The iron nitrogenase reduces carbon dioxide to formate and methane under physiological conditions: A route to feedstock chemicals.

Nitrogenases are the only known enzymes that reduce molecular nitrogen (N2) to ammonia. Recent findings have demonstrated that nitrogenases also reduce the greenhouse gas carbon dioxide (CO2), suggesting CO2 to be a competitor of N2. However, the impact of omnipresent CO2 on N2 fixation has not been investigated to date. Here, we study the competing reduction of CO2 and N2 by the two nitrogenases of Rhodobacter capsulatus, the molybdenum and the iron nitrogenase. The iron nitrogenase is almost threefold more efficient in CO2 reduction and profoundly less selective for N2 than the molybdenum isoform under mixtures of N2 and CO2. Correspondingly, the growth rate of diazotrophically grown R. capsulatus strains relying on the iron nitrogenase notably decreased after adding CO2. The in vivo CO2 activity of the iron nitrogenase facilitates the light-driven extracellular accumulation of formate and methane, one-carbon substrates for other microbes, and feedstock chemicals for a circular economy.

A nitrogenase-like enzyme is involved in the novel anaerobic assimilation pathway of a sulfonate, isethionate, in the photosynthetic bacterium Rhodobacter capsulatus.

Prokaryotes contribute to the global sulfur cycle by using diverse sulfur compounds as sulfur sources or electron acceptors. In this study, we report that a nitrogenase-like enzyme (NFL) and a radical SAM enzyme (RSE) are involved in the novel anaerobic assimilation pathway of a sulfonate, isethionate, in the photosynthetic bacterium Rhodobacter capsulatus. The nflHDK genes for NFL are localized at a locus containing genes for known sulfonate metabolism in the genome. A gene nflB encoding an RSE is present just upstream of nflH, forming a small gene cluster nflBHDK. Mutants lacking any nflBHDK genes are incapable of growing with isethionate as the sole sulfur source under anaerobic photosynthetic conditions, indicating that all four NflBHDK proteins are essential for the isethionate assimilation pathway. Heterologous expression of the islAB genes encoding a known isethionate lyase that degrades isethionate to sulfite and acetaldehyde restored the isethionate-dependent growth of a mutant lacking nflDK, indicating that the enzyme encoding nflBHDK is involved in an isethionate assimilation reaction to release sulfite. Furthermore, the heterologous expression of nflBHDK and ssuCAB encoding an isethionate transporter in the closely related species R. sphaeroides, which does not have nflBHDK and cannot grow with isethionate as the sole sulfur source, conferred isethionate-dependent growth ability to this species. We propose to rename nflBHDK as isrBHDK (isethionate reductase). The isrBHDK genes are widely distributed among various prokaryote phyla. Discovery of the isethionate assimilation pathway by IsrBHDK provides a missing piece for the anaerobic sulfur cycle and for understanding the evolution of ancient sulfur metabolism.IMPORTANCENitrogenase is an important enzyme found in prokaryotes that reduces atmospheric nitrogen to ammonia and plays a fundamental role in the global nitrogen cycle. It has been noted that nitrogenase-like enzymes (NFLs), which share an evolutionary origin with nitrogenase, have evolved to catalyze diverse reactions such as chlorophyll biosynthesis (photosynthesis), coenzyme F430 biosynthesis (methanogenesis), and methionine biosynthesis. In this study, we discovered that an NFL with unknown function in the photosynthetic bacterium Rhodobacter capsulatus is a novel isethionate reductase (Isr), which catalyzes the assimilatory degradation of isethionate, a sulfonate, releasing sulfite used as the sulfur source under anaerobic conditions. Isr is widely distributed among various bacterial phyla, including intestinal bacteria, and is presumed to play an important role in sulfur metabolism in anaerobic environments such as animal guts and microbial mats. This finding provides a clue for understanding ancient metabolism that evolved under anaerobic environments at the dawn of life.

TED: Enhancing Translation Efficiency in Bacterial Expression Systems.

