Purple Bacterium Rhodobacter Capsulatus Research Articles

Intact photosynthetic bacteria-based electrodes for self-powered metal ions monitoring

The low-cost and early monitoring of metal ion contaminants is paramount to prevent widespread contamination of water environments. Self-powered microbial electrochemical sensors represent an interesting approach to achieving this goal. Purple non-sulfur bacteria have a versatile metabolism and a well-characterized photosynthetic system, making them an ideal candidate for developing biohybrid technologies. In this work, we report the use of these bacteria in biophotoelectrodes to develop self-powered monitoring systems for two common pollutants, NiCl2 and CuSO4. The microbial biophotoelectrode was obtained on a homemade poly-hydroxybutyrate-carbon nanofibers electrode modified with a redox-adhesive polydopamine matrix-based entrapping the purple bacterium Rhodobacter capsulatus. The presence of 500 μM NiCl2 resulted in a 60 % decrease in current density, while the simultaneous presence of 100 μM NiCl2 and 100 mM CuSO4 led to an 83 % current inhibition. Given the implementation of the biophotoelectrode in the field, the biohybrid system was tested in a complex matrix containing beer, demonstrating the promising ability of the photoelectrochemical system to act as an efficient biosensor in complex solutions. Finally, the biohybrid electrode was coupled to a cathode performing oxygen reduction, which allowed obtaining a self-powered monitoring system, paving the way for the future implementation of a low-cost monitoring system for widespread metal ions contaminant monitoring.

Properties of Mutant Photosynthetic Reaction Centers of Purple Non-Sulfur Bacteria Cereibacter sphaeroides with M206 Ile→Gln Substitution

Properties of Mutant Photosynthetic Reaction Centers of Purple Non-Sulfur Bacteria Cereibacter sphaeroides with M206 Ile→Gln Substitution

Probing Carboxylate Anolytes for Photo-Biofuel Cells through Combination of Bioinformatics and Electrochemistry

Photoheterotrophic purple bacterium Rhodobacter capsulatus has recently gained attention for its high halotolerance and its photo-enhanced extracellular electron transfer via exogenous quinone redox mediators, opening opportunity for self-powered decontamination of saline wastewater. Biodegradation of malate and succinate in R. capsulatus electrochemical systems has undergone prior examination, although a consistent bioelectrochemical system to comparatively study multiple carbon sources has not previously been developed. In this study, electrochemical techniques, biological assays, and computational tools were combined to evaluate malate, succinate, propionate, and lactate as oxidizable fuels in photo-enhanced microbial electrochemical systems. Specifically, cyclic voltammetry and amperometry data demonstrated that R. capsulatus generates distinctive photo-enhanced current densities dependent on both fuel identity and concentration. Bacterial growth curve studies were in agreement with the electrochemical data, indicating that lactate, which yielded the greatest bio-anodic current density (9.5 ± 1.9 µA cm-2), allowed the bacteria to grow more rapidly than the other substrates, suggesting its effectiveness as a fuel. Moreover, propionate appeared to be the fuel least efficiently utilized by R. capsulatus, having the slowest growth curve and the lowest current density generation (1.3 ± 0.2 µA cm-2). Finally, high-throughput differential gene expression analysis allowed for illuminating the physiological underpinnings of the observed differences between substrates. Most remarkably, cells grown in lactate were found to overexpress light harvesting complex II proteins and to underexpress flagellar motility proteins, which both correspond to the high photo-enhanced current density in lactate bioanodes compared to malate and propionate.

Structural Analyses of CrtJ and Its B12-Binding Co-Regulators SAerR and LAerR from the Purple Photosynthetic Bacterium Rhodobacter capsulatus.

