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Sample records for a-proteobacterium rhodobacter sphaeroides

  1. The architecture of Rhodobacter sphaeroides chromatophores.

    PubMed

    Scheuring, Simon; Nevo, Reinat; Liu, Lu-Ning; Mangenot, Stéphanie; Charuvi, Dana; Boudier, Thomas; Prima, Valerie; Hubert, Pierre; Sturgis, James N; Reich, Ziv

    2014-08-01

    The chromatophores of Rhodobacter (Rb.) sphaeroides represent a minimal bio-energetic system, which efficiently converts light energy into usable chemical energy. Despite extensive studies, several issues pertaining to the morphology and molecular architecture of this elemental energy conversion system remain controversial or unknown. To tackle these issues, we combined electron microscope tomography, immuno-electron microscopy and atomic force microscopy. We found that the intracellular Rb. sphaeroides chromatophores form a continuous reticulum rather than existing as discrete vesicles. We also found that the cytochrome bc1 complex localizes to fragile chromatophore regions, which most likely constitute the tubular structures that interconnect the vesicles in the reticulum. In contrast, the peripheral light-harvesting complex 2 (LH2) is preferentially hexagonally packed within the convex vesicular regions of the membrane network. Based on these observations, we propose that the bc1 complexes are in the inter-vesicular regions and surrounded by reaction center (RC) core complexes, which in turn are bounded by arrays of peripheral antenna complexes. This arrangement affords rapid cycling of electrons between the core and bc1 complexes while maintaining efficient excitation energy transfer from LH2 domains to the RCs.

  2. Bioremediation of lead contaminated soil with Rhodobacter sphaeroides.

    PubMed

    Li, Xiaomin; Peng, Weihua; Jia, Yingying; Lu, Lin; Fan, Wenhong

    2016-08-01

    Bioremediation with microorganisms is a promising technique for heavy metal contaminated soil. Rhodobacter sphaeroides was previously isolated from oil field injection water and used for bioremediation of lead (Pb) contaminated soil in the present study. Based on the investigation of the optimum culturing conditions and the tolerance to Pb, we employed the microorganism for the remediation of Pb contaminated soil simulated at different contamination levels. It was found that the optimum temperature, pH, and inoculum size for R. sphaeroides is 30-35 °C, 7, and 2 × 10(8) mL(-1), respectively. Rhodobacter sphaeroides did not remove the Pb from soil but did change its speciation. During the bioremediation process, more available fractions were transformed to less accessible and inert fractions; in particular, the exchangeable phase was dramatically decreased while the residual phase was substantially increased. A wheat seedling growing experiment showed that Pb phytoavailability was reduced in amended soils. Results inferred that the main mechanism by which R. sphaeroides treats Pb contaminated soil is the precipitation formation of inert compounds, including lead sulfate and lead sulfide. Although the Pb bioremediation efficiency on wheat was not very high (14.78% root and 24.01% in leaf), R. sphaeroides remains a promising alternative for Pb remediation in contaminated soil. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Electron transport-dependent taxis in Rhodobacter sphaeroides.

    PubMed

    Gauden, D E; Armitage, J P

    1995-10-01

    Rhodobacter sphaeroides showed chemotaxis to the terminal electron acceptors oxygen and dimethyl sulfoxide, and the responses to these effectors were shown to be influenced by the relative activities of the different electron transport pathways. R. sphaeroides cells tethered by their flagella showed a step-down response to a decrease in the oxygen or dimethyl sulfoxide concentration when using them as terminal acceptors. Bacteria using photosynthetic electron transport, however, showed a step-down response to oxygen addition. Addition of the proton ionophore carbonyl cyanide 4-trifluoromethoxyphenylhydrazone did not cause a transient behavioral response, although it decreased the electrochemical proton gradient (delta p) and increased the rate of electron transport. However, removal of the ionophore, which caused an increase in delta p and a decrease in the electron transport rate, resulted in a step-down response. Together, these data suggest that behavioral responses of R. sphaeroides to electron transport effectors are caused by changes in the rate of electron transport rather than changes in delta p.

  4. Hydrogen production by Rhodobacter sphaeroides DSM 158 under intense irradiation.

    PubMed

    Krujatz, Felix; Härtel, Paul; Helbig, Karsten; Haufe, Nora; Thierfelder, Simone; Bley, Thomas; Weber, Jost

    2015-01-01

    To identify optimal hydrogen production conditions using growing cultures of Rhodobacter sphaeroides DSM 158 the effects of varying the reactor's volumetric power input (0.01-1.4kWm(-3)) and irradiation intensity (5-2500Wm(-2)) were investigated in batch and continuous production modes. Irradiation intensity had a greater effect on hydrogen production than volumetric power input. Hydrogen production and photofermentative biomass formation were maximized by irradiation at 2250Wm(-2) with a volumetric power input of 0.55kWm(-3). The bacterial dry weight (2.64gL(-1)) and rate of hydrogen production (195mLL(-1)h(-1)) achieved under these conditions were greater than any that have previously been reported for batch-mode hydrogen production by R. sphaeroides. Continuous mode experiments (D=0.1h(-1)) yielded a bacterial dry weight, hydrogen production rate, productivity and hydrogen yield of 2.35±0.18gL(-1), 165±6.2mLL(-1)h(-1), 3.96LL(-1)d(-1) and 36.6%, respectively.

  5. Global insights into energetic and metabolic networks in Rhodobacter sphaeroides

    PubMed Central

    2013-01-01

    Background Improving our understanding of processes at the core of cellular lifestyles can be aided by combining information from genetic analyses, high-throughput experiments and computational predictions. Results We combined data and predictions derived from phenotypic, physiological, genetic and computational analyses to dissect the metabolic and energetic networks of the facultative photosynthetic bacterium Rhodobacter sphaeroides. We focused our analysis on pathways crucial to the production and recycling of pyridine nucleotides during aerobic respiratory and anaerobic photosynthetic growth in the presence of an organic electron donor. In particular, we assessed the requirement for NADH/NADPH transhydrogenase enzyme, PntAB during respiratory and photosynthetic growth. Using high-throughput phenotype microarrays (PMs), we found that PntAB is essential for photosynthetic growth in the presence of many organic electron donors, particularly those predicted to require its activity to produce NADPH. Utilizing the genome-scale metabolic model iRsp1095, we predicted alternative routes of NADPH synthesis and used gene expression analyses to show that transcripts from a subset of the corresponding genes were conditionally increased in a ΔpntAB mutant. We then used a combination of metabolic flux predictions and mutational analysis to identify flux redistribution patterns utilized in the ΔpntAB mutant to compensate for the loss of this enzyme. Data generated from metabolic and phenotypic analyses of wild type and mutant cells were used to develop iRsp1140, an expanded genome-scale metabolic reconstruction for R. sphaeroides with improved ability to analyze and predict pathways associated with photosynthesis and other metabolic processes. Conclusions These analyses increased our understanding of key aspects of the photosynthetic lifestyle, highlighting the added importance of NADPH production under these conditions. It also led to a significant improvement in the

  6. Structural Characterization of the Fla2 Flagellum of Rhodobacter sphaeroides

    PubMed Central

    de la Mora, Javier; Uchida, Kaoru; del Campo, Ana Martínez; Camarena, Laura; Aizawa, Shin-Ichi

    2015-01-01

    ABSTRACT Rhodobacter sphaeroides is a free-living alphaproteobacterium that contains two clusters of functional flagellar genes in its genome: one acquired by horizontal gene transfer (fla1) and one that is endogenous (fla2). We have shown that the Fla2 system is normally quiescent and under certain conditions produces polar flagella, while the Fla1 system is always active and produces a single flagellum at a nonpolar position. In this work we purified and characterized the structure and analyzed the composition of the Fla2 flagellum. The number of polar filaments per cell is 4.6 on average. By comparison with the Fla1 flagellum, the prominent features of the ultra structure of the Fla2 HBB are the absence of an H ring, thick and long hooks, and a smoother zone at the hook-filament junction. The Fla2 helical filaments have a pitch of 2.64 μm and a diameter of 1.4 μm, which are smaller than those of the Fla1 filaments. Fla2 filaments undergo polymorphic transitions in vitro and showed two polymorphs: curly (right-handed) and coiled. However, in vivo in free-swimming cells, we observed only a bundle of filaments, which should probably be left-handed. Together, our results indicate that Fla2 cell produces multiple right-handed polar flagella, which are not conventional but exceptional. IMPORTANCE R. sphaeroides possesses two functional sets of flagellar genes. The fla1 genes are normally expressed in the laboratory and were acquired by horizontal transfer. The fla2 genes are endogenous and are expressed in a Fla1− mutant grown phototrophically and in the absence of organic acids. The Fla1 system produces a single lateral or subpolar flagellum, and the Fla2 system produces multiple polar flagella. The two kinds of flagella are never expressed simultaneously, and both are used for swimming in liquid media. The two sets of genes are certainly ready for responding to specific environmental conditions. The characterization of the Fla2 system will help us to understand

  7. The two photocycles of photoactive yellow protein from Rhodobacter sphaeroides.

    PubMed

    Haker, Andrea; Hendriks, Johnny; van Stokkum, Ivo H M; Heberle, Joachim; Hellingwerf, Klaas J; Crielaard, Wim; Gensch, Thomas

    2003-03-07

    The absorption spectrum of the photoactive yellow protein from Rhodobacter sphaeroides (R-PYP) shows two maxima, absorbing at 360 nm (R-PYP(360)) and 446 nm (R-PYP(446)), respectively. Both forms are photoactive and part of a temperature- and pH-dependent equilibrium (Haker, A., Hendriks, J., Gensch, T., Hellingwerf, K. J., and Crielaard, W. (2000) FEBS Lett. 486, 52-56). At 20 degrees C, for PYP characteristic, the 446-nm absorbance band displays a photocycle, in which the depletion of the 446-nm ground state absorption occurs in at least three phases, with time constants of <30 ns, 0.5 micros, and 17 micros. Intermediates with both blue- and red-shifted absorption maxima are transiently formed, before a blue-shifted intermediate (pB(360), lambda(max) = 360 nm) is established. The photocycle is completed with a monophasic recovery of the ground state with a time constant of 2.5 ms. At 7 degrees C these photocycle transitions are slowed down 2- to 3-fold. Upon excitation of R-PYP(360) with a UV-flash (330 +/- 50 nm) a species with a difference absorption maximum at approximately 435 nm is observed that returns to R-PYP(360) on a minute time scale. Recovery can be accelerated by a blue light flash (450 nm). R-PYP(360) and R-PYP(446) differ in their overall protein conformation, as well as in the isomerization and protonation state of the chromophore, as determined with the fluorescent polarity probe Nile Red and Fourier Transform Infrared spectroscopy, respectively.

  8. Hydrogen Production by Co-cultures of Rhizopus oryzae and a Photosynthetic Bacterium, Rhodobacter sphaeroides RV

    NASA Astrophysics Data System (ADS)

    Asada, Yasuo; Ishimi, Katsuhiro; Nagata, Yoko; Wakayama, Tatsuki; Miyake, Jun; Kohno, Hideki

    Hydrogen production with glucose by using co-immobilized cultures of a fungus, Rhizopus oryzae NBRC5384, and a photosynthetic bacterium, Rhodobacter sphaeroides RV, in agar gels was studied. The co-immobilized cultures converted glucose to hydrogen via lactate in a high molar yield of about 8moles of hydrogen per glucose at a maximum under illuminated conditions.

  9. Three separate proteins constitute the magnesium chelatase of Rhodobacter sphaeroides.

    PubMed

    Willows, R D; Gibson, L C; Kanangara, C G; Hunter, C N; von Wettstein, D

    1996-01-15

    The insertion of magnesium into protoporphyrin IX is the first step unique to chlorophyll production and is catalyzed by magnesium chelatase. The Rhodobacter sphaeroides genes, bchI and bchD together, and bchH alone, were cloned and expressed with the pET3a vector in Escherichia coli strain BL21 (DE3). The 40-kDa BchI protein was synthesized in greater abundance compared to the 70-kDa BchD protein when both were expressed together from the same plasmid. The production of large amounts of the 140-kDa BchH protein in E. coli was accompanied by an accumulation of protoporphyrin IX. The accumulated protoporphyrin IX was bound specifically to BchH in an approximate molar ratio of 1:1. All three recombinant proteins were soluble; BchH was monomeric, Bchl was dimeric, while BchD appeared to be polymeric with a molecular mass of approximately 550 kDa. The BchH and BchI proteins were purified to apparent homogeneity while BchD was separated from BchI and partially purified. Magnesium was inserted into protoporphyrin IX and deuteroporphyrin by combining these three proteins in the presence of ATP. One monomer of BchH to one dimer of BchI gave the optimal magnesium chelatase activity and the activity was dependent on the amount of partially purified BchD added to the assay at the optimum BchH:BchI ratio. The reaction was dissected into two parts with an activation step requiring BchI, BchD, and Mg2+-ATP, and a metal-insertion step which in addition requires Mg2+, protoporphyrin IX, and BchH. The stoichiometric binding of protoporphyrin IX to BchH in vitro is direct evidence for BchH carrying out such a role in vivo whereas the other two proteins are involved in ATP activation and magnesium insertion.

  10. The Rhodobacter sphaeroides flagellar motor is a variable-speed rotor.

    PubMed

    Packer, H L; Lawther, H; Armitage, J P

    1997-06-02

    The rotation rate of the unidirectional stop/start motor of Rhodobacter sphaeroides was investigated using computerised motion analysis of tethered cells. The R. sphaeroides motor was found to have a variable rotation rate compared to the virtually constant-speed motor of wild-type and CheR mutant (smooth swimming) Escherichia coli. In addition, the dynamics of the R. sphaeroides motor during stopping was analysed with no consistent correlation behaviour. The motor could go from full rotation to stop, or stop to full rotation within one video frame, i.e. 0.02 s, but it could also slow down into a stop or restart slowly, taking up to 0.25 s. The R. sphaeroides motor under chemokinetic stimulation was also analysed and was found to show increased torque generation and reduced variation in rotation rate.

  11. Removal of cadmium and zinc from contaminated wastewater using Rhodobacter sphaeroides.

    PubMed

    Li, Xiaomin; Peng, Weihua; Jia, Yingying; Lu, Lin; Fan, Wenhong

    2017-06-01

    Rhodobacter sphaeroides was used for bioremediation of wastewater polluted with cadmium (Cd) and zinc (Zn). The tolerance of the microorganism to selected heavy metals (HMs), as well as the effects of pH, temperature and inoculum size on the removal rate, was investigated. The remediation effects of R. sphaeroides were analysed at different initial concentrations of HMs. Bioremediation mechanisms were thoroughly discussed based on the results from the cell characterisation analysis. Cd and Zn could inhibit the growth of R. sphaeroides. However, Cd was more toxic than Zn, with corresponding EC50 values of 5.34 and 69.79 mg L(-1). Temperature and pH had greater influence on the removal rate of HMs than inoculum size. The optimal conditions for temperature and pH were 35 °C-40 °C and pH 7, respectively. Initial concentration of HMs and remediation time also affected the removal rate. Rhodobacter sphaeroides had a relatively higher remediation effect under the present experimental conditions. The removal rates for Cd and Zn reached 97.92% and 97.76%, respectively. Results showed that biosorption and HM precipitation were the main bioremediation mechanisms. This information is necessary to better understand the removal mechanism of R. sphaeroides, and is significant for its pilot test and future practical application.

  12. Ultrafast exciton relaxation in the B850 antenna complex of Rhodobacter sphaeroides.

    PubMed

    Nagarajan, V; Alden, R G; Williams, J C; Parson, W W

    1996-11-26

    Spectral changes were measured with femtosecond resolution following low-intensity, broad-band excitation of the peripheral antenna complex of the purple photosynthetic bacterium Rhodobacter sphaeroides. Absorption anisotropy decays also were measured. We identified a 35-fs relaxation of the absorption and emission spectra of the excited state, as well as a 20-fs anisotropy decay. We interpret these results as interlevel relaxation and dephasing, respectively, of extensively delocalized exciton states of the circular bacteriochlorophyll aggregate.

  13. Gene co-expression network analysis in Rhodobacter capsulatus and application to comparative expression analysis of Rhodobacter sphaeroides

    SciTech Connect

    Pena-Castillo, Lourdes; Mercer, Ryan; Gurinovich, Anastasia; Callister, Stephen J.; Wright, Aaron T.; Westbye, Alexander; Beatty, J. T.; Lang, Andrew S.

    2014-08-28

    The genus Rhodobacter contains purple nonsulfur bacteria found mostly in freshwater environments. Representative strains of two Rhodobacter species, R. capsulatus and R. sphaeroides, have had their genomes fully sequenced and both have been the subject of transcriptional profiling studies. Gene co-expression networks can be used to identify modules of genes with similar expression profiles. Functional analysis of gene modules can then associate co-expressed genes with biological pathways, and network statistics can determine the degree of module preservation in related networks. In this paper, we constructed an R. capsulatus gene co-expression network, performed functional analysis of identified gene modules, and investigated preservation of these modules in R. capsulatus proteomics data and in R. sphaeroides transcriptomics data. Results: The analysis identified 40 gene co-expression modules in R. capsulatus. Investigation of the module gene contents and expression profiles revealed patterns that were validated based on previous studies supporting the biological relevance of these modules. We identified two R. capsulatus gene modules preserved in the protein abundance data. We also identified several gene modules preserved between both Rhodobacter species, which indicate that these cellular processes are conserved between the species and are candidates for functional information transfer between species. Many gene modules were non-preserved, providing insight into processes that differentiate the two species. In addition, using Local Network Similarity (LNS), a recently proposed metric for expression divergence, we assessed the expression conservation of between-species pairs of orthologs, and within-species gene-protein expression profiles. Conclusions: Our analyses provide new sources of information for functional annotation in R. capsulatus because uncharacterized genes in modules are now connected with groups of genes that constitute a joint functional

  14. Biohydrogen Production from Tofu Wastewater with Glutamine Auxotrophic Mutant of Rhodobacter sphaeroides

    NASA Astrophysics Data System (ADS)

    Zheng, G. H.; Kang, Z. H.; Qian, Y. F.; Wang, L.; Zhou, Q.; Zhu, H. G.

    2008-02-01

    Hydrogen production from organic wastewater by photo-bacteria has been attracted more attention, not only because hydrogen is a clean energy, but also because it can be a process for organic wastewater pre-treatment. However NH4+, which normally is the integrant in organic wastewater, is the inhibitor to hydrogen production with photo-bacteria. In this study, the NH4+ effect on biohydrogen generation and nitrogenase activity of anoxygenic phototrophic bacteria-Rhodobacter sphaeroides was studied. Biohydrogen generation with wild-type R. sphaeroides was found to be more sensitive to NH4+ due to the obvious inhibition of NH4+ to its nitrogenase. For avoiding inhibition of NH4+ to biohydrogen generation of R. sphaeroides, a glutamine auxotrophic mutant R. sphaeroides AR-3 was obtained by EMS treatment. The mutant could generate biohydrogen efficiently in the medium with higher NH4+ concentration. Under suitable conditions, AR-3 produced biohydrogen from tofu wastewater with an average generation rate of 14.2 ml L-1h-1, it was increased by more than 100% compared with that from wild-type R. sphaeroides.

  15. Inhibited growth of Clostridium butyricum in efficient H2-producing co-culture with Rhodobacter sphaeroides.

    PubMed

    Laurinavichene, Tatyana; Laurinavichius, Kestutis; Shastik, Evgeny; Tsygankov, Anatoly

    2016-12-01

    Cell number of Clostridium butyricum and Rhodobacter sphaeroides in co-culture was measured using q-PCR approach. During efficient H2 photoproduction from starch (6.2 mol H2/mol glucose), Clostridia growth and starch-hydrolyzing activity was partly suppressed. Apparently, the effect of R. sphaeroides towards C. butyricum was not attributed to altered Eh or pH values in the presence of purple bacteria. Further, disk-diffusion test proved that R. sphaeroides was capable of producing inhibitors against another purple bacterium, Rhodospirillum rubrum, but not against C. butyricum. We suggested that at initial cell number ratio C. butyricum:R. sphaeroides 1:1 purple bacteria outcompeted C. butyricum for yeast extract at its low concentration (80 mg/L). Under these conditions, the H2 yield was rather high (5.7 mol/mol). When the yeast extract concentration increased to 320 mg/L, this process was replaced by the low-yield H2 production (1.8 mol/mol) characteristic of Clostridia. However, increased percentage of purple bacteria in inoculum under these conditions prevented this shift. The outcome of competition depended on both the yeast extract concentration and cell number ratio. Apparently, the competition for yeast extract helped to maintain balance between fast-growing C. butyricum and slower-growing R. sphaeroides for efficient H2 photoproduction.

  16. A global signal transduction system regulates aerobic and anaerobic CO2 fixation in Rhodobacter sphaeroides.

    PubMed Central

    Qian, Y; Tabita, F R

    1996-01-01

    Complementation of a mutant of Rhodobacter sphaeroides defective in photosynthetic CO2 reduction led to the identification of a gene which encodes a protein that is related to a class of sensor kinases involved in bacterial signal transduction. The nucleotide sequence and deduced amino acid sequence led to the finding that the gene which complemented the mutant is the regB (prrB) gene, previously isolated from both R. sphaeroides and Rhodobacter capsulatus and shown to regulate the anaerobic expression of structural genes required for the synthesis of the reaction center and light-harvesting systems of these organisms. The current investigation indicates that in addition to its role in the regulation of photosystem biosynthesis, regB (prrB) of R. sphaeroides is intimately involved in the positive regulation of the cbbI and cbbII Calvin cycle CO2 fixation operons. In addition to regulating the expression of structural genes encoding enzymes of the primary pathway for CO2 fixation in R. sphaeroides, regB was also found to be required for the expression of a gene(s) important for the putative alternative CO2 fixation pathway(s) of this organism. A mutation in regB also blocked expression of structural genes of the cbb regulon in a strain of R. sphaeroides capable of aerobic CO2-dependent growth in the dark. It is thus apparent that regB is part of a two-component system and encodes a sensor kinase involved in the global regulation of both anoxygenic light-dependent- and oxygenic light-independent CO2 fixation as well as anoxygenic photosystem biosynthesis. PMID:8550404

  17. iRsp1095: A genome-scale reconstruction of the Rhodobacter sphaeroides metabolic network

    PubMed Central

    2011-01-01

    Background Rhodobacter sphaeroides is one of the best studied purple non-sulfur photosynthetic bacteria and serves as an excellent model for the study of photosynthesis and the metabolic capabilities of this and related facultative organisms. The ability of R. sphaeroides to produce hydrogen (H2), polyhydroxybutyrate (PHB) or other hydrocarbons, as well as its ability to utilize atmospheric carbon dioxide (CO2) as a carbon source under defined conditions, make it an excellent candidate for use in a wide variety of biotechnological applications. A genome-level understanding of its metabolic capabilities should help realize this biotechnological potential. Results Here we present a genome-scale metabolic network model for R. sphaeroides strain 2.4.1, designated iRsp1095, consisting of 1,095 genes, 796 metabolites and 1158 reactions, including R. sphaeroides-specific biomass reactions developed in this study. Constraint-based analysis showed that iRsp1095 agreed well with experimental observations when modeling growth under respiratory and phototrophic conditions. Genes essential for phototrophic growth were predicted by single gene deletion analysis. During pathway-level analyses of R. sphaeroides metabolism, an alternative route for CO2 assimilation was identified. Evaluation of photoheterotrophic H2 production using iRsp1095 indicated that maximal yield would be obtained from growing cells, with this predicted maximum ~50% higher than that observed experimentally from wild type cells. Competing pathways that might prevent the achievement of this theoretical maximum were identified to guide future genetic studies. Conclusions iRsp1095 provides a robust framework for future metabolic engineering efforts to optimize the solar- and nutrient-powered production of biofuels and other valuable products by R. sphaeroides and closely related organisms. PMID:21777427

  18. [High-order derivative spectroscopy of infrared absorption spectra of the reaction centers from Rhodobacter sphaeroides].

    PubMed

    2005-01-01

    The infrared absorption spectra of reduced and chemically oxidized reaction center preparations from the purple bacterium Rhodobacter sphaeroides were investigated by means of high-order derivative spectroscopy. The model Gaussian band with a maximum at 810 nm and a half-band of 15 nm found in the absorption spectrum of the reduced reaction center preparation is eliminated after the oxidation of photoactive bacteriochlorophyll dimer (P). This band was related to the absorption of the P(+)y excitonic band of P. On the basis of experimental results, it was concluded that the bleaching of the P(+)y absorption band at 810 nm in the oxidized reaction center preparations gives the main contribution to the blue shift of the 800 nm absorption band of Rb. sphaeroides reaction centers.

  19. Modifying the endogenous electron fluxes of Rhodobacter sphaeroides 2.4.1 for improved electricity generation.

    PubMed

    Wong, Man Tung; Cheng, Danhui; Wang, Ri; Hsing, I-Ming

    2016-05-01

    The purple bacteria Rhodobacter sphaeroides serve as a promising biocatalyst in the photo-microbial fuel cell system (photo-MFC). This gram-negative species performs highly efficient anoxygenic photosynthesis that ensures an anaerobic environment in the anode compartment. Previous studies incorporating R. sphaeroides into photo-MFC were conducted using platinum as the anode electrode. In this study, we detected a steady current generation of R. sphaeroides in a bioelectrochemical system where glassy carbon was the working electrode and a typical growth medium was the electrolyte. The bioelectricity generation synchronized with the supplementation of reduced carbon source and showed immediate response to illumination, which strongly indicated the correlation between the observed current and the cytoplasmic quinone activity. Modifications of the endogenous electron flows mediated by quinone pool are shown to have significantly enhanced the bioelectricity generation. We anticipate that the findings in this study would advance future optimization of R. sphaeroides as an anode strain, as well as facilitate the study of bioenergetics in photosynthetic bacteria.

  20. Interaction of Product Analogues With the Active Site of Rhodobacter Sphaeroides Dimethyl Sulfoxide Reductase

    SciTech Connect

    George, G.N.; Nelson, K.J.; Harris, H.H.; Doonan, C.J.; Rajagopalan, K.V.; /Saskatchewan U. /Duke U. /Sydney U.

    2007-07-09

    We report a structural characterization using X-ray absorption spectroscopy of Rhodobacter sphaeroides dimethylsulfoxide (DMSO) reductase reduced with trimethylarsine, and show that this is structurally analogous to the physiologically relevant dimethylsulfide-reduced DMSO reductase. Our data unambiguously indicate that these species should be regarded as formal MoIV species, and indicate a classical coordination complex of trimethylarsine oxide, with no special structural distortions. The similarity of the trimethylarsine and dimethylsulfide complexes suggests in turn that the dimethylsulfide reduced enzyme possesses a classical coordination of DMSO with no special elongation of the S-O bond, as previously suggested.

  1. Absorbance changes accompanying the fast fluorescence induction in the purple bacterium Rhodobacter sphaeroides.

    PubMed

    Bína, David; Litvín, Radek; Vácha, Frantisek

    2010-08-01

    The authors present a study of the fluorescence and absorbance transients occurring in whole cells of purple nonsulfur bacterium Rhodobacter sphaeroides on the millisecond timescale under pulsed actinic illumination. The fluorescence induction curve is interpreted in terms of combination of effects of redox changes in the reaction center and the membrane potential. The results of this study support the view that the membrane potential act predominantly to increase the fluorescence yield. Advantages of the pulsed actinic illumination for study of the operation of the electron transport chain in vivo are discussed.

  2. In Vivo Analysis of Cobinamide Salvaging in Rhodobacter sphaeroides Strain 2.4.1 ▿ †

    PubMed Central

    Gray, Michael J.; Escalante-Semerena, Jorge C.

    2009-01-01

    The genome of Rhodobacter sphaeroides encodes the components of two distinct pathways for salvaging cobinamide (Cbi), a precursor of adenosylcobalamin (AdoCbl, coenzyme B12). One pathway, conserved among bacteria, depends on a bifunctional kinase/guanylyltransferase (CobP) enzyme to convert adenosylcobinamide (AdoCbi) to AdoCbi-phosphate (AdoCbi-P), an intermediate in de novo AdoCbl biosynthesis. The other pathway, of archaeal origin, depends on an AdoCbi amidohydrolase (CbiZ) enzyme to generate adenosylcobyric acid (AdoCby), which is converted to AdoCbi-P by the AdoCbi-P synthetase (CobD) enzyme. Here we report that R. sphaeroides strain 2.4.1 synthesizes AdoCbl de novo and that it salvages Cbi using both of the predicted Cbi salvaging pathways. AdoCbl produced by R. sphaeroides was identified and quantified by high-performance liquid chromatography and bioassay. The deletion of cobB (encoding an essential enzyme of the de novo corrin ring biosynthetic pathway) resulted in a strain of R. sphaeroides that would not grow on acetate in the absence of exogenous corrinoids. The results from a nutritional analysis showed that the presence of either CbiZ or CobP was necessary and sufficient for Cbi salvaging, that CbiZ-dependent Cbi salvaging depended on the presence of CobD, and that CobP-dependent Cbi salvaging occurred in a cbiZ+ strain. Possible reasons why R. sphaeroides maintains two distinct pathways for Cbi salvaging are discussed. PMID:19376876

  3. Rhodobacter sphaeroides LexA has dual activity: optimising and repressing recA gene transcription

    PubMed Central

    Tapias, Angels; Fernández, Silvia; Alonso, Juan C.; Barbé, Jordi

    2002-01-01

    Transcription of the Rhodobacter sphaeroides recA promoter (PrecA) is induced upon DNA damage in a lexA-dependent manner. In vivo experiments demonstrate that LexA protein represses and might also activate transcription of PrecA. Purified R.sphaeroides LexA protein specifically binds the SOS boxes located within the PrecA region. In vitro transcription analysis, using Escherichia coli RNA polymerase (RNAP), indicated that the presence of LexA may stimulate and repress transcription of PrecA. EMSA and DNase I footprinting experiments show that LexA and RNAP can bind simultaneously to PrecA. At low LexA concentrations it enhances RNAP binding to PrecA, stimulates open complex formation and strand separation beyond the transcription start site. At high LexA concentrations, however, RNAP-promoted strand separation is not observed beyond the +5 region. LexA might repress transcription by interfering with the clearance process instead of blocking the access of RNAP to the promoter region. Based on these findings we propose that the R.sphaeroides LexA protein performs fine tuning of the SOS response, which might provide a physiological advantage by enhancing transcription of SOS genes and delaying full activation of the response. PMID:11917014

  4. Wheat straw degradation and production of alternative substrates for nitrogenase of Rhodobacter sphaeroides.

    PubMed

    Dziga, Dariusz; Jagiełło-Flasińska, Dominika

    2015-01-01

    Cellulose is a major component of plant biomass and could be applied in the production of biofuels, especially bioethanol. An alternative approach is production of a clean fuel - hydrogen from cellulosic biomass. In this paper an innovatory model of cellulosic waste degradation has been proposed to verify the possibility of utilization of cellulose derivatives by purple non-sulfur bacteria. The concept is based on a two-step process of wheat straw conversion by bacteria in order to obtain an organic acid mixture. In the next stage such products are consumed by Rhodobacter sphaeroides, the known producer of hydrogen. It has been documented that Cellulomonas uda expresses cellulolytic activity in the presence of wheat straw as an only source of carbon. R. sphaeroides applied in this research can effectively consume organic acids released from straw by C. uda and Lactobacillus rhamnosus and is able to grow in the presence of these substrates. Additionally, an increased nitrogenase activity of R. sphaeroides has been indicated when bacteria were cultivated in the presence of cellulose derivatives which suggests that hydrogen production occurs.

  5. Response kinetics of tethered Rhodobacter sphaeroides to changes in light intensity.

    PubMed

    Berry, R M; Armitage, J P

    2000-03-01

    Rhodobacter sphaeroides can swim toward a wide range of attractants (a process known as taxis), propelled by a single rotating flagellum. The reversals of motor direction that cause tumbles in Eschericia coli taxis are replaced by brief motor stops, and taxis is controlled by a complex sensory system with multiple homologues of the E. coli sensory proteins. We tethered photosynthetically grown cells of R. sphaeroides by their flagella and measured the response of the flagellar motor to changes in light intensity. The unstimulated bias (probability of not being stopped) was significantly larger than the bias of tethered E. coli but similar to the probability of not tumbling in swimming E. coli. Otherwise, the step and impulse responses were the same as those of tethered E. coli to chemical attractants. This indicates that the single motor and multiple sensory signaling pathways in R. sphaeroides generate the same swimming response as several motors and a single pathway in E. coli, and that the response of the single motor is directly observable in the swimming pattern. Photo-responses were larger in the presence of cyanide or the uncoupler carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP), consistent with the photo-response being detected via changes in the rate of electron transport.

  6. Rhodobacter sphaeroides adaptation to high concentrations of cobalt ions requires energetic metabolism changes.

    PubMed

    Volpicella, Mariateresa; Costanza, Alessandra; Palumbo, Orazio; Italiano, Francesca; Claudia, Leoni; Placido, Antonio; Picardi, Ernesto; Carella, Massimo; Trotta, Massimo; Ceci, Luigi R

    2014-05-01

    Rhodobacter sphaeroides has for a long time been investigated for its adaptive capacities to different environmental and nutritional conditions, including presence of heavy metals, which make it a valuable model organism for understanding bacterial adaptation to metal stress conditions and future environmental applications, such as bioremediation of polluted sites. To further characterize the capability of R. sphaeroides to cope with high cobalt ion concentrations, we combined the selection of adaptive defective mutants, carried out by negative selection of transposon insertional libraries on 5 mM Co(2+) -enriched solid medium, with the analysis of growing capacities and transcriptome profiling of a selected mutant (R95). A comparative analysis of results from the mutant and wild-type strains clearly indicated that the adaptive ability of R. sphaeroides strongly relies on its ability to exploit any available energy-supplying metabolisms, being able to behave as photo- or chemotrophic microorganism. The selected R95 mutant, indeed, exhibits a severe down-expression of an ABC sugar transporter, which results nonpermissive for its growth in cobalt-enriched media under aerobic conditions. Interestingly, the defective expression of the transporter does not have dramatic effects on the growth ability of the mutant when cultivated under photosynthetic conditions. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  7. Cloning and heterologous expression of chlorophyll a synthase in Rhodobacter sphaeroides.

    PubMed

    Ipekoğlu, Emre M; Göçmen, Koray; Öz, Mehmet T; Gürgan, Muazzez; Yücel, Meral

    2017-03-01

    Rhodobacter sphaeroides is a purple non-sulfur bacterium which photoheterotrophically produces hydrogen from organic acids under anaerobic conditions. A gene coding for putative chlorophyll a synthase (chlG) from cyanobacterium Prochlorococcus marinus was amplified by nested polymerase chain reaction and cloned into an inducible-expression plasmid which was subsequently transferred to R. sphaeroides for heterologous expression. Induced expression of chlG in R. sphaeroides led to changes in light absorption spectrum within 400-700 nm. The hydrogen production capacity of the mutant strain was evaluated on hydrogen production medium with 15 mM malate and 2 mM glutamate. Hydrogen yield and productivity were increased by 13.6 and 22.6%, respectively, compared to the wild type strain. The results demonstrated the feasibility of genetic engineering to combine chlorophyll and bacteriochlorophyll biosynthetic pathways which utilize common intermediates. Heterologous expression of key enzymes from biosynthetic pathways of various pigments is proposed here as a general strategy to improve absorption spectra and yield of photosynthesis and hydrogen gas production in bacteria. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Dimerization of core complexes as an efficient strategy for energy trapping in Rhodobacter sphaeroides.

    PubMed

    Chenchiliyan, Manoop; Timpmann, Kõu; Jalviste, Erko; Adams, Peter G; Hunter, C Neil; Freiberg, Arvi

    2016-06-01

    In the purple phototrophic bacterium Rhodobacter sphaeroides, light harvesting LH2 complexes transfer absorbed solar energy to RC-LH1-PufX core complexes, which are mainly found in the dimeric state. Many other purple phototrophs have monomeric core complexes and the basis for requiring dimeric cores is not fully established, so we analysed strains of Rba. sphaeroides that contain either native dimeric core complexes or altered monomeric cores harbouring a deletion of the first 12 residues from the N-terminus of PufX, which retains the PufX polypeptide but removes the major determinant of core complex dimerization. Membranes were purified from strains with dimeric or monomeric cores, and with either high or low levels of the LH2 complex. Samples were interrogated with absorption, steady-state fluorescence, and picosecond time-resolved fluorescence kinetic spectroscopies to reveal their light-harvesting and energy trapping properties. We find that under saturating excitation light intensity the photosynthetic membranes containing LH2 and monomeric core complexes have fluorescence lifetimes nearly twice that of membranes with LH2 plus dimeric core complexes. This trend of increased lifetime is maintained with RCs in the open state as well, and for two different levels of LH2 content. Thus, energy trapping is more efficient when photosynthetic membranes of Rba. sphaeroides consist of RC-LH1-PufX dimers and LH2 complexes. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Advancing Rhodobacter sphaeroides as a platform for expression of functional membrane proteins.

    PubMed

    Erbakan, Mustafa; Curtis, Brandon S; Nixon, B Tracy; Kumar, Manish; Curtis, Wayne R

    2015-11-01

    Membrane protein overexpression is often hindered by toxic effects on the expression host, limiting achievable volumetric productivity. Moreover, protein structure and function may be impaired due to inclusion body formation and proteolytic degradation. To address these challenges, we employed the photosynthetic bacterium, Rhodobacter sphaeroides for expression of challenging membrane proteins including human aquaporin 9 (hAQP9), human tight junction protein occludin (Occ), Escherichia coli toxin peptide GhoT, cellulose synthase enzyme complex (BcsAB) of R. sphaeroides and cytochrome-cy (Cyt-cy) from Rhodobacter capsulatus. Titers of 47 mg/L for Cyt-cy, 7.5 mg/L for Occ, 1.5 mg/L for BcsAB and 0.5 mg/L for hAQP9 were achieved from affinity purification. While purification of GhoT was not successful, transformants displayed a distinct growth phenotype that correlated with GhoT expression. We also evaluated the functionality of these proteins by performing water transport studies for hAQP9, peroxidase activity for cytochrome-cy, and in vitro cellulose synthesis activity assay for BcsAB. While previous studies with Rhodobacter have utilized oxygen-limited semi-aerobic growth for membrane protein expression, substantial titer improvements are achieved as a result of a 3-fold increase in biomass yield using the anaerobic photoheterotrophic growth regime, which utilizes the strong native puc promoter. This versatile platform is shown to enable recovery of a wide variety of difficult-to-express membrane proteins in functional form.

  10. Effect of changes in the composition of cellular fatty acids on membrane fluidity of Rhodobacter sphaeroides.

    PubMed

    Kim, Eui-Jin; Lee, Jeong K

    2015-02-01

    The cellular fatty acid composition is important for metabolic plasticity in Rhodobacter sphaeroides. We explored the effects of changing the cellular ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs) in R. sphaeroides by overexpressing several key fatty acid biosynthetic enzymes through the use of expression plasmid pRK415. Bacteria containing the plasmid pRKfabI1 with the fabI1 gene that encodes enoyl-acyl carrier protein (ACP) reductase showed a reduction in the cellular UFA to SFA ratio from 4 (80% UFA) to 2 (65% UFA) and had decreased membrane fluidity and reduced cell growth. Additionally, the ratio of UFA to SFA of the chromatophore vesicles from pRKfabI1 -containing cells was similarly lowered, and the cell had decreased levels of light-harvesting complexes, but no change in intracytoplasmic membrane (ICM) content or photosynthetic (PS) gene expression. Both inhibition of enoyl- ACP reductase with diazaborine and addition of exogenous UFA restored membrane fluidity, cell growth, and the UFA to SFA ratio to wild-type levels in this strain. R. sphaeroides containing the pRKfabB plasmid with the fabB gene that encodes the enzyme β-ketoacyl-ACP synthase I exhibited an increased UFA to SFA ratio from 4 (80% UFA) to 9 (90% UFA), but showed no change in membrane fluidity or growth rate relative to control cells. Thus, membrane fluidity in R. sphaeroides remains fairly unchanged when membrane UFA levels are between 80% and 90%, whereas membrane fluidity, cell growth, and cellular composition are affected when UFA levels are below 80%.

  11. Changes in morphology, cell wall composition and soluble proteome in Rhodobacter sphaeroides cells exposed to chromate.

    PubMed

    Italiano, Francesca; Rinalducci, Sara; Agostiano, Angela; Zolla, Lello; De Leo, Francesca; Ceci, Luigi R; Trotta, Massimo

    2012-10-01

    The response of the carotenoidless Rhodobacter sphaeroides mutant R26 to chromate stress under photosynthetic conditions is investigated by biochemical and spectroscopic measurements, proteomic analysis and cell imaging. Cell cultures were found able to reduce chromate within 3-4 days. Chromate induces marked changes in the cellular dimension and morphology, as revealed by atomic force microscopy, along with compositional changes in the cell wall revealed by infrared spectroscopy. These effects are accompanied by significant changes in the level of several proteins: 15 proteins were found up-regulated and 15 down-regulated. The protein content found in chromate exposed cells is in good agreement with the biochemical, spectroscopic and microscopic results. Moreover at the present stage no specific chromate-reductase could be found in the soluble proteome, indicating that detoxification of the pollutant proceeds via aspecific reductants.

  12. Crystallization and preliminary X-ray crystallographic analysis of phosphoribulokinase from Rhodobacter sphaeroides.

    PubMed Central

    Roberts, D. L.; Runquist, J. A.; Miziorko, H. M.; Kim, J. J.

    1995-01-01

    A recombinant form of Rhodobacter sphaeroides phosphoribulokinase (PRK), expressed in Escherichia coli and isolated by affinity chromatography, was crystallized by the sitting drop vapor diffusion technique using NH4H2PO4 (pH 5.6) as the precipitating agent. PRK crystallizes in the cubic space group P432, with unit cell parameters a = b = c = 129.55 A. Based on the assumption of one 32-kDa monomer per asymmetric unit, the Vm value is 2.83 A3/Da. The octameric molecular symmetry is consistent with two planar tetramers stacked in a nearly eclipsed arrangement. A native data set has been collected to 2.6 A resolution. PMID:8563645

  13. Phosphoribulokinase mediates nitrogenase-induced carbon dioxide fixation gene repression in Rhodobacter sphaeroides

    PubMed Central

    Farmer, Ryan M.

    2015-01-01

    In many organisms there is a balance between carbon and nitrogen metabolism. These observations extend to the nitrogen-fixing, nonsulfur purple bacteria, which have the classic family of P(II) regulators that coordinate signals of carbon and nitrogen status to regulate nitrogen metabolism. Curiously, these organisms also possess a reverse mechanism to regulate carbon metabolism based on cellular nitrogen status. In this work, studies in Rhodobacter sphaeroides firmly established that the activity of the enzyme that catalyses nitrogen fixation, nitrogenase, induces a signal that leads to repression of genes encoding enzymes of the Calvin–Benson–Bassham (CBB) CO2 fixation pathway. Additionally, genetic and metabolomic experiments revealed that NADH-activated phosphoribulokinase is an intermediate in the signalling pathway. Thus, nitrogenase activity appears to be linked to cbb gene repression through phosphoribulokinase. PMID:26306848

  14. [On the electron stabilization within the quinone acceptor part of Rhodobacter sphaeroides photosynthetic reaction centers].

    PubMed

    Noks, P P; Krasil'nikov, P M; Mamonov, P A; Seĭfullina, N Kh; Uchoa, A F; Baptista, M S

    2008-01-01

    The time evolution of the photoinduced differential absorption spectrum of isolated Rhodobacter sphaeroides photosynthetic reaction centers was investigated. The measurements were carried out in the spectral region of 400-500 nm on the time scale of up to 200 microseconds. The spectral changes observed can be interpreted in terms of the effects of proton shift along hydrogen bonds between the primary quinone acceptor and the protein. A theoretical analysis of the spectrum time evolution was performed, which is based on the consideration of the kinetics of proton tunneling along the hydrogen bond. It was shown that the stabilization of the primary quinone electronic state occurs within the first several tens of microseconds after quinone reduction. It slows down upon the deuteration of reaction centers as well as after adding 90% of glycerol; on the other hand, it accelerates as temperature rises up to 40 degrees C.

  15. Time-resolved tryptophan fluorescence in photosynthetic reaction centers from Rhodobacter sphaeroides

    NASA Technical Reports Server (NTRS)

    Godik, V. I.; Blankenship, R. E.; Causgrove, T. P.; Woodbury, N.

    1993-01-01

    Tryptophan fluorescence of reaction centers isolated from Rhodobacter sphaeroides, both stationary and time-resolved, was studied. Fluorescence kinetics were found to fit best a sum of four discrete exponential components. Half of the initial amplitude was due to a component with a lifetime of congruent to 60 ps, belonging to Trp residues, capable of efficient transfer of excitation energy to bacteriochlorophyll molecules of the reaction center. The three other components seem to be emitted by Trp ground-state conformers, unable to participate in such a transfer. Under the influence of intense actinic light, photooxidizing the reaction centers, the yield of stationary fluorescence diminished by congruent to 1.5 times, while the number of the kinetic components and their life times remained practically unchanged. Possible implications of the observed effects for the primary photosynthesis events are considered.

  16. Femtosecond infrared spectroscopy of reaction centers from Rhodobacter sphaeroides between 1000 and 1800 cm-1.

    PubMed Central

    Hamm, P; Zurek, M; Mäntele, W; Meyer, M; Scheer, H; Zinth, W

    1995-01-01

    Time-resolved pump-and-probe experiments of reaction centers of the purple bacterium Rhodobacter sphaeroides (R26) in the mid-IR region between 1000 and 1800 cm-1 are recorded with a time resolution of 300-400 fs. The difference spectra of the states P*, P+HA-, and P+QA- with respect to the ground state P predominantly reflect changes of the special pair. They show positive and negative bands due to changes of distinct vibrational modes superimposed on a broad background of enhanced absorption. A number of certain bands can be assigned to the special pair P, to the bacteriopheophytin HA, and to the quinone QA. The temporal evolution of the IR absorbance changes is well described by the time constants known from femtosecond spectroscopy of the electronic states. Differences occur only at very early times, which are indicative of fast vibrational relaxation with a time constant of a few hundred femtoseconds. PMID:7892185

  17. Time-resolved tryptophan fluorescence in photosynthetic reaction centers from Rhodobacter sphaeroides

    NASA Technical Reports Server (NTRS)

    Godik, V. I.; Blankenship, R. E.; Causgrove, T. P.; Woodbury, N.

    1993-01-01

    Tryptophan fluorescence of reaction centers isolated from Rhodobacter sphaeroides, both stationary and time-resolved, was studied. Fluorescence kinetics were found to fit best a sum of four discrete exponential components. Half of the initial amplitude was due to a component with a lifetime of congruent to 60 ps, belonging to Trp residues, capable of efficient transfer of excitation energy to bacteriochlorophyll molecules of the reaction center. The three other components seem to be emitted by Trp ground-state conformers, unable to participate in such a transfer. Under the influence of intense actinic light, photooxidizing the reaction centers, the yield of stationary fluorescence diminished by congruent to 1.5 times, while the number of the kinetic components and their life times remained practically unchanged. Possible implications of the observed effects for the primary photosynthesis events are considered.

  18. Cytotoxic activities of extracts and compounds from Viscum coloratum and its transformation products by Rhodobacter sphaeroides.

    PubMed

    Yang, Guan-E; Chen, Bainian; Zhang, Zhaoming; Gong, Jun; Bai, Hongjun; Li, Jiankuan; Wang, Yufen; Li, Baozhen

    2009-03-01

    The bioassay-oriented fractionation of mistletoe crude extracts (MCEE) using 75% ethanol and culture products of mistletoe transformed by Rhodobacter sphaeroides, a photosynthetic bacterium (PSBT), revealed that the high cytotoxic activities were due to the petroleum ether extracts (PEs) and the acid-precipitated proteins from the aqueous extracts (AQs) of MCEE and PSBT. The isolated triterpenes may account for the activities of the PEs of MCEE and PSBT, respectively. Extraction of MCEE using petroleum ether led to the isolation of 3-epi-betulinic acid (1), betulonic acid (2), oleanolic acid (3), and beta-amyrin acetate (4), while petroleum ether extraction of PSBT led to the isolation of 1,3,4,betulinic acid (5), erythrodiol (6), and (3beta)-olean-12-ene-3,23-diol (7). The PE of PSBT exerted higher cytotoxicity than the PE of MCEE, which was due to the different triterpene contents of these two extracts. The cytotoxic activities of all compounds were tested, and the results revealed that compounds 1, 2, 3, 5, 6, and 7 contributed significantly to the cytotoxicities of both PEs. The AQ of the PSBT exerted almost the same cytotoxic activity and lower toxicity compared to the AQ of the MCEE. These findings indicate that mistletoe products biotransformed by R. sphaeroides could be used to treat cancers, since they have lower toxicities and higher antitumor activities compared to standard treatments.

  19. The N Terminus of FliM Is Essential To Promote Flagellar Rotation in Rhodobacter sphaeroides

    PubMed Central

    Poggio, Sebastian; Osorio, Aurora; Corkidi, Gabriel; Dreyfus, Georges; Camarena, Laura

    2001-01-01

    FliM is part of the flagellar switch complex. Interaction of this protein with phospho-CheY (CheY-P) through its N terminus constitutes the main information relay point between the chemotactic system and the flagellum. In this work, we evaluated the role of the N terminus of FliM in the swimming behavior of Rhodobacter sphaeroides. Strains expressing the FliM protein with substitutions in residues previously reported in Escherichia coli as being important for interaction with CheY showed an increased stop frequency compared with wild-type cells. In accordance, we observed that R. sphaeroides cells expressing FliM lacking either the first 13 or 20 amino acids from the N terminus showed a stopped phenotype. We show evidence that FliMΔ13 and FliMΔ20 are stable proteins and that cells expressing them allow flagellin export at levels indistinguishable from those detected for the wild-type strain. These results suggest that the N-terminal region of FliM is required to promote swimming in this bacterium. The role of CheY in controlling flagellar rotation in this organism is discussed. PMID:11325943

  20. Photoresponses of the purple nonsulfur bacteria Rhodospirillum centenum and Rhodobacter sphaeroides.

    PubMed Central

    Sackett, M J; Armitage, J P; Sherwood, E E; Pitta, T P

    1997-01-01

    We have measured the photoresponse of two purple nonsulfur bacteria, Rhodobacter sphaeroides and Rhodospirillum centenum, under defined conditions in a light beam propagating at 90 degrees to the optical axis of the microscope. This beam presented cells with a steep gradient of intensity perpendicular to the direction of propagation and a shallow gradient in the direction of light propagation. R. centenum, a species that reverses to change direction, accumulated in the light beam, as expected for a "scotophobic" response, while R. sphaeroides, which stops rather than reverses, accumulated outside the light beam. We also compared the behavior of liquid-grown R. centenum, which swims by using a single polar flagellum, to that of surface-grown R. centenum, which swarms over agar by using many lateral flagella and has been shown to move as colonies toward specific wavelengths of light. When suspended in liquid medium, both liquid- and surface-grown R. centenum showed similar responses to the light gradient. In all cases, free-swimming cells responded to the steep gradient of intensity but not to the shallow gradient, indicating they cannot sense the direction of light propagation but only its intensity. In a control experiment, the known phototactic alga Chlamydamonas reinhardtii was shown to swim in the direction of light propagation. PMID:9352928

  1. A molecular brake, not a clutch, stops the Rhodobacter sphaeroides flagellar motor

    PubMed Central

    Pilizota, Teuta; Brown, Mostyn T.; Leake, Mark C.; Branch, Richard W.; Berry, Richard M.; Armitage, Judith P.

    2009-01-01

    Many bacterial species swim by employing ion-driven molecular motors that power the rotation of helical filaments. Signals are transmitted to the motor from the external environment via the chemotaxis pathway. In bidirectional motors, the binding of phosphorylated CheY (CheY-P) to the motor is presumed to instigate conformational changes that result in a different rotor-stator interface, resulting in rotation in the alternative direction. Controlling when this switch occurs enables bacteria to accumulate in areas favorable for their survival. Unlike most species that swim with bidirectional motors, Rhodobacter sphaeroides employs a single stop-start flagellar motor. Here, we asked, how does the binding of CheY-P stop the motor in R. sphaeroides—using a clutch or a brake? By applying external force with viscous flow or optical tweezers, we show that the R. sphaeroides motor is stopped using a brake. The motor stops at 27–28 discrete angles, locked in place by a relatively high torque, approximately 2–3 times its stall torque. PMID:19571004

  2. A cryptochrome-like protein is involved in the regulation of photosynthesis genes in Rhodobacter sphaeroides.

    PubMed

    Hendrischk, Anne-Kathrin; Frühwirth, Sebastian Walter; Moldt, Julia; Pokorny, Richard; Metz, Sebastian; Kaiser, Gebhard; Jäger, Andreas; Batschauer, Alfred; Klug, Gabriele

    2009-11-01

    Blue light receptors belonging to the cryptochrome/photolyase family are found in all kingdoms of life. The functions of photolyases in repair of UV-damaged DNA as well as of cryptochromes in the light-dependent regulation of photomorphogenetic processes and in the circadian clock in plants and animals are well analysed. In prokaryotes, the only role of members of this protein family that could be demonstrated is DNA repair. Recently, we identified a gene for a cryptochrome-like protein (CryB) in the alpha-proteobacterium Rhodobacter sphaeroides. The protein lacks the typical C-terminal extension of cryptochromes, and is not related to the Cry DASH family. Here we demonstrate that CryB binds flavin adenine dinucleotide that can be photoreduced by blue light. CryB binds single-stranded DNA with very high affinity (K(d) approximately 10(-8) M) but double-stranded DNA and single-stranded RNA with far lower affinity (K(d) approximately 10(-6) M). Despite of that, no in vitro repair activity for pyrimidine dimers in single-stranded DNA could be detected. However, we show that CryB clearly affects the expression of genes for pigment-binding proteins and consequently the amount of photosynthetic complexes in R. sphaeroides. Thus, for the first time a role of a bacterial cryptochrome in gene regulation together with a biological function is demonstrated.

  3. Comparison of aerobic and photosynthetic Rhodobacter sphaeroides 2.4.1 proteomes

    SciTech Connect

    Callister, Stephen J.; Nicora, Carrie D.; Zeng, Xiaohua; Roh, Jung Hyeob; Dominguez, Migual; Tavano, Christine; Monroe, Matthew E.; Kaplan, Samuel; Donohue, Timothy; Smith, Richard D.; Lipton, Mary S.

    2006-07-05

    Proteomes from aerobic and photosynthetic grown Rhodobacter sphaeroides 2.4.1 cell cultures were characterized using liquid chromatography-mass spectrometry in conjunction with an accurate mass and elution time (AMT) tag approach. Roughly 8000 high quality peptides were detected that represented 1,445 gene products and 34% of the predicted proteins. The identified proteins corresponded primarily to open reading frames (ORFs) contained within the two chromosomal elements of this bacterium, but a significant number were also observed from ORFs associated with 5 naturally occurring plasmids. Data mining of peptides revealed a number of proteins uniquely detected within the photosynthetic cell culture. Proteins observed in both aerobic respiratory and photosynthetic grown cultures were analyzed semi-quantitatively by comparing their estimated abundances to provide insights into bioenergetic models for aerobic respiration and photosynthesis. Additional emphasis was placed on gene products annotated as hypothetical to gain information as to their potential roles within these two growth conditions. Where possible, transcriptome data for R. sphaeroides obtained under the same culture conditions were compared with these results. This comparative study demonstrated the applicability of the AMT tag approach for high-throughput proteomic analyses of pathways associated with the photosynthetic lifestyle.

  4. Modelling and analysis of bacterial tracks suggest an active reorientation mechanism in Rhodobacter sphaeroides.

    PubMed

    Rosser, Gabriel; Baker, Ruth E; Armitage, Judith P; Fletcher, Alexander G

    2014-08-06

    Most free-swimming bacteria move in approximately straight lines, interspersed with random reorientation phases. A key open question concerns varying mechanisms by which reorientation occurs. We combine mathematical modelling with analysis of a large tracking dataset to study the poorly understood reorientation mechanism in the monoflagellate species Rhodobacter sphaeroides. The flagellum on this species rotates counterclockwise to propel the bacterium, periodically ceasing rotation to enable reorientation. When rotation restarts the cell body usually points in a new direction. It has been assumed that the new direction is simply the result of Brownian rotation. We consider three variants of a self-propelled particle model of bacterial motility. The first considers rotational diffusion only, corresponding to a non-chemotactic mutant strain. Two further models incorporate stochastic reorientations, describing 'run-and-tumble' motility. We derive expressions for key summary statistics and simulate each model using a stochastic computational algorithm. We also discuss the effect of cell geometry on rotational diffusion. Working with a previously published tracking dataset, we compare predictions of the models with data on individual stopping events in R. sphaeroides. This provides strong evidence that this species undergoes some form of active reorientation rather than simple reorientation by Brownian rotation. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  5. Phototrophic utilization of taurine by the purple nonsulfur bacteria Rhodopseudomonas palustris and Rhodobacter sphaeroides.

    PubMed

    Novak, Ryan T; Gritzer, Rachel F; Leadbetter, Edward R; Godchaux, Walter

    2004-06-01

    Taurine metabolism by two phototrophically grown purple nonsulfur bacteria enrichment isolates has been examined. Rhodopseudomonas palustris (strain Tau1) grows with taurine as a sole electron donor, sulfur and nitrogen source during photoautotrophic growth. Rhodobacter sphaeroides (strain Tau3) grows on the compound as sole electron donor, sulfur and nitrogen source, and partial carbon source, in the presence of CO(2) during photoheterotrophic growth. Both organisms utilize an inducible taurine-pyruvate aminotransferase and a sulfoacetaldehyde acetyltransferase. The products of this metabolism are bisulfite and acetyl phosphate. Bisulfite ultimately was oxidized to sulfate, but this was not an adequate source of electrons for photometabolism. Experiments using either [U-(14)C]taurine or (14)CO(2) demonstrated that Rb. sphaeroides Tau3 assimilated the carbon from approximately equimolar amounts of taurine and exogenous CO(2). The taurine-carbon assimilation was not diminished by excess non-radioactive bicarbonate. Malate synthase (but not isocitrate lyase) was induced in these taurine-grown cells. It is concluded that assimilation of taurine carbon occurs through an intermediate other than CO(2). Similar labelling experiments with Rp. palustris Tau1 determined that taurine is utilized only as an electron donor for the reduction of CO(2), which contributes all the cell carbon. Photoautotrophic metabolism was confirmed in this organism by the absence of either malate synthase or isocitrate lyase in taurine+CO(2)-grown cells. Culture collection strains of these two bacteria did not utilize taurine in these fashions.

  6. Color-sensitive motility and methanol release responses in Rhodobacter sphaeroides.

    PubMed

    Kort, R; Crielaard, W; Spudich, J L; Hellingwerf, K J

    2000-06-01

    Blue-light-induced repellent and demethylation responses, characteristic of behavioral adaptation, were observed in Rhodobacter sphaeroides. They were analyzed by computer-assisted motion analysis and through the release of volatile tritiated compounds from [methyl-(3)H]methionine-labeled cells, respectively. Increases in the stop frequency and the rate of methanol release were induced by exposure of cells to repellent light signals, such as an increase in blue- and a decrease in infrared-light intensity. At a lambda of >500 nm the amplitude of the methanol release response followed the absorbance spectrum of the photosynthetic pigments, suggesting that they function as photosensors for this response. In contrast to the previously reported motility response to a decrease in infrared light, the blue-light response reported here does not depend on the number of photosynthetic pigments per cell, suggesting that it is mediated by a separate sensor. Therefore, color discrimination in taxis responses in R. sphaeroides involves two photosensing systems: the photosynthetic pigments and an additional photosensor, responding to blue light. The signal generated by the former system could result in the migration of cells to a light climate beneficial for photosynthesis, while the blue-light system could allow cells to avoid too-high intensities of (harmful) blue light.

  7. Assembly of functional photosystem complexes in Rhodobacter sphaeroides incorporating carotenoids from the spirilloxanthin pathway.

    PubMed

    Chi, Shuang C; Mothersole, David J; Dilbeck, Preston; Niedzwiedzki, Dariusz M; Zhang, Hao; Qian, Pu; Vasilev, Cvetelin; Grayson, Katie J; Jackson, Philip J; Martin, Elizabeth C; Li, Ying; Holten, Dewey; Neil Hunter, C

    2015-02-01

    Carotenoids protect the photosynthetic apparatus against harmful radicals arising from the presence of both light and oxygen. They also act as accessory pigments for harvesting solar energy, and are required for stable assembly of many light-harvesting complexes. In the phototrophic bacterium Rhodobacter (Rba.) sphaeroides phytoene desaturase (CrtI) catalyses three sequential desaturations of the colourless carotenoid phytoene, extending the number of conjugated carbon-carbon double bonds, N, from three to nine and producing the yellow carotenoid neurosporene; subsequent modifications produce the yellow/red carotenoids spheroidene/spheroidenone (N=10/11). Genomic crtI replacements were used to swap the native three-step Rba. sphaeroides CrtI for the four-step Pantoea agglomerans enzyme, which re-routed carotenoid biosynthesis and culminated in the production of 2,2'-diketo-spirilloxanthin under semi-aerobic conditions. The new carotenoid pathway was elucidated using a combination of HPLC and mass spectrometry. Premature termination of this new pathway by inactivating crtC or crtD produced strains with lycopene or rhodopin as major carotenoids. All of the spirilloxanthin series carotenoids are accepted by the assembly pathways for LH2 and RC-LH1-PufX complexes. The efficiency of carotenoid-to-bacteriochlorophyll energy transfer for 2,2'-diketo-spirilloxanthin (15 conjugated CC bonds; N=15) in LH2 complexes is low, at 35%. High energy transfer efficiencies were obtained for neurosporene (N=9; 94%), spheroidene (N=10; 96%) and spheroidenone (N=11; 95%), whereas intermediate values were measured for lycopene (N=11; 64%), rhodopin (N=11; 62%) and spirilloxanthin (N=13; 39%). The variety and stability of these novel Rba. sphaeroides antenna complexes make them useful experimental models for investigating the energy transfer dynamics of carotenoids in bacterial photosynthesis.

  8. Assembly of functional photosystem complexes in Rhodobacter sphaeroides incorporating carotenoids from the spirilloxanthin pathway

    DOE PAGES

    Chi, Shuang C.; Mothersole, David J.; Dilbeck, Preston; ...

    2014-10-27

    Carotenoids protect the photosynthetic apparatus against harmful radicals arising from the presence of both light and oxygen. They also act as accessory pigments for harvesting solar energy, and are required for stable assembly of many light-harvesting complexes. In the phototrophic bacterium Rhodobacter (Rba.) sphaeroides phytoene desaturase (CrtI) catalyses three sequential desaturations of the colourless carotenoid phytoene, extending the number of conjugated carbon-carbon double bonds, N, from three to nine and producing the yellow carotenoid neurosporene; subsequent modifications produce the yellow/red carotenoids spheroidene/spheroidenone (N=10/11). Genomic crtI replacements were used to swap the native three-step Rba. sphaeroides CrtI for the four-step Pantoeamore » agglomerans enzyme, which re-routed carotenoid biosynthesis and culminated in the production of 2,2'-diketo-spirilloxanthin under semi-aerobic conditions. The new carotenoid pathway was elucidated using a combination of HPLC and mass spectrometry. Premature termination of this new pathway by inactivating crtC or crtD produced strains with lycopene or rhodopin as major carotenoids. All of the spirilloxanthin series carotenoids are accepted by the assembly pathways for LH2 and RC-LH1-PufX complexes. The efficiency of carotenoid-to-bacteriochlorophyll energy transfer for 2,2'-diketo-spirilloxanthin (15 conjugated CC bonds; N=15) in LH2 complexes is low, at 35%. High energy transfer efficiencies were obtained for neurosporene (N=9; 94%), spheroidene (N=10; 96%) and spheroidenone (N=11; 95%), whereas intermediate values were measured for lycopene (N=11; 64%), rhodopin (N=11; 62%) and spirilloxanthin (N=13; 39%). In conclusion, the variety and stability of these novel Rba. sphaeroides antenna complexes make them useful experimental models for investigating the energy transfer dynamics of carotenoids in bacterial photosynthesis.« less

  9. Skin Anti-Aging Activities of Bacteriochlorophyll a from Photosynthetic Bacteria, Rhodobacter sphaeroides.

    PubMed

    Kim, Nam Young; Yim, Tae Bin; Lee, Hyeon Yong

    2015-10-01

    In this work, the anti-aging skin effects of bacteriochlorophyll a isolated from Rhodobacter sphaeroides are first reported, with notably low cytotoxicity in the range of 1% to 14% in adding 0.00078 (% (w/w)) of the extracts, compared with the normal growth of both human dermal fibroblast and keratinocyte cells without any treatment as a control. The highest production of procollagen from human fibroblast cells (CCD-986sk) was observed as 221.7 ng/ml with 0.001 (% (w/w)) of bacteriochlorophyll a, whereas 150 and 200 ng/ml of procollagen production resulted from addition of 0.001 (% (w/w)) of the photosynthetic bacteria. The bacteriochlorophylla- induced TNF-α production increased to 63.8%, which was lower secretion from HaCaT cells than that from addition of 0.00005 (% (w/w)) of bacteriochlorophyll a. Additionally, bacteriochlorophyll a upregulated the expression of genes related to skin anti-aging (i.e., keratin 10, involucrin, transglutaminase-1, and MMPs), by up to 4-15 times those of the control. However, crude extracts from R. sphaeroides did not enhance the expression level of these genes. Bacteriochlorophyll a showed higher antioxidant activity of 63.8% in DPPH free radical scavenging than those of water, ethanol, and 70% ethanol extracts (14.0%, 57.2%, and 12.6%, respectively). It was also shown that the high antioxidant activity could be attributed to the skin anti-aging effect of bacteriochlorophyll a, although R. sphaeroides itself would not exhibit significant anti-aging activities.

  10. Combining Genome-Scale Experimental and Computational Methods To Identify Essential Genes in Rhodobacter sphaeroides

    PubMed Central

    Burger, Brian T.; Imam, Saheed; Scarborough, Matthew J.; Noguera, Daniel R.

    2017-01-01

    ABSTRACT Rhodobacter sphaeroides is one of the best-studied alphaproteobacteria from biochemical, genetic, and genomic perspectives. To gain a better systems-level understanding of this organism, we generated a large transposon mutant library and used transposon sequencing (Tn-seq) to identify genes that are essential under several growth conditions. Using newly developed Tn-seq analysis software (TSAS), we identified 493 genes as essential for aerobic growth on a rich medium. We then used the mutant library to identify conditionally essential genes under two laboratory growth conditions, identifying 85 additional genes required for aerobic growth in a minimal medium and 31 additional genes required for photosynthetic growth. In all instances, our analyses confirmed essentiality for many known genes and identified genes not previously considered to be essential. We used the resulting Tn-seq data to refine and improve a genome-scale metabolic network model (GEM) for R. sphaeroides. Together, we demonstrate how genetic, genomic, and computational approaches can be combined to obtain a systems-level understanding of the genetic framework underlying metabolic diversity in bacterial species. IMPORTANCE Knowledge about the role of genes under a particular growth condition is required for a holistic understanding of a bacterial cell and has implications for health, agriculture, and biotechnology. We developed the Tn-seq analysis software (TSAS) package to provide a flexible and statistically rigorous workflow for the high-throughput analysis of insertion mutant libraries, advanced the knowledge of gene essentiality in R. sphaeroides, and illustrated how Tn-seq data can be used to more accurately identify genes that play important roles in metabolism and other processes that are essential for cellular survival. Author Video: An author video summary of this article is available. PMID:28744485

  11. Free Fe(3+)/Fe(2+) improved the biomass resource recovery and organic matter removal in Rhodobacter sphaeroides purification of sewage.

    PubMed

    Liu, Rijia; Wu, Pan; Lang, Lang; Xu, Changru; Wang, Yanling

    2016-01-01

    The enhancement in biomass production and organic matter removal of Rhodobacter sphaeroides (R. sphaeroides) through iron ions in soybean protein wastewater treatment was investigated. Different dosages of ferric ions were introduced in the reactors under light-anaerobic conditions. Free ferric and ferrous ions in wastewater were formed and their concentrations were the optimal for the growth of R. sphaeroides when the total Fe dosage was 20 mg/L. At the optimal dosage, biomass production (4000 mg/L) and protease activity improved by 50% and 48% when compared to the controls, respectively. The organic matter and protein removal reached above 90% and hydraulic retention time was shortened from 96 to 72 h. A mechanism analysis indicated that iron ions can effectively improve the adenosine triphosphate production, which may furthermore encourage the synthesis of protease and the cellular material.

  12. A Cardiolipin-Deficient Mutant of Rhodobacter sphaeroides Has an Altered Cell Shape and Is Impaired in Biofilm Formation

    PubMed Central

    Lin, Ti-Yu; Santos, Thiago M. A.; Kontur, Wayne S.

    2015-01-01

    ABSTRACT Cell shape has been suggested to play an important role in the regulation of bacterial attachment to surfaces and the formation of communities associated with surfaces. We found that a cardiolipin synthase (Δcls) mutant of the rod-shaped bacterium Rhodobacter sphaeroides—in which synthesis of the anionic, highly curved phospholipid cardiolipin (CL) is reduced by 90%—produces ellipsoid-shaped cells that are impaired in biofilm formation. Reducing the concentration of CL did not cause significant defects in R. sphaeroides cell growth, swimming motility, lipopolysaccharide and exopolysaccharide production, surface adhesion protein expression, and membrane permeability. Complementation of the CL-deficient mutant by ectopically expressing CL synthase restored cells to their rod shape and increased biofilm formation. Treating R. sphaeroides cells with a low concentration (10 μg/ml) of the small-molecule MreB inhibitor S-(3,4-dichlorobenzyl)isothiourea produced ellipsoid-shaped cells that had no obvious growth defect yet reduced R. sphaeroides biofilm formation. This study demonstrates that CL plays a role in R. sphaeroides cell shape determination, biofilm formation, and the ability of the bacterium to adapt to its environment. IMPORTANCE Membrane composition plays a fundamental role in the adaptation of many bacteria to environmental stress. In this study, we build a new connection between the anionic phospholipid cardiolipin (CL) and cellular adaptation in Rhodobacter sphaeroides. We demonstrate that CL plays a role in the regulation of R. sphaeroides morphology and is important for the ability of this bacterium to form biofilms. This study correlates CL concentration, cell shape, and biofilm formation and provides the first example of how membrane composition in bacteria alters cell morphology and influences adaptation. This study also provides insight into the potential of phospholipid biosynthesis as a target for new chemical strategies designed to

  13. A Rhodobacter sphaeroides Protein Mechanistically Similar to Escherichia coli DksA Regulates Photosynthetic Growth

    PubMed Central

    Lennon, Christopher W.; Lemmer, Kimberly C.; Irons, Jessica L.; Sellman, Max I.; Donohue, Timothy J.; Ross, Wilma

    2014-01-01

    ABSTRACT DksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the “stringent response” to nutrient starvation in the gammaproteobacterium Escherichia coli and for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly related Rhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length to E. coli DksA but lacks the Zn finger motif of the E. coli DksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of an E. coli strain lacking the dksA gene and modulates transcription in vitro with E. coli RNA polymerase (RNAP) similarly to E. coli DksA. RSP2654 reduces RNAP-promoter complex stability in vitro with RNAPs from E. coli or R. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity to E. coli DksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsp has distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp. PMID:24781745

  14. 5-Aminolevulinate production by Escherichia coli containing the Rhodobacter sphaeroides hemA gene

    SciTech Connect

    Van Der Werf, M.J.; Zeikus, J.G. |

    1996-10-01

    The Rhodobacter sphaeroides hemA gene codes for 5-aminolevulinate (ALA) synthase. This enzyme catalyzes the pyridoxal phosphate-dependent condensation of succinyl coenzyme A and glycine-forming ALA. The R. sphaeroides hemA gene in the pUC18/19 vector system was transformed into Escherichia coli. The effects of both genetic and physiological factors on the expression of ALA synthase and the production of ALA were studied. ALA synthase activity levels were maximal when hemA had the same transcription direction as the lac promoter. The distance between the lac promoter and hemA affected the expression of ALA synthase on different growth substrates. The E. coli host strain used had an enormous effect on the ALA synthase activity level and on the production of ALA, with E. coli DH1 being best suited. The ALA synthase activity level was also dependent on the carbon source. Succinate, L-malate, fumarate, and L-aspartate gave the highest levels of ALA synthase activity, while the use of lactose as a carbon source resulted in a repression of ALA synthase. After growth on succinate, ALA synthase represented {approx}5% of total cellular protein. The ALA synthase activity level was also dependent on the pH of the medium, with maximal activity occurring at pH 6.5. ALA production by whole cells was limited by the availability of glycine, and the addition of 2 g of glycine per liter to the growth medium increased the production of ALA fivefold, to 2.25 mM. In recombinant E. coli extracts, up to 22 mM ALA was produced from succinate, glycine, and ATP. 58 refs., 4 figs., 7 tabs.