Genetic engineering enables the forced expression of desired products in bacteria, which can then be used for a variety of applications, including functional analysis and pharmaceuticals. Here, we describe a method for tuning translation in bacteria, including Escherichia coli and Rhodobacter capsulatus, based on a phenomenon known as TED (translation enhancement by a Dictyostelium gene sequence). This method promotes translation of mRNA encoded by downstream genes by inserting a short nucleotide sequence into the 5' untranslated region between the promoter and the Shine-Dalgarno (SD) sequence. Various expression levels can be observed depending on the inserted sequence and its length, even with an identical promoter.

Two pathways to understanding electron transfer in reaction centers from photosynthetic bacteria: A comparison of Rhodobacter sphaeroides and Rhodobacter capsulatus mutants.

The rates, yields, mechanisms and directionality of electron transfer (ET) are explored in twelve pairs of Rhodobacter (R.) sphaeroides and R. capsulatus mutant RCs designed to defeat ET from the excited primary donor (P*) to the A-side cofactors and re-direct ET to the normally inactive mirror-image B-side cofactors. In general, the R. sphaeroides variants have larger P+HB- yields (up to ∼90%) than their R. capsulatus analogs (up to ∼60%), where HB is the B-side bacteriopheophytin. Substitution of Tyr for Phe at L-polypeptide position L181 near BB primarily increases the contribution of fast P* â†’ P+BB- â†’ P+HB- two-step ET, where BB is the "bridging" B-side bacteriochlorophyll. The second step (∼6-8 ps) is slower than the first (∼3-4 ps), unlike A-side two-step ET (P* â†’ P+BA- â†’ P+HA-) where the second step (∼1 ps) is faster than the first (∼3-4 ps) in the native RC. Substitutions near HB, at L185 (Leu, Trp or Arg) and at M-polypeptide site M133/131 (Thr, Val or Glu), strongly affect the contribution of slower (20-50 ps) P* â†’ P+HB- one-step superexchange ET. Both ET mechanisms are effective in directing electrons "the wrong way" to HB and both compete with internal conversion of P* to the ground state (∼200 ps) and ET to the A-side cofactors. Collectively, the work demonstrates cooperative amino-acid control of rates, yields and mechanisms of ET in bacterial RCs and how A- vs. B-side charge separation can be tuned in both species.

Insights into conformational changes in cytochrome b during the early steps of its maturation.

Membrane proteins carrying redox cofactors are key subunits of respiratory chain complexes, yet the exact path of their folding and maturation remains poorly understood. Here, using cryo-EM and structure prediction via Alphafold2, we generated models of early assembly intermediates of cytochrome b (Cytb), a central subunit of complex III. The predicted structure of the first assembly intermediate suggests how the binding of Cytb to the assembly factor Cbp3-Cbp6 imposes an open configuration to facilitate the acquisition of its heme cofactors. Moreover, structure predictions of the second intermediate indicate how hemes get stabilized by binding of the assembly factor Cbp4, with a concomitant weakening of the contact between Cbp3-Cbp6 and Cytb, preparing for the release of the fully hemylated protein from the assembly factors.

Changing the Electron Acceptor Specificity of Rhodobacter capsulatus Formate Dehydrogenase from NAD+ to NADP.

Formate dehydrogenases catalyze the reversible oxidation of formate to carbon dioxide. These enzymes play an important role in CO2 reduction and serve as nicotinamide cofactor recycling enzymes. More recently, the CO2-reducing activity of formate dehydrogenases, especially metal-containing formate dehydrogenases, has been further explored for efficient atmospheric CO2 capture. Here, we investigate the nicotinamide binding site of formate dehydrogenase from Rhodobacter capsulatus for its specificity toward NAD+ vs. NADP+ reduction. Starting from the NAD+-specific wild-type RcFDH, key residues were exchanged to enable NADP+ binding on the basis of the NAD+-bound cryo-EM structure (PDB-ID: 6TG9). It has been observed that the lysine at position 157 (Lys157) in the ß-subunit of the enzyme is essential for the binding of NAD+. RcFDH variants that had Glu259 exchanged for either a positively charged or uncharged amino acid had additional activity with NADP+. The FdsBL279R and FdsBK276A variants also showed activity with NADP+. Kinetic parameters for all the variants were determined and tested for activity in CO2 reduction. The variants were able to reduce CO2 using NADPH as an electron donor in a coupled assay with phosphite dehydrogenase (PTDH), which regenerates NADPH. This makes the enzyme suitable for applications where it can be coupled with other enzymes that use NADPH.

