Cryo-EM structure of the four-subunit Rhodobacter sphaeroides cytochrome bc1 complex in styrene maleic acid nanodiscs.

Cytochrome bc1 complexes are ubiquinol:cytochrome c oxidoreductases, and as such, they are centrally important components of respiratory and photosynthetic electron transfer chains in many species of bacteria and in mitochondria. The minimal complex has three catalytic components, which are cytochrome b, cytochrome c1, and the Rieske iron-sulfur subunit, but the function of mitochondrial cytochrome bc1 complexes is modified by up to eight supernumerary subunits. The cytochrome bc1 complex from the purple phototrophic bacterium Rhodobacter sphaeroides has a single supernumerary subunit called subunit IV, which is absent from current structures of the complex. In this work we use the styrene-maleic acid copolymer to purify the R. sphaeroides cytochrome bc1 complex in native lipid nanodiscs, which retains the labile subunit IV, annular lipids, and natively bound quinones. The catalytic activity of the four-subunit cytochrome bc1 complex is threefold higher than that of the complex lacking subunit IV. To understand the role of subunit IV, we determined the structure of the four-subunit complex at 2.9 Å using single particle cryogenic electron microscopy. The structure shows the position of the transmembrane domain of subunit IV, which lies across the transmembrane helices of the Rieske and cytochrome c1 subunits. We observe a quinone at the Qo quinone-binding site and show that occupancy of this site is linked to conformational changes in the Rieske head domain during catalysis. Twelve lipids were structurally resolved, making contacts with the Rieske and cytochrome b subunits, with some spanning both of the two monomers that make up the dimeric complex.

Sulfoquinovosyl diacylglycerol is required for dimerisation of the Rhodobacter sphaeroides reaction centre-light harvesting 1 core complex.

The reaction centre-light harvesting 1 (RC-LH1) core complex is indispensable for anoxygenic photosynthesis. In the purple bacterium Rhodobacter (Rba.) sphaeroides RC-LH1 is produced both as a monomer, in which 14 LH1 subunits form a C-shaped antenna around 1 RC, and as a dimer, where 28 LH1 subunits form an S-shaped antenna surrounding 2 RCs. Alongside the five RC and LH1 subunits, an additional polypeptide known as PufX provides an interface for dimerisation and also prevents LH1 ring closure, introducing a channel for quinone exchange that is essential for photoheterotrophic growth. Structures of Rba. sphaeroides RC-LH1 complexes revealed several new components; protein-Y, which helps to form the quinone channel; protein-Z, of unknown function and seemingly unique to dimers; and a tightly bound sulfoquinovosyl diacylglycerol (SQDG) lipid that interacts with two PufX arginine residues. This lipid lies at the dimer interface alongside weak density for a second molecule, previously proposed to be an ornithine lipid. In this work we have generated strains of Rba. sphaeroides lacking protein-Y, protein-Z, SQDG or ornithine lipids to assess the roles of these previously unknown components in the assembly and activity of RC-LH1. We show that whilst the removal of either protein-Y, protein-Z or ornithine lipids has only subtle effects, SQDG is essential for the formation of RC-LH1 dimers but its absence has no functional effect on the monomeric complex.

Acute effects of the environmental probiotics Rhodobacter sphaeroides on intestinal bacteria and transcriptome in shrimp Penaeusvannamei.