  15. delta-Aminolevulinate couples cycA transcription to changes in heme availability in Rhodobacter sphaeroides.

    PubMed

    Schilke, B A; Donohue, T J

    1992-07-05

    In this paper, the response of the transcriptional control region of the Rhodobacter sphaeroides cytochrome c2 gene, cycA, to intermediates in heme biosynthesis was studied. To determine if cycA transcription was regulated by heme availability, several precursors or analogs of tetrapyrroles were tested. Addition of delta-aminolevulinate (ALA), the first committed intermediate in heme biosynthesis, was shown to inhibit cycA transcription initiation at both the upstream and downstream promoter regions. In addition, an ALA auxotroph, which can grow in the presence of high levels of ALA, showed a 5 to 7-fold reduction in steady-state transcription from cycA::lacZYA operon fusions. To identify genetic elements responsible for negative regulation by ALA, trans-acting mutants with increased expression of cycA were isolated that were resistant to growth inhibition by the heme analog cohemin. These cohemin-resistant mutants (Chr) have elevated levels of several cycA transcripts and they contain cycA transcripts that had not previously been detected in wild-type cells. In addition, cycA transcription in the Chr mutants continues after the addition of ALA. Finally, we found that Chr mutants have increased ALA synthase activity, suggesting that synthesis of cytochrome c2 and ALA synthase are controlled by a common gene product whose activity has been modified in these mutants. A model is presented to explain how changes in tetrapyrrole intermediates could provide an effective signal to control both cycA transcription and ALA synthase synthesis in R. sphaeroides.

  16. Role of norEF in Denitrification, Elucidated by Physiological Experiments with Rhodobacter sphaeroides

    PubMed Central

    Bergaust, Linda L.; Hartsock, Angela; Liu, Binbin; Bakken, Lars R.

    2014-01-01

    Many denitrifying organisms contain the norEF gene cluster, which codes for two proteins that are thought to be involved in denitrification because they are expressed during the reduction of nitrite and nitric oxide. The products of both genes are predicted to be membrane associated, and the norE product is a member of the cytochrome c oxidase subunit III family. However, the specific role of norEF is unknown. The denitrification phenotypes of Rhodobacter sphaeroides strains with and without norEF genes were studied, and it was found that loss of norEF lowered the rate of denitrification from nitrate and resulted in accumulation of micromolar concentrations of nitric oxide during denitrification from nitrite. norEF appears to have no direct role in the reduction of nitric oxide; however, since deletion of norEF in the wild-type 2.4.3 strain had essentially no influence on the kinetics of potential nitric oxide reduction (Vmax and Ks), as measured by monitoring the depletion of a bolus of nitric oxide injected into anoxic cultures without any other electron acceptors. However, norEF-deficient cells that had undergone a more chronic exposure to micromolar concentrations of nitric oxide showed an ∼50% reduction in Vmax but no change in apparent Ks. These results can explain the occurrence of norEF in the 2.4.3 strain of R. sphaeroides, which can reduce nitrate to nitrous oxide, and their absence from strains such as 2.4.1, which likely use nitric oxide reductase to mitigate stress due to episodic exposure to nitric oxide from exogenous sources. PMID:24706737

  17. Energy transfer properties of Rhodobacter sphaeroides chromatophores during adaptation to low light intensity.

    PubMed

    Driscoll, B; Lunceford, C; Lin, S; Woronowicz, K; Niederman, R A; Woodbury, N W

    2014-08-28

    Time-resolved fluorescence spectroscopy was used to explore the pathway and kinetics of energy transfer in photosynthetic membrane vesicles (chromatophores) isolated from Rhodobacter (Rba.) sphaeroides cells harvested 2, 4, 6 or 24 hours after a transition from growth in high to low level illumination. As previously observed, this light intensity transition initiates the remodeling of the photosynthetic apparatus and an increase in the number of light harvesting 2 (LH2) complexes relative to light harvesting 1 (LH1) and reaction center (RC) complexes. It has generally been thought that the increase in LH2 complexes served the purpose of increasing the overall energy transmission to the RC. However, fluorescence lifetime measurements and analysis in terms of energy transfer within LH2 and between LH2 and LH1 indicate that, during the remodeling time period measured, only a portion of the additional LH2 generated are well connected to LH1 and the reaction center. The majority of the additional LH2 fluorescence decays with a lifetime comparable to that of free, unconnected LH2 complexes. The presence of large LH2-only domains has been observed by atomic force microscopy in Rba. sphaeroides chromatophores (Bahatyrova et al., Nature, 2004, 430, 1058), providing structural support for the existence of pools of partially connected LH2 complexes. These LH2-only domains represent the light-responsive antenna complement formed after a switch in growth conditions from high to low illumination, while the remaining LH2 complexes occupy membrane regions containing mixtures of LH2 and LH1-RC core complexes. The current study utilized a multi-parameter approach to explore the fluorescence spectroscopic properties related to the remodeling process, shedding light on the structure-function relationship of the photosynthetic assembles. Possible reasons for the accumulation of these largely disconnected LH2-only pools are discussed.

  18. Assembly of functional photosystem complexes in Rhodobacter sphaeroides incorporating carotenoids from the spirilloxanthin pathway

    PubMed Central

    Chi, Shuang C.; Mothersole, David J.; Dilbeck, Preston; Niedzwiedzki, Dariusz M.; Zhang, Hao; Qian, Pu; Vasilev, Cvetelin; Grayson, Katie J.; Jackson, Philip J.; Martin, Elizabeth C.; Li, Ying; Holten, Dewey; Neil Hunter, C.

    2015-01-01

    Carotenoids protect the photosynthetic apparatus against harmful radicals arising from the presence of both light and oxygen. They also act as accessory pigments for harvesting solar energy, and are required for stable assembly of many light-harvesting complexes. In the phototrophic bacterium Rhodobacter (Rba.) sphaeroides phytoene desaturase (CrtI) catalyses three sequential desaturations of the colourless carotenoid phytoene, extending the number of conjugated carbon–carbon double bonds, N, from three to nine and producing the yellow carotenoid neurosporene; subsequent modifications produce the yellow/red carotenoids spheroidene/spheroidenone (N = 10/11). Genomic crtI replacements were used to swap the native three-step Rba. sphaeroides CrtI for the four-step Pantoea agglomerans enzyme, which re-routed carotenoid biosynthesis and culminated in the production of 2,2′-diketo-spirilloxanthin under semi-aerobic conditions. The new carotenoid pathway was elucidated using a combination of HPLC and mass spectrometry. Premature termination of this new pathway by inactivating crtC or crtD produced strains with lycopene or rhodopin as major carotenoids. All of the spirilloxanthin series carotenoids are accepted by the assembly pathways for LH2 and RC–LH1–PufX complexes. The efficiency of carotenoid-to-bacteriochlorophyll energy transfer for 2,2′-diketo-spirilloxanthin (15 conjugated C 000000000000 000000000000 000000000000 111111111111 000000000000 111111111111 000000000000 000000000000 000000000000 C bonds; N = 15) in LH2 complexes is low, at 35%. High energy transfer efficiencies were obtained for neurosporene (N = 9; 94%), spheroidene (N = 10; 96%) and spheroidenone (N = 11; 95%), whereas intermediate values were measured for lycopene (N = 11; 64%), rhodopin (N = 11; 62%) and spirilloxanthin (N = 13; 39%). The variety and stability of these novel Rba. sphaeroides antenna complexes make them useful experimental models for investigating the

  19. Native Mass Spectrometry Characterizes the Photosynthetic Reaction Center Complex from the Purple Bacterium Rhodobacter sphaeroides

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; Harrington, Lucas B.; Lu, Yue; Prado, Mindy; Saer, Rafael; Rempel, Don; Blankenship, Robert E.; Gross, Michael L.

    2017-01-01

    Native mass spectrometry (MS) is an emerging approach to study protein complexes in their near-native states and to elucidate their stoichiometry and topology. Here, we report a native MS study of the membrane-embedded reaction center (RC) protein complex from the purple photosynthetic bacterium Rhodobacter sphaeroides. The membrane-embedded RC protein complex is stabilized by detergent micelles in aqueous solution, directly introduced into a mass spectrometer by nano-electrospray (nESI), and freed of detergents and dissociated in the gas phase by collisional activation. As the collision energy is increased, the chlorophyll pigments are gradually released from the RC complex, suggesting that native MS introduces a near-native structure that continues to bind pigments. Two bacteriochlorophyll a pigments remain tightly bound to the RC protein at the highest collision energy. The order of pigment release and their resistance to release by gas-phase activation indicates the strength of pigment interaction in the RC complex. This investigation sets the stage for future native MS studies of membrane-embedded photosynthetic pigment-protein and related complexes.

  20. Molecular Cloning, Overexpression and Characterization of a Novel Water Channel Protein from Rhodobacter sphaeroides

    PubMed Central

    Erbakan, Mustafa; Shen, Yue-xiao; Grzelakowski, Mariusz; Butler, Peter J.; Kumar, Manish; Curtis, Wayne R.

    2014-01-01

    Aquaporins are highly selective water channel proteins integrated into plasma membranes of single cell organisms; plant roots and stromae; eye lenses, renal and red blood cells in vertebrates. To date, only a few microbial aquaporins have been characterized and their physiological importance is not well understood. Here we report on the cloning, expression and characterization of a novel aquaporin, RsAqpZ, from a purple photosynthetic bacterium, Rhodobacter sphaeroides ATCC 17023. The protein was expressed homologously at a high yield (∼20 mg/L culture) under anaerobic photoheterotrophic growth conditions. Stopped-flow light scattering experiments demonstrated its high water permeability (0.17±0.05 cm/s) and low energy of activation for water transport (2.93±0.60 kcal/mol) in reconstituted proteoliposomes at a protein to lipid ratio (w/w) of 0.04. We developed a fluorescence correlation spectroscopy based technique and utilized a fluorescent protein fusion of RsAqpZ, to estimate the single channel water permeability of RsAqpZ as 1.24 (±0.41) x 10−12 cm3/s or 4.17 (±1.38)×1010 H2O molecules/s, which is among the highest single channel permeability reported for aquaporins. Towards application to water purification technologies, we also demonstrated functional incorporation of RsAqpZ in amphiphilic block copolymer membranes. PMID:24497982

  1. Bioremediation of petroleum hydrocarbon contaminated soil by Rhodobacter sphaeroides biofertilizer and plants.

    PubMed

    Jiao, Haihua; Luo, Jinxue; Zhang, Yiming; Xu, Shengjun; Bai, Zhihui; Huang, Zhanbin

    2015-09-01

    Bio-augmentation is a promising technique for remediation of polluted soils. This study aimed to evaluate the bio-augmentation effect of Rhodobacter sphaeroides biofertilizer (RBF) on the bioremediation of total petroleum hydrocarbons (TPH) contaminated soil. A greenhouse pot experiment was conducted over a period of 120 days, three methods for enhancing bio-augmentation were tested on TPH contaminated soils, including single addition RBF, planting, and combining of RBF and three crop species, such as wheat (W), cabbage (C) and spinach (S), respectively. The results demonstrated that the best removal of TPH from contaminated soil in the RBF bio-augmentation rhizosphere soils was found to be 46.2%, 65.4%, 67.5% for W+RBF, C+RBF, S+RBF rhizosphere soils respectively. RBF supply impacted on the microbial community diversity (phospholipid fatty acids, PLFA) and the activity of soil enzymes, such as dehydrogenase (DH), alkaline phosphatase (AP) and urease (UR). There were significant difference among the soil only containing crude oil (CK), W, C and S rhizosphere soils and RBF bio-augmentation soils. Moreover, the changes were significantly distinct depended on crops species. It was concluded that the RBF is a valuable material for improving effect of remediation of TPH polluted soils.

  2. Redox potential tuning through differential quinone binding in the photosynthetic reaction center of Rhodobacter sphaeroides

    DOE PAGES

    Vermaas, Josh V.; Taguchi, Alexander T.; Dikanov, Sergei A.; ...

    2015-03-03

    Ubiquinone forms an integral part of the electron transport chain in cellular respiration and photosynthesis across a vast number of organisms. Prior experimental results have shown that the photosynthetic reaction center (RC) from Rhodobacter sphaeroides is only fully functional with a limited set of methoxy-bearing quinones, suggesting that specific interactions with this substituent are required to drive electron transport and the formation of quinol. The nature of these interactions has yet to be determined. Through parameterization of a CHARMM-compatible quinone force field and subsequent molecular dynamics simulations of the quinone-bound RC, in this paper we have investigated and characterized themore » interactions of the protein with the quinones in the QA and QB sites using both equilibrium simulation and thermodynamic integration. In particular, we identify a specific interaction between the 2-methoxy group of ubiquinone in the QB site and the amide nitrogen of GlyL225 that we implicate in locking the orientation of the 2-methoxy group, thereby tuning the redox potential difference between the quinones occupying the QA and QB sites. Finally, disruption of this interaction leads to weaker binding in a ubiquinone analogue that lacks a 2-methoxy group, a finding supported by reverse electron transfer electron paramagnetic resonance experiments of the QA–QB– biradical and competitive binding assays.« less

  3. Kinetic analysis of the thermal stability of the photosynthetic reaction center from Rhodobacter sphaeroides.

    PubMed

    Hughes, Arwel V; Rees, Paul; Heathcote, Peter; Jones, Michael R

    2006-06-01

    The temperature-induced denaturation of the photosynthetic reaction center from Rhodobacter sphaeroides has been studied through the changes that occur in the absorption spectrum of the bound chromophores on heating. At elevated temperatures, the characteristic absorbance bands of the bacteriochlorins bound to the polypeptides within the reaction center are lost, and are replaced by features typical of unbound bacteriochlorophyll and bacteriopheophytin. The kinetics of the spectral changes cannot be explained by a direct conversion from the functional to the denatured form of the protein, and require the presence of at least one intermediate. Possible mechanisms for the transformation via an intermediate are examined using a global analysis of the kinetic data, and the most likely mechanism is shown to involve a reversible transformation between the native state and an off-pathway intermediate, coupled to an irreversible transformation to the denatured state. The activation energies for the transformations between the three components are calculated from the effect of temperature on the individual rate constants, and the likely structural changes of the protein during the temperature-induced transformation are discussed.

  4. Sequence analysis of the cbb3 oxidases and an atomic model for the Rhodobacter sphaeroides enzyme.

    PubMed

    Sharma, Vivek; Puustinen, Anne; Wikström, Mårten; Laakkonen, Liisa

    2006-05-09

    The cbb3-type oxidases are members of the heme-copper oxidase superfamily, distant by sequence comparisons, but sharing common functional characteristics. To understand the minimal common properties of the superfamily, and to learn about cbb3-type oxidases specifically, we have analyzed a wide set of heme-copper oxidase sequences and built a homology model of the catalytic subunit of the cbb3 oxidase from Rhodobacter sphaeroides. We conclude that with regard to the active site surroundings, the cbb3 oxidases greatly resemble the structurally known oxidases, while major differences are found in three segments: the additional N-terminal stretch of ca. 60 amino acids, the segment following helix 3 to the end of helix 5, and the C-terminus from helix 11 onward. The conserved core contains the active site tyrosine and also an analogue of the K-channel of proton transfer, but centered on a well-conserved histidine in the lower part of helix 7. Modeling the variant parts of the enzyme suggests that two periplasmic loops (between helices 3 and 4 and between helices 11 and 12) could interact with each other as a part of the active site structure and might have an important role in proton pumping. An analogue of the D-channel is not found, but an alternative channel might form around helix 9. A preliminary packing model of the trimeric enzyme is also presented.

  5. Pentitol metabolism of Rhodobacter sphaeroides Si4: purification and characterization of a ribitol dehydrogenase.

    PubMed

    Kahle, C; Schneider, K H; Giffhorn, F

    1992-06-01

    The phototrophic bacterium Rhodobacter sphaeroides strain Si4 induced ribitol dehydrogenase (EC 1.1.1.56) when grown on ribitol- or xylitol-containing medium. This ribitol dehydrogenase was purified to apparent homogeneity by ammonium sulphate precipitation, affinity chromatography on Procion red, and chromatography on Q-Sepharose. For the native enzyme an isoelectric point of pH 6.1 and an apparent M(r) of 50,000 was determined. SDS-PAGE yielded a single peptide band of M(r) 25,000 suggesting a dimeric enzyme structure. The ribitol dehydrogenase was specific for NAD+ but unspecific as to its polyol substrate. In order of decreasing activity ribitol, xylitol, erythritol, D-glucitol and D-arabitol were oxidized. The pH optimum of substrate oxidation was 10, and that of substrate reduction was 6.5. The equilibrium constant of the interconversion of ribitol to D-ribulose was determined to be 0.33 nM at pH 7.0 and 25 degrees C. The Km-values determined for ribitol, ribulose, xylitol and NAD+ (in the presence of ribitol) were 6.3, 12.5, 77 and 0.077 mM, respectively. Because of the favourable Km for ribitol, a method for quantitative ribitol determination was elaborated.

  6. ChrR positively regulates transcription of the Rhodobacter sphaeroides cytochrome c2 gene.

    PubMed Central

    Schilke, B A; Donohue, T J

    1995-01-01

    Transcription of the Rhodobacter sphaeroides cytochrome c2 gene (cycA) is negatively regulated by both the presence of oxygen and intermediates in tetrapyrrole biosynthesis. A mutation responsible for uncoupling cycA transcription from tetrapyrrole availability was localized to a gene (chrR) that encodes a 357-amino-acid protein. Analysis of a defined chrR null mutation indicated that this protein positively regulated cycA transcription. From this and other results, it appeared that the positive action of ChrR on cycA transcription is blocked by altering the availability of either heme or some intermediate in tetrapyrrole biosynthesis. A single missense mutation which substitutes an Arg for a Cys at residue 182 of ChrR (C182R) was shown to be necessary and sufficient for the increased cycA transcription seen in the mutant strain Chr4. Thus, it appears that this C182R substitution generated an altered-function form of ChrR. In addition, by analyzing cycA transcription in delta ChrR strains, we showed that ChrR was not required for increased cycA transcription under anaerobic conditions. Instead, our results indicated that ChrR and the response regulator PrrA (J. M. Eraso and S. Kaplan, J. Bacteriol. 176:32-43, 1994) functioned independently at the upstream cycA promoter that is activated under anaerobic conditions. PMID:7721683

  7. ChrR positively regulates transcription of the Rhodobacter sphaeroides cytochrome c2 gene.

    PubMed

    Schilke, B A; Donohue, T J

    1995-04-01

    Transcription of the Rhodobacter sphaeroides cytochrome c2 gene (cycA) is negatively regulated by both the presence of oxygen and intermediates in tetrapyrrole biosynthesis. A mutation responsible for uncoupling cycA transcription from tetrapyrrole availability was localized to a gene (chrR) that encodes a 357-amino-acid protein. Analysis of a defined chrR null mutation indicated that this protein positively regulated cycA transcription. From this and other results, it appeared that the positive action of ChrR on cycA transcription is blocked by altering the availability of either heme or some intermediate in tetrapyrrole biosynthesis. A single missense mutation which substitutes an Arg for a Cys at residue 182 of ChrR (C182R) was shown to be necessary and sufficient for the increased cycA transcription seen in the mutant strain Chr4. Thus, it appears that this C182R substitution generated an altered-function form of ChrR. In addition, by analyzing cycA transcription in delta ChrR strains, we showed that ChrR was not required for increased cycA transcription under anaerobic conditions. Instead, our results indicated that ChrR and the response regulator PrrA (J. M. Eraso and S. Kaplan, J. Bacteriol. 176:32-43, 1994) functioned independently at the upstream cycA promoter that is activated under anaerobic conditions.

  8. Carbon allocation in wild-type and Glc+ Rhodobacter sphaeroides under photoheterotrophic conditions.

    PubMed Central

    Macler, B A; Bassham, J A

    1988-01-01

    The photosynthetic bacterium Rhodobacter sphaeroides is capable of producing H2 via nitrogenase when grown photoheterotrophically in the absence of N2. By using 14C-labeled malate, it was found that greater than 95% of this substrate was catabolized completely to CO2 during H2 production. About 60% of this catabolism was associated with H2 biosynthesis, while almost 40% provided reductant for other cellular purposes. Thus, only a small fraction of malate provided carbon skeletons. The addition of ammonium, which inhibited nitrogenase activity, increased substrate conversion into carbon skeletons threefold. Catabolism of malate occurred primarily via the tricarboxylic acid cycle, but gluconeogenesis was also observed. The wild-type organism grew poorly on glucose, accumulated gluconate and 2-keto-3-deoxygluconate, and did not produce H2. More than 50% of metabolized glucose appeared in carbon skeletons or in storage compounds. A glucose-utilizing mutant was five times more effective in utilizing this substrate. This mutant produced H2 from glucose, using 74% of metabolized substrate for this purpose. Glucose converted to storage products or to other carbon skeletons was reduced to 8%. Fixation of CO2 competed directly with H2 production for reducing equivalents and ATP. Refixation of CO2 released from these substrates under H2-producing conditions was, at most, 10 to 12%. Addition of ammonium increased refixation of respired CO2 to 83%. Patterns of carbon flow of fixation products were associated with the particular strains and culture conditions. PMID:3145710

  9. Absorption and fluorescence spectroscopic characterization of BLUF domain of AppA from Rhodobacter sphaeroides

    NASA Astrophysics Data System (ADS)

    Zirak, P.; Penzkofer, A.; Schiereis, T.; Hegemann, P.; Jung, A.; Schlichting, I.

    2005-08-01

    The BLUF domain of the transcriptional anti-repressor protein AppA from the non-sulfur anoxyphototrophic purple bacterium Rhodobacter sphaeroides was characterized by absorption and emission spectroscopy. The BLUF domain constructs AppA 148 (consisting of amino-acid residues 1-148) and AppA 126 (amino-acid residues 1-126) are investigated. The cofactor of the investigated domains is found to consist of a mixture of the flavins riboflavin, FMN, and FAD. The dark-adapted domains exist in two different active receptor conformations (receptor states) with different sub-nanosecond fluorescence lifetimes (BLUF r,f and BLUF r,sl) and a small non-interacting conformation (BLUF nc). The active receptor conformations are transformed to putative signalling states (BLUF s,f and BLUF s,sl) of low fluorescence efficiency and picosecond fluorescence lifetime by blue-light excitation (light-adapted domains). In the dark at room temperature both signalling states recover back to the initial receptor states with a time constant of about 17 min. A quantum yield of signalling state formation of about 25% was determined by intensity dependent transmission measurements. A photo-cycle scheme is presented including photo-induced charge transfer complex formation, charge recombination, and protein binding pocket reorganisation.

  10. The Extract of Rhodobacter sphaeroides Inhibits Melanogenesis through the MEK/ERK Signaling Pathway

    PubMed Central

    Liu, Wen-Sheng; Kuan, Yu-Diao; Chiu, Kuo-Hsun; Wang, Wei-Kuang; Chang, Fu-Hsin; Liu, Chen-Hsun; Lee, Che-Hsin

    2013-01-01

    Reducing hyperpigmentation has been a big issue for years. Even though pigmentation is a normal mechanism protecting skin from UV-causing DNA damage and oxidative stress, it is still an aesthetic problem for many people. Bacteria can produce some compounds in response to their environment. These compounds are widely used in cosmetic and pharmaceutical applications. Some probiotics have immunomodulatory activities and modulate the symptoms of several diseases. Previously, we found that the extracts of Rhodobacter sphaeroides (Lycogen™) inhibited nitric oxide production and inducible nitric-oxide synthase expression in activated macrophages. In this study, we sought to investigate an anti-melanogenic signaling pathway in α-melanocyte stimulating hormone (α-MSH)-treated B16F10 melanoma cells and zebrafish. Treatment with Lycogen™ inhibited the cellular melanin contents and expression of melanogenesis-related protein, including microphthalmia-associated transcription factor (MITF) and tyrosinase in B16F10 cells. Moreover, Lycogen™ reduced phosphorylation of MEK/ERK without affecting phosphorylation of p38. Meanwhile, Lycogen™ decreased zebrafish melanin expression in a dose-dependent manner. These findings establish Lycogen™ as a new target in melanogenesis and suggest a mechanism of action through the ERK signaling pathway. Our results suggested that Lycogen™ may have potential cosmetic usage in the future. PMID:23736765

  11. Optimization of Influencing Factors on Biomass Accumulation and 5-Aminolevulinic Acid (ALA) Yield in Rhodobacter sphaeroides Wastewater Treatment.

    PubMed

    Liu, Shuli; Li, Xiangkun; Zhang, Guangming; Zhang, Jie

    2015-11-01

    This study aimed to optimize four factors affecting biomass accumulation and 5-aminolevulinic acid (ALA) yield together with pollutants removal in Rhodobacter sphaeroides wastewater treatment. Results showed that it was feasible to produce biomass and ALA in R. sphaeroides wastewater treatment. Microaerobic, 1,000-3,000 lux, and pH 7.0 were optimal conditions for the highest ALA yield of 4.5 ± 0.5 mg/g-biomass. Under these conditions, COD removal and biomass production rate were 93.3 ± 0.9% and 31.8 ± 0.5 mg/l/h, respectively. In addition, trace elements Fe(2+), Mg(2+), Ni(2+), and Zn(2+) further improved the ALA yield, COD removal, and biomass production rate. Specifically, the highest ALA yield (12.5 ± 0.6 mg/g-biomass) was achieved with Fe(2+) addition.

  12. Energy transfer and aggregate size effects in the inhomogeneously broadened core light-harvesting complex of Rhodobacter sphaeroides

    NASA Astrophysics Data System (ADS)

    van Mourik, F.; Visschers, R. W.; van Grondelle, R.

    1992-05-01

    The 4 K fluorescence emission spectrum of the core light-harvesting complex of the photosynthetic purple bacterium Rhodobacter sphaeroides is shown to depend strongly on the wavelength of excitation. It is concluded that the dominant factor determining the absorption spectrum is inhomogeneous broadening. The site-selected emission spectra are interpreted in terms of energy transfer in a cluster of weakly coupled pigments. Each individual cluster is supposed to be a random sample from the total inhomogeneously broadened pigment pool. For small clusters, with efficient energy transfer leading to fast thermalization, the effects of site-selection depend on the cluster-size only. It is shown that site-selective excitation can yield information about the number of coupled pigments in such a cluster. For the purified LH-1 of Rb. sphaeroides the cluster size was estimated to be 16 pigments.

  13. Glutamate transport in Rhodobacter sphaeroides is mediated by a novel binding protein-dependent secondary transport system

    PubMed Central

    Jacobs, Mariken H. J.; van der Heide, Tiemen; Driessen, Arnold J. M.; Konings, Wil N.

    1996-01-01

    Growth of a glutamate transport-deficient mutant of Rhodobacter sphaeroides on glutamate as sole carbon and nitrogen source can be restored by the addition of millimolar amounts of Na+. Uptake of glutamate (Kt of 0.2 μM) by the mutant strictly requires Na+ (Km of 25 mM) and is inhibited by ionophores that collapse the proton motive force (pmf). The activity is osmotic-shock-sensitive and can be restored in spheroplasts by the addition of osmotic shock fluid. Transport of glutamate is also observed in membrane vesicles when Na+, a proton motive force, and purified glutamate binding protein are present. Both transport and binding is highly specific for glutamate. The Na+-dependent glutamate transporter of Rb. sphaeroides is an example of a secondary transport system that requires a periplasmic binding protein and may define a new family of bacterial transport proteins. PMID:8917497

  14. Glutamate transport in Rhodobacter sphaeroides is mediated by a novel binding protein-dependent secondary transport system.

    PubMed

    Jacobs, M H; van der Heide, T; Driessen, A J; Konings, W N

    1996-11-12

    Growth of a glutamate transport-deficient mutant of Rhodobacter sphaeroides on glutamate as sole carbon and nitrogen source can be restored by the addition of millimolar amounts of Na+. Uptake of glutamate (Kt of 0.2 microM) by the mutant strictly requires Na+ (K(m) of 25 mM) and is inhibited by ionophores that collapse the proton motive force (pmf). The activity is osmotic-shock-sensitive and can be restored in spheroplasts by the addition of osmotic shock fluid. Transport of glutamate is also observed in membrane vesicles when Na+, a proton motive force, and purified glutamate binding protein are present. Both transport and binding is highly specific for glutamate. The Na(+)-dependent glutamate transporter of Rb. sphaeroides is an example of a secondary transport system that requires a periplasmic binding protein and may define a new family of bacterial transport proteins.

  15. Isolation of SIP, a protein that interacts with SPB, a possible transcriptional regulatory factor in Rhodobacter sphaeroides.

    PubMed

    Shimada, Hiroshi; Ishida, Kazuhiro; Machiya, Yasuhiro; Takamiya, Ken-Ichiro

    2007-10-01

    SPB is a transcriptional factor in Rhodobacter sphaeroides that represses expression of the puf operon under aerobic or semi-aerobic light conditions. Here, we identified a 17,500 Da protein designated SIP (SPB interaction protein) that interacts with SPB, as determined by binding to an SPB-His(x6) fusion protein-Ni column. The SPB-SIP interaction in vivo was confirmed by an immunoprecipitation assay. The level of transcripts and protein of SIP did not differ for all growth conditions tested, indicating that regulation of the SIP-SPB interaction, if any, is not through modulation of sip or spb expression but rather by modification of the proteins.

  16. Multi-PAS domain-mediated protein oligomerization of PpsR from Rhodobacter sphaeroides

    SciTech Connect

    Heintz, Udo; Meinhart, Anton; Winkler, Andreas

    2014-03-01

    Crystal structures of two truncated variants of the transcription factor PpsR from R. sphaeroides are presented that enabled the phasing of a triple PAS domain construct. Together, these structures reveal the importance of α-helical PAS extensions for multi-PAS domain-mediated protein oligomerization and function. Per–ARNT–Sim (PAS) domains are essential modules of many multi-domain signalling proteins that mediate protein interaction and/or sense environmental stimuli. Frequently, multiple PAS domains are present within single polypeptide chains, where their interplay is required for protein function. Although many isolated PAS domain structures have been reported over the last decades, only a few structures of multi-PAS proteins are known. Therefore, the molecular mechanism of multi-PAS domain-mediated protein oligomerization and function is poorly understood. The transcription factor PpsR from Rhodobacter sphaeroides is such a multi-PAS domain protein that, in addition to its three PAS domains, contains a glutamine-rich linker and a C-terminal helix–turn–helix DNA-binding motif. Here, crystal structures of two N-terminally and C-terminally truncated PpsR variants that comprise a single (PpsR{sub Q-PAS1}) and two (PpsR{sub N-Q-PAS1}) PAS domains, respectively, are presented and the multi-step strategy required for the phasing of a triple PAS domain construct (PpsR{sub ΔHTH}) is illustrated. While parts of the biologically relevant dimerization interface can already be observed in the two shorter constructs, the PpsR{sub ΔHTH} structure reveals how three PAS domains enable the formation of multiple oligomeric states (dimer, tetramer and octamer), highlighting that not only the PAS cores but also their α-helical extensions are essential for protein oligomerization. The results demonstrate that the long helical glutamine-rich linker of PpsR results from a direct fusion of the N-cap of the PAS1 domain with the C-terminal extension of the N-domain that

  17. The hydrogen-bond switch reaction of the Blrb Bluf domain of Rhodobacter sphaeroides.

    PubMed

    Mathes, Tilo; van Stokkum, Ivo H M; Bonetti, Cosimo; Hegemann, Peter; Kennis, John T M

    2011-06-23

    The BlrB protein from Rhodobacter sphaeroides is a small 136 amino acid photoreceptor belonging to the BLUF family of blue light receptors. It contains merely the conserved BLUF fold responsible for binding the flavin pigment and a short C-terminal extension of unknown function. We investigated the primary photoreactions of BlrB by picosecond fluorescence and transient absorption spectroscopy. After excitation of the flavin the fluorescence decays in an H/D isotope independent manner with time constants of 21 and 390 ps, indicating a BLUF characteristic heterogeneous excited state quenched by electron transfer. By transient absorption spectroscopy, we observed a rapid relaxation of a vibrationally hot excited state within 6 ps upon excitation at 400 nm. The relaxed excited state evolves biexponentially with 18 ps (27%) and 216 ps (73%) into the signaling state spectrum indicated by a growing absorptive feature at 492 nm. Additionally, a broad triplet feature is observed as residual absorbance at a delay of 5 ns, which we attribute to derive from a significant fraction of free flavin in the sample. The photochemistry of BlrB is similar to other small BLUF proteins in respect to the fast formation of the photoproduct but does not resolve any further intermediates. We compare the photoreaction with other BLUF proteins on the basis of available spectroscopic data and crystal structures. An arginine close to the C2═O carbonyl of the flavin is likely to be a key determinant for the fast electron transfer in BlrB. Additionally, the orientation of the electron-donating tyrosine in respect to the flavin might play a role in the so far unique kinetic separation of the semiquinonic intermediates in Slr1694.

  18. Characterization and localization of phosphatidylglycerophosphate and phosphatidylserine synthases in Rhodobacter sphaeroides.

    PubMed

    Radcliffe, C W; Steiner, F X; Carman, G M; Niederman, R A

    1989-01-01

    Catalytic properties and membrane associations of the phosphatidylglycerophosphate (PGP) and phosphatidylserine (PS) synthases of Rhodobacter sphaeroides were examined to further characterize sites of phospholipid biosynthesis. In preparations of cytoplasmic membrane (CM) enriched in these activities, apparent Km values of PGP synthase were 90 microM for sn-glycerol-3-phosphate and 60 microM for CDP-diacylglycerol; the apparent Km of PS synthase for L-serine was near 165 microM. Both enzymes required Triton X-100 with optimal PS synthase activity at a detergent/CDP-diacylglycerol (mol/mol) ratio of 7.5:1.0, while for optimal PGP synthase, a range of 10-50:1.0 was observed. Unlike the enzyme in Escherichia coli and several other Gram-negative bacteria, the PS synthase activity had a specific requirement for magnesium and was tightly associated with membranes rather than ribosomes in crude cell extracts. Sedimentation studies suggested that the PGP synthase was distributed uniformly over the CM in both chemoheterotrophically and photoheterotrophically grown cells, while the PS synthase was confined mainly to a vesicular CM fraction. Solubilized PGP synthase activity migrated as a single band with a pI value near 5.5 in a chromato-focusing column and 5.8 on isoelectric focusing; in the latter procedure, the pI was shifted to 5.3 in the presence of CDP-diacylglycerol. The PGP synthase activity gave rise to a single polypeptide band in lithium dodecyl sulfate-polyacrylamide gel electrophoresis at 4 degrees C.

  19. Multi-PAS domain-mediated protein oligomerization of PpsR from Rhodobacter sphaeroides

    PubMed Central

    Heintz, Udo; Meinhart, Anton; Winkler, Andreas

    2014-01-01

    Per–ARNT–Sim (PAS) domains are essential modules of many multi-domain signalling proteins that mediate protein interaction and/or sense environmental stimuli. Frequently, multiple PAS domains are present within single polypeptide chains, where their interplay is required for protein function. Although many isolated PAS domain structures have been reported over the last decades, only a few structures of multi-PAS proteins are known. Therefore, the molecular mechanism of multi-PAS domain-mediated protein oligomerization and function is poorly understood. The transcription factor PpsR from Rhodobacter sphaeroides is such a multi-PAS domain protein that, in addition to its three PAS domains, contains a glutamine-rich linker and a C-terminal helix–turn–helix DNA-binding motif. Here, crystal structures of two N-terminally and C-terminally truncated PpsR variants that comprise a single (PpsRQ-PAS1) and two (PpsRN-Q-PAS1) PAS domains, respectively, are presented and the multi-step strategy required for the phasing of a triple PAS domain construct (PpsRΔHTH) is illustrated. While parts of the biologically relevant dimerization interface can already be observed in the two shorter constructs, the PpsRΔHTH structure reveals how three PAS domains enable the formation of multiple oligomeric states (dimer, tetramer and octamer), highlighting that not only the PAS cores but also their α-helical extensions are essential for protein oligomerization. The results demonstrate that the long helical glutamine-rich linker of PpsR results from a direct fusion of the N-cap of the PAS1 domain with the C-­terminal extension of the N-domain that plays an important role in signal transduction. PMID:24598755

  20. Transient grating spectroscopy and the calorimetry of photosynthesis in Rhodobacter sphaeroides

    NASA Astrophysics Data System (ADS)

    McCauley, Micah John

    2001-07-01

    Enormous numbers and varieties of experiments have been performed to elucidate the mechanisms of photosynthesis. Within the last twenty years, a host of new studies were enabled by the techniques of ultrafast laser spectroscopy. This work continues to explore the exchange of energy on the picosecond and even the femtosecond regime. Yet in even the relatively simple and determined structure of the photosynthetic centers in the bacteria Rhodobacter sphaeroides, the mechanisms and energetics are unclear. The early events in photosynthesis allow energy trapping through resonance energy transfer and charge separation, in what appears to be a mixture of classical and quantum mechanical motion with strong interactions with the surrounding environment. The technique of transient thermal grating spectroscopy offers unique insight into the mechanisms of energy relaxation in photosynthesis. A pair of laser pulse are tuned to the excitation wavelength and crossed inside the sample. The resulting pattern of interference drives excitation and the subsequent relaxation forms a density grating which diffracts a time-delayed third beam. Thus the energies and the rates of motion may be discerned. The peripheral light harvesting antenna (LH2) is composed of polypeptides that coordinate rings of bacteriochlorophyll. Upon excitation, energy migrates around the ring until intra-ring transfer may take place. Samples of LH2 were purified and studied. While characteristic times of energy release were determined, a volume change due to solvent interaction was found as well. In the membrane spanning protein known as the reaction center, the energy of the light harvesting rings is ultimately transferred to a pair of chlorophyll. From here, the energy is localized onto an electron, which rapidly (picoseconds) transfers to other active pigments. Samples of wild-type and the mutated (M)214H reaction centers were purified and examined. By studying the temperature dependence of the signals, the

  1. Photo dynamics of BLUF domain mutant H44R of AppA from Rhodobacter sphaeroides

    NASA Astrophysics Data System (ADS)

    Zirak, P.; Penzkofer, A.; Hegemann, P.; Mathes, T.

    2007-05-01

    The photo-cycle dynamics of the H44R mutant of the BLUF domain of the transcriptional anti-repressor protein AppA (AppA-H44R) from the non-sulfur anoxyphototropic purple bacterium Rhodobacter sphaeroides is studied in order to gain information on the involvement of His44 in the photo-cyclic mechanism of the AppA BLUF domain and to add information to the involved processes. The amino acid residue histidine at position 44 is replaced by arginine. A 12 nm red-shifted signalling state is formed upon blue-light excitation, while in wild-type AppA (AppA-wt) the red-shift is 16 nm. The recovery to the receptor dark state is approximately a factor of 2.5 faster ( τrec ≈ 6.5 min) than the recovery of the wild-type counterpart. Extended light exposure of the mutant causes photo-degradation of flavin (mainly free flavin conversion to lumichrome and re-equilibration between free and non-covalently bound flavin) and protein aggregation (showing up as light scattering). No photo-degradation was observed for AppA-wt. The quantum efficiency of signalling-state formation determined by intensity dependent absorption measurements is found to be ϕs ≈ 0.3 (for AppA-wt: ϕs ≈ 0.24). A two-component single-exponential fluorescence relaxation was observed, which is interpreted as fast fluorescence quenching to an equilibrium value by photo-induced electron transfer followed by slower fluorescence decay due to charge recombination. Based on the experimental findings, an extended photo-cycle model for BLUF domains is proposed.

  2. Light-Induced Subunit Dissociation by a LOV domain Photoreceptor from Rhodobacter sphaeroides

    PubMed Central

    Conrad, Karen S.; Bilwes, Alexandrine M.; Crane, Brian R.

    2013-01-01

    Light-oxygen-voltage (LOV) domains bind a flavin chromophore to serve as blue light sensors in a wide range of eukaryotic and prokaryotic proteins. LOV domains are associated with a variable effector domain or a separate protein signaling partner to execute a wide variety of functions that include regulation of kinases, generation of antisigma-factor antagonists, and regulation of circadian clocks. Here we present the crystal structure, photocycle kinetics, association properties, and spectroscopic features of a full-length LOV protein from Rhodobacter sphaeroides (RsLOV). RsLOV exhibits N-terminal and C-terminal helical extensions that form an unusual helical bundle at its dimer interface with some resemblance to the helical transducer of sensory rhodopsin II. The blue light-induced conformational changes of RsLOV revealed from a comparison of light and dark state crystal structures support a shared signaling mechanism of LOV domain proteins that originates with the light-induced formation of a flavin-cysteinyl photoadduct. Adduct formation disrupts hydrogen bonding in the active site and propagates structural changes through the LOV domain core to the N- and C-terminal extensions. Single residue variants in the active site and dimer interface of RsLOV alter photoadduct lifetimes and induce structural changes that perturb oligomeric state. Size exclusion chromatography, multi-angle light scattering, small-angle X-ray scattering and cross-linking studies indicate that RsLOV dimerizes in the dark, but upon light excitation, dissociates into monomers. This light-induced switch in oligomeric state may prove useful for engineering molecular associations in controlled cellular settings. PMID:23252338

  3. Isolation of a thermotolerant photosynthetic bacterium, Rhodobacter sphaeroides Strain, NAT, and its capacity for oil and chemical oxygen demand removal at high temperatures.

    PubMed

    Yamaoka, Yosuke; Takeno, Kenji; Shinkawa, Hidenori; Noparatnaraporn, Napavarn; Sasaki, Ken

    2008-06-01

    A thermotolerant photosynthetic bacterium NAT identified as Rhodobacter sphaeroides was isolated. When alginate-immobilized cells of strain NAT were used in high-temperature treatment of artificial sewage wastewater containing oil, the chemical oxygen demand (COD) decreased by 80% and 76% of the oil was removed after 96 h of treatment at 55 degrees C. Lipase activity was observed in the culture.

  4. Structural model and excitonic properties of the dimeric RC-LH1-PufX complex from Rhodobacter sphaeroides

    PubMed Central

    Şener, Melih; Hsin, Jen; Trabuco, Leonardo G.; Villa, Elizabeth; Qian, Pu; Hunter, C. Neil; Schulten, Klaus

    2009-01-01

    The light-harvesting apparatus of the purple bacterial photosynthetic unit consists of a pool of peripheral light-harvesting complexes that transfer excitation energy to a reaction center (RC) via the surrounding pigment-protein complex LH1. Recent electron microscopy and atomic force microscopy studies have revealed that RC-LH1 units of Rhodobacter sphaeroides form membrane-bending dimeric complexes together with the polypeptide PufX. We present a structural model for these RC-LH1-PufX dimeric complexes constructed using the molecular dynamics flexible fitting method based on an EM density map. The arrangement of the LH1 BChls displays a distortion near the proposed location of the PufX polypeptide. The resulting atomic model for BChl arrays is used to compute the excitonic properties of the dimeric RC-LH1 complex. A comparison is presented between the structural and excitonic features of the S-shaped dimeric BChl array of Rhodobacter sphaeroides and the circular BChl arrangement found in other purple bacteria. PMID:20161332

  5. Genetic evidence for the role of isocytochrome c2 in photosynthetic growth of Rhodobacter sphaeroides Spd mutants.

    PubMed

    Rott, M A; Witthuhn, V C; Schilke, B A; Soranno, M; Ali, A; Donohue, T J

    1993-01-01

    In Rhodobacter sphaeroides, cytochrome c2 (cyt c2)-deficient mutants are photosynthetically incompetent (PS-). However, mutations which suppress the photosynthetic deficiency (spd mutations) of cyt c2 mutants increase the levels of a cyt c2 isoform, isocyt c2. To determine whether isocyt c2 was required for photosynthetic growth of Spd mutants, we used Tn5 mutagenesis to generate a PS- mutant (TP39) that lacks both cyt c2 and isocyt c2. DNA sequence analysis of wild-type DNA that restores isocyt c2 production and photosynthetic growth to TP39 indicates that it encodes the isocyt c2 structural gene, cycI. The Tn5 insertion in TP39 is approximately 1.5 kb upstream of cycI, and our results show that it is polar onto cycI. The cycI gene has been physically mapped to a region of chromosome I that is approximately 700 kb from the R. sphaeroides photosynthetic gene cluster. Construction of a defined cycI null mutant and complementation of several mutants with the cycI gene under the control of the cyt c2 promoter region indicate that an increase in the levels of isocyt c2 alone is necessary and sufficient for photosynthetic growth in the absence of cyt c2. The data are discussed in terms of the obligate role of isocyt c2 in cyt c2-independent photosynthesis of R. sphaeroides.

  6. Genetic evidence for the role of isocytochrome c2 in photosynthetic growth of Rhodobacter sphaeroides Spd mutants.

    PubMed Central

    Rott, M A; Witthuhn, V C; Schilke, B A; Soranno, M; Ali, A; Donohue, T J

    1993-01-01

    In Rhodobacter sphaeroides, cytochrome c2 (cyt c2)-deficient mutants are photosynthetically incompetent (PS-). However, mutations which suppress the photosynthetic deficiency (spd mutations) of cyt c2 mutants increase the levels of a cyt c2 isoform, isocyt c2. To determine whether isocyt c2 was required for photosynthetic growth of Spd mutants, we used Tn5 mutagenesis to generate a PS- mutant (TP39) that lacks both cyt c2 and isocyt c2. DNA sequence analysis of wild-type DNA that restores isocyt c2 production and photosynthetic growth to TP39 indicates that it encodes the isocyt c2 structural gene, cycI. The Tn5 insertion in TP39 is approximately 1.5 kb upstream of cycI, and our results show that it is polar onto cycI. The cycI gene has been physically mapped to a region of chromosome I that is approximately 700 kb from the R. sphaeroides photosynthetic gene cluster. Construction of a defined cycI null mutant and complementation of several mutants with the cycI gene under the control of the cyt c2 promoter region indicate that an increase in the levels of isocyt c2 alone is necessary and sufficient for photosynthetic growth in the absence of cyt c2. The data are discussed in terms of the obligate role of isocyt c2 in cyt c2-independent photosynthesis of R. sphaeroides. Images PMID:8380401

  7. Quantifying the effects of light intensity on bioproduction and maintenance energy during photosynthetic growth of Rhodobacter sphaeroides.

    PubMed

    Imam, Saheed; Fitzgerald, Colin M; Cook, Emily M; Donohue, Timothy J; Noguera, Daniel R

    2015-02-01

    Obtaining a better understanding of the physiology and bioenergetics of photosynthetic microbes is an important step toward optimizing these systems for light energy capture or production of valuable commodities. In this work, we analyzed the effect of light intensity on bioproduction, biomass formation, and maintenance energy during photoheterotrophic growth of Rhodobacter sphaeroides. Using data obtained from steady-state bioreactors operated at varying dilution rates and light intensities, we found that irradiance had a significant impact on biomass yield and composition, with significant changes in photopigment, phospholipid, and biopolymer storage contents. We also observed a linear relationship between incident light intensity and H2 production rate between 3 and 10 W m(-2), with saturation observed at 100 W m(-2). The light conversion efficiency to H2 was also higher at lower light intensities. Photosynthetic maintenance energy requirements were also significantly affected by light intensity, with links to differences in biomass composition and the need to maintain redox homeostasis. Inclusion of the measured condition-dependent biomass and maintenance energy parameters and the measured photon uptake rate into a genome-scale metabolic model for R. sphaeroides (iRsp1140) significantly improved its predictive performance. We discuss how our analyses provide new insights into the light-dependent changes in bioenergetic requirements and physiology during photosynthetic growth of R. sphaeroides and potentially other photosynthetic organisms.

  8. The lipidome of the photosynthetic bacterium Rhodobacter sphaeroides R26 is affected by cobalt and chromate ions stress.

    PubMed

    Calvano, Cosima Damiana; Italiano, Francesca; Catucci, Lucia; Agostiano, Angela; Cataldi, Tommaso R I; Palmisano, Francesco; Trotta, Massimo

    2014-02-01

    A detailed characterization of membrane lipids of the photosynthetic bacterium Rhodobacter (R.) sphaeroides was accomplished by thin-layer chromatography coupled with matrix-assisted laser desorption ionization mass spectrometry. Such an approach allowed the identification of the main membrane lipids belonging to different classes, namely cardiolipins (CLs), phosphatidylethanolamines, phosphatidylglycerols (PGs), phosphatidylcholines, and sulfoquinovosyldiacylglycerols (SQDGs). Thus, the lipidomic profile of R. sphaeroides R26 grown in abiotic stressed conditions by exposure to bivalent cobalt cation and chromate oxyanion, was investigated. Compared to bacteria grown under control conditions, significant lipid alterations take place under both stress conditions; cobalt exposure stress results in the relative content increase of CLs and SQDGs, most likely compensating the decrease in PGs content, whereas chromate stress conditions result in the relative content decrease of both PGs and SQDGs, leaving CLs unaltered. For the first time, the response of R. sphaeroides to heavy metals as Co(2+) and CrO4 (2-) is reported and changes in membrane lipid profiles were rationalised.

  9. The role of cysteine 160 in thiamine diphosphate binding of the Calvin-Benson-Bassham cycle transketolase of Rhodobacter sphaeroides.

    PubMed

    Bobst, Cedric E; Tabita, F Robert

    2004-06-01

    The transketolase gene (cbbT) that encodes the Calvin-Benson-Bassham pathway transketolase (CbbT) of Rhodobacter sphaeroides was overexpressed in Escherichia coli and the recombinant protein purified to homogeneity. Like other transketolases, R. sphaeroides CbbT was found to be inactivated in the presence of oxygen. At its optimal pH of 7.8, CbbT displays a specific activity of 37 U/mg, a KR5P of 949 microM, a KXu5P of 11 microM, and a KThDP of 1.8 microM. Cysteine 160, equivalent to Cys159 of the yeast enzyme, is found within the active site and is loosely conserved amongst several sources of transketolase. To investigate the role of cysteine 160 found in the active site of R. sphaeroides CbbT, this residue was targeted for mutagenesis. Cys160 was changed to alanine, serine, aspartate, and glutamate. To compare the effect of these mutations on ThDP binding, spectral techniques were employed in addition to analysis by enzymatic activity. Fluorescence quenching was used to measure both equilibrium binding constants as well as first order rates of binding. The results of these studies indicated that Cys160 played an important and substantial role in cofactor binding, revealing the importance of this loosely conserved residue. In addition, the Cys160 mutants did not appear to alter oxygen-mediated inactivation.

  10. Weak temperature dependence of P (+) H A (-) recombination in mutant Rhodobacter sphaeroides reaction centers.

    PubMed

    Gibasiewicz, Krzysztof; Białek, Rafał; Pajzderska, Maria; Karolczak, Jerzy; Burdziński, Gotard; Jones, Michael R; Brettel, Klaus

    2016-06-01

    In contrast with findings on the wild-type Rhodobacter sphaeroides reaction center, biexponential P (+) H A (-)  → PH A charge recombination is shown to be weakly dependent on temperature between 78 and 298 K in three variants with single amino acids exchanged in the vicinity of primary electron acceptors. These mutated reaction centers have diverse overall kinetics of charge recombination, spanning an average lifetime from ~2 to ~20 ns. Despite these differences a protein relaxation model applied previously to wild-type reaction centers was successfully used to relate the observed kinetics to the temporal evolution of the free energy level of the state P (+) H A (-) relative to P (+) B A (-) . We conclude that the observed variety in the kinetics of charge recombination, together with their weak temperature dependence, is caused by a combination of factors that are each affected to a different extent by the point mutations in a particular mutant complex. These are as follows: (1) the initial free energy gap between the states P (+) B A (-) and P (+) H A (-) , (2) the intrinsic rate of P (+) B A (-)  → PB A charge recombination, and (3) the rate of protein relaxation in response to the appearance of the charge separated states. In the case of a mutant which displays rapid P (+) H A (-) recombination (ELL), most of this recombination occurs in an unrelaxed protein in which P (+) B A (-) and P (+) H A (-) are almost isoenergetic. In contrast, in a mutant in which P (+) H A (-) recombination is relatively slow (GML), most of the recombination occurs in a relaxed protein in which P (+) H A (-) is much lower in energy than P (+) H A (-) . The weak temperature dependence in the ELL reaction center and a YLH mutant was modeled in two ways: (1) by assuming that the initial P (+) B A (-) and P (+) H A (-) states in an unrelaxed protein are isoenergetic, whereas the final free energy gap between these states following the protein relaxation is large (~250 meV or

  11. Integration of energy and electron transfer processes in the photosynthetic membrane of Rhodobacter sphaeroides

    PubMed Central

    Cartron, Michaël L.; Olsen, John D.; Sener, Melih; Jackson, Philip J.; Brindley, Amanda A.; Qian, Pu; Dickman, Mark J.; Leggett, Graham J.; Schulten, Klaus; Hunter, C. Neil

    2014-01-01

    Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll–protein complexes responsible for light absorption, photochemistry and quinol (QH2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc1 (cytbc1) complexes that oxidise QH2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc1 complexes with gold nanobeads, each attached by a Histidine10 (His10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc1 complexes occur as dimers in the membrane. The cytbc1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC-LH1-PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC-LH1-PufX complexes tended to co-purify with cytbc1, whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC-LH1-PufX arrays, but not with a fixed, stoichiometric cytbc1-RC-LH1-PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc1, ATP synthase, cytaa3 and cytcbb3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1-RC-PufX dimers & 2 RC-LH1-PufX monomers, 4 cytbc1 dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities. PMID:24530865

  12. Identification of key residues that confer Rhodobacter sphaeroides LPS activity at horse TLR4/MD-2.

    PubMed

    Irvine, Katherine L; Gangloff, Monique; Walsh, Catherine M; Spring, David R; Gay, Nicholas J; Bryant, Clare E

    2014-01-01

    The molecular determinants underpinning how hexaacylated lipid A and tetraacylated precursor lipid IVa activate Toll-like receptor 4 (TLR4) are well understood, but how activation is induced by other lipid A species is less clear. Species specificity studies have clarified how TLR4/MD-2 recognises different lipid A structures, for example tetraacylated lipid IVa requires direct electrostatic interactions for agonism. In this study, we examine how pentaacylated lipopolysaccharide from Rhodobacter sphaeroides (RSLPS) antagonises human TLR4/MD-2 and activates the horse receptor complex using a computational approach and cross-species mutagenesis. At a functional level, we show that RSLPS is a partial agonist at horse TLR4/MD-2 with greater efficacy than lipid IVa. These data suggest the importance of the additional acyl chain in RSLPS signalling. Based on docking analysis, we propose a model for positioning of the RSLPS lipid A moiety (RSLA) within the MD-2 cavity at the TLR4 dimer interface, which allows activity at the horse receptor complex. As for lipid IVa, RSLPS agonism requires species-specific contacts with MD-2 and TLR4, but the R2 chain of RSLA protrudes from the MD-2 pocket to contact the TLR4 dimer in the vicinity of proline 442. Our model explains why RSLPS is only partially dependent on horse TLR4 residue R385, unlike lipid IVa. Mutagenesis of proline 442 into a serine residue, as found in human TLR4, uncovers the importance of this site in RSLPS signalling; horse TLR4 R385G/P442S double mutation completely abolishes RSLPS activity without its counterpart, human TLR4 G384R/S441P, being able to restore it. Our data highlight the importance of subtle changes in ligand positioning, and suggest that TLR4 and MD-2 residues that may not participate directly in ligand binding can determine the signalling outcome of a given ligand. This indicates a cooperative binding mechanism within the receptor complex, which is becoming increasingly important in TLR

  13. Identification of Key Residues That Confer Rhodobacter sphaeroides LPS Activity at Horse TLR4/MD-2

    PubMed Central

    Walsh, Catherine M.; Spring, David R.; Gay, Nicholas J.; Bryant, Clare E.

    2014-01-01

    The molecular determinants underpinning how hexaacylated lipid A and tetraacylated precursor lipid IVa activate Toll-like receptor 4 (TLR4) are well understood, but how activation is induced by other lipid A species is less clear. Species specificity studies have clarified how TLR4/MD-2 recognises different lipid A structures, for example tetraacylated lipid IVa requires direct electrostatic interactions for agonism. In this study, we examine how pentaacylated lipopolysaccharide from Rhodobacter sphaeroides (RSLPS) antagonises human TLR4/MD-2 and activates the horse receptor complex using a computational approach and cross-species mutagenesis. At a functional level, we show that RSLPS is a partial agonist at horse TLR4/MD-2 with greater efficacy than lipid IVa. These data suggest the importance of the additional acyl chain in RSLPS signalling. Based on docking analysis, we propose a model for positioning of the RSLPS lipid A moiety (RSLA) within the MD-2 cavity at the TLR4 dimer interface, which allows activity at the horse receptor complex. As for lipid IVa, RSLPS agonism requires species-specific contacts with MD-2 and TLR4, but the R2 chain of RSLA protrudes from the MD-2 pocket to contact the TLR4 dimer in the vicinity of proline 442. Our model explains why RSLPS is only partially dependent on horse TLR4 residue R385, unlike lipid IVa. Mutagenesis of proline 442 into a serine residue, as found in human TLR4, uncovers the importance of this site in RSLPS signalling; horse TLR4 R385G/P442S double mutation completely abolishes RSLPS activity without its counterpart, human TLR4 G384R/S441P, being able to restore it. Our data highlight the importance of subtle changes in ligand positioning, and suggest that TLR4 and MD-2 residues that may not participate directly in ligand binding can determine the signalling outcome of a given ligand. This indicates a cooperative binding mechanism within the receptor complex, which is becoming increasingly important in TLR

  14. Integration of energy and electron transfer processes in the photosynthetic membrane of Rhodobacter sphaeroides

    DOE PAGES

    Cartron, Michaël L.; Olsen, John D.; Sener, Melih; ...

    2014-02-13

    Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll–protein complexes responsible for light absorption, photochemistry and quinol (QH2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc1 (cytbc1) complexes that oxidise QH2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc1 complexes with gold nanobeads, each attached by a Histidine10 (His10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc1more » complexes occur as dimers in the membrane. The cytbc1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC-LH1-PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC-LH1-PufX complexes tended to co-purify with cytbc1, whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC-LH1-PufX arrays, but not with a fixed, stoichiometric cytbc1-RC-LH1- PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc1, ATP synthase, cytaa3 and cytcbb3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1-RC-PufX dimers & 2 RC-LH1-PufX monomers, 4 cytbc1 dimers and 2 ATP synthases. In conclusion, simulation of the interconnected energy, electron and proton transfer processes showed a halfmaximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities.« less

  15. Biocontrol activity and patulin-removal effects of Bacillus subtilis, Rhodobacter sphaeroides and Agrobacterium tumefaciens against Penicillium expansum.

    PubMed

    Wang, Y; Yuan, Y; Liu, B; Zhang, Z; Yue, T

    2016-11-01

    This study was conducted to evaluate the biocontrol potential of Bacillus subtilis CICC 10034, Rhodobacter sphaeroides CGMCC 1.2182 and Agrobacterium tumefaciens CGMCC 1.2554 against patulin (PAT)-producer Penicillium expansum and their ability to remove PAT. Bacillus subtilis effectively inhibited P. expansum both on apples and in in vitro experiments, which reduced the rot diameter on apples by 38% compared with the control. The reduction was followed by those induced by A. tumefaciens (27·63%) and R. sphaeroides (23·67%). None of the cell-free supernatant (CFS) was able to prevent pathogen growth. Three antagonists could suppress PAT production by P. expansum on apples by 98·5, 93·7 and 94·99% after treatment with B. subtilis, R. sphaeroides and A. tumefaciens respectively. In addition, the three strains led to a 0·56-1·47 log CFU g(-1) reduction in colony number of P. expansum on apples. Survival of antagonists on apple wounds revealed their tolerance to PAT. Furthermore, both live and autoclaved cells of three strains efficiently adsorbed artificially spiked PAT from medium. The selected antagonists could be applied before harvesting to control apple infection by PAT-producing fungi and also during processing to act as PAT detoxifiers. Since little information related to the capability of R. sphaeroides and A. tumefaciens to inhibit P. expansum is currently available, the results of this study provide some new perspectives to the biocontrol field. © 2016 The Society for Applied Microbiology.

  16. (1)H, (13)C and (15)N resonance assignments for the response regulator CheY3 from Rhodobacter sphaeroides.

    PubMed

    Varela, Lorena; Bell, Christian H; Armitage, Judith P; Redfield, Christina

    2016-10-01

    Rhodobacter sphaeroides has emerged as a model system for studies of the complex chemotaxis pathways that are a hallmark of many non-enteric bacteria. The genome of R. sphaeroides encodes two sets of flagellar genes, fla1 and fla2, that are controlled by three different operons. Each operon encodes homologues of most of the proteins required for the well-studied E. coli chemotaxis pathway. R. sphaeroides has six homologues of the response regulator CheY that are localized to and are regulated by different clusters of chemosensory proteins in the cell and have different effects on chemotaxis. CheY6 is the major CheY stopping the fla1 flagellar motor and associated with a cytoplasmically localised chemosensory pathway. CheY3 and CheY4 are associated with a membrane localised polar chemosensory cluster, and can bind to but not stop the motor. CheY6 and either CheY3 or CheY4 are required for chemotaxis. We are using NMR spectroscopy to characterise and compare the structure and dynamics of CheY3 and CheY6 in solution. We are interested in defining the conformational changes that occur upon activation of these two proteins and to identify differences in their properties that can explain the different functions they play in chemotaxis in R. sphaeroides. Here we present the (1)H, (13)C and (15)N assignments for CheY3 in its active, inactive and Mg(2+)-free apo form. These assignments provide the starting point for detailed investigations of the structure and function of CheY3.

  17. Characterization of D-tagatose-3-epimerase from Rhodobacter sphaeroides that converts D-fructose into D-psicose.

    PubMed

    Zhang, Longtao; Mu, Wanmeng; Jiang, Bo; Zhang, Tao

    2009-06-01

    A non-characterized gene, previously proposed as the D-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with D-fructose and decreased for other substrates in the order: D-tagatose, D-psicose, D-ribulose, D-xylulose and D-sorbose. Its activity was maximal at pH 9 and 40 degrees C while being enhanced by Mn(2+). At pH 9 and 40 degrees C, 118 g D-psicose l(-1) was produced from 700 g D-fructose l(-1) after 3 h.

  18. Direct Visualization of Exciton Reequilibration in the LH1 and LH2 Complexes of Rhodobacter sphaeroides by Multipulse Spectroscopy

    SciTech Connect

    Stuart, Thomas A. Cohen; Vengris, Mikas; Novoderezhkin, Vladimir I.; Cogdell, Richard J.; Hunter, C. Neil; van Grondelle, Rienk

    2011-01-01

    The dynamics of the excited states of the light-harvesting complexes LH1 and LH2 of Rhodobacter sphaeroides are governed, mainly, by the excitonic nature of these ring-systems. In a pump-dump-probe experiment, the first pulse promotes LH1 or LH2 to its excited state and the second pulse dumps a portion of the excited state. By selective dumping, we can disentangle the dynamics normally hidden in the excited-state manifold. We find that by using this multiple-excitation technique we can visualize a 400-fs reequilibration reflecting relaxation between the two lowest exciton states that cannot be directly explored by conventional pump-probe. An oscillatory feature is observed within the exciton reequilibration, which is attributed to a coherent motion of a vibrational wavepacket with a period of ~150 fs. Our disordered exciton model allows a quantitative interpretation of the observed reequilibration processes occurring in these antennas.

  19. Reversible inactivation and characterization of purified inactivated form I ribulose 1,5-bisphosphate carboxylase/oxygenase of Rhodobacter sphaeroides.

    PubMed

    Wang, X; Tabita, F R

    1992-06-01

    Form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) from Rhodobacter sphaeroides is inactivated upon the addition of organic acids to photolithoautotrophically grown cultures. Activity recovers after the dissipation of the organic acid from the culture. The inactivation process depends on both the concentration of the organic compound and the nitrogen status of the cells. The inactivated RubisCO has been purified and was shown to exhibit mobility on both nondenaturing and sodium dodecyl sulfate gels different from that of the active enzyme prepared from cells not treated with organic acids. However, the Michaelis constants for ribulose 1,5-bisphosphate and CO2 or O2 were not dramatically altered. Purified inactivated RubisCO could be activated in vitro by increasing the temperature or the levels of Mg(II), and this activation was accompanied by changes in the electrophoretic mobility of the protein. When foreign bacterial RubisCO genes were expressed in an R. sphaeroides host strain lacking the ability to synthesize endogenous RubisCO, only slight inactivation of RubisCO activity was attained.

  20. The relationship between carotenoid biosynthesis and the assembly of the light-harvesting LH2 complex in Rhodobacter sphaeroides.

    PubMed Central

    Lang, H P; Hunter, C N

    1994-01-01

    Coloured carotenoids play some undefined role in the assembly of a functional light-harvesting 2 (LH2) complex in photosynthetic bacteria. We have used a series of transposon Tn5 insertion mutants disrupted at various stages of the carotenoid-biosynthetic pathway, together with an LH2 deletion/insertion mutant, to investigate this effect in Rhodobacter sphaeroides. Mutants were initially characterized by low-temperature absorbance spectroscopy and ultrastructural analysis: Northern-blot analysis demonstrated normal pucBA transcripts for LH2 polypeptides in all the carotenoid mutants. Analysis of translation of the puc transcript and investigation of the fate of any resulting LH2 polypeptides by SDS/PAGE, Western-blot and pulse-chase experiments clearly demonstrated that, in the absence of coloured carotenoids, the LH2 alpha- and beta-polypeptides are synthesized but are rapidly turned over and do not become stably integrated into the membrane. Complementation of mutants with lesions in the crtB and crtI genes, encoding phytoene synthase and phytoene desaturase respectively, with the cloned R. sphaeroides crtI gene, resulted in restoration of carotenoid biosynthesis and stable assembly of the LH2 complex in the crtI mutant but not in the crtB mutant, despite the presence of the CrtI protein. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 Figure 8 PMID:8129720

  1. Accumulation of a novel glycolipid and a betaine lipid in cells of Rhodobacter sphaeroides grown under phosphate limitation.

    PubMed

    Benning, C; Huang, Z H; Gage, D A

    1995-02-20

    Cells of the photosynthetic bacterium Rhodobacter sphaeroides grown under phosphate-limiting conditions accumulated nonphosphorous glycolipids and lipids carrying head groups derived from amino acids. Concomitantly, the relative amount of phosphoglycerolipids decreased from 90 to 22 mol% of total polar lipids in the membranes. Two lipids, not detectable in cells grown under standard conditions, were synthesized during phosphate-limited growth. Fast atom bombardment mass spectroscopy, exact mass measurements, 1H NMR spectroscopy, sugar composition analysis, and methylation analysis of the predominant glycolipid led to the identification of the novel compound 1,2-di-O-acyl-3-O-[alpha-D-glucopyranosyl-(1-->4)-O-beta-D-galactopyr anosyl]glycerol. The second lipid was identified as the betaine lipid 1,2-di-O-acyl-[4'-(N,N,N-trimethyl)-homoserine]glycerol by cochromatography employing an authentic standard from Chlamydomonas reinhardtii, fast atom bombardment mass spectroscopy, exact mass measurements, and 1H NMR spectroscopy. Prior to this observation, the occurrence of this lipid was thought to be restricted to lower plants and algae. Apparently, these newly synthesized nonphosphorous lipids, in addition to the sulfo- and the ornithine lipid also found in R. sphaeroides grown under optimal conditions, take over the role of phosphoglycerolipids in phosphate-deprived cells.

  2. The sRNA SorY confers resistance during photooxidative stress by affecting a metabolite transporter in Rhodobacter sphaeroides

    PubMed Central

    Adnan, Fazal; Weber, Lennart; Klug, Gabriele

    2015-01-01

    Exposure to oxygen and light generates photooxidative stress by the bacteriochlorophyll a mediated formation of singlet oxygen (1O2) in the facultative photosynthetic bacterium Rhodobacter sphaeroides. We have identified SorY as an sRNA, which is induced under several stress conditions and confers increased resistance against 1O2. SorY by direct interaction affects the takP mRNA, encoding a TRAP-T transporter. We present a model in which SorY reduces the metabolite flux into the tricarboxylic acid cycle (TCA cycle) by reducing malate import through TakP. It was previously shown that oxidative stress in bacteria leads to switch from glycolysis to the pentose phosphate pathway and to reduced activity of the TCA cycle. As a consequence the production of the prooxidant NADH is reduced and production of the protective NADPH is enhanced. In R. sphaeroides enzymes for glycolysis, pentose phosphate pathway, Entner–Doudoroff pathway and gluconeogenesis are induced in response to 1O2 by the alternative sigma factor RpoHII. The same is true for the sRNA SorY. By limiting malate import SorY thus contributes to the balance of the metabolic fluxes under photooxidative stress conditions. This assigns a so far unknown function to an sRNA in oxidative stress response. PMID:25833751

  3. Characteristics of Pos19 – A Small Coding RNA in the Oxidative Stress Response of Rhodobacter sphaeroides

    PubMed Central

    Eisenhardt, Benjamin D.; Remes, Bernhard; Klug, Gabriele

    2016-01-01

    The phototrophic bacterium Rhodobacter sphaeroides induces several small RNAs (sRNAs) when singlet oxygen (1O2) levels are elevated, a situation also referred to as photo-oxidative stress. An RNA-seq study identified the RSs0019 sRNA, which is renamed Pos19 (photo-oxidative stress induced sRNA 19). Pos19 is part of the RpoE regulon and consequently induced upon 1O2 and peroxide stress. The 219 nt long Pos19 transcript contains a small open reading frame (sORF) of 150 nt, which is translated in vivo. Over-expression of Pos19 results in reduced mRNA levels for several genes, of which numerous are involved in sulfur metabolism. The negative effect on the potential targets is maintained even when translation of the sORF is abolished, arguing that regulation is entailed by the sRNA itself. Reporter studies further revealed that regulation of the most affected mRNA, namely RSP_0557, by Pos19 is Hfq-dependent. Direct binding of Pos19 to Hfq was shown by co-immunoprecipitation. Physiological experiments indicated Pos19 to be involved in the balance of glutathione biosynthesis. Moreover, a lack of Pos19 leads to elevated reactive oxygen species levels. Taken together our data identify the sRNA Pos19 as a coding sRNA with a distinct expression pattern and potential role under oxidative stress in the phototrophic bacterium R. sphaeroides. PMID:27669425

  4. Biohydrogen and polyhydroxyalkanoate co-production by Enterobacter aerogenes and Rhodobacter sphaeroides from Calophyllum inophyllum oil cake.

    PubMed

    Arumugam, A; Sandhya, M; Ponnusami, V

    2014-07-01

    The feasibility of coupled biohydrogen and polyhydroxyalkanoate production by Enterobacter aerogenes and Rhodobacter sphaeroides using Calophyllum inophyllum oil cake was studied under dark and photo fermentation conditions. The utilization of a non-edible acidic oil cake (C. inophyllum), and exploitation of a modified minimal salt media led to reduction in the cost of media. Cost of fermentation is reduced by implementation of alternate dark-photo fermentative periods and through the use of a co-culture consisting of a dark fermentative (E. aerogenes) and a photo fermentative (R. sphaeroides) bacterium. The biohydrogen and polyhydroxyalkanoate produced were 7.95 L H2/L media and 10.73 g/L media, respectively, under alternate dark and photo fermentation and were 3.23 L H2/L media and 5.6g/L media, respectively under complete dark fermentation. The characteristics of the oil cake and alternate dark (16 h) and photo (8h) fermentative conditions were found to be supportive in producing high biohydrogen and polyhydroxyalkanoate (PHA) yield.

  5. The sulfolipid sulfoquinovosyldiacylglycerol is not required for photosynthetic electron transport in Rhodobacter sphaeroides but enhances growth under phosphate limitation

    SciTech Connect

    Benning, C.; Somerville, C.R. ); Beatty, J.T. ); Prince, R.C. )

    1993-02-15

    All photosynthetic organisms, with the exception of several species of photosynthetic bacteria, are thought to contain the sulfolipid 6-sulfo-[alpha]-D-quinovosyldiacylglycerol. The association of this lipid with photosynthetic membranes has led to the assumption that it plays some role in photosynthesis. Stable null mutants of the photosynthetic bacterium Rhodobacter sphaeroides completely lacking sulfolipid were obtained by disruption of the sqdB gene. The ratios of the various components of the photosynthetic electron transport chain, as well as the electron transfer rates during cyclic electron transport, were not altered in the mutants, when grown under optimal conditions. Growth rates of wild type and mutants were identical under a variety of growth conditions, with the exception of phosphate limitation, which resulted in reduced growth of the mutants. Phosphate limitation of the wild type a used a significant reduction in the amount of all phospholipids and an increased amount of sulfolipid. By contrast, the sulfolipid-deficient mutant had reduced levels of phosphatidylcholine and phosphatidylethanolamine but maintained a normal level of phosphatidylglycerol. In addition, two unidentified lipids lacking phosphorus accumulated in the membranes of both wild-type and mutant strains under phosphate limitation. We conclude that sulfolipid plays no significant unique role in photoheterotrophic growth or photosynthetic electron transport in R. sphaeroides but may function as a surrogate for phospholipids, particularly phosphatidylglycerol, under phosphate-limiting conditions. 34 refs., 5 figs., 1 tab.