Extracellular Polysaccharide Receptor and Receptor-Binding Proteins of the Rhodobacter capsulatus Bacteriophage-like Gene Transfer Agent RcGTA.

A variety of prokaryotes produce a bacteriophage-like gene transfer agent (GTA), and the alphaproteobacterial Rhodobacter capsulatus RcGTA is a model GTA. Some environmental isolates of R. capsulatus lack the ability to acquire genes transferred by the RcGTA (recipient capability). In this work, we investigated the reason why R. capsulatus strain 37b4 lacks recipient capability. The RcGTA head spike fiber and tail fiber proteins have been proposed to bind extracellular oligosaccharide receptors, and strain 37b4 lacks a capsular polysaccharide (CPS). The reason why strain 37b4 lacks a CPS was unknown, as was whether the provision of a CPS to 37b4 would result in recipient capability. To address these questions, we sequenced and annotated the strain 37b4 genome and used BLAST interrogations of this genome sequence to search for homologs of genes known to be needed for R. capsulatus recipient capability. We also created a cosmid-borne genome library from a wild-type strain, mobilized the library into 37b4, and used the cosmid-complemented strain 37b4 to identify genes needed for a gain of function, allowing for the acquisition of RcGTA-borne genes. The relative presence of CPS around a wild-type strain, 37b4, and cosmid-complemented 37b4 cells was visualized using light microscopy of stained cells. Fluorescently tagged head spike fiber and tail fiber proteins of the RcGTA particle were created and used to measure the relative binding to wild-type and 37b4 cells. We found that strain 37b4 lacks recipient capability because of an inability to bind RcGTA; the reason it is incapable of binding is that it lacks CPS, and the absence of CPS is due to the absence of genes previously shown to be needed for CPS production in another strain. In addition to the head spike fiber, we found that the tail fiber protein also binds to the CPS.

Rhodobacter capsulatus forms a compact crescent-shaped LH1-RC photocomplex.

Rhodobacter (Rba.) capsulatus has been a favored model for studies of all aspects of bacterial photosynthesis. This purple phototroph contains PufX, a polypeptide crucial for dimerization of the light-harvesting 1-reaction center (LH1-RC) complex, but lacks protein-U, a U-shaped polypeptide in the LH1-RC of its close relative Rba. sphaeroides. Here we present a cryo-EM structure of the Rba. capsulatus LH1-RC purified by DEAE chromatography. The crescent-shaped LH1-RC exhibits a compact structure containing only 10 LH1 αß-subunits. Four αß-subunits corresponding to those adjacent to protein-U in Rba. sphaeroides were absent. PufX in Rba. capsulatus exhibits a unique conformation in its N-terminus that self-associates with amino acids in its own transmembrane domain and interacts with nearby polypeptides, preventing it from interacting with proteins in other complexes and forming dimeric structures. These features are discussed in relation to the minimal requirements for the formation of LH1-RC monomers and dimers, the spectroscopic behavior of both the LH1 and RC, and the bioenergetics of energy transfer from LH1 to the RC.

Cryo-EM structure of a monomeric RC-LH1-PufX supercomplex with high-carotenoid content from Rhodobacter capsulatus.

In purple photosynthetic bacteria, the photochemical reaction center (RC) and light-harvesting complex 1 (LH1) assemble to form monomeric or dimeric RC-LH1 membrane complexes, essential for bacterial photosynthesis. Here, we report a 2.59-Å resolution cryoelectron microscopy (cryo-EM) structure of the RC-LH1 supercomplex from Rhodobacter capsulatus. We show that Rba. capsulatus RC-LH1 complexes are exclusively monomers in which the RC is surrounded by a 15-subunit LH1 ring. Incorporation of a transmembrane polypeptide PufX leads to a large opening within the LH1 ring. Each LH1 subunit associates two carotenoids and two bacteriochlorophylls, which is similar to Rba. sphaeroides RC-LH1 but more than one carotenoid per LH1 in Rba. veldkampii RC-LH1 monomer. Collectively, the unique Rba. capsulatus RC-LH1-PufX represents an intermediate structure between Rba. sphaeroides and Rba. veldkampii RC-LH1-PufX. Comparison of PufX from the three Rhodobacter species indicates the important residues involved in dimerization of RC-LH1.