Among purple photosynthetic bacteria, the transcription factor CrtJ is a major regulator of photosystem gene expression. Depending on growing conditions, CrtJ can function as an aerobic repressor or an anaerobic activator of photosystem genes. Recently, CrtJ’s activity was shown to be modulated by two size variants of a B12 binding co-regulator called SAerR and LAerR in Rhodobacter capsulatus. The short form, SAerR, promotes CrtJ repression, while the longer variant, LAerR, converts CrtJ into an activator. In this study, we solved the crystal structure of R. capsulatus SAerR at a 2.25 Å resolution. Hydroxycobalamin bound to SAerR is sandwiched between a 4-helix bundle cap, and a Rossman fold. This structure is similar to a AerR-like domain present in CarH from Thermus termophilus, which is a combined photoreceptor/transcription regulator. We also utilized AlphaFold software to predict structures for the LAerR, CrtJ, SAerR-CrtJ and LAerR-CrtJ co-complexes. These structures provide insights into the role of B12 and an LAerR N-terminal extension in regulating the activity of CrtJ.

Understanding metabolic bioelectrocatalysis of the purple bacterium Rhodobacter capsulatus through substrate modulation

Photoheterotrophic purple bacterium Rhodobacter capsulatus has recently gained attention for its halotolerance and photo-enhanced extracellular electron transfer, which is opening opportunities for self-powered decontamination of saline wastewater. Biodegradation of malate and succinate in R. capsulatus electrochemical systems has undergone prior examination, although a consistent bioelectrochemical system to comparatively study multiple carbon sources has not previously been developed. In this study, electrochemical techniques, biological assays, and genetic studies were combined to evaluate malate, succinate, propionate, and lactate as oxidizable fuels in photo-enhanced microbial electrochemical systems. Specifically, cyclic voltammetry and amperometry data demonstrated that R. capsulatus generates distinctive current densities dependent on both fuel identity and concentration. Bacterial growth curve studies agreed with electrochemical data, indicating that lactate, which yielded the greatest bio-anodic current density (9.5 ± 1.9 µA cm−2), allowed the bacteria to grow more rapidly than the other substrates, suggesting its effectiveness as a fuel. Finally, differential gene expression analysis allowed for illumination of the physiological underpinnings of the observed differences between substrates. Most remarkably, cells grown in lactate were found to overexpress light harvesting complex II proteins and to underexpress flagellar motility proteins, which both correspond to the higher relative anodic current density in lactate bioanodes.

Structure and mechanism of the RNA dependent RNase Cas13a from Rhodobacter capsulatus

Cas13a are single-molecule effectors of the Class II, Type VI family of CRISPR-Cas systems that are part of the bacterial and archaeal defense systems. These RNA-guided and RNA-activated RNA endonucleases are characterized by their ability to cleave target RNAs complementary to the crRNA-spacer sequence, as well as bystander RNAs in a sequence-unspecific manner. Due to cleavage of cellular transcripts they induce dormancy in the host cell and thus protect the bacterial population by aborting the infectious cycle of RNA-phages. Here we report the structural and functional characterization of a Cas13a enzyme from the photo-auxotrophic purple bacteria Rhodobacter capsulatus. The X-ray crystal structure of the RcCas13a-crRNA complex reveals its distinct crRNA recognition mode as well as the enzyme in its contracted, pre-activation conformation. Using site-directed mutagenesis in combination with mass spectrometry, we identified key residues responsible for pre-crRNA processing by RcCas13a in its distinct catalytic site, and elucidated the acid-base mediated cleavage reaction mechanism. In addition, RcCas13a cleaves target-RNA as well as bystander-RNAs in Escherichia coli which requires its catalytic active HEPN (higher eukaryotes and prokaryotes nucleotide binding) domain nuclease activity. Our data provide further insights into the molecular mechanisms and function of this intriguing family of RNA-dependent RNA endonucleases that are already employed as efficient tools for RNA detection and regulation of gene expression.

Reliable determination of the growth and hydrogen production parameters of the photosynthetic bacterium Rhodobacter capsulatus in fed batch culture using a combination of the Gompertz function and the Luedeking-Piret model

In this study, experimental results of hydrogen producing process based on anaerobic photosynthesis using the purple non-sulfur bacterium Rhodobacter capsulatus are scrutinized. The bacterial culture was carried out in a photo-bioreactor operated in a quasi-continuous mode, using lactate as a carbon source. The method is based on the continuous stirred tank reactors (CSTR) technique to access kinetic parameters. The dynamic evolution of hydrogen production as a function of time was accurately simulated using Luedeking-Piret model and the growth of R. capsulatus was computed using Gompertz model. The combination of both models was successfully applied to determine the relevant parameters (λ, μmax, α and β) for two R. capsulatus strains studied: the wild-type strain B10 and the H2 over-producing mutant IR3. The mathematical description indicates that the photofermentation is more promising than dark fermentation for the conversion of organic substrates into biogas.

