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The photosynthetic α-proteobacterium Rhodobacter capsulatus reduces and thereby fixes atmospheric dinitrogen (N 2 ) by a molybdenum (Mo)-nitrogenase and an iron-only (Fe)-nitrogenase. Differential expression of the structural genes of Mo-nitrogenase (nifHDK) and Fe-nitrogenase (anfHDGK) is strictly controlled and activated by NifA and AnfA, respectively. In contrast to NifA-binding sites, AnfA-binding sites are poorly defined. Here, we identified two highly similar AnfA-binding sites in the R. capsulatus anfH promoter by studying the effects of promoter mutations on in vivo anfH expression and in vitro promoter binding by AnfA. Comparison of the experimentally determined R. capsulatus AnfA-binding sites and presumed AnfA-binding sites from other α-proteobacteria revealed a consensus sequence of dyad symmetry, TAC-N 6 -GTA, suggesting that AnfA proteins bind their target promoters as dimers. Chromosomal replacement of the anfH promoter by the nifH promoter restored anfHDGK expression and Fe-nitrogenase activity in an R. capsulatus strain lacking AnfA suggesting that AnfA is required for AnfHDGK production, but dispensable for biosynthesis of the iron-only cofactor and electron delivery to Fe-nitrogenase, pathways activated by NifA. These observations strengthen our model, in which the Fe-nitrogenase system in R. capsulatus is largely integrated into the Mo-nitrogenase system., (© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Rhodobacter capsulatus fixes atmospheric nitrogen (N 2 ) by a molybdenum (Mo)-nitrogenase and a Mo-free iron (Fe)-nitrogenase, whose production is induced or repressed by Mo, respectively. At low nanomolar Mo concentrations, both isoenzymes are synthesized and contribute to nitrogen fixation. Here we examined the regulatory interplay of the central transcriptional activators NifA and AnfA by proteome profiling. As expected from earlier studies, synthesis of the structural proteins of Mo-nitrogenase (NifHDK) and Fe-nitrogenase (AnfHDGK) required NifA and AnfA, respectively, both of which depend on the alternative sigma factor RpoN to activate expression of their target genes. Unexpectedly, NifA was found to be essential for the synthesis of Fe-nitrogenase, electron supply to both nitrogenases, biosynthesis of their cofactors, and production of RpoN. Apparently, RpoN is the only NifA-dependent factor required for target gene activation by AnfA, since plasmid-borne rpoN restored anfH transcription in a NifA-deficient strain. However, plasmid-borne rpoN did not restore Fe-nitrogenase activity in this strain. Taken together, NifA requirement for synthesis and activity of both nitrogenases suggests that Fe-nitrogenase functions as a complementary nitrogenase rather than an alternative isoenzyme in R. capsulatus., (© 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Biological nitrogen fixation, the reduction of chemically inert dinitrogen to bioavailable ammonia, is a central process in the global nitrogen cycle highly relevant for life on earth. N 2 reduction to NH 3 is catalyzed by nitrogenases exclusively synthesized by diazotrophic prokaryotes. All diazotrophs have a molybdenum nitrogenase containing the unique iron-molybdenum cofactor FeMoco. In addition, some diazotrophs encode one or two alternative Mo-free nitrogenases that are less efficient at reducing N 2 than Mo-nitrogenase. To permit biogenesis of Mo-nitrogenase and other molybdoenzymes when Mo is scarce, bacteria synthesize the high-affinity molybdate transporter ModABC. Generally, Mo supports expression of Mo-nitrogenase genes, while it represses production of Mo-free nitrogenases and ModABC. Since all three nitrogenases and ModABC can reach very high levels at suitable Mo concentrations, tight Mo-mediated control saves considerable resources and energy. This review outlines the similarities and differences in Mo-responsive regulation of nitrogen fixation and molybdate transport in diverse diazotrophs., (© 2018 John Wiley & Sons Ltd.)

Rhodobacter capsulatus synthesizes the high-affinity ABC transporters CysTWA and ModABC to specifically import the chemically related oxyanions sulfate and molybdate, respectively. In addition, R. capsulatus has the low-affinity permease PerO acting as a general oxyanion transporter, whose elimination increases tolerance to molybdate and tungstate. Although PerO-like permeases are widespread in bacteria, their function has not been examined in any other species to date. Here, we present evidence that PerO permeases from the alphaproteobacteria Agrobacterium tumefaciens , Dinoroseobacter shibae , Rhodobacter sphaeroides , and Sinorhizobium meliloti and the gammaproteobacterium Pseudomonas stutzeri functionally substitute for R. capsulatus PerO in sulfate uptake and sulfate-dependent growth, as shown by assimilation of radioactively labeled sulfate and heterologous complementation. Disruption of perO genes in A. tumefaciens , R. sphaeroides , and S. meliloti increased tolerance to tungstate and, in the case of R. sphaeroides , to molybdate, suggesting that heterometal oxyanions are common substrates of PerO permeases. This study supports the view that bacterial PerO permeases typically transport sulfate and related oxyanions and, hence, form a functionally conserved permease family. IMPORTANCE Despite the widespread distribution of PerO-like permeases in bacteria, our knowledge about PerO function until now was limited to one species, Rhodobacter capsulatus In this study, we showed that PerO proteins from diverse bacteria are functionally similar to the R. capsulatus prototype, suggesting that PerO permeases form a conserved family whose members transport sulfate and related oxyanions., (Copyright © 2017 American Society for Microbiology.)

Many enzymes require the molybdenum cofactor, Moco. Under Mo-limiting conditions, the high-affinity ABC transporter ModABC permits molybdate uptake and Moco biosynthesis in bacteria. Under Mo-replete conditions, Escherichia coli represses modABC transcription by the one-component regulator, ModE, consisting of a DNA-binding and a molybdate-sensing domain. Instead of a full-length ModE protein, many bacteria have a shorter ModE protein, ModE(S) , consisting of a DNA-binding domain only. Here, we asked how such proteins sense the intracellular molybdenum status. We show that the Agrobacterium tumefaciens ModE(S) protein Atu2564 is essential for modABC repression. ModE(S) binds two Mo-boxes in the modA promoter as shown by electrophoretic mobility shift assays. Northern analysis revealed cotranscription of modE(S) with the upstream gene, atu2565, which was dispensable for ModE(S) activity. To identify genes controlling ModE(S) function, we performed transposon mutagenesis. Tn5 insertions resulting in derepressed modA transcription mapped to the atu2565-modE(S) operon and several Moco biosynthesis genes. We conclude that A. tumefaciens ModE(S) activity responds to Moco availability rather than to molybdate concentration directly, as is the case for E. coli ModE. Similar results in Sinorhizobium meliloti suggest that Moco dependence is a common feature of ModE(S) regulators., (© 2016 John Wiley & Sons Ltd.)

