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Nitrogen fixation is tightly regulated in Rhodospirillum rubrum at two different levels: transcriptional regulation of nif expression and posttranslational regulation of dinitrogenase reductase by reversible ADP-ribosylation catalyzed by the DRAT-DRAG (dinitrogenase reductase ADP-ribosyltransferase-dinitrogenase reductase-activating glycohydrolase) system. We report here the characterization of glnB, glnA, and nifA mutants and studies of their relationship to the regulation of nitrogen fixation. Two mutants which affect glnB (structural gene for P(II)) were constructed. While P(II)-Y51F showed a lower nitrogenase activity than that of wild type, a P(II) deletion mutant showed very little nif expression. This effect of P(II) on nif expression is apparently the result of a requirement of P(II) for NifA activation, whose activity is regulated by NH(4)(+) in R. rubrum. The modification of glutamine synthetase (GS) in these glnB mutants appears to be similar to that seen in wild type, suggesting that a paralog of P(II) might exist in R. rubrum and regulate the modification of GS. P(II) also appears to be involved in the regulation of DRAT activity, since an altered response to NH(4)(+) was found in a mutant expressing P(II)-Y51F. The adenylylation of GS plays no significant role in nif expression or the ADP-ribosylation of dinitrogenase reductase, since a mutant expressing GS-Y398F showed normal nitrogenase activity and normal modification of dinitrogenase reductase in response to NH(4)(+) and darkness treatments.

The Klebsiella pneumoniae nitrogen fixation (nif) mRNAs are unusually stable, with half-lives of 20 to 30 min under conditions favorable to nitrogen fixation (limiting nitrogen, anaerobiosis, temperatures of 30 degrees C). Addition of O2 or fixed nitrogen or temperature increases to 37 degrees C or more result in the dramatic destabilization of the nif mRNAs, decreasing the half-lives by a factor of 3 to 5. A plasmid expression system, independent of nif transcriptional regulation, was used to define cis determinants required for the regulated stability of the 5.2-kb nifHDKTY mRNA and to test the model suggested by earlier work that NifA is required in trans to stabilize nif mRNA under nif-derepressing conditions. O2 regulation of nifHDKTY mRNA stability is impaired in a plasmid containing a deletion of a 499-bp region of nifH, indicating that a site(s) required for the O2-regulated stability of the mRNA is located within this region. The simple model suggested from earlier work that NifA is required for stabilizing nif mRNA under conditions favorable for nitrogen fixation was disproved, and in its place, a more complicated model involving the sensing of nitrogenase activity as a component of the system regulating mRNA stability is proposed. Analysis of nifY mutants and overexpression suggests a possible involvement of the protein in this sensing process.

The iron-molybdenum cofactor (FeMo-co) of nitrogenase contains molybdenum, iron, sulfur, and homocitrate in a ratio of 1:7:9:1. In vitro synthesis of FeMo-co has been established, and the reaction requires an ATP-regenerating system, dithionite, molybdate, homocitrate, and at least NifB-co (the metabolic product of NifB), NifNE, and dinitrogenase reductase (NifH). The typical in vitro FeMo-co synthesis reaction involves mixing extracts from two different mutant strains of Azotobacter vinelandii defective in the biosynthesis of cofactor or an extract of a mutant strain complemented with the purified missing component. Surprisingly, the in vitro synthesis of FeMo-co with only purified components failed to generate significant FeMo-co, suggesting the requirement for one or more other components. Complementation of these assays with extracts of various mutant strains demonstrated that NifX has a role in synthesis of FeMo-co. In vitro synthesis of FeMo-co with purified components is stimulated approximately threefold by purified NifX. Complementation of these assays with extracts of A. vinelandii DJ42. 48 (DeltanifENX DeltavnfE) results in a 12- to 15-fold stimulation of in vitro FeMo-co synthesis activity. These data also demonstrate that apart from the NifX some other component(s) is required for the cofactor synthesis. The in vitro synthesis of FeMo-co with purified components has allowed the detection, purification, and identification of an additional component(s) required for the synthesis of cofactor.