DNA Gyrase Inhibitors Increase the Frequency of Bacteriophage-like RcGTA-Mediated Gene Transfer in Rhodobacter capsulatus.

Rhodobacter capsulatus produces a bacteriophage-like particle called the gene transfer agent (RcGTA) that mediates horizontal gene transfer. RcGTA particles transfer random ~4.5-kb fragments of genomic DNA that integrate into recipient genomes by allelic replacement. This work addresses the effect of sub-inhibitory concentrations of antibiotics on gene transfer by RcGTA. A transduction assay was developed to test the effects of various substances on gene transfer. Using this assay, low concentrations of DNA gyrase inhibitors were found to increase the frequency of gene transfer. Novobiocin was studied in more detail, and it was found that this antibiotic did not influence the production or release of RcGTA but instead appeared to act on the recipient cells. The target of novobiocin in other species has been shown to be the GyrB subunit of DNA gyrase (a heterotetramer of 2GyrA and 2GyrB). R. capsulatus encodes GyrA and GyrB homologues, and a GyrB overexpression plasmid was created and found to confer resistance to novobiocin. The presence of the overexpression plasmid in recipient cells greatly diminished the novobiocin-mediated increase in gene transfer, confirming that this effect is due to the binding of novobiocin by GyrB. The results of this work show that antibiotics affect gene transfer in R. capsulatus and may be relevant to microbial genetic exchange in natural ecosystems.

Redox Brake Regulator RedB and FnrL Function as Yin-Yang Regulators of Anaerobic-Aerobic Metabolism in Rhodobacter capsulatus.

We recently described a new member of the CRP (cyclic AMP receptor protein)/FNR (fumarate and nitrate reductase regulatory protein) family called RedB, an acronym for redox brake, that functions to limit the production of ATP and NADH. This study shows that the RedB regulon significantly overlaps the FnrL regulon, with 199 genes being either directly or indirectly regulated by both of these global regulatory proteins. Among these 199 coregulated genes, 192 are divergently regulated, indicating that RedB functions as an antagonist of FnrL. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis indicates that RedB and Fnr directly coregulate only 4 out of 199 genes. The primary mechanism for the divergent regulation of target genes thus involves indirect regulation by both RedB and FnrL (156 cases). Additional regulation involves direct binding by RedB and indirect regulation by FnrL (36 cases) or direct binding by FnrL and indirect regulation by RedB (3 cases). Analysis of physiological pathways under direct and indirect control by these global regulators demonstrates that RedB functions primarily to limit energy production, while FnrL functions to enhance energy production. This regulation includes glycolysis, gluconeogenesis, photosynthesis, hydrogen oxidation, electron transport, carbon fixation, lipid biosynthesis, and protein synthesis. Finally, we show that 75% of genomes from diverse species that code for RedB proteins also harbor genes coding for FNR homologs. This cooccurrence indicates that RedB likely has an important role in buffering FNR-mediated energy production in a broad range of species. IMPORTANCE The CRP/FNR family of regulatory proteins constitutes a large collection of related transcription factors, several of which globally regulate cellular energy production. A well-characterized example is FNR (called FnrL in Rhodobacter capsulatus), which is responsible for regulating the expression of numerous genes that promote maximal energy production and growth under anaerobic conditions. In a companion article (N. Ke, J. E. Kumka, M. Fang, B. Weaver, et al., Microbiol Spectr 10:e02353-22, 2022, https://doi.org/10.1128/Spectrum02353-22), we identified a new subgroup of the CRP/FNR family and demonstrated that a member of this new subgroup, called RedB, has a role in limiting cellular energy production. In this study, we show that numerous genes encompassing the RedB regulon significantly overlap genes that are members of the FnrL regulon. Furthermore, 97% of the genes that are members of both the RedB and FnrL regulons are divergently regulated by these two transcription factors. RedB thus functions as a buffer limiting the amount of energy production that is promoted by FnrL.

The archetypal gene transfer agent RcGTA is regulated via direct interaction with the enigmatic RNA polymerase omega subunit.