Unlabelled: Rhodobacter capsulatus is capable of synthesizing two nitrogenases, a molybdenum-dependent nitrogenase and an alternative Mo-free iron-only nitrogenase, enabling this diazotroph to grow with molecular dinitrogen (N2) as the sole nitrogen source. Here, the Mo responses of the wild type and of a mutant lacking ModABC, the high-affinity molybdate transporter, were examined by proteome profiling, Western analysis, epitope tagging, and lacZ reporter fusions. Many Mo-controlled proteins identified in this study have documented or presumed roles in nitrogen fixation, demonstrating the relevance of Mo control in this highly ATP-demanding process. The levels of Mo-nitrogenase, NifHDK, and the Mo storage protein, Mop, increased with increasing Mo concentrations. In contrast, Fe-nitrogenase, AnfHDGK, and ModABC, the Mo transporter, were expressed only under Mo-limiting conditions. IscN was identified as a novel Mo-repressed protein. Mo control of Mop, AnfHDGK, and ModABC corresponded to transcriptional regulation of their genes by the Mo-responsive regulators MopA and MopB. Mo control of NifHDK and IscN appeared to be more complex, involving different posttranscriptional mechanisms. In line with the simultaneous control of IscN and Fe-nitrogenase by Mo, IscN was found to be important for Fe-nitrogenase-dependent diazotrophic growth. The possible role of IscN as an A-type carrier providing Fe-nitrogenase with Fe-S clusters is discussed., Importance: Biological nitrogen fixation is a central process in the global nitrogen cycle by which the abundant but chemically inert dinitrogen (N2) is reduced to ammonia (NH3), a bioavailable form of nitrogen. Nitrogen reduction is catalyzed by nitrogenases found in diazotrophic bacteria and archaea but not in eukaryotes. All diazotrophs synthesize molybdenum-dependent nitrogenases. In addition, some diazotrophs, including Rhodobacter capsulatus, possess catalytically less efficient alternative Mo-free nitrogenases, whose expression is repressed by Mo. Despite the importance of Mo in biological nitrogen fixation, this is the first study analyzing the proteome-wide Mo response in a diazotroph. IscN was recognized as a novel member of the molybdoproteome in R. capsulatus. It was dispensable for Mo-nitrogenase activity but supported diazotrophic growth under Mo-limiting conditions., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Rhodobacter capsulatus fixes atmospheric dinitrogen via two nitrogenases, Mo- and Fe-nitrogenase, which operate under different conditions. Here, we describe the functions in nitrogen fixation and regulation of the rcc00574 (cooA) and rcc00575 (cowN) genes, which are located upstream of the structural genes of Mo-nitrogenase, nifHDK. Disruption of cooA or cowN specifically impaired Mo-nitrogenase-dependent growth at carbon monoxide (CO) concentrations still tolerated by the wild type. The cooA gene was shown to belong to the Mo-nitrogenase regulon, which is exclusively expressed when ammonium is limiting. Its expression was activated by NifA1 and NifA2, the transcriptional activators of nifHDK. AnfA, the transcriptional activator of Fe-nitrogenase genes, repressed cooA, thereby counteracting NifA activation. CooA activated cowN expression in response to increasing CO concentrations. Base substitutions in the presumed CooA binding site located upstream of the cowN transcription start site abolished cowN expression, indicating that cowN regulation by CooA is direct. In conclusion, a transcription factor-based network controls cowN expression to protect Mo-nitrogenase (but not Fe-nitrogenase) under appropriate conditions., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Rhodobacter capsulatus is able to grow with N2 as the sole nitrogen source using either a molybdenum-dependent or a molybdenum-free iron-only nitrogenase whose expression is strictly inhibited by ammonium. Disruption of the fdxD gene, which is located directly upstream of the Mo-nitrogenase genes, nifHDK, abolished diazotrophic growth via Mo-nitrogenase at oxygen concentrations still tolerated by the wild type, thus demonstrating the importance of FdxD under semiaerobic conditions. In contrast, FdxD was not beneficial for diazotrophic growth depending on Fe-nitrogenase. These findings suggest that the 2Fe2S ferredoxin FdxD specifically supports the Mo-nitrogenase system, probably by protecting Mo-nitrogenase against oxygen, as previously shown for its Azotobacter vinelandii counterpart, FeSII. Expression of fdxD occurred under nitrogen-fixing conditions, but not in the presence of ammonium. Expression of fdxD strictly required NifA1 and NifA2, the transcriptional activators of the Mo-nitrogenase genes, but not AnfA, the transcriptional activator of the Fe-nitrogenase genes. Expression of the fdxD and nifH genes, as well as the FdxD and NifH protein levels, increased with increasing molybdate concentrations. Molybdate induction of fdxD was independent of the molybdate-sensing regulators MopA and MopB, which repress anfA transcription at micromolar molybdate concentrations. In this report, we demonstrate the physiological relevance of an fesII-like gene, fdxD, and show that the cellular nitrogen and molybdenum statuses are integrated to control its expression.

To identify copper homeostasis genes in Rhodobacter capsulatus, we performed random transposon Tn5 mutagenesis. Screening of more than 10,000 Tn5 mutants identified tellurite resistance gene trgB as a so far unrecognized major copper tolerance determinant. The trgB gene is flanked by tellurite resistance gene trgA and cysteine synthase gene cysK2. While growth of trgA mutants was only moderately restricted by tellurite, trgB and cysK2 mutants were severely affected by tellurite, which implies that viability under tellurite stress requires increased cysteine levels. Mutational analyses revealed that trgB was the only gene in this chromosomal region conferring cross-tolerance towards copper. Expression of the monocistronic trgB gene required promoter elements overlapping the trgA coding region as shown by nested deletions. Neither copper nor tellurite affected trgB transcription as demonstrated by reverse transcriptase PCR and trgB-lacZ fusions. Addition of tellurite or copper gave rise to increased cellular tellurium and copper concentrations, respectively, as determined by inductively coupled plasma-optical emission spectroscopy. By contrast, cellular iron concentrations remained fairly constant irrespective of tellurite or copper addition. This is the first study demonstrating a direct link between copper and tellurite response in bacteria.

Copper is an essential cofactor of various enzymes, but free copper is highly toxic to living cells. To maintain cellular metabolism at different ambient copper concentrations, bacteria have evolved specific copper homeostasis systems that mostly act as defence mechanisms. As well as under free-living conditions, copper defence is critical for virulence in pathogenic bacteria. Most bacteria synthesize P-type copper export ATPases as principal defence determinants when copper concentrations exceed favourable levels. In addition, many bacteria utilize resistance-nodulation-cell division (RND)-type efflux systems and multicopper oxidases to cope with excess copper. This review summarizes our current knowledge on copper-sensing transcriptional regulators, which we assign to nine different classes. Widespread one-component regulators are CueR, CopY and CsoR, which were initially identified in Escherichia coli, Enterococcus hirae and Mycobacterium tuberculosis, respectively. CueR activates homeostasis gene expression at elevated copper concentrations, while CopY and CsoR repress their target genes under copper-limiting conditions. Besides these one-component systems, which sense the cytoplasmic copper status, many Gram-negative bacteria utilize two-component systems, which sense periplasmic copper concentrations. In addition to these well-studied transcriptional factors, copper control mechanisms acting at the post-transcriptional and the post-translational levels will be discussed.

The copper-regulated Rhodobacter capsulatus cutO (multicopper oxidase) gene confers copper tolerance and is carried in the tricistronic orf635-cutO-cutR operon. Transcription of cutO strictly depends on the promoter upstream of orf635, as demonstrated by lacZ reporter fusions to nested promoter fragments. Remarkably, orf635 expression was not affected by copper availability, whereas cutO and cutR were expressed only in the presence of copper. Differential regulation was abolished by site-directed mutations within the orf635-cutO intergenic region, suggesting that this region encodes a copper-responsive mRNA element. Bioinformatic predictions and RNA structure probing experiments revealed an intergenic stem-loop structure as the candidate mRNA element. This is the first posttranscriptional copper response mechanism reported in bacteria.