In recent years, a substantial number of studies have been dedicated to exploring the potential benefits of probiotics in aquaculture. Rhodobacter sphaeroides can be used in aquaculture-related environmental bioremediation, and its protein is also used as a feed additive in Penaeus vannamei culture. To investigate the effects of releasing R. sphaeroides as environmental probiotics on P. vannamei, we employed 16S rRNA gene and mRNA transcriptome sequencing. Our study focused on assessing alterations in intestinal bacteria and intestinal gene expression in P. vannamei, establishing correlations between them. Our findings revealed a significant increase in the relative abundances of Rhodobacter, Paracoccus, Sulfitobacter, and other bacterial OTUs within the intestinal bacterial community. Additionally, we observed enhanced complexity and stability in the intestinal bacterial correlation network, indicating improved synergy among bacteria and reduced competition. Moreover, the introduction of R. sphaeroides resulted in the down-regulation of certain immune genes and the up-regulation of genes linked to growth and metabolism in the intestinal tissues of P. vannamei. Importantly, we identified a noteworthy correlation between the changes in intestinal bacteria and these alterations in intestinal tissue gene expressions. By conducting analyses of the intestinal bacterial community and intestinal tissue transcriptome, this study revealed the effects of releasing R. sphaeroides as sediment probiotics in P. vannamei culture water. These results serve as vital scientific references for the application of R. sphaeroides in P. vannamei aquaculture.

Rhodobacter sphaeroides supplementation improves defense ability of Chinese mitten crab Eriocheir sinensis against Shewanella putrefaciens infection via intestinal flora and metabolism regulation.

Shewanella putrefaciens is a vital bacterial pathogen implicated in serious diseases in Chinese mitten crab Eriocheir sinensis. Yet the use of probiotics to improve the defense ability of E. sinensis against S. putrefaciens infection remains poorly understood. In the present study, the protective effect of dietary R. sphaeroides against S. putrefaciens infection in E. sinensis was evaluated through antioxidant capability, immune response, and survival under bacterial challenge assays, and its protective mechanism was further explored using a combination of intestinal flora and metabolome assays. Our results indicated that dietary R. sphaeroides could significantly improve immunity and antioxidant ability of Chinese mitten crabs, thereby strengthening their disease resistance with the relative percentage survival of 81.09% against S. putrefaciens. In addition, dietary R. sphaeroides could significantly alter the intestinal microbial composition and intestinal metabolism of crabs, causing not only the reduction of potential threatening pathogen load but also the increase of differential metabolites in tryptophan metabolism, pyrimidine metabolism, and glycerophospholipid metabolism. Furthermore, the regulation of differential metabolites such as N-Acetylserotonin positively correlated with beneficial Rhodobacter could be a potential protection strategy for Shewanella infection. To the best of our knowledge, this is the first study to illustrate the protective effect and mechanism of R. sphaeroides supplementation to protect E. sinensis against S. putrefaciens infection.

Highly Efficient DSSCs Sensitized Using NIR Responsive Bacteriopheophytine-a and Its Derivatives Extracted from Rhodobacter Sphaeroides Photobacteria.

Employing naturally extracted dyes and their derivatives as photosensitizers towards the construction of dye-sensitized solar cells (DSSCs) has been recently emerging for establishing sustainable energy conversion devices. In this present work, Rhodobacter Sphaeroides Photobacteria (Rh. Sphaeroides) was used as a natural source from which Bacteriopheophytine-a (Bhcl) dye was extracted. Further, two cationic derivatives of Bhcl, viz., Guanidino-bacteriopheophorbide-a (Gua-Bhcl) and (2-aminoethyl)triphenylphosphono-bacteriopheophorbide-a (2AETPPh-Bhcl) were synthesized. The thus obtained Bhcl, Gua-Bhcl and 2AETPPh-Bhcl were characterized using liquid chromatography-mass spectrometry (LC-MS) and their photophysical properties were investigated using excitation and emission studies. All three near-infrared (NIR) responsive dyes were employed as natural sensitizers towards the construction of DSSC devices, using platinum as a photocathode, dye-sensitized P25-TiO2 as a photoanode and I-/I3- as an electrolyte. DSSCs fabricated using all three dyes have shown reasonably good photovoltaic performance, among which 2AETPPh-Bhcl dye has shown a relatively higher power conversion efficiency (η) of 0.38% with a short circuit photocurrent density (JSC) of 1.03 mA cm-2. This could be attributed to the dye's natural optimal light absorption in the visible and NIR region and uniform dispersion through the electrostatic interaction of the cationic derivatives on the TiO2 photoanode. Furthermore, the atomic force microscopy studies and electrochemical investigations using cyclic voltammetry, electrochemical impedance spectroscopy and Bode's plot also supported the enhancement in performance attained with 2AETPPh-Bhcl dye.