Gene transfer agents (GTAs) are small virus-like particles that indiscriminately package and transfer any DNA present in their host cell, with clear implications for bacterial evolution. The first transcriptional regulator that directly controls GTA expression, GafA, was recently discovered, but its mechanism of action has remained elusive. Here, we demonstrate that GafA controls GTA gene expression via direct interaction with the RNA polymerase omega subunit (Rpo-ω) and also positively autoregulates its own expression by an Rpo-ω-independent mechanism. We show that GafA is a modular protein with distinct DNA and protein binding domains. The functional domains we observe in Rhodobacter GafA also correspond to two-gene operons in Hyphomicrobiales pathogens. These data allow us to produce the most complete regulatory model for a GTA and point toward an atypical mechanism for RNA polymerase recruitment and specific transcriptional activation in the Alphaproteobacteria.

Heterologous Production of Plant Terpenes in the Photosynthetic Bacterium Rhodobacter capsulatus.

Terpenes are one of the largest classes of secondary metabolites that occur in all kingdoms of life and offer diverse valuable properties for food and pharma industry including pleasant odor or taste as well as antimicrobial or anticancer activities. A multitude of terpene biosynthesis pathways are known, but their efficient biotechnological exploitation requires an adequate microorganism as host which can ideally provide an optimal supply with biosynthetic isoprene precursors. Rhodobacter capsulatus, a Gram-negative, facultative anaerobic, photosynthetic non-sulfur purple bacterium belonging to the α-proteobacteria represents such a host particularly suitable for terpene production. Here, we describe methods for the expression of terpene biosynthetic enzymes in R. capsulatus and the extraction of products for analysis. At the same time, we summarize the current strategies to adjust the biosynthetic precursor supply via isoprenoid biosynthetic pathways.

Hydrogen bonding rearrangement by a mitochondrial disease mutation in cytochrome bc1 perturbs heme bH redox potential and spin state.

Hemes are common elements of biological redox cofactor chains involved in rapid electron transfer. While the redox properties of hemes and the stability of the spin state are recognized as key determinants of their function, understanding the molecular basis of control of these properties is challenging. Here, benefiting from the effects of one mitochondrial disease-related point mutation in cytochrome b, we identify a dual role of hydrogen bonding (H-bond) to the propionate group of heme bH of cytochrome bc1, a common component of energy-conserving systems. We found that replacing conserved glycine with serine in the vicinity of heme bH caused stabilization of this bond, which not only increased the redox potential of the heme but also induced structural and energetic changes in interactions between Fe ion and axial histidine ligands. The latter led to a reversible spin conversion of the oxidized Fe from 1/2 to 5/2, an effect that potentially reduces the electron transfer rate between the heme and its redox partners. We thus propose that H-bond to the propionate group and heme-protein packing contribute to the fine-tuning of the redox potential of heme and maintaining its proper spin state. A subtle balance is needed between these two contributions: While increasing the H-bond stability raises the heme potential, the extent of increase must be limited to maintain the low spin and diamagnetic form of heme. This principle might apply to other native heme proteins and can be exploited in engineering of artificial heme-containing protein maquettes.

Cysteine Mutants of the Major Facilitator Superfamily-Type Transporter CcoA Provide Insight into Copper Import.

CcoA belongs to the widely distributed bacterial copper (Cu) importer subfamily CalT (CcoA-like Transporters) of the Major Facilitator Superfamily (MFS) and provides cytoplasmic Cu needed for cbb3-type cytochrome c oxidase (cbb3-Cox) biogenesis. Earlier studies have supported a 12-transmembrane helix (TMH) topology of CcoA with the well-conserved Met233xxxMet237 and His261xxxMet265 motifs in its TMH7 and TMH8, respectively. Of these residues, Met233 and His261 are essential for Cu uptake and cbb3-Cox production, whereas Met237 and Met265 contribute partly to these processes. CcoA also contains five Cys residues of unknown role and, remarkably, its structural models predict that three of these are exposed to the highly oxidizing periplasm. Here, we first demonstrate that elimination of both Met237 and Met265 completely abolishes Cu uptake and cbb3-Cox production, indicating that CcoA requires at least one of these two Met residues for activity. Second, using scanning mutagenesis to probe plausible metal-interacting Met, His, and Cys residues of CcoA, we found that the periplasm-exposed Cys49 located at the end of TMH2, the Cys247 on a surface loop between TMH7 and THM8, and the C367 located at the end of TMH11 are important for CcoA function. Analyses of the single and double Cys mutants revealed the occurrence of a disulfide bond in CcoA in vivo, possibly related to conformational changes it undergoes during Cu import as MFS-type transporter. Our overall findings suggest a model linking Cu import for cbb3-Cox biogenesis with a thiol:disulfide oxidoreduction step, advancing our understanding of the mechanisms of CcoA function. IMPORTANCE Copper (Cu) is a redox-active micronutrient that is both essential and toxic. Its cellular homeostasis is critical for supporting cuproprotein maturation while avoiding excessive oxidative stress. The Cu importer CcoA is the prototype of the widespread CalT subfamily of the MFS-type transporters. Hence, understanding its molecular mechanism of function is significant. Here, we show that CcoA undergoes a thiol:disulfide oxidoreduction cycle, which is important for its Cu import activity.