The LysR-type regulator MopB represses transcription of several target genes (including the nitrogen-fixation gene anfA) in Rhodobacter capsulatus at high molybdenum concentrations. In this study, the isolated DNA-binding domain of MopB (MopBHTH) was overexpressed in Escherichia coli. Purified MopBHTH bound the anfA promoter as shown by DNA mobility-shift assays, demonstrating the function of the isolated regulator domain. MopBHTH was crystallized using the sitting-drop vapour-diffusion method in the presence of 0.2 M lithium sulfate, 0.1 M phosphate/citrate pH 4.2, 20%(w/v) PEG 1000 at 291 K. The crystal belonged to space group P3(1)21 or P3(2)21, with unit-cell parameters a=b=61.84, c=139.64 Å, α=β=90, γ=120°, and diffracted to 3.3 Å resolution at a synchrotron source.

Molybdenum (Mo) is an important trace element that is toxic at high concentrations. To resolve the mechanisms underlying Mo toxicity, Rhodobacter capsulatus mutants tolerant to high Mo concentrations were isolated by random transposon Tn5 mutagenesis. The insertion sites of six independent isolates mapped within the same gene predicted to code for a permease of unknown function located in the cytoplasmic membrane. During growth under Mo-replete conditions, the wild-type strain accumulated considerably more Mo than the permease mutant. For mutants defective for the permease, the high-affinity molybdate importer ModABC, or both transporters, in vivo Mo-dependent nitrogenase (Mo-nitrogenase) activities at different Mo concentrations suggested that ModABC and the permease import molybdate in nanomolar and micromolar ranges, respectively. Like the permease mutants, a mutant defective for ATP sulfurylase tolerated high Mo concentrations, suggesting that ATP sulfurylase is the main target of Mo inhibition in R. capsulatus. Sulfate-dependent growth of a double mutant defective for the permease and the high-affinity sulfate importer CysTWA was reduced compared to those of the single mutants, implying that the permease plays an important role in sulfate uptake. In addition, permease mutants tolerated higher tungstate and vanadate concentrations than the wild type, suggesting that the permease acts as a general oxyanion importer. We propose to call this permease PerO (for oxyanion permease). It is the first reported bacterial molybdate transporter outside the ABC transporter family.

Either of two related molybdenum-responsive regulators, MopA and MopB, of Rhodobacter capsulatus is sufficient to repress the nitrogen-fixation gene anfA. In contrast, MopA (but not MopB) activates mop, which codes for a molybdate (Mo)-binding molbindin. Both regulators bind to conserved cis-regulatory elements called Mo-boxes. Single-base substitution of two highly conserved nucleotides within the anfA-Mo-box (T21C and C24T) had little effect on regulator binding and anfA expression as shown by DNA mobility shift assays and reporter gene fusions, respectively. In contrast to C24T, mutation C24A strongly diminished binding and repression by MopA and MopB, showing that different nucleotide substitutions at the same position may have very different effects. A triple mutation destroying the left half-site of the mop-Mo-box completely abolished mop expression by MopA, demonstrating the importance of the mop-Mo-box for mop activation. Two point mutations (T23A and T24C) still allowed binding by MopA, but abolished mop activation, most likely because these nucleotides overlap with the RNA polymerase-binding site. A mutant mop promoter, in which the mop-Mo-box was exchanged against the anfA-Mo-box, allowed activation by MopA, showing that a former repressor-binding site may act as an activator-binding site depending on its location relative to the other promoter elements.

The vast majority of the purple nonsulfur photosynthetic bacteria are diazotrophs, but the details of the complex regulation of the nitrogen fixation process are well understood only for a few species. Here we review what is known of the well-studied Rhodobacter capsulatus, which contains two different nitrogenases, a standard Mo-nitrogenase and an alternative Fe-nitrogenase, and which has overlapping transcriptional control mechanisms with regard to the presence of fixed nitrogen, oxygen, and molybdenum as well as the capability for the post-translational control of both nitrogenases in response to ammonium. R. capsulatus has two PII proteins, GlnB and GlnK, which play key roles in nitrogenase regulation at each of three different levels: activation of transcription of the nif-specific activator NifA, the post-translational control of NifA activity, and the regulation of nitrogenase activity through either ADP-ribosylation of NifH or an ADP-ribosylation-independent pathway. We also review recent work that has led to a detailed characterization of the molybdenum transport and regulatory system in R. capsulatus that ensures activity of the Mo-nitrogenase and repression of the Fe-nitrogenase, down to extremely low levels of molybdenum.

The phototrophic purple bacterium Rhodobacter capsulatus encodes two transcriptional regulators, MopA and MopB, with partially overlapping and specific functions in molybdate-dependent gene regulation. Both MopA and MopB consist of an N-terminal DNA-binding helix-turn-helix domain and a C-terminal molybdate-binding di-MOP domain. They formed homodimers as apo-proteins and in the molybdate-bound state as shown by yeast two-hybrid (Y2H) studies, glutaraldehyde cross-linking, gel filtration chromatography, and copurification experiments. Y2H studies suggested that both the DNA-binding and the molybdate-binding domains contribute to dimer formation. Analysis of molybdate binding to MopA and MopB revealed a binding stoichiometry of four molybdate oxyanions per homodimer. Specific interaction partners of MopA and MopB were the molybdate transporter ATPase ModC and the molbindin-like Mop protein, respectively. Like other molbindins, the R. capsulatus Mop protein formed hexamers, which were stabilized by binding of six molybdate oxyanions per hexamer. Heteromer formation of MopA and MopB was shown by Y2H studies and copurification experiments. Reporter gene activity of a strictly MopA-dependent mop-lacZ fusion in mutant strains defective for either mopA, mopB, or both suggested that MopB negatively modulates expression of the mop promoter. We propose that depletion of the active MopA homodimer pool by formation of MopA-MopB heteromers might represent a fine-tuning mechanism controlling mop gene expression.

Rhodobacter capsulatus can efficiently grow with taurine as the sole sulfur source. The products of the tpa-tauR-xsc gene region are essential for this activity. TauR, a MocR-like member of the GntR superfamily of transcriptional regulators, activates tpa transcription, as shown by analysis of wild-type and tauR mutant strains carrying a tpa-lacZ reporter fusion. Activation of the tpa promoter requires taurine but is not inhibited by sulfate, which is the preferred sulfur source. TauR directly binds to the tpa promoter, as demonstrated by DNA mobility shift assays. As expected for a transcriptional activator, the TauR binding site is located upstream of the transcription start site, which has been determined by primer extension. Site-directed promoter mutations reveal that TauR binds to direct repeats, an unusual property that has to date been shown for only one other member of the MocR subfamily, namely, GabR from Bacillus subtilis. In contrast, all other members of the GntR family analyzed so far bind to inverted repeats.

Both Rhodobacter capsulatus PII homologs GlnB and GlnK were found to be necessary for the proper regulation of nitrogenase activity and modification in response to an ammonium shock. As previously reported for several other bacteria, ammonium addition triggered the AmtB-dependent association of GlnK with the R. capsulatus membrane. Native polyacrylamide gel electrophoresis analysis indicates that the modification/demodification of one PII homolog is aberrant in the absence of the other. In a glnK mutant, more GlnB was found to be membrane associated under these conditions. In a glnB mutant, GlnK fails to be significantly sequestered by AmtB, even though it appears to be fully deuridylylated. Additionally, the ammonium-induced enhanced sequestration by AmtB of the unmodifiable GlnK variant GlnK-Y51F follows the wild-type GlnK pattern with a high level in the cytoplasm without the addition of ammonium and an increased level in the membrane fraction after ammonium treatment. These results suggest that factors other than PII modification are driving its association with AmtB in the membrane in R. capsulatus.