Metabolic flux analysis of coenzyme Q10 synthesized by Rhodobacter sphaeroides under the influence of different pH regulators.

Coenzyme Q10 (CoQ10) is crucial for human beings, especially in the fields of biology and medicine. The aim of this experiment was to investigate the conditions for increasing CoQ10 production. At present, microbial fermentation is the main production method of CoQ10, and the production process of microbial CoQ10 metabolism control fermentation is very critical. Metabolic flux is one of the most important determinants of cell physiology in metabolic engineering. Metabolic flux analysis (MFA) is used to estimate the intracellular flux in metabolic networks. In this experiment, Rhodobacter sphaeroides was used as the research object to analyze the effects of aqueous ammonia (NH3·H2O) and calcium carbonate (CaCO3) on the metabolic flux of CoQ10. When CaCO3 was used to adjust the pH, the yield of CoQ10 was 274.43 mg·L-1 (8.71 mg·g-1 DCW), which was higher than that of NH3·H2O adjustment. The results indicated that when CaCO3 was used to adjust pH, more glucose-6-phosphate (G6P) entered the pentose phosphate (HMP) pathway and produced more NADPH, which enhanced the synthesis of CoQ10. At the chorismic acid node, more metabolic fluxes were involved in the synthesis of p-hydroxybenzoic acid (pHBA; the synthetic precursor of CoQ10), enhancing the anabolic flow of CoQ10. In addition, Ca2+ produced by the reaction of CaCO3 with organic acids promotes the synthesis of CoQ10. In summary, the use of CaCO3 adjustment is more favorable for the synthesis of CoQ10 by R. sphaeroides than NH3·H2O adjustment. The migration of metabolic flux caused by the perturbation of culture conditions was analyzed to compare the changes in the distribution of intracellular metabolic fluxes for the synthesis of CoQ10. Thus, the main nodes of the metabolic network were identified as G6P and chorismic acid. This provides a theoretical basis for the modification of genes related to the CoQ10 synthesis pathway.

A majority of Rhodobacter sphaeroides promoters lack a crucial RNA polymerase recognition feature, enabling coordinated transcription activation.

Using an in vitro transcription system with purified RNA polymerase (RNAP) to investigate rRNA synthesis in the photoheterotrophic α-proteobacterium Rhodobacter sphaeroides, we identified a surprising feature of promoters recognized by the major holoenzyme. Transcription from R. sphaeroides rRNA promoters was unexpectedly weak, correlating with absence of -7T, the very highly conserved thymine found at the last position in -10 elements of promoters in most bacterial species. Thymine substitutions for adenine at position -7 in the three rRNA promoters strongly increased intrinsic promoter activity, indicating that R. sphaeroides RNAP can utilize -7T when present. rRNA promoters were activated by purified R. sphaeroides CarD, a transcription factor found in many bacterial species but not in ß- and γ-proteobacteria. Overall, CarD increased the activity of 15 of 16 native R. sphaeroides promoters tested in vitro that lacked -7T, whereas it had no effect on three of the four native promoters that contained -7T. Genome-wide bioinformatic analysis of promoters from R. sphaeroides and two other α-proteobacterial species indicated that 30 to 43% contained -7T, whereas 90 to 99% of promoters from non-α-proteobacteria contained -7T. Thus, promoters lacking -7T appear to be widespread in α-proteobacteria and may have evolved away from consensus to enable their coordinated regulation by transcription factors like CarD. We observed a strong reduction in R. sphaeroides CarD levels when cells enter stationary phase, suggesting that reduced activation by CarD may contribute to inhibition of rRNA transcription when cells enter stationary phase, the stage of growth when bacterial ribosome synthesis declines.