High-Yielding Terpene-Based Biofuel Production in Rhodobacter capsulatus.

Energy crisis and global climate change have driven an increased effort toward biofuel synthesis from renewable feedstocks. Herein, purple nonsulfur photosynthetic bacterium (PNSB) of Rhodobacter capsulatus was explored as a platform for high-titer production of a terpene-based advanced biofuel-bisabolene. A multilevel engineering strategy such as promoter screening, improving the NADPH availability, strengthening the precursor supply, suppressing the side pathways, and introducing a heterologous mevalonate pathway, was used to improve the bisabolene titer in R. capsulatus. The above strategies enabled a 35-fold higher titer of bisabolene than that of the starting strain, reaching 1089.7 mg/L from glucose in a shake flask. The engineered strain produced 9.8 g/L bisabolene with a yield of >0.196 g/g-glucose under the two-phase fed-batch fermentation, which corresponds to >78% of theoretical maximum. Taken together, our work represents one of the pioneering studies to demonstrate PNSB as a promising platform for terpene-based advanced biofuel production.

Cryo-EM structures of engineered active bc1-cbb3 type CIII2CIV super-complexes and electronic communication between the complexes.

Respiratory electron transport complexes are organized as individual entities or combined as large supercomplexes (SC). Gram-negative bacteria deploy a mitochondrial-like cytochrome (cyt) bc1 (Complex III, CIII2), and may have specific cbb3-type cyt c oxidases (Complex IV, CIV) instead of the canonical aa3-type CIV. Electron transfer between these complexes is mediated by soluble (c2) and membrane-anchored (cy) cyts. Here, we report the structure of an engineered bc1-cbb3 type SC (CIII2CIV, 5.2 Å resolution) and three conformers of native CIII2 (3.3 Å resolution). The SC is active in vivo and in vitro, contains all catalytic subunits and cofactors, and two extra transmembrane helices attributed to cyt cy and the assembly factor CcoH. The cyt cy is integral to SC, its cyt domain is mobile and it conveys electrons to CIV differently than cyt c2. The successful production of a native-like functional SC and determination of its structure illustrate the characteristics of membrane-confined and membrane-external respiratory electron transport pathways in Gram-negative bacteria.

The CckA-ChpT-CtrA Phosphorelay Controlling Rhodobacter capsulatus Gene Transfer Agent Production Is Bidirectional and Regulated by Cyclic di-GMP.

