Reversible regulation of the nitrogenase iron protein from Rhodospirillum rubrum by ADP-ribosylation in vitro.

Nitrogenase activity in the photosynthetic bacterium Rhodospirillum rubrum is reversibly regulated by interconversion of the Fe protein between a modified and an unmodified form. Since the discovery of the activation process in 1976, investigators have been unable to demonstrate the inactivation (modification) reaction in vitro. In this study, NAD-dependent modification and concomitant inactivation of the Fe protein were demonstrated in crude extracts of R. rubrum. Activation of the in vitro-modified Fe protein by activating enzyme and structural similarity between the in vivo and in vitro modifications are presented as evidence that the in vitro modification is the physiologically relevant ADP-ribosylation reaction. Using a partially purified preparation, we showed that the inactivating enzyme activity is stimulated by divalent metal ions and ADP, that O2-denatured Fe protein will not serve as a substrate, and that dithionite inhibits the modification reaction.     

Effect of an ntrBC mutation on the posttranslational regulation of nitrogenase activity in Rhodospirillum rubrum.

Homologs of ntrB and ntrC genes from Rhodospirillum rubrum were cloned and sequenced. A mutant lacking ntrBC was constructed, and this mutant has normal nitrogenase activity under nif-derepressing conditions, indicating that ntrBC are not necessary for the expression of the nif genes in R. rubrum. However, the post-translational regulation of nitrogenase activity by ADP-ribosylation in response to NH4+ was partially abolished in this mutant. More surprisingly, the regulation of nitrogenase activity in response to darkness was also affected, suggesting a physiological link between the ntr system and energy signal transduction in R. rubrum. The expression of glutamine synthetase, as well as its posttranslational regulation, was also altered in this ntrBC mutant.     

Correlation of activity regulation and substrate recognition of the ADP-ribosyltransferase that regulates nitrogenase activity in Rhodospirillum rubrum

In Rhodospirillum rubrum, nitrogenase activity is regulated posttranslationally through the ADP-ribosylation of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT). Several DRAT variants that are altered both in the posttranslational regulation of DRAT activity and in the ability to recognize variants of dinitrogenase reductase have been found. This correlation suggests that these two properties are biochemically connected.     

Cloning, sequencing, mutagenesis, and functional characterization of draT and draG genes from Azospirillum brasilense.

The Azospirillum brasilense draT gene, encoding dinitrogenase reductase ATP-ribosyltransferase, and draG gene, encoding dinitrogenase reductase activating glycohydrolase, were cloned and sequenced. Two genes were contiguous on the A. brasilense chromosome and showed extensive similarity to the same genes from Rhodospirillum rubrum. Analysis of mutations introduced into the dra region on the A. brasilense chromosome showed that mutants affected in draT were incapable of regulating nitrogenase activity in response to ammonium. In contrast, a mutant with an insertion in draG was still capable of ADP-ribosylating dinitrogenase reductase in response to ammonium but was no longer able to recover activity after ammonium depletion. Plasmid-borne draTG genes from A. brasilense were introduced into dra mutants of R. rubrum and restored these mutants to an apparently wild-type phenotype. It is particularly interesting that dra mutants of R. rubrum containing draTG of A. brasilense can respond to darkness and light, since A. brasilense is a nonphotosynthetic bacterium and its dra system does not normally possess that regulatory response. The nifH gene of A. brasilense, encoding dinitrogenase reductase (the substrate of dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase), is located 1.9 kb from the start of draT and is divergently transcribed. Two insertion mutations in the region between draT and nifH showed no significant effect on nitrogenase activity or its regulation.     

Necessity of a membrane component for nitrogenase activity in Rhodospirillum rubrum.

Acetylene reduction catalyzed by nitrogenase from Rhodospirillum rubrum has low activity and exhibits a lag phase. The activity can be increased by the addition of a chromatophore membrane component and the lag eliminated by preincubation with this component, which can be solubilized from chromatophores by treatment with NaCl. It is both trypsin- and oxygen-sensitive. Titration of the membrane component with nitrogenase and vice versa shows a saturation point. The membrane component interacts specifically with the Fe protein of nitrogenase, the interaction being ATP- and Mg2+-dependent.     

Reversible ADP-ribosylation of dinitrogenase reductase in a nifD- mutant of Rhodospirillum rubrum.