Construction of a Rhodobacter sphaeroides Strain That Efficiently Produces Hydrogen Gas from Acetate without Poly(ß-Hydroxybutyrate) Accumulation: Insight into the Role of PhaR in Acetate Metabolism.

The purple nonsulfur phototrophic bacterium Rhodobacter sphaeroides produces hydrogen gas (H2) from acetate. An approach to improve the H2 production is preventing accumulation of an intracellular energy storage molecule known as poly(ß-hydroxybutyrate) (PHB), which competes with H2 production for reducing power. However, disruption of PHB biosynthesis has been reported to severely impair the acetate assimilation depending on the genetic backgrounds and/or culture conditions. To solve this problem, we analyzed the relationship between PHB accumulation and acetate metabolism in R. sphaeroides. Gene deletion analyses based on the wild-type strain revealed that among the two polyhydroxyalkanoate synthase genes in the genome, phaC1, but not phaC2, is essential for PHB accumulation, and the phaC1 deletion mutant exhibited slow growth with acetate. On the other hand, a strain with the deletion of phaC1 together with phaR, which encodes a transcriptional regulator capable of sensing PHB accumulation, exhibited growth comparable to that of the wild-type strain despite no accumulation of PHB. These results suggest that PHB accumulation is required for normal growth with acetate by altering the expression of genes under the control of phaR. This hypothesis was supported by a transcriptome sequencing (RNA-seq) analysis revealing that phaR is involved in the regulation of the ethylmalonyl coenzyme A pathway for acetate assimilation. Consistent with these findings, deletion of phaC1 in a genetically engineered H2-producing strain resulted in lower H2 production from acetate due to growth defects, whereas deletion of phaR together with phaC1 restored growth with acetate and increased H2 production from acetate without PHB accumulation. IMPORTANCE This study provides a novel approach for increasing the yield of photofermentative H2 production from acetate by purple nonsulfur phototrophic bacteria. This study further suggests that polyhydroxyalkanoate is not only a storage substance for carbon and energy in bacteria, but may also act as a signaling molecule that mediates bacterial metabolic adaptations to specific environments. This notion will be helpful for understanding the physiology of polyhydroxyalkanoate-producing bacteria, as well as for their metabolic engineering via synthetic biology.

Photofermentative biohydrogen generation from organic acids by Rhodobacter sphaeroides O.U.001: Computational fluid dynamics modeling of hydrodynamics and temperature.

Hydrogen gas is a clean-burning fuel suitable for powering public vehicles. Hydrogen fuel has the highest energy density (143 MJ kg-1 ). This research paper emphasizes three-dimensional hydrodynamics and temperature distribution during photobiohydrogen generation by Rhodobacter sphaeroides strain O.U.001 in a triple-jacketed 1 L photobioreactor (PBR). The fermentation broth has turbulent flow conditions and light gradients among various layers, which affect the light conversion efficiency of the PBR. From the carbon source (malic acid), various organic acids are produced within fermentation (lactate, acetate, and formate). Modeling and simulation studies by computational fluid dynamics confirmed uniform fluid dynamics and heat transfer throughout the annular PBR. The modified Gompertz equation gave good simulated fitting with an experimental value for H2 generation. R. sphaeroides O.U. 001 gave good simulated results for H2 generation with mathematical modeling of substrate consumption kinetics and substrate utilization for biomass.

On the Mechanism of Selective Chemical Exchange of Bacteriopheophytins in the Reaction Centers of Rhodobacter sphaeroides R-26.