The phototrophic purple bacterium Rhodobacter capsulatus encodes two similar but functionally not identical molybdenum-dependent regulator proteins (MopA and MopB), which are known to replace each other in repression of the modABC genes (coding for an ABC-type high-affinity Mo transport system) and anfA (coding for the transcriptional activator of Fe-nitrogenase genes). We identified further Mo-regulated (mor) genes coding for a putative ABC-type transport system of unknown function (MorABC) and a putative Mo-binding protein (Mop). The genes coding for MopA and the ModABC transporter form part of a single transcriptional unit, mopA-modABCD, as shown by reverse transcriptase PCR. Immediately upstream of mopA and transcribed in the opposite direction is mopB. The genes coding for the putative MorABC transporter belong to two divergently transcribed operons, morAB and morC. Expression studies based on lacZ reporter gene fusions in mutant strains defective for either MopA, MopB, or both revealed that the regulators substitute for each other in Mo-dependent repression of morAB and morC. Specific Mo-dependent activation of the mop gene by MopA, but not MopB, was found to control the putative Mo-binding protein. Both MopA and MopB are thought to bind to conserved DNA sequences with dyad symmetry in the promoter regions of all target genes. The positions of these so-called Mo boxes relative to the transcription start sites (as determined by primer extension analyses) differed between Mo-repressed genes and the Mo-activated mop gene. DNA mobility shift assays showed that MopA and MopB require molybdenum to bind to their target sites with high affinity.

Rhodobacter capsulatus NtrB/NtrC two-component regulatory system controls expression of genes involved in nitrogen metabolism including urease and nitrogen fixation genes. The ntrY-ntrX genes, which are located immediately downstream of the nifR3-ntrB-ntrC operon, code for a two-component system of unknown function. Transcription of ntrY starts within the ntrC-ntrY intergenic region as shown by primer extension analysis, but maximal transcription requires, in addition, the promoter of the nifR3-ntrB-ntrC operon. While ntrB and ntrY single mutant strains were able to grow with either urea or N2 as sole nitrogen source, a ntrB/ntrY double mutant (like a ntrC-deficient strain) was no longer able to use urea or N2. These findings suggest that the histidine kinases NtrB and NtrY can substitute for each other as phosphodonors towards the response regulator NtrC.

The cutO gene of the photosynthetic purple bacterium Rhodobacter capsulatus codes for a multicopper oxidase as demonstrated by the ability of the recombinant Strep-tagged protein to oxidize several mono- and diphenolic compounds known as substrates of Escherichia coli CueO and multicopper oxidases from other organisms. The R. capsulatus cutO gene was shown to form part of a tri-cistronic operon, orf635-cutO-cutR. Expression of the cutO operon was repressed under low copper conditions by the product of the cutR gene. CutO conferred copper tolerance not only under aerobic conditions, as described for the well-characterized E. coli multicopper oxidase CueO, but also under anaerobic conditions.

Growth of Rhodobacter capsulatus with molecular dinitrogen as the sole N source via the alternative Fe-only nitrogenase requires all seven gene products of the anfHDGK-1-2-3 operon. In contrast to mutant strains carrying lesions in the structural genes of nitrogenase (anfH, anfD, anfG, and anfK), strains defective for either anf1, anf2, or anf3 are still able to reduce the artificial substrate acetylene, although with diminished activity. To obtain further information on the role of Anf1, we screened an R. capsulatus genomic library designed for use in yeast two-hybrid studies with Anf1 as bait. Two genes, which we propose to call ranR and ranT (for genes related to alternative nitrogenase), coding for products that interact with Anf1 were identified. A ranR mutant exhibited a phenotype similar to that of an anf1 mutant strain (no growth with N2 in the absence of molybdenum, but significant reduction of acetylene via the Fe-only nitrogenase), whereas a ranT mutant retained the ability to grow diazotrophically, but growth was clearly delayed compared to the parental strain. In contrast to the situation for anf1, expression of neither ranR nor ranT was regulated by ammonium or molybdenum. A putative role for Anf1, RanR, and RanT in the acquisition and/or processing of iron in connection with the Fe-only nitrogenase system is discussed.

Rhodobacter capsulatus contains two PII-like proteins, GlnB and GlnK, which play central roles in controlling the synthesis and activity of nitrogenase in response to ammonium availability. Here we used the yeast two-hybrid system to probe interactions between these PII-like proteins and proteins known to be involved in regulating nitrogen fixation. Analysis of defined protein pairs demonstrated the following interactions: GlnB-NtrB, GlnB-NifA1, GlnB-NifA2, GlnB-DraT, GlnK-NifA1, GlnK-NifA2, and GlnK-DraT. These results corroborate earlier genetic data and in addition show that PII-dependent ammonium regulation of nitrogen fixation in R. capsulatus does not require additional proteins, like NifL in Klebsiella pneumoniae. In addition, we found interactions for the protein pairs GlnB-GlnB, GlnB-GlnK, NifA1-NifA1, NifA2-NifA2, and NifA1-NifA2, suggesting that fine tuning of the nitrogen fixation process in R. capsulatus may involve the formation of GlnB-GlnK heterotrimers as well as NifA1-NifA2 heterodimers. In order to identify new proteins that interact with GlnB and GlnK, we constructed an R. capsulatus genomic library for use in yeast two-hybrid studies. Screening of this library identified the ATP-dependent helicase PcrA as a new putative protein that interacts with GlnB and the Ras-like protein Era as a new protein that interacts with GlnK.

In most bacteria, nitrogen metabolism is tightly regulated and P(II) proteins play a pivotal role in the regulatory processes. Rhodobacter capsulatus possesses two genes (glnB and glnK) encoding P(II)-like proteins. The glnB gene forms part of a glnB-glnA operon and the glnK gene is located immediately upstream of amtB, encoding a (methyl-) ammonium transporter. Expression of glnK is activated by NtrC under nitrogen-limiting conditions. The synthesis and activity of the molybdenum and iron nitrogenases of R. capsulatus are regulated by ammonium on at least three levels, including the transcriptional activation of nifA1, nifA2 and anfA by NtrC, the regulation of NifA and AnfA activity by two different NtrC-independent mechanisms, and the post-translational control of the activity of both nitrogenases by reversible ADP-ribosylation of NifH and AnfH as well as by ADP-ribosylation independent switch-off. Mutational analysis revealed that both P(II)-like proteins are involved in the ammonium regulation of the two nitrogenase systems. A mutation in glnB results in the constitutive expression of nifA and anfA. In addition, the post-translational ammonium inhibition of NifA activity is completely abolished in a glnB-glnK double mutant. However, AnfA activity was still suppressed by ammonium in the glnB-glnK double mutant. Furthermore, the P(II)-like proteins are involved in ammonium control of nitrogenase activity via ADP-ribosylation and the switch-off response. Remarkably, in the glnB-glnK double mutant, all three levels of the ammonium regulation of the molybdenum (but not of the alternative) nitrogenase are completely circumvented, resulting in the synthesis of active molybdenum nitrogenase even in the presence of high concentrations of ammonium.

