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.     

用PCR方法扩增深红红螺菌的draT基因

聚合酶链反应⁽¹⁾是最近几年分子生物学领域中一项重大的技术突破。典型的PCR引物内应含50％G+c,且没有自身互补序列,特别是在其3’－末端不应有内部二级结构。引物与靶DNA的退火温度一般为50℃,但当已有的引物不太符合要求时,就要调整退火温度。本文报道了用PCR方法扩增深红红螺菌draT基因,介绍了当引物Gc含量较低时,可采用适当降低退火温度,然后梯度升温的方法获得PCR产物。     

Effects of perturbations of the nitrogenase electron transfer chain on reversible ADP-ribosylation of nitrogenase Fe protein in Klebsiella pneumoniae strains bearing the Rhodospirillum rubrum dra operon

The redox state of nitrogenase Fe protein is shown to affect regulation of ADP-ribosylation in Klebsiella pneumoniae strains transformed by plasmids carrying dra genes from Rhodospirillum rubrum. The dra operon encodes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase, enzymes responsible for the reversible inactivation, via ADP-ribosylation, of nitrogenase Fe protein in R. rubrum. In bacteria containing the dra operon in their chromosomes, inactivation occurs in response to energy limitation or nitrogen sufficiency. The dra gene products, expressed at a low level in K. pneumoniae, enable transformants to reversibly ADP-ribosylate nitrogenase Fe protein in response to the presence of fixed nitrogen. The activities of both regulatory enzymes are regulated in vivo as described in R. rubrum. Genetic perturbations of the nitrogenase electron transport chain were found to affect the rate of inactivation of Fe protein. Strains lacking the electron donors to Fe protein (NifF or NifJ) were found to inactivate Fe protein more quickly than a strain with wild-type background. Deletion of nifD, which encodes a subunit of nitrogenase MoFe protein, was found to result in a slower inactivation response. No variation was found in the reactivation responses of these strains. It is concluded that the redox state of the Fe protein contributes to the regulation of the ADP-ribosylation of Fe protein.     

The regulation of ferredoxin-dependent nitrogenase activity in Rhodospirillum rubrum and Rhodopseudomonas capsulata

Abstract The regulatory properties of Rhodospirillum rubrum nitrogenase reduced by either the endogenous electron donor (ferredoxin) or an artificial donor (dithionite) were examined. The nitrogenase obtained from glutamate-grown cells required activating enzyme for maximum activity with either reductant. The activating enzyme requirement of ferredoxin-dependent nitrogenase activity implies a physiological significance of the activating enzyme in R. rubrum. Rhodopseudomonas capsulata nitrogenase also required activating enzyme when dithionite was the reductant, but there appeared to be no activating enzyme requirement with ferredoxin as the reductant. Because the catalytic activity of the enzyme was very low under these conditions, the physiological significance of activating enzyme in this organism remains in question.     

Inhibition of nitrogenase activity by NH+4 in Rhodospirillum rubrum.

Nitrogenase activities and the patterns of in vivo inhibition of nitrogenase by NH+4 were compared in Rhodospirillum rubrum grown under several conditions of nitrogen availability. In cells grown on N2 or glutamate plus N2, nitrogenase activity was relatively low and was totally inhibited by added NH+4 in 15 to 20 min. In contrast, cells grown on glutamate alone displayed higher nitrogenase activity, and NH+4 had very little effect. Cells grown on limiting amounts of NH+4 had lower nitrogenase activity, but NH+4 produced little inhibitory effect. Uptake of NH+4 could be demonstrated under all of these conditions, and this uptake was blocked by DL-methionine-dl-sulfoximine. The data indicated that cells not recently exposed to NH+4 had no mechanism for rapidly turning off nitrogenase activity in response to sudden additions of NH+4. In contrast, cells grown in the presence of N2, which form NH+4 internally, inhibited nitrogenase activity relatively quickly in response to added NH+4.     

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.     

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.     

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.     

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.     

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.     

Properties of the nitrogenase system from a photosynthetic bacterium, Rhodospirillum rubrum.

Soluble nitrogenase from Rhodospirillum rubrum has been isolated and separated into its two components, the MoFe protein and the Fe protein. The MoFe protein has been purified to near homogeneity and has a molecular weight or 215 000. It contains two Mo, 25--30 Fe and 19--22 acid-labile sulphide and consists of four subunits, Mw 56 000. The Fe protein has a molecular weight 65 000. It contains approximately four Fe and four acid-labile sulphide and consists of two subunits, Mw 31 500. The highest specific activities for the purified components are 920 and 1260 nmol ethylene produced per min per mg protein, respectively. The purified components require the membrane component for activity (Nordlund, S., Eriksson, U. and Baltscheffsky, H. (1977) Biochim. Biophys. Acta 462, 187--195). Titration of the MoFe protein with the Fe protein shows saturation and excess MoFe protein over Fe protein is inhibitory. Addition of Fe2+ or Mn2+ to the reaction mixture increases the activity apparently through interaction with the membrane component.     

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.     

Purification and Mn2+ activation of Rhodospirillum rubrum nitrogenase activating enzyme.

The Fe protein activating enzyme for Rhodospirillum rubrum nitrogenase was purified to approximately 90% homogeneity, using DE52-cellulose chromatography and sucrose density gradient centrifugation. Activating enzyme consists of a single polypeptide of molecular weight approximately 24,000. ATP was required for catalytic activity, but was relatively ineffective in the absence of Mg2+. When the concentration of MgATP2- was held in excess, there was an additional requirement for a free divalent metal ion (Mn2+) for enzyme activity. Kinetic experiments showed that the presence of Mg2+ influenced the apparent binding of Mn2+ by the enzyme, resulting in a lowering of the concentration of Mn2+ required to give half-maximum activity (K alpha) as the free Mg2+ concentration was increased. A low concentration of Mn2+ had a sparing effect on the requirement for free Mg2+. There is apparently a single metal-binding site on activating enzyme which preferentially binds Mn2+ as a positive effector, and free Mg2+ can compete for this site.     

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.     

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.     

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.