To elucidate the mechanism of site-selective chemical replacement of chromophores in the reaction centers (RCs) of photosynthetic bacteria by external pigments, we investigated how the efficiency of incorporation of plant pheophytin a (Pheo) into the binding sites for bacteriopheophytin a molecules (BPheo) in the isolated Rhodobacter sphaeroides R-26 RCs depended on the incubation medium temperature, Pheo aggregation state, and the presence of organic solvent (acetone). When Pheo was in a form of monomers in free detergent micelles in a water-detergent incubation medium, the degree of selective replacement of photochemically inactive BPheo HB molecules upon incubation of the RC/Pheo mixture at 5°C was ~15%. The exchange efficiency increased to 40% upon incubation at 25°C and reached 100% at the same temperature when 10% acetone was added to the incubation medium. At both 5 and 25°C, the degree of pigment exchange increased approximately twice, when a mixture of Pheo monomers and dimers in the presence of 10% acetone was used as the incubation medium. The removal of acetone from this medium with the preservation of pigment forms led to a significant decrease in the efficiency of Pheo incorporation. The effect of acetone on the pigment exchange was also observed at an elevated incubation temperature (43.5°C), when functionally active BPheo HA molecules were partially replaced. The results are discussed in terms of the mechanism according to which (i) the temperature-dependent internal movements of the RC protein facilitate the release of the BPheo molecule from the binding site with simultaneous insertion of the Pheo molecule into the same site in a coupled process, (ii) the role of temperature largely depends on the steric accessibility of binding pockets in the RC protein, (iii) the incorporation of Pheo occurs from a pool of monomeric molecules included in the RC-detergent micelles, and (iv) the presence of acetone in the incubation medium facilitates the exchange of Pheo monomers between micelles in the solution and the detergent belt of the RC complex.

A Small RNA, UdsC, Interacts with the RpoHII mRNA and Affects the Motility and Stress Resistance of Rhodobacter sphaeroides.

sRNAs have an important role in the regulation of bacterial gene expression. The sRNA, UdsC, of Rhodobacter sphaeroides is derived from the 3' UTR of the RSP_7527 mRNA, which encodes a hypothetical protein. Here, we showed the effect of UdsC on the resistance of Rhodobacter sphaeroides to hydrogen peroxide and on its motility. In vitro binding assays supported the direct interaction of UdsC with the 5' UTR of the rpoHII mRNA. RpoHII is an alternative sigma factor with an important role in stress responses in R. sphaeroides, including its response to hydrogen peroxide. We also demonstrated that RpoHII controls the expression of the torF gene, which encodes an important regulator of motility genes. This strongly suggested that the observed effect of UdsC on TorF expression is indirect and mediated by RpoHII.

Modulation of the Enzymatic Activity of the Flagellar Lytic Transglycosylase SltF by Rod Components and the Scaffolding Protein FlgJ in Rhodobacter sphaeroides.

Macromolecular cell-envelope-spanning structures such as the bacterial flagellum must traverse the cell wall. Lytic transglycosylase enzymes are capable of enlarging gaps in the peptidoglycan meshwork to allow the efficient assembly of supramolecular complexes. In the periplasmic space, the assembly of the flagellar rod requires the scaffold protein FlgJ, which includes a muramidase domain in the canonical models Salmonella enterica and Escherichia coli. In contrast, in Rhodobacter sphaeroides, FlgJ and the dedicated flagellar lytic transglycosylase SltF are separate entities that interact in the periplasm. In this study, we show that sltF is expressed, along with the genes encoding the early components of the flagellar hierarchy that include the hook-basal body proteins, making SltF available during the rod assembly. Protein-protein interaction experiments demonstrated that SltF interacts with the rod proteins FliE, FlgB, FlgC, FlgF, and FlgG through its C-terminal region. A deletion analysis that divides the C terminus in two halves revealed that the interacting regions for most of the rod proteins are not redundant. Our results also show that the presence of the rod proteins FliE, FlgB, FlgC, and FlgF displace the previously reported SltF-FlgJ interaction. In addition, we observed modulation of the transglycosylase activity of SltF mediated by FlgB and FlgJ that could be relevant to coordinate rod assembly with cell wall remodeling. In summary, different mechanisms regulate the flagellar lytic transglycosylase, SltF, ensuring a timely transcription, a proper localization and a controlled enzymatic activity. IMPORTANCE Several mechanisms participate in the assembly of cell-envelope-spanning macromolecular structures. The sequential expression of substrates to be exported, selective export, and a specific order of incorporation are some of the mechanisms that stand out to drive an efficient assembly process. Here, we analyze how the structural rod proteins, the scaffold protein FlgJ and the flagellar lytic enzyme SltF, interact in an orderly fashion to assemble the flagellar rod into the periplasmic space. A complex arrangement of transient interactions directs a dedicated flagellar muramidase toward the flagellar rod. All of these interactions bring this protein to the proximity of the peptidoglycan wall while also modulating its enzymatic activity. This study suggests how a dynamic network of interactions participates in controlling SltF, a prominent component for flagellar formation.