Protein phosphorylation is a universal mechanism for transducing cellular signals in prokaryotes and eukaryotes. The histidine kinase CckA, the histidine phosphotransferase ChpT, and the response regulator CtrA are conserved throughout the alphaproteobacteria. In Rhodobacter capsulatus, these proteins are key regulators of the gene transfer agent (RcGTA), which is present in several alphaproteobacteria. Using purified recombinant R. capsulatus proteins, we show in vitro autophosphorylation of CckA protein, and phosphotransfer to ChpT and thence to CtrA, to demonstrate biochemically that they form a phosphorelay. The secondary messenger cyclic di-GMP changed CckA from a kinase to a phosphatase, resulting in reversal of the phosphotransfer flow in the relay. The substitutions of two residues in CckA greatly affected the kinase or phosphatase activity of the protein in vitro, and production of mutant CckA proteins in vivo confirmed the importance of kinase but not phosphatase activity for the lytic release of RcGTA. However, phosphatase activity was needed to produce functional RcGTA particles. The binding of cyclic di-GMP to the wild-type and mutant CckA proteins was evaluated directly using a pulldown assay based on biotinylated cyclic di-GMP and streptavidin-linked beads.IMPORTANCE The CckA, ChpT, and CtrA phosphorelay proteins are widespread in the alphaproteobacteria, and there are two groups of organisms that differ in terms of whether this pathway is essential for cell viability. Little is known about the biochemical function of these proteins in organisms where the pathway is not essential, a group that includes Rhodobacter capsulatus This work demonstrates biochemically that CckA, ChpT, and CtrA also form a functional phosphorelay in the latter group and that the direction of phosphotransfer is reversed by cyclic di-GMP. It is important to improve understanding of more representatives of this pathway in order to obtain deeper insight into the function, composition, and evolutionary significance of a wider range of bacterial regulatory networks.

Repressor Activity of SqrR, a Master Regulator of Persulfide-Responsive Genes, Is Regulated by Heme Coordination.

Reactive sulfur species (RSS) are involved in bioactive regulation via persulfidation of proteins. However, how cells regulate RSS-based signaling and RSS metabolism is poorly understood, despite the importance of universal regulation systems in biology. We previously showed that the persulfide-responsive transcriptional factor SqrR acts as a master regulator of sulfide-dependent photosynthesis in proteobacteria. Here, we demonstrated that SqrR also binds heme at a near one-to-one ratio with a binding constant similar to other heme-binding proteins. Heme does not change the DNA-binding pattern of SqrR to the target gene promoter region; however, DNA-binding affinity of SqrR is reduced by the binding of heme, altering its regulatory activity. Circular dichroism spectroscopy clearly showed secondary structural changes in SqrR by the heme binding. Incremental change in the intracellular heme concentration is associated with small, but significant reduction in the transcriptional repression by SqrR. Overall, these results indicate that SqrR has an ability to bind heme to modulate its DNA-binding activity, which may be important for the precise regulation of RSS metabolism in vivo.

Mg-protoporphyrin IX monomethyl ester cyclase from Rhodobacter capsulatus: radical SAM-dependent synthesis of the isocyclic ring of bacteriochlorophylls.

During bacteriochlorophyll a biosynthesis, the oxygen-independent conversion of Mg-protoporphyrin IX monomethyl ester (Mg-PME) to protochlorophyllide (Pchlide) is catalyzed by the anaerobic Mg-PME cyclase termed BchE. Bioinformatics analyses in combination with pigment studies of cobalamin-requiring Rhodobacter capsulatus mutants indicated an unusual radical S-adenosylmethionine (SAM) and cobalamin-dependent BchE catalysis. However, in vitro biosynthesis of the isocyclic ring moiety of bacteriochlorophyll using purified recombinant BchE has never been demonstrated. We established a spectroscopic in vitro activity assay which was subsequently validated by HPLC analyses and H218O isotope label transfer onto the carbonyl-group (C-131-oxo) of the isocyclic ring of Pchlide. The reaction product was further converted to chlorophyllide in the presence of light-dependent Pchlide reductase. BchE activity was stimulated by increasing concentrations of NADPH or SAM, and inhibited by S-adenosylhomocysteine. Subcellular fractionation experiments revealed that membrane-localized BchE requires an additional, heat-sensitive cytosolic component for activity. BchE catalysis was not sustained in chimeric experiments when a cytosolic extract from E. coli was used as a substitute. Size-fractionation of the soluble R. capsulatus fraction indicated that enzymatic activity relies on a specific component with an estimated molecular mass between 3 and 10 kDa. A structure guided site-directed mutagenesis approach was performed on the basis of a three-dimensional homology model of BchE. A newly established in vivo complementation assay was used to investigate 24 BchE mutant proteins. Potential ligands of the [4Fe-4S] cluster (Cys204, Cys208, Cys211), of SAM (Phe210, Glu308 and Lys320) and of the proposed cobalamin cofactor (Asp248, Glu249, Leu29, Thr71, Val97) were identified.