Genetic analyses based on chromosomal lac fusions to nitrogen fixation (nif) genes demonstrated that NifA-dependent transcriptional activation of expression of Rhodobacter capsulatus nifH and nifB1 was negatively modulated by HvrA, whereas regulation of rpoN, nifA1, and nifA2 was independent of HvrA. Expression of hvrA itself was not influenced by a mutation in ntrC, which is absolutely essential for N(2) fixation. Furthermore, HvrA accumulated to comparable levels in the presence and absence of ammonium, suggesting that the amount of HvrA in the cells does not differ under nitrogenase-repressing or -derepressing conditions. In addition, competitive gel retardation studies with HvrA-His(6) purified from R. capsulatus were carried out, demonstrating preferential binding of HvrA to the nifH promoter region.

The Rhodobacter capsulatus nrfA gene product exhibits extensive similarity to the nif (nitrogen fixation) regulatory factor NrfA of Azorhizobium caulinodans and the nucleoid-associated protein Hfq of Escherichia coli. Mutational analysis revealed that, in contrast to the situation in A. caulinodans, NrfA is not essential for diazotrophic growth of R. capsulatus, but it is required for maximal growth rates with N(2) as sole nitrogen source via either molybdenum nitrogenase or the alternative nitrogenase. NrfA was shown to control N(2) fixation in R. capsulatus at the level of expression of the regulatory genes nifA1, nifA2 and anfA, encoding the transcriptional activators of all the other nitrogen fixation genes.

In R. capsulatus synthesis and activity of the molybdenum and the alternative nitrogenase is controlled at three levels by the environmental factors ammonium, molybdenum, light, and oxygen. At the first level, transcription of the nifA1, nifA2, and anfA genes--which encode the transcriptional activators of all other nif and anf genes, respectively--is controlled by the Ntr system in dependence on ammonium availability. Mutations in ginB (coding for the signal transduction protein PII) result in significant expression of nifA and anfA in the presence of ammonium. In contrast to GlnB, the PII-paralogue GlnK is not involved in the Ntr signal transduction mechanism. In addition to ammonium control, transcription of anfA is inhibited by traces of molybdenum via the molybdate-dependent repressor proteins MopA and MopB. At the second level of regulation, activity of NifA1, NifA2, and AnfA is inhibited by ammonium in an NtrC-independent manner. This post-translational ammonium control of NifA activity is partially released in the absence of GlnK, and completely abolished in a glnB/glnK double mutant. In contrast, AnfA activity is still inhibited by ammonium in the glnB/glnK mutant background. At the third level of regulation, both GlnB and GlnK as well as the (methyl)-ammonium transporter AmtB are involved in ammonium control of the DraT/DraG system, which mediates reversible ADP-ribosylation of both nitrogenase reductases (NifH and AnfH) in response to changes in ammonium availability or light intensity. Most remarkably, in a glnB/glnK double mutant ammonium control of the molybdenum (but not of the alternative) nitrogenase is completely relieved, leading to synthesis of active nitrogenase in the presence of high concentrations of ammonium.

Rhodobacter capsulatus was shown to grow efficiently with taurine as sole source of sulfur. We identified a gene region exhibiting similarity to the Escherichia coli tauABC genes coding for a taurine-specific ABC transporter. The R. capsulatus tauABC genes were flanked by two putative operons (orf459-484-590 and cysE-srpI-nifS2) both reading in opposite direction relative to tauABC. Orf459 shows strong similarity to taurine:pyruvate aminotransferase (Tpa) from Bilophila wadsworthia catalyzing the initial transamination during anaerobic taurine degradation, and Orf590 exhibits clear similarity to sulfoacetaldehyde sulfo-lyase from Desulfonispora thiosulfatigenes probably catalyzing the step following the taurine:pyruvate aminotransferase (Tpa) reaction, whereas nifS2 might code for a putative cysteine desulfurase. Expression of R. capsulatus tauABC and nifS2 was inhibited by sulfate, suggesting that tauABC and nifS2 might belong to the same regulon. In contrast, transcription of orf459 was not inhibited by sulfate but was induced by taurine. A tauAB deletion mutant showed significantly reduced growth compared to the wild-type with taurine as sole sulfur source in the presence of serine as a nitrogen source, whereas normal growth was observed in the presence of taurine and ammonium. Deletion of orf459-484-590 completely abolished growth with taurine/serine. Single mutations in any of the three genes resulted in the same phenotype, indicating that all three genes of this putative operon are essential for taurine sulfur utilization in the presence of serine. A model for anaerobic taurine sulfur assimilation in R. capsulatus is discussed.

Expression of nitrogen fixation genes in Rhodobacter capsulatus is repressed by ammonium at different regulatory levels including an NtrC-independent mechanism controlling NifA activity. In contrast to R. capsulatus NifA, heterologous NifA proteins of Klebsiella pneumoniae and Rhizobium meliloti, respectively, were not subjected to this posttranslational ammonium control in R. capsulatus. The characterization of ammonium-tolerant R. capsulatus NifA1 mutants indicated that the N-terminal domain of NifA was involved in posttranslational regulation. Analysis of a double mutant carrying amino acid substitutions in both the N-terminal domain and the C-terminal DNA-binding domain gave rise to the hypothesis that an interaction between these two domains might be involved in ammonium regulation of NifA activity. Western analysis demonstrated that both constitutively expressed wild-type and ammonium-tolerant NifA1 proteins exhibited high stability and accumulated to comparable levels in cells grown in the presence of ammonium excluding the possibility that proteolytic degradation was responsible for ammonium-dependent inactivation of NifA.

The phototrophic nonsulfur purple bacterium Rhodobacter capsulatus can use urea as a sole source of nitrogen. Three transposon Tn5-induced mutations (Xan-9, Xan-10, and Xan-19), which led to a Ure(-) phenotype, were mapped to the ureF and ureC genes, whereas two other Tn5 insertions (Xan-20 and Xan-22) were located within the ntrC and ntrB genes, respectively. As in Klebsiella aerogenes and other bacteria, the genes encoding urease (ureABC) and the genes required for assembly of the nickel metallocenter (ureD and ureEFG) are clustered in R. capsulatus (ureDABC-orf136-ureEFG). No homologues of Orf136 were found in the databases, and mutational analysis demonstrated that orf136 is not essential for urease activity or growth on urea. Analysis of a ureDA-lacZ fusion showed that maximum expression of the ure genes occurred under nitrogen-limiting conditions (e.g., serine or urea as the sole nitrogen source), but ure gene expression was not substrate (urea) inducible. Expression of the ure genes was strictly dependent on NtrC, whereas sigma(54) was not essential for urease activity. Expression of the ure genes was lower (by a factor of 3.5) in the presence of ammonium than under nitrogen-limiting conditions, but significant transcription was also observed in the presence of ammonium, approximately 10-fold higher than in an ntrC mutant background. Thus, ure gene expression in the presence of ammonium also requires NtrC. Footprint analyses demonstrated binding of NtrC to tandem binding sites upstream of the ureD promoter. Phosphorylation of NtrC increased DNA binding by at least eightfold. Although urea is effectively used as a nitrogen source in an NtrC-dependent manner, nitrogenase activity was not repressed by urea.