Impact of PNPase on the transcriptome of Rhodobacter sphaeroides and its cooperation with RNase III and RNase E.

BACKGROUND: The polynucleotide phosphorylase (PNPase) is conserved among both Gram-positive and Gram-negative bacteria. As a core part of the Escherichia coli degradosome, PNPase is involved in maintaining proper RNA levels within the bacterial cell. It plays a major role in RNA homeostasis and decay by acting as a 3'-to-5' exoribonuclease. Furthermore, PNPase can catalyze the reverse reaction by elongating RNA molecules in 5'-to-3' end direction which has a destabilizing effect on the prolonged RNA molecule. RNA degradation is often initiated by an endonucleolytic cleavage, followed by exoribonucleolytic decay from the new 3' end. RESULTS: The PNPase mutant from the facultative phototrophic Rhodobacter sphaeroides exhibits several phenotypical characteristics, including diminished adaption to low temperature, reduced resistance to organic peroxide induced stress and altered growth behavior. The transcriptome composition differs in the pnp mutant strain, resulting in a decreased abundance of most tRNAs and rRNAs. In addition, PNPase has a major influence on the half-lives of several regulatory sRNAs and can have both a stabilizing or a destabilizing effect. Moreover, we globally identified and compared differential RNA 3' ends in RNA NGS sequencing data obtained from PNPase, RNase E and RNase III mutants for the first time in a Gram-negative organism. The genome wide RNA 3' end analysis revealed that 885 3' ends are degraded by PNPase. A fair percentage of these RNA 3' ends was also identified at the same genomic position in RNase E or RNase III mutant strains. CONCLUSION: The PNPase has a major influence on RNA processing and maturation and thus modulates the transcriptome of R. sphaeroides. This includes sRNAs, emphasizing the role of PNPase in cellular homeostasis and its importance in regulatory networks. The global 3' end analysis indicates a sequential RNA processing: 5.9% of all RNase E-dependent and 9.7% of all RNase III-dependent RNA 3' ends are subsequently degraded by PNPase. Moreover, we provide a modular pipeline which greatly facilitates the identification of RNA 5'/3' ends. It is publicly available on GitHub and is distributed under ICS license.

The periplasmic component of the DctPQM TRAP-transporter is part of the DctS/DctR sensory pathway in Rhodobacter sphaeroides.

Rhodobacter sphaeroides can use C4-dicarboxylic acids to grow heterotrophically or photoheterotropically, and it was previously demonstrated in Rhodobacter capsulatus that the DctPQM transporter system is essential to support growth using these organic acids under heterotrophic but not under photoheterotrophic conditions. In this work we show that in R. sphaeroides this transporter system is essential for photoheterotrophic and heterotrophic growth, when C4-dicarboxylic acids are used as a carbon source. We also found that over-expression of dctPQM is detrimental for photoheterotrophic growth in the presence of succinic acid in the culture medium. In agreement with this, we observed a reduction of the dctPQM promoter activity in cells growing under these conditions, indicating that the amount of DctPQM needs to be reduced under photoheterotrophic growth. It has been reported that the two-component system DctS and DctR activates the expression of dctPQM. Our results demonstrate that in the absence of DctR, dctPQM is still expressed albeit at a low level. In this work, we have found that the periplasmic component of the transporter system, DctP, has a role in both transport and in signalling the DctS/DctR two-component system.

