresis suggested that Mo is required for the conversion of a high-molecular-weight precursor to component I in Plectonema.     

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.     

Activation of Inactive Nitrogenase by Acid-Treated Component I

When Azotobacter vinelandii was derepressed for nitrogenase synthesis in a N-free medium containing tungstate instead of molybdate, an inactive component I was synthesized. Although this inactive component I could be activated in vivo upon addition of molybdate to the medium, it could not be activated in vitro when molybdate was added to the extracts. Activation occurred, however, when an acid-treated component I was added to extracts of cells derepressed in medium containing tungstate. Acid treatment completely abolished component I activity. Mutant strains UW45 and UW10 were unable to fix N(2). Both strains synthesized normal levels of component II but produced inactive component I. Acid-treated component I activated inactive component I in extracts of mutant strain UW45 but not mutant strain UW10. This activating factor could be obtained from N(2)-fixing Klebsiella pneumoniae, Clostridium pasteurianum, and Rhodospirillum rubrum.     

Molybdenum and vanadium nitrogenases of Azotobacter chroococcum. Low temperature favours N2 reduction by vanadium nitrogenase.

A comparison of the effect of temperature on the reduction of N2 by purified molybdenum nitrogenase and vanadium nitrogenase of Azotobacter chroococcum showed differences in behaviour. As the assay temperature was lowered from 30 degrees C to 5 degrees C N2 remained an effective substrate for V nitrogenase, but not Mo nitrogenase, since the specific activity for N2 reduction by Mo nitrogenase decreased 10-fold more than that of V nitrogenase. Activity cross-reactions between nitrogenase components showed the enhanced low-temperature activity to be associated with the Fe protein of V nitrogenase. The lower activity of homologous Mo nitrogenase components, although dependent on the ratio of MoFe protein to Fe protein, did not equal that of V nitrogenase even under conditions of high electron flux obtained at a 12-fold molar excess of Fe protein.     

Nitrogenase V The effect of Mo,W and V on the synthesis of nitrogenase components in Azotobacter vinelandii

When Azotobacter vinelandii OP is derepressed for nitrogenase synthesis in a medium containing no added Mo, component II but not component I is detected. Derepression with W in the medium allows the cells to produce inactive component I that can be activated by addition of molybdate to the medium. This activation does not require protein synthesis. Addition of V to Mo-free media does not increase the synthesis of component I protein. It is possible that Mo is an inducer of component I synthesis and that W also is capable of inducing component I, but that this component I is inactive because it lacks Mo.     

Nitrogen fixation system of tungsten-resistant mutants of Azotobacter vinelandii.

Mutants of Azotobacter vinelandii ATCC 12837 were isolated which could fix N2 in the presence of high tungsten concentrations. The most studied of these mutants (WD2) grew well in N-free modified Burk broth containing 10 mM W, whereas the wild type would not grow in this medium. WD2 would also grow in Burk N-free broth at about the same rate as the wild type. WD2 in broth containing W exhibited 22% of the whole cell acetylene reduction activity of the wild type in broth containing Mo and showed a lowered affinity for acetylene. Two-dimensional gel electrophoresis experiments showed that N2-fixing cells of WD2 from broth containing W or Mo did not produce significant amounts of component I of native nitrogenase protein. Electron spin resonance spectra of whole cells and cell-free extracts of WD2 from broth containing W lacked any trace of the g = 3.6 resonance associated with FeMoCo.     

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.     

In Vivo Kinetics of Nitrogenase Formation in Clostridium pasteurianum

Clostridium pasteurianum exhibits diauxic growth when grown in the presence of both NH(3) and N(2); no nitrogenase activity or formation was detected either serologically or by activity during growth on NH(3). During the 60-min lag that ensued after NH(3) was consumed and before growth resumed, molybdoferredoxin and azoferredoxin were first detected by activity measurements and serologically at 25 and 40 min, respectively. With the use of rifampin and dactinomycin, it was found that azoferredoxin messenger ribonucleic acid was initiated between 25 and 30 min after the inception of the lag and was completed by 38 min. An explanation of these results and their relation to possible models for the regulation of nitrogenase is given.     

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.     

