Molybdenum is the only element in the second transition series that has a biological role (Frailsto da Silva and Williams 1993; Pope et al., 1980; Stiefel 1993). It is required for cofactors of redox enzymes that catalyse reactions at carbon, sulfur and nitrogen atoms, including nitrogenase and nitrate reductase which are key enzymes in the nitrogen cycle. There are two distinct types of molybdoenzymes. Molybdenum nitrogenase has a unique molybdenum-iron-sulfur cofactor, the [Fe4S3]-(bridging-S)3-[MoFe3S3] center called FeMoco (see Chapter 8). It catalyses the reduction of atmospheric dinitrogen to ammonia. All other molybdoenzymes are oxidoreductases that transfer an oxo group or two electrons to or from the substrate. They have a molybdenum cofactor, Moco, in which molybdenum is coordinated to a dithiolene group on the 6-alkyl sidechain of a pterin called molybdopterin (Rajagopalan, 1991; 1993; 1996). The side chain of molybdopterin is phosphorylated and is linked to a nucleotide in most procaryotes, but not in eukaryotes. Crystal structures have shown that the molybdenum cofactor in aldehyde oxido-reductase and dimethyl sulfoxide reductase has a tricyclic structure in which the bicyclic pterin is fused to a monocyclic pyran formed by the closure of the side-chain hydroxyl with the pterin ring (Romdo et al., 1995; Schindelin et al., 1996). The conformation and environment of the pterin cofactor(s) in these enzymes are different, and illustrate how the molybdenum cofactor can catalyse a wide variety of reactions.

Genetics and Regulation of Nitrogen-Fixing Bacteria This book is the second volume of a seven-volume series, which covers all fields of research related to nitrogen fixation - from basic studies throu

ABSTRACT 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.

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 N2 as sole nitrogen source via either molybdenum nitrogenase or the alternative nitrogenase. NrfA was shown to control N2 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.

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.

The phototrophic non-sulphur purple bacterium R. capsulatus can use urea as sole source of nitrogen. Five mutants (Xan-9, Xan-10, Xan-19, Xan-20, and Xan-22) unable to grow with urea as the only N source were isolated by random transposon Tn5 mutagenesis (Leimkuhler et al., 1998). Three Tn5 insertion sites were mapped within the urease gene cluster (ure), whereas the remaining two mutations were located within the ntrB-C (nifR2-R1) operon. DNA sequence analysis of the R. capsulatus ure gene region demonstrated the presence of the ureDABC-orf136-ureEFG genes, which are assumed to form one transcriptional unit due to the close linkage of these eight genes. The deduced gene products of the ure genes show high similarity to corresponding proteins from other bacteria, whereas orf136 seems to be specific for R. capsulatus, since homologues were neither found in other ure gene regions nor elsewhere in the databases.

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.

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.

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.

In all diazotrophic micro-organisms investigated so far, mutations in nifE, one of the genes involved in the biosynthesis of the FeMo cofactor (FeMoco), resulted in the accumulation of cofactorless inactive dinitrogenase. In this study, we have found that strains of the phototrophic non-sulfur purple bacterium Rhodobacter capsulatus with mutations in nifE, as well as in the operon harbouring the nifE gene, were capable of reducing acetylene and growing diazotrophically, although at distinctly lower rates than the wild-type strain. The diminished rates of substrate reduction were found to correlate with the decreased amounts of the dinitrogenase component (MoFe protein) expressed in R. capsulatus. The in vivo activity, as measured by the routine acetylene-reduction assay, was strictly Mo-dependent. Maximal activity was achieved under diazotrophic growth conditions and by supplementing the growth medium with molybdate (final concentration 20-50 microM). Moreover, in these strains a high proportion of ethane was produced from acetylene ( approximately 10% of ethylene) in vivo. However, in in vitro measurements with cell-free extracts as well as purified dinitrogenase, ethane production was always found to be less than 1%. The isolation and partial purification of the MoFe protein from the nifE mutant strain by Q-Sepharose chromatography and subsequent analysis by EPR spectroscopy and inductively coupled plasma MS revealed that FeMoco is actually incorporated into the protein (1.7 molecules of FeMoco per tetramer). On the basis of the results presented here, the role of NifNE in the biosynthetic pathway of the FeMoco demands reconsideration. It is shown for the first time that NifNE is not essential for biosynthesis of the cofactor, although its presence guarantees formation of a higher content of intact FeMoco-containing MoFe protein molecules. The implications of our findings for the biosynthesis of the FeMoco will be discussed.

ABSTRACT The phototrophic bacterium Rhodobacter capsulatus is able to reduce 2,4-dinitrophenol (DNP) to 2-amino-4-nitrophenol enzymatically and thus can grow in the presence of this uncoupler. DNP reduction was switched off by glutamine or ammonium, but this short-term regulation did not take place in a draTGdeletion mutant. Nevertheless, the target of DraTG does not seem to be the nitrophenol reductase itself since the ammonium shock did not inactivate the enzyme. In addition to this short-term regulation, ammonium or glutamine repressed the DNP reduction system. Mutants ofR. capsulatus affected in ntrC orrpoN exhibited a 10-fold decrease in nitroreductase activity in vitro but almost no DNP activity in vivo. In addition, mutants affected in rnfA or rnfC, which are also under NtrC control and encode components involved in electron transfer to nitrogenase, were unable to metabolize DNP. These results indicate that NtrC regulates dinitrophenol reduction in R. capsulatus, either directly or indirectly, by controlling expression of the Rnf proteins. Therefore, the Rnf complex seems to supply electrons for both nitrogen fixation and DNP reduction.

ABSTRACT The phototrophic nonsulfur purple bacterium Rhodobacter capsulatus can use urea as a sole source of nitrogen. Three transposon Tn 5 -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 Tn 5 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 ς 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.