Dinitrogenase reductase from a Rhodospirillum rubrum strain lacking dinitrogenase was reversibly ADP-ribosylated in vivo in response to dark-light transitions. Addition of ammonia also led to ADP-ribosylation in this strain. These results demonstrate that reduced dinitrogenase is a satisfactory substrate for the reversible ADP-ribosylation system of R. rubrum in vivo.     

Purification and characterization of the alternative nitrogenase from the photosynthetic bacterium Rhodospirillum rubrum.

The alternative nitrogenase from a nifH mutant of the photosynthetic bacterium Rhodospirillum rubrum has been purified and characterized. The dinitrogenase protein (ANF1) contains three subunits in an apparent alpha2beta2gamma2 structure and contains Fe but no Mo or V. A factor capable of activating apo-dinitrogenase (lacking the FeMo cofactor) from Azotobacter vinelandii was extracted from the alternative dinitrogenase protein with N-methylformamide. The electron paramagnetic resonance (EPR) signal of the dinitrogenase protein is not characteristic of the EPR signals of molybdenum- or vanadium-containing dinitrogenases. The alternative dinitrogenase reductase (ANF2) was purified as an alpha2 dimer containing an Fe4S4 cluster and exhibited an EPR spectrum characteristic of dinitrogenase reductases. The enzyme complex reduces protons to H2 very well but reduces N2 to ammonium poorly. Acetylene is reduced to a mixture of ethylene and ethane.     

Amino acid concentrations in Rhodospirillum rubrum during expression and switch-off of nitrogenase activity.

The amino acid concentrations in the phototrophic bacterium Rhodospirillum rubrum were measured during growth under nif-repressing and nif-derepressing conditions. The effects of ammonium, glutamine, darkness, phenazine methosulfate, and the inhibitors methionine sulfoximine and azaserine on amino acid levels of cells were tested. The changes were compared to changes in whole-cell nitrogenase activity and ADP-ribosylation of dinitrogenase reductase. Glutamate was the dominant amino acid under every growth condition. Glutamine levels were equivalent when cells were grown on high-ammonia (nif-repressing) medium or glutamate (nif-derepressing) medium. Thus, glutamine is not the solitary agent that controls nif expression. No other amino acid correlated with nif expression. Glutamine concentrations rose sharply when either glutamate-grown or N-starved cells were treated with ammonia, glutamine, or azaserine. Glutamine levels showed little change upon treatment of the cells with darkness or ammonium plus methionine sulfoximine. Treatment with phenazine methosulfate resulted in a decrease in glutamine concentration. The glutamine concentration varied independently of dinitrogenase reductase ADP-ribosylation, and it is concluded that an increase in glutamine concentration is neither necessary nor sufficient to initiate the modification of dinitrogenase reductase. No other amino acid exhibited changes in concentration that correlated consistently with modification. Glutamine synthetase activity and nitrogenase activity were not coregulated under all conditions, and thus the two regulatory cascades perceive different signal(s) under at least some conditions.     

Changes in the regulatory form of Rhodospirillum rubrum nitrogenase as influenced by nutritional and environmental factors.

The photosynthetic bacterium Rhodospirillum rubrum regulates the activity of its nitrogenase (N2ase) by interconverting the enzyme into three distinct enzymatic species: N2ase A (a fully active form) and two regulatory forms, N2ase Ractive and N2ase Rinactive. N2ase R is distinguished from N2ase A in vitro by the requirement of its Fe protein for activation by a Mn2+-dependent activating factor. N2ase is converted from the A to the R form in response to certain environmental factors such as carbon starvation, depletion of intracellular adenosine triphosphate, or the addition of NH4+ (or glutamate) to a culture of N-starved cells. The rapid inhibition of R. rubrum N2ase in vivo by NH4+ was shown to result from the conversion of N2ase A to N2ase Rinactive. On depletion of NH4+ from the culture, whole-cell N2ase activity returned; however, the enzyme remained in the R form. Unlike the effect of NH4+, adding glutamate to cells containing N2ase A did not inhibit in vivo activity, but converted the enzyme to the R form (N2ase Ractive). Although glutamate-induced N2ase R formation was much slower than the NH4+-induced reaction, it occurred in the presence of rifampin, indicating that de novo protein synthesis was not involved. This suggested that N2ase R was formed by a modification of N2ase A. Although glutamine synthetase in involved in the conversion of N2ase A to R, the adenylylation state of glutamine synthetase appears not to be involved in regulating this nitrogenase reaction.     