A Rhodobacter capsulatus reporter strain, carrying a constitutively expressed nifA gene and a nifH-lacZ gene fusion, was used for random transposon Tn5 mutagenesis to search for genes required for the NtrC-independent ammonium repression of NifA activity. A mutation in hvrA, which is known to be involved in low-light activation of the photosynthetic apparatus, released both ammonium and oxygen control of nifH expression in this reporter strain, demonstrating a regulatory link of nitrogen fixation and photosynthesis via HvrA. In addition, a significant increase in bacteriochlorophyll alpha (BChl alpha) content was found in cells under nitrogen-fixing conditions. HvrA was not involved in this up-regulation of BChl alpha. Instead, the presence of active nitrogenase seemed to be sufficient for this process, since no increase in BChl alpha content was observed in different nif mutants.

When iron becomes limiting, Synechocystis 6803 induces the synthesis of flavodoxin. As a basis for genetic analysis, the flavodoxin-encoding isiB gene of Synechocystis 6803 was cloned and sequenced. The isiB gene was disrupted by insertion of an interposon within the isiB coding region resulting in two Synechocystis 6803 mutant strains, CKF-I and CKF-II. They were distinguished from each other by the orientation of the kanamycin resistance cassette. Photoautotrophic growth of the mutant strains under iron limiting conditions, which are sufficient for induction of flavodoxin in the wild-type cells, demonstrated that IsiB was not essential for Synechocystis 6803.

In contrast to that of Anabaena sp. PCC7120, the fdxN gene in Anabaena variabilis ATCC 29413 is not interrupted by a 55-kb DNA element, making this strain more suitable for genetic analysis of fdxN independent of the developmentally regulated excision during heterocyst formation. As a basis for mutational analysis, the fdxN gene of A. variabilis was cloned and sequenced. The deduced FdxN protein sequence was highly homologous to the Anabaena 7120 fdxN gene product including eight cysteine residues that are known to be conserved among ferredoxins containing two [4Fe-4S] clusters. The fdxN gene of A. variabilis was disrupted by insertion of an interposon within the fdxN coding region resulting in mutant strain KG29. Diazotrophic growth and in vivo nitrogenase activity of KG29 were similar to those of the wild-type, indicating that FdxN was not essential for N 2 fixation in A. variabilis.

The nifV and leuA genes, which encode homocitrate synthase and alpha-isopropylmalate synthase, respectively, were cloned from the filamentous cyanobacterium Anabaena sp. strain PCC 7120 by a PCR-based strategy. Since the N-terminal parts of NifV and LeuA from other bacteria are highly similar to each other, a single pair of PCR primers was used to amplify internal fragments of both Anabaena strain 7120 genes. Sequence analysis of cloned PCR products confirmed the presence of two different nifV-like DNA fragments, which were subsequently used as nifV- and leuA-specific probes, respectively, to clone XbaI fragments of 2.1 kbp (pOST4) and 2.6 kbp (pOST2). Plasmid pOST4 carried the Anabaena strain 7120 nifV-nifZ-nifT genes, whereas pOST2 contained the leuA and dapF genes. The nifVZT genes were not located in close proximity to the main nif gene cluster in Anabaena strain 7120, and therefore nifVZT forms a second nif gene cluster in this strain. Overlaps between the nifV and nifZ genes and between the nifZ and nifT genes and the presence of a 1.8-kb transcript indicated that nifVZT might form one transcriptional unit. Transcripts of nifV were induced not only in a nitrogen-depleted culture but also by iron depletion irrespective of the nitrogen status. The nifV gene in Anabaena strain 7120 was interrupted by an interposon insertion (mutant strain BMB105) and by a plasmid integration via a single crossover with a nifV internal fragment as a site for recombination (mutant strain BMB106). Both mutant strains were capable of diazotrophic growth, and their growth rates were only slightly impaired compared to that of the wild type. Heterologous complementation of the Rhodobacter capsulatus nifV mutant R229I by the Anabaena strain 7120 nifV gene corroborated the assumption that Anabaena strain 7120 nifV also encodes a homocitrate synthase. In contrast, the Anabaena strain 7120 leuA gene did not complement the nifV mutation of R229I efficiently.

Nucleotide sequence analysis of the DNA region carrying transposon Tn5-1087b from the Anabaena 7120 nitrogen fixation-deficient mutant YC16 revealed the presence of a novel repeated DNA element in cyanobacteria designated long tandemly repeated repetitive (LTRR) sequence. The LTRR element is 37 bp long and contains an inverted repeat sequence. 17 copies of the LTRR element, 13 of which were completely identical, were identified within a 1.3 kb DNA fragment, which was flanked by two divergently transcribed genes homologous to bacteriophage T4 'gene 15' and Rhizobium meliloti exoD, respectively. LTRR-like sequences occur in several DNA regions in Anabaena 7120 and in other cyanobacteria. Furthermore, the presence of an LTRR-like DNA region in mitochondrial plasmids of Vicia faba indicates strong conservation of such structures during evolution.

A novel type of L-amino acid oxidase from Synechococcus PCC6301 was purified and subjected to amino acid sequence analysis. Since the N-terminus of the L-amino acid oxidase protein was not accessible for Edman degradation, the protein was partially hydrolysed and a contiguous sequence of 17 amino acid residues was obtained from an endogenous peptide fragment. Based on the partial peptide sequence two oligonucleotides were designed, which were used as probes in Southern hybridization experiments in order to identify the corresponding aoxA gene. The aoxA gene was isolated from a size-fractionated genomic library of Synechococcus PCC6301 and subsequently sequenced. From the nucleotide sequence (data base accession number Z48565) it can be deduced that the L-amino acid protein consists of 355 amino acid residues resulting in a molar mass of 39.2 kDa. The calculated isoelectric point of the protein is 9.81. The L-amino acid oxidase from Synechococcus PCC6301 shows low homologies to other flavin oxidases/dehydrogenases, especially amine oxidases, but no homologies to other so far sequenced L- or D-amino acid oxidases.

A gene (phoV) encoding an alkaline phosphatase from Synechococcus sp. strain PCC 7942 was isolated by screening a plasmid gene bank for expression of alkaline phosphatase activity in Escherichia coli JM103. Two independent clones carrying the same alkaline-phosphatase-encoding gene were isolated. One of these clones (pKW1) was further analysed and the nucleotide sequence of a contiguous 3234 bp DNA fragment was determined. Two complete open reading frames (ORF1 and phoV) and an incomplete ORF3 were identified reading in the same direction. The deduced phoV gene product showed 34% identity to the alkaline phosphatase PhoA from Zymomonas mobilis, and the N-terminal part of the putative ORF3 protein exhibited 57% identity to a protein of unknown function from Frankia sp. Insertional inactivation of the Synechococcus PCC 7942 phoV gene failed, indicating an essential role for either the phoV or the ORF3 gene product. PhoV consists of 550 amino acid residues, resulting in a molecular mass of 61.3 kDa. To overexpress the Synechococcus PCC 7942 phoV gene in E. coli, plasmid pKW1 was transformed into a phoA mutant of E. coli (CC118). In E. coli strain CC118(pKW1) PhoV was expressed constitutively with high rates of activity, and was shown to be membrane associated in the periplasmic space. After partial purification of the recombinant PhoV, it was shown that, like other alkaline phosphatases, the Synechococcus PhoV had a broad pH optimum in the alkaline region and a broad substrate specificity for phosphomonoesters, required Zn2+ for activity, and was inhibited by phosphate. In contrast to several other alkaline phosphatases, PhoV was inhibited by Mn2+. Due to the lack of a Synechococcus PCC 7942 phoV mutant strain, the function of PhoV remains uncertain. However, the present results show that Synechococcus PCC 7942 has a second, probably phosphate-irrepressible, alkaline phosphatase (PhoV, 61.3 kDa) in addition to the phosphate-repressible enzyme (PhoA, 145 kDa) already described.