  6. [Study of phototrophic purple bacterium Rhodobacter sphaeroides cell morphology of wild-type and ipt-transformant by atomic force and electron microscopy].

    PubMed

    Machulin, A V; Smolygina, L D; Suzina, N E; Serdiuk, O P

    2012-01-01

    A comparative study of phototrophic purple bacterium Rhodobacter sphaeroides cell morphology of wild-type and ipt-transformant was done by atomic force and electron microscopy. It was shown that transformation led to a decrease in the number or total disappearance of the flagella, as well as to changes in the structure of the outer membrane of the bacteria cell wall. On the wild-type cell surface phage-like structures were found, and in transformed cells at their places hollows were identified. This study significantly extends an understanding of the changes occurring in the ipt-transformants of phototrophic purple bacterium Rhodobacter sphaeroides. This investigation not only confirmed earlier obtained data about the differences in the wild-type and ipt-transformant phototrophic purple bacteria cell wall, but also showed fine changes in the structure of its outer membrane.

  7. Structural and functional proteomics of intracytoplasmic membrane assembly in Rhodobacter sphaeroides.

    PubMed

    Woronowicz, Kamil; Harrold, John W; Kay, Joseph M; Niederman, Robert A

    2013-01-01

    The results of a detailed structural and functional proteomic analysis of intracytoplasmic membrane (ICM) assembly in the model purple phototrophic bacterium Rhodobacter sphaeroides are reviewed in this report. Proteomics approaches have focused upon identification of membrane proteins temporally expressed during ICM development and spatially localized within the internal cell membranes, together with their structural and functional correlates. For the examination of temporal protein expression, procedures were established for the induction of ICM formation at low oxygen tension and for ICM remodeling in cells adapting to low intensity illumination, which permitted isolation by rate-zone sedimentation of ICM growth initiation sites (CM invaginations) in an upper-pigmented band (UPB), together with more mature ICM vesicles (chromatophores) as the main band. Nondenaturing clear native gel electrophoresis of the chromatophore fraction gave rise to four pigmented bands: the top and bottom bands contained the reaction center-light-harvesting 1 (RC-LH1) core complex and the LH2 peripheral antenna, respectively, while two bands of intermediate migration exhibited distinct associations of LH2 and core complexes. Proteomic analysis of the gel bands revealed developmental changes including increasing levels of LH2 polypeptides relative to those of core complexes as ICM development proceeded, as well as a large array of other associated proteins including high spectral counts for the F1FO-ATP synthase subunits, and the cytochrome bc1 complex. High counts were also observed for RSP6124, a protein of unknown function, that were correlated with increasing LH2 levels. RC-LH1-containing clear native electrophoresis gel bands from the UPB were enriched in cytoplasmic membrane (CM) markers, including electron transfer and transport proteins, as well as general membrane assembly factors (viz., preprotein translocases YidC, YajC and SecY, bacterial type 1 signal peptidase and twin arg

  8. Biohydrogen production by purple non-sulfur bacteria Rhodobacter sphaeroides: Effect of low-intensity electromagnetic irradiation.

    PubMed

    Gabrielyan, Lilit; Sargsyan, Harutyun; Trchounian, Armen

    2016-09-01

    The present work was focused on the effects of low-intensity (the flux capacity was of 0.06mWcm(-2)) electromagnetic irradiation (EMI) of extremely high frequencies or millimeter waves on the growth and hydrogen (H2) photoproduction by purple non-sulfur bacteria Rhodobacter sphaeroides MDC6521 (from Armenian mineral springs). After exposure of R. sphaeroides, grown under anaerobic conditions upon illumination, to EMI (51.8GHz and 53.0GHz) for 15min an increase of specific growth rate by ~1.2-fold, in comparison with control (non-irradiated cells), was obtained. However, the effect of EMI depends on the duration of irradiation: the exposure elongation up to 60min caused the delay of the growth lag phase and the decrease specific growth rate by ~1.3-fold, indicating the bactericidal effect of EMI. H2 yield of the culture, irradiated by EMI for 15min, determined during 72h growth, was ~1.2-fold higher than H2 yield of control cells, whereas H2 production by cultures, irradiated by EMI for 60min was not observed during 72h growth. This difference in the effects of extremely high frequency EMI indicates a direct effect of radiation on the membrane transfer and the enzymes of these bacteria. Moreover, EMI increased DCCD-inhibited H(+) fluxes across the bacterial membrane and DCCD-sensitive ATPase activity of membrane vesicles, indicating that the proton FoF1-ATPase is presumably a basic target for extremely high frequency EMI related to H2 production by cultures. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Mutagenic analysis of Cox11 of Rhodobacter sphaeroides: Insights into the assembly of CuB of cytochrome c oxidase

    PubMed Central

    Thompson, Audie K.; Smith, Daniel; Gray, Jimmy; Carr, Heather S.; Liu, Aimin; Winge, Dennis R.; Hosler, Jonathan P.

    2010-01-01

    The Cu(I) chaperone Cox11 is required for the insertion of CuB into cytochrome c oxidase (CcO) of mitochondria and many bacteria, including Rhodobacter sphaeroides. Exploration of the copper binding stoichiometry of R. sphaeroides Cox11 led to the finding that an apparent tetramer of both mitochondrial and bacterial Cox11 binds more copper than the sum of the dimers, providing another example of the flexibility of copper binding by Cu(I)-S clusters. Site-directed mutagenesis has been used to identify components of Cox11 that are not required for copper binding but are absolutely required for the assembly of CuB, including conserved Cys-35 and Lys-123. In contrast to earlier proposals, Cys-35 is not required for dimerization of Cox11 or for copper binding. These findings, plus the location of Cys-35 at the C terminus of the predicted transmembrane helix and thereby close to the surface of the membrane, allows a proposal that Cys-35 is involved in the transfer of copper from the Cu(I) cluster of Cox11 to the CuB ligands His-333/334 during the folding of CcO subunit I. Lys-123 is located near the Cu(I) cluster of Cox11, in an area otherwise devoid of charged residues. From the analysis of several Cox11 mutants, including K123E, L and R, we conclude that a previous proposal that Lys-123 provides charge balance for the stabilization of the Cu(I) cluster is unlikely to account for its absolute requirement for Cox11 function. Rather, consideration of the properties of Lys-123 and the apparent specificity of Cox11 suggests that Lys-123 plays a role in the interaction of Cox11 with its target. PMID:20524628

  10. Photobiological transformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) using Rhodobacter sphaeroides.

    PubMed

    Millerick, Kayleigh A; Johnston, Juliet T; Finneran, Kevin T

    2016-09-01

    Pump-and-treat strategies for groundwater containing explosives may be necessary when the contaminated water approaches sensitive receptors. This project investigated bacterial photosynthesis as a strategy for ex situ treatment, using light as the primary energy source to facilitate RDX transformation. The objective was to characterize the ability of photosynthetic Rhodobacter sphaeroides (strain ATCC(®) 17023 ™) to transform the high-energy explosive RDX. R. sphaeroides transformed 30 μM RDX within 40 h under light conditions; RDX was not fully transformed in the dark (non-photosynthetic conditions), suggesting that photosynthetic electron transfer was the primary mechanism. Experiments with RDX demonstrated that succinate and malate were the most effective electron donors for photosynthesis, but glycerol was also utilized as a photosynthetic electron donor. RDX was transformed irrespective of the presence of carbon dioxide. The electron shuttling compound anthraquinone-2,6-disulfonate (AQDS) increased transformation kinetics in the absence of CO2, when the cells had excess NADPH that needed to be re-oxidized because there was limited CO2 for carbon fixation. When CO2 was added, the cells generated more biomass, and AQDS had no stimulatory effect. End products indicated that RDX carbon became CO2, biomass, and a soluble, uncharacterized aqueous metabolite, determined using (14)C-labeled RDX. These data are the first to suggest that photobiological explosives transformation is possible and will provide a framework for which phototrophy can be used in environmental restoration of explosives contaminated water. Copyright © 2016. Published by Elsevier Ltd.

  11. Multiple chromosomes in bacteria. The yin and yang of trp gene localization in Rhodobacter sphaeroides 2.4.1.

    PubMed Central

    Mackenzie, C; Simmons, A E; Kaplan, S

    1999-01-01

    The existence of multiple chromosomes in bacteria has been known for some time. Yet the extent of functional solidarity between different chromosomes remains unknown. To examine this question, we have surveyed the well-described genes of the tryptophan biosynthetic pathway in the multichromosomal photosynthetic eubacterium Rhodobacter sphaeroides 2.4.1. The genome of this organism was mutagenized using Tn5, and strains that were auxotrophic for tryptophan (Trp(-)) were isolated. Pulsed-field gel mapping indicated that Tn5 insertions in both the large (3 Mb CI) and the small (0.9 Mb CII) chromosomes created a Trp(-) phenotype. Sequencing the DNA flanking the sites of the Tn5 insertions indicated that the genes trpE-yibQ-trpGDC were at a locus on CI, while genes trpF-aroR-trpB were at locus on CII. Unexpectedly, trpA was not found downstream of trpB. Instead, it was placed on the CI physical map at a locus 1.23 Mb away from trpE-yibQ-trpGDC. To relate the context of the R. sphaeroides trp genes to those of other bacteria, the DNA regions surrounding the trp genes on both chromosomes were sequenced. Of particular significance was the finding that rpsA1, which encodes ribosomal protein S1, and cmkA, which encodes cytidylate monophosphate kinase, were on CII. These genes are considered essential for translation and chromosome replication, respectively. Southern blotting suggested that the trp genes and rpsA1 exist in single copy within the genome. To date, this topological organization of the trp "operon" is unique within a bacterial genome. When taken with the finding that CII encodes essential housekeeping functions, the overall impression is one of close regulatory and functional integration between these chromosomes. PMID:10511537

  12. A comparison of the surface nanostructure from two different types of gram-negative cells: Escherichia coli and Rhodobacter sphaeroides.

    PubMed

    Oestreicher, Zachery; Taoka, Azuma; Fukumori, Yoshihiro

    2015-05-01

    Bacteria have been studied using different microscopy methods for many years. Recently, the developments of high-speed atomic force microscopy have opened the doors to study bacteria in new ways due to the fact that it uses much less force on the sample while imaging. This makes the high-speed atomic force microscope an indispensable technique for imaging the surface of living bacterial cells because it allows for the high-resolution visualization of surface proteins in their natural condition without disrupting the cell or the activity of the proteins. Previous work examining living cells of Magnetospirillum magneticum AMB-1 demonstrated that the surface of these bacteria was covered with a net-like structure that is mainly composed of porin molecules. However, it was unclear whether or not this feature was unique to other living bacteria. In this study we used the high-speed atomic force microscope to examine the surface of living cells of Escherichia coli and Rhodobacter sphaeroides to compare their structure with that of M. magneticum. Our research clearly demonstrated that both of these types of cells have an outer surface that is covered in a network of nanometer-sized holes similar to M. magneticum. The diameter of the holes was 8.0±1.5 nm for E. coli and 6.6±1.1 nm for R. sphaeroides. The results in this paper confirm that this type of outer surface structure exists in other types of bacteria and it is not unique to Magnetospirillum. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Aerobic and anaerobic regulation in Rhodobacter sphaeroides 2.4.1: the role of the fnrL gene.

    PubMed

    Zeilstra-Ryalls, J H; Kaplan, S

    1995-11-01

    In Rhodobacter sphaeroides 2.4.1, the cellular requirements for 5-aminolevulinic acid (ALA) are in part regulated by the level of ALA synthase activity, which is encoded by the hemA and hemT genes. Under standard growth conditions, only the hemA gene is transcribed, and the level of ALA synthase activity varies in response to oxygen tension. The presence of an FNR consensus sequence upstream of hemA suggested that oxygen regulation of hemA expression could be mediated, in part, through a homolog of the fnr gene. Two independent studies, one detailed here, identified a region of the R. sphaeroides 2.4.1 genome containing extensive homology to the fix region of the symbiotic nitrogen-fixing bacteria Rhizobium meliloti and Bradyrhizobium japonicum. Within this region that maps to 443 kbp on chromsome I, we have identified an fnr homolog (fnrL), as well as a gene that codes for an anaerobic coproporphyrinogen III oxidase, the second such gene identified in this organism. We also present an analysis of the role of fnrL in the physiology of R. sphaeroides 2.4.1 through the construction and characterization of fnrL-null strains. Our results further show that fnrL is essential for both photosynthetic and anaerobic-dark growth with dimethyl sulfoxide. Analysis of hemA expression, with hemA::lacZ transcriptional fusions, suggests that FnrL is an activator of hemA under anaerobic conditions. On the other hand, the open reading frame immediately upstream of hemA appears to be an activator of hemA transcription regardless of either the presence or the absence of oxygen or FnrL. Given the lack of hemT expression under these conditions, we consider FnrL regulation of hemA expression to be a major factor in bringing about changes in the level of ALA synthase activity in response to changes in oxygen tension.

  14. Coenzyme Q10 production in a 150-l reactor by a mutant strain of Rhodobacter sphaeroides.

    PubMed

    Kien, Nguyen Ba; Kong, In-Soo; Lee, Min-Gyu; Kim, Joong Kyun

    2010-05-01

    For the commercial production of CoQ(10), batch-type fermentations were attempted in a 150-l fermenter using a mutant strain of R. sphaeroides. Optimum temperature and initial aeration rate were found to be 30 degrees C and 2 vvm, respectively. Under optimum fermentation conditions, the maximum value of specific CoQ(10) content was achieved reproducibly as 6.34 mg/g DCW after 24 h, with 3.02 g/l of DCW. During the fermentation, aeration shift (from the adequate aeration at the early growth phase to the limited aeration in active cellular metabolism) was a key factor in CoQ(10) production for scale-up. A higher value of the specific CoQ(10) content (8.12 mg/g DCW) was achieved in fed-batch fermentation and comparable to those produced by the pilot-scale fed-batch fermentations of A. tumefaciens, which indicated that the mutant strain of R. sphaeroides used in this study was a potential high CoQ(10) producer. This is the first detailed study to demonstrate a pilot-scale production of CoQ(10) using a mutant strain of R. sphaeroides.

  15. The role of Cercospora zeae-maydis homologs of Rhodobacter sphaeroides 1O2-resistance genes in resistance to the photoactivated toxin cercosporin.

    PubMed

    Beseli, Aydin; Goulart da Silva, Marilia; Daub, Margaret E

    2015-01-01

    The photosynthetic bacterium Rhodobacter sphaeroides and plant pathogenic fungus Cercospora nicotianae have been used as models for understanding resistance to singlet oxygen ((1)O(2)), a highly toxic reactive oxygen species. In Rhodobacter and Cercospora, (1)O(2) is derived, respectively, from photosynthesis and from the (1)O(2)-generating toxin cercosporin which the fungus produces to parasitize plants. We identified common genes recovered in transcriptome studies of putative (1)O(2)-resistance genes in these two systems, suggesting common (1)O(2)-resistance mechanisms. To determine if the Cercospora homologs of R. sphaeroides (1)O(2)-resistance genes are involved in resistance to cercosporin, we expressed the genes in the cercosporin-sensitive fungus Neurospora crassa and assayed for increases in cercosporin resistance. Neurospora crassa transformants expressing genes encoding aldo/keto reductase, succinyl-CoA ligase, O-acetylhomoserine (thiol) lyase, peptide methionine sulphoxide reductase and glutathione S-transferase did not have elevated levels of cercosporin resistance. Several transformants expressing aldehyde dehydrogenase were significantly more resistant to cercosporin. Expression of the transgene and enzyme activity did not correlate with resistance, however. We conclude that although the genes tested in this study are important in (1)O(2) resistance in R. sphaeroides, their Cercospora homologs are not involved in resistance to (1)O(2) generated from cercosporin.

  16. Growth, pigmentation, and expression of the puf and puc operons in a light-responding-repressor (SPB)-disrupted Rhodobacter sphaeroides.

    PubMed

    Nishimura, K; Shimada, H; Hatanaka, S; Mizoguchi, H; Ohta, H; Masuda, T; Takamiya, K

    1998-04-01

    We previously cloned a trans-repressor, SPB, for the puf operon of Rhodobacter sphaeroides (Shimada et al. 1996) and revealed that SPB was a putative genetic counterpart to HvrA in Rhodobacter capsulatus, a trans-activator for the puf and puh operons (Mizoguchi et al. 1997). In this study we constructed a spb-disrupted R. sphaeroides, strain L-7, to elucidate the function of SPB. This disruption of the spb gene increased the photosynthetic growth rate and the cellular levels of photopigments under low-intensity light conditions. The disruption also derepressed the expression of the puf and puc operons under high-intensity light conditions. In strain L-7, however, strong illumination still reduced the cellular levels of photopigments as it did in the wild strain, suggesting that SPB did not directly affect the formation of photopigments. These results support our previous suggestion that SPB functions as a high-light repressor for puf operon in R. sphaeroides in striking contrast to HvrA, which is a low-light activator for puf and puh operons in R. capsulatus, even though SPB and HvrA are highly homologous. Disruption of spb gene had no effect on the oxygen-mediated regulation of the pigmentation or the expression of puf and puc operons.

  17. An Alternative Pathway of Light-Induced Transmembrane Electron Transfer in Photosynthetic Reaction Centers of Rhodobacter sphaeroides.

    PubMed

    Khatypov, R A; Khristin, A M; Fufina, T Yu; Shuvalov, V A

    2017-06-01

    In the absorption spectrum of Rhodobacter sphaeroides reaction centers, a minor absorption band was found with a maximum at 1053 nm. The amplitude of this band is ~10,000 times less and its half-width is comparable to that of the long-wavelength absorption band of the primary electron donor P870. When the primary electron donor is excited by femtosecond light pulses at 870 nm, the absorption band at 1053 nm is increased manifold during the earliest stages of charge separation. The growth of this absorption band in difference absorption spectra precedes the appearance of stimulated emission at 935 nm and the appearance of the absorption band of anion-radical BA(-) at 1020 nm, reported earlier by several researchers. When reaction centers are illuminated with 1064 nm light, the absorption spectrum undergoes changes indicating reduction of the primary electron acceptor QA, with the primary electron donor P870 remaining neutral. These photoinduced absorption changes reflect the formation of the long-lived radical state PBAHAQA(-).

  18. Augmenting light coverage for photosynthesis through YFP-enhanced charge separation at the Rhodobacter sphaeroides reaction centre

    NASA Astrophysics Data System (ADS)

    Grayson, Katie J.; Faries, Kaitlyn M.; Huang, Xia; Qian, Pu; Dilbeck, Preston; Martin, Elizabeth C.; Hitchcock, Andrew; Vasilev, Cvetelin; Yuen, Jonathan M.; Niedzwiedzki, Dariusz M.; Leggett, Graham J.; Holten, Dewey; Kirmaier, Christine; Neil Hunter, C.

    2017-01-01

    Photosynthesis uses a limited range of the solar spectrum, so enhancing spectral coverage could improve the efficiency of light capture. Here, we show that a hybrid reaction centre (RC)/yellow fluorescent protein (YFP) complex accelerates photosynthetic growth in the bacterium Rhodobacter sphaeroides. The structure of the RC/YFP-light-harvesting 1 (LH1) complex shows the position of YFP attachment to the RC-H subunit, on the cytoplasmic side of the RC complex. Fluorescence lifetime microscopy of whole cells and ultrafast transient absorption spectroscopy of purified RC/YFP complexes show that the YFP-RC intermolecular distance and spectral overlap between the emission of YFP and the visible-region (QX) absorption bands of the RC allow energy transfer via a Förster mechanism, with an efficiency of 40+/-10%. This proof-of-principle study demonstrates the feasibility of increasing spectral coverage for harvesting light using non-native genetically-encoded light-absorbers, thereby augmenting energy transfer and trapping in photosynthesis.

  19. Concomitant biohydrogen and poly-β-hydroxybutyrate production from dark fermentation effluents by adapted Rhodobacter sphaeroides and mixed photofermentative cultures.

    PubMed

    Ghimire, Anish; Valentino, Serena; Frunzo, Luigi; Pirozzi, Francesco; Lens, Piet N L; Esposito, Giovanni

    2016-10-01

    This work aimed at investigating concomitant production of biohydrogen and poly-β-hydroxybutyrate (PHB) by photofermentation (PF) using dark fermentation effluents (DFE). An adapted culture of Rhodobacter sphaeroides AV1b (pH 6.5, 24±2°C) achieved H2 and PHB yields of 256 (±2) NmLH2/g Chemical Oxygen Demand (COD) and 273.8mgPHB/gCOD (32.5±3% of the dry cells weight (DCW)), respectively. When a diluted (1:2) DFE medium was applied to the adapted pure and mixed photofermentative culture, the respective H2 yields were 164.0 (±12) and 71.3 (±6) NmLH2/gCOD and the PHB yields were 212.1 (±105.2) and 50.7 (±2.7) mgPHB/gCOD added, corresponding to 24 (±0.7) and 6.3 (±0) % DCW, respectively. The concomitant H2 and PHB production from the PF process gave a good DFE post treatment achieving up to 80% COD removal from the initial DFE.

  20. Insights into the species-specific TLR4 signaling mechanism in response to Rhodobacter sphaeroides lipid A detection

    NASA Astrophysics Data System (ADS)

    Anwar, Muhammad Ayaz; Panneerselvam, Suresh; Shah, Masaud; Choi, Sangdun

    2015-01-01

    TLR4 in complex with MD2 senses the presence of lipid A (LA) and initiates a signaling cascade that curb the infection. This complex is evolutionarily conserved and can initiate the immune system in response to a variety of LAs. In this study, molecular dynamics simulation (25 ns) was performed to elucidate the differential behavior of TLR4/MD2 complex in response to Rhodobacter sphaeroides lipid A (RsLA). Penta-acyl chain-containing RsLA is at the verge of agonist (6 acyl-chains) and antagonist (4 acyl-chains) structure, and activates the TLR4 pathway in horses and hamsters, while inhibiting in humans and murine. In the time-evolved coordinates, the promising factors that dictated the differential response included the local and global mobility pattern of complexes, solvent-accessible surface area of ligand, and surface charge distributions of TLR4 and MD2. We showed that the GlcN1-GlcN2 backbone acquires agonist (3FXI)-like configurations in horses and hamsters, while acquiring antagonist (2E59)-like configurations in humans and murine systems. Moreover, analysis of F126 behavior in the MD2 F126 loop (amino acids 123-129) and loop EF (81-89) suggested that certain sequence variations also contribute to species-specific response. This study underlines the TLR4 signaling mechanism and provides new therapeutic opportunities.

  1. Inhibitor-complexed Structures of the Cytochrome bc[subscript 1] from the Photosynthetic Bacterium Rhodobacter sphaeroides

    SciTech Connect

    Esser, Lothar; Elberry, Maria; Zhou, Fei; Yu, Chang-An; Yu, Linda; Xia, Di

    2008-06-30

    The cytochrome bc{sub 1} complex (bc{sub 1}) is a major contributor to the proton motive force across the membrane by coupling electron transfer to proton translocation. The crystal structures of wild type and mutant bc{sub 1} complexes from the photosynthetic purple bacterium Rhodobacter sphaeroides (Rsbc{sub 1}), stabilized with the quinol oxidation (Q{sub P}) site inhibitor stigmatellin alone or in combination with the quinone reduction (Q{sub N}) site inhibitor antimycin, were determined. The high quality electron density permitted assignments of a new metal-binding site to the cytochrome c1 subunit and a number of lipid and detergent molecules. Structural differences between Rsbc{sub 1} and its mitochondrial counterparts are mostly extra membranous and provide a basis for understanding the function of the predominantly longer sequences in the bacterial subunits. Functional implications for the bc{sub 1} complex are derived from analyses of 10 independent molecules in various crystal forms and from comparisons with mitochondrial complexes.

  2. Redox potential tuning through differential quinone binding in the photosynthetic reaction center of Rhodobacter sphaeroides

    SciTech Connect

    Vermaas, Josh V.; Taguchi, Alexander T.; Dikanov, Sergei A.; Wraight, Colin A.; Tajkhorshid, Emad

    2015-03-03

    Ubiquinone forms an integral part of the electron transport chain in cellular respiration and photosynthesis across a vast number of organisms. Prior experimental results have shown that the photosynthetic reaction center (RC) from Rhodobacter sphaeroides is only fully functional with a limited set of methoxy-bearing quinones, suggesting that specific interactions with this substituent are required to drive electron transport and the formation of quinol. The nature of these interactions has yet to be determined. Through parameterization of a CHARMM-compatible quinone force field and subsequent molecular dynamics simulations of the quinone-bound RC, in this paper we have investigated and characterized the interactions of the protein with the quinones in the QA and QB sites using both equilibrium simulation and thermodynamic integration. In particular, we identify a specific interaction between the 2-methoxy group of ubiquinone in the QB site and the amide nitrogen of GlyL225 that we implicate in locking the orientation of the 2-methoxy group, thereby tuning the redox potential difference between the quinones occupying the QA and QB sites. Finally, disruption of this interaction leads to weaker binding in a ubiquinone analogue that lacks a 2-methoxy group, a finding supported by reverse electron transfer electron paramagnetic resonance experiments of the QA–QB– biradical and competitive binding assays.

  3. Optimization of Biomass and 5-Aminolevulinic Acid Production by Rhodobacter sphaeroides ATCC17023 via Response Surface Methodology.

    PubMed

    Liu, Shuli; Zhang, Guangming; Li, Jianzheng; Li, Xiangkun; Zhang, Jie

    2016-06-01

    Microbial 5-aminolevulinic acid (ALA) produced from wastewater is considered as potential renewable energy. However, many hurdles are needed to be overcome such as the regulation of key influencing factors on ALA yield. Biomass and ALA production by Rhodobacter sphaeroides was optimized using response surface methodology. The culturing medium was artificial volatile fatty acids wastewater. Three additives were optimized, namely succinate and glycine that are precursors of ALA biosynthesis, and D-glucose that is an inhibitor of ALA dehydratase. The optimal conditions were achieved by analyzing the response surface plots. Statistical analysis showed that succinate at 8.56 mmol/L, glycine at 5.06 mmol/L, and D-glucose at 7.82 mmol/L were the best conditions. Under these optimal conditions, the highest biomass production and ALA yield of 3.55 g/L and 5.49 mg/g-biomass were achieved. Subsequent verification experiments at optimal values had the maximum biomass production of 3.41 ± 0.002 g/L and ALA yield of 5.78 ± 0.08 mg/g-biomass.

  4. Augmenting light coverage for photosynthesis through YFP-enhanced charge separation at the Rhodobacter sphaeroides reaction centre

    DOE PAGES

    Grayson, Katie J.; Faries, Kaitlyn M.; Huang, Xia; ...

    2017-01-05

    Photosynthesis uses a limited range of the solar spectrum, so enhancing spectral coverage could improve the efficiency of light capture. Here, we show that a hybrid reaction centre (RC)/yellow fluorescent protein (YFP) complex accelerates photosynthetic growth in the bacterium Rhodobacter sphaeroides. The structure of the RC/YFP-light-harvesting 1 (LH1) complex shows the position of YFP attachment to the RC-H subunit, on the cytoplasmic side of the RC complex. Fluorescence lifetime microscopy of whole cells and ultrafast transient absorption spectroscopy of purified RC/YFP complexes show that the YFP–RC intermolecular distance and spectral overlap between the emission of YFP and the visible-region (QX)more » absorption bands of the RC allow energy transfer via a Fo¨rster mechanism, with an efficiency of 40±10%. Finally, this proof-of-principle study demonstrates the feasibility of increasing spectral coverage for harvesting light using non-native genetically-encoded light-absorbers, thereby augmenting energy transfer and trapping in photosynthesis.« less

  5. Modeling the light- and redox-dependent interaction of PpsR/AppA in Rhodobacter sphaeroides.

    PubMed

    Pandey, Rakesh; Flockerzi, Dietrich; Hauser, Marcus J B; Straube, Ronny

    2011-05-18

    Facultative photosynthetic bacteria switch their energy generation mechanism from respiration to photosynthesis depending on oxygen tension and light. Part of this transition is mediated by the aerobic transcriptional repressor PpsR. In Rhodobacter sphaeroides, the repressive action of PpsR is antagonized by the redox- and blue-light-sensitive flavoprotein AppA which results in a unique phenotype: the repression of photosynthesis genes at intermediate oxygen levels and high light intensity, which is believed to reduce the risk of photooxidative stress. To analyze the underlying mechanism we developed a simple mathematical model based on the AppA-dependent reduction of a disulfide bond in PpsR and the light-sensitive complex formation between the reduced forms of AppA and PpsR. A steady-state analysis shows that high light repression can indeed occur at intermediate oxygen levels if PpsR is reduced on a faster timescale than AppA and if the electron transfer from AppA to PpsR is effectively irreversible. The model further predicts that if AppA copy numbers exceed those of PpsR by at least a factor of two, the transition from aerobic to anaerobic growth mode can occur via a bistable regime. We provide necessary conditions for the emergence of bistability and discuss possible experimental verifications. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  6. Probing energy transfer events in the light harvesting complex 2 (LH2) of Rhodobacter sphaeroides with two-dimensional spectroscopy.

    PubMed

    Fidler, Andrew F; Singh, Ved P; Long, Phillip D; Dahlberg, Peter D; Engel, Gregory S

    2013-10-21

    Excitation energy transfer events in the photosynthetic light harvesting complex 2 (LH2) of Rhodobacter sphaeroides are investigated with polarization controlled two-dimensional electronic spectroscopy. A spectrally broadened pulse allows simultaneous measurement of the energy transfer within and between the two absorption bands at 800 nm and 850 nm. The phased all-parallel polarization two-dimensional spectra resolve the initial events of energy transfer by separating the intra-band and inter-band relaxation processes across the two-dimensional map. The internal dynamics of the 800 nm region of the spectra are resolved as a cross peak that grows in on an ultrafast time scale, reflecting energy transfer between higher lying excitations of the B850 chromophores into the B800 states. We utilize a polarization sequence designed to highlight the initial excited state dynamics which uncovers an ultrafast transfer component between the two bands that was not observed in the all-parallel polarization data. We attribute the ultrafast transfer component to energy transfer from higher energy exciton states to lower energy states of the strongly coupled B850 chromophores. Connecting the spectroscopic signature to the molecular structure, we reveal multiple relaxation pathways including a cyclic transfer of energy between the two rings of the complex.

  7. Enhanced production of coenzyme Q10 by self-regulating the engineered MEP pathway in Rhodobacter sphaeroides.

    PubMed

    Lu, Wenqiang; Ye, Lidan; Xu, Haoming; Xie, Wenping; Gu, Jiali; Yu, Hongwei

    2014-04-01

    Fine-tuning the expression level of an engineered pathway is crucial for the metabolic engineering of a host toward a desired phenotype. However, most engineered hosts suffer from nonfunctional protein expression, metabolic imbalance, cellular burden or toxicity from intermediates when an engineered pathway is first introduced, which can decrease production of the desired product. To circumvent these obstacles, we developed a self-regulation system utilizing the trc/tac promoter, LacI(q) protein and ribosomal binding sites (RBS). With the purpose of improving coenzyme Q10 (CoQ10 ) production by increasing the decaprenyl diphosphate supplement, enzymes DXS, DXR, IDI, and IspD were constitutively overexpressed under the control of the trc promoter in Rhodobacter sphaeroides. Then, a self-regulation system combining a set of RBSs for adjusting the expression of the LacI(q) protein was applied to tune the expression of the four genes, resulting in improved CoQ10 production. Finally, another copy of the tac promoter with the UbiG gene (involved in the ubiquinone pathway of CoQ10 biosynthesis) was introduced into the engineered pathway. By optimizing the expression level of both the upstream and downstream pathway, CoQ10 production in the mutants was improved up to 93.34 mg/L (7.16 mg/g DCW), about twofold of the wild-type (48.25 mg/L, 3.24 mg/g DCW).

  8. The Genome of Rhodobacter sphaeroides Strain 2.4.1 Encodes Functional Cobinamide Salvaging Systems of Archaeal and Bacterial Origins

    PubMed Central

    Gray, Michael J.; Tavares, Norbert K.; Escalante-Semerena, Jorge C.

    2008-01-01

    Bacteria and archaea use distinct pathways for salvaging exogenous cobinamide (Cbi), a precursor of adenosylcobalamin (AdoCbl, coenzyme B12). The bacterial pathway depends on a bifunctional enzyme with kinase and guanylyltransferase activities (CobP in aerobic AdoCbl synthesizers) to convert AdoCbi to AdoCbi-GDP via an AdoCbi-P intermediate. Archaea lack CobP, and use a different strategy for the synthesis of AdoCbi-GDP. Archaea cleave off the aminopropanol (AP) group of AdoCbi using the CbiZ AdoCbi amidohydrolase to generate adenosylcobyric acid (AdoCby), which is converted to AdoCbi-P by the CbiB synthetase, and to AdoCbi-GDP by the CobY guanylyltransferase. We report phylogenetic, in vivo, and in vitro evidence that the genome of Rhodobacter sphaeroides encodes functional enzymes for Cbi salvaging systems of both bacterial and archaeal origin. Products of the reactions were identified by high performance liquid chromatography, UV-visible spectroscopy, and bioassay. The cbiZ genes of several bacteria and archaea restored Cbi salvaging in a strain of Salmonella enterica unable to salvage Cbi. Phylogenetic data led us to conclude that CbiZ is an enzyme of archaeal origin that was horizontally transferred to bacteria. Reasons why some bacteria may contain both types of Cbi salvaging system are discussed. PMID:18808385

  9. Probing energy transfer events in the light harvesting complex 2 (LH2) of Rhodobacter sphaeroides with two-dimensional spectroscopy

    SciTech Connect

    Fidler, Andrew F.; Singh, Ved P.; Engel, Gregory S.; Long, Phillip D.; Dahlberg, Peter D.

    2013-10-21

    Excitation energy transfer events in the photosynthetic light harvesting complex 2 (LH2) of Rhodobacter sphaeroides are investigated with polarization controlled two-dimensional electronic spectroscopy. A spectrally broadened pulse allows simultaneous measurement of the energy transfer within and between the two absorption bands at 800 nm and 850 nm. The phased all-parallel polarization two-dimensional spectra resolve the initial events of energy transfer by separating the intra-band and inter-band relaxation processes across the two-dimensional map. The internal dynamics of the 800 nm region of the spectra are resolved as a cross peak that grows in on an ultrafast time scale, reflecting energy transfer between higher lying excitations of the B850 chromophores into the B800 states. We utilize a polarization sequence designed to highlight the initial excited state dynamics which uncovers an ultrafast transfer component between the two bands that was not observed in the all-parallel polarization data. We attribute the ultrafast transfer component to energy transfer from higher energy exciton states to lower energy states of the strongly coupled B850 chromophores. Connecting the spectroscopic signature to the molecular structure, we reveal multiple relaxation pathways including a cyclic transfer of energy between the two rings of the complex.

  10. Coupling of cytochrome and quinone turnovers in the photocycle of reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides.

    PubMed Central

    Osváth, S; Maróti, P

    1997-01-01

    A minimal kinetic model of the photocycle, including both quinone (Q-6) reduction at the secondary quinone-binding site and (mammalian) cytochrome c oxidation at the cytochrome docking site of isolated reaction centers from photosynthetic purple bacteria Rhodobacter sphaeroides, was elaborated and tested by cytochrome photooxidation under strong continuous illumination. The typical rate of photochemical excitation by a laser diode at 810 nm was 2.200 s-1, and the rates of stationary turnover of the reaction center (one-half of that of cytochrome photooxidation) were 600 +/- 70 s-1 at pH 6 and 400 +/- 50 s-1 at pH 8. The rate of turnover showed strong pH dependence, indicating the contribution of different rate-limiting processes. The kinetic limitation of the photocycle was attributed to the turnover of the cytochrome c binding site (pH < 6), light intensity and quinone/quinol exchange (6 < pH < 8), and proton-coupled second electron transfer in the quinone acceptor complex (pH > 8). The analysis of the double-reciprocal plot of the rate of turnover versus light intensity has proved useful in determining the light-independent (maximum) turnover rate of the reaction center (445 +/- 50 s-1 at pH 7.8). PMID:9251814

  11. Physiological Roles for Two Periplasmic Nitrate Reductases in Rhodobacter sphaeroides 2.4.3 (ATCC 17025)▿

    PubMed Central

    Hartsock, Angela; Shapleigh, James P.

    2011-01-01

    The metabolically versatile purple bacterium Rhodobacter sphaeroides 2.4.3 is a denitrifier whose genome contains two periplasmic nitrate reductase-encoding gene clusters. This work demonstrates nonredundant physiological roles for these two enzymes. One cluster is expressed aerobically and repressed under low oxygen while the second is maximally expressed under low oxygen. Insertional inactivation of the aerobically expressed nitrate reductase eliminated aerobic nitrate reduction, but cells of this strain could still respire nitrate anaerobically. In contrast, when the anaerobic nitrate reductase was absent, aerobic nitrate reduction was detectable, but anaerobic nitrate reduction was impaired. The aerobic nitrate reductase was expressed but not utilized in liquid culture but was utilized during growth on solid medium. Growth on a variety of carbon sources, with the exception of malate, the most oxidized substrate used, resulted in nitrite production on solid medium. This is consistent with a role for the aerobic nitrate reductase in redox homeostasis. These results show that one of the nitrate reductases is specific for respiration and denitrification while the other likely plays a role in redox homeostasis during aerobic growth. PMID:21949073

  12. Nucleotide sequence and functional analysis of cbbR, a positive regulator of the Calvin cycle operons of Rhodobacter sphaeroides.

    PubMed Central

    Gibson, J L; Tabita, F R

    1993-01-01

    Structural genes encoding Calvin cycle enzymes in Rhodobacter sphaeroides are duplicated and organized within two physically distinct transcriptional units, the form I and form II cbb operons. Nucleotide sequence determination of the region upstream of the form I operon revealed a divergently transcribed open reading frame, cbbR, that showed significant similarity to the LysR family of transcriptional regulatory proteins. Mutants containing an insertionally inactivated cbbR gene were impaired in photoheterotrophic growth and completely unable to grow photolithoautotrophically with CO2 as the sole carbon source. In the cbbR strain, expression of genes within the form I operon was completely abolished and that of the form II operon was reduced to about 30% of the wild-type level. The cloned cbbR gene complemented the mutant for wild-type growth characteristics, and normal levels of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) were observed. However, rocket immunoelectrophoresis revealed that the wild-type level of RubisCO was due to overexpression of the form II enzyme, whereas expression of the form I RubisCO was 10% of that of the wild-type strain. The cbbR insertional inactivation did not appear to affect aerobic expression of either CO2 fixation operon, but preliminary evidence suggests that the constitutive expression of the form II operon observed in the cbbR strain may be subject to repression during aerobic growth. PMID:8376325

  13. Augmenting light coverage for photosynthesis through YFP-enhanced charge separation at the Rhodobacter sphaeroides reaction centre.

    PubMed

    Grayson, Katie J; Faries, Kaitlyn M; Huang, Xia; Qian, Pu; Dilbeck, Preston; Martin, Elizabeth C; Hitchcock, Andrew; Vasilev, Cvetelin; Yuen, Jonathan M; Niedzwiedzki, Dariusz M; Leggett, Graham J; Holten, Dewey; Kirmaier, Christine; Neil Hunter, C

    2017-01-05

    Photosynthesis uses a limited range of the solar spectrum, so enhancing spectral coverage could improve the efficiency of light capture. Here, we show that a hybrid reaction centre (RC)/yellow fluorescent protein (YFP) complex accelerates photosynthetic growth in the bacterium Rhodobacter sphaeroides. The structure of the RC/YFP-light-harvesting 1 (LH1) complex shows the position of YFP attachment to the RC-H subunit, on the cytoplasmic side of the RC complex. Fluorescence lifetime microscopy of whole cells and ultrafast transient absorption spectroscopy of purified RC/YFP complexes show that the YFP-RC intermolecular distance and spectral overlap between the emission of YFP and the visible-region (QX) absorption bands of the RC allow energy transfer via a Förster mechanism, with an efficiency of 40±10%. This proof-of-principle study demonstrates the feasibility of increasing spectral coverage for harvesting light using non-native genetically-encoded light-absorbers, thereby augmenting energy transfer and trapping in photosynthesis.

  14. The equine TLR4/MD-2 complex mediates recognition of lipopolysaccharide from Rhodobacter sphaeroides as an agonist.

    PubMed

    Lohmann, Katharina L; Vandenplas, Michel L; Barton, Michelle H; Bryant, Clare E; Moore, James N

    2007-01-01

    Lipopolysaccharide (LPS) antagonists inhibit the response of inflammatory cells to LPS, presumably by competitive inhibition, and may be of therapeutic value in the treatment of endotoxemia and sepsis. The inhibitory effects of some LPS antagonists are restricted to certain host species, however, as the same molecules can have significant endotoxic activity in other species. This species-specific recognition appears to be mediated by Toll-like receptor 4 (TLR4) and/or MD-2. We have shown previously that LPS from Rhodobacter sphaeroides ( RsLPS) is an LPS antagonist in human cells but an agonist (or LPS mimetic) in equine cells. In the present study, HEK293 cells were transfected with combinations of human and equine CD14, TLR4 and MD-2, and incubated with either RsLPS or with LPS from Escherichia coli as an endotoxin control. NF-kappaB activation was measured in a dual luciferase assay as an indicator of cellular activation. Our results indicate that E. colic LPS activated NF-kappaB in cells transfected with all combinations of the three receptor proteins, whereas RsLPS activated NF-kappaB only in cells expressing the single combination of equine TLR4 and equine MD-2. We conclude that the TLR4/MD-2 complex is responsible for recognition of RsLPS as an agonist in equine cells.

  15. Augmenting light coverage for photosynthesis through YFP-enhanced charge separation at the Rhodobacter sphaeroides reaction centre

    PubMed Central

    Grayson, Katie J.; Faries, Kaitlyn M.; Huang, Xia; Qian, Pu; Dilbeck, Preston; Martin, Elizabeth C.; Hitchcock, Andrew; Vasilev, Cvetelin; Yuen, Jonathan M.; Niedzwiedzki, Dariusz M.; Leggett, Graham J.; Holten, Dewey; Kirmaier, Christine; Neil Hunter, C.

    2017-01-01

    Photosynthesis uses a limited range of the solar spectrum, so enhancing spectral coverage could improve the efficiency of light capture. Here, we show that a hybrid reaction centre (RC)/yellow fluorescent protein (YFP) complex accelerates photosynthetic growth in the bacterium Rhodobacter sphaeroides. The structure of the RC/YFP-light-harvesting 1 (LH1) complex shows the position of YFP attachment to the RC-H subunit, on the cytoplasmic side of the RC complex. Fluorescence lifetime microscopy of whole cells and ultrafast transient absorption spectroscopy of purified RC/YFP complexes show that the YFP–RC intermolecular distance and spectral overlap between the emission of YFP and the visible-region (QX) absorption bands of the RC allow energy transfer via a Förster mechanism, with an efficiency of 40±10%. This proof-of-principle study demonstrates the feasibility of increasing spectral coverage for harvesting light using non-native genetically-encoded light-absorbers, thereby augmenting energy transfer and trapping in photosynthesis. PMID:28054547

  16. Magnesium-protoporphyrin chelatase of Rhodobacter sphaeroides: reconstitution of activity by combining the products of the bchH, -I, and -D genes expressed in Escherichia coli.

    PubMed

    Gibson, L C; Willows, R D; Kannangara, C G; von Wettstein, D; Hunter, C N

    1995-03-14

    Magnesium-protoporphyrin chelatase lies at the branch point of the heme and (bacterio)chlorophyll biosynthetic pathways. In this work, the photosynthetic bacterium Rhodobacter sphaeroides has been used as a model system for the study of this reaction. The bchH and the bchI and -D genes from R. sphaeroides were expressed in Escherichia coli. When cell-free extracts from strains expressing BchH, BchI, and BchD were combined, the mixture was able to catalyze the insertion of Mg into protoporphyrin IX in an ATP-dependent manner. This was possible only when all three genes were expressed. The bchH, -I, and -D gene products are therefore assigned to the Mg chelatase step in bacteriochlorophyll biosynthesis. The mechanism of the Mg chelation reaction and the implications for chlorophyll biosynthesis in plants are discussed.

  17. Whole-genome shotgun optical mapping of Rhodobacter sphaeroides strain 2.4. 1 and its use for whole-genome shotgun sequence assembly

    SciTech Connect

    Shou, S.; Kvikstad, E.; Kile, A.; Severin, J.; Forrest, D.; Runnheim, R.; Churas, C.; Hickman, J. W.; Mackenzie, C.; Choudhary, M.; Donohue, T.; Kaplan, S.; Schwartz, D. C.

    2003-09-01

    Rhodobacter sphaeroides 2.4.1 is a facultative photoheterotrophic bacterium with tremendous metabolic diversity, which has significantly contributed to our understanding of the molecular genetics of photosynthesis, photoheterotrophy, nitrogen fixation, hydrogen metabolism, carbon dioxide fixation, taxis, and tetrapyrrole biosynthesis. To further understand this remarkable bacterium, and to accelerate an ongoing sequencing project, two whole-genome restriction maps (EcoRI and HindIII) of R. sphaeroides strain 2.4.1 were constructed using shotgun optical mapping. The approach directly mapped genomic DNA by the random mapping of single molecules. The two maps were used to facilitate sequence assembly by providing an optical scaffold for high-resolution alignment and verification of sequence contigs. Our results show that such maps facilitated the closure of sequence gaps by the early detection of nascent sequence contigs during the course of the whole-genome shotgun sequencing process.

  18. Acetate-dependent photoheterotrophic growth and the differential requirement for the Calvin-Benson-Bassham reductive pentose phosphate cycle in Rhodobacter sphaeroides and Rhodopseudomonas palustris.

    PubMed

    Laguna, Rick; Tabita, F Robert; Alber, Birgit E

    2011-02-01

    Rhodobacter sphaeroides ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)-deletion strain 16 was capable of photoheterotrophic growth with acetate, while Rhodopseudomonas palustris RubisCO-deletion strain 2040 could not grow under these conditions. The reason for this difference lies in the fact that Rba. sphaeroides and Rps. palustris use different pathways for acetate assimilation, the ethylmalonyl-CoA pathway, and glyoxylate-bypass cycle, respectively. The ethylmalonyl-CoA pathway is distinct from the glyoxylate cycle as one molecule of CO(2) and one molecule of HCO(3) (-) per three molecules of acetyl-CoA are co-assimilated to form two malate molecules. The glyoxylate cycle directly converts two acetyl-CoA molecules to malate. Each pathway, therefore, also dictates at what point, CO(2) and reductant are consumed, thereby determining the requirement for the Calvin-Benson-Bassham reductive pentose phosphate cycle.

  19. Characteristics of light-harvesting complex II mutant of Rhodobacter sphaeroides with alterations at the transmembrane helices of beta-subunit.

    PubMed

    Wang, Wanneng; Hu, Zongli; Li, Jinzhe; Chen, Xuqing; Chen, Guoping

    2009-07-01

    The peripheral light-harvesting complex II (LHII) is an important component of the photosynthetic apparatus of Rhodobacter sphaeroides. In this study, genetic, biochemical, and spectroscopic approaches were applied to investigate the spectral properties and functions of LHII in which two amino acid residues Phe32 and Leu42 in the transmembrane helix domain of pucB-encoded beta-apoprotein were replaced by Leu and Pro. The mutated LHII complex showed blue shift of absorbance peaks in the near infrared region at approximately 801-845 nm in R. sphaeroides. It should be noted that the B800 peak was much lower than that of the native LHII, and transfer energy was efficient from the B800 to the B850 pigments in the LHII complex. The results suggest that the mutated pucB could be expressed in R. sphaeroides, and the functional LHII was assembled into the membrane of R. sphaeroides notwithstanding with the different spectral properties. These mutated residues were indeed critical for the modulation of characteristics and function of LHII complex.

  20. In vivo sensitivity of blue-light-dependent signaling mediated by AppA/PpsR or PrrB/PrrA in Rhodobacter sphaeroides.

    PubMed

    Metz, Sebastian; Jäger, Andreas; Klug, Gabriele

    2009-07-01

    Formation of photosynthesis complexes in Rhodobacter sphaeroides is regulated in a redox- and light-dependent manner by the AppA/PpsR and PrrB/PrrA systems. While on the one hand, blue light is sensed by the flavin adenine dinucleotide-binding BLUF domain of AppA, on the other, light is absorbed by bacteriochlorophyll signals through PrrB/PrrA. We show that much smaller quantities initiate the AppA-mediated response to blue light than the bacteriochlorophyll-mediated response.

  1. Stable Carbon Isotope Discrimination by Form IC Rubisco Enzymes of the Extremely Metabolically Versatile Rhodobacter sphaeroides and Ralstonia eutropha}

    NASA Astrophysics Data System (ADS)

    Thomas, P. J.; Boller, A. J.; Zhao, Z.; Tabita, F. R.; Cavanaugh, C. M.; Scott, K. M.

    2006-12-01

    Variations in the relative amounts of 12C and 13C in microbial biomass can be used to infer the pathway(s) autotrophs use to fix and assimilate dissolved inorganic carbon. Discrimination against 13C by the enzymes catalyzing autotrophic carbon fixation is a major factor dictating biomass stable carbon isotopic compositions (δ13C = {[13C/12Csample/13C/12Cstandard] - 1} × 1000). Five different forms of RubisCO (IA, IB, IC, ID, and II) are utilized by algae and autotrophic bacteria reliant on the Calvin-Benson cycle for carbon fixation. To date, isotope discrimination has been measured for form IA, IB, and II RubisCOs, and their ɛ values (={[12k/13k] - 1} × 1000; 12k and 13k = rates of 12C and 13C fixation) range from 18 to 29‰, explaining the variation in biomass δ13C values of autotrophs utilizing these enzymes. Isotope discrimination by form IC RubisCO has not been measured, despite the presence of this enzyme in many proteobacteria of ecological interest, including marine manganese-oxidizing bacteria, some nitrifying and nitrogen-fixing bacteria, and extremely metabolically versatile organisms such as Rhodobacter sphaeroides and Ralstonia eutropha. The purpose of this work was to determine the ɛ values for form IC RubisCO enzymes from R. sphaeroides and R. eutropha. Recombinant form IC RubisCOs were purified by conventional column chromatography procedures. Assay conditions (pH, dissolved inorganic carbon concentration) were tested to determine which parameters were conducive to the high rates of carbon fixation necessary for ɛ determination. Under standard conditions (pH 8.5 and 5 mM DIC), form IC RubisCO activities were sufficient for ɛ determination. Experiments are currently being conducted to measure the ɛ values of these enzymes. Sampling the full phylogenetic breadth of RubisCO enzymes for isotopic discrimination makes it possible to constrain the range of δ13C values of organisms fixing carbon via the Calvin-Benson cycle. These results are

  2. Decoherence dynamics of coherent electronic excited states in the photosynthetic purple bacterium Rhodobacter sphaeroides

    NASA Astrophysics Data System (ADS)

    Liang, Xian-Ting; Zhang, Wei-Min; Zhuo, Yi-Zhong

    2010-01-01

    In this paper, we present a theoretical description to the quantum coherence and decoherence phenomena of energy transfer in photosynthesis observed in a recent experiment [Science 316, 1462 (2007)]. As a successive two-color laser pulses with selected frequencies cast on a sample of the photosynthetic purple bacterium Rb. sphaeroides two resonant excitations of electrons in chromophores can be generated. However, this effective two-level subsystem will interact with its protein environment and decoherence is inevitable. We describe this subsystem coupled with its environment as a dynamical spin-boson model. The non-Markovian decoherence dynamics is described using a quasiadiabatic propagator path integral (QUAPI) approach. With the photon-induced effective time-dependent level splitting energy and level flip coupling coefficient between the two excited states and the environment-induced non-Markovian decoherence dynamics, our theoretical result is in good agreement with the experimental data.

  3. New insights into the photochemistry of carotenoid spheroidenone in light-harvesting complex 2 from the purple bacterium Rhodobacter sphaeroides.

    PubMed

    Niedzwiedzki, Dariusz M; Dilbeck, Preston L; Tang, Qun; Martin, Elizabeth C; Bocian, David F; Hunter, C Neil; Holten, Dewey

    2017-03-01

    Light-harvesting complex 2 (LH2) from the semi-aerobically grown purple phototrophic bacterium Rhodobacter sphaeroides was studied using optical (static and time-resolved) and resonance Raman spectroscopies. This antenna complex comprises bacteriochlorophyll (BChl) a and the carotenoid spheroidenone, a ketolated derivative of spheroidene. The results indicate that the spheroidenone-LH2 complex contains two spectral forms of the carotenoid: (1) a minor, "blue" form with an S2 (1(1)B u(+) ) spectral origin band at 522 nm, shifted from the position in organic media simply by the high polarizability of the binding site, and (2) the major, "red" form with the origin band at 562 nm that is associated with a pool of pigments that more strongly interact with protein residues, most likely via hydrogen bonding. Application of targeted modeling of excited-state decay pathways after carotenoid excitation suggests that the high (92%) carotenoid-to-BChl energy transfer efficiency in this LH2 system, relative to LH2 complexes binding carotenoids with comparable double-bond conjugation lengths, derives mainly from resonance energy transfer from spheroidenone S2 (1(1)B u(+) ) state to BChl a via the Qx state of the latter, accounting for 60% of the total transfer. The elevated S2 (1(1)B u(+) ) → Qx transfer efficiency is apparently associated with substantially decreased energy gap (increased spectral overlap) between the virtual S2 (1(1)B u(+) ) → S0 (1(1)A g(-) ) carotenoid emission and Qx absorption of BChl a. This reduced energetic gap is the ultimate consequence of strong carotenoid-protein interactions, including the inferred hydrogen bonding.

  4. High yield of B-branch electron transfer in a quadruple reaction center mutant of the photosynthetic bacterium Rhodobacter sphaeroides.

    PubMed

    de Boer, Arjo L; Neerken, Sieglinde; de Wijn, Rik; Permentier, Hjalmar P; Gast, Peter; Vijgenboom, Erik; Hoff, Arnold J

    2002-03-05

    A new reaction center (RC) quadruple mutant, called LDHW, of Rhodobacter sphaeroides is described. This mutant was constructed to obtain a high yield of B-branch electron transfer and to study P(+)Q(B)(-) formation via the B-branch. The A-branch of the mutant RC contains two monomer bacteriochlorophylls, B(A) and beta, as a result of the H mutation L(M214)H. The latter bacteriochlorophyll replaces bacteriopheophytin H(A) of wild-type RCs. As a result of the W mutation A(M260)W, the A-branch does not contain the ubiquinone Q(A); this facilitates the study of P(+)Q(B)(-) formation. Furthermore, the D mutation G(M203)D introduces an aspartic acid residue near B(A). Together these mutations impede electron transfer through the A-branch. The B-branch contains two bacteriopheophytins, Phi(B) and H(B), and a ubiquinone, Q(B.) Phi(B) replaces the monomer bacteriochlorophyll B(B) as a result of the L mutation H(M182)L. In the LDHW mutant we find 35-45% B-branch electron transfer, the highest yield reported so far. Transient absorption spectroscopy at 10 K, where the absorption bands due to the Q(X) transitions of Phi(B) and H(B) are well resolved, shows simultaneous bleachings of both absorption bands. Although photoreduction of the bacteriopheophytins occurs with a high yield, no significant (approximately 1%) P(+)Q(B)(-) formation was found.

  5. Role of phospholipids of subunit III in the regulation of structural rearrangements in cytochrome c oxidase of Rhodobacter sphaeroides.

    PubMed

    Alnajjar, Khadijeh S; Cvetkov, Teresa; Prochaska, Lawrence

    2015-02-03

    Subunit III of cytochrome c oxidase possesses structural domains that contain conserved phospholipid binding sites. Mutations within these domains induce a loss of phospholipid binding, coinciding with decreased electron transfer activity. Functional and structural roles for phospholipids in the enzyme from Rhodobacter sphaeroides have been investigated. Upon the removal of intrinsic lipids using phospholipase A2, electron transfer activity was decreased 30-50%. Moreover, the delipidated enzyme exhibited turnover-induced, suicide inactivation, which was reversed by the addition of exogenous lipids, most specifically by cardiolipin. Cardiolipin exhibited two sites of interaction with the delipidated enzyme, a high-affinity site (Km = 0.14 μM) and a low-affinity site (Km = 26 μM). Subunit I of the delipidated enzyme exhibited a faster digestion rate when it was treated with α-chymotrypsin compared to that of the wild-type enzyme, suggesting that lipid removal induces a conformational change to expose the digestion sites further. Upon reaction of subunit III of the enzyme with a fluorophore (AEDANS), fluorescence anisotropy showed an increased rotational rate of the fluorophore in the absence of lipids, indicating increased flexibility of subunit III within the enzyme's tertiary structure. Additionally, Förster resonance energy transfer between AEDANS and a fluorescently labeled cardiolipin revealed that cardiolipin binds in the v-shaped cleft of subunit III in the delipidated enzyme and that it moves closer to the active site in subunit I upon a change in the redox state of the enzyme. In conclusion, these results show that the phospholipids regulate events occurring during electron transfer activity by maintaining the structural integrity of the enzyme at the active site.

  6. An extended model for the repression of photosynthesis genes by the AppA/PpsR system in Rhodobacter sphaeroides.

    PubMed

    Pandey, Rakesh; Flockerzi, Dietrich; Hauser, Marcus J B; Straube, Ronny

    2012-09-01

    Purple bacteria derive energy from aerobic respiration or photosynthesis depending on the availability of oxygen and light. Under aerobic conditions, photosynthesis genes are specifically repressed by the PpsR protein. In Rhodobacter sphaeroides, the repressive action of PpsR is antagonized by the blue-light and redox-sensitive flavoprotein AppA, which sequesters PpsR under anaerobic conditions into transcriptionally inactive complexes. However, under semi-aerobic conditions, blue-light excitation of AppA causes the AppA-PpsR complexes to dissociate, again leading to a repression of photosynthesis genes. We have recently developed a simple mathematical model suggesting that this phenotype arises from the formation of a maximum in the response curve of reduced PpsR at intermediate oxygen concentrations. However, this model focused mainly on the oxygen-dependent interactions whereas light regulation was only implemented in a simplified manner. In the present study, we incorporate a more detailed mechanism for the light-dependent interaction between AppA and PpsR, which now allows for a direct comparison with experiments. Specifically, we take into account that, upon blue-light excitation, AppA undergoes a conformational change, creating a long-lived signalling state causing the dissociation of the AppA-PpsR complexes. The predictions of the extended model are found to be in good agreement with experimental results on the light-dependent repression of photosynthesis genes under semi-aerobic conditions. We also identify the potential kinetic and stoichiometric constraints that the interplay between light and redox regulation imposes on the functionality of the AppA/PpsR system, especially with respect to a possible bistable response. © 2012 The Authors Journal compilation © 2012 FEBS.

  7. Interactions between Cytochrome c2 and the Photosynthetic Reaction Center from Rhodobacter sphaeroides : The Cation-pi Interaction†

    PubMed Central

    Paddock, M. L.; Weber, K. H.; Chang, C.; Okamura, M. Y.

    2008-01-01

    The cation-pi interaction between positively charged and aromatic groups is a common feature of many proteins and protein complexes. The structure of the complex between cytochrome c2 (cyt c2) and photosynthetic reaction center (RC) from Rhodobacter sphaeroides exhibits a cation-pi complex formed between Arg-C32 on cyt c2 and Tyr-M295 on the RC (Axelrod et. al (2002) J. Mol. Biol. 319, 501–515). The importance of the cation-pi interaction for binding and electron transfer was studied by mutating Tyr-M295 and Arg-C32. The first and second order rates for electron transfer were not affected by mutating Tyr-M295 to Ala indicating that the cation-pi complex does not greatly affect the association process or structure of the state active in electron transfer. The dissociation constant KD showed a greater increase when Try-M295 was replaced by non-aromatic Ala (3-fold) than by aromatic Phe (1.2-fold) characteristic of a cation-pi interaction. Replacement of Arg-C32 by Ala increased KD (80-fold) largely due to removal of electrostatic interactions with negatively charged residues on the RC. Replacement by Lys, increased KD (6-fold) indicating that Lys does not form a cation-pi complex. This specificity for Arg may be due to a solvation effect. Double mutant analysis indicates interaction energy between Tyr-M295 and Arg-C32 of about −24 meV (−0.6 kcal/mole). This energy is surprisingly small considering the widespread occurrence of cation-pi complexes and may be due to the trade-off between the favorable cation-pi binding energy and the unfavorable desolvation energy needed to bury Arg-C32 in the short-range contact region between the two proteins. PMID:16008347

  8. Expression of the Rhodobacter sphaeroides hemA and hemT genes, encoding two 5-aminolevulinic acid synthase isozymes.

    PubMed Central

    Neidle, E L; Kaplan, S

    1993-01-01

    The nucleotide sequences of the Rhodobacter sphaeroides hemA and hemT genes, encoding 5-aminolevulinic acid (ALA) synthase isozymes, were determined. ALA synthase catalyzes the condensation of glycine and succinyl coenzyme A, the first and rate-limiting step in tetrapyrrole biosynthesis. The hemA and hemT structural gene sequences were 65% identical to each other, and the deduced HemA and HemT polypeptide sequences were 53% identical, with an additional 16% of aligned amino acids being similar. HemA and HemT were homologous to all characterized ALA synthases, including two human ALA synthase isozymes. In addition, they were evolutionarily related to 7-keto-8-aminopelargonic acid synthetase (BioF) and 2-amino-3-ketobutyrate coenzyme A ligase (Kbl), enzymes which catalyze similar reactions. Two hemA transcripts were identified, both expressed under photosynthetic conditions at levels approximately three times higher than those found under aerobic conditions. A single transcriptional start point was identified for both transcripts, and a consensus sequence at this location indicated that an Fnr-like protein may be involved in the transcriptional regulation of hemA. Transcription of hemT was not detected in wild-type cells under the physiological growth conditions tested. In a mutant strain in which the hemA gene had been inactivated, however, hemT was expressed. In this mutant, hemT transcripts were characterized by Northern (RNA) hybridization, primer extension, and ribonuclease protection techniques. A small open reading frame of unknown function was identified upstream of, and transcribed in the same direction as, hemA. Images PMID:8468290

  9. Physical and genetic mapping of the Rhodobacter sphaeroides 2.4.1 genome: genome size, fragment identification, and gene localization.

    PubMed Central

    Suwanto, A; Kaplan, S

    1989-01-01

    Four restriction endonucleases, AseI (5'-ATTAAT), SpeI (5'-ACTAGT), DraI (5'-TTTAAA), and SnaBI (5'-TACGTA), generated DNA fragments of suitable size distributions for mapping the genome of Rhodobacter sphaeroides by transverse alternating field electrophoresis. AseI produced 17 fragments, ranging in size from 3 to 1,105 kilobases (kb), SpeI yielded 16 fragments (12 to 1,645 kb), DraI yielded at least 25 fragments (6 to 800 kb), and SnaBI generated 10 fragments (12 to 1,225 kb). A total genome size of approximately 4,400 +/- 112 kb was determined by summing the fragment lengths in each of the digests generated by using the different restriction endonucleases. The total genomic DNA consisted of chromosomal DNA (3,960 +/- 112 kb) and the five endogenous plasmids (approximately 450 kb total) whose cognate DNA fragments have been unambiguously identified. A number of genes have been physically mapped to the AseI-generated restriction endonuclease fragments of total genomic DNA by Southern hybridization analysis with either homologous or heterologous specific gene probes or, in the case of several auxotrophic and pigment-biosynthetic mutants apparently generated by Tn5, a Tn5-specific probe. Other genes have been mapped by a comparison with wild-type patterns of the electrophoretic banding patterns of the AseI-digested genomic DNA derived from mutants generated by the insertion of either kanamycin or spectinomycin-streptomycin resistance cartridges. The relative orientations, distance, and location of the pufBALMX, puhA, cycA, and pucBA operons have also been determined, as have been the relative orientations between prkB and hemT and between prkA and the fbc operon. Images PMID:2553662

  10. Quenching Capabilities of Long-Chain Carotenoids in Light-Harvesting-2 Complexes from Rhodobacter sphaeroides with an Engineered Carotenoid Synthesis Pathway.

    PubMed

    Dilbeck, Preston L; Tang, Qun; Mothersole, David J; Martin, Elizabeth C; Hunter, C Neil; Bocian, David F; Holten, Dewey; Niedzwiedzki, Dariusz M

    2016-06-23

    Six light-harvesting-2 complexes (LH2) from genetically modified strains of the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. These strains were engineered to incorporate carotenoids for which the number of conjugated groups (N = NC═C + NC═O) varies from 9 to 15. The Rb. sphaeroides strains incorporate their native carotenoids spheroidene (N = 10) and spheroidenone (N = 11), as well as longer-chain analogues including spirilloxanthin (N = 13) and diketospirilloxantion (N = 15) normally found in Rhodospirillum rubrum. Measurements of the properties of the carotenoid first singlet excited state (S1) in antennas from the Rb. sphaeroides set show that carotenoid-bacteriochlorophyll a (BChl a) interactions are similar to those in LH2 complexes from various other bacterial species and thus are not significantly impacted by differences in polypeptide composition. Instead, variations in carotenoid-to-BChl a energy transfer are primarily regulated by the N-determined energy of the carotenoid S1 excited state, which for long-chain (N ≥ 13) carotenoids is not involved in energy transfer. Furthermore, the role of the long-chain carotenoids switches from a light-harvesting supporter (via energy transfer to BChl a) to a quencher of the BChl a S1 excited state B850*. This quenching is manifested as a substantial (∼2-fold) reduction of the B850* lifetime and the B850* fluorescence quantum yield for LH2 housing the longest carotenoids.

  11. The cobinamide amidohydrolase (cobyric acid-forming) CbiZ enzyme: a critical activity of the cobamide remodelling system of Rhodobacter sphaeroides.

    PubMed

    Gray, Michael J; Escalante-Semerena, Jorge C

    2009-12-01

    The chemical structures of cobamides [cobalamin (Cbl)-like compounds] are the same, except for the lower ligand, which in adenosylcobalamin (AdoCbl) is 5,6-dimethylbenzimidazole, and in adenosylpseudocobalamin (AdopseudoCbl) is adenine. Why the lower ligand of cobamides varies and what the mechanism of lower ligand replacement is are long-standing questions in the field of B(12) biosynthesis. Work reported here uncovers the strategy used by the photosynthetic alpha-proteobacterium Rhodobacter sphaeroides to procure the cobamide it needs to grow on acetate as a carbon and energy source. On the basis of genetic and biochemical evidence we conclude that, in R. sphaeroides, the activity of the cobyric acid-producing amidohydrolase CbiZ enzyme is essential for the conversion of AdopseudoCbl into AdoCbl, the cobamide needed for the catabolism of acetate. The CbiZ enzyme uses AdopseudoCbl as a substrate, but not AdoCbl. Implications of these findings for cobamide remodelling in R. sphaeroides and in other CbiZ-containing microorganisms are discussed.

  12. DegS and RseP Homologous Proteases Are Involved in Singlet Oxygen Dependent Activation of RpoE in Rhodobacter sphaeroides

    PubMed Central

    Nuss, Aaron M.; Adnan, Fazal; Weber, Lennart; Berghoff, Bork A.; Glaeser, Jens; Klug, Gabriele

    2013-01-01

    Singlet oxygen (1O2) is the main agent of photooxidative stress and is generated by photosensitizers as (bacterio)chlorophylls. It leads to the damage of cellular macromolecules and therefore photosynthetic organisms have to mount an adaptive response to 1O2 formation. A major player of the photooxidative stress response in Rhodobacter sphaeroides is the alternative sigma factor RpoE, which is inactivated under non-stress conditions by its cognate anti-sigma factor ChrR. By using random mutagenesis we identified RSP_1090 to be required for full activation of the RpoE response under 1O2 stress, but not under organic peroxide stress. In this study we show that both RSP_1090 and RSP_1091 are required for full resistance towards 1O2. Moreover, we revealed that the DegS and RseP homologs RSP_3242 and RSP_2710 contribute to 1O2 resistance and promote ChrR proteolysis. The RpoE signaling pathway in R. sphaeroides is therefore highly similar to that of Escherichia coli, although very different anti-sigma factors control RpoE activity. Based on the acquired results, the current model for RpoE activation in response to 1O2 exposure in R. sphaeroides was extended. PMID:24223961

  13. The cobinamide amidohydrolase (cobyric acid-forming) CbiZ enzyme: A critical activity of the cobamide remodeling system of Rhodobacter sphaeroides

    PubMed Central

    Gray, Michael J.; Escalante-Semerena, Jorge C.

    2010-01-01

    SUMMARY The chemical structures of cobamides (cobalamin [Cbl]-like compounds) are the same, except for the lower ligand, which in adenosylcobalamin (AdoCbl) is 5,6-dimethylbenzimidazole (DMB), and in adenosylpseudocobalamin (AdopseudoCbl) is adenine. Why the lower ligand of cobamides varies and what the mechanism of lower ligand replacement is are long-standing questions in the field of B12 biosynthesis. Work reported here uncovers the strategy used by the photosynthetic α-proteobacterium Rhodobacter sphaeroides to procure the cobamide it needs to grow on acetate as a carbon and energy source. On the basis of genetic and biochemical evidence we conclude that, in R. sphaeroides, the activity of the cobyric acid-producing amidohydrolase CbiZ enzyme is essential for the conversion of AdopseudoCbl into AdoCbl, the cobamide needed for the catabolism of acetate. The CbiZ enzyme uses AdopseudoCbl as a substrate, but not AdoCbl. Implications of these findings for cobamide remodeling in R. sphaeroides and in other CbiZ-containing microorganisms are discussed. PMID:19889098

  14. Cardiolipin Deficiency in Rhodobacter sphaeroides Alters the Lipid Profile of Membranes and of Crystallized Cytochrome Oxidase, but Structure and Function Are Maintained

    SciTech Connect

    Zhang, Xi; Tamot, Banita; Hiser, Carrie; Reid, Gavin E.; Benning, Christoph; Ferguson-Miller, Shelagh

    2012-05-08

    Many recent studies highlight the importance of lipids in membrane proteins, including in the formation of well-ordered crystals. To examine the effect of changes in one lipid, cardiolipin, on the lipid profile and the production, function, and crystallization of an intrinsic membrane protein, cytochrome c oxidase, we mutated the cardiolipin synthase (cls) gene of Rhodobacter sphaeroides, causing a >90% reduction in cardiolipin content in vivo and selective changes in the abundances of other lipids. Under these conditions, a fully native cytochrome c oxidase (CcO) was produced, as indicated by its activity, spectral properties, and crystal characteristics. Analysis by MALDI tandem mass spectrometry (MS/MS) revealed that the cardiolipin level in CcO crystals, as in the membranes, was greatly decreased. Lipid species present in the crystals were directly analyzed for the first time using MS/MS, documenting their identities and fatty acid chain composition. The fatty acid content of cardiolipin in R. sphaeroides CcO (predominantly 18:1) differs from that in mammalian CcO (18:2). In contrast to the cardiolipin dependence of mammalian CcO activity, major depletion of cardiolipin in R. sphaeroides did not impact any aspect of CcO structure or behavior, suggesting a greater tolerance of interchange of cardiolipin with other lipids in this bacterial system.

  15. Quenching Capabilities of Long-Chain Carotenoids in Light-Harvesting-2 Complexes from Rhodobacter sphaeroides with an Engineered Carotenoid Synthesis Pathway

    PubMed Central

    2016-01-01

    Six light-harvesting-2 complexes (LH2) from genetically modified strains of the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. These strains were engineered to incorporate carotenoids for which the number of conjugated groups (N = NC=C + NC=O) varies from 9 to 15. The Rb. sphaeroides strains incorporate their native carotenoids spheroidene (N = 10) and spheroidenone (N = 11), as well as longer-chain analogues including spirilloxanthin (N = 13) and diketospirilloxantion (N = 15) normally found in Rhodospirillum rubrum. Measurements of the properties of the carotenoid first singlet excited state (S1) in antennas from the Rb. sphaeroides set show that carotenoid-bacteriochlorophyll a (BChl a) interactions are similar to those in LH2 complexes from various other bacterial species and thus are not significantly impacted by differences in polypeptide composition. Instead, variations in carotenoid-to-BChl a energy transfer are primarily regulated by the N-determined energy of the carotenoid S1 excited state, which for long-chain (N ≥ 13) carotenoids is not involved in energy transfer. Furthermore, the role of the long-chain carotenoids switches from a light-harvesting supporter (via energy transfer to BChl a) to a quencher of the BChl a S1 excited state B850*. This quenching is manifested as a substantial (∼2-fold) reduction of the B850* lifetime and the B850* fluorescence quantum yield for LH2 housing the longest carotenoids. PMID:27285777

  16. Proton displacements coupled to primary electron transfer in the Rhodobacter sphaeroides reaction center.

    PubMed

    Eisenmayer, Thomas J; Lasave, Jorge A; Monti, Adriano; de Groot, Huub J M; Buda, Francesco

    2013-09-26

    Using first-principles molecular dynamics (AIMD) and constrained density functional theory (CDFT) we identify the pathway of primary electron transfer in the R. Sphaeroides reaction center from the special pair excited state (P*) to the accessory bacteriochlorophyll (BA). Previous AIMD simulations on the special pair (PLPM) predicted a charge-transfer intermediate formation through the excited-state relaxation along a reaction coordinate characterized by the rotation of an axial histidine (HisM202). To account for the full electron transfer we extend the model to include the primary acceptor BA. In this extended model, the LUMO is primarily localized on the acceptor BA and extends over an interstitial water (water A) that is known to influence the rate of electron transfer (Potter et al. Biochemistry 2005 280, 27155-27164). A vibrational analysis of the dynamical trajectories gives a frequency of 30-35 cm(-1) for a molecular motion involving the hydrogen-bond network around water A, in good agreement with experimental findings (Yakovlev et al. Biochemistry, 2003, 68, 603-610). In its binding pocket water A can act as a switch by breaking and forming hydrogen bonds. With CDFT we calculate the energy required to the formation of the charge-separated state and find it to decrease along the predicted anisotropic reaction coordinate. Furthermore, we observe an increased coupling between the ground and charge-separated state. Water A adapts its hydrogen-bonding network along this reaction coordinate and weakens the hydrogen bond with HisM202. We also present AIMD simulations on the radical cation (P(•+)) showing a weakening of the hydrogen bond between HisL168 and the 3(1)-acetyl of PL. This work demonstrates how proton displacements are crucially coupled to the primary electron transfer and characterizes the reaction coordinate of the initial photoproduct formation.