Evaluating Organic Acids As Oxidizable Fuels in R. Capsulatus-Based Bioelectrochemical Systems

The development of microbial fuel cells (MFCs) for wastewater treatment has recently gained significant attention in the bioremediation and renewable energy industries. Bacteria can be integrated into the anodes of MFCs to oxidize organic wastes, simultaneously producing electrical current and decontaminating wastewater. The photoheterotrophic purple bacterium Rhodobacter capsulatus has demonstrated photo-catalyzed electron transfer to carbon electrodes via exogenous quinone redox mediators, and tolerance to high salinity, opening opportunities for solar-powered MFC systems for treatment of saline wastewater. While the consumption of malate by R. capsulatus has been electrochemically analyzed previously, in this project, alternative target carbon sources were examined in order to improve electrochemical performance of these R. capsulatus systems. A combination of electrochemical techniques and biological assays were used to evaluate the relative viability of malate, lactate, succinate and propionate as oxidizable fuels. Specifically, cyclic voltammetry and amperometry data demonstrated that R. capsulatus generates distinctive bio-photocurrent densities depending on the fuel metabolized. Bioanodes with immobilized R. capsulatus cells oxidizing lactate (9.49 ± 1.89 µA cm-2) as the fuel yielded the greatest mediated bio-photocurrent densities compared to the other fuels, while those metabolizing propionate (1.33 ± 0.18 µA cm-2) generated the smallest current densities. In tandem with electrochemistry methods, growth curve assays and RNA sequencing were performed to further delve into the biological effects of altering the carbon source.

Engineered Rhodobacter capsulatus as a Phototrophic Platform Organism for the Synthesis of Plant Sesquiterpenoids.

Sesquiterpenoids are a large class of natural compounds offering manifold properties valuable for food, cosmetics, agriculture, and pharma industry. Production in microorganisms is a sustainable approach to provide sesquiterpenoids for research and industrial use independent of their natural sources. This requires the functional transfer of the respective biocatalytic pathways in an adequate host microorganism offering a sufficient supply of precursors that is ideally adjusted to the individual demand of the recombinant biosynthesis route. The phototrophic purple bacterium Rhodobacter capsulatus offers unique physiological properties that are favorable for biosynthesis of hydrophobic terpenes. Under phototrophic conditions, it develops a large intracytoplasmic membrane suitable for hosting membrane-bound enzymes and metabolites of respective biosynthetic pathways. In addition, Rhodobacter harbors an intrinsic carotenoid biosynthesis that can be engineered toward the production of foreign terpenes. Here, we evaluate R. capsulatus as host for the production of plant sesquiterpenoids under phototrophic conditions using patchoulol and valencene as a proof of concept. The heterologous expression of patchoulol synthase PcPS from Pogostemon cablin as well as the valencene synthases CsVS from Citrus sinensis and CnVS from Callitropsis nootkatensis led to the production of the respective sesquiterpenoids in R. capsulatus. To analyze, if gradually adjustable formation of the key precursor farnesylpyrophosphate (FPP) is beneficial for sesquiterpene synthesis under phototrophic conditions, the intrinsic 1-deoxy-D-xylulose 5-phosphate (DXP) pathway genes as well as the heterologous mevalonate pathway genes were modularly expressed in various combinations. To this end, different plasmids and chromosomally integrated expression tools were developed harboring the strong and tightly controlled Pnif promoter for heterologous gene expression. Notably, comparative studies identified a distinct combination of precursor biosynthetic genes as best-performing setup for each of the tested sesquiterpene synthases. In summary, we could demonstrate that R. capsulatus is a promising alternative platform organism that is suited for sustainable sesquiterpenoid formation under phototrophic cultivation conditions. A modular engineering of R. capsulatus strains via tailored co-expression of FPP biosynthetic genes further allowed adaptation of sesquiterpene precursor formation to its catalytic conversion by different plant terpene synthases.