The effect of some antiseptic drugs on the energy transfer in chromatophore photosynthetic membranes of purple non-sulfur bacteria Rhodobacter sphaeroides.

Chromatophores of purple non-sulfur bacteria (PNSB) are invaginations of the cytoplasmic membrane that contain a relatively simple system of light-harvesting protein-pigment complexes, a photosynthetic reaction center (RC), a cytochrome complex, and ATP synthase, which transform light energy into the energy of synthesized ATP. The high content of negatively charged phosphatidylglycerol (PG) and cardiolipin (CL) in PNSB chromatophore membranes makes these structures potential targets that bind cationic antiseptics. We used the methods of stationary and kinetic fluorescence spectroscopy to study the effect of some cationic antiseptics (chlorhexidine, picloxydine, miramistin, and octenidine at concentrations up to 100 µM) on the spectral and kinetic characteristics of the components of the photosynthetic apparatus of Rhodobacter sphaeroides chromatophores. Here we present the experimental data on the reduced efficiency of light energy conversion in the chromatophore membranes isolated from the photosynthetic bacterium Rb. sphaeroides in the presence of cationic antiseptics. The addition of antiseptics did not affect the energy transfer between the light-harvesting LH1 complex and reaction center (RC). However, it significantly reduced the efficiency of the interaction between the LH2 and LH1 complexes. The effect was maximal with 100 µM octenidine. It has been proved that molecules of cationic antiseptics, which apparently bind to the heads of negatively charged cardiolipin molecules located in the rings of light-harvesting pigments on the cytoplasmic surface of the chromatophores, can disturb the optimal conditions for efficient energy migration in chromatophore membranes.

Pigment-modified reaction centers of Chloroflexus aurantiacus: chemical exchange of bacteriopheophytins with plant-type pheophytins.

The pigment composition of isolated reaction centers (RCs) of the green filamentous bacterium Chloroflexus (Cfl.) aurantiacus was changed by chemical exchange of native bacteriopheophytin a (BPheo) molecules with externally added pheophytin a (Pheo) or [3-acetyl]-Pheo upon incubation of RC/pheophytin mixtures at room temperature and 45 °C. The modified RCs were characterized by Vis/NIR absorption spectroscopy, and the effect of pigment exchange on RC photochemical activity was assessed by measuring the photoaccumulation of the reduced pigment at the binding site HA. It is shown that both pheophytins can be exchanged into the HA site instead of BPheo by incubation at room temperature. While the newly introduced Pheo molecule is not active in electron transfer, the [3-acetyl]-Pheo molecule is able to replace functionally the photoreducible HA BPheo molecule with the formation of the [3-acetyl]-Pheo- radical anion instead of the BPheo-. After incubation at 45 °C, the majority (~ 90%) of HA BPheo molecules is replaced by both Pheo and [3-acetyl]-Pheo. Only a partial replacement of inactive BPheo molecules with pheophytins is observed even when the incubation temperature is raised to 50 °C. The results are discussed in terms of (i) differences in the accessibility of BPheo binding sites for extraneous pigments depending on structural constraints and incubation temperature and (ii) the effect of the reduction potential of pigments introduced into the HA site on the energetics of the charge separation process. The possible implication of Pheo-exchanged preparations for studying early electron-transfer events in Cfl. aurantiacus RCs is considered.

The transition of Rhodobacter sphaeroides into a microbial cell factory.