Four Rhodobacter capsulatus mutants unable to grow with proline as the sole nitrogen source were isolated by random Tn5 mutagenesis. The Tn5 insertions were mapped within two adjacent chromosomal EcoRI fragments. DNA sequence analysis of this region revealed three open reading frames designated selD, putR, and putA. The putA gene codes for a protein of 1,127 amino acid residues which is homologous to PutA of Salmonella typhimurium and Escherichia coli. The central part of R. capsulatus PutA showed homology to proline dehydrogenase of Saccharomyces cerevisiae (Put1) and Drosophila melanogaster (SlgA). The C-terminal part of PutA exhibited homology to Put2 (pyrroline-5-carboxylate dehydrogenase) of S. cerevisiae and to aldehyde dehydrogenases from different organisms. Therefore, it seems likely that in R. capsulatus, as in enteric bacteria, both enzymatic steps for proline degradation are catalyzed by a single polypeptide (PutA). The deduced amino acid sequence of PutR (154 amino acid residues) showed homology to the small regulatory proteins Lrp, BkdR, and AsnC. The putR gene, which is divergently transcribed from putA, is essential for proline utilization and codes for an activator of putA expression. The expression of putA was induced by proline and was not affected by ammonia or other amino acids. In addition, putA expression was autoregulated by PutA itself. Mutations in glnB, nifR1 (ntrC), and NifR4 (ntrA encoding sigma 54) had no influence on put gene expression. The open reading frame located downstream of R. capsulatus putR exhibited strong homology to the E. coli selD gene, which is involved in selenium metabolism. R. capsulatus selD mutants exhibited a Put+ phenotype, demonstrating that selD is required neither for viability nor for proline utilization.

The fdxN gene from Rhizobium meliloti encoding a bacterial-type ferredoxin (FdxN) was expressed in Escherichia coli under the control of the lac promoter. The fdxN gene product was purified under anaerobic conditions by ion-exchange chromatography and gel-filtration steps using an antiserum raised against an FdxN-LacZ fusion protein as a detection system. The purified ferredoxin was shown to be identical to the predicted R. meliloti FdxN protein in its amino acid composition and N-terminal amino acid sequence. Chemical determination of the iron content revealed 8.6 +/- 0.6 mol Fe/mol FdxN. The ultraviolet/visible absorption spectrum of the FdxN protein in the oxidized form exhibited maxima at 284 nm and 378 nm, with an A378/A284 ratio of 0.7. EPR spectroscopy revealed a rhombic signal when FdxN was partially reduced, and a broad signal indicative of spin-spin interaction when fully reduced, suggesting the presence of two Fe-S cluster/ferredoxin polypeptide. Our data suggest that FdxN contains two [4Fe-4S] clusters. Purified FdxN was able to mediate electron transport between illuminated chloroplasts and Rhodobacter capsulatus nitrogenase in vitro.

DNA sequence analysis of a 12236 bp fragment, which is located upstream of nifE in Rhodobacter capsulatus nif region A, revealed the presence of ten open reading frames. With the exception of fdxC and fdxN, which encode a plant-type and a bacterial-type ferredoxin, the deduced products of these coding regions exhibited no significant homology to known proteins. Analysis of defined insertion and deletion mutants demonstrated that six of these genes were required for nitrogen fixation. Therefore, we propose to call these genes rnfA, rnfB, rnfC, rnfD, rnfE and rnfF (for Rhodobacter nitrogen fixation). Secondary structure predictions suggested that the rnf genes encode four potential membrane proteins and two putative iron-sulphur proteins, which contain cysteine motifs (C-X2-C-X2-C-X3-C-P) typical for [4Fe--4S] proteins. Comparison of the in vivo and in vitro nitrogenase activities of fdxN and rnf mutants suggested that the products encoded by these genes are involved in electron transport to nitrogenase. In addition, these mutants were shown to contain significantly reduced amounts of nitrogenase. The hypothesis that this new class of nitrogen fixation genes encodes components of an electron transfer system to nitrogenase was corroborated by analysing the effect of metronidazole. Both the fdxN and rnf mutants had higher growth yields in the presence of metronidazole than the wild type, suggesting that these mutants contained lower amounts of reduced ferredoxins.

Synthetic oligonucleotides, which were designed according to amino acid sequences conserved between Rhodospirillum rubrum and Azospirillum brasilense DraT and DraG, respectively, were used to identify the corresponding genes of Rhodobacter capsulatus. Sequence analysis of a 1904 bp DNA fragment proved the existence of R. capsulatus draT and draG. These two genes were separated by 11 bp only, suggesting that R. capsulatus draT and draG were part of one transcriptional unit. In contrast to R. rubrum, A. brasilense and Azospirillum lipoferum, the R. capsulatus draTG genes were not located upstream of the structural genes of nitrogenase nifHDK but close to the dctP gene at a distance of about 1000 kb from the nifHDK genes. Deletion mutations in the draTG gene region were constructed and introduced into R. capsulatus wild-type and a nifHDK deletion strain. The resulting mutant strains were examined for post-translational regulation of the molybdenum and the alternative nitrogenase in response to ammonia and darkness. Under 'switch-off' conditions the modified (ADP-ribosylated) and the non-modified forms of component II of both the molybdenum and the alternative nitrogenase were detected in a draTG wild-type background by immunoblot analysis, whereas only the non-modified forms were present in the draTG deletion strains. Nitrogenase activity in these strains was followed by the acetylene reduction assay. In contrast to the wild-type, draTG mutants were not affected in nitrogenase activity in response to ammonia or darkness. These results demonstrated that the draTG genes are required for post-translational regulation of both the molybdenum and the heterometal-free nitrogenase in R. capsulatus.

The expression of nif genes in Rhodobacter capsulatus depends on the two regulatory genes, rpoN and nifA, encoding a nif-specific alternative sigma factor of RNA polymerase and a nif-specific transcriptional activator, respectively. The expression of the rpoN gene itself is also RPON/NIFA dependent. In order to better characterize the regulation of nif gene induction, chromosomal nifH-, rpoN-, nifA1- and nifA2- lacZ fusions were constructed and the expression of these different nif-lacZ fusions was determined under photoheterotrophic conditions at different starting ammonium concentrations. The two nifA genes were found to be induced first, followed by nifH and finally by rpoN upon weak, medium and strong nitrogen starvation, respectively. This induction profile and the correlation between the expression of the different nif genes suggested that nifA1 expression is the limiting factor for nif gene induction. This hypothesis was tested by construction of different nifA1 overexpressing mutants. Contrary to the current model of nif gene expression in R. capsulatus, which predicted constitutive nif gene expression in such mutants, a strong repression of nifH and rpoN was found at high ammonium concentration. The low nifH expression under these conditions is unaffected by nifA2 and is not increased in a ntrC mutant, ruling out any role of NTRC as a mediator of this repression. This finding implies an additional, so far unidentified, regulation by fixed nitrogen in R. capsulatus. Changing the expression level of rpoN indicated that low levels of RPON are already sufficient for full nifH induction. The nifA1 and rpoN expression mutants were also tested for diazotrophic growth. Similar generation times were determined for the mutants and for the wild type, but diazotrophic growth of the nifA1 over-expressing ntrC mutant RCM14 did not start until after a prolonged lag phase of two to three days.