  17. Transcriptional Activation of the Rhodobacter sphaeroides Cytochrome c2 Gene P2 Promoter by the Response Regulator PrrA

    PubMed Central

    Comolli, James C.; Carl, Audrey J.; Hall, Christine; Donohue, Timothy

    2002-01-01

    Anoxygenic photosynthetic growth of Rhodobacter sphaeroides, a member of the α subclass of the class Proteobacteria, requires the response regulator PrrA. PrrA and the sensor kinase PrrB are part of a two-component signaling pathway that influences a wide range of processes under oxygen-limited conditions. In this work we characterized the pathway of transcription activation by PrrB and PrrA by purifying these proteins, analyzing them in vitro, and characterizing a mutant PrrA protein in vivo and in vitro. When purified, a soluble transmitter domain of PrrB (cPrrB) could autophosphorylate, rapidly transfer phosphate to PrrA, and stimulate dephosphorylation of phospho-PrrA. Unphosphorylated PrrA activated transcription from a target cytochrome c2 gene (cycA) promoter, P2, which contained sequences from −73 to +22 relative to the transcription initiation site. However, phosphorylation of PrrA increased its activity since activation of cycA P2 was enhanced up to 15-fold by treatment with the low-molecular-weight phosphodonor acetyl phosphate. A mutant PrrA protein containing a single amino acid substitution in the presumed phosphoacceptor site (PrrA-D63A) was not phosphorylated in vitro but also was not able to stimulate cycA P2 transcription. PrrA-D63A also had no apparent in vivo activity, demonstrating that aspartate 63 is necessary both for the function of PrrA and for its phosphorylation-dependent activation. The cellular level of wild-type PrrA was negatively autoregulated so that less PrrA was present in the absence of oxygen, conditions in which the activities of many PrrA target genes increase. PrrA-D63A failed to repress expression of the prrA gene under anaerobic conditions, suggesting that this single amino acid change also eliminated PrrA function in vivo. PMID:11751815

  18. Mutational analysis of the C-terminal domain of the Rhodobacter sphaeroides response regulator PrrA

    PubMed Central

    Jones, Denise F.; Stenzel, Rachelle A.; Donohue, Timothy J.

    2009-01-01

    The Rhodobacter sphaeroides response regulator PrrA directly activates transcription of genes necessary for energy conservation at low O2 tensions and under anaerobic conditions. It is proposed that PrrA homologues contain a C-terminal DNA-binding domain (PrrA-CTD) that lacks significant amino acid sequence similarity to those found in other response regulators. To test this hypothesis, single amino acid substitutions were created at 12 residues in the PrrA-CTD. These mutant PrrA proteins were purified and tested for the ability to be phosphorylated by the low-molecular-mass phosphate donor acetyl phosphate, to activate transcription and to bind promoter DNA. Each mutant PrrA protein accepted phosphate from 32P-labelled acetyl phosphate. At micromolar concentrations of acetyl phosphate-treated wild-type PrrA, a single 20 bp region in the PrrA-dependent cycA P2 promoter was protected from DNase I digestion. Of the mutant PrrA proteins tested, only acetyl phosphate-treated PrrA-N168A and PrrA-I177A protected cycA P2 from DNase I digestion at similar protein concentrations compared to wild-type PrrA. The use of in vitro transcription assays with the PrrA-dependent cycA P2 and puc promoters showed that acetyl phosphate-treated PrrA-N168A produced transcript levels similar to that of wild-type PrrA at comparable protein concentrations. Using concentrations of acetyl phosphate-treated PrrA that are saturating for the wild-type protein, PrrA-H170A and PrrA-I177A produced<45%as much transcript as wild-type PrrA. Under identical conditions, the remaining mutant PrrA proteins produced little or no detectable transcripts from either promoter in vitro. Explanations are presented for why these amino acid side chains in the PrrA-CTD are important for its ability to activate transcription. PMID:16339955

  19. Isolation, size estimates, and spectral heterogeneity of an oligomeric series of light-harvesting 1 complexes from Rhodobacter sphaeroides.

    PubMed

    Westerhuis, Willem H J; Sturgis, James N; Ratcliffe, Emma C; Hunter, C Neil; Niederman, Robert A

    2002-07-09

    A series of light-harvesting 1 (LH1) complexes was isolated by lithium dodecyl sulfate-polyacrylamide gel electrophoresis at 4 degrees C from Rhodobacter sphaeroides M21, which lacks the peripheral light-harvesting 2 (LH2) complex. This ladder of LH1 bands was also demonstrated in the wild type, partially superimposed upon a smaller number of LH2 complexes. An assessment of electrophoretic mobility vs acrylamide concentration, in which the reaction center LM particle and annular LH1 and LH2 complexes were used as standards of known structure, indicated that the LH1 gel bands 2 to 10 represent regular oligomers of an alpha beta heterodimeric unit, that vary in size from (alpha beta)(2-3) to (alpha beta)(10-11). The isolated LH1 complexes exhibited oligomeric state dependent optical properties, characterized by red shifts in near-IR absorption and emission maxima at 77 K of approximately 6 nm as aggregate sizes increased from approximately 3 to 7-8 alpha beta-heterodimers, accompanied by shifts in highly polarized fluorescence from the blue to the red side of the absorption band. This has been explained by the oligomerization of heterodimers to form a curvilinear array of excitonically coupled chromophores, with the anisotropic long-wavelength component, designated originally as B896, corresponding to low energy excitonic transitions arising from interactions within inhomogeneous BChl clusters [Westerhuis et al. (1999) J. Phys. Chem. B 103, 7733-7742]. Differences in electrophoretic profiles of LH1 bands between strains M21 and M2192, an LH1-only strain that also lacks PufX, further suggested that the more rapidly migrating bands represent arced fragments of the curvilinear array of LH1 complexes thought to exist as a large closed circular structure only in the latter strain. The electrophoretic banding pattern also indicated that the LH1 complex may be located at the peripheries of dimeric intramembrane particle arrays seen in freeze-fracture replicas of tubular M21

  20. B-branch electron transfer in reaction centers of Rhodobacter sphaeroides assessed with site-directed mutagenesis.

    PubMed

    de Boer, Arjo L; Neerken, Sieglinde; de Wijn, Rik; Permentier, Hjalmar P; Gast, Peter; Vijgenboom, Erik; Hoff, Arnold J

    2002-01-01

    Mutants of Rhodobacter (Rba.) sphaeroides are described which were designed to study electron transfer along the so-called B-branch of reaction center (RC) cofactors. Combining the mutation L(M214)H, which results in the incorporation of a bacteriochlorophyll, beta, for H(A) [Kirmaier et al. (1991) Science 251: 922-927] with two mutations, G(M203)D and Y(M210)W, near B(A), we have created a double and a triple mutant with long lifetimes of the excited state P(*) of the primary donor P, viz. 80 and 160 ps at room temperature, respectively. The yield of P(+)Q(A) (-) formation in these mutants is reduced to 50 and 30%, respectively, of that in wildtype RCs. For both mutants, the quantum yield of P(+)H(B) (-) formation was less than 10%, in contrast to the 15% B-branch electron transfer demonstrated in RCs of a similar mutant of Rba. capsulatus with a P(*) lifetime of 15 ps [Heller et al. (1995) Science 269: 940-945]. We conclude that the lifetime of P(*) is not a governing factor in switching to B-branch electron transfer. The direct photoreduction of the secondary quinone, Q(B), was studied with a triple mutant combining the G(M203)D, L(M214)H and A(M260)W mutations. In this triple mutant Q(A) does not bind to the reaction center [Ridge et al. (1999) Photosynth Res 59: 9-26]. It is shown that B-branch electron transfer leading to P(+)Q(B) (-) formation occurs to a minor extent at both room temperature and at cryogenic temperatures (about 3% following a saturating laser flash at 20 K). In contrast, in wildtype RCs P(+)Q(B) (-) formation involves the A-branch and does not occur at all at cryogenic temperatures. Attempts to accumulate the P(+)Q(B) (-) state under continuous illumination were not successful. Charge recombination of P(+)Q(B) (-) formed by B-branch electron transfer in the new mutant is much faster (seconds) than has been previously reported for charge recombination of P(+)Q(B) (-) trapped in wildtype RCs (10(5) s) [Kleinfeld et al. (1984b) Biochemistry 23

  1. An RpoHI-Dependent Response Promotes Outgrowth after Extended Stationary Phase in the Alphaproteobacterium Rhodobacter sphaeroides.

    PubMed

    Remes, B; Rische-Grahl, T; Müller, K M H; Förstner, K U; Yu, Sung-Huan; Weber, L; Jäger, A; Peuser, V; Klug, G

    2017-07-15

    Under unfavorable growth conditions, bacteria enter stationary phase and can maintain cell viability over prolonged periods with no increase in cell number. To obtain insights into the regulatory mechanisms that allow bacteria to resume growth when conditions become favorable again (outgrowth), we performed global transcriptome analyses at different stages of growth for the alphaproteobacterium Rhodobacter sphaeroides The majority of genes were not differentially expressed across growth phases. After a short stationary phase (about 20 h after growth starts to slow down), only 7% of the genes showed altered expression (fold change of >1.6 or less than -1.6, corresponding to a log2 fold change of >0.65 or less than -0.65, respectively) compared to expression at exponential phase. Outgrowth induced a distinct response in gene expression which was strongly influenced by the length of the preceding stationary phase. After a long stationary phase (about 64 h after growth starts to slow down), a much larger number of genes (15.1%) was induced in outgrowth than after a short stationary phase (1.7%). Many of those genes are known members of the RpoHI/RpoHII regulons and have established functions in stress responses. A main effect of RpoHI on the transcriptome in outgrowth after a long stationary phase was confirmed. Growth experiments with mutant strains further support an important function in outgrowth after prolonged stationary phase for the RpoHI and RpoHII sigma factors.IMPORTANCE In natural environments, the growth of bacteria is limited mostly by lack of nutrients or other unfavorable conditions. It is important for bacterial populations to efficiently resume growth after being in stationary phase, which may last for long periods. Most previous studies on growth-phase-dependent gene expression did not address outgrowth after stationary phase. This study on growth-phase-dependent gene regulation in a model alphaproteobacterium reveals, for the first time, that the

  2. Enhanced photo-fermentative H2 production using Rhodobacter sphaeroides by ethanol addition and analysis of soluble microbial products

    PubMed Central

    2014-01-01

    Background Biological fermentation routes can provide an environmentally friendly way of producing H2 since they use renewable biomass as feedstock and proceed under ambient temperature and pressure. In particular, photo-fermentation has superior properties in terms of achieving high H2 yield through complete degradation of substrates. However, long-term H2 production data with stable performance is limited, and this data is essential for practical applications. In the present work, continuous photo-fermentative H2 production from lactate was attempted using the purple non-sulfur bacterium, Rhodobacter sphaeroides KD131. As a gradual drop in H2 production was observed, we attempted to add ethanol (0.2% v/v) to the medium. Results As continuous operation went on, H2 production was not sustained and showed a negligible H2 yield (< 0.5 mol H2/mol lactateadded) within two weeks. Electron balance analysis showed that the reason for the gradual drop in H2 production was ascribed to the increase in production of soluble microbial products (SMPs). To see the possible effect of ethanol addition, a batch test was first conducted. The presence of ethanol significantly increased the H2 yield from 1.15 to 2.20 mol H2/mol lactateadded, by suppressing the production of SMPs. The analysis of SMPs by size exclusion chromatography showed that, in the later period of fermentation, more than half of the low molecular weight SMPs (< 1 kDa) were consumed and used for H2 production when ethanol had been added, while the concentration of SMPs continuously increased in the absence of ethanol. It was found that the addition of ethanol facilitated the utilization of reducing power, resulting in an increase in the cellular levels of NAD+ and NADP+. In continuous operation, ethanol addition was effective, such that stable H2 production was attained with an H2 yield of 2.5 mol H2/mol lactateadded. Less than 15% of substrate electrons were used for SMP production, whereas 35% were used in

  3. B-side electron transfer promoted by absorbance of multiple photons in Rhodobacter sphaeroides R-26 reaction centers

    SciTech Connect

    Lin, S.; Jackson, J.A.; Taguchi, A.K.W.; Woodbury, N.W.

    1999-06-03

    Femtosecond transient absorbance spectra of quinone-depleted Rhodobacter sphaeroides R-26 reaction centers in the Q{sub X} transition region have been measured at 15 K under various excitation conditions. This study focuses on the excitation wavelength dependence and excitation intensity dependence of the formation of charge-separated states on the A- and B-side of the reaction center, judging from the bleaching of the 533 nm (B-side) and 544 nm (A-side) ground-state transitions of the reaction center bacteriopheophytins (H{sub A} and H{sub B}). Upon low-intensity selective excitation directly into the bacteriopheophytin Q{sub Y} transitions (near 760 nm), bleaching of both ground-state bacteriopheophytin Q{sub X} transitions appeared immediately, showing that initially either the A- or B-side bacteriopheophytin could be excited. However, both excited states ultimately resulted in P{sup +}H{sub A}{sup {minus}} formation under these conditions. Low-intensity excitation at any of the various wavelengths showed no difference in the kinetics of the A-side charge separation forming P{sup +}H{sub A}{sup {minus}} and no substantial formation of the B-side charge-separated state, P{sup +}H{sub B}{sup {minus}}. In contrast, high-intensity 595 nm excitation resulted in substantial long-lived bleaching of the B-side bacteriopheophytin ground-state transition at 533 nm. This 533 nm bleaching was formed with essentially the same time constant as the bleaching at 544 nm due to A-side charge separation. Both bleaching bands persisted at the longest times measured in quinone-removed reaction centers. The long-lived bleaching at 533 nm using high-intensity excitation most likely represents the formation of P{sup +}H{sub B}{sup {minus}} with a relative yield of nearly 40%. One possible mechanism for B-side electron transfer is that two-photon excitation of the reaction center resulting in the state P{sup *}B{sub B}{sup *} makes P{sup +}B{sub B}{sup {minus}} thermodynamically

  4. IscR of Rhodobacter sphaeroides functions as repressor of genes for iron-sulfur metabolism and represents a new type of iron-sulfur-binding protein

    PubMed Central

    Remes, Bernhard; Eisenhardt, Benjamin D; Srinivasan, Vasundara; Klug, Gabriele

    2015-01-01

    IscR proteins are known as transcriptional regulators for Fe–S biogenesis. In the facultatively phototrophic bacterium, Rhodobacter sphaeroides IscR is the product of the first gene in the isc-suf operon. A major role of IscR in R. sphaeroides iron-dependent regulation was suggested in a bioinformatic study (Rodionov et al., PLoS Comput Biol 2:e163, 2006), which predicted a binding site in the upstream regions of several iron uptake genes, named Iron-Rhodo-box. Most known IscR proteins have Fe–S clusters featuring (Cys)3(His)1 ligation. However, IscR proteins from Rhodobacteraceae harbor only a single-Cys residue and it was considered unlikely that they can ligate an Fe–S cluster. In this study, the role of R. sphaeroides IscR as transcriptional regulator and sensor of the Fe–S cluster status of the cell was analyzed. A mutant lacking IscR is more impaired in growth under iron limitation than the wild-type and exhibits significantly increased ROS levels in iron-replete and iron-deplete conditions. Expression studies reveal that R. sphaeroides IscR in its cluster-bound form functions as transcriptional repressor of genes involved in iron metabolism by direct binding to the promoter region of genes preceded by the motif. A total of 110 genes are directly or indirectly affected by IscR. Furthermore, IscR possesses a unique Fe–S cluster ligation scheme with only a single cysteine involved. PMID:26235649

  5. Use of new strains of Rhodobacter sphaeroides and a modified simple culture medium to increase yield and facilitate purification of the reaction centre.

    PubMed

    Jun, D; Saer, R G; Madden, J D; Beatty, J T

    2014-05-01

    A new gene expression system was developed in Rhodobacter sphaeroides, replacing a pRK415-based system used previously. The broad host-range IPTG-inducible plasmid pIND4 was used to create the plasmid pIND4-RC1 for expression of the puhA and pufQBALMX genes, encoding the reaction centre (RC) and light-harvesting complex 1 (LH1) proteins. The strain R. sphaeroides ΔRCLH was used to make a knockout of the rshI restriction endonuclease gene, enabling electroporation of DNA into the bacterium; a subsequent knockout of ppsR was made, creating the strain R. sphaeroides RCx lacking this oxygen-sensing repressor of the photosynthesis gene cluster. Using pIND4-RC1, LH1 levels were increased by a factor of about 8 over pRS1 per cell in cultures grown semi-aerobically. In addition, the ppsR knockout allowed for photosynthetic pigment-protein complex synthesis in the presence of high concentrations of molecular oxygen; here, LH1 levels per cell increased by 20 % when grown under high aeration conditions. A new medium (called RLB) is the E. coli medium LB supplemented with MgCl2 and CaCl2, which was found to increase growth rates and final cell culture densities, with an increase of 30 % of LH1 per cell detected in R. sphaeroides RCx(pIND4-RC1) grown in RLB versus LB medium. Furthermore, cell density was about three times greater in RLB compared to semi-aerobic conditions. The combination of all the modifications resulted in an increase of LH1 and RC per mL of culture volume by approximately 35-fold, and a decrease in the length of culture incubation time from about 5 days to ~36 h.

  6. Structural and preliminary molecular dynamics studies of the Rhodobacter sphaeroides reaction center and its mutant form L(M196)H + H(M202)L

    NASA Astrophysics Data System (ADS)

    Klyashtorny, V. G.; Fufina, T. Yu.; Vasilieva, L. G.; Shuvalov, V. A.; Gabdulkhakov, A. G.

    2014-07-01

    Pigment-protein interactions are responsible for the high efficiency of the light-energy transfer and conversion in photosynthesis. The reaction center (RC) from the purple bacterium Rhodobacter sphaeroides is the most convenient model for studying the mechanisms of primary processes of photosynthesis. Site-directed mutagenesis can be used to study the effect of the protein environment of electron-transfer cofactors on the optical properties, stability, pigment composition, and functional activity of RC. The preliminary analysis of RC was performed by computer simulation of the amino acid substitutions L(M196)H + H(M202)L at the pigment-protein interface and by estimating the stability of the threedimensional structure of the mutant RC by the molecular dynamics method. The doubly mutated reaction center was overexpressed, purified, and crystallized. The three-dimensional structure of this mutant was determined by X-ray crystallography and compared with the molecular dynamics model.

  7. Crystallization and preliminary X-ray analysis of the periplasmic nitrate reductase (NapA-NapB complex) from Rhodobacter sphaeroides f. sp. denitrificans.

    PubMed

    Pignol, D; Adriano, J M; Fontecilla-Camps, J C; Sabaty, M

    2001-12-01

    The periplasmic nitrate reductase of Rhodobacter sphaeroides f. sp. denitrificans is a heterodimer responsible for the first step of reduction in the denitrification process by the conversion of nitrate to nitrite. It consists of a 91 kDa molybdenum-containing catalytic subunit (NapA) and a 17 kDa dihaem cytochrome c (NapB). Crystals of the NapA-NapB complex were obtained by the vapour-diffusion method using ammonium sulfate as precipitant. They belong to the P6(1)22 space group, with unit-cell parameters a = b = 151.9, c = 255.8 A, and contain a single complex in the asymmetric unit. A complete native data set was collected at a synchrotron source to 3.1 A resolution.

  8. New tetragonal form of reaction centers from Rhodobacter sphaeroides and the involvement of a manganese ion at a crystal contact point

    PubMed Central

    Uyeda, G.; Cámara-Artigas, A.; Williams, J. C.; Allen, J. P.

    2005-01-01

    Crystals have been obtained of wild-type reaction centers from Rhodobacter sphaeroides using manganese chloride as a precipitating agent. The crystals belong to the tetragonal space group P4222, with unit-cell parameters a = b = 207.8, c = 107.5 Å. The crystal structure has been determined to a resolution limit of 4.6 Å using a previously determined structure of the reaction center as a molecular-replacement model. The calculated electron-density maps show the presence of a manganese ion at one of the crystal contact points bridging two symmetry-related histidine residues, suggesting that the metal plays a key role in facilitating the crystallization of the protein in this form. PMID:16511142

  9. New tetragonal form of reaction centers from Rhodobacter sphaeroides and the involvement of a manganese ion at a crystal contact point.

    PubMed

    Uyeda, G; Cámara-Artigas, A; Williams, J C; Allen, J P

    2005-08-01

    Crystals have been obtained of wild-type reaction centers from Rhodobacter sphaeroides using manganese chloride as a precipitating agent. The crystals belong to the tetragonal space group P4(2)22, with unit-cell parameters a = b = 207.8, c = 107.5 A. The crystal structure has been determined to a resolution limit of 4.6 A using a previously determined structure of the reaction center as a molecular-replacement model. The calculated electron-density maps show the presence of a manganese ion at one of the crystal contact points bridging two symmetry-related histidine residues, suggesting that the metal plays a key role in facilitating the crystallization of the protein in this form.

  10. Isolation and ultrastructural study of the flagellar basal body complex from Rhodobacter sphaeroides WS8 (wild type) and a polyhook mutant PG.

    PubMed

    West, M A; Dreyfus, G

    1997-09-29

    Filament-Hook-Basal Body (FHBB) complexes were isolated from the purple non-sulphur facultative anaerobic bacterium Rhodobacter sphaeroides (WS8) by lysozyme digestion of the cells followed by an alkaline treatment and ultracentrifugation, and they were analysed by electron microscopy. The structure is composed of a filament linked through an enlarged junction to the hook and a basal body composed of L and P rings, a rod, and a less well-defined cytoplasmic ring that has evidence of additional attached structures. Hook-basal body complexes isolated from a mutant (PG) which produces an extended hook but no filament shows basal body structures identical to those of wild-type FHBBs.

  11. Absence of the cbb3 Terminal Oxidase Reveals an Active Oxygen-Dependent Cyclase Involved in Bacteriochlorophyll Biosynthesis in Rhodobacter sphaeroides

    PubMed Central

    Chen, Guangyu E.; Martin, Elizabeth C.; Hunter, C. Neil

    2016-01-01

    ABSTRACT The characteristic green color associated with chlorophyll pigments results from the formation of an isocyclic fifth ring on the tetrapyrrole macrocycle during the biosynthesis of these important molecules. This reaction is catalyzed by two unrelated cyclase enzymes employing different chemistries. Oxygenic phototrophs such as plants and cyanobacteria utilize an oxygen-dependent enzyme, the major component of which is a diiron protein named AcsF, while BchE, an oxygen-sensitive [4Fe-4S] cluster protein, dominates in phototrophs inhabiting anoxic environments, such as the purple phototrophic bacterium Rhodobacter sphaeroides. We identify a potential acsF in this organism and assay for activity of the encoded protein in a strain lacking bchE under various aeration regimes. Initially, cells lacking bchE did not demonstrate AcsF activity under any condition tested. However, on removal of a gene encoding a subunit of the cbb3-type respiratory terminal oxidase, cells cultured under regimes ranging from oxic to micro-oxic exhibited cyclase activity, confirming the activity of the oxygen-dependent enzyme in this model organism. Potential reasons for the utilization of an oxygen-dependent enzyme in anoxygenic phototrophs are discussed. IMPORTANCE The formation of the E ring of bacteriochlorophyll pigments is the least well characterized step in their biosynthesis, remaining enigmatic for over 60 years. Two unrelated enzymes catalyze this cyclization step; O2-dependent and O2-independent forms dominate in oxygenic and anoxygenic phototrophs, respectively. We uncover the activity of an O2-dependent enzyme in the anoxygenic purple phototrophic bacterium Rhodobacter sphaeroides, initially by inactivation of the high-affinity terminal respiratory oxidase, cytochrome cbb3. We propose that the O2-dependent form allows for the biosynthesis of a low level of bacteriochlorophyll under oxic conditions, so that a rapid initiation of photosynthetic processes is possible for

  12. Dimerisation of the Rhodobacter sphaeroides RC-LH1 photosynthetic complex is not facilitated by a GxxxG motif in the PufX polypeptide.

    PubMed

    Crouch, Lucy I; Holden-Dye, Katherine; Jones, Michael R

    2010-11-01

    In purple photosynthetic bacteria the initial steps of light energy transduction take place in an RC-LH1 complex formed by the photochemical reaction centre (RC) and the LH1 light harvesting pigment-protein. In Rhodobacter sphaeroides, the RC-LH1 complex assembles in a dimeric form in which two RCs are surrounded by an S-shaped LH1 antenna. There is currently debate over the detailed architecture of this dimeric RC-LH1 complex, with particular emphasis on the location and precise function of a minor polypeptide component termed PufX. It has been hypothesised that the membrane-spanning helical region of PufX contains a GxxxG dimerisation motif that facilitates the formation of a dimer of PufX at the interface of the RC-LH1 dimer, and more specifically that the formation of this PufX dimer seeds assembly of the remaining RC-LH1 dimer (J. Busselez et al., 2007). In the present work this hypothesis was tested by site directed mutagenesis of the glycine residues proposed to form the GxxxG motif. Mutation of these glycines to leucine did not decrease the propensity of the RC-LH1 complex to assemble in a dimeric form, as would be expected from experimental studies of the effect of mutation on GxxxG motifs in other membrane proteins. Indeed increased yields of dimer were seen in two of the glycine-to-leucine mutants constructed. It is concluded that the PufX from Rhodobacter sphaeroides does not contain a genuine GxxxG helix dimerisation motif.

  13. Control of hemA Expression in Rhodobacter sphaeroides 2.4.1: Effect of a Transposon Insertion in the hbdA Gene

    PubMed Central

    Fales, Linda; Kryszak, Luiza; Zeilstra-Ryalls, Jill

    2001-01-01

    The common precursor to all tetrapyrroles is 5-aminolevulinic acid (ALA), and in Rhodobacter sphaeroides its formation occurs via the Shemin pathway. ALA synthase activity is encoded by two differentially regulated genes in R. sphaeroides 2.4.1: hemA and hemT. In our investigations of hemA regulation, we applied transposon mutagenesis under aerobic conditions, followed by a selection that identified transposon insertion mutants in which hemA expression is elevated. One of these mutants has been characterized previously (J. Zeilstra-Ryalls and S. Kaplan, J. Bacteriol. 178:985–993, 1996), and here we describe our analysis of a second mutant strain. The transposon inserted into the coding sequences of hbdA, coding for S-(+)-β-hydroxybutyryl–coenzyme A dehydrogenase and catalyzing an NAD-dependent reaction. We provide evidence that the hbdA gene product participates in polyhydroxybutyrate (PHB) metabolism and, based on our findings, we discuss possibilities as to how defective PHB metabolism might alter the level of hemA expression. PMID:11160087

  14. Physical Mapping of bchG, orf427, and orf177 in the Photosynthesis Gene Cluster of Rhodobacter sphaeroides: Functional Assignment of the Bacteriochlorophyll Synthetase Gene

    PubMed Central

    Addlesee, Hugh A.; Fiedor, Leszek; Hunter, C. Neil

    2000-01-01

    The purple photosynthetic bacterium Rhodobacter sphaeroides has within its genome a cluster of photosynthesis-related genes approximately 41 kb in length. In an attempt to identify genes involved in the terminal esterification stage of bacteriochlorophyll biosynthesis, a previously uncharacterized 5-kb region of this cluster was sequenced. Four open reading frames (ORFs) were identified, and each was analyzed by transposon mutagenesis. The product of one of these ORFs, bchG, shows close homologies with (bacterio)chlorophyll synthetases, and mutants in this gene were found to accumulate bacteriopheophorbide, the metal-free derivative of the bacteriochlorophyll precursor bacteriochlorophyllide, suggesting that bchG is responsible for the esterification of bacteriochlorophyllide with an alcohol moiety. This assignment of function to bchG was verified by the performance of assays demonstrating the ability of BchG protein, heterologously synthesized in Escherichia coli, to esterify bacteriochlorophyllide with geranylgeranyl pyrophosphate in vitro, thereby generating bacteriochlorophyll. This step is pivotal to the assembly of a functional photosystem in R. sphaeroides, a model organism for the study of structure-function relationships in photosynthesis. A second gene, orf177, is a member of a large family of isopentenyl diphosphate isomerases, while sequence homologies suggest that a third gene, orf427, may encode an assembly factor for photosynthetic complexes. The function of the remaining ORF, bchP, is the subject of a separate paper (H. Addlesee and C. N. Hunter, J. Bacteriol. 181:7248–7255, 1999). An operonal arrangement of the genes is proposed. PMID:10809697

  15. Functional importance of a pair of conserved glutamic acid residues and of Ca(2+) binding in the cbb(3)-type oxygen reductases from Rhodobacter sphaeroides and Vibrio cholerae.

    PubMed

    Ouyang, Hanlin; Han, Huazhi; Roh, Jung H; Hemp, James; Hosler, Jonathan P; Gennis, Robert B

    2012-09-18

    The cbb(3)-type cytochrome c oxidases are members of the family of heme-copper proton pumping respiratory oxygen reductases. The structure of the cbb(3)-type oxidase from Pseudomonas stutzeri reveals that, in addition to the six redox-active metal centers (two b-type hemes, three c-type hemes, and Cu(B)), the enzyme also contains at least one Ca(2+). The calcium bridges two propionate carboxyls at the interface between the low-spin heme b and the active-site heme b(3) and, in addition, is ligated to a serine in subunit CcoO and by a glutamate in subunit CcoN. The glutamate that is ligated to Ca(2+) is one of a pair of glutamic acid residues that has previously been suggested to be part of a proton exit pathway for pumped protons. In this work, mutations of these glutamates are investigated in the cbb(3)-type oxidases from Vibrio cholerae and Rhodobacter sphaeroides. Metal analysis shows that each of these wild-type enzymes contains Ca(2+). Mutations of the glutamate expected to ligate the Ca(2+) in each of these enzymes (E126 in V. cholerae and E180 in R. sphaeroides) result in a loss of activity as well as a loss of Ca(2+). Mutations of the nearby glutamate (E129 in V. cholerae and E183 in R. sphaeroides) also resulted in a loss of oxidase activity and a loss of Ca(2+). It is concluded that the Ca(2+) is essential for assembly of the fully functional enzyme and that neither of the glutamates is likely to be part of a pathway for pumped protons within the cbb(3)-type oxygen reductases. A more likely role for these glutamates is the maintenance of the structural integrity of the active conformation of the enzyme.

  16. Quenching capabilities of long-chain carotenoids in light-harvesting-2 complexes from Rhodobacter sphaeroides with an engineered carotenoid synthesis pathway

    DOE PAGES

    Dilbeck, Preston L.; Tang, Qun; Mothersole, David J.; ...

    2016-06-10

    Here, six light-harvesting-2 complexes (LH2) from genetically modified strains of the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. These strains were engineered to incorporate carotenoids for which the number of conjugated groups (N = NC=C + NC=O) varies from 9 to 15. The Rb. sphaeroides strains incorporate their native carotenoids spheroidene (N = 10) and spheroidenone (N = 11), as well as longer-chain analogues including spirilloxanthin (N = 13) and diketospirilloxantion (N = 15) normally found in Rhodospirillum rubrum. Measurements of the properties of the carotenoid first singlet excitedmore » state (S1) in antennas from the Rb. sphaeroides set show that carotenoid-bacteriochlorophyll a (BChl a) interactions are similar to those in LH2 complexes from various other bacterial species and thus are not significantly impacted by differences in polypeptide composition. Instead, variations in carotenoid-to-BChl a energy transfer are primarily regulated by the N-determined energy of the carotenoid S1 excited state, which for long-chain (N ≥ 13) carotenoids is not involved in energy transfer. Furthermore, the role of the long-chain carotenoids switches from a lightharvesting supporter (via energy transfer to BChl a) to a quencher of the BChl a S1 excited state B850*. This quenching is manifested as a substantial (~2-fold) reduction of the B850* lifetime and the B850* fluorescence quantum yield for LH2 housing the longest carotenoids« less

  17. Functional characteristics of spirilloxanthin and keto-bearing Analogues in light-harvesting LH2 complexes from Rhodobacter sphaeroides with a genetically modified carotenoid synthesis pathway.

    PubMed

    Niedzwiedzki, Dariusz M; Dilbeck, Preston L; Tang, Qun; Mothersole, David J; Martin, Elizabeth C; Bocian, David F; Holten, Dewey; Hunter, C Neil

    2015-01-01

    Light-harvesting 2 (LH2) complexes from a genetically modified strain of the purple photosynthetic bacterium Rhodobacter (Rba.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. Carotenoid synthesis in the Rba. sphaeroides strain was engineered to redirect carotenoid production away from spheroidene into the spirilloxanthin synthesis pathway. The strain assembles LH2 antennas with substantial amounts of spirilloxanthin (total double-bond conjugation length N=13) if grown anaerobically and of keto-bearing long-chain analogs [2-ketoanhydrorhodovibrin (N=13), 2-ketospirilloxanthin (N=14) and 2,2'-diketospirilloxanthin (N=15)] if grown semi-aerobically (with ratios that depend on growth conditions). We present the photophysical, electronic, and vibrational properties of these carotenoids, both isolated in organic media and assembled within LH2 complexes. Measurements of excited-state energy transfer to the array of excitonically coupled bacteriochlorophyll a molecules (B850) show that the mean lifetime of the first singlet excited state (S1) of the long-chain (N≥13) carotenoids does not change appreciably between organic media and the protein environment. In each case, the S1 state appears to lie lower in energy than that of B850. The energy-transfer yield is ~0.4 in LH2 (from the strain grown aerobically or semi-aerobically), which is less than half that achieved for LH2 that contains short-chain (N≤11) analogues. Collectively, the results suggest that the S1 excited state of the long-chain (N≥13) carotenoids participates little if at all in carotenoid-to-BChl a energy transfer, which occurs predominantly via the carotenoid S2 excited state in these antennas.

  18. A self-transmissible, narrow-host-range endogenous plasmid of Rhodobacter sphaeroides 2.4.1: physical structure, incompatibility determinants, origin of replication, and transfer functions.

    PubMed Central

    Suwanto, A; Kaplan, S

    1992-01-01

    Rhodobacter sphaeroides 2.4.1 naturally harbors five cryptic endogenous plasmids (C. S. Fornari, M. Watkins, and S. Kaplan, Plasmid 11:39-47, 1984). The smallest plasmid (pRS241e), with a molecular size of 42 kb, was observed to be a self-transmissible plasmid which can transfer only to certain strains of R. sphaeroides. Transfer frequencies can be as high as 10(-2) to 10(-3) per donor under optimal mating conditions in liquid media in the absence of oxygen. pRS241e, designated the S factor, was also shown to possess a narrow host range, failing either to replicate or to be maintained in Escherichia coli, Agrobacterium tumefaciens, and Rhizobium meliloti. It was further revealed that one of the remaining four endogenous plasmids, pRS241d, was also transmissible at a frequency similar to that of the S. factor. As a cointegrate with pSUP203, S was maintained in E. coli, providing sufficient DNA from which a physical map of S could be constructed. Progressive subcloning of S-factor DNA, in conjunction with assays of plasmid transfer, led to the localization and identification of oriV (IncA), IncB, and the putative oriT locus. The DNA sequence of the 427 bp containing oriTs revealed topological similarity to other described oriT sequences, consisting of an A-T-rich DNA region, several direct and inverted repeats, and putative integration host factor (IHF)-binding sites, and was shown to be functional in promoting plasmid transfer. Images PMID:1735707

  19. Magnesium chelatase from Rhodobacter sphaeroides: initial characterization of the enzyme using purified subunits and evidence for a BchI-BchD complex.

    PubMed

    Gibson, L C; Jensen, P E; Hunter, C N

    1999-01-15

    The enzyme magnesium-protoporphyrin IX chelatase (Mg chelatase) catalyses the insertion of Mg into protoporphyrin IX, the first committed step in (bacterio)chlorophyll biosynthesis. In the photosynthetic bacterium Rhodobacter sphaeroides, this reaction is catalysed by the products of the bchI, bchD and bchH genes. These genes have been expressed in Escherichia coli so that the BchI, BchD and BchH proteins are produced with N-terminal His6 affinity tags, which has led to the production of large amounts of highly purified, highly active Mg chelatase subunits from a single chromatography step. Furthermore, BchD has been purifed free of contamination with the chaperone GroEL, which had proven to be a problem in the past. BchD, present largely as an insoluble protein in E. coli, was purified in 6 M urea and refolded by addition of BchI, MgCl2 and ATP, yielding highly active protein. BchI/BchD mixtures prepared in this way were used in conjunction with BchH to determine the kinetic parameters of R. sphaeroides Mg chelatase for its natural substrates. We have been able to demonstrate for the first time that BchI and BchD form a complex, and that Mg2+ and ATP are required to establish and maintain this complex. Gel filtration data suggest that BchI and BchD form a complex of molecular mass 200 kDa in the presence of Mg2+ and ATP. Our data suggest that, in vivo, BchD is only folded correctly and maintained in its correct conformation in the presence of BchI, Mg2+ and ATP.

  20. The C-terminus of PufX plays a key role in dimerisation and assembly of the reaction center light-harvesting 1 complex from Rhodobacter sphaeroides.

    PubMed

    Qian, Pu; Martin, Elizabeth C; Ng, Irene W; Hunter, C Neil

    2017-09-01

    In bacterial photosynthesis reaction center-light-harvesting 1 (RC-LH1) complexes trap absorbed solar energy by generating a charge separated state. Subsequent electron and proton transfers form a quinol, destined to diffuse to the cytochrome bc1 complex. In bacteria such as Rhodobacter (Rba.) sphaeroides and Rba. capsulatus the PufX polypeptide creates a channel for quinone/quinol traffic across the LH1 complex that surrounds the RC, and it is therefore essential for photosynthetic growth. PufX also plays a key role in dimerization of the RC-LH1-PufX core complex, and the structure of the Rba. sphaeroides complex shows that the PufX C-terminus, particularly the region from X49-X53, likely mediates association of core monomers. To investigate this putative interaction we analysed mutations PufX R49L, PufX R53L, PufX R49/53L and PufX G52L by measuring photosynthetic growth, fractionation of detergent-solubilised membranes, formation of 2-D crystals and electron microscopy. We show that these mutations do not affect assembly of PufX within the core or photosynthetic growth but they do prevent dimerization, consistent with predictions from the RC-LH1-PufX structure. We obtained low resolution structures of monomeric core complexes with and without PufX, using electron microscopy of negatively stained single particles and 3D reconstruction; the monomeric complex with PufX corresponds to one half of the dimer structure whereas LH1 completely encloses the RC if the gene encoding PufX is deleted. On the basis of the insights gained from these mutagenesis and structural analyses we propose a sequence for assembly of the dimeric RC-LH1-PufX complex. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  1. A Cluster of Four Homologous Small RNAs Modulates C1 Metabolism and the Pyruvate Dehydrogenase Complex in Rhodobacter sphaeroides under Various Stress Conditions

    PubMed Central

    Billenkamp, Fabian; Peng, Tao; Berghoff, Bork A.

    2015-01-01

    ABSTRACT In bacteria, regulatory RNAs play an important role in the regulation and balancing of many cellular processes and stress responses. Among these regulatory RNAs, trans-encoded small RNAs (sRNAs) are of particular interest since one sRNA can lead to the regulation of multiple target mRNAs. In the purple bacterium Rhodobacter sphaeroides, several sRNAs are induced by oxidative stress. In this study, we focused on the functional characterization of four homologous sRNAs that are cotranscribed with the gene for the conserved hypothetical protein RSP_6037, a genetic arrangement described for only a few sRNAs until now. Each of the four sRNAs is characterized by two stem-loops that carry CCUCCUCCC motifs in their loops. They are induced under oxidative stress, as well as by various other stress conditions, and were therefore renamed here sRNAs CcsR1 to CcsR4 (CcsR1–4) for conserved CCUCCUCCC motif stress-induced RNAs 1 to 4. Increased CcsR1–4 expression decreases the expression of genes involved in C1 metabolism or encoding components of the pyruvate dehydrogenase complex either directly by binding to their target mRNAs or indirectly. One of the CcsR1–4 target mRNAs encodes the transcriptional regulator FlhR, an activator of glutathione-dependent methanol/formaldehyde metabolism. Downregulation of this glutathione-dependent pathway increases the pool of glutathione, which helps to counteract oxidative stress. The FlhR-dependent downregulation of the pyruvate dehydrogenase complex reduces a primary target of reactive oxygen species and reduces aerobic electron transport, a main source of reactive oxygen species. Our findings reveal a previously unknown strategy used by bacteria to counteract oxidative stress. IMPORTANCE Phototrophic organisms have to cope with photo-oxidative stress due to the function of chlorophylls as photosensitizers for the formation of singlet oxygen. Our study assigns an important role in photo-oxidative stress resistance to a

  2. A cluster of four homologous small RNAs modulates C1 metabolism and the pyruvate dehydrogenase complex in Rhodobacter sphaeroides under various stress conditions.

    PubMed

    Billenkamp, Fabian; Peng, Tao; Berghoff, Bork A; Klug, Gabriele

    2015-05-01

    In bacteria, regulatory RNAs play an important role in the regulation and balancing of many cellular processes and stress responses. Among these regulatory RNAs, trans-encoded small RNAs (sRNAs) are of particular interest since one sRNA can lead to the regulation of multiple target mRNAs. In the purple bacterium Rhodobacter sphaeroides, several sRNAs are induced by oxidative stress. In this study, we focused on the functional characterization of four homologous sRNAs that are cotranscribed with the gene for the conserved hypothetical protein RSP_6037, a genetic arrangement described for only a few sRNAs until now. Each of the four sRNAs is characterized by two stem-loops that carry CCUCCUCCC motifs in their loops. They are induced under oxidative stress, as well as by various other stress conditions, and were therefore renamed here sRNAs CcsR1 to CcsR4 (CcsR1-4) for conserved CCUCCUCCC motif stress-induced RNAs 1 to 4. Increased CcsR1-4 expression decreases the expression of genes involved in C1 metabolism or encoding components of the pyruvate dehydrogenase complex either directly by binding to their target mRNAs or indirectly. One of the CcsR1-4 target mRNAs encodes the transcriptional regulator FlhR, an activator of glutathione-dependent methanol/formaldehyde metabolism. Downregulation of this glutathione-dependent pathway increases the pool of glutathione, which helps to counteract oxidative stress. The FlhR-dependent downregulation of the pyruvate dehydrogenase complex reduces a primary target of reactive oxygen species and reduces aerobic electron transport, a main source of reactive oxygen species. Our findings reveal a previously unknown strategy used by bacteria to counteract oxidative stress. Phototrophic organisms have to cope with photo-oxidative stress due to the function of chlorophylls as photosensitizers for the formation of singlet oxygen. Our study assigns an important role in photo-oxidative stress resistance to a cluster of four homologous s

  3. The role of dor gene products in controlling the P2 promoter of the cytochrome c2 gene, cycA, in Rhodobacter sphaeroides

    PubMed Central

    Tavano, Christine L.; Comolli, James C.; Donohue, Timothy J.

    2009-01-01

    This study explores the regulatory networks controlling anaerobic energy production by the facultative phototroph Rhodobacter sphaeroides. The specific aim was to determine why activity of the P2 promoter for the gene (cycA) encoding the essential photosynthetic electron carrier, cytochrome c2, is decreased when the alternative electron acceptor DMSO is added to photosynthetically grown cells. The presence of DMSO is believed to activate the DorR response regulator, which controls expression of proteins required to reduce DMSO. A DorR− strain showed no change in cycA P2 promoter activity when DMSO was added to photosynthetic cells, indicating that DorR was required for the decreased expression in wild-type cells. To test if DorR acted directly at this promoter to change gene expression, recombinant DorR was purified and studied in vitro. Preparations of DorR that were active at other target promoters showed no detectable interaction with cycA P2, suggesting that this protein is not a direct regulator of this promoter. We also found that cycA P2 activity in a DorA− strain was not decreased by the addition of DMSO to photosynthetic cells. A model is presented to explain why the presence of a functional DMSO reductase (DorA) is required for DMSO to decrease cycA P2 expression under photosynthetic conditions. PMID:15184575

  4. Effects of the cryptochrome CryB from Rhodobacter sphaeroides on global gene expression in the dark or blue light or in the presence of singlet oxygen.

    PubMed

    Frühwirth, Sebastian; Teich, Kristin; Klug, Gabriele

    2012-01-01

    Several regulators are controlling the formation of the photosynthetic apparatus in the facultatively photosynthetic bacterium Rhodobacter sphaeroides. Among the proteins affecting photosynthesis gene expression is the blue light photoreceptor cryptochrome CryB. This study addresses the effect of CryB on global gene expression. The data reveal that CryB does not only influence photosynthesis gene expression but also genes for the non-photosynthetic energy metabolism like citric acid cycle and oxidative phosphorylation. In addition several genes involved in RNA processing and in transcriptional regulation are affected by a cryB deletion. Although CryB was shown to undergo a photocycle it does not only affect gene expression in response to blue light illumination but also in response to singlet oxygen stress conditions. While there is a large overlap in these responses, some CryB-dependent effects are specific for blue-light or photooxidative stress. In addition to protein-coding genes some genes for sRNAs show CryB-dependent expression. These findings give new insight into the function of bacterial cryptochromes and demonstrate for the first time a function in the oxidative stress response.

  5. Eukaryotic behaviour of a prokaryotic energy-transducing membrane: fully detached vesicular organelles arise by budding from the Rhodobacter sphaeroides intracytoplasmic photosynthetic membrane.

    PubMed

    Niederman, Robert A

    2010-05-01

    A major feature that distinguishes prokaryotic organisms from eukaryotes is their less complex internal structure, in which all membrane-associated functions are thought to be present within a continuous lipid-protein bilayer, rather than with distinct organelles. Contrary to this notion, as described by Tucker and co-workers in this issue of Molecular Microbiology, the application of cryo-electron tomography to the purple bacterium Rhodobacter sphaeroides has demonstrated a heretofore unrecognized ultrastructural complexity within the intracytoplasmic membrane (ICM) housing the photosynthetic apparatus. In addition to distinguishing invaginations of the cytoplasmic membrane (CM) and interconnected vesicular structures still attached to the CM, a eukaryote-like ICM budding process was revealed, which results in the formation of fully detached vesicular structures. These bacterial organelles are able to carry out both the light-harvesting and light-driven energy transduction activities necessary for the cells to assume a photosynthetic lifestyle. Their formation is shown to represent the final stage in a membrane invagination and growth process, originating with small CM indentations, which after cell disruption give rise to a membrane fraction that can be separated from mature ICM vesicles by rate-zone sedimentation.

  6. Action Spectroscopy on Dense Samples of Photosynthetic Reaction Centers of Rhodobacter sphaeroides WT Based on Nanosecond Laser-Flash C Photo-CIDNP MAS NMR.

    PubMed

    Daviso, Eugenio; Diller, Anna; Gast, Peter; Alia, A; Lugtenburg, Johan; Müller, Marc G; Matysik, Jörg

    2010-03-01

    Photochemically induced dynamic nuclear polarization magic-angle spinning nuclear magnetic resonance (photo-CIDNP MAS NMR) allows for the investigation of the electronic structure of the photochemical machinery of photosynthetic reaction centers (RCs) at atomic resolution. For such experiments, either continuous radiation from white xenon lamps or green laser pulses are applied to optically dense samples. In order to explore their optical properties, optically thick samples of isolated and quinone-removed RCs of the purple bacteria of Rhodobacter sphaeroides wild type are studied by nanosecond laser-flash (13)C photo-CIDNP MAS NMR using excitation wavelengths between 720 and 940 nm. Action spectra of both the transient nuclear polarization as well as the nuclear hyperpolarization, remaining in the electronic ground state at the end of the photocycle, are obtained. It is shown that the signal intensity is limited by the amount of accessible RCs and that the different mechanisms of the photo-CIDNP production rely on the same photophysical origin, which is the photocycle induced by one single photon.

  7. Coproporphyrin Excretion and Low Thiol Levels Caused by Point Mutation in the Rhodobacter sphaeroides S-Adenosylmethionine Synthetase Gene ▿ †

    PubMed Central

    Sabaty, Monique; Adryanczyk, Géraldine; Roustan, Chloë; Cuiné, Stephan; Lamouroux, Christine; Pignol, David

    2010-01-01

    A spontaneous mutant of Rhodobacter sphaeroides f. sp. denitrificans IL-106 was found to excrete a large amount of a red compound identified as coproporphyrin III, an intermediate in bacteriochlorophyll and heme synthesis. The mutant, named PORF, is able to grow under phototrophic conditions but has low levels of intracellular cysteine and glutathione and overexpresses the cysteine synthase CysK. The expression of molybdoenzymes such as dimethyl sulfoxide (DMSO) and nitrate reductases is also affected under certain growth conditions. Excretion of coproporphyrin and overexpression of CysK are not directly related but were both found to be consequences of a diminished synthesis of the key metabolite S-adenosylmethionine (SAM). The wild-type phenotype is restored when the gene metK encoding SAM synthetase is supplied in trans. The metK gene in the mutant strain has a mutation leading to a single amino acid change (H145Y) in the encoded protein. This point mutation is responsible for a 70% decrease in intracellular SAM content which probably affects the activities of numerous SAM-dependent enzymes such as coproporphyrinogen oxidase (HemN); uroporphyrinogen III methyltransferase (CobA), which is involved in siroheme synthesis; and molybdenum cofactor biosynthesis protein A (MoaA). We propose a model showing that the attenuation of the activities of SAM-dependent enzymes in the mutant could be responsible for the coproporphyrin excretion, the low cysteine and glutathione contents, and the decrease in DMSO and nitrate reductase activities. PMID:20038586

  8. Properties of Rhodobacter sphaeroides photosynthetic reaction center with double amino acid substitution I(L177)H+H(M182)L.

    PubMed

    Fufina, T Yu; Vasilieva, L G; Khatypov, R A; Shuvalov, V A

    2011-04-01

    Histidine M182 in the reaction center (RC) of Rhodobacter sphaeroides serves as the fifth ligand of the bacteriochlorophyll (BChl) B(B) Mg atom. When this His is substituted by an amino acid that is not able to coordinate Mg, bacteriopheophytin appears in the B(B) binding site instead of BChl (Katilius, E., et al. (1999) J. Phys. Chem. B, 103, 7386-7389). We have shown that in the presence of the additional mutation I(L177)H the coordination of the BChl B(B) Mg atom in the double mutant I(L177)H+H(M182)L RC still remains. Changes in the double mutant RC absorption spectrum attributed to BChl absorption suggest that BChl B(B) Mg atom axial ligation might be realized not from the usual α-side of the BChl macrocycle, but from the opposite, β-side. Weaker coordination of BChl B(B) Mg atom compared to the other mutant RC BChl molecules suggests that not an amino acid residue but a water molecule might be a possible ligand. The results are discussed in the light of the structural changes that occurred in the RC upon Ile/His substitution in the L177 position.

  9. Partial reversion of the electrogenic reaction in the ubiquinol: cytochrome c2-oxidoreductase of Rhodobacter sphaeroides chromatophores under neutral and alkaline conditions.

    PubMed

    Mulkidjanian AYa; Mamedov, M D; Semenov AYu; Shinkarev, V P; Verkhovsky, M I; Drachev, L A

    1990-12-17

    The interaction of the photosynthetic reaction center (RC)-generated ubiquinol with the ubiquinone-reducing center C of ubiquinol:cytochrome c2-oxidoreductase (bc1-complex) has been studied electrometrically in Rhodobacter sphaeroides chromatophores. The addition of myxothiazol inhibited the ubiquinol-oxidizing center Z, suppressing the phases of membrane potential generation by the bc1-complex, but at the same time induced an electrogenic phase of opposite polarity, sensitive to antimycin A, the inhibitor of center C. The rise time of this reverse phase varied from 3 ms at pH 6.0 to 1 ms at pH 9.5. At pH greater than 9.5 the reverse phase was limited by the rate of ubiquinol formation in RC. The magnitude of the reverse phase was constant within the pH range 7.5-10.0. It is assumed that the reverse phase is due to the electrogenic deprotonation reaction which takes place after the binding of the RC-generated ubiquinol to center C.

  10. Effects of the Cryptochrome CryB from Rhodobacter sphaeroides on Global Gene Expression in the Dark or Blue Light or in the Presence of Singlet Oxygen

    PubMed Central

    Frühwirth, Sebastian; Teich, Kristin; Klug, Gabriele

    2012-01-01

    Several regulators are controlling the formation of the photosynthetic apparatus in the facultatively photosynthetic bacterium Rhodobacter sphaeroides. Among the proteins affecting photosynthesis gene expression is the blue light photoreceptor cryptochrome CryB. This study addresses the effect of CryB on global gene expression. The data reveal that CryB does not only influence photosynthesis gene expression but also genes for the non-photosynthetic energy metabolism like citric acid cycle and oxidative phosphorylation. In addition several genes involved in RNA processing and in transcriptional regulation are affected by a cryB deletion. Although CryB was shown to undergo a photocycle it does not only affect gene expression in response to blue light illumination but also in response to singlet oxygen stress conditions. While there is a large overlap in these responses, some CryB-dependent effects are specific for blue-light or photooxidative stress. In addition to protein-coding genes some genes for sRNAs show CryB-dependent expression. These findings give new insight into the function of bacterial cryptochromes and demonstrate for the first time a function in the oxidative stress response. PMID:22496766

  11. Chronic exposure to Rhodobacter sphaeroides extract Lycogen™ prevents UVA-induced malondialdehyde accumulation and procollagen I down-regulation in human dermal fibroblasts.

    PubMed

    Yang, Tsai-Hsiu; Lai, Ying-Hsiu; Lin, Tsuey-Pin; Liu, Wen-Sheng; Kuan, Li-Chun; Liu, Chia-Chyuan

    2014-01-23

    UVA contributes to the pathogenesis of skin aging by downregulation of procollagen I content and induction of matrix metalloproteinase (MMP)-associated responses. Application of antioxidants such as lycopene has been demonstrated as a convenient way to achieve protection against skin aging. Lycogen™, derived from the extracts of Rhodobacter sphaeroides, exerts several biological effects similar to that of lycopene whereas most of its anti-aging efficacy remains uncertain. In this study, we attempted to examine whether Lycogen™ could suppress malondialdehyde (MDA) accumulation and restore downregulated procollagen I expression induced by UVA exposure. In human dermal fibroblasts Hs68 cells, UVA repressed cell viability and decreased procollagen I protein content accompanied with the induction of MMP-1 and MDA accumulation. Remarkably, incubation with 50 µM Lycogen™ for 24 h ameliorated UVA-induced cell death and restored UVA-induced downregulation of procollagen in a dose-related manner. Lycogen™ treatment also prevented the UVA-induced MMP-1 upregulation and intracellular MDA generation in Hs68 cells. Activation of NFκB levels, one of the downstream events induced by UVA irradiation and MMP-1 induction, were also prevented by Lycogen™ administration. Taken together, our findings demonstrate that Lycogen™ may be an alternative agent that prevents UVA-induced skin aging and could be used in cosmetic and pharmaceutical applications.

  12. In vivo effects on photosynthesis gene expression of base pair exchanges in the gene encoding the light-responsive BLUF domain of AppA in Rhodobacter sphaeroides.

    PubMed

    Metz, Sebastian; Hendriks, Johnny; Jäger, Andreas; Hellingwerf, Klaas; Klug, Gabriele

    2010-01-01

    The Rhodobacter sphaeroides protein AppA has the unique quality of sensing and transmitting light and redox signals. By acting as antirepressor to the PpsR protein, it acts as a major regulator in photosynthesis gene expression. In this study, we show that by introducing amino acid exchanges into the AppA protein, the in vivo activity as an antirepressor can be greatly altered. The tryptophan 104 to phenylalanine (W104F) base exchange greatly diminished blue-light sensitivity of the BLUF domain. From the obtained in vivo data, the difference in thermal recovery rate of the signaling state of the BLUF domain between the wild type and mutated protein was calculated, predicting an about 10-fold faster recovery in the mutant, which is consistent with in vitro data. Introduction of a tyrosine 21 to phenylalanine (Y21F) or to cysteine (Y21C) mutation led to a complete loss of AppA antirepressor activity, while additionally leading to an increase of photosynthesis gene expression after illumination with high blue-light quantities. Interestingly, this effect is not visible in a W104F/Y21F double mutant that again shows a wild-type-like behavior of the BLUF domain after blue-light illumination, thus restoring the activity of AppA.

  13. pH modulates the quinone position in the photosynthetic reaction center from Rhodobacter sphaeroides in the neutral and charge separated states.

    PubMed

    Koepke, Juergen; Krammer, Eva-Maria; Klingen, Astrid R; Sebban, Pierre; Ullmann, G Matthias; Fritzsch, Günter

    2007-08-10

    The structure of the photosynthetic reaction-center from Rhodobacter sphaeroides has been determined at four different pH values (6.5, 8.0, 9.0, 10.0) in the neutral and in charge separated states. At pH 8.0, in the neutral state, we obtain a resolution of 1.87 A, which is the best ever reported for the bacterial reaction center protein. Our crystallographic data confirm the existence of two different binding positions of the secondary quinone (QB). We observe a new orientation of QB in its distal position, which shows no ring-flip compared to the orientation in the proximal position. Datasets collected for the different pH values show a pH-dependence of the population of the proximal position. The new orientation of QB in the distal position and the pH-dependence could be confirmed by continuum electrostatics calculations. Our calculations are in agreement with the experimentally observed proton uptake upon charge separation. The high resolution of our crystallographic data allows us to identify new water molecules and external residues being involved in two previously described hydrogen bond proton channels. These extended proton-transfer pathways, ending at either of the two oxo-groups of QB in its proximal position, provide additional evidence that ring-flipping is not required for complete protonation of QB upon reduction.

  14. Carotenoid to bacteriochlorophyll energy transfer in the RC-LH1-PufX complex from Rhodobacter sphaeroides containing the extended conjugation keto-carotenoid diketospirilloxanthin.

    PubMed

    Šlouf, Václav; Keşan, Gürkan; Litvín, Radek; Swainsbury, David J K; Martin, Elizabeth C; Hunter, C Neil; Polívka, Tomáš

    2017-05-20

    RC-LH1-PufX complexes from a genetically modified strain of Rhodobacter sphaeroides that accumulates carotenoids with very long conjugation were studied by ultrafast transient absorption spectroscopy. The complexes predominantly bind the carotenoid diketospirilloxanthin, constituting about 75% of the total carotenoids, which has 13 conjugated C=C bonds, and the conjugation is further extended to two terminal keto groups. Excitation of diketospirilloxanthin in the RC-LH1-PufX complex demonstrates fully functional energy transfer from diketospirilloxanthin to BChl a in the LH1 antenna. As for other purple bacterial LH complexes having carotenoids with long conjugation, the main energy transfer route is via the S2-Qx pathway. However, in contrast to LH2 complexes binding diketospirilloxanthin, in RC-LH1-PufX we observe an additional, minor energy transfer pathway associated with the S1 state of diketospirilloxanthin. By comparing the spectral properties of the S1 state of diketospirilloxanthin in solution, in LH2, and in RC-LH1-PufX, we propose that the carotenoid-binding site in RC-LH1-PufX activates the ICT state of diketospirilloxanthin, resulting in the opening of a minor S1/ICT-mediated energy transfer channel.

  15. Protonation of interacting residues in a protein by a Monte Carlo method: application to lysozyme and the photosynthetic reaction center of Rhodobacter sphaeroides.

    PubMed Central

    Beroza, P; Fredkin, D R; Okamura, M Y; Feher, G

    1991-01-01

    We used Monte Carlo methods to treat statistical problem of electrostatic interactions among many titrating amino acids and applied these methods to lysozyme and the photosynthetic reaction center of Rhodobacter sphaeroides, including all titrating sites. We computed the average protonation of residues as a function of pH from an equilibrium distribution of states generated by random sampling. Electrostatic energies were calculated from a finite difference solution to the linearized Poisson-Boltzmann equation using the coordinates from solved protein structures. For most calculations we used the Metropolis algorithm to sample protonation states; for strongly coupled sites, we substantially reduced sampling errors by using a modified algorithm that allows multiple site transitions. The Monte Carlo method agreed with calculations for a small test system, lysozyme, for which the complete partition function was calculated. We also calculated the pH dependence of the free energy change associated with electron transfer from the primary to the secondary quinone in the photosynthetic reaction center. The shape of the resulting curve agreed fairly well with experiment, but the proton uptake from which the free energy was calculated agreed only to within a factor of two with the observed values. We believe that this discrepancy resulted from errors in the individual electrostatic energy calculations rather than from errors in the Monte Carlo sampling. PMID:2062860

  16. The Photoheterotrophic Growth of Bacteriochlorophyll Synthase-Deficient Mutant of Rhodobacter sphaeroides Is Restored by I44F Mutant Chlorophyll Synthase of Synechocystis sp. PCC 6803.

    PubMed

    Kim, Eui-Jin; Kim, Hyeonjun; Lee, Jeong K

    2016-05-28

    Chlorophyll synthase (ChlG) and bacteriochlorophyll synthase (BchG) have a high degree of substrate specificity. The BchG mutant of Rhodobacter sphaeroides, BG1 strain, is photosynthetically incompetent. When BG1 harboring chlG of Synechocystis sp. PCC 6803 was cultured photoheterotrophically, colonies arose at a frequency of approximately 10(-8). All the suppressor mutants were determined to have the same mutational change, ChlGI44F. The mutated enzyme ChlGI44F showed BchG activity. Remarkably, BchGF28I, which has the substitution of F at the corresponding 28(th) residue to I, showed ChlG activity. The Km values of ChlGI44F and BchGF28I for their original substrates, chlorophyllide (Chlide) a and bacteriochlorophyllide (Bchlide) a, respectively, were not affected by the mutations, but the Km values of ChlGI44F and BchGF28I for the new substrates Bchlide a and Chlide a, respectively, were more than 10-fold larger than those for their original substrates, suggesting the lower affinities for new substrates. Taken together, I44 and F28 are important for the substrate specificities of ChlG and BchG, respectively. The BchG activity of ChlGI44F and the ChlG activity of BchGF28I further suggest that ChlG and BchG are evolutionarily related enzymes.

  17. Light-dependent regulation of photosynthesis genes in Rhodobacter sphaeroides 2.4.1 is coordinately controlled by photosynthetic electron transport via the PrrBA two-component system and the photoreceptor AppA.

    PubMed

    Happ, Hendrik N; Braatsch, Stephan; Broschek, Vera; Osterloh, Lisa; Klug, Gabriele

    2005-11-01

    Formation of the photosynthetic apparatus in Rhodobacter is regulated by oxygen tension and light intensity. Here we show that in anaerobically grown Rhodobacter cells a light-dependent increase in expression of the puc and puf operons encoding structural proteins of the photosynthetic complexes requires an active photosynthetic electron transport. The redox-sensitive CrtJ/PpsR repressor of photosynthesis genes, which was suggested to mediate electron transport-dependent signals, is not involved in this light-dependent signal chain. Our data reveal that the signal initiated in the photosynthetic reaction centre is transmitted via components of the electron transport chain and the PrrB/PrrA two-component system in Rhodobacter sphaeroides. Under blue light illumination in the absence of oxygen this signal leads to activation of photosynthesis genes and interferes with a blue-light repression mediated by the AppA photoreceptor and the PpsR transcriptional repressor in R. sphaeroides. Thus, light either sensed by a photoreceptor or initiating photosynthetic electron transport has opposite effects on the transcription of photosynthesis genes. Both signalling pathways involve redox-dependent steps that finally determine the effect of light on gene expression.

  18. Transcriptional Regulation by the Short-Chain Fatty Acyl Coenzyme A Regulator (ScfR) PccR Controls Propionyl Coenzyme A Assimilation by Rhodobacter sphaeroides

    PubMed Central

    Carter, Michael S.

    2015-01-01

    ABSTRACT Propionyl coenzyme A (propionyl-CoA) assimilation by Rhodobacter sphaeroides proceeds via the methylmalonyl-CoA pathway. The activity of the key enzyme of the pathway, propionyl-CoA carboxylase (PCC), was upregulated 20-fold during growth with propionate compared to growth with succinate. Because propionyl-CoA is an intermediate in acetyl-CoA assimilation via the ethylmalonyl-CoA pathway, acetate growth also requires the methylmalonyl-CoA pathway. PCC activities were upregulated 8-fold in extracts of acetate-grown cells compared to extracts of succinate-grown cells. The upregulation of PCC activities during growth with propionate or acetate corresponded to increased expression of the pccB gene, which encodes a subunit of PCC. PccR (RSP_2186) was identified to be a transcriptional regulator required for the upregulation of pccB transcript levels and, consequently, PCC activity: growth substrate-dependent regulation was lost when pccR was inactivated by an in-frame deletion. In the pccR mutant, lacZ expression from a 215-bp plasmid-borne pccB upstream fragment including 27 bp of the pccB coding region was also deregulated. A loss of regulation as a result of mutations in the conserved motifs TTTGCAAA-X4-TTTGCAAA in the presence of PccR allowed the prediction of a possible operator site. PccR, together with homologs from other organisms, formed a distinct clade within the family of short-chain fatty acyl coenzyme A regulators (ScfRs) defined here. Some members from other clades within the ScfR family have previously been shown to be involved in regulating acetyl-CoA assimilation by the glyoxylate bypass (RamB) or propionyl-CoA assimilation by the methylcitrate cycle (MccR). IMPORTANCE Short-chain acyl-CoAs are intermediates in essential biosynthetic and degradative pathways. The regulation of their accumulation is crucial for appropriate cellular function. This work identifies a regulator (PccR) that prevents the accumulation of propionyl-CoA by controlling

  19. The site-directed mutation I(L177)H in Rhodobacter sphaeroides reaction center affects coordination of P(A) and B(B) bacteriochlorophylls.

    PubMed

    Vasilieva, L G; Fufina, T Y; Gabdulkhakov, A G; Leonova, M M; Khatypov, R A; Shuvalov, V A

    2012-08-01

    To explore the influence of the I(L177)H single mutation on the properties of the nearest bacteriochlorophylls (BChls), three reaction centers (RCs) bearing double mutations were constructed in the photosynthetic purple bacterium Rhodobacter sphaeroides, and their properties and pigment content were compared with those of the correspondent single mutant RCs. Each pair of the mutations comprised the amino acid substitution I(L177)H and another mutation altering histidine ligand of BChl P(A) or BChl B(B). Contrary to expectations, the double mutation I(L177)H+H(L173)L does not bring about a heterodimer RC but causes a 46nm blue shift of the long-wavelength P absorbance band. The histidine L177 or a water molecule were suggested as putative ligands for P(A) in the RC I(L177)H+H(L173)L although this would imply a reorientation of the His backbone and additional rearrangements in the primary donor environment or even a repositioning of the BChl dimer. The crystal structure of the mutant I(L177)H reaction center determined to a resolution of 2.9Å shows changes at the interface region between the BChl P(A) and the monomeric BChl B(B). Spectral and pigment analysis provided evidence for β-coordination of the BChl B(B) in the double mutant RC I(L177)H+H(M182)L and for its hexacoordination in the mutant reaction center I(L177)H. Computer modeling suggests involvement of two water molecules in the β-coordination of the BChl B(B). Possible structural consequences of the L177 mutation affecting the coordination of the two BChls P(A) and B(B) are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

  20. Examination of stability of mutant photosynthetic reaction center of Rhodobacter sphaeroides I(L177)H and determination of location of bacteriochlorophyll covalently bound to the protein.

    PubMed

    Fufina, T Y; Vasilieva, L G; Shuvalov, V A

    2010-02-01

    We demonstrated earlier that as a result of the I(L177)H mutation in the photosynthetic reaction center (RC) of the bacterium Rhodobacter sphaeroides, one of the bacteriochlorophylls (BChl) binds with the L-subunit, simultaneously raising coordination stability of the central magnesium atom of the bacteriochlorophyll associated with the protein. In this study, spectral properties of wild type RC and I(L177)H in the presence of urea and SDS as well as at 48 degrees C were examined. It is shown that the I(L177)H mutation decreases the RC stability. Under denaturing conditions, some changes indicating breakdown of oligomeric structure of the complex and loss of interaction between pigments and their protein environment are observed in I(L177)H RC spectra. In addition, pheophytinization of bacteriochlorophylls occurs in both types of RC in the presence of SDS. However, an 811-nm band is observed in the spectrum of the mutant RC under these conditions, which indicates retention of one of the BChl molecules in the protein binding site and stable coordination of its central magnesium atom. It is shown that in both types of RC, monomeric BChl B(B) can be modified by sodium borohydride treatment and then extracted by acetone-methanol mixture. Spectral properties of the BChl covalently bound with the protein in I(L177)H RC do not change. The results demonstrate that BChl P(A) is the molecule of BChl tightly bound with the L-subunit in mutant RC as it was supposed earlier.

  1. Role of B800 in carotenoid-bacteriochlorophyll energy and electron transfer in LH2 complexes from the purple bacterium Rhodobacter sphaeroides.

    PubMed

    Polívka, Tomas; Niedzwiedzki, Dariusz; Fuciman, Marcel; Sundström, Villy; Frank, Harry A

    2007-06-28

    The role of the B800 in energy and electron transfer in LH2 complexes has been studied using femtosecond time-resolved transient absorption spectroscopy. The B800 site was perturbed by application of lithium dodecyl sulfate (LDS), and comparison of treated and untreated LH2 complexes from Rhodobacter sphaeroides incorporating carotenoids neurosporene, spheroidene, and spheroidenone was used to explore the role of B800 in carotenoid to bacteriochlorophyll-a (BChla) energy transfer and carotenoid radical formation. Efficiencies of the S1-mediated energy transfer in the LDS-treated complexes were 86, 61, and 57% in the LH2 complexes containing neurosporene, spheroidene, and spheroidenone, respectively. Analysis of the carotenoid S1 lifetimes in solution, LDS-treated, and untreated LH2 complexes allowed determination of B800/B850 branching ratio in the S1-mediated energy transfer. It is shown that B800 is a major acceptor, as approximately 60% of the energy from the carotenoid S1 state is accepted by B800. This value is nearly independent of conjugation length of the carotenoid. In addition to its role in energy transfer, the B800 BChla is the only electron acceptor in the event of charge separation between carotenoid and BChla in LH2 complexes, which is demonstrated by prevention of carotenoid radical formation in the LDS-treated LH2 complexes. In the untreated complexes containing neurosporene and spheroidene, the carotenoid radical is formed with a time constant of 300-400 fs. Application of different excitation wavelengths and intensity dependence of the carotenoid radical formation showed that the carotenoid radical can be formed only after excitation of the S2 state of carotenoid, although the S2 state itself is not a precursor of the charge-separated state. Instead, either a hot S1 state or a charge-transfer state lying between S2 and S1 states of the carotenoid are discussed as potential precursors of the charge-separated state.

  2. Electron-Transfer Secondary Reaction Matrices for MALDI MS Analysis of Bacteriochlorophyll a in Rhodobacter sphaeroides and Its Zinc and Copper Analogue Pigments

    NASA Astrophysics Data System (ADS)

    Calvano, Cosima Damiana; Ventura, Giovanni; Trotta, Massimo; Bianco, Giuliana; Cataldi, Tommaso R. I.; Palmisano, Francesco

    2017-01-01

    Bacteriochlorophyll a ( BChl a), a photosynthetic pigment performing the same functions of chlorophylls in plants, features a bacteriochlorin macrocycle ring (18 π electrons) with two reduced pyrrole rings along with a hydrophobic terpenoid side chain (i.e., the phytol residue). Chlorophylls analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is not so straightforward since pheophytinization (i.e., release of the central metal ion) and cleavage of the phytol-ester linkage are invariably observed by employing protonating matrices such as 2,5-dihydroxybenzoic acid, sinapinic acid, and α-cyano-4-hydroxycinnamic acid. Using BChl a from Rhodobacter sphaeroides R26 strain as a model system, different electron-transfer (ET) secondary reaction matrices, leading to the formation of almost stable radical ions in both positive ([M]+•) and negative ([M]-•) ionization modes at m/z 910.55, were evaluated. Compared with ET matrices such as trans-2-[3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene]malononitrile (DCTB), 2,2':5',2''-terthiophene (TER), anthracene (ANT), and 9,10-diphenylanthracene (DP-ANT), 1,5-diaminonaphthalene (DAN) was found to provide the highest ionization yield with a negligible fragmentation. DAN also displayed excellent ionization properties for two metal ion-substituted bacteriochlorophylls, (i.e., Zn- and Cu-BChl a at m/z 950.49 and 949.49), respectively. MALDI MS/MS of both radical charged molecular species provide complementary information, thus making analyte identification more straightforward.

