Bernd Masepohl's research while affiliated with Ruhr-Universität Bochum and other places

The photosynthetic α‐proteobacterium Rhodobacter capsulatus reduces and thereby fixes atmospheric dinitrogen (N2) 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–N6–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. Rhodobacter capsulatus fixes nitrogen from air by a molybdenum‐dependent and an iron‐only nitrogenase. Here, we present evidence that the transcriptional activator AnfA is required for AnfHDGK production, but dispensable for biosynthesis of the Fe‐only cofactor and electron delivery.

Rhodobacter capsulatus fixes atmospheric nitrogen (N2) 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. In Rhodobacter capsulatus, biological nitrogen fixation is catalyzed by the molybdenum nitrogenase and a Mo-free iron-only nitrogenase. Here, we show that NifA controls synthesis of both nitrogenases by controlling synthesis of the sigma factor RpoN.

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. N2 reduction to NH3 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 N2 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. This article is protected by copyright. All rights reserved.

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 widespread in bacteria, the function of PerO-like permeases 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-depending growth as shown by assimilation of radioactive labeled sulfate and heterologous complementation. Disruption of perO genes in A. tumefaciens , R. sphaeroides , and S. meliloti increased tolerance to tungstate, and in 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, until now our knowledge about PerO function is limited to one species, Rhodobacter capsulatus . In this study, we show 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.

Biological nitrogen fixation (BNF) is the nitrogenase-catalyzed process in which dinitrogen (N2) is reduced to ammonia (NH3), the preferred nitrogen source in bacteria. All N2-fixing or diazotrophic bacteria have molybdenum-nitrogenases. In addition, some diazotrophs possess one or two alternative Mo-free nitrogenases, namely a vanadium and/or an iron-only nitrogenase, which are less efficient than Mo-nitrogenase in terms of ATP-consumption per N2 reduced. BNF is widespread in photosynthetic purple nonsulfur bacteria, which are capable of using light energy to generate ATP for nitrogenase activity. This review focusses on BNF regulation in the purple nonsulfur bacteria Rhodobacter capsulatus, Rhodopseudomonas palustris, and Rhodospirillum rubrum. Rp. palustris is one of few diazotrophs having both alternative nitrogenases, whereas Rb. capsulatus and Rs. rubrum have Fe-nitrogenases but no V-nitrogenase. Purple nonsulfur bacteria regulate BNF in response to ammonium, molybdenum, iron, oxygen, and light. BNF regulation involves common regulatory proteins including the two-component nitrogen regulatory system NtrB-NtrC, the transcriptional activator NifA, the nitrogen-specific sigma factor RpoN, the DraT-DraG system for posttranslational nitrogenase regulation, and at least two PII signal transduction proteins. When ammonium is limiting, NtrB phosphorylates NtrC, which in turn activates expression of nifA and other BNF-related genes. NifA and its homologs VnfA and AnfA activate expression of Mo, V, and Fe-nitrogenase genes, respectively, in concert with RpoN. DraT mediates nitrogenase switch-off by ADP-ribosylation upon ammonium addition or light deprivation, the latter condition causing energy depletion. DraG reactivates nitrogenase upon ammonium consumption or reillumination. PII-like proteins integrate the cellular nitrogen, carbon, and energy levels, and control activity of NtrB, NifA, DraT, and DraG. Beside these similarities in BNF regulation, there are species-specific differences. NifA is active as synthesized in Rb. capsulatus, but requires activation by PII in Rp. palustris and Rs. rubrum. Reversible ADP-ribosylation is the only mechanism regulating nitrogenase in Rs. rubrum, whereas Rb. capsulatus and Rp. palustris have additional ADP-ribosylation-independent mechanisms. Last but not least, molybdate directly represses anfA transcription and hence, Fe-nitrogenase expression in Rb. capsulatus, whereas expression of the alternative nitrogenases in Rp. palustris and Rs. rubrum respond to Mo-nitrogenase activity rather than to molybdate directly.

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. This article is protected by copyright. All rights reserved.

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 Mo-repressed. 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.

Rhodobacter capsulatus grows efficiently with atmospheric dinitrogen as sole nitrogen source using two nitrogenases, the molybdenum nitrogenase and an alternative Mo-independent iron-only nitrogenase. The ABC transporter ModABC and the oxyanion permease PerO import molybdate required for the iron-molybdenum cofactor of Mo-nitrogenase at ambient nanomolar and micromolar concentrations, respectively. Expression of both nitrogenases is strongly inhibited by ammonium and, in addition, synthesis of Fe-nitrogenase is repressed by molybdate. This review summarizes our current knowledge on the regulatory mechanisms controlling nitrogen fixation and molybdate transport involving more than 20 regulatory proteins. Under nitrogen-fixing conditions, expression of Mo- and Fe-nitrogenase genes is induced by a regulatory cascade comprised of the two-component nitrogen regulation system NtrBC and the transcriptional activators NifA1, NifA2, and AnfA. Ammonium addition to a nitrogen-fixing culture immediately inhibits further transcription of nitrogen fixation genes and nitrogenase activity by inhibition of NtrC, NifA1, NifA2, and AnfA, and switch-off of Mo- and Fe-nitrogenases. In addition, the one-component molybdate-responsive regulators MopA and MopB repress the anfA and mopA-modABC genes confining expression of Fe-nitrogenase and the ModABC transporter to Mo-limiting conditions.

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.

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.