Regulation of nitrate reductase in cyanobacteria. Repression-derepression control of nitrate reductase apoprotein in the cyanobacterium Nostoc muscorum

The repression-derepression control of Nostoc muscorum nitrate reductase was studied with regard to the Mo-cofactor and apoprotein levels. It was found that the synthesis of Mo-cofactor is constitutive but the apoprotein is subject to the repression-derepression control. In NH₄⁺ medium apoprotein synthesis was repressed and in N₂ and NO₃^? media apoprotein synthesis was derepressed. The apoprotein levels were similar in NO₃^? and N₂ media; however, the nitrate reductase activity was lower in N₂ medium due to lower Mo-cofactor activity. The lower Mo-cofactor activity in N₂-fixing conditions as compared to that in non-N₂-fixing conditions was consistent with the earlier view that the Mo-cofactor of nitrate reductase may be a precursor for FeMo-cofactor of nitrogenase.     

Effect of Molybdenum Starvation and Tungsten on the Synthesis of Nitrogenase Components in Klebsiella pneumoniae

Klebsiella pneumoniae M5a1 grows well in the presence or absence of molybdenum in media containing excess NH(4) (+). However, growth on N(2) is completely dependent on the presence of molybdenum in the medium. Tungstate competes with the molybdate requirement during growth on N(2). In molybdenum-depleted medium, neither protein component of nitrogenase is active and neither component can be detected antigenically. These data provide evidence that molybdenum is an inducer of nitrogenase synthesis.     

Expression of an alternative nitrogen fixation system in Azotobacter vinelandii.

Nitrogenase activities were determined from maximum acetylene reduction rates for mutant strains of Azotobacter vinelandii which are unable to fix N2 in the presence of molybdenum (Nif-) but undergo phenotypic reversal to Nif+ under conditions of Mo deficiency. The system responsible for N2 fixation under these conditions is thought to be an alternative N2 fixation system (Bishop et al., Proc. Natl. Acad. Sci. U.S.A. 77:7342-7346, 1980). Phenotypic reversal of Nif- strains to Nif+ strains was also observed in N-free medium without Mo but with either V or Re. Two protein patterns were found on two-dimensional gels of proteins from the extracts of wild-type cells cultured in N-free medium without Mo and with or without V or Re. The expression of each protein pattern in the wild-type strain of A. vinelandii seemed to depend upon the physiological state of the N2-fixing culture. Electron paramagnetic resonance experiments were conducted on whole cells of A. vinelandii grown under conditions of Mo deprivation in the absence of fixed N. No g = 3.65 signal (an electron paramagnetic resonance signal characteristic of the Mo-containing component of nitrogenase) was detectable in these cells, regardless of whether V or Re was present during growth of these cells, These results are discussed from the perspective that the well-known effect of V on N2 fixation by A. vinelandii may involve an alternative N2 fixation system.     

Molybdenum accumulation and storage in Klebsiella pneumoniae and Azotobacter vinelandii.

In Klebsiella pneumoniae, Mo accumulation appeared to be coregulated with nitrogenase synthesis. O2 and NH+4, which repressed nitrogenase synthesis, also prevented Mo accumulation. In Azotobacter vinelandii, Mo accumulation did not appear to be regulated Mo was accumulated to levels much higher than those seen in K. pneumoniae even when nitrogenase synthesis was repressed. Accumulated Mo was bound mainly to a Mo storage protein, and it could act as a supply for the Mo needed in component I synthesis when extracellular Mo had been exhausted. When A. vinelandii was grown in the presence of WO2-(4) rather than MoO2-(4), it synthesized a W-containing analog of the Mo storage protein. The Mo storage protein was purified from both NH+4 and N2-grown cells of A. vinelandii and found to be a tetramer of two pairs of different subunits binding a minimum of 15 atoms of Mo per tetramer.     

A nitrogen-fixation gene (nifC) in Clostridium pasteurianum with sequence similarity to chlJ of Escherichia coli

The molybdenum-containing nitrogenase contains an iron-molybdenum cofactor, whose synthesis involves at least six nif genes. Genes corresponding to nifE, N, B, and V occur in proximity in Clostridium pasteurianum, with nifN-B occurring as one gene and with nifV omega and nifV alpha in place of nifV. Between nifN-B and nifV omega V alpha, we found a gene whose sequence is similar to chlJ of Escherichia coli. chlJ is part of the chlD locus, which is involved in Mo transport. C. pasteurianum actively accumulates Mo in a process coregulated with nitrogen fixation. We propose that nifC is involved in Mo transport. The expression of nifC may be coregulated with nitrogen fixation because of the presence of nif-distinctive promoter and upstream sequences preceding nifC-nifV omega-nifV alpha. NifC contains a region typical of integral membrane proteins. Our findings suggest the involvement of a membrane-located nif gene product in Mo transport in C. pasteurianum.