Dark fermentation effluent as substrate for hydrogen production from Rhodobacter capsulatus highlighting the performance of different fermentation systems.

Hydrogen production by biological route is a potentially sustainable alternative. Nowadays, energy production from sustainable sources has become urgent for several countries as well as for international policies. In this perspective, hydrogen has gained substantial global attention as clean, sustainable, and versatile energy carrier. In the current work, the resulting effluent from dark fermentation, rich in organic acids, was used as substrate for the purple non-sulfur bacteria (PNS) Rhodobacter capsulatus. In the first stage, experiments were carried out in bioreactors of 50mL to check the influence of the composition of the effluent dark fermentation. The results proved that the provision of a sugar source improved bio-H2 production. The lactose and lactic acid concentrations exceeding 4.4 and 12g/L, respectively, resulted in a productivity of up to 37.14mmol H2/Ldays. Based on initial conditions obtained on the previous assays, in the second stage, a photo-fermentation in enlarged scale (1.5L) was performed with the purpose to monitor the production of hydrogen and metabolites, sugar consumption and growth cells during the process. It was observed that the maximum productivity obtained was 98.23mmol H2/Ldays in 26h of process.

Understanding Biophotocurrent Generation in Photosynthetic Purple Bacteria

Establishing an efficient extracellular electron transfer (EET) process between photoelectroactive microorganisms and an electrode surface is critical for the development of photobioelectrocatalysis. Soluble and immobilized redox mediators have been applied with the purple bacterium Rhodobacter capsulatus for this purpose. However, detailed information on its EET with an electrode surface is not available and, therefore, choice of mediators has been by trial and error. Herein, we experimentally evaluated the capability of different soluble, quinone-based redox mediators to support EET and compared the experimental data with a computational model based on density functional theory calculations. We show that computed electrochemical redox properties of redox mediators in a lipophilic environment correlate to EET processes of Rhodobacter capsulatus, suggesting that intermembrane mediator characteristics are more diagnostic than redox properties of the mediators in an aqueous solution, and that the limiting e...

Inhibition of bacteriochlorophyll biosynthesis in the purple phototrophic bacteria Rhodospirillumrubrum and Rhodobacter capsulatus grown in the presence of a toxic concentration of selenite

Background In many works, the chemical composition of bacterially-produced elemental selenium nanoparticles (Se0-nanoparticles) was investigated using electron dispersive X-ray analysis. The results suggest that these particles should be associated with organic compounds. However, a complete analysis of their chemical composition is still missing. Aiming at identifying organic compounds associated with the Se0-nanoparticles produced by the purple phototrophic bacteria Rhodospirillum rubrum and Rhodobacter capsulatus (α group of the proteobacteria), we used MALDI-TOF spectrometry.Results This technic revealed that numerous signals obtained from particles produced by both species of bacteria were from metabolites of the photosynthetic system. Furthermore, not only bacteriochlorophyll a, bacteriopheophytin a, and bacteriopheophorbide a, which are known to accumulate in stationary phase cultures of these bacteria grown phototrophically in the absence of selenite, were identified. The particles were also associated with intermediary metabolites of the bacteriochlorophyll a biosynthesis pathway such as protoporphyrin IX, protoporphyrin IX monomethyl ester, bacteriochlorophyllide a and, most likely, Mg-protoporphyrin IX-monomethyl ester, as well as with oxidation products of the substrates of protochlorophyllide reductase and chlorin reductase.Conclusion Accumulation of intermediary metabolites of the bacteriochlorophyll biosynthesis pathway in these purple phototrophic bacteria was attributed to inhibition of oxygen-sensitive enzymes involved in this pathway. Consistent with this interpretation it has been reported that these bacteria reduce selenite intracellularly, that they contain high levels of glutathione and that the reduction of selenite with glutathione is a very fast reaction accompanied by the production of reactive oxygen species. As many enzymes involved in the biosynthesis of bacteriochlorophyll contain [Fe-S] clusters in their active site, which are known to be degraded in the presence of reactive oxygen species as well as in the presence of molecular oxygen, we concluded that the substrates of these enzymes accumulate in cells during selenite reduction.Association of metabolites of bacteriochlorophyll biosynthesis and degradation with the Se0-nanoparticles produced by Rhodospirillum rubrum and Rhodobacter capsulatus is proposed to result from coating of the nanoparticles with the intracytoplasmic membrane of these bacteria, where the photochemical apparatus is concentrated.