In the nitrogenase system of Klebsiella pneumoniae, nifT is located between nifDK, the structural genes for dinitrogenase, and nifY, whose product is involved in nitrogenase maturation. It is, therefore, a reasonable hypothesis that the NifT protein might also have a role in the maturation of nitrogenase. However, the phenotypic characterization of nifT and nifT-overexpressing strains for effects on the regulation, maturation, and activity of nitrogenase identified no properties that were distinct from those of the wild type. We conclude that the K. pneumoniae NifT protein is not essential for nitrogen fixation under the conditions examined.

In the presence of carbon monoxide, the photosynthetic bacterium Rhodospirillum rubrum induces expression of proteins which allow the organism to metabolize carbon monoxide in the net reaction CO + H2O --> CO2 + H2. These proteins include the enzymes carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. In this paper, we present the complete amino acid sequence for the large subunit of this hydrogenase and describe the properties of the crude enzyme in relation to other known hydrogenases. The amino acid sequence deduced from the CO-induced hydrogenase large-subunit gene (cooH) shows significant similarity to large subunits of other Ni-Fe hydrogenases. The closest similarity is with HycE (58% similarity and 37% identity) from Escherichia coli, which is the large subunit of an Ni-Fe hydrogenase (isoenzyme 3). The properties of the CO-induced hydrogenase are unique. It is exceptionally resistant to inhibition by carbon monoxide. It also exhibits a very high ratio of H2 evolution to H2 uptake activity compared with other known hydrogenases. The CO-induced hydrogenase is tightly membrane bound, and its inhibition by nonionic detergents is described. Finally, the presence of nickel in the hydrogenase is addressed. Analysis of wild-type R. rubrum grown on nickel-depleted medium indicates a requirement for nickel for hydrogenase activity. However, analysis of strain UR294 (cooC insertion mutant defective in nickel insertion into CODH) shows that independent nickel insertion mechanisms are utilized by hydrogenase and CODH. CooH lacks the C-terminal peptide that is found in other Ni-Fe hydrogenases; in other systems, this peptide is cleaved during Ni processing.

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.

A second nitrogenase activity has been demonstrated in Rhodospirillum rubrum. This nitrogenase is expressed whenever a strain lacks an active Mo nitrogenase because of physiological or genetic inactivation. The alternative nitrogenase is able to support growth on N2 in the absence of fixed N. V does not stimulate, nor does Mo or W inhibit, growth or activity under the conditions tested. The proteins responsible for this activity were identified by electrophoretic and immunological properties. The synthesis of these proteins was repressed by NH4+. The alternative nitrogenase reductase is ADP ribosylated in response to darkness by the system that regulates the activity of the Mo nitrogenase. The genes for the alternative nitrogenase have been cloned, and the alternative nitrogenase reductase has been expressed in an in vitro transcription-translation system.

Dinitrogenase reductase (the nifH product) from Rhodospirillum rubrum is regulated by a post-translational modification system encoded by draTG. As demonstrated in this report, the cloning, sequencing, and functional characterization of the nifH gene provides a basis for further analysis as well as revealing interesting features of gene organization. The coding regions of nifH and draT are separated by only 400 bp, though the genes are divergently transcribed and differentially regulated. The construction of a nifH insertion caused a Nif- phenotype and destroyed the mutant's ability to synthesize both dinitrogenase and dinitrogenase reductase, verifying functionality and transcriptional organization of the nifHDK genes.

The primary product of biological nitrogen fixation, ammonia, reversibly regulates nitrogenase activity in a variety of diazotrophs by a process called "NH4(+)-switch-off/on." Strong correlative evidence from work in Azospirillum lipoferum and Rhodospirillum rubrum indicates that this regulation involves both the inactivation of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase and the reactivation by dinitrogenase reductase activating glycohydrolase. The genes encoding these two enzymes, draT and draG, have been cloned from these two organisms, so that direct genetic evidence can be marshaled to test this model in vivo. The draT/G system has been transferred to and monitored in the enteric nitrogen-fixing bacterium Klebsiella pneumoniae, an organism normally devoid of such a regulatory mechanism. The expressed draT and draG genes allowed K. pneumoniae to respond to NH4Cl with a reversible regulation of nitrogenase activity that was correlated with the reversible ADP-ribosylation of dinitrogenase reductase in vivo. Thus, the expression of draT and draG genes in K. pneumoniae is necessary and sufficient to support NH4(+)-switch-off/on, and ADP-ribosylation serves as a reversible regulatory mechanism for controlling nitrogenase activity in prokaryotes.