  3. PrrC from Rhodobacter sphaeroides, a homologue of eukaryotic Sco proteins, is a copper-binding protein and may have a thiol-disulfide oxidoreductase activity.

    PubMed

    McEwan, Alastair G; Lewin, Allison; Davy, Sharon L; Boetzel, Ruth; Leech, Andrew; Walker, Daniel; Wood, Tania; Moore, Geoffrey R

    2002-05-08

    PrrC from Rhodobacter sphaeroides provides the signal input to a two-component signal transduction system that senses changes in oxygen tension and regulates expression of genes involved in photosynthesis (Eraso, J.M. and Kaplan, S. (2000) Biochemistry 39, 2052-2062; Oh, J.-I. and Kaplan, S. (2000) EMBO J. 19, 4237-4247). It is also a homologue of eukaryotic Sco proteins and each has a C-x-x-x-C-P sequence. In mitochondrial Sco proteins these cysteines appear to be essential for the biogenesis of the CuA centre of respiratory cytochrome oxidase. Overexpression and purification of a water-soluble and monomeric form of PrrC has provided sufficient material for a chemical and spectroscopic study of the properties of the four cysteine residues of PrrC, and its ability to bind divalent cations, including copper. PrrC expressed in the cytoplasm of Escherichia coli binds Ni2+ tightly and the data are consistent with a mononuclear metal site. Following removal of Ni2+ and formation of renatured metal-free rPrrC (apo-PrrC), Cu2+ could be loaded into the reduced form of PrrC to generate a protein with a distinctive UV-visible spectrum, having absorbance with a lambda(max) of 360 nm. The copper:PrrC ratio is consistent with the presence of a mononuclear metal centre. The cysteines of metal-free PrrC oxidise in the presence of air to form two intramolecular disulfide bonds, with one pair being extremely reactive. The cysteine thiols with extreme O2 sensitivity are involved in copper binding in reduced PrrC since the same copper-loaded protein could not be generated using oxidised PrrC. Thus, it appears that PrrC, and probably Sco proteins in general, could have both a thiol-disulfide oxidoreductase function and a copper-binding role.

  4. Characterization of mutations in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides that affect the quinone reductase site (Qc).

    PubMed

    Hacker, B; Barquera, B; Crofts, A R; Gennis, R B

    1993-04-27

    The cytochrome b subunit of the bc1 complex contains two heme components, cytochrome bL and cytochrome bH, and is the locus of both a quinol oxidizing site (Qo or Qz) and a quinone reducing site (Qi or Qc). The quinone reductase site has been previously characterized as the site of interaction for a set of inhibitors including antimycin A, diuron, funiculosin, and HQNO. In this paper, four highly conserved residues in the cytochrome b subunit of Rhodobacter sphaeroides (A52, H217, K251, and D252) were targeted for site-directed mutagenesis. These residues were chosen as being likely to be at or near the quinone reductase site, on the basis of known locations of missense mutations in the homologous yeast subunit that confer resistance to Qc-directed inhibitors. The site-directed mutants all exhibit a normal rate of reduction of cytochrome bH, suggesting a fully functional quinol oxidizing site. However, each of the mutants is impaired, to varying degrees, in the rate of reoxidation of cytochrome bH. Two mutants (H217A and D252A) are unable to grow photosynthetically, indicating a severe defect in the bc1 complex. In both cases, the cause of the defect is the lack of reoxidation of cytochrome bH by ubiquinone. This is the first report of mutations that selectively impair the rate of electron transfer from cytochrome bH to the Qc-site. This set of mutations will be useful not only for modeling the structure of the quinone reducing site but also in elucidating the catalytic mechanism of this portion of the Q-cycle.

  5. Electron-Transfer Secondary Reaction Matrices for MALDI MS Analysis of Bacteriochlorophyll a in Rhodobacter sphaeroides and Its Zinc and Copper Analogue Pigments.

    PubMed

    Calvano, Cosima Damiana; Ventura, Giovanni; Trotta, Massimo; Bianco, Giuliana; Cataldi, Tommaso R I; Palmisano, Francesco

    2017-01-01

    Bacteriochlorophyll a (BChl a), a photosynthetic pigment performing the same functions of chlorophylls in plants, features a bacteriochlorin macrocycle ring (18 π electrons) with two reduced pyrrole rings along with a hydrophobic terpenoid side chain (i.e., the phytol residue). Chlorophylls analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is not so straightforward since pheophytinization (i.e., release of the central metal ion) and cleavage of the phytol-ester linkage are invariably observed by employing protonating matrices such as 2,5-dihydroxybenzoic acid, sinapinic acid, and α-cyano-4-hydroxycinnamic acid. Using BChl a from Rhodobacter sphaeroides R26 strain as a model system, different electron-transfer (ET) secondary reaction matrices, leading to the formation of almost stable radical ions in both positive ([M](+•)) and negative ([M](-•)) ionization modes at m/z 910.55, were evaluated. Compared with ET matrices such as trans-2-[3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene]malononitrile (DCTB), 2,2':5',2''-terthiophene (TER), anthracene (ANT), and 9,10-diphenylanthracene (DP-ANT), 1,5-diaminonaphthalene (DAN) was found to provide the highest ionization yield with a negligible fragmentation. DAN also displayed excellent ionization properties for two metal ion-substituted bacteriochlorophylls, (i.e., Zn- and Cu-BChl a at m/z 950.49 and 949.49), respectively. MALDI MS/MS of both radical charged molecular species provide complementary information, thus making analyte identification more straightforward. Graphical Abstract ᅟ.

  6. The Rhodobacter sphaeroides ECF sigma factor, sigma(E), and the target promoters cycA P3 and rpoE P1.

    PubMed

    Newman, J D; Falkowski, M J; Schilke, B A; Anthony, L C; Donohue, T J

    1999-11-26

    Rhodobacter sphaeroides rpoE encodes a 19.2 kDa protein, sigma(E), related to members of the extra-cytoplasmic function subfamily of eubacterial RNA polymerase sigma factors. We demonstrate that sigma(E) directs transcription from rpoE P1, the promoter for the rpoEchrR operon, and from cycA P3, a promoter for the cytochrome c2 structural gene. Comparison of these sigma(E)-dependent promoters reveals significant sequence conservation in their -35 and -10 regions; however, rpoE P1 is over 80-fold stronger than cycA P3. Both promoters contain identical -35 hexamers, (-36)TGATCC(-31), that appear to constitute the preferred sequence, since any single base mutation in this region of cycA P3 reduces promoter function. The higher activity of rpoE P1 appears to reflect a better -10 region, (-13)TAAGA(-9), as it contains four out of five of the nucleotides found to be important to sigma(E)-dependent transcription. We also propose that ChrR acts as an inhibitor of sigma(E), since these two proteins can form a complex, and DeltachrR mutations increase sigma(E)-dependent transcription. ChrR is believed to respond to a signal from tetrapyrrole biosynthesis because loss of function mutations in chrR lead to cohemin resistance. Based on our observations, we present a model in which cohemin resistance is conferred by increasing sigma(E) activity. Copyright 1999 Academic Press.

  7. Spectral Identification of Intermediates Generated during the Reaction of Dioxygen with the Wild-Type and EQ(I-286) Mutant of Rhodobacter sphaeroides Cytochrome c Oxidase

    PubMed Central

    Szundi, Istvan; Funatogawa, Chie; Cassano, Jennifer; McDonald, William; Ray, Jayashree; Hiser, Carrie; Ferguson-Miller, Shelagh; Gennis, Robert B.; Einarsdóttir, Ólöf

    2012-01-01

    Cytochrome c oxidase from Rhodobacter sphaeroides (Rs) is frequently used to model the more complex mitochondrial enzyme. The O2 reduction in both enzymes is generally described by a unidirectional mechanism involving the sequential formation of the ferrous-oxy complex (compound A), the PR state, the oxyferryl F form, and the oxidized state. In this study we investigated the reaction of dioxygen with the wild-type reduced Rs cytochrome oxidase and the EQ(I-286) mutant using the CO flow-flash technique. Singular value decomposition and multi-exponential fitting of the time-resolved optical absorption difference spectra showed that three apparent lifetimes, 18 μs, 53 μs, and 1.3 ms, are sufficient to fit the kinetics of the O2 reaction of the wild-type enzyme. A comparison of the experimental intermediate spectra with the corresponding intermediate spectra of the bovine enzyme revealed that PR is not present in the reaction mechanism of the wild-type Rs aa3. Transient absorbance changes at 440 and 610 nm support this conclusion. For the EQ(I-286) mutant, in which a key glutamic residue in the D proton pathway is replaced by glutamine, two lifetimes, 16 and 108 μs, were observed. A spectral analysis of the intermediates shows that the O2 reaction in the EQ(I-286) mutant terminates at the PR state, with 70% of heme a becoming oxidized. These results indicate significant differences in the kinetics of O2 reduction between the bovine and wild-type Rs aa3 oxidases, which may arise from differences in the relative rates of internal electron and proton movements in the two enzymes. PMID:23057757

  8. Assembly of photosynthetic apparatus in Rhodobacter sphaeroides as revealed by functional assessments at different growth phases and in synchronized and greening cells.

    PubMed

    Kis, M; Asztalos, E; Sipka, G; Maróti, P

    2014-12-01

    The development of photosynthetic membranes of intact cells of Rhodobacter sphaeroides was tracked by light-induced absorption spectroscopy and induction and relaxation of the bacteriochlorophyll fluorescence. Changes in membrane structure were induced by three methods: synchronization of cell growth, adjustment of different growth phases and transfer from aerobic to anaerobic conditions (greening) of the bacteria. While the production of the bacteriochlorophyll and carotenoid pigments and the activation of light harvesting and reaction center complexes showed cell-cycle independent and continuous increase with characteristic lag phases, the accumulation of phospholipids and membrane potential (electrochromism) exhibited stepwise increase controlled by cell division. Cells in the stationary phase of growth demonstrated closer packing and tighter energetic coupling of the photosynthetic units (PSU) than in their early logarithmic stage. The greening resulted in rapid (within 0-4 h) induction of BChl synthesis accompanied with a dominating role for the peripheral light harvesting system (up to LH2/LH1 ~2.5), significantly increased rate (~7·10(4) s(-1)) and yield (F v/F max ~0.7) of photochemistry and modest (~2.5-fold) decrease of the rate of electron transfer (~1.5·10(4) s(-1)). The results are discussed in frame of a model of sequential assembly of the PSU with emphasis on crowding the LH2 complexes resulting in an increase of the connectivity and yield of light capture on the one hand and increase of hindrance to diffusion of mobile redox agents on the other hand.

  9. Overexpression of Rhodobacter sphaeroides PufX-bearing maltose-binding protein and its effect on the stability of reconstituted light-harvesting core antenna complex.

    PubMed

    Sakai, Shunnsuke; Hiro, Akito; Kondo, Masaharu; Mizuno, Toshihisa; Tanaka, Toshiki; Dewa, Takehisa; Nango, Mamoru

    2012-03-01

    The PufX protein, encoded by the pufX gene of Rhodobacter sphaeroides, plays a key role in the organization and function of the core antenna (LH1)-reaction centre (RC) complex, which collects photons and triggers primary photochemical reactions. We synthesized a PufX/maltose-binding protein (MBP) fusion protein to study the effect of the PufX protein on the reconstitution of B820 subunit-type and LH1-type complexes. The fusion protein was synthesized using an Escherichia coli expression system and purified by affinity chromatography. Reconstitution experiments demonstrated that the MBP-PufX protein destabilizes the subunit-type complex (20°C), consistent with previous reports. Interestingly, however, the preformed LH1-type complex was stable in the presence of MBP-PufX. The MBP-PufX protein did not influence the preformed LH1-type complexes (4°C). The LH1-type complex containing MBP-PufX showed a unique temperature-dependent structural transformation that was irreversible. The predominant form of the complex at 4°C was the LH1-type. When shifted to 20°C, subunit-type complexes became predominant. Upon subsequent cooling back to 4°C, instead of re-forming the LH1-type complexes, the predominant form remained the subunit-type complexes. In contrast, reversible transformation of LH1 (4°C) and subunit-type complexes (20°C) occurs in the absence of PufX. These results are consistent with the suggestion that MBP-PufX interacts with the LH1α- polypeptide in the subunit (α/β)-type complex (at 20°C), preventing oligomerization of the subunit to form LH1-type complexes.

  10. Femtosecond stage of electron transfer in reaction centers of the triple mutant SL178K/GM203D/LM214H of Rhodobacter sphaeroides.

    PubMed

    Yakovlev, A G; Shkuropatova, T A; Shkuropatova, V A; Shuvalov, V A

    2010-04-01

    Coherent processes in an initial phase of charge transfer in reaction centers (RCs) of the triple mutant S(L178)K/G(M203)D/L(M214)H of Rhodobacter sphaeroides were investigated by difference (light - dark) absorption spectroscopy with 18 fsec time resolution. Electron transfer in the B cofactor branch is activated in this mutant, while the A-branch electron transfer is slowed in comparison with native RCs of Rba. sphaeroides. A bulk of absorption difference spectra was analyzed in the 940-1060 nm range (stimulated emission of excited bacteriochlorophyll dimer P* and absorption of bacteriochlorophyll anions B(A)(-) and beta(-), where beta is a bacteriochlorophyll substituting the native bacteriopheophytin H(A)) and in the 735-775 nm range (bleaching of the absorption band of the bacteriopheophytin H(B) in the B-branch) in the -0.1 to 4 psec range of delays with respect to the moment of photoexcitation of P at 870 nm. Spectra were measured at 293 and 90 K. The kinetics of P* stimulated emission at 940 nm shows its decay with a time constant of approximately 14 psec at 90 K and approximately 18 psec at 293 K, which is accompanied by oscillations with a frequency of approximately 150 cm(-1). A weak absorption band is found at 1018 nm that is formed approximately 100 fsec after excitation of P and reflects the electron transfer from P* to beta and/or B(A) with accumulation of the P(+)beta(-) and/or P(+)B(A)(-) states. The kinetics of DeltaA at 1018 nm contains the oscillations at approximately 150 cm(-1) and distinct low-frequency oscillations at 20-100 cm(-1); also, the amplitude of the oscillations at 150 cm(-1) is much smaller at 293 than at 90 K. The oscillations in the kinetics of the 1018 nm band do not contain a 32 cm(-1) mode that is characteristic for native Rba. sphaeroides RCs having water molecule HOH55 in their structure. The DeltaA kinetics at 751 nm reflects the electron transfer to H(B) with formation of the P(+)H(B)(-) state. The oscillatory part of

  11. Nucleotide sequence and transcriptional analysis of the flanking region of the gene (spb) for the trans-acting factor that controls light-mediated expression of the puf operon in Rhodobacter sphaeroides.

    PubMed

    Mizoguchi, H; Masuda, T; Nishimura, K; Shimada, H; Ohta, H; Shioi, Y; Takamiya, K

    1997-05-01

    We recently reported the existence of a trans-acting factor (SPB) in Rhodobacter sphaeroides that repressed the expression of the puf operon during illumination. SPB was somewhat homologous to HvrA of Rhodobacter capsulatus, but these proteins appear to have functionally different properties. We now report an analysis of the flanking region of spb in the genome of R.sphaeroides, and we show that spb is a positional counterpart of hvrA of R. capsulatus. The region directly downstream of spb was found to contain three genes, two of which were highly homologous to orf5 and ahcY in R. capsulatus. However, a gene corresponding to hvrB, which controls the expression of orf5 and ahcY in R. capsulatus, was absent in R. sphaeroides. The level of the transcript of ahcY did not change in cells grown under photosynthetic and by respiratory conditions. By contrast, orf5 was transcribed at a higher rate in photosynthetically grown cells under high-intensity light than under low-intensity light, indicating features of transcription different from those in R. capsulatus. A third gene, orf318, which was absent in the corresponding region of R. capsulatus, encoded an amino acid sequence that was significantly homologous to the consensus sequence of RfaI and RfaJ of E.coli, which are glycosyl transferases involved in the synthesis of lipopolysaccharide. orf318 was transcribed in the opposite direction to ahcY, and at only a low level, under all conditions tested.

  12. Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase. The coxII/coxIII operon codes for structural and assembly proteins homologous to those in yeast.

    PubMed

    Cao, J; Hosler, J; Shapleigh, J; Revzin, A; Ferguson-Miller, S

    1992-12-05

    The coxII/coxIII operon of Rhodobacter sphaeroides cytochrome c oxidase has been sequenced and characterized by insertional inactivation/complementation analysis. The organization of the genes in this locus (coxII.orf1.orf3.coxIII) is the same as that of the equivalent operon of Paracoccus denitrificans (ctaC.ctaB.ctaG.ctaE), but unlike that of other bacteria whose cytochrome oxidase genes have been characterized so far. The predicted amino acid sequence homology with eukaryotic oxidases is also higher for Rb. sphaeroides (and P. denitrificans) than for other bacterial versions of the enzyme. The inactivation of coxII results in loss of the characteristic cytochrome oxidase spectrum from membranes of the mutant strain. Full recovery requires introduction into the bacterium of the complete operon containing coxII.orf1.orf3.coxIII; partial complementation yielding a spectrally altered enzyme is achieved with a plasmid containing coxII or coxII.orf1.orf3. These results indicate that the peptides ORF1, ORF3, and COXIII are all required for assembly of native cytochrome c oxidase, suggesting an oxidase-specific assembly or chaperonin function for the ORFs in Rb. sphaeroides similar to that observed for the homologous gene products in yeast, COX10 and COX11.

  13. PucC and LhaA direct efficient assembly of the light‐harvesting complexes in Rhodobacter sphaeroides

    PubMed Central

    Mothersole, David J.; Jackson, Philip J.; Vasilev, Cvetelin; Tucker, Jaimey D.; Brindley, Amanda A.; Dickman, Mark J.

    2015-01-01

    Summary The mature architecture of the photosynthetic membrane of the purple phototroph R hodobacter sphaeroides has been characterised to a level where an atomic‐level membrane model is available, but the roles of the putative assembly proteins LhaA and PucC in establishing this architecture are unknown. Here we investigate the assembly of light‐harvesting LH2 and reaction centre‐light‐harvesting1‐PufX (RC‐LH1‐PufX) photosystem complexes using spectroscopy, pull‐downs, native gel electrophoresis, quantitative mass spectrometry and fluorescence lifetime microscopy to characterise a series of lha A and puc C mutants. LhaA and PucC are important for specific assembly of LH1 or LH2 complexes, respectively, but they are not essential; the few LH1 subunits found in Δlha A mutants assemble to form normal RC‐LH1‐PufX core complexes showing that, once initiated, LH1 assembly round the RC is cooperative and proceeds to completion. LhaA and PucC form oligomers at sites of initiation of membrane invagination; LhaA associates with RCs, bacteriochlorophyll synthase (BchG), the protein translocase subunit YajC and the YidC membrane protein insertase. These associations within membrane nanodomains likely maximise interactions between pigments newly arriving from BchG and nascent proteins within the SecYEG‐SecDF‐YajC‐YidC assembly machinery, thereby co‐ordinating pigment delivery, the co‐translational insertion of LH polypeptides and their folding and assembly to form photosynthetic complexes. PMID:26419219

  14. Effect of light intensity and various organic acids on the growth of Rhodobacter sphaeroides LHII-deficient mutant in a turbidostat culture.

    PubMed

    Eltsova, Zinaida; Bolshakov, Maxim; Tsygankov, Anatoly

    2016-12-01

    The composition of photosynthetic apparatus of Rhodobacter sphaeroides wild strain 2.4.1 and its LHII-deficient mutant DBCΩ was compared. The absence of LHII in the mutant was confirmed by comparison of chromatophores spectra and by the absence of electrophoretic band corresponding to LHII complex. Continuous turbidostat cultures of wild strain and its LHII-deficient mutant were compared in response to different light intensities. Cultures were grown using lactate, mixture of lactate and acetate or succinate as carbon source. For comparative analysis, an approximation of experimental data by Monod and Gompertz equations were used. Cultures of DBCΩ had lower growth rates than wild strain when grown on lactate as electron donor and carbon source. Cultures of both strains grown on lactate and acetate or on succinate had similar growth rates. The cultures showed maximum growth rates when grown with succinate. Bacteriochlorophyll a content increased in both strains with decrease of incident light intensity. However, the variation of Bchl a content in wild strain was much more significant. Under light-limiting conditions, bacteriochlorophyll a content in DBCΩ was 4-5 times lower than in the wild strain. Under light-saturating conditions, it was only 1.5-2.5 times lower. Growing with lactate or with lactate and acetate, the mutant switched from light limitation under low light intensities to limitation by organic acids under higher light, whereas the parental strain had similar switch of limiting factor only when growing with lactate and acetate mixture. DBCΩ mutant has higher minimal light intensity enabling growth on any organic acid as a substrate. When growing with lactate or with lactate and acetate, the mutant reached maximum growth rate at lower light intensities than the wild strain. This phenomenon was observed for the first time. Taking into account the concentration of BChl a under light-limiting conditions, the thickness of the suspension capable of

  15. A Second and Unusual pucBA Operon of Rhodobacter sphaeroides 2.4.1: Genetics and Function of the Encoded Polypeptides

    PubMed Central

    Zeng, Xiaohua; Choudhary, Madhu; Kaplan, Samuel

    2003-01-01

    A new operon (designated the puc2BA operon) displaying a high degree of similarity to the original pucBA genes of Rhodobacter sphaeroides 2.4.1 (designated puc1) was identified and studied genetically and biochemically. The puc2B-encoded polypeptide is predicted to exhibit 94% identity with the original β-apoprotein. The puc2A-encoded polypeptide is predicted to be much larger (263 amino acids) than the 54-amino-acid puc1A-encoded polypeptide. In the first 48 amino acids of the puc2A-encoded polypeptide there is 58% amino acid sequence identity to the original puc1A-encoded polypeptide. We found that puc2BA is expressed, and DNA sequence data suggested that puc2BA is regulated by the PpsR/AppA repressor-antirepressor and FnrL. Employing genetic and biochemical approaches, we obtained evidence that the puc2B-encoded polypeptide is able to enter into LH2 complex formation, but neither the full-length puc2A-encoded polypeptide nor its N-terminal 48-amino-acid derivative is able to enter into LH2 complex formation. Thus, the sole source of α-polypeptides for the LH2 complex is puc1A. The role of the puc1C-encoded polypeptide was also determined. We found that the presence of this polypeptide is essential for normal levels of transcription and translation of the puc1 operon but not for transcription and translation of the puc2 operon. Thus, the puc1C gene product appears to have both transcriptional and posttranscriptional roles in LH2 formation. Finally, the absence of any LH2 complex when puc1B was deleted in frame was surprising since we know that in the presence of functional puc2BA, approximately 30% of the LH2 complexes normally observed contain a puc2B-encoded β-polypeptide. PMID:14526029

  16. Partial steps of charge translocation in the nonpumping N139L mutant of Rhodobacter sphaeroides cytochrome c oxidase with a blocked D-channel.

    PubMed

    Siletsky, Sergey A; Zhu, Jiapeng; Gennis, Robert B; Konstantinov, Alexander A

    2010-04-13

    The N139L substitution in the D-channel of cytochrome oxidase from Rhodobacter sphaeroides results in an approximately 15-fold decrease in the turnover number and a loss of proton pumping. Time-resolved absorption and electrometric assays of the F --> O transition in the N139L mutant oxidase result in three major findings. (1) Oxidation of the reduced enzyme by O(2) shows approximately 200-fold inhibition of the F --> O step (k approximately 2 s(-1) at pH 8) which is not compatible with enzyme turnover ( approximately 30 s(-1)). Presumably, an abnormal intermediate F(deprotonated) is formed under these conditions, one proton-deficient relative to a normal F state. In contrast, the F --> O transition in N139L oxidase induced by single-electron photoreduction of intermediate F, generated by reaction of the oxidized enzyme with H(2)O(2), decelerates to an extent compatible with enzyme turnover. (2) In the N139L mutant, the protonic phase of Deltapsi generation coupled to the flash-induced F --> O transition greatly decreases in rate and magnitude and can be assigned to the movement of a proton from E286 to the binuclear site, required for reduction of heme a(3) from the Fe(4+) horizontal lineO(2-) state to the Fe(3+)-OH(-) state. Electrogenic reprotonation of E286 from the inner aqueous phase is missing from the F --> O step in the mutant. (3) In the N139L mutant, the KCN-insensitive rapid electrogenic phase may be composed of two components with lifetimes of approximately 10 and approximately 40 mus and a magnitude ratio of approximately 3:2. The 10 mus phase matches vectorial electron transfer from Cu(A) to heme a, whereas the 40 mus component is assigned to intraprotein proton displacement across approximately 20% of the membrane dielectric thickness. This proton displacement might be triggered by rotation of the charged K362 side chain coupled to heme a reduction. The two components of the rapid electrogenic phase have been resolved subsequently with other D

  17. Effects of ionizable residues on the absorption spectrum and initial electron-transfer kinetics in the photosynthetic reaction center of Rhodobacter sphaeroides.

    PubMed

    Johnson, E T; Nagarajan, V; Zazubovich, V; Riley, K; Small, G J; Parson, W W

    2003-11-25

    Effects of ionizable amino acids on spectroscopic properties and electron-transfer kinetics in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides are investigated by site-directed mutations designed to alter the electrostatic environment of the bacteriochlorophyll dimer that serves as the photochemical electron donor (P). Arginine residues at homologous positions in the L and M subunits (L135 and M164) are changed independently: Arg L135 is replaced by Lys, Leu, Glu, and Gln and Arg M164 by Leu and Glu. Asp L155 also is mutated to Asn, Tyr L164 to Phe, and Cys L247 to Lys and Asp. The mutations at L155, L164, and M164 have little effect on the absorption spectrum, whereas those at L135 and L247 shift the long-wavelength absorption band of P to higher energies. Fits to the ground-state absorption and hole-burned spectra indicate that the blue shift and increased width of the absorption band in the L135 mutants are due partly to changes in the distribution of energies for the zero-phonon absorption line and partly to stronger electron-phonon coupling. The initial electron-transfer kinetics are not changed significantly in most of the mutants, but the time constant increases from 3.0 +/- 0.2 in wild-type RCs to 4.7 +/- 0.2 in C(L247)D and 7.0 +/- 0.3 ps in C(L247)K. The effects of the mutations on the solvation free energies of the product of the initial electron-transfer reaction (P(+)) and the charge-transfer states that contribute to the absorption spectrum ( and ) were calculated by using a distance-dependent electrostatic screening factor. The results are qualitatively in accord with the view that electrostatic interactions of the bacteriochlorophylls with ionized residues of the protein are strongly screened and make only minor contributions to the energetics and dynamics of charge separation. However, the slowing of electron transfer in the Cys L247 mutants and the blue shift of the spectrum in some of the Arg L135 and Cys L247 mutants cannot be

  18. Protonated rhodosemiquinone at the Q(B) binding site of the M265IT mutant reaction center of photosynthetic bacterium Rhodobacter sphaeroides.

    PubMed

    Maróti, Ágnes; Wraight, Colin A; Maróti, Péter

    2015-03-31

    The second electron transfer from primary ubiquinone Q(A) to secondary ubiquinone Q(B) in the reaction center (RC) from Rhodobacter sphaeroides involves a protonated Q(B)(-) intermediate state whose low pK(a) makes direct observation impossible. Here, we replaced the native ubiquinone with low-potential rhodoquinone at the Q(B) binding site of the M265IT mutant RC. Because the in situ midpoint redox potential of Q(A) of this mutant was lowered approximately the same extent (≈100 mV) as that of Q(B) upon exchange of ubiquinone with low-potential rhodoquinone, the inter-quinone (Q(A) → Q(B)) electron transfer became energetically favorable. After subsequent saturating flash excitations, a period of two damped oscillations of the protonated rhodosemiquinone was observed. The Q(B)H(•) was identified by (1) the characteristic band at 420 nm of the absorption spectrum after the second flash and (2) weaker damping of the oscillation at 420 nm (due to the neutral form) than at 460 nm (attributed to the anionic form). The appearance of the neutral semiquinone was restricted to the acidic pH range, indicating a functional pK(a) of <5.5, slightly higher than that of the native ubisemiquinone (pK(a) < 4.5) at pH 7. The analysis of the pH and temperature dependencies of the rates of the second electron transfer supports the concept of the pH-dependent pK(a) of the semiquinone at the Q(B) binding site. The local electrostatic potential is severely modified by the strongly interacting neighboring acidic cluster, and the pK(a) of the semiquinone is in the middle of the pH range of the complex titration. The kinetic and thermodynamic data are discussed according to the proton-activated electron transfer mechanism combined with the pH-dependent functional pK(a) of the semiquinone at the Q(B) site of the RC.

  19. Identification of the first steps in charge separation in bacterial photosynthetic reaction centers of Rhodobacter sphaeroides by ultrafast mid-infrared spectroscopy: electron transfer and protein dynamics.

    PubMed

    Pawlowicz, Natalia P; van Grondelle, Rienk; van Stokkum, Ivo H M; Breton, Jacques; Jones, Michael R; Groot, Marie Louise

    2008-08-01

    Time-resolved visible pump/mid-infrared (mid-IR) probe spectroscopy in the region between 1600 and 1800 cm(-1) was used to investigate electron transfer, radical pair relaxation, and protein relaxation at room temperature in the Rhodobacter sphaeroides reaction center (RC). Wild-type RCs both with and without the quinone electron acceptor Q(A), were excited at 600 nm (nonselective excitation), 800 nm (direct excitation of the monomeric bacteriochlorophyll (BChl) cofactors), and 860 nm (direct excitation of the dimer of primary donor (P) BChls (P(L)/P(M))). The region between 1600 and 1800 cm(-1) encompasses absorption changes associated with carbonyl (C=O) stretch vibrational modes of the cofactors and protein. After photoexcitation of the RC the primary electron donor P excited singlet state (P*) decayed on a timescale of 3.7 ps to the state P(+)B(L)(-) (where B(L) is the accessory BChl electron acceptor). This is the first report of the mid-IR absorption spectrum of P(+)B(L)(-); the difference spectrum indicates that the 9-keto C=O stretch of B(L) is located around 1670-1680 cm(-1). After subsequent electron transfer to the bacteriopheophytin H(L) in approximately 1 ps, the state P(+)H(L)(-) was formed. A sequential analysis and simultaneous target analysis of the data showed a relaxation of the P(+)H(L)(-) radical pair on the approximately 20 ps timescale, accompanied by a change in the relative ratio of the P(L)(+) and P(M)(+) bands and by a minor change in the band amplitude at 1640 cm(-1) that may be tentatively ascribed to the response of an amide C=O to the radical pair formation. We conclude that the drop in free energy associated with the relaxation of P(+)H(L)(-) is due to an increased localization of the electron hole on the P(L) half of the dimer and a further consequence is a reduction in the electrical field causing the Stark shift of one or more amide C=O oscillators.

  20. Three-dimensional structure of the Rhodobacter sphaeroides RC-LH1-PufX complex: dimerization and quinone channels promoted by PufX.

    PubMed

    Qian, Pu; Papiz, Miroslav Z; Jackson, Philip J; Brindley, Amanda A; Ng, Irene W; Olsen, John D; Dickman, Mark J; Bullough, Per A; Hunter, C Neil

    2013-10-29

    Reaction center-light harvesting 1 (RC-LH1) complexes are the fundamental units of bacterial photosynthesis, which use solar energy to power the reduction of quinone to quinol prior to the formation of the proton gradient that drives ATP synthesis. The dimeric RC-LH1-PufX complex of Rhodobacter sphaeroides is composed of 64 polypeptides and 128 cofactors, including 56 LH1 bacteriochlorophyll a (BChl a) molecules that surround and donate energy to the two RCs. The 3D structure was determined to 8 Å by X-ray crystallography, and a model was built with constraints provided by electron microscopy (EM), nuclear magnetic resonance (NMR), mass spectrometry (MS), and site-directed mutagenesis. Each half of the dimer complex consists of a RC surrounded by an array of 14 LH1 αβ subunits, with two BChls sandwiched between each αβ pair of transmembrane helices. The N- and C-terminal extrinsic domains of PufX promote dimerization by interacting with the corresponding domains of an LH1 β polypeptide from the other half of the RC-LH1-PufX complex. Close contacts between PufX, an LH1 αβ subunit, and the cytoplasmic domain of the RC-H subunit prevent the LH1 complex from encircling the RC and create a channel connecting the RC QB site to an opening in the LH1 ring, allowing Q/QH₂ exchange with the external quinone pool. We also identified a channel that connects the two halves of the dimer, potentially forming a long-range pathway for quinone migration along rows of RC-LH1-PufX complexes in the membrane. The structure of the RC-LH1-PufX complex explains the crucial role played by PufX in dimer formation, and it shows how quinone traffic traverses the LH1 complex as it shuttles between the RC and the cytochrome bc₁ complex.

  1. Heterologous Production of the Photosynthetic Reaction Center and Light Harvesting 1 Complexes of the Thermophile Thermochromatium tepidum in the Mesophile Rhodobacter sphaeroides and Thermal Stability of a Hybrid Core Complex.

    PubMed

    Jun, D; Huang, V; Beatty, J T

    2017-10-15

    The photosynthetic complexes of the thermophile Thermochromatium tepidum are of considerable interest in biohybrid solar cell applications because of the ability of thermophilic proteins to tolerate elevated temperatures. Synthetic operons encoding reaction center (RC) and light harvesting 1 (LH1) pigment-protein complexes of T. tepidum were expressed in the mesophile Rhodobacter sphaeroides The T. tepidum RC (TRC) was assembled and was found to be functional with the addition of menadione to populate the QA pocket. The production of T. tepidum LH1 (TLH1) was increased by selection of a phototrophy-capable mutant after UV irradiation mutagenesis, which yielded a hybrid RC-TLH1 core complex consisting of the R. sphaeroides RC and T. tepidum TLH1, confirmed by the absorbance peak of TLH1 at 915 nm. Affinity chromatography partial purification and subsequent sucrose gradient analysis of the hybrid RC-TLH1 core complex indicated that this core complex assembled as a monomer. Furthermore, the RC-TLH1 hybrid core complex was more tolerant of a temperature of 70°C than the R. sphaeroides RC-LH1 core complexes in both the dimeric and monomeric forms; after 1 h, the hybrid complex retained 58% of the initial starting value, compared to values of 11% and 53% for the R. sphaeroides RC-LH1 dimer and monomer forms, respectively.IMPORTANCE This work is important because it is a new approach to bioengineering of photosynthesis proteins for potential use in biophotovoltaic solar energy capture. The work establishes a proof of principle for future biohybrid solar cell applications. Copyright © 2017 American Society for Microbiology.

  2. Identification of intrinsic high-level resistance to rare-earth oxides and oxyanions in members of the class Proteobacteria: characterization of tellurite, selenite, and rhodium sesquioxide reduction in Rhodobacter sphaeroides.

    PubMed Central

    Moore, M D; Kaplan, S

    1992-01-01

    We have identified intrinsic high-level resistance (HLR) to tellurite, selenite, and at least 15 other rare-earth oxides and oxyanions in the facultative photoheterotroph Rhodobacter sphaeroides grown either chemoheterotrophically or photoheterotrophically. Other members of the class Proteobacteria, including members of the alpha-2 and alpha-3 phylogenetic subgroups, were also shown to effect the reduction of many of these compounds, although genera from the alpha-1, beta-1, and gamma-3 subgroups did not express HLR to the oxyanions examined. Detailed analyses employing R. sphaeroides have shown that HLR to at least one class of these oxyanions, the tellurite class (e.g., tellurate, tellurite, selenate, selenite, and rhodium sesquioxide), occurred via intracellular oxyanion reduction and resulted in deposition of metal in the cytoplasmic membrane. The concomitant evolution of hydrogen gas from cells grown photoheterotrophically in the presence of these oxyanions was also observed. HLR to tellurite class oxyanions in R. sphaeroides was not affected by exogenous methionine or phosphate but was reduced 40-fold by the addition of cysteine to growth media. In contrast HLR to the periodate class oxyanions (e.g., periodate, siliconate, and siliconite) was inhibited by extracellular PO4(3-) but did not result in metal deposition or gas evolution. Finally, we observed that HLR to arsenate class oxyanions (e.g., arsenate, molybdate, and tungstate) occurred by a third, distinct mechanism, as evidenced by the lack of intracellular metal deposition and hydrogen gas evolution and an insensitivity to extracellular PO4(3-) or cysteine. Examination of a number of R. sphaeroides mutants has determined the obligate requirement for an intact CO2 fixation pathway and the presence of a functional photosynthetic electron transport chain to effect HLR to K2TeO3 under photosynthetic growth conditions, whereas functional cytochromes bc1 and c2 were required under aerobic growth conditions

  3. Trapping kinetics in mutants of the photosynthetic purple bacterium Rhodobacter sphaeroides: influence of the charge separation rate and consequences for the rate-limiting step in the light-harvesting process.

    PubMed

    Beekman, L M; van Mourik, F; Jones, M R; Visser, H M; Hunter, C N; van Grondelle, R

    1994-03-22

    The primary light-harvesting processes, energy transfer in the light-harvesting antenna, and trapping of the excited states by reaction centers were studied in several mutant strains of the photosynthetic purple bacterium Rhodobacter sphaeroides. The mutants had reaction centers in which the rates of electron transfer were modified by site-directed mutations at the M210 position. Low-intensity pump-probe laser spectroscopy was used to monitor the absorbance transients in the Qy region of the antenna pigments, and it was found that despite a wide variation in charge separation rates within the RC, produced by the alterations at Tyr M210, there was relatively little corresponding variation in the overall trapping rate. These effects of the mutations on the trapping kinetics demonstrate that the rate-limiting step of the overall light-harvesting process is the transfer of the excitations from the antenna to the reaction center.

  4. The functional importance of a pair of conserved glutamic acid residues and of Ca2+ binding in the cbb3–type oxygen reductases from Rhodobacter sphaeroides and Vibrio cholerae

    PubMed Central

    Ouyang, Hanlin; Han, Huazhi; Roh, Jung H.; Hemp, James; Hosler, Jonathan P.; Gennis, Robert B.

    2012-01-01

    The cbb3-type cytochrome c oxidases are members of the heme-copper proton pumping respiratory oxygen reductases. The structure of the cbb3-type oxidase from Pseudomonas stutzeri reveals that, in addition to the six redox-active metal centers (two hemes b, three hemes c and CuB), the enzyme also contains at least one Ca2+. The calcium bridges two propionate carboxyls at the interface between the low-spin heme b and the active-site heme b3 and, in addition, is ligated to a serine in subunit CcoO and by a glutamate in CcoN. The glutamate that is ligated to Ca2+ is one of a pair of glutamic acid residues that has previously been suggested to be part of a proton exit pathway for pumped protons. In the current work, mutants in these glutamates are investigated in the cbb3-type oxidases from Vibrio cholerae and from Rhodobacter sphaeroides. Metal analysis shows that each of these wild type enzymes contains Ca2+. Mutations of the glutamate expected to ligate the Ca2+ in each of these enzymes (E126 in V. cholerae; E180 in R. sphaeroides) result in the loss of activity as well as loss of Ca2+. Mutations in the nearby glutamate (E129 in V. cholerae; E183 in R. sphaeroides) also resulted in loss of oxidase activity and loss of Ca2+. It is concluded that the Ca2+ is essential for assembly of the fully functional enzyme and that neither of the glutamates is likely to be part of a pathway for pumped protons within the cbb3-type oxygen reductases. A more likely role for these glutamates is the maintenance of the structural integrity of the active conformation of the enzyme. PMID:22913716

  5. Regulation of a polyamine transporter by the conserved 3' UTR-derived sRNA SorX confers resistance to singlet oxygen and organic hydroperoxides in Rhodobacter sphaeroides.

    PubMed

    Peng, Tao; Berghoff, Bork A; Oh, Jeong-Il; Weber, Lennart; Schirmer, Jasmin; Schwarz, Johannes; Glaeser, Jens; Klug, Gabriele

    2016-10-02

    Singlet oxygen is generated by bacteriochlorophylls when light and oxygen are simultaneously present in Rhodobacter sphaeroides. Singlet oxygen triggers a specific response that is partly regulated by the alternative sigma factor RpoHI/HII. The sRNA RSs2461 has previously been identified as an RpoHI/HII-dependent sRNA and is derived from the 3' UTR of the mRNA for an OmpR-type transcriptional regulator. Similar to the RpoHI/HII-dependent CcsR and SorY sRNAs, RSs2461 affects the resistance of R. sphaeroides against singlet oxygen and was therefore renamed here SorX. Furthermore, SorX has a strong impact on resistance against organic hydroperoxides that usually occur as secondary damages downstream of singlet oxygen. The 75-nt SorX 3' fragment, which is generated by RNase E cleavage and highly conserved among related species, represents the functional entity. A target search identified potA mRNA, which encodes a subunit of a polyamine transporter, as a direct SorX target and stress resistance via SorX could be linked to potA. The PotABCD transporter is an uptake system for spermidine in E. coli. While spermidine is generally described as beneficial during oxidative stress, we observed significantly increased sensitivity of R. sphaeroides to organic hydroperoxides in the presence of spermidine. We therefore propose that the diminished import of spermidine, due to down-regulation of potA by SorX, counteracts oxidative stress. Together with results from other studies this underlines the importance of regulated transport to bacterial stress defense.

  6. Regulation of a polyamine transporter by the conserved 3′ UTR-derived sRNA SorX confers resistance to singlet oxygen and organic hydroperoxides in Rhodobacter sphaeroides

    PubMed Central

    Peng, Tao; Berghoff, Bork A.; Oh, Jeong-Il; Weber, Lennart; Schirmer, Jasmin; Schwarz, Johannes; Glaeser, Jens; Klug, Gabriele

    2016-01-01

    ABSTRACT Singlet oxygen is generated by bacteriochlorophylls when light and oxygen are simultaneously present in Rhodobacter sphaeroides. Singlet oxygen triggers a specific response that is partly regulated by the alternative sigma factor RpoHI/HII. The sRNA RSs2461 has previously been identified as an RpoHI/HII-dependent sRNA and is derived from the 3′ UTR of the mRNA for an OmpR-type transcriptional regulator. Similar to the RpoHI/HII-dependent CcsR and SorY sRNAs, RSs2461 affects the resistance of R. sphaeroides against singlet oxygen and was therefore renamed here SorX. Furthermore, SorX has a strong impact on resistance against organic hydroperoxides that usually occur as secondary damages downstream of singlet oxygen. The 75-nt SorX 3′ fragment, which is generated by RNase E cleavage and highly conserved among related species, represents the functional entity. A target search identified potA mRNA, which encodes a subunit of a polyamine transporter, as a direct SorX target and stress resistance via SorX could be linked to potA. The PotABCD transporter is an uptake system for spermidine in E. coli. While spermidine is generally described as beneficial during oxidative stress, we observed significantly increased sensitivity of R. sphaeroides to organic hydroperoxides in the presence of spermidine. We therefore propose that the diminished import of spermidine, due to down-regulation of potA by SorX, counteracts oxidative stress. Together with results from other studies this underlines the importance of regulated transport to bacterial stress defense. PMID:27420112

  7. An investigation of slow charge separation in a tyrosine M210 to tryptophan mutant of the Rhodobacter sphaeroides reaction center by femtosecond mid-infrared spectroscopy.

    PubMed

    Pawlowicz, Natalia P; van Stokkum, Ivo H M; Breton, Jacques; van Grondelle, Rienk; Jones, Michael R

    2010-03-20

    Energy and electron transfer in a tyrosine M210 to tryptophan (YM210W) mutant of the Rhodobacter sphaeroides reaction center (RC) were investigated through time-resolved visible pump/mid-infrared (mid-IR) probe spectroscopy at room temperature, with the aim to further characterize the primary charge separated states in the RC. This mutant is known to display slow and multi-exponential charge separation, and was used in earlier work to prove the existence of an alternative route for charge separation starting from the accessory bacteriochlorophyll in the active branch, B(L). The mutant RCs were excited at 860 nm (direct excitation of the primary donor (P) BChls (P(L)/P(M))), 600 nm (unselective excitation), 805 nm (direct excitation of both accessory bacteriochlorophyll cofactors B(L) and B(M)) and 795 nm (direct excitation of B(L)). Absorption changes associated with carbonyl (C=O) stretch vibrational modes of the cofactors and protein were recorded in the region between 1600 and 1775 cm(-1), and both a sequential analysis and simultaneous target analysis of the data were performed. The decay of P* in the YM210W mutant was multi-exponential with lifetimes of 29 and 63.5 ps. The decay of P(+)B(L)(-) state was approximately 10 times longer in the YM210W RC than in the R-26 RC (approximately 7 ps vs. approximately 0.7 ps), and in the mid-IR difference absorption spectrum of P(+)B(L)(-) the stretching frequency of the 9-keto C=O group of B(L) in the ground state was located around 1675-1680 cm(-1), consistent with the presence of a hydrogen bond donated by an adjacent water molecule. Excitation at 795 nm produced a small amount of B(L)*-driven charge separation, as assessed from the excitation wavelength dependence of the raw difference spectra recorded during the first few ps after excitation. This process led to the formation of P(+)B(L)(-). Only the relaxed form of the P(+)H(L)(-) radical pair was observed in the YM210W mutant, and the mid-IR difference absorption

  8. Evolutionary implications of phylogenetic analyses of the gene transfer agent (GTA) of Rhodobacter capsulatus.

    PubMed

    Lang, Andrew S; Taylor, Terumi A; Beatty, J Thomas

    2002-11-01

    The gene transfer agent (GTA) of the a-proteobacterium Rhodobacter capsulatus is a cell-controlled genetic exchange vector. Genes that encode the GTA structure are clustered in a 15-kb region of the R. capsulatus chromosome, and some of these genes show sequence similarity to known bacteriophage head and tail genes. However, the production of GTA is controlled at the level of transcription by a cellular two-component signal transduction system. This paper describes homologues of both the GTA structural gene cluster and the GTA regulatory genes in the a-proteobacteria Rhodopseudomonas palustris, Rhodobacter sphaeroides, Caulobacter crescentus, Agrobacterium tumefaciens and Brucella melitensis. These sequences were used in a phylogenetic tree approach to examine the evolutionary relationships of selected GTA proteins to these homologues and (pro)phage proteins, which was compared to a 16S rRNA tree. The data indicate that a GTA-like element was present in a single progenitor of the extant species that contain both GTA structural cluster and regulatory gene homologues. The evolutionary relationships of GTA structural proteins to (pro)phage proteins indicated by the phylogenetic tree patterns suggest a predominantly vertical descent of GTA-like sequences in the a-proteobacteria and little past gene exchange with (pro)phages.

  9. Application of the Accurate Mass and Time Tag Approach to the Proteome Analysis of Sub-cellular Fractions Obtained from Rhodobacter sphaeroides 2.4.1 Aerobic and Photosynthetic Cell Cultures

    SciTech Connect

    Callister, Stephen J.; Dominguez, Migual; Nicora, Carrie D.; Zeng, Xiaohua; Tavano, Christine; Kaplan, Samuel; Donohue, Timothy; Smith, Richard D.; Lipton, Mary S.

    2006-08-04

    Abstract The high-throughput accurate mass and time tag (AMT) proteomic approach was utilized to characterize the proteomes for cytoplasm, cytoplasmic membrane, periplasm, and outer membrane fractions from aerobic and photosynthetic cultures of the gram-nagtive bacterium Rhodobacter sphaeroides 2.4.1. In addition, we analyzed the proteins within purified chromatophore fractions that house the photosynthetic apparatus from photosynthetically grown cells. In total, 8300 peptides were identified with high confidence from at least one sub-cellular fraction from either cell culture. These peptides were derived from 1514 genes or 35% percent of proteins predicted to be encoded by the genome. A significant number of these proteins were detected within a single sub-cellular fraction and their localization was compared to in-silico predictions. However, the majority of proteins were observed in multiple sub-cellular fractions, and the most likely sub-cellular localization for these proteins was investigated using a Z-score analysis of peptide abundance along with clustering techniques. Good (81%) agreement was observed between the experimental results and in-silico predictions. The AMT tag approach provides localization evidence for those proteins that have no predicted localization information, those annotated as putative proteins, and/or for those proteins annotated as hypothetical and conserved hypothetical.

  10. Blue shifts in bacteriochlorophyll absorbance correlate with changed hydrogen bonding patterns in light-harvesting 2 mutants of Rhodobacter sphaeroides with alterations at alpha-Tyr-44 and alpha-Tyr-45.

    PubMed

    Fowler, G J; Sockalingum, G D; Robert, B; Hunter, C N

    1994-05-01

    A combination of Fourier-Transform (FT) resonance Raman spectroscopy and site-directed mutagenesis has been used to examine the function of two highly conserved aromatic residues, alpha-Tyr-44 and alpha-Tyr-45, in the light-harvesting 2 (LH2) complex of the photosynthetic bacterium Rhodobacter sphaeroides. In LH2 complexes, aromatic residues located at positions alpha-44 and alpha-45 are thought to be located near the putative binding site for bacteriochlorophyll, and alterations at these positions are known to produce blue shifts in bacteriochlorophyll absorbance. In the present work, mutant LH2 complexes carrying the alterations alpha-Tyr-44-->Phe, alpha-Tyr-45-->Phe and alpha-Tyr-44,-45-->Phe,Leu were examined. FT resonance Raman spectroscopy of the resulting complexes shows the breakage of a hydrogen bond to the 2-acetyl carbonyl group of one of the B850 bacteriochlorophylls in the LH2 complex; in the double mutant, breakage of a second bond is probable. These results suggest that one of these hydrogen bonds is to alpha-Tyr-44, placing this residue in close proximity to ring I of one of the B850 bacteriochlorophyll a pigments. The breakage of one, then two, 2-acetyl carbonyl hydrogen bonds correlates well with the shift in the absorbance of the B850 pigments of 11 nm then 26 nm at 77 K. Thus a consistency between literature theoretical calculations and the observations from both absorption and FT resonance Raman spectroscopy is demonstrated.

  11. The Semiquinone at the Qi Site of the bc1 Complex Explored Using HYSCORE Spectroscopy and Specific Isotopic Labeling of Ubiquinone in Rhodobacter sphaeroides via 13C Methionine and Construction of a Methionine Auxotroph

    PubMed Central

    2015-01-01

    Specific isotopic labeling at the residue or substituent level extends the scope of different spectroscopic approaches to the atomistic level. Here we describe 13C isotopic labeling of the methyl and methoxy ring substituents of ubiquinone, achieved through construction of a methionine auxotroph in Rhodobacter sphaeroides strain BC17 supplemented with l-methionine with the side chain methyl group 13C-labeled. Two-dimensional electron spin echo envelope modulation (HYSCORE) was applied to study the 13C methyl and methoxy hyperfine couplings in the semiquinone generated in situ at the Qi site of the bc1 complex in its membrane environment. The data were used to characterize the distribution of unpaired spin density and the conformations of the methoxy substituents based on density functional theory calculations of 13C hyperfine tensors in the semiquinone of the geometry-optimized X-ray structure of the bc1 complex (Protein Data Bank entry 1PP9) with the highest available resolution. Comparison with other proteins indicates individual orientations of the methoxy groups in each particular case are always different from the methoxy conformations in the anion radical prepared in a frozen alcohol solution. The protocol used in the generation of the methionine auxotroph is more generally applicable and, because it introduces a gene deletion using a suicide plasmid, can be applied repeatedly. PMID:25184535

  12. Application of the Accurate Mass and Time Tag Approach to the Proteome Analysis of Sub-cellular Fractions Obtained from Rhodobacter sphaeroides 2.4.1. Aerobic and Photosynthetic Cell Cultures

    PubMed Central

    Callister, Stephen J.; Dominguez, Miguel A.; Nicora, Carrie D.; Zeng, Xiaohua; Tavano, Christine L.; Kaplan, Samuel; Donohue, Timothy J.; Smith, Richard D.; Lipton, Mary S.

    2009-01-01

    The high-throughput accurate mass and time (AMT) tag proteomic approach was utilized to characterize the proteomes for cytoplasm, cytoplasmic membrane, periplasm, and outer membrane fractions from aerobic and photosynthetic cultures of the gram-nagtive bacterium Rhodobacter sphaeroides 2.4.1. In addition, we analyzed the proteins within purified chromatophore fractions that house the photosynthetic apparatus from photosynthetically grown cells. In total, 8300 peptides were identified with high confidence from at least one subcellular fraction from either cell culture. These peptides were derived from 1514 genes or 35% percent of proteins predicted to be encoded by the genome. A significant number of these proteins were detected within a single subcellular fraction and their localization was compared to in silico predictions. However, the majority of proteins were observed in multiple subcellular fractions, and the most likely subcellular localization for these proteins was investigated using a Z-score analysis of estimated protein abundance along with clustering techniques. Good (81%) agreement was observed between the experimental results and in silico predictions. The AMT tag approach provides localization evidence for those proteins that have no predicted localization information, those annotated as putative proteins, and/or for those proteins annotated as hypothetical and conserved hypothetical. PMID:16889416

  13. Mechanism of energy transfer from carotenoids to bacteriochlorophyll : light-harvesting by carotenoids having different extents of {pi}-electron conjugation incorporated into the B850 antenna complex from the carotenoidless bacterium Rhodobacter sphaeroides R-26.1.

    SciTech Connect

    Desamero, R. Z. B.; Chynwat, V.; van der Hoef, I.; Jansen, F. J.; Lugtenburg, J.; Gosztola, D.; Wasielewski, M. R.; Cua, A.; Bocian, D. F.; Frank, H. A.; Univ. of Connecticut; Leiden Univ.; Northwestern Univ.; Univ. of California; Univ. of connecticut

    1998-10-15

    Spheroidene and a series of spheroidene analogues with extents of p-electron conjugation ranging from 7 to 13 carbon-carbon double bonds were incorporated into the B850 light-harvesting complex of Rhodobacter sphaeroides R-26.1. The structures and spectroscopic properties of the carotenoids and the dynamics of energy transfer from the carotenoid to bacteriochlorophyll (BChl) in the B850 complex were studied by using steady-state absorption, fluorescence, fluorescence excitation, resonance Raman, and time-resolved absorption spectroscopy. The spheroidene analogues used in this study were 5',6'-dihydro-7',8'-didehydrospheroidene, 7',8'-didehydrospheroidene, and 1',2'-dihydro-3',4',7',8'-tetradehydrospheroidene. These data, taken together with results from 3,4,7,8-tetrahydrospheroidene, 3,4,5,6-tetrahydrospheroidene, 3,4-dihydrospheroidene, and spheroidene already published (Frank, H. A.; Farhoosh, R.; Aldema, M. L.; DeCoster, B.; Christensen, R. L.; Gebhard, R.; Lugtenburg, J. Photochem. Photobiol. 1993, 57, 49. Farhoosh, R.; Chynwat, V.; Gebhard, R.; Lugtenburg, J.; Frank, H. A. Photosynth. Res. 1994, 42, 157), provide a systematic series of molecules for understanding the molecular features that determine the mechanism of energy transfer from carotenoids to BChl in photosynthetic bacterial light-harvesting complexes. The data support the hypothesis that only carotenoids having 10 or less carbon-carbon double bonds transfer energy via their 21Ag (S1) states to BChl to any significant degree. Energy transfer via the 11Bu (S2) state of the carotenoid becomes more important than the S1 route as the number of conjugated carbon-carbon double bonds increases. The results also suggest that the S2 state associated with the Qx transition of the B850 BChl is the most likely acceptor state for energy transfer originating from both the 2{sup 1}A{sub g} (S{sub 1}) and 1{sup 1}B{sub u} (S{sub 2}) states of all carotenoids.

  14. Blue shifts in bacteriochlorophyll absorbance correlate with changed hydrogen bonding patterns in light-harvesting 2 mutants of Rhodobacter sphaeroides with alterations at alpha-Tyr-44 and alpha-Tyr-45.

    PubMed Central

    Fowler, G J; Sockalingum, G D; Robert, B; Hunter, C N

    1994-01-01

    A combination of Fourier-Transform (FT) resonance Raman spectroscopy and site-directed mutagenesis has been used to examine the function of two highly conserved aromatic residues, alpha-Tyr-44 and alpha-Tyr-45, in the light-harvesting 2 (LH2) complex of the photosynthetic bacterium Rhodobacter sphaeroides. In LH2 complexes, aromatic residues located at positions alpha-44 and alpha-45 are thought to be located near the putative binding site for bacteriochlorophyll, and alterations at these positions are known to produce blue shifts in bacteriochlorophyll absorbance. In the present work, mutant LH2 complexes carrying the alterations alpha-Tyr-44-->Phe, alpha-Tyr-45-->Phe and alpha-Tyr-44,-45-->Phe,Leu were examined. FT resonance Raman spectroscopy of the resulting complexes shows the breakage of a hydrogen bond to the 2-acetyl carbonyl group of one of the B850 bacteriochlorophylls in the LH2 complex; in the double mutant, breakage of a second bond is probable. These results suggest that one of these hydrogen bonds is to alpha-Tyr-44, placing this residue in close proximity to ring I of one of the B850 bacteriochlorophyll a pigments. The breakage of one, then two, 2-acetyl carbonyl hydrogen bonds correlates well with the shift in the absorbance of the B850 pigments of 11 nm then 26 nm at 77 K. Thus a consistency between literature theoretical calculations and the observations from both absorption and FT resonance Raman spectroscopy is demonstrated. Images Figure 2 PMID:8192657

  15. Catalytically-relevant electron transfer between two hemes bL in the hybrid cytochrome bc1-like complex containing a fusion of Rhodobacter sphaeroides and capsulatus cytochromes b.

    PubMed

    Czapla, Monika; Cieluch, Ewelina; Borek, Arkadiusz; Sarewicz, Marcin; Osyczka, Artur

    2013-06-01

    To address mechanistic questions about the functioning of dimeric cytochrome bc1 new genetic approaches have recently been developed. They were specifically designed to enable construction of asymmetrically-mutated variants suitable for functional studies. One approach exploited a fusion of two cytochromes b that replaced the separate subunits in the dimer. The fusion protein, built from two copies of the same cytochrome b of purple bacterium Rhodobacter capsulatus, served as a template to create a series of asymmetrically-mutated cytochrome bc1-like complexes (B-B) which, through kinetic studies, disclosed several important principles of dimer engineering. Here, we report on construction of another fusion protein complex that adds a new tool to investigate dimeric function of the enzyme through the asymmetrically mutated forms of the protein. This complex (BS-B) contains a hybrid protein that combines two different cytochromes b: one coming from R. capsulatus and the other - from a closely related species, R. sphaeroides. With this new fusion we addressed a still controversial issue of electron transfer between the two hemes bL in the core of dimer. Kinetic data obtained with a series of BS-B variants provided new evidence confirming the previously reported observations that electron transfer between those two hemes occurs on a millisecond timescale, thus is a catalytically-relevant event. Both types of the fusion complexes (B-B and BS-B) consistently implicate that the heme-bL-bL bridge forms an electronic connection available for inter-monomer electron transfer in cytochrome bc1. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. 2D ESEEM of the [sup 15]N-labeled radical cations of bacteriochlorophyll a and of the primary donor in reaction centers of Rhodobacter sphaeroides

    SciTech Connect

    Kaess, H.; Rautter, J.; Boenigk, B.; Lubitz, W. ); Hoefer, P. )

    1995-01-05

    The radical cations of [[sup 15]N]bacteriochlorophyll a and of the primary donor P[sub 865] in [sup 15]N-labeled reaction centers of Rhodobacter spaeroides R-26.1 were investigated in frozen solutions using two-dimensional ESEEM methods. Both three-pulse (stimulated echo) and four-pulse (HYSCORE) sequences were employed to avoid ambiguities in data analysis. Computer simulations of the experimental powder spectra were performed, yielding a complete set of nitrogen hyperfine coupling tensors for both systems. The obtained tensor values are compared with those from ENDOR measurements and from semiempirical INDO-type MO calculations. The results obtained from 2D stimulated echo ESEEM and HYSCORE for P[sub 865][sup [center dot]+] are interpreted in terms of an asymmetric spin density distribution over the halves of the bacteriochlorophyll dimer ([open quote]special pair[close quote]) in the reaction center with a ratio of approximately 5:1. This asymmetry is considerably larger at low temperatures in the frozen state than at room temperature. It is postulated that two different conformational states of the dimer exist at these temperatures with different spin density distributions. 60 refs., 9 figs., 4 tabs.

  17. Respiratory Syncytial Virus Fusion Protein-Induced Toll-Like Receptor 4 (TLR4) Signaling Is Inhibited by the TLR4 Antagonists Rhodobacter sphaeroides Lipopolysaccharide and Eritoran (E5564) and Requires Direct Interaction with MD-2

    PubMed Central

    Rallabhandi, Prasad; Phillips, Rachel L.; Boukhvalova, Marina S.; Pletneva, Lioubov M.; Shirey, Kari Ann; Gioannini, Theresa L.; Weiss, Jerrold P.; Chow, Jesse C.; Hawkins, Lynn D.; Vogel, Stefanie N.; Blanco, Jorge C. G.

    2012-01-01

    ABSTRACT Respiratory syncytial virus (RSV) is a leading cause of infant mortality worldwide. Toll-like receptor 4 (TLR4), a signaling receptor for structurally diverse microbe-associated molecular patterns, is activated by the RSV fusion (F) protein and by bacterial lipopolysaccharide (LPS) in a CD14-dependent manner. TLR4 signaling by LPS also requires the presence of an additional protein, MD-2. Thus, it is possible that F protein-mediated TLR4 activation relies on MD-2 as well, although this hypothesis has not been formally tested. LPS-free RSV F protein was found to activate NF-κB in HEK293T transfectants that express wild-type (WT) TLR4 and CD14, but only when MD-2 was coexpressed. These findings were confirmed by measuring F-protein-induced interleukin 1β (IL-1β) mRNA in WT versus MD-2−/− macrophages, where MD-2−/− macrophages failed to show IL-1β expression upon F-protein treatment, in contrast to the WT. Both Rhodobacter sphaeroides LPS and synthetic E5564 (eritoran), LPS antagonists that inhibit TLR4 signaling by binding a hydrophobic pocket in MD-2, significantly reduced RSV F-protein-mediated TLR4 activity in HEK293T-TLR4–CD14–MD-2 transfectants in a dose-dependent manner, while TLR4-independent NF-κB activation by tumor necrosis factor alpha (TNF-α) was unaffected. In vitro coimmunoprecipitation studies confirmed a physical interaction between native RSV F protein and MD-2. Further, we demonstrated that the N-terminal domain of the F1 segment of RSV F protein interacts with MD-2. These data provide new insights into the importance of MD-2 in RSV F-protein-mediated TLR4 activation. Thus, targeting the interaction between MD-2 and RSV F protein may potentially lead to novel therapeutic approaches to help control RSV-induced inflammation and pathology. PMID:22872782

  18. The effect of exchange of bacteriopheophytin a with plant pheophytin a on charge separation in Y(M210)W mutant reaction centers of Rhodobacter sphaeroides at low temperature.

    PubMed

    Shkuropatov, Anatoli Ya; Neerken, Sieglinde; Permentier, Hjalmar P; de Wijn, Rik; Schmidt, Kristiane A; Shuvalov, Vladimir A; Aartsma, Thijs J; Gast, Peter; Hoff, Arnold J

    2003-03-06

    The bacteriopheophytin a molecules at the H(A) and H(B) binding sites of reaction centers (RCs) of the Y(M210)W mutant of Rhodobacter sphaeroides were chemically exchanged with plant pheophytin a. The Y(M210)W mutation slows down the formation of H(A)(-), presumably by raising the free energy level of the P(+)B(A)(-) state above that of P* due to increasing the oxidation potential of the primary electron donor P and lowering the reduction potential of the accessory bacteriochlorophyll B(A). Exchange of the bacteriopheophytins with pheophytin a on the contrary lowers the redox potential of H(A), inhibiting its reduction. A combination of the mutation and pigment exchange was therefore expected to make the A-side of the RC incapable of electron transfer and cause the excited state P* to deactivate directly to the ground state or through the B-side, or both. Time-resolved absorption difference spectroscopy at 10 K on the RCs that were modified in this way showed a lifetime of P* lengthened to about 500 ps as compared to about 200 ps measured in the original Y(M210)W RCs. We show that the decay of P* in the pheophytin-exchanged preparations is accompanied by both return to the ground state and formation of a new charge-separated state, the absorption difference spectrum of which is characterized by bleachings at 811 and 890 nm. This latter state was formed with a time constant of ca. 1.7 ns and a yield of about 30%, and lasted a few nanoseconds. On the basis of spectroscopic observations these bands at 811 and 890 nm are tentatively attributed to the presence of the P(+)B(B)(-) state, where B(B) is the accessory bacteriochlorophyll in the "inactive" B-branch of the cofactors. The B(B) molecules in Y(M210)W RCs are suggested to be spectrally heterogeneous, absorbing in the Q(y) region at 813 or 806 nm. The results are discussed in terms of perturbation of the free energy level of the P(+)B(B)(-) state and absorption properties of the B(B) bacteriochlorophyll in the

  19. Electron-nuclear and electron-electron double resonance spectroscopies show that the primary quinone acceptor QA in reaction centers from photosynthetic bacteria Rhodobacter sphaeroides remains in the same orientation upon light-induced reduction.

    PubMed

    Flores, Marco; Savitsky, Anton; Paddock, Mark L; Abresch, Edward C; Dubinskii, Alexander A; Okamura, Melvin Y; Lubitz, Wolfgang; Möbius, Klaus

    2010-12-23

    Reaction centers (RCs) from the photosynthetic bacterium Rhodobacter (Rb.) sphaeroides R-26 exhibit changes in the recombination kinetics of the charge-separated radical-pair state, P(·+) Q(A)(·-), composed of the dimeric bacteriochlorophyll donor P and the ubiquinone-10 acceptor Q(A), depending on whether the RCs are cooled to cryogenic temperatures in the dark or under continuous illumination (Kleinfeld et al. Biochemistry 1984, 23, 5780-5786). Structural changes near redox-active cofactors have been postulated to be responsible for these changes in kinetics and to occur in the course of light-induced oxidation and reduction of the cofactors thereby assuring a high quantum yield. Here we investigated such potential light-induced structural changes, associated with the formation of P(·+) Q(A)(·-), via pulsed electron-nuclear double resonance (ENDOR) at Q-band (34 GHz) and pulsed electron-electron double resonance (PELDOR) at W-band (95 GHz). Two types of light excitation have been employed for which identical RC samples were prepared: (a) one sample was frozen in the dark and then illuminated to generate transient P(·+) Q(A)(·-), and (b) one was frozen under illumination which resulted in both trapped and transient P(·+) Q(A)(·-) at 80 K. The hyperfine interactions between Q(A)(·-) and the protein were found to be the same in RCs frozen in the dark as in RCs frozen under illumination. Furthermore, these interactions are completely consistent with those observed in RC crystals frozen in the dark. Thus, QA remains in its binding site with the same position and orientation upon reduction. This conclusion is consistent with the result of our orientation-resolving PELDOR experiments on transient P(·+) Q(A)(·-) radical pairs. However, these findings are incompatible with the recently proposed ~60° reorientation of Q(A) upon its photoreduction, as deduced from an analysis of Q-band quantum-beat oscillations (Heinen et al. J. Am. Chem. Soc. 2007, 129, 15935

  20. Electron spin echo envelope modulation spectroscopy supports the suggested coordination of two histidine ligands to the Rieske Fe-S centers of the cytochrome b sub 6 f complex of spinach and the cytochrome bc sub 1 complexes of Rhodospirillum rubrum, Rhodobacter sphaeroides R-26, and bovine heart mitochondria

    SciTech Connect

    Britt, R.D.; Sauer, K.; Klein, M.P. ); Knaff, D.B.; Kriauciunas, A. ); Yu, Changan; Yu, Linda ); Malkin, R. )

    1991-02-19

    Electron spin echo envelope modulation (ESEEM) experiments performed on the Rieske Fe-S clusters of the cytochrome b{sub 6}f complex of spinach chloroplasts and of the cytochrome bc{sub 1} complexes of Rhodospirillum rubrum, Rhodobacter sphaeroides R-26, and bovine heart mitochondria show modulation components resulting from two distinct classes of {sup 14}N ligands. At the g = 1.92 region of the Rieske EPR spectrum of the cytochrome b{sub 6}f complex, the measured hyperfine couplings for the two classes of coupled nitrogens are A{sub 1} = 4.6 MHz and A{sub 2} = 3.8 MHz. Similar couplings are observed for the Rieske centers in the three cytochrome bc{sub 1} complexes. These ESEEM results indicate a nitrogen coordination environment for these Rieske Fe-S centers that is similar to that of the Fe-S cluster of a bacterial dioxygenase enzyme with two coordinated histidine ligands. The Rieske Fe-S cluster lacks modulation components from a weakly coupled peptide nitrogen observed in water-soluble spinach ferredoxin. Treatment with the quinone analogue inhibitor DBMIB causes a shift in the Rieske EPR spectrum to g = 1.95 with no alteration in the magnetic couplings to the two nitrogen atoms. However, the ESEEM pattern of the DBMIB-altered Rieske EPR signal shows evidence of an additional weakly coupled nitrogen similar to that observed in the spinach ferrodoxin ESEEM patterns.

  1. Analysis of the kinetics of P+ HA- recombination in membrane-embedded wild-type and mutant Rhodobacter sphaeroides reaction centers between 298 and 77 K indicates that the adjacent negatively charged QA ubiquinone modulates the free energy of P+ HA- and may influence the rate of the protein dielectric response.

    PubMed

    Gibasiewicz, Krzysztof; Pajzderska, Maria; Dobek, Andrzej; Brettel, Klaus; Jones, Michael R

    2013-09-26

    Time-resolved spectroscopic studies of recombination of the P(+)HA(-) radical pair in photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides give an opportunity to study protein dynamics triggered by light and occurring over the lifetime of P(+)HA(-). The state P(+)HA(-) is formed after the ultrafast light-induced electron transfer from the primary donor pair of bacteriochlorophylls (P) to the acceptor bacteriopheophytin (HA). In order to increase the lifetime of this state, and thus increase the temporal window for the examination of protein dynamics, it is possible to block forward electron transfer from HA(-) to the secondary electron acceptor QA. In this contribution, the dynamics of P(+)HA(-) recombination were compared at a range of temperatures from 77 K to room temperature, electron transfer from HA(-) to QA being blocked either by prereduction of QA or by genetic removal of QA. The observed P(+)HA(-) charge recombination was significantly slower in the QA-deficient RCs, and in both types of complexes, lowering the temperature from RT to 77 K led to a slowing of charge recombination. The effects are explained in the frame of a model in which charge recombination occurs via competing pathways, one of which is thermally activated and includes transient formation of a higher-energy state, P(+)BA(-). An internal electrostatic field supplied by the negative charge on QA increases the free energy levels of the state P(+)HA(-), thus decreasing its energetic distance to the state P(+)BA(-). In addition, the dielectric response of the protein environment to the appearance of the state P(+)HA(-) is accelerated from ∼50-100 ns in the QA-deficient mutant RCs to ∼1-16 ns in WT RCs with a negatively charged QA(-). In both cases, the temperature dependence of the protein dynamics is weak.

  2. Iron homeostasis in the Rhodobacter genus

    PubMed Central

    Zappa, Sébastien; Bauer, Carl E.