Repeated batch cycles as an alternative for hydrogen production by co-culture photofermentation

The use of hydrogen as an energy source represents an alternative to reduce the environmental impact since water is the only major by-product in its combustion. It can be used as an on-board fuel for motive power through internal combustion engine or fuel cell that converts chemical energy (an electro-chemical device) and can be applied in several transportation devices. The hybrid system for hydrogen production by combining the dark fermentation followed by photofermentation has great potential. In the current work, the production of hydrogen was investigated in repeated-batch processes using dark fermentation effluent as substrate to pure culture (Rhodopseudomonas palustris) and co-culture purple non-sulfur bacteria (Rhodopseudomonas palustris and Rhodobacter capsulatus). The influence of glucose and milk whey permeate (MWP) on the hydrogen production was studied. The results showed that the system employing the co-culture and glucose kept the culture activity for a long time (679 h) and the amount of accumulated hydrogen was 0.98 ± 0.02 mol and maximum productivity reached 287.39 ± 5.75 mmol of H2/L·day. The findings from alternating feeding between glucose and milk whey permeate, showed higher results for the amount of accumulated hydrogen (1.41 ± 0.01 mol). Besides, maximum productivity was of 266.60 ± 5.33 mmol of H2/L·day.

Primary processes in the bacterial reaction center probed by two-dimensional electronic spectroscopy

In the initial steps of photosynthesis, reaction centers convert solar energy to stable charge-separated states with near-unity quantum efficiency. The reaction center from purple bacteria remains an important model system for probing the structure-function relationship and understanding mechanisms of photosynthetic charge separation. Here we perform 2D electronic spectroscopy (2DES) on bacterial reaction centers (BRCs) from two mutants of the purple bacterium Rhodobacter capsulatus, spanning the Q y absorption bands of the BRC. We analyze the 2DES data using a multiexcitation global-fitting approach that employs a common set of basis spectra for all excitation frequencies, incorporating inputs from the linear absorption spectrum and the BRC structure. We extract the exciton energies, resolving the previously hidden upper exciton state of the special pair. We show that the time-dependent 2DES data are well-represented by a two-step sequential reaction scheme in which charge separation proceeds from the excited state of the special pair (P*) to P+HA- via the intermediate P+BA- When inhomogeneous broadening and Stark shifts of the B* band are taken into account we can adequately describe the 2DES data without the need to introduce a second charge-separation pathway originating from the excited state of the monomeric bacteriochlorophyll BA*.

Absence of Thiol-Disulfide Oxidoreductase DsbA Impairs cbb3-Type Cytochrome c Oxidase Biogenesis in Rhodobacter capsulatus.

The thiol-disulfide oxidoreductase DsbA carries out oxidative folding of extra-cytoplasmic proteins by catalyzing the formation of intramolecular disulfide bonds. It has an important role in various cellular functions, including cell division. The purple non-sulfur bacterium Rhodobacter capsulatus mutants lacking DsbA show severe temperature-sensitive and medium-dependent respiratory growth defects. In the presence of oxygen, at normal growth temperature (35°C), DsbA− mutants form colonies on minimal medium, but they do not grow on enriched medium where cells elongate and lyse. At lower temperatures (i.e., 25°C), cells lacking DsbA grow normally in both minimum and enriched media, however, they do not produce the cbb3-type cytochrome c oxidase (cbb3-Cox) on enriched medium. Availability of chemical oxidants (e.g., Cu2+ or a mixture of cysteine and cystine) in the medium becomes critical for growth and cbb3-Cox production in the absence of DsbA. Indeed, addition of Cu2+ to the enriched medium suppresses, and conversely, omission of Cu2+ from the minimal medium induces, growth and cbb3-Cox defects. Alleviation of these defects by addition of redox-active chemicals indicates that absence of DsbA perturbs cellular redox homeostasis required for the production of an active cbb3-Cox, especially in enriched medium where bioavailable Cu2+ is scarce. This is the first report describing that DsbA activity is required for full respiratory capability of R. capsulatus, and in particular, for proper biogenesis of its cbb3-Cox. We propose that absence of DsbA, besides impairing the maturation of the c-type cytochrome subunits, also affects the incorporation of Cu into the catalytic subunit of cbb3-Cox. Defective high affinity Cu acquisition pathway, which includes the MFS-type Cu importer CcoA, and lower production of the c-type cytochrome subunits lead together to improper assembly and degradation of cbb3-Cox.