An insertional mutation was made in the nifX gene of Klebsiella pneumoniae. This mutation had little effect on the nitrogenase activity of the strain, as measured by acetylene reduction. However, on the addition of NH4+ or O2 (conditions which block nif protein synthesis by transcriptional and posttranscriptional mechanisms), the NifX- mutant synthesized nitrogenase proteins longer and had more accumulated nifHDKTY mRNA than did the wild-type K. pneumoniae at all time points tested. Conversely, overexpression of the wild-type nifX region blocked nif protein synthesis, protein accumulation, and nifHDKTY mRNA accumulation. These complementary results indicate that a product of the nifX region has a role in the negative regulation of nif regulation in response to NH4+ and O2.

A genomic library of Azospirillum lipoferum was constructed with phage lambda EMBL4 as vector. From this library, the genes encoding dinitrogenase reductase ADP-ribosyltransferase (DRAT), draT, and dinitrogenase reductase-activating glycohydrolase (DRAG), draG, were cloned by hybridization with the heterologous probes of Rhodospirillum rubrum. As in R. rubrum, draT is located between draG and nifH, the gene encoding dinitrogenase reductase (a substrate for the DRAG/DRAT system). In the crude extract of Escherichia coli harboring the expression vector for this region, DRAT and DRAG enzyme activities were detected, confirming the identity of the cloned genes. Southern hybridization with genomic DNA from different Azospirillum spp., demonstrated a correlation between observable draTG hybridization and the biochemical demonstration of this covalent modification system.

Nitrogen fixation activity in the photosynthetic bacterium Rhodospirillum rubrum is controlled by the reversible ADP-ribosylation of the dinitrogenase reductase component of the nitrogenase enzyme complex. This report describes the cloning and characterization of the genes encoding the ADP-ribosyltransferase (draT) and the ADP-ribosylglycohydrolase (draG) involved in this regulation. These genes are shown to be contiguous on the R. rubrum chromosome and highly linked to the nifHDK genes. Sequence analysis revealed the use of TTG as the initiation codon of the draT gene as well as a potential open reading frame immediately downstream of draG. The mono-ADP-ribosylation system in R. rubrum is the first in which both the target protein and modifying enzymes as well as their structural genes have been isolated, making it the model system of choice for analysis of this post-translational regulatory mechanism.

The function of the cloned draT gene of Rhodospirillum rubrum was studied by placing it under the control of the tac promoter in the vector, pKK223-3. After induction with isopropyl-beta-D-thiogalactopyranoside, dinitrogenase reductase ADP-ribosyltransferase (DRAT) activity was detected in crude extracts of the heterologous hosts Escherichia coli and Klebsiella pneumoniae. In addition, the expression of draT produced a Nif- phenotype in the otherwise wild-type K. pneumoniae strains, the result of the ADP-ribosylation of accumulated dinitrogenase reductase (DR). DR from a nifF- background was also susceptible to ADP-ribosylation, indicating that the oxidized form of DR will serve as a substrate for DRAT in vivo. A mutation that changes the Arg-101 residue of DR, the ADP-ribose attaching site, eliminates the ADP-ribosylation of DR in vivo, confirming the necessity of this residue for modification.

The nifN and -E gene products are involved in the synthesis of the iron-molybdenum cofactor of dinitrogenase, the enzyme responsible for the reduction of dinitrogen to ammonia. By using the in vitro iron-molybdenum cofactor biosynthesis assay, we have followed the purification of these gene products 450-fold to greater than 95% purity. An overall recovery of 20% was obtained with the purified protein having a specific activity of 6900 units/mg of protein. The protein (hereafter referred to as NIFNE) was found to contain equimolar amounts of the nifN and -E gene products and have a native molecular mass of 200 +/- 10 kDa, which indicates an alpha 2 beta 2 structure. NIFNE was oxygen labile with a half-life of 1 min in air. A UV-visible spectrum of the dye-oxidized protein showed an absorption maximum at 425 nm that could be bleached by reduction of NIFNE with sodium dithionite, suggesting the presence of an Fe center in NIFNE.

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.