Expression of the activating enzyme and Fe protein of nitrogenase from Rhodospirillum rubrum.

Activating enzyme (AE) is responsible for the in vitro activation of inactive Fe protein of nitrogenase from Rhodospirillum rubrum cells cultured anaerobically with glutamate as the N source. The expression of Fe protein and AE was examined in R. rubrum cultured photosynthetically or aerobically on media containing malate as the carbon source. One of the following N sources was used in each culture: glutamate, glutamine, limiting ammonia, high ammonia, glutamate plus histidine, and high ammonia plus histidine. Chromatophores from every culture exhibited AE activity; activity was highest in glutamate-grown cells. Fe protein was observed by rocket immunoelectrophoresis in cultures with nitrogenase activity. Several Nif-, Gln-, and His- mutants of R. rubrum were assayed for AE activity, nitrogenase activity, and Fe protein. Every mutant expressed AE activity, and Fe protein was observed in those cultures with nitrogenase activity. AE from every preparation was O2 labile, and each O2-denatured AE preparation inhibited activation by active AE.     

Adenine nucleotide levels in Rhodospirillum rubrum during switch-off of whole-cell nitrogenase activity.

Adenine nucleotide pools were measured in Rhodospirillum rubrum cultures that contained nitrogenase. The average energy charge [([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP])] was found to be 0.66 and 0.62 in glutamate-grown and N-limited cultures respectively. Treatment of glutamate-grown cells with darkness, ammonia, glutamine, carbonyl cyanide m-chlorophenylhydrazone, or phenazine methosulphate resulted in perturbations in the adenine nucleotide pools, and led to loss of whole-cell nitrogenase activity and modification in vivo of the Fe protein. Treatment of N-limited cells resulted in similar changes in adenine nucleotide pools but not enzyme modification. No correlations were found between changes in adenine nucleotide pools or ratios of these pools and switch-off of nitrogenase activity by Fe protein modification in vivo. Phenazine methosulphate inhibited whole-cell activity at low concentrations. The effect on nitrogenase activity was apparently independent of Fe protein modification.     

Posttranslational regulation of nitrogenase in Rhodospirillum rubrum strains overexpressing the regulatory enzymes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase activating glycohydrolase.

Rhodospirillum rubrum strains that overexpress the enzymes involved in posttranslational nitrogenase regulation, dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG), were constructed, and the effect of this overexpression on in vivo DRAT and DRAG regulation was investigated. Broad-host-range plasmid constructs containing a fusion of the R. rubrum nifH promoter and translation initiation sequences to the second codon of draT, the first gene of the dra operon, were constructed. Overexpression plasmid constructs which overexpressed (i) only functional DRAT, (ii) only functional DRAG and presumably the putative downstream open reading frame (ORF)-encoded protein, or (iii) all three proteins were generated and introduced into wild-type R. rubrum. Overexpression of DRAT still allowed proper regulation of nitrogenase activity, with ADP-ribosylation of dinitrogenase reductase by DRAT occurring only upon dark or ammonium stimuli, suggesting that DRAT is still regulated upon overexpression. However, overexpression of DRAG and the downstream ORF altered nitrogenase regulation such that dinitrogenase reductase did not accumulate in the ADP-ribosylated form under inactivation conditions, suggesting that DRAG was constitutively active and that therefore DRAG regulation is altered upon overexpression. Proper DRAG regulation was observed in a strain overexpressing DRAT, DRAG, and the downstream ORF, suggesting that a proper balance of DRAT and DRAG levels is required for proper DRAG regulation.     

Light-induced reversible pH changes in chromatophores from Rhodospirillum rubrum

Light-induced pH changes with chromatophores from Rhodospirillum rubrum have been studied as a function of pH, chromatophore and salt concentrations, and light intensity. Optimal reaction conditions have been determined. It has been demonstrated that light-induced pH changes may also be obtained at high light intensities by addition of phenzaine methosulfate after a lag period in a system inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide.     