Generation of semiquinone-[2Fe-2S]+ spin-coupled center at the Qo site of cytochrome bc1 in redox-poised, illuminated photosynthetic membranes from Rhodobacter capsulatus

One of the less understood parts of the catalytic cycle of cytochrome bc1/b6f complexes is the mechanism of electronic bifurcation occurring within the hydroquinone oxidation site (Qo site). Several models describing this mechanism invoke a phenomenon of formation of an unstable semiquinone. Recent studies with isolated cytochrome bc1 or b6f revealed that a relatively stable semiquinone spin-coupled to the reduced Rieske cluster (SQ-FeS) is generated at the Qo site during the oxidation of ubi- or plastohydroquinone analogs under conditions of continuous turnover. Here, we identified the EPR transition of SQ-FeS formed upon oxidation of ubihydroquinone in native photosynthetic membranes from purple bacterium Rhodobacter capsulatus. We observed a significant amount of SQ-FeS generated when the antimycin-inhibited enzyme experiences conditions of non-equilibrium caused by the continuous light activation of the reaction center. We also noted that SQ-FeS cannot be detected under equilibrium redox titrations in dark. The non-equilibrium redox titrations of SQ-FeS indicate that this center has a higher apparent redox midpoint potential when compared to the redox midpoint potential of the quinone pool. This suggests that SQ-FeS is stabilized, which corroborates a recently proposed mechanism in which the SQ-FeS state is metastable and functions to safely hold electrons at the local energy minimum during the oxidation of ubihydroquinone and limits superoxide formation. Our results open new possibilities to study the formation and properties of this state in cytochromes bc under close to physiological conditions in which non-equilibrium is attained by the light activation of bacterial reaction centers or photosystems.

Rapid C8-vinyl reduction of divinyl-chlorophyllide a by BciA from Rhodobacter capsulatus

Abstract Divinyl-chlorophyllide a (DV-Chlide a ) is a universal precursor for chlorophyll or bacteriochlorophyll biosynthesis in all photosynthetic organisms. Previous mutational analyses revealed that BciA works for reduction of the C8-vinyl group of DV-Chlide a. Chlorophyllide oxidoreductase (COR) reduces the C7=C8 double bond of Chlide a bearing the C8-ethyl group, but also potentially catalyzes the reduction of the C8-vinyl group of DV-Chlide a. In this study, we prepared a recombinant BciA protein from the purple photosynthetic bacterium Rhodobacter capsulatus and analyzed its C8-vinyl reduction activity towards DV-Chlide a . BciA formed a functional oligomeric complex consisting of at least three rigid dimers. BciA required NADPH as an electron donor for its C8-vinyl reduction activity. The enzymatic activity of BciA towards the substrate DV-Chlide a was much higher than that of COR towards Chlide a . Phylogenetic distribution and the enzymatic parameters of BciA and COR suggest that BciA is a more recently acquired auxiliary C8-vinyl reductase, attained to meet the high demand for bacteriochlorophylls used to produce large amounts of light-harvesting complexes.