Microbial cell factories are the workhorses of industrial biotechnology and improving their performances can significantly optimize industrial bioprocesses. Microbial strain engineering is often employed for increasing the competitiveness of bio-based product synthesis over more classical petroleum-based synthesis. Recently, efforts for strain optimization have been standardized within the iterative concept of "design-build-test-learn" (DBTL). This approach has been successfully employed for the improvement of traditional cell factories like Escherichia coli and Saccharomyces cerevisiae. Within the past decade, several new-to-industry microorganisms have been investigated as novel cell factories, including the versatile α-proteobacterium Rhodobacter sphaeroides. Despite its history as a laboratory strain for fundamental studies, there is a growing interest in this bacterium for its ability to synthesize relevant compounds for the bioeconomy, such as isoprenoids, poly-ß-hydroxybutyrate, and hydrogen. In this study, we reflect on the reasons for establishing R. sphaeroides as a cell factory from the perspective of the DBTL concept. Moreover, we discuss current and future opportunities for extending the use of this microorganism for the bio-based economy. We believe that applying the DBTL pipeline for R. sphaeroides will further strengthen its relevance as a microbial cell factory. Moreover, the proposed use of strain engineering via the DBTL approach may be extended to other microorganisms that have not been critically investigated yet for industrial applications.

Characterization of a Rhodobacter sphaeroides primary fatty acid kinase.

Widely distributed among prokaryotes, short chain fatty acid kinases provide a path for fatty acid entry into central metabolic pathways. These enzymes catalyze the reversible, ATP-dependent synthesis of acyl-phosphates, which leads to the production of acyl-CoA derivatives by a coordinate acyltransferase. To date, characterized representatives of short chain fatty acid kinases exhibit relatively narrow substrate specificity. In this work, biochemical characterization of a predicted acetate kinase from Rhodobacter sphaeroides reveals a novel enzyme with broad substrate specificity for primary fatty acids of varying lengths (C2--C8).

Effects of low-molecular-weight polyols on the hydration status of the light-harvesting complex 2 from Rhodobacter sphaeroides 2.4.1.

Low-molecular-weight (MW) polyols are organic osmolytes influencing water activity. We have investigated the effects of polyol molecules (glycerol and sorbitol) on the optical and triplet excitation dynamics of light-harvesting complex 2 (LH2) from Rhodobacter (Rba.) sphaeroides in buffer-detergent solutions. The resonance Raman spectroscopy demonstrated that, on increasing glycerol and sorbitol volume fractions ranging from 0 to 80% (v/v) (accompanied by the decreasing water activities), the planar and all-trans conformation of carotenoids (Crts) remained unchanged, and the bacteriochlorophyll a (BChl) Qy absorption intensity decreased. The B850 fluorescence amplitude elevated in the 20-80% v/v sorbitol and 20-40% v/v glycerol solution, but decreased in 80% v/v glycerol solution. The change of 3[Crt*-BChl] interaction bands caused by 3Crt*-BChl interaction had no obvious correlation with water activities against polyol volume fractions, which are rationalized by the water activity sensitive of C- and N-termini of protein which binding with BChls. The results suggest that Rba. sphaeroides LH2 is more sensitive to low-molecular-weight polyols compared with that of the thermophiles purple bacterium Thermochromatium (Tch.) tepidum we had investigated before.

Effect of Detergents and Osmolytes on Thermal Stability of Native and Mutant Rhodobacter sphaeroides Reaction Centers.

Photosynthetic reaction center (RC) of the purple bacterium Rhodobacter sphaeroides is one of the most well-studied transmembrane pigment-protein complexes. It is a relatively stable protein with established conditions for its isolation from membranes, purification, and storage. However, it has been shown that some amino acid substitutions can affect stability of the RC, which results in a decrease of the RCs yield during its isolation and purification, disturbs spectral properties of the RCs during storage, and can lead to sample heterogeneity. To optimize conditions for studying mutant RCs, the effect of various detergents and osmolytes on thermal stability of the complex was examined. It was shown that trehalose and, to a lesser extent, sucrose, maltose, and hydroxyectoin at 1 M concentration slow down thermal denaturation of RCs. Sodium cholate was found to have significant stabilizing effect on the structure of native and genetically modified RCs. The use of sodium cholate as a detergent has several advantages and can be recommended for the storage and investigation of the unstable mutant membrane complexes of purple bacteria in long-term experiments.