Properties and regulation of glutamine synthetase from Rhodospirillum rubrum.

Glutamine synthetase from Rhodospirillum rubrum was purified and characterized with respect to its pH optimum and the effect of Mg2+ on its active and inactive forms. Both adenine and phosphorus were incorporated into the inactive form of the enzyme, indicating covalent modification by AMP. The modification could not be removed by phosphodiesterase. Evidence for regulation of the enzyme by oxidation was obtained. Extracts from oxygen-treated cells had lower specific activities than did extracts from cells treated anaerobically. Glutamine synthetase activity was found to decrease in the dark in phototrophically grown cells; activity was recovered on re-illumination.     

The fixABCX genes in Rhodospirillum rubrum encode a putative membrane complex participating in electron transfer to nitrogenase

In our efforts to identify the components participating in electron transport to nitrogenase in Rhodospirillum rubrum, we used mini-Tn5 mutagenesis followed by metronidazole selection. One of the mutants isolated, SNT-1, exhibited a decreased growth rate and about 25% of the in vivo nitrogenase activity compared to the wild-type values. The in vitro nitrogenase activity was essentially wild type, indicating that the mutation affects electron transport to nitrogenase. Sequencing showed that the Tn5 insertion is located in a region with a high level of similarity to fixC, and extended sequencing revealed additional putative fix genes, in the order fixABCX. Complementation of SNT-1 with the whole fix gene cluster in trans restored wild-type nitrogenase activity and growth. Using Western blotting, we demonstrated that expression of fixA and fixB occurs only under conditions under which nitrogenase also is expressed. SNT-1 was further shown to produce larger amounts of both ribulose 1,5-bisphosphate carboxylase/oxygenase and polyhydroxy alkanoates than the wild type, indicating that the redox status is affected in this mutant. Using Western blotting, we found that FixA and FixB are soluble proteins, whereas FixC most likely is a transmembrane protein. We propose that the fixABCX genes encode a membrane protein complex that plays a central role in electron transfer to nitrogenase in R. rubrum. Furthermore, we suggest that FixC is the link between nitrogen fixation and the proton motive force generated in the photosynthetic reactions.     

Studies on the activating enzyme for iron protein of nitrogenase from Rhodospirillum rubrum

Removal of ADP-ribose from the iron protein of nitrogenase by activating enzyme resulted in the activation of the inactive iron protein. A radioassay that directly measured the initial velocity of the activation was developed using iron protein radiolabeled with either [8-3H]- or [G-32P]ADP-ribose. The release of radiolabeled ADP-ribose by activating enzyme was linearly correlated with the increase in the specific activity of the iron protein as measured by acetylene reduction. Both ATP and MnCl2 were required for the activation of inactive iron protein. The optimal ratio of [MnCl2]/[ATP] in the radioassay was 2:1, and the optimal concentrations were 4 mM and 2 mM for [MnCl2] and [ATP], respectively. The Km for inactive iron protein was 74 microM and the Vmax was 628 pmol of [32P] ADP-ribose released min-1 microgram of activating enzyme-1. Adenosine, cytidine, guanosine, or uridine mono-, di-, or triphosphates did not substitute for ATP in the activation of native iron protein. Activating enzyme removed ADP-ribose from oxygen-denatured iron protein in the absence of ATP. ADP, ADP-ribose, pyrophosphate, and high concentrations of NaCl inhibited activating enzyme activity.     

Studies on the effect of NAD(H) on nitrogenase activity in Rhodospirillum rubrum

The effect of NAD(P) and analogs of this nucleotide on nitrogenase activity in Rhodospirillum rubrum has been studied. Addition of NAD⁺ to nitrogen fixing Rsp. rubrum leads to inhibition of nitrogenase. NADP⁺ has the same effect but NADH or analogs modified in the nicotinamide portion do not cause inhibition. In contrast to ammonium ions, addition of NAD⁺ leads to inhibition of nitrogenase in cells that have been N-starved under argon. The inhibitory effect of NAD⁺ is more pronounced at lower light intensities. Addition of NAD⁺ also leads to inhibition of glutamine synthetase, a phenomenon also occurring when “switchoff” is produced by the addition of effectors such as ammonium ions or glutamine. It is also shown that NAD⁺ is taken up by Rsp. rubrum cells.