    2013-01-01

    Metals are utilized for a variety of critical cellular functions and are essential for survival. However cells are faced with the conundrum of needing metals coupled with e fact that some metals, iron in particular are toxic if present in excess. Maintaining metal homeostasis is therefore of critical importance to cells. In this review we have systematically analyzed sequenced genomes of three members of the Rhodobacter genus, R. capsulatus SB1003, R. sphaeroides 2.4.1 and R. ferroxidans SW2 to determine how these species undertake iron homeostasis. We focused our analysis on elemental ferrous and ferric iron uptake genes as well as genes involved in the utilization of iron from heme. We also discuss how Rhodobacter species manage iron toxicity through export and sequestration of iron. Finally we discuss the various putative strategies set up by these Rhodobacter species to regulate iron homeostasis and the potential novel means of regulation. Overall, this genomic analysis highlights surprisingly diverse features involved in iron homeostasis in the Rhodobacter genus. PMID:24382933

  3. A Complete Set of Flagellar Genes Acquired by Horizontal Transfer Coexists with the Endogenous Flagellar System in Rhodobacter sphaeroides▿ †

    PubMed Central

    Poggio, Sebastian; Abreu-Goodger, Cei; Fabela, Salvador; Osorio, Aurora; Dreyfus, Georges; Vinuesa, Pablo; Camarena, Laura

    2007-01-01

    Bacteria swim in liquid environments by means of a complex rotating structure known as the flagellum. Approximately 40 proteins are required for the assembly and functionality of this structure. Rhodobacter sphaeroides has two flagellar systems. One of these systems has been shown to be functional and is required for the synthesis of the well-characterized single subpolar flagellum, while the other was found only after the genome sequence of this bacterium was completed. In this work we found that the second flagellar system of R. sphaeroides can be expressed and produces a functional flagellum. In many bacteria with two flagellar systems, one is required for swimming, while the other allows movement in denser environments by producing a large number of flagella over the entire cell surface. In contrast, the second flagellar system of R. sphaeroides produces polar flagella that are required for swimming. Expression of the second set of flagellar genes seems to be positively regulated under anaerobic growth conditions. Phylogenic analysis suggests that the flagellar system that was initially characterized was in fact acquired by horizontal transfer from a γ-proteobacterium, while the second flagellar system contains the native genes. Interestingly, other α-proteobacteria closely related to R. sphaeroides have also acquired a set of flagellar genes similar to the set found in R. sphaeroides, suggesting that a common ancestor received this gene cluster. PMID:17293429

  4. Cross-species investigation of the functions of the Rhodobacter PufX polypeptide and the composition of the RC-LH1 core complex.

    PubMed

    Crouch, Lucy I; Jones, Michael R

    2012-02-01

    In well-characterised species of the Rhodobacter (Rba.) genus of purple photosynthetic bacteria it is known that the photochemical reaction centre (RC) is intimately-associated with an encircling LH1 antenna pigment protein, and this LH1 antenna is prevented from completely surrounding the RC by a single copy of the PufX protein. In Rba. veldkampii only monomeric RC-LH1 complexes are assembled in the photosynthetic membrane, whereas in Rba. sphaeroides and Rba. blasticus a dimeric form is also assembled in which two RCs are surrounded by an S-shaped LH1 antenna. The present work established that dimeric RC-LH1 complexes can also be isolated from Rba. azotoformans and Rba. changlensis, but not from Rba. capsulatus or Rba. vinaykumarii. The compositions of the monomers and dimers isolated from these four species of Rhodobacter were similar to those of the well-characterised RC-LH1 complexes present in Rba. sphaeroides. Pigment proteins were also isolated from strains of Rba. sphaeroides expressing chimeric RC-LH1 complexes. Replacement of either the Rba. sphaeroides LH1 antenna or PufX with its counterpart from Rba. capsulatus led to a loss of the dimeric form of the RC-LH1 complex, but the monomeric form had a largely unaltered composition, even in strains in which the expression level of LH1 relative to the RC was reduced. The chimeric RC-LH1 complexes were also functional, supporting bacterial growth under photosynthetic conditions. The findings help to tease apart the different functions of PufX in different species of Rhodobacter, and a specific protein structural arrangement that allows PufX to fulfil these three functions is proposed.

  5. Nitrate reductase from Rhodopseudomonas sphaeroides.

    PubMed Central

    Kerber, N L; Cardenas, J

    1982-01-01

    The facultative phototroph Rhodopseudomonas sphaeroides DSM158 was incapable of either assimilating or dissimilating nitrate, although the organism could reduce it enzymatically to nitrite either anaerobically in the light or aerobically in the dark. Reduction of nitrate was mediated by a nitrate reductase bound to chromatophores that could be easily solubilized and functioned with chemically reduced viologens or photochemically reduced flavins as electron donors. The enzyme was solubilized, and some of its kinetic and molecular parameters were determined. It seemed to be nonadaptive, ammonia did not repress its synthesis, and its activity underwent a rapid decline when the cells entered the stationary growth phase. Studies with inhibitors and with metal antagonists indicated that molybdenum and possibly iron participate in the enzymatic reduction of nitrate. The conjectural significance of this nitrate reductase in phototrophic bacteria is discussed. PMID:6978883

  6. Time-Resolved Surface-Enhanced IR-Absorption Spectroscopy of Direct Electron Transfer to Cytochrome c Oxidase from R. sphaeroides

    PubMed Central

    Schwaighofer, Andreas; Steininger, Christoph; Hildenbrandt, David M.; Srajer, Johannes; Nowak, Christoph; Knoll, Wolfgang; Naumann, Renate L.C.

    2013-01-01

    Time-resolved surface-enhanced IR-absorption spectroscopy triggered by electrochemical modulation has been performed on cytochrome c oxidase from Rhodobacter sphaeroides. Single bands isolated from a broad band in the amide I region using phase-sensitive detection were attributed to different redox centers. Their absorbances changing on the millisecond timescale could be fitted to a model based on protonation-dependent chemical reaction kinetics established previously. Substantial conformational changes of secondary structures coupled to redox transitions were revealed. PMID:24359742

  7. Rhodopseudomonas sphaeroides lipid A derivatives block in vitro induction of tumor necrosis factor and endotoxin tolerance by smooth lipopolysaccharide and monophosphoryl lipid A.

    PubMed Central

    Henricson, B E; Perera, P Y; Qureshi, N; Takayama, K; Vogel, S N

    1992-01-01

    Rhodopseudomonas (Rhodobacter) sphaeroides diphosphoryl lipid A is a relatively inert species of lipid A but has been shown to antagonize the effects of toxic lipopolysaccharide (LPS) both in vivo and in vitro. The antagonist and its monophosphoryl derivative were examined for the ability to block tumor necrosis factor synthesis and reverse tolerance induction in vitro in macrophage cultures stimulated with bioactive preparations of smooth LPS, rough LPS, diphosphoryl lipid A, and monophosphoryl lipid A. Inhibition of agonist activity and reversal of tolerance by these novel penta-acylated lipid A antagonists provides new insight into macrophage-LPS interactions. PMID:1398939

  8. In Vivo Sensitivity of Blue-Light-Dependent Signaling Mediated by AppA/PpsR or PrrB/PrrA in Rhodobacter sphaeroides▿

    PubMed Central

    Metz, Sebastian; Jäger, Andreas; Klug, Gabriele

    2009-01-01

    Formation of photosynthesis complexes in Rhodobacter sphaeroides is regulated in a redox- and light-dependent manner by the AppA/PpsR and PrrB/PrrA systems. While on the one hand, blue light is sensed by the flavin adenine dinucleotide-binding BLUF domain of AppA, on the other, light is absorbed by bacteriochlorophyll signals through PrrB/PrrA. We show that much smaller quantities initiate the AppA-mediated response to blue light than the bacteriochlorophyll-mediated response. PMID:19395480

  9. Description of Rhodobacter azollae sp. nov. and Rhodobacter lacus sp. nov.

    PubMed

    Suresh, G; Sailaja, B; Ashif, A; Dave, Bharti P; Sasikala, Ch; Ramana, Ch V

    2017-09-01

    Three strains (JA826T, JA912T and JA913), which were yellowish brown colour, rod to oval shaped, Gram-stain-negative, motile, phototrophic bacteria with a vesicular architecture of intracytoplasmic membranes, were isolated from different pond samples. The DNA G+C content of the three strains was between 64.6 and 65.5 mol%. The highest 16S rRNA gene sequence similarity of all three strains was with the type strains of the genus Rhodobacter sensu stricto in the family Rhodobacteraceae. Strain JA826T had highest sequence similarity with Rhodobacter maris JA276T (98.5 %), Rhodobacter viridis JA737T (97.5 %) and other members of the genus Rhodobacter (<97 %). Strain JA912T had highest sequence similarity with Rhodobacter viridis JA737T (99.6 %), Rhodobacter sediminis N1T (99.3 %), Rhodobacter capsulatus ATCC 11166T (98.8 %) and less than 97 % similarity with other members of the genus Rhodobacter. The 16S rRNA gene sequence similarity between strains JA826T and JA912T was 96.9 %. DNA-DNA hybridization showed that strains JA826T and JA912T (values among themselves and between the type strains of nearest members <44 %) did not belong to any of the nearest species of the genus Rhodobacter. However, strains JA912T and JA913 were closely related (DNA-DNA hybridization value >90 %). The genomic distinction was also supported by differences in phenotypic and chemotaxonomic characteristics in order to propose strains JA826T (=KCTC 15478T=LMG 28758T) and JA912T (=KCTC 15475T=LMG 28748T) as new species in the genus Rhodobacter sensu stricto with the names Rhodobacter lacus and Rhodobacter azollae, respectively.

  10. Structural and spectroscopic consequences of hexacoordination of a bacteriochlorophyll cofactor in the Rhodobacter sphaeroides reaction center .

    PubMed

    Frolov, Dmitrij; Marsh, May; Crouch, Lucy I; Fyfe, Paul K; Robert, Bruno; van Grondelle, Rienk; Hadfield, Andrea; Jones, Michael R

    2010-03-09

    The structural and functional consequences of changing the coordination state of one of the bacteriochlorophyll (BChl) cofactors in the purple bacterial reaction center have been explored. A combination of steady state spectroscopy and X-ray crystallography was used to demonstrate that mutagenesis of residue 181 of the L-polypeptide from Phe to Arg (FL181R) causes the BChl at the accessory (B(B)) position on the so-called inactive cofactor branch to become hexacoordinated, with no significant changes to the structure of the surrounding protein. This change was accompanied by the appearance of a distinctive absorbance band at 631 nm in the room-temperature absorbance spectrum. The ligand donor was not the Arg side chain but rather an intervening water molecule, and contrary to expectations, the Mg of B(B) did not adopt a more in-plane geometry in response to hexacoordination. The mutation caused a disturbance to the detailed conformation of the BChl macrocycle that manifested in a number of subtle changes to the resonance Raman spectrum. Hexacoordination of B(B) produced a small increase in the lifetime of the excited electronic state of the primary donor bacteriochlorophylls (P*), indicating some disturbance to light-driven energy and/or electron transfer events on the time scale of a few picoseconds after light excitation. The B(B) bacteriochlorophyll returned to a pentacoordinated state in a double mutant where the FL181R mutation was combined with removal of the native axial ligand through mutation of His M182 to Leu. Experimental evidence of hexacoordinated bacteriochlorophylls in the literature on antenna proteins is considered, and possible reasons why hexacoordinated bacteriochlorophylls and chlorophylls appear to be avoided in photosynthetic proteins are discussed.

  11. The proton collecting function of the inner surface of cytochrome c oxidase from Rhodobacter sphaeroides.

    PubMed

    Marantz, Y; Nachliel, E; Aagaard, A; Brzezinski, P; Gutman, M

    1998-07-21

    The experiments presented in this study address the problem of how the cytoplasmic surface (proton-input side) of cytochrome c oxidase interacts with protons in the bulk. For this purpose, the cytoplasmic surface of the enzyme was labeled with a fluorescein (Flu) molecule covalently bound to Cys223 of subunit III. Using the Flu as a proton-sensitive marker on the surface and phiOH as a soluble excited-state proton emitter, the dynamics of the acid-base equilibration between the surface and the bulk was measured in the time-resolved domain. The results were analyzed by using a rigorous kinetic analysis that is based on numeric integration of coupled nonliner differential rate equations in which the rate constants are used as adjustable parameters. The analysis of 11 independent measurements, carried out under various initial conditions, indicated that the protonation of the Flu proceeds through multiple pathways involving diffusion-controlled reactions and proton exchange among surface groups. The surface of the protein carries an efficient system made of carboxylate and histidine moieties that are sufficiently close to each other as to form a proton-collecting antenna. It is the passage of protons among these sites that endows cytochrome c oxidase with the capacity to pick up protons from the buffered cytoplasmic matrix within a time frame compatible with the physiological turnover of the enzyme.

  12. Alternative initial proton acceptors for the D pathway of Rhodobacter sphaeroides cytochrome c oxidase

    PubMed Central

    Varanasi, Lakshman; Hosler, Jonathan

    2011-01-01

    In order to characterize protein structures that control proton uptake, forms of cytochrome c oxidase (CcO) containing a carboxyl or a thiol group in line with the initial, internal waters of the D pathway for proton transfer have been assayed in the presence and absence of subunit III. Subunit III provides approximately half of the protein surrounding the entry region of the D pathway. The mutant N139D-D132N contains a carboxyl group 6Å within the D pathway and lacks the normal, surface-exposed proton acceptor, Asp-132. With subunit III, the steady-state activity of this mutant is slow but once subunit III is removed its activity is the same as wild-type CcO lacking subunit III (∼1800 H+ s-1). Thus, a carboxyl group ∼25% within the pathway enhances proton uptake even though the carboxyl has no direct contact with bulk solvent. Protons from solvent apparently move to internal Asp-139 through a short file of waters, normally blocked by subunit III. Cysteine-139 also supports rapid steady-state proton uptake, demonstrating that an anion other than a carboxyl can attract and transfer protons into the D pathway. When both Asp-132 and Asp/Cys-139 are present, the removal of subunit III increases CcO activity to rates greater than that of normal CcO due to simultaneous proton uptake by two initial acceptors. The results show how the environment of the initial proton acceptor for the D pathway in these CcO forms dictates the pH range of CcO activity, with implications for the function of Asp-132, the normal proton acceptor. PMID:21344856

  13. ATPases and phosphate exchange activities in magnesium chelatase subunits of Rhodobacter sphaeroides.

    PubMed

    Hansson, M; Kannangara, C G

    1997-11-25

    Three separate proteins, BchD, BchH, and BchI, together with ATP, insert magnesium into protoporphyrin IX. An analysis of ATP utilization by the subunits revealed the following: BchH catalyzed ATP hydrolysis at the rate of 0.9 nmol per min per mg of protein. BchI and BchD, tested individually, had no ATPase activity but, when combined, hydrolyzed ATP at the rate of 117.9 nmol/min per mg of protein. Magnesium ions were required for the ATPase activities of both BchH and BchI+D, and these activities were inhibited 50% by 2 mM o-phenanthroline. BchI additionally catalyzed a phosphate exchange reaction from ATP and ADP. We conclude that ATP hydrolysis by BchI+D is required for an activation step in the magnesium chelatase reaction, whereas ATPase activity of BchH and the phosphate exchange activity of BchI participate in subsequent reactions leading to the insertion of Mg2+ into protoporphyrin IX.

  14. Mutations to R. sphaeroides Reaction Center Perturb Energy Levels and Vibronic Coupling but Not Observed Energy Transfer Rates.

    PubMed

    Flanagan, Moira L; Long, Phillip D; Dahlberg, Peter D; Rolczynski, Brian S; Massey, Sara C; Engel, Gregory S

    2016-03-10

    The bacterial reaction center is capable of both efficiently collecting and quickly transferring energy within the complex; therefore, the reaction center serves as a convenient model for both energy transfer and charge separation. To spectroscopically probe the interactions between the electronic excited states on the chromophores and their intricate relationship with vibrational motions in their environment, we examine coherences between the excited states. Here, we investigate this question by introducing a series of point mutations within 12 Å of the special pair of bacteriochlorophylls in the Rhodobacter sphaeroides reaction center. Using two-dimensional spectroscopy, we find that the time scales of energy transfer dynamics remain unperturbed by these mutations. However, within these spectra, we detect changes in the mixed vibrational-electronic coherences in these reaction centers. Our results indicate that resonance between bacteriochlorophyll vibrational modes and excitonic energy gaps promote electronic coherences and support current vibronic models of photosynthetic energy transfer.

  15. Kinetics of cytochrome c oxidase from R. sphaeroides initiated by direct electron transfer followed by tr-SEIRAS.

    PubMed

    Steininger, Christoph; Reiner-Rozman, Ciril; Schwaighofer, Andreas; Knoll, Wolfgang; Naumann, Renate L C

    2016-12-01

    Time-resolved surface-enhanced IR-absorption spectroscopy (tr-SEIRAS) has been performed on cytochrome c oxidase from Rhodobacter sphaeroides. The enzyme was converted electrochemically into the fully reduced state. Thereafter, in the presence of oxygen, the potential was switched to open circuit potential (OCP). Under these conditions, the enzyme is free to undergo enzymatic oxidation in the absence of an external electric field. Tr-SEIRAS was performed using the step-scan technique, triggered by periodic potential pulses switching between - 800mV and OCP. Single bands were resolved in a broad band in the amide I region using phase sensitive detection. Amplitudes of these bands were analyzed as a function of time. Time constants in the ms time scale were considered in terms of conformational changes of the protein secondary structures associated with the enzymatic turnover of the protein.

  16. Thioredoxin 2 is involved in oxidative stress defence and redox-dependent expression of photosynthesis genes in Rhodobacter capsulatus.

    PubMed

    Li, Kuanyu; Härtig, Elisabeth; Klug, Gabriele

    2003-02-01

    Thioredoxins are small ubiquitous proteins that display different functions mainly via redox-mediated processes. The facultatively photosynthetic bacterium Rhodobacter capsulatus harbours at least two genes for thioredoxin 1 and 2, trxA and trxC. It is demonstrated that thioredoxin 2 of R. capsulatus can partially replace the thioredoxin 1 function as a hydrogen donor for methionine sulfoxide reductase but cannot replace thioredoxin 1 as a subunit of phage T7 DNA polymerase. By inactivating the trxC gene in R. capsulatus, it is shown that thioredoxin 2 is involved in resistance against oxidative stress. As thioredoxin 1 of Rhodobacter sphaeroides, R. capsulatus thioredoxin 2 affects the oxygen-dependent expression of photosynthesis genes, albeit in an opposite way. The trxC mutant of R. capsulatus shows a stronger increase in photosynthesis gene expression after a decrease in oxygen tension than the isogenic wild-type strain. The expression of the trxC gene is downregulated by oxygen.

  17. The Flagellar Protein FliL Is Essential for Swimming in Rhodobacter sphaeroides▿ †

    PubMed Central

    Suaste-Olmos, Fernando; Domenzain, Clelia; Mireles-Rodríguez, José Cruz; Poggio, Sebastian; Osorio, Aurora; Dreyfus, Georges; Camarena, Laura

    2010-01-01

    In this work we characterize the function of the flagellar protein FliL in Rhodobacter sphaeroides. Our results show that FliL is essential for motility in this bacterium and that in its absence flagellar rotation is highly impaired. A green fluorescent protein (GFP)-FliL fusion forms polar and lateral fluorescent foci that show different spatial dynamics. The presence of these foci is dependent on the expression of the flagellar genes controlled by the master regulator FleQ, suggesting that additional components of the flagellar regulon are required for the proper localization of GFP-FliL. Eight independent pseudorevertants were isolated from the fliL mutant strain. In each of these strains a single nucleotide change in motB was identified. The eight mutations affected only three residues located on the periplasmic side of MotB. Swimming of the suppressor mutants was not affected by the presence of the wild-type fliL allele. Pulldown and yeast two-hybrid assays showed that that the periplasmic domain of FliL is able to interact with itself but not with the periplasmic domain of MotB. From these results we propose that FliL could participate in the coupling of MotB with the flagellar rotor in an indirect fashion. PMID:20889747

  18. Overlapping Alternative Sigma Factor Regulons in the Response to Singlet Oxygen in Rhodobacter sphaeroides▿ †

    PubMed Central

    Nuss, Aaron M.; Glaeser, Jens; Berghoff, Bork A.; Klug, Gabriele

    2010-01-01

    Organisms performing photosynthesis in the presence of oxygen have to cope with the formation of highly reactive singlet oxygen (1O2) and need to mount an adaptive response to photooxidative stress. Here we show that the alternative sigma factors RpoHI and RpoHII are both involved in the 1O2 response and in the heat stress response in Rhodobacter sphaeroides. We propose RpoHII to be the major player in the 1O2 response, whereas RpoHI is more important for the heat stress response. Mapping of the 5′ ends of RpoHII- and also RpoHI/RpoHII-dependent transcripts revealed clear differences in the −10 regions of the putative promoter sequences. By using bioinformatic tools, we extended the RpoHII regulon, which includes genes induced by 1O2 exposure. These genes encode proteins which are, e.g., involved in methionine sulfoxide reduction and in maintaining the quinone pool. Furthermore, we identified small RNAs which depend on RpoHI and RpoHII and are likely to contribute to the defense against photooxidative stress and heat stress. PMID:20304993

  19. RpoHII Activates Oxidative-Stress Defense Systems and Is Controlled by RpoE in the Singlet Oxygen-Dependent Response in Rhodobacter sphaeroides▿ †

    PubMed Central

    Nuss, Aaron M.; Glaeser, Jens; Klug, Gabriele

    2009-01-01

    Photosynthetic organisms need defense systems against photooxidative stress caused by the generation of highly reactive singlet oxygen (1O2). Here we show that the alternative sigma factor RpoHII is required for the expression of important defense factors and that deletion of rpoHII leads to increased sensitivity against exposure to 1O2 and methylglyoxal in Rhodobacter sphaeroides. The gene encoding RpoHII is controlled by RpoE, and thereby a sigma factor cascade is constituted. We provide the first in vivo study that identifies genes controlled by an RpoHII-type sigma factor, which is widely distributed in the Alphaproteobacteria. RpoHII-dependent genes encode oxidative-stress defense systems, including proteins for the degradation of methylglyoxal, detoxification of peroxides, 1O2 scavenging, and redox and iron homeostasis. Our experiments indicate that glutathione (GSH)-dependent mechanisms are involved in the defense against photooxidative stress in photosynthetic bacteria. Therefore, we conclude that systems pivotal for the organism's defense against photooxidative stress are strongly dependent on GSH and are specifically recognized by RpoHII in R. sphaeroides. PMID:18978062

  20. Salt Stress-Induced Changes in the Transcriptome, Compatible Solutes, and Membrane Lipids in the Facultatively Phototrophic Bacterium Rhodobacter sphaeroides▿†

    PubMed Central

    Tsuzuki, Minoru; Moskvin, Oleg V.; Kuribayashi, Masayuki; Sato, Kiichi; Retamal, Susana; Abo, Mitsuru; Zeilstra-Ryalls, Jill; Gomelsky, Mark

    2011-01-01

    Responses to NaCl stress were investigated in phototrophically grown Alphaproteobacterium Rhodobacter sphaeroides by transcriptome profiling, mutational analysis, and measurements of compatible solutes and membrane phospholipids. After exposure to salt stress, genes encoding two putative glycine betaine uptake systems, proVWX and betS, were highly upregulated. Mutational analysis revealed that BetS, not ProVWX, was the primary transporter of this compatible solute. Upon the addition of salt, exogenous glycine betaine was taken up rapidly, and maximal intracellular levels were reached within minutes. In contrast, synthesis of another important compatible solute in R. sphaeroides, trehalose, increased slowly following salt stress, reaching maximal levels only after several hours. This accumulation pattern was consistent with the more gradual increase in salt-induced transcription of the trehalose biosynthesis operon otsBA. Several genes encoding putative transcription factors were highly induced by salt stress. Multiple copies of one of these factors, crpO (RSP1275), whose product is a member of the cyclic AMP receptor protein/fumarate and nitrate reduction regulator (CRP/FNR) family, improved NaCl tolerance. When crpO was provided in multicopy, expression of genes for synthesis or transport of compatible solutes was unaltered, but the membrane phospholipid composition became biased toward that found in salt-stressed cells. Collectively, this study characterized transcriptional responses to salt stress, correlated changes in transcription with compatible solute accumulation rates, identified the main glycine betaine transporter and trehalose synthase, characterized salt-induced changes in phospholipid composition, and uncovered a transcription factor associated with changes in phospholipids. These findings set the stage for deciphering the salt stress-responsive regulatory network in R. sphaeroides. PMID:21908636

  1. The RegA regulon exhibits variability in response to altered growth conditions and differs markedly between Rhodobacter species

    PubMed Central

    Schindel, Heidi S.

    2016-01-01

    The RegB/RegA two-component system from Rhodobacter capsulatus regulates global changes in gene expression in response to alterations in oxygen levels. Studies have shown that RegB/RegA controls many energy-generating and energy-utilizing systems such as photosynthesis, nitrogen fixation, carbon fixation, hydrogen utilization, respiration, electron transport and denitrification. In this report, we utilized RNA-seq and ChIP-seq to analyse the breadth of genes indirectly and directly regulated by RegA. A comparison of mRNA transcript levels in wild type cells relative to a RegA deletion strain shows that there are 257 differentially expressed genes under photosynthetic defined minimal growth medium conditions and 591 differentially expressed genes when grown photosynthetically in a complex rich medium. ChIP-seq analysis also identified 61 unique RegA binding sites with a well-conserved recognition sequence, 33 of which exhibit changes in neighbouring gene expression. These transcriptome results define new members of the RegA regulon including genes involved in iron transport and motility. These results also reveal that the set of genes that are regulated by RegA are growth medium specific. Similar analyses under dark aerobic conditions where RegA is thought not to be phosphorylated by RegB reveal 40 genes that are differentially expressed in minimal medium and 20 in rich medium. Finally, a comparison of the R. capsulatus RegA regulon with the orthologous PrrA regulon in Rhodobacter sphaeroides shows that the number of photosystem genes regulated by RegA and PrrA are similar but that the identity of genes regulated by RegA and PrrA beyond those involved in photosynthesis are quite distinct. PMID:28348828

  2. Regulation of the primary quinone binding conformation by the H subunit in reaction centers from Rhodobacter sphaeroides.

    PubMed

    Sun, Chang; Taguchi, Alexander T; Beal, Nathan J; O'Malley, Patrick J; Dikanov, Sergei A; Wraight, Colin A

    2015-11-19

    Unlike photosystem II (PSII) in higher plants, bacterial photosynthetic reaction centers (bRCs) from Proteobacteria have an additional peripheral membrane subunit "H". The H subunit is necessary for photosynthetic growth, but can be removed chemically in vitro. The remaining LM dimer retains its activity to perform light-induced charge separation. Here we investigate the influence of the H subunit on interactions between the primary semiquinone and the protein matrix, using a combination of site-specific isotope labeling, pulsed electron paramagnetic resonance (EPR), and density functional theory (DFT) calculations. The data reveal substantially weaker binding interactions between the primary semiquinone and the LM dimer than observed for the intact bRC; the amount of electron spin transferred to the nitrogen hydrogen bond donors is significantly reduced, the methoxy groups are more free to rotate, and the spectra indicate a heterogeneous mixture of bound semiquinone states. These results are consistent with a loosening of the primary quinone binding pocket in the absence of the H subunit.

  3. Effects of the measuring light on the photochemistry of the bacterial photosynthetic reaction center from Rhodobacter sphaeroides.

    PubMed

    Husu, Ivan; Giustini, Mauro; Colafemmina, Giuseppe; Palazzo, Gerardo; Mallardi, Antonia

    2011-09-01

    The bacterial reaction center (RC) has become a reference model in the study of the diverse interactions of quinones with electron transfer complexes. In these studies, the RC functionality was probed through flash-induced absorption changes where the state of the primary donor is probed by means of a continuous measuring beam and the electron transfer is triggered by a short intense light pulse. The single-beam set-up implies the use as reference of the transmittance measured before the light pulse. Implicit in the analysis of these data is the assumption that the measuring beam does not elicit the protein photochemistry. At variance, measuring beam is actinic in nature at almost all the suitable wavelengths. In this contribution, the analytical modelling of the time evolution of neutral and charge-separated RCs has been performed. The ability of measuring light to elicit RC photochemistry induces a first order growth of the charge-separated state up to a steady state that depends on the light intensity and on the occupation of the secondary quinone (Q(B)) site. Then the laser pulse pumps all the RCs in the charge-separated state. The following charge recombination is still affected by the measuring beam. Actually, the kinetics of charge recombination measured in RC preparation with the Q(B) site partially occupied are two-exponential. The rate constant of both fast and slow phases depends linearly on the intensity of the measuring beam while their relative weights depend not only on the fractions of RC with the Q(B) site occupied but also on the measuring light intensity itself.

  4. Structural Insight into Substrate Differentiation of the Sugar-metabolizing Enzyme Galactitol Dehydrogenase from Rhodobacter sphaeroides D*

    PubMed Central

    Carius, Yvonne; Christian, Henning; Faust, Annette; Zander, Ulrich; Klink, Björn U.; Kornberger, Petra; Kohring, Gert-Wieland; Giffhorn, Friedrich; Scheidig, Axel J.

    2010-01-01

    Galactitol 2-dehydrogenase (GatDH) belongs to the protein superfamily of short-chain dehydrogenases. As an enzyme capable of the stereo- and regioselective modification of carbohydrates, it exhibits a high potential for application in biotechnology as a biocatalyst. We have determined the crystal structure of the binary form of GatDH in complex with its cofactor NAD(H) and of the ternary form in complex with NAD(H) and three different substrates. The active form of GatDH constitutes a homo-tetramer with two magnesium-ion binding sites each formed by two opposing C termini. The catalytic tetrad is formed by Asn116, Ser144, Tyr159, and Lys163. GatDH structurally aligns well with related members of the short-chain dehydrogenase family. The substrate binding pocket can be divided into two parts of different size and polarity. In the smaller part, the side chains of amino acids Ser144, Ser146, and Asn151 are important determinants for the binding specificity and the orientation of (pro-) chiral compounds. The larger part of the pocket is elongated and flanked by polar and non-polar residues, enabling a rather broad substrate spectrum. The presented structures provide valuable information for a rational design of this enzyme to improve its stability against pH, temperature, or solvent concentration and to optimize product yield in bioreactors. PMID:20410293

  5. Electrostatic dominoes: long distance propagation of mutational effects in photosynthetic reaction centers of Rhodobacter capsulatus.

    PubMed

    Sebban, P; Maróti, P; Schiffer, M; Hanson, D K

    1995-07-04

    Two point mutants from the purple bacterium Rhodobacter capsulatus, both modified in the M protein of the photosynthetic reaction center, have been studied by flash-induced absorbance spectroscopy. These strains carry either the M231Arg --> Leu or M43ASN --> Asp mutations, which are located 9 and 15 A, respectively, from the terminal electron acceptor QB. In the wild-type Rb. sphaeroides structure, M231Arg is involved in a conserved salt bridge with H125Glu and H232Glu and M43Asn is located among several polar residues that form or surround the QB binding site. These substitutions were originally uncovered in phenotypic revertants isolated from the photosynthetically incompetent L212Glu-L213Asp --> Ala-Ala site-specific double mutant. As second-site suppressor mutations, they have been shown to restore the proton transfer function that is interrupted in the L212Ala-L213Ala double mutant. The electrostatic effects that are induced in reaction centers by the M231Arg --> Leu and M43Asn --> Asp substitutions are roughly the same in either the double-mutant or wild-type backgrounds. In a reaction center that is otherwise wild type in sequence, they decrease the free energy gap between the QA- and QB- states by 24 +/- 5 and 45 +/- 5 meV, respectively. The pH dependences of K2, the QA-QB <--> QAQB- equilibrium constant, are altered in reaction centers that carry either of these substitutions, revealing differences in the pKas of titratable groups compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. High-level soluble expression of the hemA gene from Rhodobacter capsulatus and comparative study of its enzymatic properties.

    PubMed

    Lou, Jia-wei; Zhu, Li; Wu, Mian-bin; Yang, Li-rong; Lin, Jian-ping; Cen, Pei-lin

    2014-05-01

    The Rhodobacter capsulatus hemA gene, which encodes 5-aminolevulinic acid synthase (ALAS), was expressed in Escherichia coli Rosetta (DE3) and the enzymatic properties of the purified recombinant ALAS (RC-ALAS) were studied. Compared with ALASs encoded by hemA genes from Agrobacterium radiobacter (AR-ALAS) and Rhodobacter sphaeroides (RS-ALAS), the specific activity of RC-ALAS reached 198.2 U/mg, which was about 31.2% and 69.5% higher than those of AR-ALAS (151.1 U/mg) and RS-ALAS (116.9 U/mg), respectively. The optimum pH values and temperatures of the three above mentioned enzymes were all pH 7.5 and 37 °C, respectively. Moreover, RC-ALAS was more sensitive to pH, while the other two were sensitive to temperature. The effects of metals, ethylene diamine tetraacetic acid (EDTA), and sodium dodecyl sulfate (SDS) on the three ALASs were also investigated. The results indicate that they had the same effects on the activities of the three ALASs. SDS and metal ions such as Co(2+), Zn(2+), and Cu(2+) strongly inhibited the activities of the ALASs, while Mn(2+) exerted slight inhibition, and K(+), Ca(2+), Ba(2+), Mg(2+), or EDTA had no significant effect. The specificity constant of succinyl coenzyme A [(kcat/Km)(S-CoA)] of RC-ALAS was 1.4989, which was higher than those of AR-ALAS (0.7456) and RS-ALAS (1.1699), showing its high catalytic efficiency. The fed-batch fermentation was conducted using the recombinant strain containing the R. capsulatus hemA gene, and the yield of 5-aminolevulinic acid (ALA) achieved was 8.8 g/L (67 mmol/L) under the appropriate conditions.

  7. The Conserved Dcw Gene Cluster of R. sphaeroides Is Preceded by an Uncommonly Extended 5’ Leader Featuring the sRNA UpsM

    PubMed Central

    Weber, Lennart; Thoelken, Clemens; Volk, Marcel; Remes, Bernhard; Lechner, Marcus; Klug, Gabriele

    2016-01-01

    Cell division and cell wall synthesis mechanisms are similarly conserved among bacteria. Consequently some bacterial species have comparable sets of genes organized in the dcw (division and cell wall) gene cluster. Dcw genes, their regulation and their relative order within the cluster are outstandingly conserved among rod shaped and gram negative bacteria to ensure an efficient coordination of growth and division. A well studied representative is the dcw gene cluster of E. coli. The first promoter of the gene cluster (mraZ1p) gives rise to polycistronic transcripts containing a 38 nt long 5’ UTR followed by the first gene mraZ. Despite reported conservation we present evidence for a much longer 5’ UTR in the gram negative and rod shaped bacterium Rhodobacter sphaeroides and in the family of Rhodobacteraceae. This extended 268 nt long 5’ UTR comprises a Rho independent terminator, which in case of termination gives rise to a non-coding RNA (UpsM). This sRNA is conditionally cleaved by RNase E under stress conditions in an Hfq- and very likely target mRNA-dependent manner, implying its function in trans. These results raise the question for the regulatory function of this extended 5’ UTR. It might represent the rarely described case of a trans acting sRNA derived from a riboswitch with exclusive presence in the family of Rhodobacteraceae. PMID:27802301

  8. The Conserved Dcw Gene Cluster of R. sphaeroides Is Preceded by an Uncommonly Extended 5' Leader Featuring the sRNA UpsM.

    PubMed

    Weber, Lennart; Thoelken, Clemens; Volk, Marcel; Remes, Bernhard; Lechner, Marcus; Klug, Gabriele

    2016-01-01

    Cell division and cell wall synthesis mechanisms are similarly conserved among bacteria. Consequently some bacterial species have comparable sets of genes organized in the dcw (division and cell wall) gene cluster. Dcw genes, their regulation and their relative order within the cluster are outstandingly conserved among rod shaped and gram negative bacteria to ensure an efficient coordination of growth and division. A well studied representative is the dcw gene cluster of E. coli. The first promoter of the gene cluster (mraZ1p) gives rise to polycistronic transcripts containing a 38 nt long 5' UTR followed by the first gene mraZ. Despite reported conservation we present evidence for a much longer 5' UTR in the gram negative and rod shaped bacterium Rhodobacter sphaeroides and in the family of Rhodobacteraceae. This extended 268 nt long 5' UTR comprises a Rho independent terminator, which in case of termination gives rise to a non-coding RNA (UpsM). This sRNA is conditionally cleaved by RNase E under stress conditions in an Hfq- and very likely target mRNA-dependent manner, implying its function in trans. These results raise the question for the regulatory function of this extended 5' UTR. It might represent the rarely described case of a trans acting sRNA derived from a riboswitch with exclusive presence in the family of Rhodobacteraceae.

  9. Complete Genome Sequences of Five Bacteriophages That Infect Rhodobacter capsulatus.

    PubMed

    Bollivar, David W; Bernardoni, Brooke; Bockman, Matthew R; Miller, Brenda M; Russell, Daniel A; Delesalle, Veronique A; Krukonis, Gregory P; Hatfull, Graham F; Cross, Madeline R; Szewczyk, Marlena M; Eppurath, Atul

    2016-05-26

    Five bacteriophages that infect the Rhodobacter capsulatus strain YW1 were isolated from stream water near Bloomington, Illinois, USA. Two distinct genome types are represented in the newly isolated bacteriophages. These genomes are different from other bacteriophage genomes previously described. Copyright © 2016 Bollivar et al.

  10. Soluble variants of Rhodobacter capsulatus membrane-anchored cytochrome cy are efficient photosynthetic electron carriers.

    PubMed

    Oztürk, Yavuz; Lee, Dong-Woo; Mandaci, Sevnur; Osyczka, Artur; Prince, Roger C; Daldal, Fevzi

    2008-05-16

    Photosynthetic (Ps) electron transport pathways often contain multiple electron carriers with overlapping functions. Here we focus on two c-type cytochromes (cyt) in facultative phototrophic bacteria of the Rhodobacter genus: the diffusible cyt c2 and the membrane-anchored cyt c(y). In species like R. capsulatus, cyt c(y) functions in both Ps and respiratory electron transport chains, whereas in other species like R. sphaeroides, it does so only in respiration. The molecular bases of this difference was investigated by producing a soluble variant of cyt c(y) (S-c(y)), by fusing genetically the cyt c2 signal sequence to the cyt c domain of cyt c(y). This novel electron carrier was unable to support the Ps growth of R. capsulatus. However, strains harboring cyt S-c(y) regained Ps growth ability by acquiring mutations in its cyt c domain. They produced cyt S-c(y) variants at amounts comparable with that of cyt c2, and conferred Ps growth. Chemical titration indicated that the redox midpoint potential of cyt S-c(y) was about 340 mV, similar to that of cyts c2 or c(y). Remarkably, electron transfer kinetics from the cyt bc1 complex to the photochemical reaction center (RC) mediated by cyt S-c(y) was distinct from those seen with the cyt c2 or cyt c(y). The kinetics exhibited a pronounced slow phase, suggesting that cyt S-c(y) interacted with the RC less tightly than cyt c2. Comparison of structural models of cyts c2 and S-c(y) revealed that several of the amino acid residues implicated in long-range electrostatic interactions promoting binding of cyt c2 to the RC are not conserved in cyt c(y), whereas those supporting short-range hydrophobic interactions are conserved. These findings indicated that attaching electron carrier cytochromes to the membrane allowed them to weaken their interactions with their partners so that they could accommodate more rapid multiple turnovers.

  11. Triazine herbicide resistance in the photosynthetic bacterium Rhodopseudomonas sphaeroides

    PubMed Central

    Brown, Alfred E.; Gilbert, Carl W.; Guy, Rachel; Arntzen, Charles J.

    1984-01-01

    The photoaffinity herbicide azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-s-triazine) selectively labels the L subunit of the reaction center of the photosynthetic bacterium Rhodopseudomonas sphaeroides. Herbicide-resistant mutants retain the L subunit and have altered binding properties for methylthio- and chloro-substituted triazines as well as altered equilibrium constants for electron transfer between primary and secondary electron acceptors. We suggest that a subtle alteration in the L subunit is responsible for herbicide resistance and that the L subunit is the functional analog of the 32-kDa QB protein of chloroplast membranes. Images PMID:16593520

  12. Triazine herbicide resistance in the photosynthetic bacterium Rhodopseudomonas sphaeroides

    SciTech Connect

    Brown, A.E.; Gilbert, C.W.; Guy, R.; Arntzen, C.J.

    1984-10-01

    The photoaffinity herbicide azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-s-triazine) selectively labels the L subunit of the reaction center of the photosynthetic bacterium Rhodopseudomonas sphaeroides. Herbicide-resistant mutants retain the L subunit and have altered binding properties for methylthio- and chloro-substituted triazines as well as altered equilibrium constants for electron transfer between primary and secondary electron acceptors. We suggest that a subtle alteration in the L subunit is responsible for herbicide resistance and that the L subunit is the functional analog of the 32-kDa Q/sub B/ protein of chloroplast membranes. 42 references, 6 figures, 1 table.

  13. An mRNA degrading complex in Rhodobacter capsulatus

    PubMed Central

    Jäger, Stephanie; Fuhrmann, Oliver; Heck, Claudia; Hebermehl, Markus; Schiltz, Emile; Rauhut, Reinhard; Klug, Gabriele

    2001-01-01

    An RNA degrading, high molecular weight complex was purified from Rhodobacter capsulatus. N-terminal sequencing, glycerol-gradient centrifugation, and immunoaffinity purification as well as functional assays were used to determine the physical and biochemical characteristics of the complex. The complex comprises RNase E and two DEAD-box RNA helicases of 74 and 65 kDa, respectively. Most surprisingly, the transcription termination factor Rho is a major, firmly associated component of the degradosome. PMID:11713307

  14. Clustering of genes necessary for hydrogen oxidation in Rhodobacter capsulatus.

    PubMed Central

    Xu, H W; Wall, J D

    1991-01-01

    Three cosmids previously shown to contain information necessary for the expression of uptake of hydrogenase in Rhodobacter capsulatus were found to be present in a cluster on the chromosome. Earlier genetic experiments suggested the presence of at least six genes essential for hydrogenase activity that are now shown to be in a region of approximately 18 kb that includes the structural genes for the enzyme. A potential response regulator gene was sequenced as a part of the hup gene region. PMID:2007559

  15. Cloning of the Rhodobacter capsulatus hemA gene.

    PubMed Central

    Biel, S W; Wright, M S; Biel, A J

    1988-01-01

    Portions of the Rhodobacter capsulatus hemA gene have been cloned from a hemA::Tn5 insertion strain into the lambda bacteriophage derivative EMBL3. A cosmid containing the wild-type R. capsulatus hemA gene was isolated by complementation of the hemA::Tn5 mutant. The cosmid contains a 1.4-kilobase EcoRI fragment that spans the hemA::Tn5 insertion site. The entire hemA gene is contained in this fragment and the adjacent 0.6-kilobase EcoRI fragment. Images PMID:2842318

  16. Purification and characterization of the cytochrome c oxidase from Rhodopseudomonas sphaeroides.

    PubMed

    Gennis, R B; Casey, R P; Azzi, A; Ludwig, B

    1982-06-15

    When grown aerobically in the dark, Rhodopseudomonas sphaeroides develops a respiratory chain similar to that in mitochondria and the photosynthetic apparatus is suppressed. The aa3-type cytochrome c oxidase from Rps. sphaeroides has been purified in Triton X-100 by affinity chromatography with Sepharose 4B coupled to yeast cytochrome c. The oxidase contains 14 nmol heme a/mg protein and is composed of three polypeptide subunits with relative molecular masses of 45000, 37000 and 35000. The enzyme is highly active in the presence of detergents, with a maximal velocity of 300 s-1/mol oxidase using either yeast or horse-heart cytochrome c. The Rps. sphaeroides oxidase is cross-reactive with antibodies directed against the oxidases from Paracoccus denitrificans and Saccharomyces cerevisiae. A particularly close relationship is indicated in the case of P. denitrificans. The Rps. sphaeroides oxidase has been incorporated into phospholipid vesicles. The resulting oxidase in these vesicles demonstrates high enzymatic activity and a respiratory control ratio of 5. Using these vesicles, no evidence for proton extrusion accompanying cytochrome c oxidation was observed. The data suggest that the Rps. sphaeroides oxidase does not function as a proton pump.

  17. Identification of a histidine-tyrosine cross-link in the active site of the cbb3-type cytochrome c oxidase from Rhodobacter sphaeroides.

    PubMed

    Rauhamäki, Virve; Baumann, Marc; Soliymani, Rabah; Puustinen, Anne; Wikström, Mårten

    2006-10-31

    The heme-copper oxidases constitute a superfamily of terminal dioxygen-reducing enzymes located in the inner mitochondrial or in the bacterial cell membrane. The presence of a mechanistically important covalent bond between a histidine ligand of the copper ion (Cu(B)) in the active site and a generally conserved tyrosine residue nearby has been shown to exist in the canonical cytochrome c oxidases. However, according to sequence alignment studies, this critical tyrosine is missing from the subfamily of cbb(3)-type oxidases found in certain bacteria. Recently, homology modeling has suggested that a tyrosine residue located in a different helix might fulfill this role in these enzymes. Here, we show directly by methods of protein chemistry and mass spectrometry that there is indeed a covalent link between this tyrosine and the copper-ligating histidine. The identity of the cross-linked tyrosine was determined by showing that the cross-link is not formed when this residue is replaced by phenylalanine, even though structural integrity is maintained. These results suggest a universal functional importance of the histidine-tyrosine cross-link in the mechanism of O(2) reduction by all heme-copper oxidases.

  18. Hydrogen production from starch by co-culture of Clostridium acetobutylicum and Rhodobacter sphaeroides in one step hybrid dark- and photofermentation in repeated fed-batch reactor.

    PubMed

    Zagrodnik, R; Łaniecki, M

    2017-01-01

    Hydrogen production from starch by a co-culture hybrid dark and photofermentation under repeated fed-batch conditions at different organic loading rates (OLR) was studied. Effective cooperation between bacteria in co-culture during initial days was observed at controlled pH 7.0. However, at pH above 6.5 dark fermentation phase was redirected from H2 formation towards production of formic acid, lactic acid and ethanol (which are not coupled with hydrogen production) with simultaneous lower starch removal efficiency. This resulted in decrease in the hydrogen production rate. The highest H2 production in co-culture process (3.23LH2/Lmedium - after 11days) was achieved at OLR of 1.5gstarch/L/day, and it was twofold higher than for dark fermentation process (1.59LH2/Lmedium). The highest H2 yield in the co-culture (2.62molH2/molhexose) was obtained at the OLR of 0.375gstarch/L/day. Different pH requirements of bacteria were proven to be a key limitation in co-culture system.

  19. Comparison of transient grating signals from spheroidene in an organic solvent and in pigment-protein complexes from Rhodobacter sphaeroides 2.4.1

    NASA Astrophysics Data System (ADS)

    Sugisaki, Mitsuru; Fujiwara, Masazumi; Kosumi, Daisuke; Fujii, Ritsuko; Nango, Mamoru; Cogdell, Richard J.; Hashimoto, Hideki

    2010-06-01

    The concurrent dynamics of the electronic excitation and vibronic oscillations of spheroidene have been investigated by means of the transient grating (TG) spectroscopy. The third-order optical responses of spheroidene in an organic solvent, in the LH2 light-harvesting antenna complexes, and in chromatophores have been compared in order to investigate the influence of the environment surrounding this photosynthetic pigment. Vibronic coherent oscillations with a period of several tens of femtosecond have been clearly observed superimposed on a slowly varying background, which reflects the electronic dynamics. The dynamics of the coherent oscillations have been analyzed by means of the wavelet analysis. Within our experimental accuracy, the decay times of the CC and CC stretching modes and CCH3 rocking mode of each specimen are very close. The experimental results have also been analyzed using a Brownian oscillator model. For these numerical calculations, the spectral density for the underdamped modes has been determined from the Raman spectrum of spheroidene. It was found that the low-frequency modes that reflect the influence of the protein environment can be approximated by the overdamped Brownian oscillator. The experimentally observed linear absorption spectra as well as the third-order optical responses, i.e., TG curves, are reproduced very well by these calculations. The close agreement between the experiments and calculations indicates that the Feynman-diagrammatic approach can be applied to express not only the internal conversion but also the intermolecular excitation energy-transfer processes. The vibronic decay rates of spheroidene in LH2 complexes and chromatophores are evaluated to be about 20% larger than in the organic solvent.

  20. Spectroscopic Studies of the AppA BLUF Domain from Rhodobacter sphaeroides: Addressing Movement of Tryptophan 104 in the Signaling State†

    PubMed Central

    Dragnea, Vladimira; Arunkumar, Alphonse I.; Yuan, Hua; Giedroc, David P.; Bauer, Carl E.

    2009-01-01

    Previous crystallographic studies of the AppA BLUF domain indicated that Trp104 is capable of undertaking alternate conformations depending on the length of the BLUF domain. A BLUF domain containing a C-terminal deletion (AppA1–126) reveals that Trp104 is partially solvent exposed while a BLUF domain containing a slightly longer carboxyl terminal region (AppA17–133) shows that Trp104 is deeply buried. This observation has led to a model proposing that Trp104 moves from a deeply buried position in the dark state to a solvent-exposed position in the light excited state. In this study we investigated whether there is indeed movement of Trp104 upon light excitation using a combination of NMR and absorption spectroscopy, steady-state fluorescence, and acrylamide quenching of tryptophan fluorescence. Our results indicate that AppA17–133 and AppA1–126 contain Trp104 in distinct alternate conformations in solution and that light absorption by the flavin causes partial movement/uncovering of Trp104. However, we conclude that light exposure does not cause dramatic change of Trp104 from “Trp-in” to “Trp-out” conformations (or vice versa) upon light absorption. These results do not support a model of Trp104 movement as a key output signal. PMID:19746968

  1. Polyhydroxyalkanoate production in Rhodobacter capsulatus: genes, mutants, expression, and physiology.

    PubMed Central

    Kranz, R G; Gabbert, K K; Locke, T A; Madigan, M T

    1997-01-01

    Like many other prokaryotes, the photosynthetic bacterium Rhodobacter capsulatus produces high levels of polyhydroxyalkanoates (PHAs) when a suitable carbon source is available. The three genes that are traditionally considered to be necessary in the PHA biosynthetic pathway, phaA (beta-ketothiolase), phaB (acetoacetylcoenzyme A reductase), and phaC (PHA synthase), were cloned from Rhodobacter capsulatus. In R. capsulatus, the phaAB genes are not linked to the phaC gene. Translational beta-galactosidase fusions to phaA and phaC were constructed and recombined into the chromosome. Both phaC and phaA were constitutively expressed regardless of whether PHA production was induced, suggesting that control is posttranslational at the enzymatic level. Consistent with this conclusion, it was shown that the R. capsulatus transcriptional nitrogen-sensing circuits were not involved in PHA synthesis. The doubling times of R. capsulatus transcriptional nitrogen-sensing circuits were not involved in PHA synthesis. The doubling times of R. capsulatus grown on numerous carbon sources were determined, indicating that this bacterium grows on C2 to C12 fatty acids. Grown on acetone, caproate, or heptanoate, wild-type R. capsulatus produced high levels of PHAs. Although a phaC deletion strain was unable to synthesize PHAs on any carbon source, phaA and phaAB deletion strains were able to produce PHAs, indicating that alternative routes for the synthesis of substrates for the synthase are present. The nutritional versatility and bioenergetic versatility of R. capsulatus, coupled with its ability to produce large amounts of PHAs and its genetic tractability, make it an attractive model for the study of PHA production. PMID:9251189

  2. Role of proton motive force in phototactic and aerotactic responses of Rhodopseudomonas sphaeroides

    SciTech Connect

    Armitage, J.P.; Ingham, C.; Evans, M.C.W.

    1985-03-01

    Rhodopseudomonas sphaeroides grown under nonrigorous anaerobic conditions in the light developed components of a branched respiratory electron transfer chain, and a photosynthetic electron transfer chain. Both respiratory pathways were sensitive to rotenone and high concentrations of cyanide, but oxygen uptake was only partially inhibited by the addition of low concentrations of cyanide or antimycin A. When incubated anaerobically in the dark, R. sphaeroides responded positively to an oxygen gradient in the absence of rotenone. In the presence of rotenone, aerotaxis only occurred when the antimycin A-sensitive branch of the pathway was functioning, although both branches still reduced oxygen. Although there was electron movement along the respiratory chain, aerotaxis only occurred in response to a change in proton motive force. When incubated anaerobically in the light, the movement of R. sphaeroides up to a light gradient depended on photosynthetic electron transport. When incubated aerobically, high-intensity actinic illumination inhibited oxygen uptake and aerotaxis. In a low-intensity light gradient the phototactic response was inhibited by oxygen. These results are discussed in relation to the interaction of the electron transfer chains and their roles in controlling tactic responses in R. sphaeroides.

  3. Photomixotrophic growth of Rhodobacter capsulatus SB1003 on ferrous iron.

    PubMed

    Kopf, S H; Newman, D K

    2012-05-01

    This study investigates the role iron oxidation plays in the purple non-sulfur bacterium Rhodobacter capsulatus SB1003. This organism is unable to grow photoautotrophically on unchelated ferrous iron [Fe(II)] despite its ability to oxidize chelated Fe(II). This apparent paradox was partly resolved by the discovery that SB1003 can grow photoheterotrophically on the photochemical breakdown products of certain ferric iron-ligand complexes, yet whether it could concomitantly benefit from the oxidation of Fe(II) to fix CO(2) was unknown. Here, we examine carbon fixation by stable isotope labeling of the inorganic carbon pool in cultures growing phototrophically on acetate with and without Fe(II). We show that R. capsulatus SB1003, an organism formally thought incapable of phototrophic growth on Fe(II), can actually harness the reducing power of this substrate and grow photomixotrophically, deriving carbon both from organic sources and from fixation of inorganic carbon. This suggests the possibility of a wider occurrence of photoferrotrophy than previously assumed. © 2011 Blackwell Publishing Ltd.

  4. ADP-ribosylation of dinitrogenase reductase in Rhodobacter capsulatus

    SciTech Connect

    Jouanneau, Y.; Roby, C.; Meyer, C.M.; Vignais, P.M. )

    1989-07-25

    In the photosynthetic bacterium Rhodobacter capsulatus, nitrogenase is regulated by a reversible covalent modification of Fe protein or dinitrogenase reductase (Rc2). The linkage of the modifying group to inactive Rc2 was found to be sensitive to alkali and to neutral hydroxylamine. Complete release of the modifying group was achieved by incubation of inactive Rc2 in 0.4 or 1 M hydroxylamine. After hydroxylamine treatment of the Rc2 preparation, the modifying group could be isolated and purified by affinity chromatography and ion-exchange HPLC. The modifying group comigrated with ADP-ribose on both ion-exchange HPLC and thin-layer chromatography. Analyses by {sup 31}P NMR spectroscopy and mass spectrometry provided further evidence that the modifying group was ADP-ribose. The NMR spectrum of inactive Rc2 exhibited signals characteristic of ADP-ribose; integration of these signals allowed calculation of a molar ration ADP-ribose/Rc2 of 0.63. A hexapeptide carrying the ADP-ribose moiety was purified from a subtilisin digest of inactive Rc2. The structure of this peptide, determined by amino acid analysis and sequencing, is Gly-Arg(ADP-ribose)-Gly-Val-Ile-Thr. This structure allows identification of the binding site for ADP-ribose as Arg 101 of the polypeptide chain of Rc2. It is concluded that nitrogenase activity in R. capsulatus is regulated by reversible ADP-ribosylation of a specific arginyl residue of dinitrogenase reductase.

  5. Expression of regulatory nif genes in Rhodobacter capsulatus.

    PubMed Central

    Hübner, P; Willison, J C; Vignais, P M; Bickle, T A

    1991-01-01

    Translational fusions of the Escherichia coli lacZ gene to Rhodobacter capsulatus nif genes were constructed in order to determine the regulatory circuit of nif gene expression in R. capsulatus, a free-living photosynthetic diazotroph. The expression of nifH, nifA (copies I and II), and nifR4 was measured in different regulatory mutant strains under different physiological conditions. The expression of nifH and nifR4 (the analog of ntrA in Klebsiella pneumoniae) depends on the NIFR1/R2 system (the analog of the ntr system in K. pneumoniae), on NIFA, and on NIFR4. The expression of both copies of nifA is regulated by the NIFR1/R2 system and is modulated by the N source of the medium under anaerobic photosynthetic growth conditions. In the presence of ammonia or oxygen, moderate expression of nifA was detectable, whereas nifH and nifR4 were not expressed under these conditions. The implications for the regulatory circuit of nif gene expression in R. capsulatus are discussed and compared with the situation in K. pneumoniae, another free-living diazotroph. PMID:1902215

  6. Culturable Rhodobacter and Shewanella species are abundant in estuarine turbidity maxima of the Columbia River.

    PubMed

    Bräuer, S L; Adams, C; Kranzler, K; Murphy, D; Xu, M; Zuber, P; Simon, H M; Baptista, A M; Tebo, B M

    2011-03-01

    Measurements of dissolved, ascorbate-reducible and total Mn by ICP-OES revealed significantly higher concentrations during estuarine turbidity maxima (ETM) events, compared with non-events in the Columbia River. Most probable number (MPN) counts of Mn-oxidizing or Mn-reducing heterotrophs were not statistically different from that of other heterotrophs (10³ -10⁴ cells ml⁻¹) when grown in defined media, but counts of Mn oxidizers were significantly lower in nutrient-rich medium (13 cells ml⁻¹). MPN counts of Mn oxidizers were also significantly lower on Mn(III)-pyrophosphate and glycerol (21 cells ml⁻¹). Large numbers of Rhodobacter spp. were cultured from dilutions of 10⁻² to 10⁻⁵, and many of these were capable of Mn(III) oxidation. Up to c. 30% of the colonies tested LBB positive, and all 77 of the successfully sequenced LBB positive colonies (of varying morphology) yielded sequences related to Rhodobacter spp. qPCR indicated that a cluster of Rhodobacter isolates and closely related strains (95-99% identity) represented approximately 1-3% of the total Bacteria, consistent with clone library results. Copy numbers of SSU rRNA genes for either Rhodobacter spp. or Bacteria were four to eightfold greater during ETM events compared with non-events. Strains of a Shewanella sp. were retrieved from the highest dilutions (10⁻⁵) of Mn reducers, and were also capable of Mn oxidation. The SSU rRNA gene sequences from these strains shared a high identity score (98%) with sequences obtained in clone libraries. Our results support previous findings that ETMs are zones with high microbial activity. Results indicated that Shewanella and Rhodobacter species were present in environmentally relevant concentrations, and further demonstrated that a large proportion of culturable bacteria, including Shewanella and Rhodobacter spp., were capable of Mn cycling in vitro.

  7. The Single Superoxide Dismutase of Rhodobacter capsulatus Is a Cambialistic, Manganese-Containing Enzyme

    PubMed Central

    Tabares, Leandro C.; Bittel, Cristian; Carrillo, Néstor; Bortolotti, Ana; Cortez, Néstor

    2003-01-01

    The phototrophic bacterium Rhodobacter capsulatus contains a single, oxygen-responsive superoxide dismutase (SODRc) homologous to iron-containing superoxide dismutase enzymes. Recombinant SODRc, however, displayed higher activity after refolding with Mn2+, especially when the pH of the assay mixture was raised. SODRc isolated from Rhodobacter cells also preferentially contains manganese, but metal discrimination depends on the culture conditions, with iron fractions increasing from 7% in aerobic cultures up to 40% in photosynthetic cultures. Therefore, SODRc behaves as a Mn-containing dismutase with cambialistic properties. PMID:12730184

  8. Nostoc sphaeroides Kützing, an excellent candidate producer for CELSS

    NASA Astrophysics Data System (ADS)

    Hao, Zongjie; Li, Dunhai; Li, Yanhui; Wang, Zhicong; Xiao, Yuan; Wang, Gaohong; Liu, Yongding; Hu, Chunxiang; Liu, Qifang

    2011-11-01

    Some phytoplankton can be regarded as possible candidates in the establishment of Controlled Ecological Life Support System (CELSS) for some intrinsic characteristics, the first characteristic is that they should grow rapidly, secondly, they should be able to endure some stress factors and develop some corresponding adaptive strategies; also it is very important that they could provide food rich in nutritious protein and vitamins for the crew; the last but not the least is they can also fulfill the other main functions of CELSS, including supplying oxygen, removing carbon dioxide and recycling the metabolic waste. According to these characteristics, Nostoc sphaeroides, a potential healthy food in China, was selected as the potential producer in CELSS. It was found that the oxygen average evolution rate of this algae is about 150 μmol O 2 mg -1 h -1, and the size of them are ranged from 2 to 20 mm. Also it can be cultured with high population density, which indicated that the potential productivity of Nostoc sphaeroides is higher than other algae in limited volume. We measured the nutrient contents of the cyanobacterium and concluded it was a good food for the crew. Based on above advantages, Nostoc sphaeroides was assumed to a suitable phytoplankton for the establishment of Controlled Ecological Life Support System. We plan to develop suitable bioreactor with the cyanobacterium for supplying oxygen and food in future space missions.

  9. Analysis of the biosynthetic pathway for sulfoquinovosyldiacylglycerol in the purple bacterium R. sphaeroides

    SciTech Connect

    Benning, C.; Somerville, C.R. )

    1990-05-01

    The membrane lipid sulfoquinovosyldiacylglycerol (SQD) can be found in all photosynthetically active membranes studied. In a green leaf, about 50% of the organic sulfur is bound in SQD. Therefore, this sulfolipid constitutes a major component of the global sulfur cycle. However, since the discovery of SQD, very little progress has been made towards the elucidation of the biosynthetic pathway. For a genetical analysis of the pathway of SQD, we selected the photosynthetic purple-nonsulfur bacterium R. sphaeroides, as our model system. We have been able to isolate several classes of mutants, which show reduced levels of SQD. Some of the mutants accumulate {sup 35}S-labeled, water soluble compounds. We expect that these compounds are related to precursors or are precursors, which accumulate due to a specific block in the biosynthetic pathway of SQD. To study the possible precursor function of these compounds, we developed an in vitro SQD biosynthesis system using cell free extracts from R. sphaeroides. In addition, we have been able to isolate cosmids prepared from WT DNA, which complement three classes of SQD mutants in R. sphaeroides. The analysis and expression of the genes encoded by these cosmids should facilitate the characterization of the proteins involved in SQD biosynthesis.

  10. Rhodobacter ovatus sp. nov., a phototrophic alphaproteobacterium isolated from a polluted pond.

    PubMed

    Srinivas, T N R; Anil Kumar, P; Sasikala, Ch; Spröer, C; Ramana, Ch V

    2008-06-01

    A novel ovoid, phototrophic, purple non-sulfur bacterium was isolated in pure culture from a sample of sediment from an industrially polluted pond (Noor Mohammad Kunta) in Hyderabad, India. Strain JA234(T) was found to be Gram-negative and non-motile and grew photoheterotrophically with a number of organic compounds serving as carbon source/electron donor. Photo-organoheterotrophic and chemo-organoheterotrophic growth were demonstrated. Biotin and thiamine were required for growth of strain JA234(T). Phylogenetic analysis on the basis of 16S rRNA gene sequences showed that strain JA234(T) is most closely related to Rhodobacter azotoformans (97.3 % sequence similarity) and clusters with other species of the genus Rhodobacter of the family Rhodobacteraceae. However, DNA-DNA hybridization with Rba. azotoformans JCM 9340(T) showed a relatedness of only 31.3 % with respect to strain JA234(T). On the basis of 16S rRNA gene sequence analysis, DNA-DNA hybridization data and morphological and physiological characteristics, strain JA234(T) represents a novel species of the genus Rhodobacter, for which the name Rhodobacter ovatus sp. nov. is proposed. The type strain is JA234(T) (=JCM 14779(T)=CCUG 55049(T)).

  11. Sequence of the indoleglycerol phosphate synthase (trpC) gene from Rhodobacter capsulatus.

    PubMed Central

    Becker-Rudzik, M; Young, D A; Marrs, B L

    1992-01-01

    We have isolated, cloned, and sequenced the indoleglycerol phosphate synthase gene (trpC) from Rhodobacter capsulatus. Normalized alignment scores comparing the trpC gene of R. capsulatus with the trpC genes of other bacterial species are reported. An unexpected degree of similarity to the trpC gene of Bacillus subtilis was found. PMID:1644778

  12. Effects of light intensity and quality on phycobiliprotein accumulation in the cyanobacterium Nostoc sphaeroides Kützing.

    PubMed

    Ma, Rui; Lu, Fan; Bi, Yonghong; Hu, Zhengyu

    2015-08-01

    To assess the effects of light intensity and quality on the growth and phycobiliproteins (PBP) accumulation in Nostoc sphaeroides Kützing (N. sphaeroides). Dry weights, dry matter, protein, chlorophyll and PBP contents were higher under 90 μmol m(-2) s(-1) than under other intensities (both higher and lower). Phycocyanin and allophycocyanin increased with light intensity while phycoerythrin decreased. Fresh weights, protein and PBP contents increased at the highest rates under blue light. Red light resulted in higher values of dry matter, phycocyanin and chlorophyll a. White light at 90 μmol m(-2) s(-1) or blue light 30 μmol m(-2) s(-1) were optimal for the growth and phycobiliprotein accumulation in N. sphaeroides.

  13. Physiological control and regulation of the Rhodobacter capsulatus cbb operons.

    PubMed

    Paoli, G C; Vichivanives, P; Tabita, F R

    1998-08-01

    The genes encoding enzymes of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway in Rhodobacter capsulatus are organized in at least two operons, each preceded by a separate cbbR gene, encoding potential LysR-type transcriptional activators. As a prelude to studies of cbb gene regulation in R. capsulatus, the nucleotide sequence of a 4,537-bp region, which included cbbRII, was determined. This region contained the following open reading frames: a partial pgm gene (encoding phosphoglucomutase) and a complete qor gene (encoding NADPH:quinone oxidoreductase), followed by cbbRII, cbbF (encoding fructose 1,6-bisphosphatase), cbbP (encoding phosphoribulokinase), and part of cbbT (encoding transketolase). Physiological control of the CBB pathway and regulation of the R. capsulatus cbb genes were studied by using a combination of mutant strains and promoter fusion constructs. Characterization of mutant strains revealed that either form I or form II ribulose 1, 5-bisphosphate carboxylase/oxygenase (RubisCO), encoded by the cbbLS and cbbM genes, respectively, could support photoheterotrophic and autotrophic growth. A strain with disruptions in both cbbL and cbbM could not grow autotrophically and grew photoheterotrophically only when dimethyl sulfoxide was added to the culture medium. Disruption of cbbP resulted in a strain that did not synthesize form II RubisCO and had a phenotype similar to that observed in the RubisCO-minus strain, suggesting that there is only one cbbP gene in R. capsulatus and that this gene is cotranscribed with cbbM. Analysis of RubisCO activity and synthesis in strains with disruptions in either cbbRI or cbbRII, and beta-galactosidase determinations from wild-type and mutant strains containing cbbIp- and cbbIIp-lacZ fusion constructs, indicated that the cbbI and cbbII operons of R. capsulatus are within separate CbbR regulons.

  14. Embryonic, larval, and early juvenile development of the tropical sea urchin, Salmacis sphaeroides (Echinodermata: Echinoidea).

    PubMed

    Rahman, M Aminur; Yusoff, Fatimah Md; Arshad, A; Shamsudin, Mariana Nor; Amin, S M N

    2012-01-01

    Salmacis sphaeroides (Linnaeus, 1758) is one of the regular echinoids, occuring in the warm Indo-West Pacific, including Johor Straits, between Malaysia and Singapore. In order to investigate the developmental basis of morphological changes in embryos and larvae, we documented the ontogeny of S. sphaeroides in laboratory condition. Gametes were obtained from adult individuals by 0.5 M KCl injection into the coelomic cavity. Fertilization rate at limited sperm concentration (10(-5) dilution) was 96.6 ± 1.4% and the resulting embryos were reared at 24°C. First cleavage (2-cell), 4-cell, 8-cell, 16-cell, 32-cell, and multicell (Morulla) stages were achieved 01.12, 02.03, 02.28, 02.51, 03.12, and 03.32 h postfertilization. Ciliated blastulae with a mean length of 174.72 ± 4.43 μm hatched 08.45 h after sperm entry. The gastrulae formed 16.15 h postfertilization and the archenteron elongated constantly while ectodermal red-pigmented cells migrated synchronously to the apical plate. Pluteus larva started to feed unicellular algae in 2 d, grew continuously, and finally attained metamorphic competence in 35 d after fertilization. Metamorphosis took approximately 1 h 30 min from attachment to the complete resorption of larval tissues and the development of complete juvenile structure with adult spines, extended tubefeet and well-developed pedicellaria, the whole event of which usually took place within 1 d postsettlement. This study represents the first successful investigation on embryonic, larval, and early juvenile development of S. sphaeroides. The findings would greatly be helpful towards the understanding of ontogeny and life-history strategies, which will facilitate us to develop the breeding, seed production, and culture techniques of sea urchins in captive condition.

  15. Embryonic, Larval, and Early Juvenile Development of the Tropical Sea Urchin, Salmacis sphaeroides (Echinodermata: Echinoidea)

    PubMed Central

    Rahman, M. Aminur; Yusoff, Fatimah Md.; Arshad, A.; Shamsudin, Mariana Nor; Amin, S. M. N.

    2012-01-01

    Salmacis sphaeroides (Linnaeus, 1758) is one of the regular echinoids, occuring in the warm Indo-West Pacific, including Johor Straits, between Malaysia and Singapore. In order to investigate the developmental basis of morphological changes in embryos and larvae, we documented the ontogeny of S. sphaeroides in laboratory condition. Gametes were obtained from adult individuals by 0.5 M KCl injection into the coelomic cavity. Fertilization rate at limited sperm concentration (10−5 dilution) was 96.6 ± 1.4% and the resulting embryos were reared at 24°C. First cleavage (2-cell), 4-cell, 8-cell, 16-cell, 32-cell, and multicell (Morulla) stages were achieved 01.12, 02.03, 02.28, 02.51, 03.12, and 03.32 h postfertilization. Ciliated blastulae with a mean length of 174.72 ± 4.43 μm hatched 08.45 h after sperm entry. The gastrulae formed 16.15 h postfertilization and the archenteron elongated constantly while ectodermal red-pigmented cells migrated synchronously to the apical plate. Pluteus larva started to feed unicellular algae in 2 d, grew continuously, and finally attained metamorphic competence in 35 d after fertilization. Metamorphosis took approximately 1 h 30 min from attachment to the complete resorption of larval tissues and the development of complete juvenile structure with adult spines, extended tubefeet and well-developed pedicellaria, the whole event of which usually took place within 1 d postsettlement. This study represents the first successful investigation on embryonic, larval, and early juvenile development of S. sphaeroides. The findings would greatly be helpful towards the understanding of ontogeny and life-history strategies, which will facilitate us to develop the breeding, seed production, and culture techniques of sea urchins in captive condition. PMID:23055824

  16. Evidence for the purely electronic character of primary electron transfer in purple bacteria Rh. Sphaeroides

    NASA Astrophysics Data System (ADS)

    Glebov, I. O.; Poddubnyy, V. V.; Eremin, V. V.

    2015-11-01

    A quantum-chemical calculation of the excited electronic states of a Rh. Sphaeroides reaction centre was performed. We discovered a new excited electronic state which can participate in electron transfer (ET). The energy gradient calculations showed that photoexcitation activates only high-frequency vibrational modes. This contradicts the widely accepted picture of ET resulting from vibrational wave packet motion. An alternative model is suggested where ET has a purely dissipative character and occurs only due to pigment--protein interaction. With this model, we demonstrate that oscillations in the femtosecond spectra can be caused by the new electronic state and non-Markovian character of dissipative dynamics.

  17. First Insights into the Genome Sequence of the Strictly Anaerobic Homoacetogenic Sporomusa sphaeroides Strain E (DSM 2875)

    PubMed Central

    Villamizar, Genis Andrés Castillo; Daniel, Rolf

    2017-01-01

    ABSTRACT Here, we report the draft genome sequence of Sporomusa sphaeroides strain E (DSM 2875), a strict anaerobic homoacetogenic bacterium. It is able to grow autotrophically on different one-carbon compounds. The strain possesses several genes of the Wood-Ljungdahl pathway. The genome consists of a single chromosome (4.98 Mb). PMID:28336590

  18. Kinetic analysis of N-acylphosphatidylserine accumulation and implications for membrane assembly in Rhodopseudomonas sphaeroides.

    PubMed Central

    Cain, B D; Donohue, T J; Kaplan, S

    1982-01-01

    The accumulation of N-acylphosphatidylserine (NAPS) in response to the inclusion of Tris in the growth medium of Rhodopseudomonas sphaeroides strain M29-5 has been examined. In the accompanying paper (Donohue et al., J. Bacteriol. 152:000--000, 1982), we show that in response to Tris, NAPS accumulated to as much as 40% of the total cellular phospholipid content. NAPS accumulation began immediately upon addition of Tris and was reflected as an abrupt 12-fold increase in the apparent rate of NAPS accumulation. We suggest that Tris altered the flow of metabolites through a preexisting and previously unknown metabolic pathway. NAPS accumulation ceased immediately upon the removal of Tris; however, accumulated NAPS remained largely metabolically stable. Importantly, under conditions in which NAPS was not accumulated, the intracytoplasmic membrane was shown to be virtually devoid of newly synthesized NAPS. The significance of this observation is discussed in terms of its physiological implications on phospholipid transfer and membrane biogenesis in R. sphaeroides. PMID:6982265

  19. Nostoc sphaeroides Kütz, a candidate producer par excellence for CELSS

    NASA Astrophysics Data System (ADS)

    Wang, Gaohong; Hao, Zongjie; Liu, Yongding

    A lot of aquatic organisms could be regarded as suitable candidates par excellence in the establishment of CELSS, since they are relatively easy and fast to grow and resistant to changes in environmental condition as well as providing nutritious, protein-and vitamin-rich foods for the crew, which can fulfill the main functions of CELSS, including supplying oxygen, water and food, removing carbon dioxide and making daily life waste reusable. Our labotory has developed mass culture of Nostoc sphaeroides Kütz, which is one of traditional healthy food in China and. The oxygen evolution rate of the cyanobacterium is about 150 molO2.mg-1.h-1, and it usually grows into colony with size between 2-20mm, which is easy to be harvested. It also can be cultured with high density, which show that the productivity of the cyanobacterium in limited volume is higher than other microalgae. We had measured the nutrient content of the cyanobacterium and developed some Chinese Dishes and Soups with Nostoc sphaeroides Kütz, which showed that it was a good food for crew. Using remote sensing technique, we also investigated its growth in Closed System under microgravity by SHENZHOU-2 spacecraft in January 2001. We plan to develop suitable bioreactor with the cyanobacterium for supplying oxygen and food to crew in future.