Characterization of redox-active cysteine residues of persulfide-responsive transcriptional repressor SqrR

ABSTRACTWe recently identified the novel persulfide sensor SqrR that functions as a master regulator of sulfide-dependent gene expression in the purple photosynthetic bacterium Rhodobacter capsulatus. SqrR binds to the promoter regions of target genes to repress their expression in the absence of sulfide, and the repressor activity is negated by sulfide treatment. SqrR has 3 cysteine residues, 2 of which are conserved in SqrR homologs from other bacteria: Cys41 and Cys107. SqrR forms an intramolecular tetrasulfide bond between Cys41 and Cys107 when exposed to persulfide, which results in loss of the DNA-binding activity in vitro. Here, we address the mechanism through which these cysteine residues are modified by persulfides. We show that the predicted pKa value of Cys107, as revealed by a putative SqrR structural model, is lower than that of Cys41. Furthermore, C41S SqrR in which Cys41 was changed to serine forms an intermolecular disulfide-bond between Cys107 of 2 SqrRs, suggesting high nucleophilic reactivity of Cy107. These data suggest that Cys107 and Cys41 function as attacking Cys and resolving Cys, respectively; this occurs during tetrasulfide-bond formation of WT SqrR, when it is exposed to persulfide.

Acetate Metabolism in the Purple Non-sulfur Bacterium Rhodobacter capsulatus.

The purple non-sulfur bacterium Rhodobacter capsulatus B10 can grow on acetate as the sole carbon source under photoheterotrophic conditions. It is known that the bacterium can use the glyoxylate cycle and, in addition to it, or alternatively to it, an unknown pathway for acetate assimilation. We analyzed the genetic potential for functioning of additional metabolic pathways of oxaloacetic acid (OAA) pool replenishment in R. capsulatus. Using published microarray data of more than 4000 transcripts of genes for R. capsulatus, the genes necessary for acetate assimilation were analyzed. The results of the analysis showed the presence of all genes necessary for functioning of the ethylmalonyl-CoA pathway, and also a combination of pathways of formation of pyruvic acid/phosphoenol pyruvate (PA/PEP) (from acetyl-CoA and formate, from acetyl-CoA and CO2, as well as from 3-phosphoglyceric acid formed in the Calvin-Benson cycle) with their subsequent carboxylation. Using expression analysis, we showed that OAA pool replenishment on acetate medium could be achieved via a combination of PA/PEP synthesis from Calvin-Benson cycle intermediates and their carboxylation (with participation of pyruvate carboxylase, two reversible malate dehydrogenases (decarboxylating) and PEP-carboxykinase) to tricarboxylic acid cycle intermediates, the glyoxylate cycle, and a modified ethylmalonyl-CoA pathway in R. capsulatus under these experimental conditions. It was found that analogs of ethylmalonyl-CoA pathway enzymes exist. These enzymes differ in their specificity for S-enantiomers.

Sulfide-responsive transcriptional repressor SqrR functions as a master regulator of sulfide-dependent photosynthesis

Sulfide was used as an electron donor early in the evolution of photosynthesis, with many extant photosynthetic bacteria still capable of using sulfur compounds such as hydrogen sulfide (H2S) as a photosynthetic electron donor. Although enzymes involved in H2S oxidation have been characterized, mechanisms of regulation of sulfide-dependent photosynthesis have not been elucidated. In this study, we have identified a sulfide-responsive transcriptional repressor, SqrR, that functions as a master regulator of sulfide-dependent gene expression in the purple photosynthetic bacterium Rhodobacter capsulatus SqrR has three cysteine residues, two of which, C41 and C107, are conserved in SqrR homologs from other bacteria. Analysis with liquid chromatography coupled with an electrospray-interface tandem-mass spectrometer reveals that SqrR forms an intramolecular tetrasulfide bond between C41 and C107 when incubated with the sulfur donor glutathione persulfide. SqrR is oxidized in sulfide-stressed cells, and tetrasulfide-cross-linked SqrR binds more weakly to a target promoter relative to unmodified SqrR. C41S and C107S R. capsulatus SqrRs lack the ability to respond to sulfide, and constitutively repress target gene expression in cells. These results establish that SqrR is a sensor of H2S-derived reactive sulfur species that maintain sulfide homeostasis in this photosynthetic bacterium and reveal the mechanism of sulfide-dependent transcriptional derepression of genes involved in sulfide metabolism.