DNA sequence analysis of a 3494-bp HindIII-BclI fragment of the Rhodobacter capsulatus nif region A revealed genes that are homologous to ORF6, nifU, nifS, nifV and nifW from Azotobacter vinelandii and Klebsiella pneumoniae. R. capsulatus nifU, which is present in two copies, encodes a novel type of NifU protein. The deduced amino acid sequences of NifUI and NifUII share homology only with the C-terminal domain of NifU from A. vinelandii and K. pneumoniae. In contrast to nifA and nifB, which are almost perfectly duplicated, the predicted amino acid sequences of the two NifU proteins showed only 39% sequence identity. Expression of the ORF6-nifUISVW operon, which is preceded by a putative sigma 54-dependent promoter, required the function of NifA and the nif-specific rpoN gene product encoded by nifR4. Analysis of defined insertion and deletion mutants demonstrated that only nifS was absolutely essential for nitrogen fixation in R. capsulatus. Strains carrying mutations in nifV were capable of very slow diazotrophic growth, whereas ORF6, nifUI and nifW mutants as well as a nifUI/nifUII double mutant exhibited a Nif+ phenotype. Interestingly, R. capsulatus nifV mutants were able to reduce acetylene not only to ethylene but also to ethane under conditions preventing the expression of the alternative nitrogenase system. Homocitrate added to the growth medium repressed ethane formation and cured the NifV phenotype in R. capsulatus. Higher concentrations of homocitrate were necessary to complement the NifV phenotype of a polar nifV mutant (NifV-NifW-), indicating a possible role of NifW either in homocitrate transport or in the incorporation of this compound into the iron-molybdenum cofactor of nitrogenase.

To analyze the overexpression of the Rhizobium meliloti fdxN gene in Escherichia coli, different translational and transcriptional fusions were constructed. The translational signals of R. meliloti fdxN were recognized in E. coli as demonstrated by the use of in-frame lac fusions. Translational fusions consisting of the lacZ or the lpp gene fused in frame to the 3' end of the entire fdxN gene were expressed at high levels in E. coli. In contrast, the wild-type R. meliloti FdxN protein without a C-terminal fusion could only be detected using the very sensitive T7 promoter-polymerase system and not in immunoblots with antibodies against an FdxN-LacZ hybrid protein. Evidently, translational fusions to the 3' end of fdxN had a stabilizing effect on the expression of the fdxN gene. A constitutively expressed transcriptional fdxN fusion, which did not mediate detectable amounts of FdxN protein either in E. coli or in free-living R. meliloti cells, complemented the Fix- phenotype of an R. meliloti fdxN::[Tc] mutant strain to wild-type levels. Therefore, either low amounts of the wild-type FdxN protein are sufficient for symbiotic nitrogen fixation or there are stabilizing factors, which are present only in R. meliloti bacteroids but not in free-living R. meliloti cells. Fusion proteins consisting of FdxN and LacZ or a partial Lpp protein restored the Fix- phenotype of an R. meliloti fdxN mutant to 3 and 11%, respectively, indicating that a C-terminal fusion did not completely abolish the function of FdxN.

The Rhizobium meliloti fdxN gene, which is part of the nifA-nifB-fdxN operon, is absolutely required for symbiotic nitrogen fixation. The deduced sequence of the FdxN protein is characterized by two cysteine motifs typical of bacterial-type ferredoxins. The Fix-phenotype of an R. meliloti fdxN::[Tc] mutant could be rescued by the R. leguminosarum fdxN gene, whereas no complementation was observed with nif-associated genes encoding ferredoxins from Bradyrhizobium japonicum, Azotobacter vinelandii, A. chroococcum and Rhodobacter capsulatus. In addition to these heterologous genes, several R. meliloti fdxN mutant genes constructed by site-directed mutagenesis were analyzed. Not only a cysteine residue within the second cysteine motif (position 42), which is known to coordinate the Fe-S cluster in homologous proteins, but also a cysteine located down-stream of this motif (position 61), was found to be essential for the activity of the R. meliloti FdxN protein. Changing the amino acid residue proline in position 56 into methionine resulted in a FdxN mutant protein with decreased activity, whereas changes in positions 35 (Asp35Glu) and 45 (Gly45Glu) had no significant effect on the function of the FdxN mutant proteins. In contrast to bacterial-type ferredoxins, which contain two identical cysteine motifs of the form C-X2-C-X2-C-X3-C, nif-associated ferredoxins, including R. meliloti FdxN, are characterized by two different cysteine motifs. Six "additional" amino acids separate the second (Cys42) and the third cysteine (Cys51) in the C-terminal motif (C-X2-C-X8-C-X3-C).(ABSTRACT TRUNCATED AT 250 WORDS)

A Synechococcus PCC7942 mutant in which the psbO gene was inactivated by insertion of a chloramphenicol interposon and which did not contain any detectable manganese stabilizing protein in immunoblot experiments, was constructed. Such a Synechococcus mutant was able to grow under photoautotrophic conditions. Isolated thylakoid membranes from the mutant required addition of CaCl2 and MnCl2 for photosynthetic O2 evolution, and the detectable L-amino acid oxidase activity in the isolated thylakoid membranes from the mutant was approximately four times higher than in wild-type thylakoids. The results are discussed with respect to our model suggesting that the water-oxidizing enzyme may have evolved from a flavoprotein with L-amino acid dehydrogenase/oxidase activity.

Rhodobacter capsulatus mutants unable to fix nitrogen were isolated by random transposon Tn5 mutagenesis. The Tn5 insertion sites of 30 Nif- mutants were mapped within three unlinked chromosomal regions designated A, B, and C. The majority of Tn5 insertions (21 mutants) map within nif region A, characterized by two ClaI fragments of 2.5 and 25 kilobases (kb). The 17-kb ClaI fragment of nif region B contains six nif::Tn5 insertions, and the three remaining mutations are located on a 32-kb ClaI fragment of nif region C. Hybridization experiments using all 17 Klebsiella pneumoniae nif genes individually as probes revealed homology to nifE, nifS, nifA, and nifB in nif region A. The nifHDK genes were localized in nif region B. About 2 kb away from this operon, a second copy of the DNA fragments homologous to nifA and nifB, originally found in nif region A, was identified.

A DNA region showing homology to Klebsiella pneumoniae nifA and nifB is duplicated in Rhodobacter capsulatus. The two copies of this region are called nifA/nifB copy I and nifA/nifB copy II. Deletion mutagenesis demonstrated that either of the two copies is sufficient for growth in nitrogen-free medium. In contrast, a double deletion mutant turned out to be deficient in nitrogen fixation. The complete nucleotide sequence of a 4838 bp fragment containing nifA/nifB copy I was determined. Two open reading frames coding for a 59,653 (NifA) and a 49,453 (NifB) dalton protein could be detected. Comparison of the amino acid sequences revealed that the R. capsulatus nifA and nifB gene products are more closely related to the NifA and NifB proteins of Rhizobium meliloti and Rhizobium leguminosarum than to those of K. pneumoniae. A rho-independent termination signal and a typical nif promoter region containing a putative NifA binding site and a consensus nif promoter are located within the region between the R. capsulatus nifA and nifB genes. The nifB sequence is followed by an open reading frame (ORF1) coding for a 27721 dalton protein in nifA/nifB copy I. DNA sequence analysis of nifA/nifB copy II showed that both copies differ in the DNA region downstream of nifB and in the noncoding sequence in front of nifA. All other regions compared, i.e. the 5' part of nifA, the intergenic region and the 3' part of nifB, are identical in both copies.