  20. The mechanisms of protection of antioxidants on Nostoc sphaeroides against UV-B radiation

    NASA Astrophysics Data System (ADS)

    Wang, G. H.

    UV radiation is one of space harmful factor for earth organisms in space exploration In the present work we studied on the role of antioxidant system in Nostoc sphaeroides K u tz Cyanobacteria and the effects of exogenous antioxidant molecules on its photosynthetic rate under UV-B radiation It was found that UV-B radiation decreased the photosynthetic activity of cyanobacterium but promoted the activity of antioxidant system to protect photosystem II PSII and exogenous antioxidant sodium nitroprusside SNP N-acetylcysteine NAC had an obvious protection on PSII activity under UV-B radiation The activity of SOD Superoxide Dismutase EC 1 15 1 1 CAT Catalase EC 1 11 1 6 POD Peroxidase EC 1 11 1 7 and content of MDA and ASC were improved by 0 5mM and 1mM SNP but 0 1mM SNP decreased the activity of antioxide system Exogenous NAC addition decreased the activity of SOD POD CAT and the content MDA and ASC but exogenous NAC addition increased the content of GSH The results suggested that exogenous SNP and NAC may protect algae by different mechanisms in which SNP maybe play double roles as sources of reactive free radicals or ROS scavengers in formation of algae s protection of PSII under UV-B radiation while NAC does function as antioxidant reagent or precursor of glutathione which could protect PSII directly from UV-B radiation Keyword antioxidant system exogenous or endogenous antioxidant Nostoc sphaeroides photosynthesis UV-B radiation

  1. Isolation of a Rhodobacter capsulatus mutant that lacks c-type cytochromes and excretes porphyrins.

    PubMed Central

    Biel, S W; Biel, A J

    1990-01-01

    A Rhodobacter capsulatus mutant lacking cytochrome oxidase activity was isolated by Tn5 mutagenesis. Difference spectroscopy of crude extracts and extracted c-type cytochromes demonstrated that this mutant completely lacked all c-type cytochromes. The strain did, however, synthesize normal amounts of b-type cytochromes and nonheme iron. This mutant also excreted large amounts of coproporphyrin and protoporphyrin and synthesized reduced amounts of bacteriochlorophyll, suggesting a link between the synthesis of c-type cytochromes and the expression of the tetrapyrrole biosynthetic pathway. Images FIG. 3 FIG. 5 PMID:2155198

  2. Isolation of a cytochrome-deficient mutant strain of Sporomusa sphaeroides not capable of oxidizing methyl groups.

    PubMed Central

    Kamlage, B; Blaut, M

    1993-01-01

    The homoacetogenic anaerobic bacterium Sporomusa sphaeroides was mutagenized with UV light. Taking advantage of the ampicillin enrichment technique and a newly developed test for the detection of heme in bacterial colonies, the cytochrome-deficient mutant strain S. sphaeroides BK824 was isolated. In contrast to the wild type, this mutant strain failed to grow on betaine, betaine plus methanol, H2 plus CO2, and methanol plus CO2. Growth on betaine plus formate, betaine plus H2, betaine plus pyruvate, methanol plus H2 and CO2, and acetoin was not impaired. All enzymes of the Wood pathway as well as hydrogenase and carbon monoxide dehydrogenase were detectable at comparable activities in both the wild type and the cytochrome-deficient mutant. Labeling experiments with [14C]methanol demonstrated the inability of S. sphaeroides BK824 to oxidize methyl groups. The role of cytochromes in electron transport steps associated with the Wood pathway enzymes and their possible role in energy conservation during autotrophic growth in acetogens are discussed. PMID:8491723

  3. Microbial photodegradation of aminoarenes. Metabolism of 2-amino-4-nitrophenol by Rhodobacter capsulatus.

    PubMed

    Witte, C P; Blasco, R; Castillo, F

    1998-03-01

    The phototrophic bacterium Rhodobacter capsulatus photoreduces 2,4-dinitrophenol to 2-amino-4-nitrophenol, which is further metabolized by an aerobic pathway that is also light-dependent. The catabolism of 2-amino-4-nitrophenol requires O2 and the presence of alternative carbon (C) and nitrogen (N) sources, preferably acetate and ammonium. Rhodobacter capsulatus B10, a bacterium unable to assimilate nitrate, releases negligible amounts of nitrite when growing with 2-amino-4-nitrophenol, thus suggesting that an oxygenase, nitrite-producing activity is not involved in the metabolization of the compound. The diazotrophic growth of R. capsulatus increases in the presence of 2-amino-4-nitrophenol, but growth with ammonium is clearly inhibited by the compound. Mutant strains of R. capsulatus B10, which are affected in nifHDK, nifR1, or nifR4 genes, unable to fix dinitrogen, do not grow with 2-amino-4-nitrophenol as the sole N source. This indicates that the compound cannot be used as a N source. The nif mutants degrade 2-amino-4-nitrophenol to the same extent as the wild-type in the presence of ammonium. The compound is not used as a C source by the bacterium, either. Aromatic stable intermediates, such as 2,4-diaminophenol or 4-nitrocatechol, are not detectable in microaerobic cultures of R. capsulatus growing with 2,4-dinitrophenol or 2-amino-4-nitrophenol.

  4. Gene transfer agent (GTA) genes reveal diverse and dynamic Roseobacter and Rhodobacter populations in the Chesapeake Bay.

    PubMed

    Zhao, Yanlin; Wang, Kui; Budinoff, Charles; Buchan, Alison; Lang, Andrew; Jiao, Nianzhi; Chen, Feng

    2009-03-01

    Within the bacterial class Alphaproteobacteria, the order Rhodobacterales contains the Roseobacter and Rhodobacter clades. Roseobacters are abundant and play important biogeochemical roles in marine environments. Roseobacter and Rhodobacter genomes contain a conserved gene transfer agent (GTA) gene cluster, and GTA-mediated gene transfer has been observed in these groups of bacteria. In this study, we investigated the genetic diversity of these two groups in Chesapeake Bay surface waters using a specific PCR primer set targeting the conserved Rhodobacterales GTA major capsid protein gene (g5). The g5 gene was successfully amplified from 26 Rhodobacterales isolates and the bay microbial communities using this primer set. Four g5 clone libraries were constructed from microbial assemblages representing different regions and seasons of the bay and yielded diverse sequences. In total, 12 distinct g5 clusters could be identified among 158 Chesapeake Bay clones, 11 fall within the Roseobacter clade, and one falls in the Rhodobacter clade. The vast majority of the clusters (10 out of 12) lack cultivated representatives. The composition of g5 sequences varied dramatically along the bay during the wintertime, and a distinct Roseobacter population composition between winter and summer was observed. The congruence between g5 and 16S rRNA gene phylogenies indicates that g5 may serve as a useful genetic marker to investigate diversity and abundance of Roseobacter and Rhodobacter in natural environments. The presence of the g5 gene in the natural populations of Roseobacter and Rhodobacter implies that genetic exchange through GTA transduction could be an important mechanism for maintaining the metabolic flexibility of these groups of bacteria.

  5. Assembly and structural organization of pigment-protein complexes in membranes of Rhodopseudomonas sphaeroides.

    PubMed

    Hunter, C N; Pennoyer, J D; Niederman, R A

    1982-01-01

    The B875 and B800-850 light-harvesting pigment-protein complexes of Rhodopseudomonas sphaeroides are characterized further by lithium dodecyl sulfate/polyacrylamide gel electrophoresis at 4 degrees C. Bacteriochlorophyll a was shown in reconstruction studies to remain complexed with its respective binding proteins during this procedure. From distributions in these gels, a quantitative description for the arrangement of the complexes is proposed. Assembly of the complexes was examined in delta-aminolevulinate-requiring mutant H-5 after a shift from high- to low-light intensity. After 10 h of delta-[3H]aminolevulinate labeling, the specific radioactivity of bacteriochlorophyll in a fraction containing putative membrane invaginations reached the maximal level, while that of the mature photosynthetic membrane was at only one-third this level. This suggests that membrane invaginations are sites of preferential bacteriochlorophyll synthesis in which completed pigment-proteins exist transiently. Analysis of the 3H distribution after electrophoretic separation further suggests that photosynthetic membranes grow mainly by addition of B800-850 to preformed membrane consisting largely of B875 and photochemical reaction centers. These results corroborate the above model for the structural organization of the light-harvesting system and indicate that the structurally and functionally discrete B800-850 pool is not completely assembled until all B875 sites for B800-850 interactions are occupied.

  6. Crystallization of a flavodoxin involved in nitrogen fixation in Rhodobacter capsulatus

    PubMed Central

    Pérez-Dorado, Inmaculada; Bortolotti, Ana; Cortez, Néstor; Hermoso, Juan A.

    2008-01-01

    Flavodoxins are small electron-transfer proteins that contain one molecule of noncovalently bound flavin mononucleotide (FMN). The flavodoxin NifF from the photosynthetic bacterium Rhodobacter capsulatus is reduced by one electron from ferredoxin/flavodoxin:NADP(H) reductase and was postulated to be an electron donor to nitrogenase in vivo. NifF was cloned and overexpressed in Escherichia coli, purified and concentrated for crystallization using the hanging-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of PEG 3350 and PEG 400 at pH 5.5 and belong to the tetragonal space group P41212, with unit-cell parameters a = b = 66.49, c = 121.32 Å. X-ray data sets have been collected to 2.17 Å resolution. PMID:18453705

  7. Identification and isolation of genes essential for H2 oxidation in Rhodobacter capsulatus.

    PubMed Central

    Xu, H W; Love, J; Borghese, R; Wall, J D

    1989-01-01

    Mutants of Rhodobacter capsulatus unable to grow photoautotrophically with H2 and CO2 were isolated. Those lacking uptake hydrogenase activity as measured by H2-dependent methylene blue reduction were analyzed genetically and used in complementation studies for the isolation of the wild-type genes. Results of further subcloning and transposon Tn5 mutagenesis suggest the involvement of a minimum of five genes. Hybridization to the 2.2-kilobase-pair SstI fragment that lies within the coding region for the large and small subunits of Bradyrhizobium japonicum uptake hydrogenase showed one region of strong homology among the R. capsulatus fragments isolated, which we interpret to mean that one or both structural genes were among the genes isolated. Images PMID:2536678

  8. An overlap between operons involved in carotenoid and bacteriochlorophyll biosynthesis in Rhodobacter capsulatus.

    PubMed

    Young, D A; Rudzik, M B; Marrs, B L

    1992-08-15

    A new example of superoperonal gene arrangement has been documented in the Rhodobacter capsulatus photosynthetic gene cluster. The promoter for the operon initiated by the bchI gene is embedded within an upstream operon for carotenoid synthesis. The stop codon for the crtA gene, the only gene in the first operon, overlaps the start codon of the downstream bchI gene. As a consequence of this overlap, the promoter(s) for the bch operon must be located within the crtA structural gene. The bchI gene is shown here for the first time to be required for the conversion of protoporphyrin IX to subsequent intermediates in bacteriochlorophyll biosynthesis.

  9. Cloning, characterization, and regulation of nifF from Rhodobacter capsulatus.

    PubMed Central

    Gennaro, G; Hübner, P; Sandmeier, U; Yakunin, A F; Hallenbeck, P C

    1996-01-01

    The Rhodobacter capsulatus nifF gene and upstream sequence were cloned by using a probe based on the N-terminal sequence of NifF. nifF was found to not be contained in the previously described nif regions I, II, and III. Comparison of the deduced amino acid sequence showed that it is highly similar to NifF from Azotobacter vinelandii and NifF from Klebsiella pneumoniae. Analysis of translational fusions demonstrated that the regulation of transcription was the same as previously reported at the protein level. Insertional mutagen esis showed that NifF contributes significantly to nitrogenase activity under normal nitrogen-fixing conditions and that it is absolutely required for nitrogen fixation under iron limitation. PMID:8682802

  10. Replacement of sugars to hydrogen production by Rhodobacter capsulatus using dark fermentation effluent as substrate.

    PubMed

    Silva, Felipe Thales Moreira; Moreira, Luiza Rojas; de Souza Ferreira, Juliana; Batista, Fabiana Regina Xavier; Cardoso, Vicelma Luiz

    2016-01-01

    Hydrogen is a promising alternative for the increased global energy demand since it has high energy density and is a clean fuel. The aim of this work was to evaluate the photo-fermentation by Rhodobacter capsulatus, using the dark fermentation effluent as substrate. Different systems were tested by changing the type of sugar in the dark fermentation, investigating the influence of supplementing DFE with sugar and adding alternate and periodically lactose and glucose throughout the process. The supplementation of the DFE with sugar resulted in higher H2 productivity and the replacement of the sugars repeatedly during the photo-fermentation process was important to maintain the cell culture active. By controlling the residual amount of sugar, bacteria inhibition was avoided; lactic acid, that was toxic to the biomass, was consumed and the metabolic route of butyric acid production was predominant. Under optimum conditions, the H2 productivity reached 208.40mmolH2/Ld in 52h.

  11. In vitro assembly of a prohead-like structure of the Rhodobacter capsulatus gene transfer agent

    SciTech Connect

    Spano, Anthony J. . E-mail: ajs6z@virginia.edu; Chen, Frank S.; Goodman, Benjamin E.; Sabat, Agnes E.; Simon, Martha N.; Wall, Joseph S.; Correia, John J.; McIvor, Wilson; Newcomb, William W.; Brown, Jay C.; Schnur, Joel M.; Lebedev, Nikolai

    2007-07-20

    The gene transfer agent (GTA) is a phage-like particle capable of exchanging double-stranded DNA fragments between cells of the photosynthetic bacterium Rhodobacter capsulatus. Here we show that the major capsid protein of GTA, expressed in E. coli, can be assembled into prohead-like structures in the presence of calcium ions in vitro. Transmission electron microscopy (TEM) of uranyl acetate staining material and thin sections of glutaraldehyde-fixed material demonstrates that these associates have spherical structures with diameters in the range of 27-35 nm. The analysis of scanning TEM images revealed particles of mass {approx} 4.3 MDa, representing 101 {+-} 11 copies of the monomeric subunit. The establishment of this simple and rapid method to form prohead-like particles permits the GTA system to be used for genome manipulation within the photosynthetic bacterium, for specific targeted drug delivery, and for the construction of biologically based distributed autonomous sensors for environmental monitoring.

  12. Mechanism of nitrogenase switch-off by oxygen. [Klebsiella pneumoniae; Rhodopseudomonas sphaeroides f. sp. denitrificans; Rhodopseudomonas capsulate

    SciTech Connect

    Goldberg, I.; Nadler, V.; Hochman, A.

    1987-02-01

    Oxygen caused a reversible inhibition (switch-off) of nitrogenase activity in whole cells of four strains of diazotrophs, the facultative anaerobe Klebsiella pneumoniae and three strains of photosynthetic bacteria (Rhodopseudomonas sphaeroides f. sp. denitrificans and Rhodopseudomonas capsulata strians AD2 and BK5). In K. pneumoniae 50% inhibition of acetylene reduction was attained at an O/sub 2/ concentration of 0.37 ..mu..M. Cyanide (90 ..mu..M), which did not affect acetylene reduction but inhibited whole-cell respiration by 60 to 70%, shifted the O/sub 2/ concentration that caused 50% inhibition of nitrogenase activity to 2.9 ..mu..M. A mutant strain of K. pneumoniae, strain AH11, has a respiration rate that is 65 to 75% higher than that of the wild type, but is nitrogenase activity is similar to wild-type activity. Acetylene reduction by whole cells of this mutant was inhibited 50% by 0.20 ..mu..M O/sub 2/. Inhibition by CN/sup -/ of 40 to 50% of the O/sub 2/ uptake in the mutant shifted the O/sub 2/ concentration that caused 50% inhibition of nitrogenase to 1.58 ..mu..M. Thus, when the respiration rates were lower, higher oxygen concentrations were required to inhibit nitrogenase. Reversible inhibition of nitrogenase activity in vivo was caused under anaerobic conditions by other electron acceptors. Addition of 2 mM sulfite to cell suspensions of R. capsulata B10 and R. sphaeroides inhibited nitrogenase activity. Nitrite also inhibited acetylene reduction in whole cells of the photodenitrifier R. sphaeroides but not in R. capsulata B10, which is not capable of enzymatic reduction of NO/sub 2//sup -/. Lower concentrations of NO/sub 2//sup -/ were required to inhibit the activity in NO/sub 3//sup -/-grown cells, which have higher activities of nitrite reductase.

  13. Growth and survival of the tropical sea urchin, Salmacis sphaeroides fed with different macroalgae in captive rearing condition.

    PubMed

    Rahman, M A; Yusoff, F M; Arshad, A; Ara, R

    2016-07-01

    An experiment was undertaken to develop appropriate culture techniques for high-valued tropical sea urchin, Salmacis sphaeroides under captive aqua-rearing conditions. Three-month-old juveniles produced through induced breeding, larval rearing and metamorphic induction were stocked with 20 juveniles in each of nine well-aerated glass aquaria (46 x 30 x 30 cm).? Juveniles fed with red alga (Amphiroa fragilissima) were designated as Treatment-1 (T1), brown alga (Sargassum polysystum) as Treatment-2 (T(2)) and sea grass (Enhalus acoroides) as Treatment-3 (T(3)). At the time of stocking, juveniles were under the same age group and batch-reared with a mean length and weight of 9.98 ? 0.56 mm and 0.49 ? 0.11 g, respectively. The juveniles were fed ad libitum, and the seawater in each rearing aquarium was changed at bi-monthly intervals. The culture was carried out for one year during which time the juveniles attained sexual maturity. Growth performances (viz., final weight, weight gain, final length, length gain, specific growth rate and daily growth rate) and survival of adults were significantly higher (P < 0.05) in T(1) than those in T(2) and T(3), respectively. Gonad production, in terms of wet gonad weight and gonad index, also followed the same trend as that for growth. Hence, of the three algal feed evaluated, red alga appeared to be the most suitable food for rearing of S. sphaeroides under captive conditions. The present study is the first demonstration of successful culturing of S. sphaeroides in a static aquarium system, the findings of which could be helpful towards the commercial sea urchin aquaculture. ?

  14. Controlling light-use by Rhodobacter capsulatus continuous cultures in a flat-panel photobioreactor.

    PubMed

    Hoekema, Sebastiaan; Douma, Rutger D; Janssen, Marcel; Tramper, Johannes; Wijffels, René H

    2006-11-05

    The main bottleneck in scale-up of phototrophic fermentation is the low efficiency of light energy conversion to the desired product, which is caused by an excessive dissipation of light energy to heat. The photoheterotrophic formation of hydrogen from acetate and light energy by the microorganism Rhodobacter capsulatus NCIMB 11773 was chosen as a case study in this work. A light energy balance was set up, in which the total bacterial light energy absorption is split up and attributed to its destinations. These are biomass growth and maintenance, generation of hydrogen and photosynthetic heat dissipation. The constants defined in the light energy balance were determined experimentally using a flat-panel photobioreactor with a 3-cm optical path. An experimental method called D-stat was applied. Continuous cultures were kept in a so-called pseudo steady state, while the dilution rate was reduced slowly and smoothly. The biomass yield and maintenance coefficients of Rhodobacter capsulatus biomass on light energy were determined at 12.4 W/m(2) (400-950 nm) and amounted to 2.58 x 10(-8) +/- 0.04 x 10(-8) kg/J and 102 +/- 3.5 W/kg, respectively. The fraction of the absorbed light energy that was dissipated to heat at 473 W/m(2) depended on the biomass concentration in the reactor and varied between 0.80 and 0.88, as the biomass concentration was increased from 2.0 to 8.0 kg/m(3). The process conditions were estimated at which a 3.7% conversion efficiency of absorbed light energy to produced hydrogen energy should be attainable at 473 W/m(2). (c) 2006 Wiley Periodicals, Inc.

  15. A structural role of the carotenoid in the light-harvesting II protein of Rhodobacter capsulatus.

    PubMed Central

    Zurdo, J; Fernandez-Cabrera, C; Ramirez, J M

    1993-01-01

    The membrane-linked light-harvesting II protein (LHII) of Rhodobacter capsulatus was partly depleted of carotenoids by selective extraction with light petroleum. Carotenoid removal was accompanied by bleaching of the Qy(S1<--S0) absorption band of bacteriochlorophyll (Bchl) a near 800 nm, by a bathochromic shift and a broadening of the other Bchl Qy band at 850 nm, and by the formation of a weak Qy band of dissociated Bchl near 770 nm. The changes in the 800 and 850 nm bands seemed to reflect alterations in only those Bchl molecules that had lost their associated carotenoids, firstly, because the extent of the changes was closely correlated to the degree of carotenoid extraction, and, secondly, because the residual fraction of carotenoid-containing LHII, which could be almost quantitatively recovered from the membrane after detergent solubilization and ion-exchange chromatography, showed an unmodified LHII absorption spectrum. The Bchl responsible for the shifted 850 nm band remained bound to protein, since its visible (Qx) transition seemed to retain the induced optical activity of the native bound pigment. Besides, the shifted Bchl could act as an efficient acceptor of singlet excitation energy from the pigments of the intact LHII fraction. The close similarity between the spectroscopic Bchl changes that accompany carotenoid extraction and the differential spectral features of carotenoidless LHII of Rhodobacter mutants, previously reported, strongly suggests that the direct cause of the spectral modifications is the absence of carotenoid and not any independent effect of the experimental manipulation of the membrane. Several interpretations of the structural changes that underlie the observed spectral changes are possible. The simplest one is to assume that carotenoid removal elicits an alteration in the angle between the Qy transition moments of two strongly interacting Bchl molecules. PMID:8452543

  16. A structural role of the carotenoid in the light-harvesting II protein of Rhodobacter capsulatus.

    PubMed

    Zurdo, J; Fernandez-Cabrera, C; Ramirez, J M

    1993-03-01

    The membrane-linked light-harvesting II protein (LHII) of Rhodobacter capsulatus was partly depleted of carotenoids by selective extraction with light petroleum. Carotenoid removal was accompanied by bleaching of the Qy(S1<--S0) absorption band of bacteriochlorophyll (Bchl) a near 800 nm, by a bathochromic shift and a broadening of the other Bchl Qy band at 850 nm, and by the formation of a weak Qy band of dissociated Bchl near 770 nm. The changes in the 800 and 850 nm bands seemed to reflect alterations in only those Bchl molecules that had lost their associated carotenoids, firstly, because the extent of the changes was closely correlated to the degree of carotenoid extraction, and, secondly, because the residual fraction of carotenoid-containing LHII, which could be almost quantitatively recovered from the membrane after detergent solubilization and ion-exchange chromatography, showed an unmodified LHII absorption spectrum. The Bchl responsible for the shifted 850 nm band remained bound to protein, since its visible (Qx) transition seemed to retain the induced optical activity of the native bound pigment. Besides, the shifted Bchl could act as an efficient acceptor of singlet excitation energy from the pigments of the intact LHII fraction. The close similarity between the spectroscopic Bchl changes that accompany carotenoid extraction and the differential spectral features of carotenoidless LHII of Rhodobacter mutants, previously reported, strongly suggests that the direct cause of the spectral modifications is the absence of carotenoid and not any independent effect of the experimental manipulation of the membrane. Several interpretations of the structural changes that underlie the observed spectral changes are possible. The simplest one is to assume that carotenoid removal elicits an alteration in the angle between the Qy transition moments of two strongly interacting Bchl molecules.

  17. Structural and phylogenetic analysis of Rhodobacter capsulatus NifF: uncovering general features of nitrogen-fixation (nif)-flavodoxins.

    PubMed

    Pérez-Dorado, Inmaculada; Bortolotti, Ana; Cortez, Néstor; Hermoso, Juan A

    2013-01-09

    Analysis of the crystal structure of NifF from Rhodobacter capsulatus and its homologues reported so far reflects the existence of unique structural features in nif flavodoxins: a leucine at the re face of the isoalloxazine, an eight-residue insertion at the C-terminus of the 50's loop and a remarkable difference in the electrostatic potential surface with respect to non-nif flavodoxins. A phylogenetic study on 64 sequences from 52 bacterial species revealed four clusters, including different functional prototypes, correlating the previously defined as "short-chain" with the firmicutes flavodoxins and the "long-chain" with gram-negative species. The comparison of Rhodobacter NifF structure with other bacterial flavodoxin prototypes discloses the concurrence of specific features of these functional electron donors to nitrogenase.

  18. Structural and Phylogenetic Analysis of Rhodobacter capsulatus NifF: Uncovering General Features of Nitrogen-fixation (nif)-Flavodoxins

    PubMed Central

    Pérez-Dorado, Inmaculada; Bortolotti, Ana; Cortez, Néstor; Hermoso, Juan A.

    2013-01-01

    Analysis of the crystal structure of NifF from Rhodobacter capsulatus and its homologues reported so far reflects the existence of unique structural features in nif flavodoxins: a leucine at the re face of the isoalloxazine, an eight-residue insertion at the C-terminus of the 50’s loop and a remarkable difference in the electrostatic potential surface with respect to non-nif flavodoxins. A phylogenetic study on 64 sequences from 52 bacterial species revealed four clusters, including different functional prototypes, correlating the previously defined as “short-chain” with the firmicutes flavodoxins and the “long-chain” with gram-negative species. The comparison of Rhodobacter NifF structure with other bacterial flavodoxin prototypes discloses the concurrence of specific features of these functional electron donors to nitrogenase. PMID:23303276

  19. Hypocholesterolemic effect of Nostoc commune var. sphaeroides Kützing, an edible blue-green alga.

    PubMed

    Rasmussen, Heather E; Blobaum, Kara R; Jesch, Elliot D; Ku, Chai Siah; Park, Young-Ki; Lu, Fan; Carr, Timothy P; Lee, Ji-Young

    2009-10-01

    Intake of an edible blue-green alga Nostoc commune var. sphaeroides Kützing (N. Commune) has been shown to lower plasma total cholesterol concentration, but the mechanisms behind the hypocholesterolemic effect have not been elucidated. To elucidate the mechanisms underlying the cholesterol-lowering effect of N. commune in mice. Male C57BL/6J mice were fed the AIN-93 M diet supplemented with 0 or 5% (wt/wt) dried N. Commune for 4 weeks. Lipid levels in the plasma and liver, intestinal cholesterol absorption and fecal sterol excretion were measured. Expression of hepatic and intestinal genes involved in cholesterol metabolism was evaluated by quantitative realtime PCR. N. commune supplementation significantly reduced total plasma cholesterol and triglyceride concentrations by approximately 20% compared to controls. Intestinal cholesterol absorption was significantly decreased, while fecal neutral sterol output was significantly increased in N. commune-fed mice. mRNA levels of the cholesterol transporters such as Niemann Pick C1 Like 1, scavenger receptor class B type 1, ATP-binding cassette transporters G5 and A1 in small intestine were not significantly different between two groups. Hepatic lipid contents including total cholesterol, triglyceride and free cholesterol in N. commune-fed mice were not significantly altered. However, the expression of cholesterol modulating genes including sterol regulatory element binding protein-2 and 3-hydroxy-3-methylglutaryl coenzyme A reductase were significantly increased in mice fed N. commune. N. commune supplementation exerted a hypocholesterolemic effect in mice, largely in part, by reducing intestinal cholesterol absorption and promoting fecal neutral sterol excretion.

  20. Isolation and partial characterization of Rhodopseudomonas sphaeroides mutants defective in the regulation of ribulose bisphosphate carboxylase/oxygenase.

    PubMed

    Weaver, K E; Tabita, F R

    1983-11-01

    Several mutants of Rhodopseudomonas sphaeroides defective in the derepression of the enzyme ribulose 1,5-bisphosphate carboxylase have been isolated by using the unstable Tn5 vectors pJB4JI and pRK340. Transpositional insertion mutants obtained with pJB4JI were demonstrated to be incapable of increasing ribulose 1,5-bisphosphate carboxylase/oxygenase levels when grown on butyrate-bicarbonate medium or under conditions of carbon starvation, whereas the wild-type strain increased activity four- to eightfold. When the wild-type strain was starved for carbon in the presence of chloramphenicol, no derepression was observed. Crude extracts from mutant and wild-type strains had distinct and consistent differences in protein content as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chromatographic evidence indicated that mutants were defective in the regulation of only one of the two forms of ribulose 1,5-bisphosphate carboxylase/oxygenase synthesized by R. sphaeroides.

  1. Transcriptional Profiling of Hydrogen Production Metabolism of Rhodobacter capsulatus under Temperature Stress by Microarray Analysis

    PubMed Central

    Gürgan, Muazzez; Afşar Erkal, Nilüfer; Özgür, Ebru; Gündüz, Ufuk; Eroglu, Inci; Yücel, Meral

    2015-01-01

    Biohydrogen is a clean and renewable form of hydrogen, which can be produced by photosynthetic bacteria in outdoor large-scale photobioreactors using sunlight. In this study, the transcriptional response of Rhodobacter capsulatus to cold (4 °C) and heat (42 °C) stress was studied using microarrays. Bacteria were grown in 30/2 acetate/glutamate medium at 30 °C for 48 h under continuous illumination. Then, cold and heat stresses were applied for two and six hours. Growth and hydrogen production were impaired under both stress conditions. Microarray chips for R. capsulatus were custom designed by Affymetrix (GeneChip®. TR_RCH2a520699F). The numbers of significantly changed genes were 328 and 293 out of 3685 genes under cold and heat stress, respectively. Our results indicate that temperature stress greatly affects the hydrogen production metabolisms of R. capsulatus. Specifically, the expression of genes that participate in nitrogen metabolism, photosynthesis and the electron transport system were induced by cold stress, while decreased by heat stress. Heat stress also resulted in down regulation of genes related to cell envelope, transporter and binding proteins. Transcriptome analysis and physiological results were consistent with each other. The results presented here may aid clarification of the genetic mechanisms for hydrogen production in purple non-sulfur (PNS) bacteria under temperature stress. PMID:26086826

  2. Field evidence for the potential of Rhodobacter capsulatus as Biofertilizer for flooded rice.

    PubMed

    Gamal-Eldin, Hosny; Elbanna, Khaled

    2011-02-01

    In a previous study, we evaluated the effects of inoculating rice plants with the phototrophic purple nonsulfur bacterium Rhodobacter capsulatus (Rc) on growth and yield of rice in pots and lysimeter experiments and the results obtained have been highly encouraging. In this study, we carried out two field experiments: one in the experimental farm of the Faculty of Agriculture, Fayoum University, and the second in a farmer's field in Kafr El-sheikh, to assess the effects of Rc on growth and yield of rice in comparison and in combination with chemical nitrogen fertilizer (CNF) and farmyard manure. The results indicated that both biological and grain yields in all the Rc inoculated treatments were significantly higher than those in the uninoculated corresponding treatments in both fields. With regard to grain yield, the major factor for determining the effectiveness of any agricultural treatment, inoculation with Rc in combination with 50% of the recommended CNF rate gave a grain yield that was statistically equivalent to that obtained with 100% of the recommended CNF rate. These results provide a clear evidence for the potential of Rc as biofertilizer for flooded rice under field conditions.

  3. Coenzyme binding and hydride transfer in Rhodobacter capsulatus ferredoxin/flavodoxin NADP(H) oxidoreductase.

    PubMed

    Bortolotti, Ana; Pérez-Dorado, Inmaculada; Goñi, Guillermina; Medina, Milagros; Hermoso, Juan A; Carrillo, Néstor; Cortez, Néstor

    2009-02-01

    Ferredoxin-NADP(H) reductases catalyse the reversible hydride/electron exchange between NADP(H) and ferredoxin/flavodoxin, comprising a structurally defined family of flavoenzymes with two distinct subclasses. Those present in Gram-negative bacteria (FPRs) display turnover numbers of 1-5 s(-1) while the homologues of cyanobacteria and plants (FNRs) developed a 100-fold activity increase. We investigated nucleotide interactions and hydride transfer in Rhodobacter capsulatus FPR comparing them to those reported for FNRs. NADP(H) binding proceeds as in FNRs with stacking of the nicotinamide on the flavin, which resulted in formation of charge-transfer complexes prior to hydride exchange. The affinity of FPR for both NADP(H) and 2'-P-AMP was 100-fold lower than that of FNRs. The crystal structure of FPR in complex with 2'-P-AMP and NADP(+) allowed modelling of the adenosine ring system bound to the protein, whereas the nicotinamide portion was either not visible or protruding toward solvent in different obtained crystals. Stabilising contacts with the active site residues are different in the two reductase classes. We conclude that evolution to higher activities in FNRs was partially favoured by modification of NADP(H) binding in the initial complexes through changes in the active site residues involved in stabilisation of the adenosine portion of the nucleotide and in the mobile C-terminus of FPR.

  4. Molecular structure of FoxE, the putative iron oxidase of Rhodobacter ferrooxidans SW2.

    PubMed

    Pereira, Luis; Saraiva, Ivo H; Oliveira, A Sofia F; Soares, Cláudio M; Louro, Ricardo O; Frazão, Carlos

    2017-10-01

    The ancient metabolism of photoferrotrophy is likely to have played a key role in the biogeochemical cycle of iron on Early Earth leading to the deposition of Banded Iron Formations prior to the emergence of oxygenic photosynthesis. Extant organisms still performing this metabolism provide a convenient window to peer into its molecular mechanisms. Here we report the molecular structure of FoxE, the putative terminal iron oxidase of Rhodobacter ferrooxidans SW2. This protein is organized as a trimer with two hemes and a disulfide bridge per monomer. The distance between hemes, their solvent exposure and the surface electrostatics ensure a controlled electron transfer rate. They also guarantee segregation between electron capture from ferrous iron and electron release to downstream acceptors, which do not favor the precipitation of ferric iron. Combined with the functional characterization of this protein, the structure reveals how iron oxidation can be performed in the periplasmic space of this Gram-negative bacterium at circumneutral pH, while minimizing the risk of mineral precipitation and cell encrustation. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Functional Characterization of the FoxE Iron Oxidoreductase from the Photoferrotroph Rhodobacter ferrooxidans SW2*

    PubMed Central

    Saraiva, Ivo H.; Newman, Dianne K.; Louro, Ricardo O.

    2012-01-01

    Photoferrotrophy is presumed to be an ancient type of photosynthetic metabolism in which bacteria use the reducing power of ferrous iron to drive carbon fixation. In this work the putative iron oxidoreductase of the photoferrotroph Rhodobacter ferrooxidans SW2 was cloned, purified, and characterized for the first time. This protein, FoxE, was characterized using spectroscopic, thermodynamic, and kinetic techniques. It is a c-type cytochrome that forms a trimer or tetramer in solution; the two hemes of each monomer are hexacoordinated by histidine and methionine. The hemes have positive reduction potentials that allow downhill electron transfer from many geochemically relevant ferrous iron forms to the photosynthetic reaction center. The reduction potentials of the hemes are different and are cross-assigned to fast and slow kinetic phases of ferrous iron oxidation in vitro. Lower reactivity was observed at high pH and may contribute to prevent ferric iron precipitation inside or at the surface of the cell. These results help fill in the molecular details of a metabolic process that likely contributed to the deposition of precambrian banded iron formations, globally important sedimentary rocks that are found on every continent today. PMID:22661703

  6. Crystallization of a flavodoxin involved in nitrogen fixation in Rhodobacter capsulatus

    SciTech Connect

    Pérez-Dorado, Inmaculada; Bortolotti, Ana; Cortez, Néstor; Hermoso, Juan A.

    2008-05-01

    The flavodoxin NifF from R. capsulatus, a candidate for nitrogenase reduction during nitrogen fixation, has been crystallized using the hanging-drop vapour-diffusion method. Preliminary X-ray data processing at 2.17 Å resolution allowed determination of the crystal system and unit-cell parameters. Flavodoxins are small electron-transfer proteins that contain one molecule of noncovalently bound flavin mononucleotide (FMN). The flavodoxin NifF from the photosynthetic bacterium Rhodobacter capsulatus is reduced by one electron from ferredoxin/flavodoxin:NADP(H) reductase and was postulated to be an electron donor to nitrogenase in vivo. NifF was cloned and overexpressed in Escherichia coli, purified and concentrated for crystallization using the hanging-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of PEG 3350 and PEG 400 at pH 5.5 and belong to the tetragonal space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 66.49, c = 121.32 Å. X-ray data sets have been collected to 2.17 Å resolution.

  7. Mutational and nucleotide sequence analysis of S-adenosyl-L-homocysteine hydrolase from Rhodobacter capsulatus.

    PubMed Central

    Sganga, M W; Aksamit, R R; Cantoni, G L; Bauer, C E

    1992-01-01

    The genetic locus ahcY, encoding the enzyme S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) from the bacterium Rhodobacter capsulatus, has been mapped by mutational analysis to within a cluster of genes involved in regulating the induction and maintenance of the bacterial photosynthetic apparatus. Sequence analysis demonstrates that ahcY encodes a 51-kDa polypeptide that displays 64% sequence identity to its human homolog. Insertion mutants in ahcY lack detectable S-adenosyl-L-homocysteine hydrolase activity and, as a consequence, S-adenosyl-L-homocysteine accumulates in the cells, resulting in a 16-fold decrease in the intracellular ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine as compared to wild-type cells. The ahcY disrupted strain fails to grow in minimal medium; however, growth is restored in minimal medium supplemented with methionine or homocysteine or in a complex medium, thereby indicating that the hydrolysis of S-adenosyl-L-homocysteine plays a key role in the metabolism of sulfur-containing amino acids. The ahcY mutant, when grown in supplemented medium, synthesizes significantly reduced levels of bacteriochlorophyll, indicating that modulation of the intracellular ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine may be an important factor in regulating bacteriochlorophyll biosynthesis. PMID:1631127

  8. A Rhodobacter capsulatus member of a universal permease family imports molybdate and other oxyanions.

    PubMed

    Gisin, Jonathan; Müller, Alexandra; Pfänder, Yvonne; Leimkühler, Silke; Narberhaus, Franz; Masepohl, Bernd

    2010-11-01

    Molybdenum (Mo) is an important trace element that is toxic at high concentrations. To resolve the mechanisms underlying Mo toxicity, Rhodobacter capsulatus mutants tolerant to high Mo concentrations were isolated by random transposon Tn5 mutagenesis. The insertion sites of six independent isolates mapped within the same gene predicted to code for a permease of unknown function located in the cytoplasmic membrane. During growth under Mo-replete conditions, the wild-type strain accumulated considerably more Mo than the permease mutant. For mutants defective for the permease, the high-affinity molybdate importer ModABC, or both transporters, in vivo Mo-dependent nitrogenase (Mo-nitrogenase) activities at different Mo concentrations suggested that ModABC and the permease import molybdate in nanomolar and micromolar ranges, respectively. Like the permease mutants, a mutant defective for ATP sulfurylase tolerated high Mo concentrations, suggesting that ATP sulfurylase is the main target of Mo inhibition in R. capsulatus. Sulfate-dependent growth of a double mutant defective for the permease and the high-affinity sulfate importer CysTWA was reduced compared to those of the single mutants, implying that the permease plays an important role in sulfate uptake. In addition, permease mutants tolerated higher tungstate and vanadate concentrations than the wild type, suggesting that the permease acts as a general oxyanion importer. We propose to call this permease PerO (for oxyanion permease). It is the first reported bacterial molybdate transporter outside the ABC transporter family.

  9. Purification and properties of a nif-specific flavodoxin from the photosynthetic bacterium Rhodobacter capsulatus.

    PubMed Central

    Yakunin, A F; Gennaro, G; Hallenbeck, P C

    1993-01-01

    A flavodoxin was isolated from iron-sufficient, nitrogen-limited cultures of the photosynthetic bacterium Rhodobacter capsulatus. Its molecular properties, molecular weight, UV-visible absorption spectrum, and amino acid composition suggest that it is similar to the nif-specific flavodoxin, NifF, of Klebsiella pneumoniae. The results of immunoblotting showed that R. capsulatus flavodoxin is nif specific, since it is absent from ammonia-replete cultures and is not synthesized by the mutant strain J61, which lacks a nif-specific regulator (NifR1). Growth of cultures under iron-deficient conditions causes a small amount of flavodoxin to be synthesized under ammonia-replete conditions and increases its synthesis under N2-fixing conditions, suggesting that its synthesis is under a dual system of control with respect to iron and fixed nitrogen availability. Here we show that flavodoxin, when supplemented with catalytic amounts of methyl viologen, is capable of efficiently reducing nitrogenase in an illuminated chloroplast system. Thus, this nif-specific flavodoxin is a potential in vivo electron carrier to nitrogenase; however, its role in the nitrogen fixation process remains to be established. Images PMID:8226618

  10. Purification of a sixth ferredoxin from Rhodobacter capsulatus. Primary structure and biochemical properties.

    PubMed

    Naud, I; Vinçon, M; Garin, J; Gaillard, J; Forest, E; Jouanneau, Y

    1994-06-15

    A new ferredoxin has been purified from the photosynthetic bacterium Rhodobacter capsulatus. It is the sixth ferredoxin to be isolated from this bacterium and it was called FdVI. Its primary structure was established based on amino acid sequence analysis of the protein and of peptides derived from it. It is composed of 106 residues including five cysteines. The calculated mass of the polypeptide is 11,402.6 Da which matches the experimental value determined by electrospray mass spectrometry. Amino acid sequence comparison revealed that ferredoxin VI (FdVI) is strikingly similar to a ferredoxin from Caulobacter crescentus and to the putidaredoxin from Pseudomonas putida. FdVI exhibited an ultraviolet-visible absorption spectrum typical for a [2Fe-2S] ferredoxin. EPR spectroscopy of the reduced protein showed a nearly axial signal similar to that of mitochondrial and P. putida ferredoxins. FdVI is biosynthesized in cells growing anaerobically under either nitrogen-sufficient or nitrogen-deficient conditions. Although the function of FdVI is unknown, its structural resemblance to [2Fe-2S] ferredoxins known to transfer electrons to oxygenases such as P-450 cytochromes, suggests that FdVI may have a similar role in R. capsulatus.

  11. Long-term biological hydrogen production by agar immobilized Rhodobacter capsulatus in a sequential batch photobioreactor.

    PubMed

    Elkahlout, Kamal; Alipour, Siamak; Eroglu, Inci; Gunduz, Ufuk; Yucel, Meral

    2017-04-01

    In this study, agar immobilization technique was employed for biological hydrogen production using Rhodobacter capsulatus DSM 1710 (wild type) and YO3 (hup-mutant) strains in sequential batch process. Different agar and glutamate concentrations were tested with defined nutrient medium. Agar concentration 4% (w/v) and 4 mM glutamate were selected for bacterial immobilization in terms of rate and longevity of hydrogen production. Acetate concentration was increased from 40 to 60-100 and 60 mM gave best results with both bacterial strains immobilized in 4% (w/v) agar. Cell concentration was increased from 2.5 to 5 mg dcw mL(-1) agar and it was found that increasing cell concentration of wild-type strain caused decrease in yield and productivity while these parameters improved by increasing cell concentration of mutant strain. Also, the hydrogen production time has extended from 17 days up to 60 days according to the process conditions and parameters. Hydrogen production by immobilized photosynthetic bacteria is a convenient technology for hydrogen production as it enables to produce hydrogen with high organic acid concentrations comparing to suspended cultures. Besides, immobilization increases the stability of the system and allowed sequential batch operation for long-term application.

  12. RegA control of bacteriochlorophyll and carotenoid synthesis in Rhodobacter capsulatus.

    PubMed

    Willett, Jonathan; Smart, James L; Bauer, Carl E

    2007-11-01

    We provide in vivo genetic and in vitro biochemical evidence that RegA directly regulates bacteriochlorophyll and carotenoid biosynthesis in Rhodobacter capsulatus. beta-Galactosidase expression assays with a RegA-disrupted strain containing reporter plasmids for Mg-protoporphyrin IX monomethyl ester oxidative cyclase (bchE), Mg-protoporphyrin IX chelatase (bchD), and phytoene dehydrogenase (crtI) demonstrate RegA is responsible for fourfold anaerobic induction of bchE, threefold induction of bchD, and twofold induction of crtI. Promoter mapping studies, coupled with DNase I protection assays, map the region of RegA binding to three sites in the bchE promoter region. Similar studies at the crtA and crtI promoters indicate that RegA binds to a single region equidistant from these divergent promoters. These results demonstrate that RegA is directly responsible for anaerobic induction of bacteriochlorophyll biosynthesis genes bchE, bchD, bchJ, bchI, bchG, and bchP and carotenoid biosynthesis genes crtI, crtB, and crtA.

  13. Electrochemical communication between heterotrophically grown Rhodobacter capsulatus with electrodes mediated by an osmium redox polymer.

    PubMed

    Hasan, Kamrul; Patil, Sunil A; Górecki, Kamil; Leech, Dónal; Hägerhäll, Cecilia; Gorton, Lo

    2013-10-01

    The metabolically versatile purple bacteria Rhodobacter capsulatus was investigated to check its possible applicability in biofuel cells and electrochemical microbial biosensors. The wild type strain ATCC 17015 and mutant strain 37b4 lacking the lipopolysaccharide capsule was compared for their ability to communicate with electrodes modified with an osmium redox polymer. In this work, aerobic heterotrophically grown R. capsulatus were used to screen for efficient cell-electrode communication for later implementation using photoheterotrophically grown bacteria. The bacterial cells embedded in the osmium polymer matrix demonstrated efficient electrical "wiring" with the electrodes and were able to generate a noticeable current with succinate as substrate. Interestingly, at 2mM succinate the wild type strain showed much better bioelectrocatalytic current generation (4.25 μA/cm(2)) than the strain lacking capsule (1.55 μA/cm(2)). The wild type strain also exhibited a stable current response for longer time, demonstrating that the bacterial lipopolysaccharide in fact enhances the stability of the polymer matrix layer of the modified electrode. Control experiments with R. capsulatus without any mediator did not show any current irrespective of the capsule presence. This demonstrates that development of photosensors and other light driven bioelectrochemical devices could be feasible using R. capsulatus and will be at focus for future studies.

  14. Hydrogen photosynthesis by Rhodobacter capsulatus and its coupling to a PEM fuel cell

    NASA Astrophysics Data System (ADS)

    He, Deliang; Bultel, Yann; Magnin, Jean-Pierre; Roux, Claude; Willison, John C.

    Four different mutant strains of Rhodobacter capsulatus (IR1, IR3, IR4 and JP91), a photosynthetic purple non-sulfur bacterium, were tested for their ability to produce hydrogen in a 3 L volume photobioreactor coupled to a small PEM fuel cell. The four mutants, together with the wild-type strain, B10, were grown at 30 °C under illumination with 30 mmol L -1DL-lactate and 5 mmol L -1L-glutamate as carbon and nitrogen source, respectively. Bacterial growth was measured by monitoring the increase in absorbance at 660 nm, and hydrogen yield, and substrate conversion efficiency were measured under the same conditions. The hydrogen production capability of the five strains was then compared and shown to be in the order: IR3 > JP91 > IR4 > B10 > IR1. The most preferment strain, IR3, showed a substrate conversion efficiency of 84.8% and a hydrogen yield of 3.9 L L -1 of culture. The biogas produced by these photobioreactor cultures was successfully used as feed for a small PEM fuel cell system, with the mutant IR3 showing the most sustained hydrogen and current production. The maximum current was similar to that obtained using pure hydrogen produced by a small electrolysis cell (High-Tec Inc.).

  15. Extracellular production of tellurium nanoparticles by the photosynthetic bacterium Rhodobacter capsulatus.

    PubMed

    Borghese, Roberto; Brucale, Marco; Fortunato, Gianuario; Lanzi, Massimiliano; Mezzi, Alessio; Valle, Francesco; Cavallini, Massimiliano; Zannoni, Davide

    2016-05-15

    The toxic oxyanion tellurite (TeO3(2-)) is acquired by cells of Rhodobacter capsulatus grown anaerobically in the light, via acetate permease ActP2 and then reduced to Te(0) in the cytoplasm as needle-like black precipitates. Interestingly, photosynthetic cultures of R. capsulatus can also generate Te(0) nanoprecipitates (TeNPs) outside the cells upon addition of the redox mediator lawsone (2-hydroxy-1,4-naphtoquinone). TeNPs generation kinetics were monitored to define the optimal conditions to produce TeNPs as a function of various carbon sources and lawsone concentration. We report that growing cultures over a 10 days period with daily additions of 1mM tellurite led to the accumulation in the growth medium of TeNPs with dimensions from 200 up to 600-700 nm in length as determined by atomic force microscopy (AFM). This result suggests that nucleation of TeNPs takes place over the entire cell growth period although the addition of new tellurium Te(0) to pre-formed TeNPs is the main strategy used by R. capsulatus to generate TeNPs outside the cells. Finally, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FT-IR) analysis of TeNPs indicate they are coated with an organic material which keeps the particles in solution in aqueous solvents.

  16. Role of Rhodobacter sp. Strain PS9, a Purple Non-Sulfur Photosynthetic Bacterium Isolated from an Anaerobic Swine Waste Lagoon, in Odor Remediation

    PubMed Central

    Do, Young S.; Schmidt, Thomas M.; Zahn, James A.; Boyd, Eric S.; de la Mora, Arlene; DiSpirito, Alan A.

    2003-01-01

    Temporal pigmentation changes resulting from the development of a purple color in anaerobic swine waste lagoons were investigated during a 4-year period. The major purple photosynthetic bacterium responsible for these color changes and the corresponding reductions in odor was isolated from nine photosynthetic lagoons. By using morphological, physiological, and phylogenetic characterization methods we identified the predominant photosynthetic bacterium as a new strain of Rhodobacter, designated Rhodobacter sp. strain PS9. Rhodobacter sp. strain PS9 is capable of photoorganotrophic growth on a variety of organic compounds, including all of the characteristic volatile organic compounds (VOC) responsible for the odor associated with swine production facilities (J. A. Zahn, A. A. DiSpirito, Y. S. Do, B. E. Brooks, E. E. Copper, and J. L. Hatfield, J. Environ. Qual. 30:624-634, 2001). The seasonal variations in airborne VOC emitted from waste lagoons showed that there was a 80 to 93% decrease in the concentration of VOC during a photosynthetic bloom. During the height of a bloom, the Rhodobacter sp. strain PS9 population accounted for 10% of the total community and up to 27% of the eubacterial community based on 16S ribosomal DNA signals. Additional observations based on seasonal variations in meteorological, biological, and chemical parameters suggested that the photosynthetic blooms of Rhodobacter sp. strain PS9 were correlated with lagoon water temperature and with the concentrations of sulfate and phosphate. In addition, the photosynthetic blooms of Rhodobacter sp. strain PS9 were inversely correlated with the concentrations of protein and fluoride. PMID:12620863

  17. Crystallization and preliminary X-ray analysis of the Rhodobacter capsulatus magnesium chelatase BchI subunit.

    PubMed

    Willows, R D; Hansson, M; Beale, S I; Laurberg, M; Al-Karadaghi, S

    1999-03-01

    The Rhodobacter capsulatus BchI protein is one of three subunits of Mg chelatase, the enzyme which catalyzes the first committed step of chlorophyll and bacteriochlorophyll biosynthesis. The BchI protein was produced with an inducible T7 RNA polymerase expression system in Escherichia coli. The protein was purified from the soluble cell-extract fraction and crystallized from polyethylene glycol solution. The crystals diffract to a minimum Bragg spacing of 2.1 A. The space group is P63 with unit-cell dimensions a = b = 90.6, c = 84.1 A.

  18. The Rhodobacter capsulatus glnB gene is regulated by NtrC at tandem rpoN-independent promoters.

    PubMed Central

    Foster-Hartnett, D; Kranz, R G

    1994-01-01

    The protein encoded by glnB of Rhodobacter capsulatus is part of a nitrogen-sensing cascade which regulates the expression of nitrogen fixation genes (nif). The expression of glnB was studied by using lacZ fusions, primer extension analysis, and in vitro DNase I footprinting. Our results suggest that glnB is transcribed from two promoters, one of which requires the R. capsulatus ntrC gene but is rpoN independent. Another promoter upstream of glnB is repressed by NtrC; purified R. capsulatus NtrC binds to sites that overlap this distal promoter region. Images PMID:8051036

  19. The bluF gene of Rhodobacter capsulatus is involved in conversion of cobinamide to cobalamin (vitamin B12).

    PubMed Central

    Pollich, M; Wersig, C; Klug, G

    1996-01-01

    The bluF gene of Rhodobacter capsulatus is the first gene of the bluFEDCB operon which is involved in late steps of the cobalamin synthesis. To determine the function of the bluF gene product, a bluF::omega-Km mutant strain was constructed and characterized. This vitamin B12 auxotrophic mutant strain shows a 3.5-times higher vitamin B12 requirement under phototrophic growth conditions than under chemotrophic growth conditions. Surprisingly, the bluF promoter activity does not respond to alterations to the oxygen tension or vitamin B12 concentration. PMID:8955417

  20. Molecular genetic and molecular evolutionary studies on the bacteriochlorophyll synthesis genes of Rhodobacter capsulatus

    SciTech Connect

    Burke-Agueero, Donald H.

    1992-08-01

    Rhodobacter capsulatus, purple bacterium capable of either aerobic or photosynthetic growth, has proven to be very useful in genetic studies of photosynthesis. Forty-four genes clustered together within a 46 kilobase region are required to establish photosynthetic ability in R. capsulatus. Approximately twenty of these genes are involved in bacteriochlorophyll synthesis of which eight ``bch`` genes are the subject of this thesis. Six of these genes were found to code for the two ring reductases. The first converts protochlorophyllide (PChlide) into a chlorin, the immediate precursor to chlorophyll a, and then into a bacteriochlorin. Each reductase is shown to be made up of three subunits. PChlide reductase is coded by the genes bchN, bchB, and bchL. Proteins with amino acid sequences markedly similar to those of bchN and bchL have been shown in other organisms to be required for chlorophyll synthesis; hence, their designation as chlN and chlB. A third chloroplast-encoded gene of heretofore unknown function shares amino acid identities with bchB and is probably the third subunit of the plant PChlide reductase. The bchA locus, which encodes the chlorin reductase, is found to be made up of three separate, translationally coupled genes, referred to as bchX, bchY, and bchZ. Amino acid similarities between bchX, bchL, and the nitrogenase reductase protein nifH suggest that all three classes of proteins share certain three-dimensional structural features, including elements that are central to the enzymatic mechanism of nifH. PChlide reductase and chlorin reductase are clearly derived from a common ancestor. Several lines of analysis suggests the ancestor of both enzyme systems reduced PChlide twice to produce bacteriochlorophyll supporting the concept bacteriochlorophyll as the ancestral reaction center pigment.

  1. Molecular genetic and molecular evolutionary studies on the bacteriochlorophyll synthesis genes of Rhodobacter capsulatus

    SciTech Connect

    Burke-Agueero, D.H.

    1992-08-01

    Rhodobacter capsulatus, purple bacterium capable of either aerobic or photosynthetic growth, has proven to be very useful in genetic studies of photosynthesis. Forty-four genes clustered together within a 46 kilobase region are required to establish photosynthetic ability in R. capsulatus. Approximately twenty of these genes are involved in bacteriochlorophyll synthesis of which eight bch'' genes are the subject of this thesis. Six of these genes were found to code for the two ring reductases. The first converts protochlorophyllide (PChlide) into a chlorin, the immediate precursor to chlorophyll a, and then into a bacteriochlorin. Each reductase is shown to be made up of three subunits. PChlide reductase is coded by the genes bchN, bchB, and bchL. Proteins with amino acid sequences markedly similar to those of bchN and bchL have been shown in other organisms to be required for chlorophyll synthesis; hence, their designation as chlN and chlB. A third chloroplast-encoded gene of heretofore unknown function shares amino acid identities with bchB and is probably the third subunit of the plant PChlide reductase. The bchA locus, which encodes the chlorin reductase, is found to be made up of three separate, translationally coupled genes, referred to as bchX, bchY, and bchZ. Amino acid similarities between bchX, bchL, and the nitrogenase reductase protein nifH suggest that all three classes of proteins share certain three-dimensional structural features, including elements that are central to the enzymatic mechanism of nifH. PChlide reductase and chlorin reductase are clearly derived from a common ancestor. Several lines of analysis suggests the ancestor of both enzyme systems reduced PChlide twice to produce bacteriochlorophyll supporting the concept bacteriochlorophyll as the ancestral reaction center pigment.

  2. Coordinated, long-range, solid substrate movement of the purple photosynthetic bacterium Rhodobacter capsulatus.

    PubMed

    Shelswell, Kristopher John; Beatty, J Thomas

    2011-05-04

    The long-range movement of Rhodobacter capsulatus cells in the glass-agar interstitial region of borosilicate Petri plates was found to be due to a subset of the cells inoculated into plates. The macroscopic appearance of plates indicated that a small group of cells moved in a coordinated manner to form a visible satellite cluster of cells. Satellite clusters were initially separated from the point of inoculation by the absence of visible cell density, but after 20 to 24 hours this space was colonized by cells apparently shed from a group of cells moving away from the point of inoculation. Cell movements consisted of flagellum-independent and flagellum-dependent motility contributions. Flagellum-independent movement occurred at an early stage, such that satellite clusters formed after 12 to 24 hours. Subsequently, after 24 to 32 hours, a flagellum-dependent dispersal of cells became visible, extending laterally outward from a line of flagellum-independent motility. These modes of taxis were found in several environmental isolates and in a variety of mutants, including a strain deficient in the production of the R. capsulatus acyl-homoserine lactone quorum-sensing signal. Although there was great variability in the direction of movement in illuminated plates, cells were predisposed to move toward broad spectrum white light. This predisposition was increased by the use of square plates, and a statistical analysis indicated that R. capsulatus is capable of genuine phototaxis. Therefore, the variability in the direction of cell movement was attributed to optical effects on light waves passing through the plate material and agar medium.

  3. Phototrophic Fe(II) oxidation promotes organic carbon acquisition by Rhodobacter capsulatus SB1003.

    PubMed

    Caiazza, Nicky C; Lies, Douglas P; Newman, Dianne K

    2007-10-01

    Anoxygenic phototrophic Fe(II) oxidation is usually considered to be a lithoautotrophic metabolism that contributes to primary production in Fe-based ecosystems. In this study, we employed Rhodobacter capsulatus SB1003 as a model organism to test the hypothesis that phototrophic Fe(II) oxidation can be coupled to organic carbon acquisition. R. capsulatus SB1003 oxidized Fe(II) under anoxic conditions in a light-dependent manner, but it failed to grow lithoautotrophically on soluble Fe(II). When the strain was provided with Fe(II)-citrate, however, growth was observed that was dependent upon microbially catalyzed Fe(II) oxidation, resulting in the formation of Fe(III)-citrate. Subsequent photochemical breakdown of Fe(III)-citrate yielded acetoacetic acid that supported growth in the light but not the dark. The deletion of genes (RRC00247 and RRC00248) that encode homologs of atoA and atoD, required for acetoacetic acid utilization, severely impaired the ability of R. capsulatus SB1003 to grow on Fe(II)-citrate. The growth yield achieved by R. capsulatus SB1003 in the presence of citrate cannot be explained by lithoautotrophic growth on Fe(II) enabled by indirect effects of the ligand [such as altering the thermodynamics of Fe(II) oxidation or preventing cell encrustation]. Together, these results demonstrate that R. capsulatus SB1003 grows photoheterotrophically on Fe(II)-citrate. Nitrilotriacetic acid also supported light-dependent growth on Fe(II), suggesting that Fe(II) oxidation may be a general mechanism whereby some Fe(II)-oxidizing bacteria mine otherwise inaccessible organic carbon sources.

  4. Zinc biosorption by the purple non-sulfur bacterium Rhodobacter capsulatus.

    PubMed

    Magnin, Jean-Pierre; Gondrexon, Nicolas; Willison, John C

    2014-12-01

    This paper presents the first report providing information on the zinc (Zn) biosorption potentialities of the purple non-sulfur bacterium Rhodobacter capsulatus. The effects of various biological, physical, and chemical parameters on Zn biosorption were studied in both the wild-type strain B10 and a strain, RC220, lacking the endogenous plasmid. At an initial Zn concentration of 10 mg·L(-1), the Zn biosorption capacity at pH 7 for bacterial biomass grown in synthetic medium containing lactate as carbon source was 17 and 16 mg Zn·(g dry mass)(-1) for strains B10 and RC220, respectively. Equilibrium was achieved in a contact time of 30-120 min, depending on the initial Zn concentration. Zn sorption by live biomass was modelled, at equilibrium, according to the Redlich-Peterson and Langmuir isotherms, in the range of 1-600 mg Zn·L(-1). The wild-type strain showed a maximal Zn uptake capacity (Qm) of 164 ± 8 mg·(g dry mass)(-1) and an equilibrium constant (Kads) of 0.017 ± 0.00085 L·(mg Zn)(-1), compared with values of 73.9 mg·(g dry mass)(-1) and 0.361 L·mg(-1) for the strain lacking the endogenous plasmid. The Qm value observed for R. capsulatus B10 is one of the highest reported in the literature, suggesting that this strain may be useful for Zn bioremediation. The lower Qm value and higher equilibrium constant observed for strain RC220 suggest that the endogenous plasmid confers an enhanced biosorption capacity in this bacterium, although no genetic determinants for Zn resistance appear to be located on the plasmid, and possible explanations for this are discussed.

  5. Inactivation of Mg chelatase during transition from anaerobic to aerobic growth in Rhodobacter capsulatus.

    PubMed

    Willows, Robert D; Lake, Vanessa; Roberts, Thomas Hugh; Beale, Samuel I

    2003-06-01

    The facultative photosynthetic bacterium Rhodobacter capsulatus can adapt from an anaerobic photosynthetic mode of growth to aerobic heterotrophic metabolism. As this adaptation occurs, the cells must rapidly halt bacteriochlorophyll synthesis to prevent phototoxic tetrapyrroles from accumulating, while still allowing heme synthesis to continue. A likely control point is Mg chelatase, the enzyme that diverts protoporphyrin IX from heme biosynthesis toward the bacteriochlorophyll biosynthetic pathway by inserting Mg(2+) to form Mg-protoporphyrin IX. Mg chelatase is composed of three subunits that are encoded by the bchI, bchD, and bchH genes in R. capsulatus. We report that BchH is the rate-limiting component of Mg chelatase activity in cell extracts. BchH binds protoporphyrin IX, and BchH that has been expressed and purified from Escherichia coli is red in color due to the bound protoporphyrin IX. Recombinant BchH is rapidly inactivated by light in the presence of O(2), and the inactivation results in the formation of a covalent adduct between the protein and the bound protoporphyrin IX. When photosynthetically growing R. capsulatus cells are transferred to aerobic conditions, Mg chelatase is rapidly inactivated, and BchH is the component that is most rapidly inactivated in vivo when cells are exposed to aerobic conditions. The light- and O(2)-stimulated inactivation of BchH could account for the rapid inactivation of Mg chelatase in vivo and provide a mechanism for inhibiting the synthesis of bacteriochlorophyll during adaptation of photosynthetically grown cells to aerobic conditions while still allowing heme synthesis to occur for aerobic respiration.

  6. Rhodobacter capsulatus magnesium chelatase subunit BchH contains an oxygen sensitive iron-sulfur cluster.

    PubMed

    Sirijovski, Nick; Mamedov, Fikret; Olsson, Ulf; Styring, Stenbjörn; Hansson, Mats

    2007-12-01

    Magnesium chelatase is the first unique enzyme of the bacteriochlorophyll biosynthetic pathway. It consists of three subunits (BchI, BchD, and BchH). Amino acid sequence analysis of the Rhodobacter capsulatus BchH revealed a novel cysteine motif (393CX2CX3CX14C) that was found in only six other proteobacteria (CX2CX3CX11-14C). The cysteine motif is likely to coordinate an unprecedented [Fe-S] cluster. Purified BchH demonstrated absorbance in the 460 nm region. This absorbance was abolished in BchH proteins with alanine substitutions at positions Cys396 and Cys414. These modified proteins were also EPR silent. In contrast, wild type BchH protein in the reduced state showed EPR signals resembling those of a [4Fe-4S] cluster with rhombic symmetry and g values at 1.90, 1.93, and 2.09, superimposed with a [3Fe-4S] cluster centered at g = 2.02. The [3Fe-4S] signal was observed independently of the [4Fe-4S] signal under oxidizing conditions. Mg-chelatase activity assays showed that the cluster is not catalytic. We suggest that the [4Fe-4S] and [3Fe-4S] signals originate from a single coordination site on the monomeric BchH protein and that the [4Fe-4S] cluster is sensitive to oxidation. It is speculated that the cluster participates in the switching between aerobic and anaerobic life of the proteobacteria.

  7. The Reductive Half-reaction of Xanthine Dehydrogenase from Rhodobacter capsulatus

    PubMed Central

    Hall, James; Reschke, Stefan; Cao, Hongnan; Leimkühler, Silke; Hille, Russ

    2014-01-01

    The kinetic properties of an E232Q variant of the xanthine dehydrogenase from Rhodobacter capsulatus have been examined to ascertain whether Glu232 in wild-type enzyme is protonated or unprotonated in the course of catalysis at neutral pH. We find that kred, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the variant, a result that is inconsistent with Glu232 being neutral in the active site of the wild-type enzyme. A comparison of the pH dependence of both kred and kred/Kd from reductive half-reaction experiments between wild-type and enzyme and the E232Q variant suggests that the ionized Glu232 of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pKa of substrate by two pH units and ensuring that at physiological pH the neutral form of substrate predominates in the Michaelis complex. A kinetic isotope study of the wild-type R. capsulatus enzyme indicates that, as previously determined for the bovine and chicken enzymes, product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release, however, is not as great in the bacterial enzyme as compared with the vertebrate forms. The results indicate that the bacterial and bovine enzymes catalyze the chemical step of the reaction to the same degree and that the faster turnover observed with the bacterial enzyme is due to a faster rate constant for product release than is seen with the vertebrate enzyme. PMID:25258317

  8. Enhanced Nitrogenase Activity in Strains of Rhodobacter capsulatus That Overexpress the rnf Genes

    PubMed Central

    Jeong, Ho-Sang; Jouanneau, Yves

    2000-01-01

    In the photosynthetic bacterium Rhodobacter capsulatus, a putative membrane-bound complex encoded by the rnfABCDGEH operon is thought to be dedicated to electron transport to nitrogenase. In this study, the whole rnf operon was cloned under the control of the nifH promoter in plasmid pNR117 and expressed in several rnf mutants. Complementation analysis demonstrated that transconjugants which integrated plasmid pNR117 directed effective biosynthesis of a functionally competent complex in R. capsulatus. Moreover, it was found that strains carrying pNR117 displayed nitrogenase activities 50 to 100% higher than the wild-type level. The results of radioactive labeling experiments indicated that the intracellular content of nitrogenase polypeptides was marginally altered in strains containing pNR117, whereas the levels of the RnfB and RnfC proteins present in the membrane were four- and twofold, respectively, higher than the wild-type level. Hence, the enhancement of in vivo nitrogenase activity was correlated with a commensurate overproduction of the Rnf polypeptides. In vitro nitrogenase assays performed in the presence of an artificial electron donor indicated that the catalytic activity of the enzyme was not increased in strains overproducing the Rnf polypeptides. It is proposed that the supply of reductants through the Rnf complex might be rate limiting for nitrogenase activity in vivo. Immunoprecipitation experiments performed on solubilized membrane proteins revealed that RnfB and RnfC are associated with each other and with additional polypeptides which may be components of the membrane-bound complex. PMID:10671439

  9. Optimizing multi-step B-side charge separation in photosynthetic reaction centers from Rhodobacter capsulatus

    SciTech Connect

    Faries, Kaitlyn M.; Kressel, Lucas L.; Dylla, Nicholas P.; Wander, Marc J.; Hanson, Deborah K.; Holten, Dewey; Laible, Philip D.; Kirmaier, Christine

    2016-02-01

    Using high-throughput methods for mutagenesis, protein isolation and charge-separation functionality, we have assayed 40 Rhodobacter capsulatus reaction center (RC) mutants for their P+ QB- yield (P is a dimer of bacteriochlorophylls and Q is a ubiquinone) as produced using the normally inactive B-side cofactors BB and HB (where B is a bacteriochlorophyll and H is a bacteriopheophytin). Two sets of mutants explore all possible residues at M131 (M polypeptide, native residue Val near HB) in tandem with either a fixed His or a fixed Asn at L181 (L polypeptide, native residue Phe near BB). A third set of mutants explores all possible residues at L181 with a fixed Glu at M131 that can form a hydrogen bond to HB. For each set of mutants, the results of a rapid millisecond screening assay that probes the yield of P+ QB- are compared among that set and to the other mutants reported here or previously. For a subset of eight mutants, the rate constants and yields of the individual B-side electron transfer processes are determined via transient absorption measurements spanning 100 fs to 50 μs. The resulting ranking of mutants for their yield of P+ QB- from ultrafast experiments is in good agreement with that obtained from the millisecond screening assay, further validating the efficient, high-throughput screen for B-side transmembrane charge separation. Results from mutants that individually show progress toward optimization of P+ HB- → P+ QB- electron transfer or initial P* → P+ HB- conversion highlight unmet challenges of optimizing both processes simultaneously.

  10. Draft Genome Sequences of Two Heat-Resistant Mutant Strains (A52 and B41) of the Photosynthetic Hydrogen-Producing Bacterium Rhodobacter capsulatus

    PubMed Central

    Gokce, Abdulmecit; Cakar, Zeynep Petek; Yucel, Meral; Ozcan, Orhan; Sencan, Sevde; Sertdemir, Ibrahim; Erguner, Bekir; Yuceturk, Betul; Sarac, Aydan; Yuksel, Bayram

    2016-01-01

    The draft genome sequences of two heat-resistant mutant strains, A52 and B41, derived from Rhodobacter capsulatus DSM 1710, and with different hydrogen production levels, are reported here. These sequences may help understand the molecular basis of heat resistance and hydrogen production in R. capsulatus. PMID:27284151

  11. Draft Genome Sequences of Two Heat-Resistant Mutant Strains (A52 and B41) of the Photosynthetic Hydrogen-Producing Bacterium Rhodobacter capsulatus.

    PubMed

    Gokce, Abdulmecit; Cakar, Zeynep Petek; Yucel, Meral; Ozcan, Orhan; Sencan, Sevde; Sertdemir, Ibrahim; Erguner, Bekir; Yuceturk, Betul; Sarac, Aydan; Yuksel, Bayram; Ozturk, Yavuz

    2016-06-09

    The draft genome sequences of two heat-resistant mutant strains, A52 and B41, derived from Rhodobacter capsulatus DSM 1710, and with different hydrogen production levels, are reported here. These sequences may help understand the molecular basis of heat resistance and hydrogen production in R. capsulatus. Copyright © 2016 Gokce et al.

  12. Bacterial Survival under Extreme UV Radiation: A Comparative Proteomics Study of Rhodobacter sp., Isolated from High Altitude Wetlands in Chile

    PubMed Central

    Pérez, Vilma; Hengst, Martha; Kurte, Lenka; Dorador, Cristina; Jeffrey, Wade H.; Wattiez, Ruddy; Molina, Veronica; Matallana-Surget, Sabine

    2017-01-01

    Salar de Huasco, defined as a polyextreme environment, is a high altitude saline wetland in the Chilean Altiplano (3800 m.a.s.l.), permanently exposed to the highest solar radiation doses registered in the world. We present here the first comparative proteomics study of a photoheterotrophic bacterium, Rhodobacter sp., isolated from this remote and hostile habitat. We developed an innovative experimental approach using different sources of radiation (in situ sunlight and UVB lamps), cut-off filters (Mylar, Lee filters) and a high-throughput, label-free quantitative proteomics method to comprehensively analyze the effect of seven spectral bands on protein regulation. A hierarchical cluster analysis of 40 common proteins revealed that all conditions containing the most damaging UVB radiation induced similar pattern of protein regulation compared with UVA and visible light spectral bands. Moreover, it appeared that the cellular adaptation of Rhodobacter sp. to osmotic stress encountered in the hypersaline environment from which it was originally isolated, might further a higher resistance to damaging UV radiation. Indeed, proteins involved in the synthesis and transport of key osmoprotectants, such as glycine betaine and inositol, were found in very high abundance under UV radiation compared to the dark control, suggesting the function of osmolytes as efficient reactive oxygen scavengers. Our study also revealed a RecA-independent response and a tightly regulated network of protein quality control involving proteases and chaperones to selectively degrade misfolded and/or damaged proteins. PMID:28694800

  13. Molecular Cloning and Expression Analysis of the Rhodobacter capsulatus sodB Gene, Encoding an Iron Superoxide Dismutase

    PubMed Central

    Cortez, Néstor; Carrillo, Néstor; Pasternak, Cécile; Balzer, Angelika; Klug, Gabriele

    1998-01-01

    Genetic complementation of a sodA sodB Escherichia coli mutant strain was used to clone Rhodobacter capsulatus genes involved in detoxification of superoxide radicals. After sequence analysis, 1 of the 16 identical clones obtained by this selection procedure was shown to contain an open reading frame with sequence similarity to that coding for Fe-containing superoxide dismutases (SodB). The R. capsulatus sodB gene was expressed in E. coli, and the nature of the metal ligand was confirmed by inhibitor sensitivity assays with lysates from both bacterial species. Activity staining of cleared Rhodobacter lysates resolved by polyacrylamide gel electrophoresis indicated that SodB was the only superoxide dismutase present in this phototrophic organism. The sodB gene was expressed at low levels in R. capsulatus cells grown under anaerobic or semiaerobic conditions, but expression was strongly induced upon exposure of the bacteria to air or to methyl viologen. Attempts to construct a sodB mutant in this organism by allelic exchange of the chromosomal copy of the gene with a suicide plasmid containing a mutated sodB gene were unsuccessful, strongly suggesting that the encoded superoxide dismutase is essential for viability of R. capsulatus in aerobic cultures. PMID:9765573

  14. Refinement of the high-resolution physical and genetic map of Rhodobacter capsulatus and genome surveys using blots of the cosmid encyclopedia.

    PubMed Central

    Fonstein, M; Koshy, E G; Nikolskaya, T; Mourachov, P; Haselkorn, R

    1995-01-01

    Cosmids from a library containing Rhodobacter capsulatus DNA fragments were previously ordered in two contigs: one corresponding to the chromosome and one to a 134 kb plasmid. This map contained 40 regions connected only by colony hybridization. To confirm the linkage and correct the map, the actual sizes of the overlaps were determined by blot-hybridization with Rhodobacter chromosomal DNA and by mapping of additional cosmids. Several revisions of the earlier map include single cosmid shifts and inversions. One additional gap in a cosmid contig was also found, raising the possibility that the chromosome is not a contiguous circle. About 2500 additional EcoRI,BamHI and HindIII restriction sites were added to the 560 EcoRV sites previously mapped onto the Rhodobacter chromosome, increasing the resolution of the physical map to the size of individual genes. Twenty-five new markers were located on the genetic map. The 48 markers now mapped represent nearly 300 genes and ORFs cloned from different species of Rhodobacter. The orientation of transcription of the four rrn operons was established using 16S rRNA- and 23S rRNA-specific probes and digestion with the rare-cutting enzyme, CeuI. Gel blots of 192 cosmids of the miniset of R.capsulatus digested with EcoRV were prepared. Such a hybridization template represents the whole genome cut into 560 DNA fragments varying in size from 0.4 to 25 kb. This template was used for high-resolution mapping of single genes, analysis of total genomic DNAs from related Rhodobacter strains and differentially expressed RNAs. Images PMID:7737133

  15. Novel Rhodobacter capsulatus genes required for the biogenesis of various c-type cytochromes.

    PubMed

    Deshmukh, M; Brasseur, G; Daldal, F

    2000-01-01

    Following chemical mutagenesis and screening for the inability to grow by photosynthesis and the absence of cyt cbb3 oxidase activity, two c-type cytochrome (cyt)-deficient mutants, 771 and K2, of Rhodobacter capsulatus were isolated. Both mutants were completely deficient in all known c-type cyts, and could not be complemented by the previously known cyt c biogenesis genes of R. capsulatus. Complementation of 771 and K2 with a wild-type chromosomal library led to the identification of two novel genes, cycJ and ccdA respectively. The cycJ is highly homologous to ccmE/cycJ, encountered in various Gram-negative species. Unlike in other species, where cycJ is a part of an operon essential for cyt c biogenesis, in R. capsulatus, it is located immediately downstream from argC, involved in arginine biosynthesis. Mutation of its universally conserved histidine residue, which is critical for its proposed haem chaperoning role, to an alanine led to loss of its function. All R. capsulatus cycJ mutants studied so far excrete copious amounts of coproporphyrin and protoporphyrin when grown on enriched media, suggesting that its product is also a component of the haem delivery branch of cyt c biogenesis in this species. In contrast, the R. capsulatus ccdA was homologous to the cyt c biogenesis gene ccdA, found in the gram-positive bacterium Bacillus subtilis, and to the central region of dipZ, encoding a protein disulphide reductase required for cyt c biogenesis in Escherichia coli. Membrane topology of CcdA was established in R. capsulatus using ccdA:phoA and ccdA :lacZ gene fusions. The deduced topology revealed that the two conserved cysteine residues of CcdA are, as predicted, membrane embedded. Mutagenesis of these cysteines showed that both are required for the function of CcdA in cyt c biogenesis. This study demonstrated for the first time that CcdA homologues are also required for cyt c biogenesis in some gram-negative bacteria such as R. capsulatus.

  16. nif gene expression studies in Rhodobacter capsulatus: ntrC-independent repression by high ammonium concentrations.

    PubMed

    Hübner, P; Masepohl, B; Klipp, W; Bickle, T A

    1993-10-01

    The expression of nif genes in Rhodobacter capsulatus depends on the two regulatory genes, rpoN and nifA, encoding a nif-specific alternative sigma factor of RNA polymerase and a nif-specific transcriptional activator, respectively. The expression of the rpoN gene itself is also RPON/NIFA dependent. In order to better characterize the regulation of nif gene induction, chromosomal nifH-, rpoN-, nifA1- and nifA2- lacZ fusions were constructed and the expression of these different nif-lacZ fusions was determined under photoheterotrophic conditions at different starting ammonium concentrations. The two nifA genes were found to be induced first, followed by nifH and finally by rpoN upon weak, medium and strong nitrogen starvation, respectively. This induction profile and the correlation between the expression of the different nif genes suggested that nifA1 expression is the limiting factor for nif gene induction. This hypothesis was tested by construction of different nifA1 overexpressing mutants. Contrary to the current model of nif gene expression in R. capsulatus, which predicted constitutive nif gene expression in such mutants, a strong repression of nifH and rpoN was found at high ammonium concentration. The low nifH expression under these conditions is unaffected by nifA2 and is not increased in a ntrC mutant, ruling out any role of NTRC as a mediator of this repression. This finding implies an additional, so far unidentified, regulation by fixed nitrogen in R. capsulatus. Changing the expression level of rpoN indicated that low levels of RPON are already sufficient for full nifH induction. The nifA1 and rpoN expression mutants were also tested for diazotrophic growth. Similar generation times were determined for the mutants and for the wild type, but diazotrophic growth of the nifA1 over-expressing ntrC mutant RCM14 did not start until after a prolonged lag phase of two to three days.

  17. Photo-biohydrogen production potential of Rhodobacter capsulatus-PK from wheat straw

    PubMed Central

    2013-01-01

    Background Biotechnological exploitation of lignocellulosic biomass is promising for sustainable and environmentally sound energy provision strategy because of the abundant availability of the renewable resources. Wheat straw (WS) comprising of 75-80% cellulose and hemicellulose is one of widely available, inexpensive and renewable lignocellulosic biomass types. The cellulosic and hemicellulose substrate can be hydrolyzed into monomeric sugars by chemical and/or biological methods. Results This study examined comparative potential of dilute acid and pre-ammonia pretreated and enzymatically hydrolyzed wheat straw (WS) for hydrogen production by purple non sulfur bacterium Rhodobacter capsulatus-PK. Gas production became noticeable after 14 h of inoculation in WS pretreated with 4% H2SO4. The detoxified liquid hydrolyzate (DLH) after overliming attained a production level of 372 mL-H2/L after 16 h under illumination of 120-150 W/m2 at 30 ± 2.0°C. Whereas the non-detoxified acid pretreated hydrolyzate (NDLH) of WS could produce only upto 254 mL-H2/L after 21 h post inoculation. Evolution of H2 became observable just after 10 ± 2.0 h of inoculation by employing 48 h age inoculum on the WS pretreated with 30% ammonia, hydrolyzed with cellulase 80 FPU/g and β-glucosidase 220 CbU/ml at 50°C. Upto 712 ml/L of culture was measured with continuous shaking for 24 h. The 47.5% and 64.2% higher hydrogen volume than the DLH and NDLH substrates, respectively appeared as a function of significantly higher monomeric sugar contents of the enzymatically hydrolyzed substrate and lesser/zero amounts of toxic derivatives including pH reducing agents. Conclusion Photofermentative hydrogen production from lignocellulosic waste is a feasible approach for eco-friendly sustainable supply of bioenergy in a cost-effective way. Results of this study provide new insight for addressing biotechnological exploitation of abundantly available and low-cost cellulosic substrates

  18. Mechanism of proton-coupled electron transfer for quinone (Q{sub B}) reduction in reaction centers of Rb sphaeroides

    SciTech Connect

    Graige, M.S.; Paddock, M.L; Feher, G.; Okamura, M.Y.; Bruce, J.M.

    1996-09-25

    The mechanism of the proton-coupled electron transfer reaction, Q{sub A}{sup -}Q{sub B}{sup -} + H{sup +} {yields} Q{sub A}(Q{sub B}H){sup -} (i.e. k{sup (2)}{sub AB}), was studied in reaction centers (RCs) from the photosynthetic bacterium Rb. sphaeroides by substituting quinones with different redox potentials into the Q{sub A} site. These substitutions change the driving force for electron transfer without affecting proton transfer rates or proton binding equilibria around the Q{sub B} site. The measured rate constants, k{sup (2)}{sub AB}, increased with increasing electron driving force (by a factor of 10 per 160 meV change in redox free energy). The proton-coupled electron transfer was modeled. The free energy dependencies of these possible mechanisms were predicted using Marcus theory and were compared to the observed dependence. The best agreement with the experimental data is given by a two-step mechanism in which fast reversible proton transfer is followed by rate limiting electron transfer. For this mechanism the observed free energy dependence for k{sup (2)}{sub AB} can be fitted using reasonable parameters of the Marcus theory. The free energy dependence predicted using a simple model for a concerted reaction also provides a reasonable fit to the data. 75 refs., 9 figs., 2 tabs.

  19. The light-induced carotenoid absorbance changes in Rhodopseudomonas sphaeroides: an analysis and interpretation of the band shifts.

    PubMed

    Symons, M; Swysen, C; Sybesma, C

    1977-12-23

    An analysis has been made of the spectrum of the carotenoid absorption band shift generated by continuous illumination of chromatophores of the GlC-mutant of Rhodopseudomonas sphaeroides at room temperature by means of three computer programs. There appears to be at least two pools of the same carotenoid, only one of which, comprising about 20% of the total carotenoid content, is responsible for the light-induced absorbance changes. The 'remaining' pool absorbs at wavelengths which were about 5 nm lower than those at which the 'changing' pool absorbs. This difference in absorption wavelength could indicate that the two pools are influenced differently by permanent local electric fields. The electrochromic origin of the absorbance changes has been demonstrated directly; the isosbestic points of the absorption difference spectrum move to shorter wavelengths upon lowering of the light-induced electric field. Band shifts up to 1.7 nm were observed. A comparison of the light-induced absorbance changes with a KCl-valinomycin-induced diffusion potential has been used to calibrate the electrochromic shifts. The calibration value appeared to be 137 +/- 6 mV per nm shift.

  20. Isolation and characterization of the pigment-protein complexes of Rhodopseudomonas sphaeroides by lithium dodecyl sulfate/polyacrylamide gel electrophoresis.

    PubMed

    Broglie, R M; Hunter, C N; Delepelaire, P; Niederman, R A; Chua, N H; Clayton, R K

    1980-01-01

    When purified photosynthetic membranes from Rhodopseudomonas sphaeroides were treated with lithium dodecyl sulfate and subjected to polyacrylamide gel electrophoresis at 4 degrees C, up to 11 pigment-protein complexes were resolved. Absorption spectra revealed that the smallest complex contained reaction center pigments and the others contained the antenna components B850 and B875 in various proportions. Of these antenna complexes, the largest was almost entirely B850 and the smallest contained only B875. After solubilization at 100 degrees C and electrophoresis on polyacrylamide gradient gels, the B850 complex gave rise to two polypeptide components migrating with apparent Mr of 10,000 and 8000, whereas with the B875 complex, two components were observed with apparent Mr of 12,000 and 8000. The reaction center complex gave rise to only the 24 and 21 kilodalton polypeptide subunits. Fluorescence emission spectra showed maxima at 872 and 902 nm for B850 and B875, respectively. Analyses of bacteriochlorophyll a and carotenoids indicated that, in the B875 complex, two molecules of each of these pigments are associated with the two polypeptides. The associations of B850 and B875 in large and small complexes obtained by lithium dodecyl sulfate treatment are consistent with models of their organization within the membrane.

  1. Electrochromic absorbance changes of photosynthetic pigments in Rhodopseudomonas sphaeroides. I. Stimulation by secondary electron transport at low temperature.

    PubMed

    de Grooth, B G; Amesz, J

    1977-11-17

    Light-induced absorbance changes were measured at temperatures between --30 and --55 degrees C in chromatophores of Rhodopseudomonas sphaeroides. Absorbance changes due to photooxidation of reaction center bacteriochlorophyll (P-870) were accompanied by a red shift of the absorption bands of a carotenoid. The red shift was inhibited by gramicidin D. The kinetics of P-870 indicated electron transport from the "primary" to a secondary electron acceptor. This electron transport was slowed down by lowering the temperature or increasing the pH of the suspension. Electron transport from soluble cytochrome c to P-870+ occurred in less purified chromatophore preparations. This electron transport was accompanied by a relatively large increase of the carotenoid absorbance change. This agrees with the hypothesis that P-870 is located inside the membrane, so that an additional membrane potential is generated upon transfer of an electron from cytochrome to P-870+. A strong stimulation of the carotenoid changes (more than 10-fold in some experiments) and pronounced band shifts of bacteriochlorophyll B-850 were observed upon illumination in the presence of artifical donor-acceptor systems. Reduced N-methylphenazonium methosulphate (PMS) and N,N,N',N'-tetramethyl-p-phenylene-diamine (TMPD) were fairly efficient donors, whereas endogenous ubiquinone and oxidized PMS acted as secondary acceptor. These results indicate the generation of large membrane potentials at low temperature, caused by sustained electron transport across the chromatophore membrane. The artificial probe, merocyanine MC-V did not show electrochromic band shifts at low temperature.

  2. High yield single stage conversion of glucose to hydrogen by photofermentation with continuous cultures of Rhodobacter capsulatus JP91.

    PubMed

    Abo-Hashesh, Mona; Desaunay, Nicolas; Hallenbeck, Patrick C

    2013-01-01

    Photofermentative hydrogen (H(2)) production from glucose with the photosynthetic bacterium Rhodobacter capsulatus JP91 (hup(-)) was examined using a photobioreactor operated in continuous mode. Stable and high hydrogen yields on glucose were obtained at three different retention times (HRTs; 24, 48 and 72 h). The H(2) production rates, varying between 0.57 and 0.81 mmol/h, and optical densities (OD(600 nm)) were similar for the different HRTs examined. However, the rate of glucose consumption was influenced by HRT being greater at HRT 24h than HRTs 48 and 72 h. The highest hydrogen yield, 9.0 ± 1.2 mol H(2)/mol glucose, was obtained at 48 h HRT. These results show that single stage photofermentative hydrogen production from glucose using photobioreactors operated in continuous culture mode gives high, nearly stoichiometric yields of hydrogen from glucose, and thus is considerably more promising than either two stage photofermentation or co-culture approaches.

  3. Tellurite uptake by cells of the facultative phototroph Rhodobacter capsulatus is a Delta pH-dependent process.

    PubMed

    Borsetti, Francesca; Toninello, Antonio; Zannoni, Davide

    2003-11-20

    The uptake by light-grown cells of Rhodobacter capsulatus of the highly toxic metalloid oxyanion tellurite (TeO(3)(2-)) was examined. We show that tellurite is rapidly taken up by illuminated cells in a process which is inhibited by the protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) and by the K(+)/H(+) exchanger nigericin. Notably, the light-driven membrane potential (Delta psi) is enhanced by K(2)TeO(3)> or =200 microM. Further, tellurite uptake is largely insensitive to valinomycin, strongly repressed by the sulfhydryl reagent N-ethylethylmaleimide (NEM) and competitively inhibited by phosphate. We conclude that tellurite is transported into cells by a Delta pH-dependent, non-electrogenic process which is likely to involve the phosphate transporter (PiT family).

  4. The response of antioxidant systems in Nostoc sphaeroides against UV-B radiation and the protective effects of exogenous antioxidants

    NASA Astrophysics Data System (ADS)

    Wang, Gaohong; Hu, Chunxiang; Li, Dunhai; Zhang, Delu; Li, Xiaoyan; Chen, Kun; Liu, Yongding

    UV radiation is one of many harmful factors found in space that are detrimental to organisms on earth in space exploration. In the present work, we examined the role of antioxidant system in Nostoc sphaeroides Kütz (Cyanobacterium) and the effects of exogenously applied antioxidant molecules on its photosynthetic rate under UV-B radiation. It was found that UV-B radiation promoted the activity of antioxidant system to protect photosystem II (PSII) and exogenously applied antioxidant: sodium nitroprusside (SNP) and N-acetylcysteine (NAC) had an obvious protection on PSII activity under UV-B radiation. The activity of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7) and content of MDA (malondialdehyde) and ASC (ascorbate) were improved by 0.5 mM and 1 mM SNP, but 0.1 mM SNP decreased the activity of antioxidant system. Addition of exogenous NAC decreased the activity of SOD, POD, CAT and the content MDA and ASC. In contrast, exogenously applied NAC increased GSH content. The results suggest that exogenous SNP and NAC may protect algae by different mechanisms: SNP may play double roles as both sources of reactive free radicals as well as ROS scavengers in mediating the protective role of PSII on algae under UV-B radiation. On the other hand, NAC functions as an antioxidant or precursor of glutathione, which could protect PSII directly from UV-B radiation.

  5. Magnesium chelatase: association with ribosomes and mutant complementation studies identify barley subunit Xantha-G as a functional counterpart of Rhodobacter subunit BchD.

    PubMed

    Kannangara, C G; Vothknecht, U C; Hansson, M; von Wettstein, D

    1997-03-18

    Magnesium chelatase catalyses the insertion of Mg2+ into protoporphyrin and is found exclusively in organisms which synthesise chlorophyll or bacteriochlorophyll. Soluble protein preparations containing >10 mg protein/ml, obtained by gentle lysis of barley plastids and Rhodobacter sphaeroplasts, inserted Mg2+ into deuteroporphyrin IX in the presence of ATP at rates of 40 and 8 pmoles/mg protein per min, respectively. With barley extracts optimal activity was observed with 40 mM Mg2+. The activity was inhibited by micromolar concentrations of chloramphenicol. Mutations in each of three genetic loci, Xantha-f, -g and -h, in barley destroyed the activity. However, Mg-chelatase activity was reconstituted in vitro by combining pairwise the plastid stroma protein preparations from non-leaky xantha-f -g and -h mutants. This establishes that, as in Rhodobacter, three proteins are required for the insertion of magnesium into protoporphyrin IX in barley. These three proteins, Xantha-F, -G and -H, are referred to as Mg-chelatase subunits and they appear to exist separate from each other in vivo. Active preparations from barley and Rhodobacter yielded pellet and supernatant fractions upon centrifugation for 90 min at 272,000 x g. The pellet and the supernatant were inactive when assayed separately, but when they were combined activity was restored. Differential distribution of the Mg-chelatase subunits in the fractions was established by in vitro complementation assays using stroma protein from the xantha-f, -g, and -h mutants. Xantha-G protein was confined to the pellet fraction, while Xantha-H was confined to the supernatant. Reconstitution assays using purified recombinant BchH, BchI and partially purified BchD revealed that the pellet fraction from Rhodobacter contained the BchD subunit. The pellet fractions from both barley and Rhodobacter contained ribosomes and had an A260:A280 ratio of 1.8. On sucrose density gradients both Xantha-G and BchD subunits migrated with the

  6. Purification and assays of Rhodobacter capsulatus RegB-RegA two-component signal transduction system.

    PubMed

    Swem, Lee R; Swem, Danielle L; Wu, Jiang; Bauer, Carl E

    2007-01-01

    Two-component signal-transduction systems, composed of a histidine-sensor kinase and a DNA-binding response regulator, allow bacteria to detect environmental changes and adjust cellular physiology to live more efficiently in a broad distribution of niches. Although many two-component signal-transduction systems are known, a limited number of signals that stimulate these systems have been discovered. This chapter describes the purification and characterization of the predominant two-component signal-transduction system utilized by Rhodobacter capsulatus, a nonsulfur purple photosynthetic bacterium. Specifically, we explain the overexpression, detergent solubilization, and purification of the full-length membrane-spanning histidine-sensor kinase RegB. We also provide a method to measure autophosphorylation of RegB and discern the effect of its signal molecule, ubiquinone, on autophosphorylation levels. In addition we describe the overexpression and purification of the cognate response regulator RegA and a technique used to visualize the phosphotransfer reaction from RegB to RegA.

  7. Role for draTG and rnf Genes in Reduction of 2,4-Dinitrophenol by Rhodobacter capsulatus

    PubMed Central

    Sáez, Lara P.; García, Patricia; Martínez-Luque, Manuel; Klipp, Werner; Blasco, Rafael; Castillo, Francisco

    2001-01-01

    The phototrophic bacterium Rhodobacter capsulatus is able to reduce 2,4-dinitrophenol (DNP) to 2-amino-4-nitrophenol enzymatically and thus can grow in the presence of this uncoupler. DNP reduction was switched off by glutamine or ammonium, but this short-term regulation did not take place in a draTG deletion mutant. Nevertheless, the target of DraTG does not seem to be the nitrophenol reductase itself since the ammonium shock did not inactivate the enzyme. In addition to this short-term regulation, ammonium or glutamine repressed the DNP reduction system. Mutants of R. capsulatus affected in ntrC or rpoN exhibited a 10-fold decrease in nitroreductase activity in vitro but almost no DNP activity in vivo. In addition, mutants affected in rnfA or rnfC, which are also under NtrC control and encode components involved in electron transfer to nitrogenase, were unable to metabolize DNP. These results indicate that NtrC regulates dinitrophenol reduction in R. capsulatus, either directly or indirectly, by controlling expression of the Rnf proteins. Therefore, the Rnf complex seems to supply electrons for both nitrogen fixation and DNP reduction. PMID:11160111

  8. Vitamin B12 regulates photosystem gene expression via the CrtJ antirepressor AerR in Rhodobacter capsulatus

    PubMed Central

    Cheng, Zhuo; Li, Keran; Hammad, Loubna A.; Karty, Jonathan A.; Bauer, Carl E.

    2014-01-01

    Summary The tetrapyrroles heme, bacteriochlorophyll and cobalamin (B12) exhibit a complex interrelationship regarding their synthesis. In this study, we demonstrate that AerR functions as an antirepressor of the tetrapyrrole regulator CrtJ. We show that purified AerR contains B12 that is bound to a conserved histidine (His145) in AerR. The interaction of AerR to CrtJ was further demonstrated in vitro by pull down experiments using AerR as bait and quantified using microscale thermophoresis. DNase I DNA footprint assays show that AerR containing B12 inhibits CrtJ binding to the bchC promoter. We further show that bchC expression is greatly repressed in a B12 auxotroph of Rhodobacter capsulatus and that B12 regulation of gene expression is mediated by AerR’s ability to function as an antirepressor of CrtJ. This study thus provides a mechanism for how the essential tetrapyrrole, cobalamin controls the synthesis of bacteriochlorophyll, an essential component of the photosystem. PMID:24329562

  9. Open reading frame 176 in the photosynthesis gene cluster of Rhodobacter capsulatus encodes idi, a gene for isopentenyl diphosphate isomerase.

    PubMed Central

    Hahn, F M; Baker, J A; Poulter, C D

    1996-01-01

    Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A database search based on probes from the highly conserved regions in three eukaryotic IPP isomerases revealed substantial similarity with ORF176 in the photosynthesis gene cluster in Rhodobacter capsulatus. The open reading frame was cloned into an Escherichia coli expression vector. The encoded 20-kDa protein, which was purified in two steps by ion exchange and hydrophobic interaction chromatography, catalyzed the interconversion of IPP and dimethylallyl diphosphate. Thus, the photosynthesis gene cluster encodes all of the enzymes required to incorporate IPP into the ultimate carotenoid and bacteriochlorophyll metabolites in R. capsulatus. More recent searches uncovered additional putative open reading frames for IPP isomerase in seed-bearing plants (Oryza sativa, Arabadopsis thaliana, and Clarkia breweri), a worm (Caenorhabiditis elegans), and another eubacterium (Escherichia coli). The R. capsulatus enzyme is the smallest of the IPP isomerases to be identified thus far and may consist mostly of a fundamental catalytic core for the enzyme. PMID:8550491

  10. Identification and mapping of nitrogen fixation genes of Rhodobacter capsulatus: duplication of a nifA-nifB region.

    PubMed Central

    Klipp, W; Masepohl, B; Pühler, A

    1988-01-01

    Rhodobacter capsulatus mutants unable to fix nitrogen were isolated by random transposon Tn5 mutagenesis. The Tn5 insertion sites of 30 Nif- mutants were mapped within three unlinked chromosomal regions designated A, B, and C. The majority of Tn5 insertions (21 mutants) map within nif region A, characterized by two ClaI fragments of 2.5 and 25 kilobases (kb). The 17-kb ClaI fragment of nif region B contains six nif::Tn5 insertions, and the three remaining mutations are located on a 32-kb ClaI fragment of nif region C. Hybridization experiments using all 17 Klebsiella pneumoniae nif genes individually as probes revealed homology to nifE, nifS, nifA, and nifB in nif region A. The nifHDK genes were localized in nif region B. About 2 kb away from this operon, a second copy of the DNA fragments homologous to nifA and nifB, originally found in nif region A, was identified. Images PMID:2828320

  11. Identification and sequence analysis of genes involved in late steps in cobalamin (vitamin B12) synthesis in Rhodobacter capsulatus.

    PubMed Central

    Pollich, M; Klug, G

    1995-01-01

    A 6.4-kb region of a 6.8-kb BamHI fragment carrying Rhodobacter capsulatus genes involved in late steps of cobalamin synthesis has been sequenced. The nucleotide sequence and genetic analysis revealed that this fragment contains eight genes arranged in at least three operons. Five of these eight genes show homology to genes involved in the cobalamin synthesis of Pseudomonas denitrificans and Salmonella typhimurium. The arrangement of these homologous genes differs considerably in the three genera. Upstream of five overlapping genes (named bluFEDCB), a promoter activity could be detected by using lacZ fusions. This promoter shows no regulation by oxygen, vitamin B12 (cobalamin), or cobinamide. Disruption of the bluE gene by a Tn5 insertion (strain AH2) results in reduced expression of the puf and puc operons, which encode pigment-binding proteins of the photosynthetic apparatus. The mutant strain AH2 can be corrected to a wild-type-like phenotype by addition of vitamin B12 or cobinamide dicyanide. Disruption of the bluB gene by an interposon (strain BB1) also disturbs the formation of the photosynthetic apparatus. The mutation of strain BB1 can be corrected by vitamin B12 but not by cobinamide. We propose that a lack of cobalamin results in deregulation and a decreased formation of the photosynthetic apparatus. PMID:7635831

  12. A mutation in a Rhodobacter capsulatus gene encoding an integration host factor-like protein impairs in vivo hydrogenase expression.

    PubMed Central

    Toussaint, B; Bosc, C; Richaud, P; Colbeau, A; Vignais, P M

    1991-01-01

    A gene capable of encoding a protein sharing 45% identical amino acids with the alpha subunit of the integration host factor (IHF) of Escherichia coli was isolated from the photosynthetic bacterium Rhodobacter capsulatus strain B10 by complementation of a hydrogenase-deficient (Hup-) mutant, IR4. A DNA fragment of 274 base pairs containing an IHF binding consensus sequence, isolated from the promoter region of the hydrogenase structural genes (hupSL), was shown by gel retardation assays to bind the IHF protein from E. coli. The product of the R. capsulatus gene was shown to bind specifically to the 274-base-pair DNA fragment from the hupSL promoter. By analogy to the E. coli himA gene, which encodes the alpha subunit of IHF, the gene complementing the IR4 mutant was named himA of R. capsulatus. The wild-type himA gene, cloned in plasmid pBO2, was introduced into the IR4 strain and shown to restore, in trans, hydrogenase activity and autotrophic growth in the mutant. In IR4, a C----T transition mutation had replaced Arg-8 by Cys-8. Gel mobility shifts of the 274-base-pair DNA fragment, not observed with the himA gene product of IR4, were restored with extracts from IR4(pBO2) cells, containing the himA gene on the recombinant plasmid pBO2. Images PMID:1961742

  13. Proteomic analysis and identification of the structural and regulatory proteins of the Rhodobacter capsulatus gene transfer agent.

    PubMed

    Chen, Frank; Spano, Anthony; Goodman, Benjamin E; Blasier, Kiev R; Sabat, Agnes; Jeffery, Erin; Norris, Andrew; Shabanowitz, Jeffrey; Hunt, Donald F; Lebedev, Nikolai

    2009-02-01

    The gene transfer agent of Rhodobacter capsulatus (GTA) is a unique phage-like particle that exchanges genetic information between members of this same species of bacterium. Besides being an excellent tool for genetic mapping, the GTA has a number of advantages for biotechnological and nanoengineering purposes. To facilitate the GTA purification and identify the proteins involved in GTA expression, assembly and regulation, in the present work we construct and transform into R. capsulatus Y262 a gene coding for a C-terminally His-tagged capsid protein. The constructed protein was expressed in the cells, assembled into chimeric GTA particles inside the cells and excreted from the cells into surrounding medium. Transmission electron micrographs of phosphotungstate-stained, NiNTA-purified chimeric GTA confirm that its structure is similar to normal GTA particles, with many particles composed both of a head and a tail. The mass spectrometric proteomic analysis of polypeptides present in the GTA recovered outside the cells shows that GTA is composed of at least 9 proteins represented in the GTA gene cluster including proteins coded for by Orf's 3, 5, 6-9, 11, 13, and 15.

  14. 15N electron nuclear double resonance of the primary donor cation radical P+.865 in reaction centers of Rhodopseudomonas sphaeroides: additional evidence for the dimer model.

    PubMed Central

    Lubitz, W; Isaacson, R A; Abresch, E C; Feher, G

    1984-01-01

    Four 15N hyperfine coupling constants, including signs, have been measured by electron nuclear double resonance (ENDOR) and electron nuclear nuclear triple resonance (TRIPLE) for the bacteriochlorophyll a radical cation, BChla+., in vitro and for the light-induced primary donor radical cation, P+.865, in reaction centers of Rhodopseudomonas sphaeroides R-26. A comparison of the data shows that the hyperfine coupling constants have the same sign in both radicals and are, on the average, smaller by a factor of 2 in P+.865. These results provide additional evidence that P+.865 is a bacteriochlorophyll dimer and are in contradiction with the monomer structure of P+.865 recently proposed by O'Malley and Babcock. The reduction factors of the individual 15N couplings, together with the evidence from proton ENDOR data and molecular orbital calculations, indicate a dimer structure in which only two rings (either I and I or III and III) of the bacteriochlorophyll macrocycles overlap. PMID:6096857

  15. Effector-Mediated Interaction of CbbRI and CbbRII Regulators with Target Sequences in Rhodobacter capsulatus

    PubMed Central

    Dubbs, Padungsri; Dubbs, James M.; Tabita, F. Robert

    2004-01-01

    In Rhodobacter capsulatus, genes encoding enzymes of the Calvin-Benson-Bassham reductive pentose phosphate pathway are located in the cbbI and cbbII operons. Each operon contains a divergently transcribed LysR-type transcriptional activator (CbbRI and CbbRII) that regulates the expression of its cognate cbb promoter in response to an as yet unidentified effector molecule(s). Both CbbRI and CbbRII were purified, and the ability of a variety of potential effector molecules to induce changes in their DNA binding properties at their target promoters was assessed. The responses of CbbRI and CbbRII to potential effectors were not identical. In gel mobility shift assays, the affinity of both CbbRI and CbbRII for their target promoters was enhanced in the presence of ribulose-1,5-bisphosphate (RuBP), phosphoenolpyruvate, 3-phosphoglycerate, 2-phosphoglycolate. ATP, 2-phosphoglycerate, and KH2PO4 were found to enhance only CbbRI binding, while fructose-1,6-bisphosphate enhanced the binding of only CbbRII. The DNase I footprint of CbbRI was reduced in the presence of RuBP, while reductions in the CbbRII DNase I footprint were induced by fructose-1,6-bisphosphate, 3-phosphoglycerate, and KH2PO4. The current in vitro results plus recent in vivo studies suggest that CbbR-mediated regulation of cbb transcription is controlled by multiple metabolic signals in R. capsulatus. This control reflects not only intracellular levels of Calvin-Benson-Bassham cycle metabolic intermediates but also the fixed (organic) carbon status and energy charge of the cell. PMID:15547275

  16. The reductive half-reaction of xanthine dehydrogenase from Rhodobacter capsulatus: the role of Glu232 in catalysis.

    PubMed

    Hall, James; Reschke, Stefan; Cao, Hongnan; Leimkühler, Silke; Hille, Russ

    2014-11-14

    The kinetic properties of an E232Q variant of the xanthine dehydrogenase from Rhodobacter capsulatus have been examined to ascertain whether Glu(232) in wild-type enzyme is protonated or unprotonated in the course of catalysis at neutral pH. We find that kred, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the variant, a result that is inconsistent with Glu(232) being neutral in the active site of the wild-type enzyme. A comparison of the pH dependence of both kred and kred/Kd from reductive half-reaction experiments between wild-type and enzyme and the E232Q variant suggests that the ionized Glu(232) of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pKa of substrate by two pH units and ensuring that at physiological pH the neutral form of substrate predominates in the Michaelis complex. A kinetic isotope study of the wild-type R. capsulatus enzyme indicates that, as previously determined for the bovine and chicken enzymes, product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release, however, is not as great in the bacterial enzyme as compared with the vertebrate forms. The results indicate that the bacterial and bovine enzymes catalyze the chemical step of the reaction to the same degree and that the faster turnover observed with the bacterial enzyme is due to a faster rate constant for product release than is seen with the vertebrate enzyme.

  17. Electrochromic responses of carotenoid absorbance bands in purified light-harvesting complexes from Rhodobacter capsulatus reconstituted into liposomes.

    PubMed

    Goodwin, M G; Jackson, J B

    1993-09-13

    Light-Harvesting Complexes I and II (LHI and LHII) were extracted from chromatophores of Rhodobacter capsulatus, purified in Triton X-100 and reconstituted into phospholipid vesicles. Application of membrane potentials (K+ diffusion potentials) to LHII proteoliposomes led to absorbance changes in the carotenoid bands which were spectrally similar to those in chromatophores. These (electrochromic) absorbance changes were linear with the applied membrane potential between -107 mV and +105 mV. The data were consistent with the existence of two forms of carotenoid in LHII. One form, comprising 2/3 of the total and with a long wavelength absorbance maximum at 510 nm, was not significantly affected by membrane potential. The other component, comprising 1/3 of the total and with a long wavelength absorbance maximum at 516.5 nm, was shifted by approx. 1.6 nm to the red by a membrane potential of 105 mV. Reduction of the B800 bacteriochlorophyll in LHII with NaBH4 before reconstitution did not affect the absorbance spectrum of the carotenoids and it did not affect their response to applied membrane potentials in proteoliposomes. Although the electrochromically-sensitive carotenoids might be associated with B800, interactions with the bacteriochlorophyll are perhaps not the cause of the polarisation of the carotenoid that is responsible for the linearity of the response. The carotenoids in reconstituted LHI complexes were not detectably electrochromic. The electrochromic absorbance changes of carotenoids in LHII could be useful for membrane potential measurement in liposomes containing ion-translocating proteins.

  18. The NprA nitroreductase required for 2,4-dinitrophenol reduction in Rhodobacter capsulatus is a dihydropteridine reductase.

    PubMed

    Pérez-Reinado, Eva; Roldán, María Dolores; Castillo, Francisco; Moreno-Vivián, Conrado

    2008-11-01

    The Rhodobacter capsulatus nprA gene codes for a putative nitroreductase. A recombinant His(6)-NprA protein was overproduced in Escherichia coli and purified by affinity chromatography. This protein contained FMN and showed nitroreductase activity with a wide range of nitroaromatic compounds, such as 2-nitrophenol, 2,4-dinitrophenol, 2,6-dinitrophenol, 2,4,6-trinitrophenol (picric acid), 2,4-dinitrobenzoate and 2,4-dinitrotoluene, and with the nitrofuran derivatives nitrofurazone and furazolidone. NADPH was the main electron donor and the ortho nitro group was preferably reduced to the corresponding amino derivative. The apparent K(m) values of NprA for NADPH, 2,4-dinitrophenol, picric acid and furazolidone were 40 microM, 78 microM, 72 microM and 83 microM, respectively, at pH and temperature optima (pH 6.5, 30 degrees C). Escherichia coli cells overproducing the NprA protein were much more sensitive to the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) used in cancer therapy than non-transformed cells. NprA showed the highest activity with the quinonoid form of 6,7-dimethyl-7,8-dihydropterine as substrate, so that NprA may be involved in the synthesis of tetrahydrobiopterin in R. capsulatus. Expression of a transcriptional nprA-lacZ gene fusion was induced by phenylalanine or tyrosine, but not by other amino acids like glutamate or alanine. Furthermore, both nitroreductase activity and phenylalanine assimilation were inhibited in vivo by ammonium. A mutant defective in the nprA gene showed better growth rate with Phe or Tyr as nitrogen source than the wild-type strain, although both strains showed similar growth in media with Glu or without added nitrogen. These results suggest that the NprA nitroreductase may act in vivo as a dihydropteridine reductase involved in aromatic amino acids metabolism.

  19. Effector-mediated interaction of CbbRI and CbbRII regulators with target sequences in Rhodobacter capsulatus.

    PubMed

    Dubbs, Padungsri; Dubbs, James M; Tabita, F Robert

    2004-12-01

    In Rhodobacter capsulatus, genes encoding enzymes of the Calvin-Benson-Bassham reductive pentose phosphate pathway are located in the cbb(I) and cbb(II) operons. Each operon contains a divergently transcribed LysR-type transcriptional activator (CbbR(I) and CbbR(II)) that regulates the expression of its cognate cbb promoter in response to an as yet unidentified effector molecule(s). Both CbbR(I) and CbbR(II) were purified, and the ability of a variety of potential effector molecules to induce changes in their DNA binding properties at their target promoters was assessed. The responses of CbbR(I) and CbbR(II) to potential effectors were not identical. In gel mobility shift assays, the affinity of both CbbR(I) and CbbR(II) for their target promoters was enhanced in the presence of ribulose-1,5-bisphosphate (RuBP), phosphoenolpyruvate, 3-phosphoglycerate, 2-phosphoglycolate. ATP, 2-phosphoglycerate, and KH(2)PO(4) were found to enhance only CbbR(I) binding, while fructose-1,6-bisphosphate enhanced the binding of only CbbR(II). The DNase I footprint of CbbR(I) was reduced in the presence of RuBP, while reductions in the CbbR(II) DNase I footprint were induced by fructose-1,6-bisphosphate, 3-phosphoglycerate, and KH(2)PO(4). The current in vitro results plus recent in vivo studies suggest that CbbR-mediated regulation of cbb transcription is controlled by multiple metabolic signals in R. capsulatus. This control reflects not only intracellular levels of Calvin-Benson-Bassham cycle metabolic intermediates but also the fixed (organic) carbon status and energy charge of the cell.

  20. A [2Fe-2S] ferredoxin (FdVI) is essential for growth of the photosynthetic bacterium Rhodobacter capsulatus.

    PubMed Central

    Armengaud, J; Meyer, C; Jouanneau, Y

    1997-01-01

    The physiological function of Rhodobacter capsulatus FdVI, a [2Fe-2S] ferredoxin, was investigated by the cloning, sequence analysis, and mutagenesis of its structural gene, called fdxE. The DNA region surrounding fdxE was mapped, and the nucleotide sequence of a 4.2-kb fragment was determined. fdxE is preceded by a sequence that is very similar to a sigma54 recognition site and is followed by a putative transcription stop signal, suggesting that fdxE forms a separate cistron. Two open reading frames were identified upstream and downstream of fdxE and were named ORFE0 and ORFE1, respectively. The former may encode a polypeptide having 34% similarity with HtrA, a serine protease found in enteric bacteria. ORFE1 is homologous to purU, a gene involved in purine biosynthesis. Interposon mutagenesis of fdxE was unsuccessful when attempted on the wild-type strain B10. Disruption of fdxE could be achieved only in strains harboring an additional copy of fdxE on a plasmid. Mutants obtained in this way and carrying a plasmid-borne copy of fdxE under the control of the nifH promoter grew only in N-free medium, thus demonstrating that fdxE expression is required for growth. Nevertheless, such mutants were found to spontaneously revert at a frequency of 5 x 10(-6) to an apparent wild-type phenotype, although they contained no detectable amount of FdVI. Taken together, the results indicate that FdVI is required for an essential metabolic function in R. capsulatus and that this FdVI dependence could be relieved by a single-mutation event. In accordance, FdVI biosynthesis was found to be constitutive in R. capsulatus. PMID:9150228

  1. Rhodobacter capsulatus Catalyzes Light-Dependent Fe(II) Oxidation under Anaerobic Conditions as a Potential Detoxification Mechanism▿

    PubMed Central

    Poulain, Alexandre J.; Newman, Dianne K.

    2009-01-01

    Diverse bacteria are known to oxidize millimolar concentrations of ferrous iron [Fe(II)] under anaerobic conditions, both phototrophically and chemotrophically. Yet whether they can do this under conditions that are relevant to natural systems is understood less well. In this study, we tested how light, Fe(II) speciation, pH, and salinity affected the rate of Fe(II) oxidation by Rhodobacter capsulatus SB1003. Although R. capsulatus cannot grow photoautotrophically on Fe(II), it oxidizes Fe(II) at rates comparable to those of bacteria that do grow photoautotrophically on Fe(II) as soon as it is exposed to light, provided it has a functional photosystem. Chelation of Fe(II) by diverse organic ligands promotes Fe(II) oxidation, and as the pH increases, so does the oxidation rate, except in the presence of nitrilotriacetate; nonchelated forms of Fe(II) are also more rapidly oxidized at higher pH. Salt concentrations typical of marine environments inhibit Fe(II) oxidation. When growing photoheterotrophically on humic substances, R. capsulatus is highly sensitive to low concentrations of Fe(II); it is inhibited in the presence of concentrations as low as 5 μM. The product of Fe(II) oxidation, ferric iron, does not hamper growth under these conditions. When other parameters, such as pH or the presence of chelators, are adjusted to promote Fe(II) oxidation, the growth inhibition effect of Fe(II) is alleviated. Together, these results suggest that Fe(II) is toxic to R. capsulatus growing under strictly anaerobic conditions and that Fe(II) oxidation alleviates this toxicity. PMID:19717624

  2. Characterization of Rhodobacter capsulatus genes encoding a molybdenum transport system and putative molybdenum-pterin-binding proteins.

    PubMed Central

    Wang, G; Angermüller, S; Klipp, W

    1993-01-01

    The alternative, heterometal-free nitrogenase of Rhodobacter capsulatus is repressed by traces of molybdenum in the medium. Strains carrying mutations located downstream of nifB copy II were able to express the alternative nitrogenase even in the presence of high molybdate concentrations. DNA sequence analysis of a 5.5-kb fragment of this region revealed six open reading frames, designated modABCD, mopA, and mopB. The gene products of modB and modC are homologous to ChlJ and ChlD of Escherichia coli and represent an integral membrane protein and an ATP-binding protein typical of high-affinity transport systems, respectively. ModA and ModD exhibited no homology to known proteins, but a leader peptide characteristic of proteins cleaved during export to the periplasm is present in ModA, indicating that ModA might be a periplasmic molybdate-binding protein. The MopA and MopB proteins showed a high degree of amino acid sequence homology to each other. Both proteins contained a tandem repeat of a domain encompassing 70 amino acid residues, which had significant sequence similarity to low-molecular-weight molybdenum-pterin-binding proteins from Clostridium pasteurianum. Compared with that for the parental nifHDK deletion strain, the molybdenum concentrations necessary to repress the alternative nitrogenase were increased 4-fold in a modD mutant and 500-fold in modA, modB, and modC mutants. No significant inhibition of the heterometal-free nitrogenase by molybdate was observed for mopA mopB double mutants. The uptake of molybdenum by mod and mop mutants was estimated by measuring the activity of the conventional molybdenum-containing nitrogenase. Molybdenum transport was not affected in a mopA mopB double mutant, whereas strains carrying lesions in the binding-protein-dependent transport system were impaired in molybdenum uptake. PMID:8491722

  3. Nucleotide sequence of the Rhodobacter capsulatus fruK gene, which encodes fructose-1-phosphate kinase: evidence for a kinase superfamily including both phosphofructokinases of Escherichia coli.

    PubMed Central

    Wu, L F; Reizer, A; Reizer, J; Cai, B; Tomich, J M; Saier, M H

    1991-01-01

    The fruK gene encoding fructose-1-phosphate kinase (FruK), located within the fructose (fru)-catabolic operon of Rhodobacter capsulatus, was sequenced. FruK of R. capsulatus (316 amino acids; molecular weight = 31,232) is the same size as and is homologous to FruK of Escherichia coli, phosphofructokinase B (PfkB) of E. coli, phosphotagatokinase of Staphylococcus aureus, and ribokinase of E. coli. These proteins therefore make up a family of homologous proteins, termed the PfkB family. A phylogenetic tree for this new family was constructed. Sequence comparisons plus chemical inactivation studies suggested the lack of involvement of specific residues in catalysis. Although the Rhodobacter FruK differed markedly from the other enzymes within the PfkB family with respect to amino acid composition, these enzymes exhibited similar predicted secondary structural features. A large internal segment of the Rhodobacter FruK was found to be similar in sequence to the domain bearing the sugar bisphosphate-binding region of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase of plants and bacteria. Proteins of the PfkB family did not exhibit statistically significant sequence identity with PfkA of E. coli. PfkA, however, is homologous to other prokaryotic and eukaryotic ATP- and PPi-dependent Pfks (the PfkA family). These eukaryotic, ATP-dependent enzymes each consist of a homotetramer (mammalian) or a heterooctamer (yeasts), with each subunit containing an internal duplication of the size of the entire PfkA protein of E. coli. In some of these enzymes, additional domains are present. A phylogenetic tree was constructed for the PfkA family and revealed that the bacterial enzymes closely resemble the N-terminal domains of the eukaryotic enzyme subunits whereas the C-terminal domains have diverged more extensively. The PPi-dependent Pfk of potato is only distantly related to the ATP-dependent enzymes. On the basis of their similar functions, sizes, predicted

  4. Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H2 production

    DOE PAGES

    Wecker, Matt S. A.; Beaton, Stephen E.; Chado, Robert A.; ...

    2016-08-17

    The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H2 as a by-product of its nitrogenase-catalyzed nitrogen-fixing activity. Such H2 production, however, is expensive from a metabolic perspective, requiring nearly four times as many photons as the equivalent algal hydrogenase-based system. Here we report the insertion of a Clostridium acetobutylicum [FeFe]-hydrogenase and its three attendant hydrogenase assembly proteins into an R. capsulatus strain lacking its native uptake hydrogenase. Further, this strain is modified to fluoresce upon sensing H2. The resulting strain photoproduces H2 and self-reports its own H2 production through fluorescence. Furthermore, this model system represents a unique method of developing hydrogenase-basedmore » H2 production in R. capsulatus, may serve as a powerful system for in vivo directed evolution of hydrogenases and hydrogenase-associated genes, and provides a means of screening for increased metabolic production of H2.« less

  5. The H-NS-like protein HvrA modulates expression of nitrogen fixation genes in the phototrophic purple bacterium Rhodobacter capsulatus by binding to selected nif promoters.

    PubMed

    Raabe, Karsten; Drepper, Thomas; Riedel, Kai Uwe; Masepohl, Bernd; Klipp, Werner

    2002-11-05

    Genetic analyses based on chromosomal lac fusions to nitrogen fixation (nif) genes demonstrated that NifA-dependent transcriptional activation of expression of Rhodobacter capsulatus nifH and nifB1 was negatively modulated by HvrA, whereas regulation of rpoN, nifA1, and nifA2 was independent of HvrA. Expression of hvrA itself was not influenced by a mutation in ntrC, which is absolutely essential for N(2) fixation. Furthermore, HvrA accumulated to comparable levels in the presence and absence of ammonium, suggesting that the amount of HvrA in the cells does not differ under nitrogenase-repressing or -derepressing conditions. In addition, competitive gel retardation studies with HvrA-His(6) purified from R. capsulatus were carried out, demonstrating preferential binding of HvrA to the nifH promoter region.

  6. In vitro and in vivo safety assessment of edible blue-green algae, Nostoc commune var. sphaeroides Kützing and Spirulina plantensis

    PubMed Central

    Yang, Yue; Park, Youngki; Cassada, David A.; Snow, Daniel D.; Rogers, Douglas G.; Lee, Jiyoung

    2011-01-01

    Blue-green algae (BGA) have been consumed as food and herbal medicine for centuries. However, safety for their consumption has not been well investigated. This study was undertaken to evaluate in vitro and in vivo toxicity of cultivated Nostoc commune var. sphaeroides Kützing (NO) and Spirulina platensis (SP). Neither NO nor SP contained detectable levels of microcystin (MC)-LA, MC-RR, MC-LW and MC-LR by LC/MS/MS. Cell viability remained ~70-80% when HepG2 cells were incubated with 0-500 μg/ml of hexane, chloroform, methanol and water-extractable fractions of NO and SP. Four-week-old male and female C57BL/6J mice were fed an AIN-93G/M diet supplemented with 0, 2.5% or 5% of NO and SP (wt/wt) for 6 months. For both genders, BGA-rich diets did not induce noticeable abnormality in weight gain and plasma alanine aminotransferase (ALT) and aspartate aminotransferase concentrations except a significant increase in plasma ALT levels by 2.5% NO supplementation in male mice at 6 month. Histopathological analysis of livers, however, indicated that BGA did not cause significant liver damage compared with controls. In conclusion, our results suggest that NO and SP are free of MC and the long-term dietary supplementation of up to 5% of the BGA may be consumed without evident toxic side-effects. PMID:21473896

  7. Hydrogen production by hup(-) mutant and wild-type strains of Rhodobacter capsulatus from dark fermentation effluent of sugar beet thick juice in batch and continuous photobioreactors.

    PubMed

    Uyar, Basar; Gürgan, Muazzez; Özgür, Ebru; Gündüz, Ufuk; Yücel, Meral; Eroglu, Inci

    2015-10-01

    Photofermentative production of hydrogen is a promising and sustainable process; however, it should be coupled to dark fermentation to become cost effective. In order to integrate dark fermentation and photofermentation, the suitability of dark fermenter effluents for the photofermentative hydrogen production must be demonstrated. In this study, thermophilic dark fermenter effluent (DFE) of sugar beet thick juice was used as a substrate in photofermentation process to compare wild-type and uptake hydrogenase-deficient (hup (-)) mutant strains of Rhodobacter capsulatus by means of hydrogen production and biomass growth. The tests were conducted in small-scale (50 mL) batch and large-scale (4 L) continuous photobioreactors in indoor conditions under continuous illumination. In small scale batch conditions, maximum cell concentrations were 0.92 gdcw/L c and 1.50 gdcw/L c, hydrogen yields were 34 % and 31 %, hydrogen productivities were 0.49 mmol/(L c·h) and 0.26 mmol/(Lc·h), for hup (-) and wild-type cells, respectively. In large-scale continuous conditions, maximum cell concentrations were 1.44 gdcw/L c and 1.87 gdcw/L c, hydrogen yields were 48 and 46 %, and hydrogen productivities were 1.01 mmol/(L c·h) and 1.05 mmol/(L c·h), for hup (-) and wild-type cells, respectively. Our results showed that Rhodobacter capsulatus hup (-) cells reached to a lower maximum cell concentration but their hydrogen yield and productivity were in the same range or superior compared to the wild-type cells in both batch and continuous operating modes. The maximum biomass concentration, yield and productivity of hydrogen were higher in continuous mode compared to the batch mode with both bacterial strains.

  8. BchJ and BchM interact in a 1 : 1 ratio with the magnesium chelatase BchH subunit of Rhodobacter capsulatus.

    PubMed

    Sawicki, Artur; Willows, Robert D

    2010-11-01

    Substrate channeling between the enzymatic steps in the (bacterio)chlorophyll biosynthetic pathway catalyzed by magnesium chelatase (BchI/ChlI, BchD/ChlD and BchH/ChlH subunits) and S-adenosyl-L-methionine:magnesium-protoporphyrin IX O-methyltransferase (BchM/ChlM) has been suggested. This involves delivery of magnesium-protoporphyrin IX from the BchH/ChlH subunit of magnesium chelatase to BchM/ChlM. Stimulation of BchM/ChlM activity by BchH/ChlH has previously been shown, and physical interaction of the two proteins has been demonstrated. In plants and cyanobacteria, there is an added layer of complexity, as Gun4 serves as a porphyrin (protoporphyrin IX and magnesium-protoporphyrin IX) carrier, but this protein does not exist in anoxygenic photosynthetic bacteria. BchJ may play a similar role to Gun4 in Rhodobacter, as it has no currently assigned function in the established pathway. Purified recombinant Rhodobacter capsulatus BchJ and BchM were found to cause a shift in the equilibrium amount of Mg-protoporphyrin IX formed in a magnesium chelatase assay. Analysis of this shift revealed that it was always in a 1 : 1 ratio with either of these proteins and the BchH subunit of the magnesium chelatase. The establishment of the new equilibrium was faster with BchM than with BchJ in a coupled magnesium chelatase assay. BchJ bound magnesium-protoporphyrin IX or formed a ternary complex with BchH and magnesium-protoporphyrin IX. These results suggest that BchJ may play a role as a general magnesium porphyrin carrier, similar to one of the roles of GUN4 in oxygenic organisms.

  9. Characterization of a Rhodobacter capsulatus reaction center mutant that enhances the distinction between spectral forms of the initial electron donor.

    PubMed

    Eastman, J E; Taguchi, A K; Lin, S; Jackson, J A; Woodbury, N W

    2000-12-05

    A large scale mutation of the Rhodobacter capsulatus reaction center M-subunit gene, sym2-1, has been constructed in which amino acid residues M205-M210 have been changed to the corresponding L subunit amino acids. Two interconvertable spectral forms of the initial electron donor are observed in isolated reaction centers from this mutant. Which conformation dominates depends on ionic strength, the nature of the detergent used, and the temperature. Reaction centers from this mutant have a ground-state absorbance spectrum that is very similar to wild-type when measured immediately after purification in the presence of high salt. However, upon subsequent dialysis against a low ionic strength buffer or the addition of positively charged detergents, the near-infrared spectral band of P (the initial electron donor) in sym2-1 reaction centers is shifted by over 30 nm to the blue, from 852 to 820 nm. Systematically varying either the ionic strength or the amount of charged detergent reveals an isobestic point in the absorbance spectrum at 845 nm. The wild-type spectrum also shifts with ionic strength or detergent with an isobestic point at 860 nm. The large spectral separation between the two dominant conformational forms of the sym2-1 reaction center makes detailed measurements of each state possible. Both of the spectral forms of P bleach in the presence of light. Electrochemical measurements of the P/P+ midpoint potential of sym2-1 reaction centers show an increase of about 30 mV upon conversion from the long-wavelength form to the short-wavelength form of the mutant. The rate constant of initial electron transfer in both forms of the mutant reaction centers is essentially the same, suggesting that the spectral characteristics of P are not critical for charge separation. The short-wavelength form of P in this mutant also converts to the long-wavelength form as a function of temperature between room temperature and 130 K, again giving rise to an isobestic point, in this

  10. Identification and sequence analysis of the hupR1 gene, which encodes a response regulator of the NtrC family required for hydrogenase expression in Rhodobacter capsulatus.

    PubMed Central

    Richaud, P; Colbeau, A; Toussaint, B; Vignais, P M

    1991-01-01

    The hupR1 gene from Rhodobacter capsulatus was cloned and sequenced. It can encode a protein of 53,843 Da which shares significant similarity with several transcriptional regulators and activates transcription of the structural hupSL genes of [NiFe]hydrogenase, as shown by the use of a translational fusion of lacZ with the hupSL promoter. A Hup- mutant having a point mutation in the hupR1 gene is described. PMID:1885559

  11. Multiconformation Continuum Electrostatics Analysis of the Effects of a Buried Asp Introduced Near Heme a in Rb. sphaeroides Cytochrome c Oxidase

    PubMed Central

    Zhang, Jun; Gunner, M.R.

    2013-01-01

    Cytochrome c oxidase (CcO) reduces O2 to water via a series of proton coupled electron transfers generating a transmembrane electrochemical gradient. Coupling electron and proton transfer requires changing buried residues pKas at each stage in the reaction cycle. Heme a is a key cofactor in the CcO electron transfer chain. Mutation of Ser44 to Asp has been reported (Mills et al Biochemistry (2008) 47, 11499-11509), changing the hydrogen bond acceptor from His102, the Heme a axial ligand in Rhodobactor sphaeroides CcO. This adds an acidic residue to the CcO interior. The electrochemical behavior of Heme a in wild type and S44D CcO is compared using the continuum electrostatics program MCCE. The introduced, deeply buried Asp remains ionized at physiological pH only when the nearby heme is oxidized. Heme a reduction is now calculated to be strongly coupled to Asp proton binding, while with Ser44 it is weakly coupled to small protonation shifts at multiple sites, increasing the pH dependence in the mutant. At pH 7, the partially ionized Asp44 is calculated to lower the Heme redox potential by 50 mV as expected given the thermodynamics of coupled electron and proton transfers. This highlights an inconsistency in the experimental results where a low Asp pKa is found together with a stabilized reduced Heme. The stabilization of a model complex heme oxidation by a hydrogen bond to the axial His ligand calculated with Continuum Electrostatics and with Density Functional Theory was in good agreement. PMID:20701325

  12. Lipid extract of Nostoc commune var. sphaeroides Kutzing, a blue-green alga, inhibits the activation of sterol regulatory element binding proteins in HepG2 cells.

    PubMed

    Rasmussen, Heather E; Blobaum, Kara R; Park, Young-Ki; Ehlers, Sarah J; Lu, Fan; Lee, Ji-Young

    2008-03-01

    Nostoc commune var. sphaeroides Kützing (N. commune), a blue-green alga, has been used as both a food ingredient and in medicine for centuries. To determine the effect of N. commune on cholesterol metabolism, N. commune lipid extract was incubated at increasing concentrations (25-100 mg/L) with HepG2 cells, a human hepatoma cell line. The addition of N. commune lipid extract markedly reduced mRNA abundance of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and LDL receptor (LDLR) (P < 0.05), with a concomitant decrease in their protein expression (P < 0.001). Reduced HMGR activity by 90% with N. commune lipid extract confirmed the inhibitory role of N. commune in cholesterol synthesis (P < 0.006). To elucidate a molecular mechanism underlying the repression of HMGR and LDLR by N. commune lipid extract, expression of sterol regulatory element binding protein 2 (SREBP-2) was assessed. Whereas mRNA for SREBP-2 remained unchanged, SREBP-2 mature protein was reduced by N. commune (P < 0.009). In addition, N. commune lipid extract also decreased SREBP-1 mature protein by approximately 30% (P < 0.002) and reduced the expression of SREBP-1-responsive genes such as fatty acid synthase and stearoyl CoA desaturase 1 (SCD-1) (P < 0.05). Therefore, our results demonstrate that N. commune lipid extract inhibits the maturation process of both SREBP-1 and -2, resulting in a decrease in expression of genes involved in cholesterol and fatty acid metabolism.

  13. The SOS Response Master Regulator LexA Regulates the Gene Transfer Agent of Rhodobacter capsulatus and Represses Transcription of the Signal Transduction Protein CckA

    PubMed Central

    Kuchinski, Kevin S.; Brimacombe, Cedric A.; Westbye, Alexander B.; Ding, Hao

    2016-01-01

    ABSTRACT The gene transfer agent of Rhodobacter capsulatus (RcGTA) is a genetic exchange element that combines central aspects of bacteriophage-mediated transduction and natural transformation. RcGTA particles resemble a small double-stranded DNA bacteriophage, package random ∼4-kb fragments of the producing cell genome, and are released from a subpopulation (<1%) of cells in a stationary-phase culture. RcGTA particles deliver this DNA to surrounding R. capsulatus cells, and the DNA is integrated into the recipient genome though a process that requires homologs of natural transformation genes and RecA-mediated homologous recombination. Here, we report the identification of the LexA repressor, the master regulator of the SOS response in many bacteria, as a regulator of RcGTA activity. Deletion of the lexA gene resulted in the abolition of detectable RcGTA production and an ∼10-fold reduction in recipient capability. A search for SOS box sequences in the R. capsulatus genome sequence identified a number of putative binding sites located 5′ of typical SOS response coding sequences and also 5′ of the RcGTA regulatory gene cckA, which encodes a hybrid histidine kinase homolog. Expression of cckA was increased >5-fold in the lexA mutant, and a lexA cckA double mutant was found to have the same phenotype as a ΔcckA single mutant in terms of RcGTA production. The data indicate that LexA is required for RcGTA production and maximal recipient capability and that the RcGTA-deficient phenotype of the lexA mutant is largely due to the overexpression of cckA. IMPORTANCE This work describes an unusual phenotype of a lexA mutant of the alphaproteobacterium Rhodobacter capsulatus in respect to the phage transduction-like genetic exchange carried out by the R. capsulatus gene transfer agent (RcGTA). Instead of the expected SOS response characteristic of prophage induction, this lexA mutation not only abolishes the production of RcGTA particles but also impairs the ability

  14. Proteome Profiling of the Rhodobacter capsulatus Molybdenum Response Reveals a Role of IscN in Nitrogen Fixation by Fe-Nitrogenase

    PubMed Central

    Hoffmann, Marie-Christine; Wagner, Eva; Langklotz, Sina; Pfänder, Yvonne; Hött, Sina; Bandow, Julia E.

    2015-01-01

    ABSTRACT Rhodobacter capsulatus is capable of synthesizing two nitrogenases, a molybdenum-dependent nitrogenase and an alternative Mo-free iron-only nitrogenase, enabling this diazotroph to grow with molecular dinitrogen (N2) as the sole nitrogen source. Here, the Mo responses of the wild type and of a mutant lacking ModABC, the high-affinity molybdate transporter, were examined by proteome profiling, Western analysis, epitope tagging, and lacZ reporter fusions. Many Mo-controlled proteins identified in this study have documented or presumed roles in nitrogen fixation, demonstrating the relevance of Mo control in this highly ATP-demanding process. The levels of Mo-nitrogenase, NifHDK, and the Mo storage protein, Mop, increased with increasing Mo concentrations. In contrast, Fe-nitrogenase, AnfHDGK, and ModABC, the Mo transporter, were expressed only under Mo-limiting conditions. IscN was identified as a novel Mo-repressed protein. Mo control of Mop, AnfHDGK, and ModABC corresponded to transcriptional regulation of their genes by the Mo-responsive regulators MopA and MopB. Mo control of NifHDK and IscN appeared to be more complex, involving different posttranscriptional mechanisms. In line with the simultaneous control of IscN and Fe-nitrogenase by Mo, IscN was found to be important for Fe-nitrogenase-dependent diazotrophic growth. The possible role of IscN as an A-type carrier providing Fe-nitrogenase with Fe-S clusters is discussed. IMPORTANCE Biological nitrogen fixation is a central process in the global nitrogen cycle by which the abundant but chemically inert dinitrogen (N2) is reduced to ammonia (NH3), a bioavailable form of nitrogen. Nitrogen reduction is catalyzed by nitrogenases found in diazotrophic bacteria and archaea but not in eukaryotes. All diazotrophs synthesize molybdenum-dependent nitrogenases. In addition, some diazotrophs, including Rhodobacter capsulatus, possess catalytically less efficient alternative Mo-free nitrogenases, whose expression

  15. Phosphate Concentration and the Putative Sensor Kinase Protein CckA Modulate Cell Lysis and Release of the Rhodobacter capsulatus Gene Transfer Agent

    PubMed Central

    Westbye, A. B.; Leung, M. M.; Florizone, S. M.; Taylor, T. A.; Johnson, J. A.; Fogg, P. C.

    2013-01-01

    The gene transfer agent of Rhodobacter capsulatus (RcGTA) is a bacteriophage-like genetic element with the sole known function of horizontal gene transfer. Homologues of RcGTA genes are present in many members of the alphaproteobacteria and may serve an important role in microbial evolution. Transcription of RcGTA genes is induced as cultures enter the stationary phase; however, little is known about cis-active sequences. In this work, we identify the promoter of the first gene in the RcGTA structural gene cluster. Additionally, gene transduction frequency depends on the growth medium, and the reason for this is not known. We report that millimolar concentrations of phosphate posttranslationally inhibit the lysis-dependent release of RcGTA from cells in both a complex medium and a defined medium. Furthermore, we found that cell lysis requires the genes rcc00555 and rcc00556, which were expressed and studied in Escherichia coli to determine their predicted functions as an endolysin and holin, respectively. Production of RcGTA is regulated by host systems, including a putative histidine kinase, CckA, and we found that CckA is required for maximal expression of rcc00555 and for maturation of RcGTA to yield gene transduction-functional particles. PMID:23995641

  16. Effect of light-dark cycles on hydrogen and poly-β-hydroxybutyrate production by a photoheterotrophic culture and Rhodobacter capsulatus using a dark fermentation effluent as substrate.

    PubMed

    Montiel Corona, Virginia; Le Borgne, Sylvie; Revah, Sergio; Morales, Marcia

    2017-02-01

    A Rhodobacter capsulatus strain and a photoheterotrophic culture (IZT) were cultivated to produce hydrogen under different light-dark cycles. A dark fermentation effluent (DFE) was used as substrate. It was found that IZT culture had an average cumulative hydrogen production (Paccum H2) of 1300±43mLH2L(-1) under continuous illumination and light-dark cycles of 30 or 60min. In contrast, R. capsulatus reduced its Paccum H2 by 20% under 30:30min light-dark cycles, but tripled its poly-β-hydroxybutyrate (PHB) content (308±2mgPHB gdw(-1)) compared to continuous illumination. The highest PHB content by IZT culture was 178±10mgPHB gdw(-1) under 15:15min light-dark cycles. PCR-DGGE analysis revealed that the IZT culture was mainly composed of Rhodopseudomonas palustris identified with high nucleotide similarity (99%). The evaluated cultures might be used for hydrogen and PHB production. They might provide energy savings by using light-dark cycles and DFE valorization.

  17. Use of hupS::lacZ gene fusion to study regulation of hydrogenase expression in Rhodobacter capsulatus: stimulation by H2.

    PubMed Central

    Colbeau, A; Vignais, P M

    1992-01-01

    The Escherichia coli beta-galactosidase enzyme was used as a reporter molecule for genetic fusions in Rhodobacter capsulatus. DNA fragments that were from the upstream region of the hydrogenase structural operon hupSLM and contained 5' hupS sequences were fused in frame to a promoterless lacZ gene, yielding fusion proteins comprising the putative signal sequence and the first 22 amino acids of the HupS protein joined to the eight amino acid of beta-galactosidase. We demonstrate the usefulness of the hupS::lacZ fusion in monitoring regulation of hydrogenase gene expression. The activities of plasmid-determined beta-galactosidase and chromosome-encoded hydrogenase changed in parallel in response to various growth conditions (light or dark, aerobiosis or anaerobiosis, and presence or absence of ammonia or of H2), showing that changes in hydrogenase activity were due to changes in enzyme synthesis. Molecular hydrogen stimulated hydrogenase synthesis in dark, aerobic cultures and in illuminated, anaerobic cultures. Analysis of hupS::lacZ expression in various mutants indicated that neither the hydrogenase structural genes nor NifR4 (sigma 54) was essential for hydrogen regulation of hydrogenase synthesis. PMID:1624420

  18. Purification of the integration host factor homolog of Rhodobacter capsulatus: cloning and sequencing of the hip gene, which encodes the beta subunit.

    PubMed Central

    Toussaint, B; Delic-Attree, I; De Sury D'Aspremont, R; David, L; Vinçon, M; Vignais, P M

    1993-01-01

    We describe a method for rapid purification of the integration host factor (IHF) homolog of Rhodobacter capsulatus that has allowed us to obtain microgram quantities of highly purified protein. R. capsulatus IHF is an alpha beta heterodimer similar to IHF of Escherichia coli. We have cloned and sequenced the hip gene, which encodes the beta subunit. The deduced amino acid sequence (10.7 kDa) has 46% identity with the beta subunit of IHF from E. coli. In gel electrophoretic mobility shift DNA binding assays, R. capsulatus IHF was able to form a stable complex in a site-specific manner with a DNA fragment isolated from the promoter of the structural hupSL operon, which contains the IHF-binding site. The mutated IHF protein isolated from the Hup- mutant IR4, which is mutated in the himA gene (coding for the alpha subunit), gave a shifted band of greater mobility, and DNase I footprinting analysis has shown that the mutated IHF interacts with the DNA fragment from the hupSL promoter region differently from the way that the wild-type IHF does. Images PMID:8407826

  19. Sequence analysis and interposon mutagenesis of the hupT gene, which encodes a sensor protein involved in repression of hydrogenase synthesis in Rhodobacter capsulatus.

    PubMed Central

    Elsen, S; Richaud, P; Colbeau, A; Vignais, P M

    1993-01-01

    The hupT gene, which represses hydrogenase gene expression in the purple photosynthetic bacterium Rhodobacter capsulatus, has been identified and sequenced. The nucleotide sequence of hupT and of the contiguous downstream open reading frame, hupU, is reported. The HupT protein of 456 amino acids (48,414 Da) has sequence similarity with the FixL, DctB, NtrB, and ArcB proteins and is predicted to be a soluble sensor kinase. Insertional inactivation of the hupT gene led to deregulation of transcriptional control, so that the hydrogenase structural operon hupSLC became overexpressed in cells grown anaerobically or aerobically. The HupT- mutants were complemented in trans by a plasmid containing an intact copy of the hupT gene. The hupU open reading frame, capable of encoding a protein of 84,879 Da, shared identity with [NiFe]hydrogenase subunits; the strongest similarity was observed with the periplasmic hydrogenase of Desulfovibrio baculatus. Images PMID:8226687

  20. Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H2 production.

    PubMed

    Wecker, Matt S A; Beaton, Stephen E; Chado, Robert A; Ghirardi, Maria L

    2017-02-01

    The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H2 as a by-product of its nitrogenase-catalyzed nitrogen-fixing activity. Such H2 production, however, is expensive from a metabolic perspective, requiring nearly four times as many photons as the equivalent algal hydrogenase-based system (Ghirardi et al., 2009 Photobiological hydrogen-producing systems. Chem Soc Rev 38(1):52-61). Here, we report the insertion of a Clostridium acetobutylicum [FeFe]-hydrogenase and its three attendant hydrogenase assembly proteins into an R. capsulatus strain lacking its native uptake hydrogenase. Further, this strain is modified to fluoresce upon sensing H2 . The resulting strain photoproduces H2 and self-reports its own H2 production through fluorescence. This model system represents a unique method of developing hydrogenase-based H2 production in R. capsulatus, may serve as a powerful system for in vivo directed evolution of hydrogenases and hydrogenase-associated genes, and provides a means of screening for increased metabolic production of H2 . Biotechnol. Bioeng. 2017;114: 291-297. © 2016 Wiley Periodicals, Inc.

  1. NifA- and CooA-Coordinated cowN Expression Sustains Nitrogen Fixation by Rhodobacter capsulatus in the Presence of Carbon Monoxide

    PubMed Central

    Hoffmann, Marie-Christine; Pfänder, Yvonne; Fehringer, Maria; Narberhaus, Franz

    2014-01-01

    Rhodobacter capsulatus fixes atmospheric dinitrogen via two nitrogenases, Mo- and Fe-nitrogenase, which operate under different conditions. Here, we describe the functions in nitrogen fixation and regulation of the rcc00574 (cooA) and rcc00575 (cowN) genes, which are located upstream of the structural genes of Mo-nitrogenase, nifHDK. Disruption of cooA or cowN specifically impaired Mo-nitrogenase-dependent growth at carbon monoxide (CO) concentrations still tolerated by the wild type. The cooA gene was shown to belong to the Mo-nitrogenase regulon, which is exclusively expressed when ammonium is limiting. Its expression was activated by NifA1 and NifA2, the transcriptional activators of nifHDK. AnfA, the transcriptional activator of Fe-nitrogenase genes, repressed cooA, thereby counteracting NifA activation. CooA activated cowN expression in response to increasing CO concentrations. Base substitutions in the presumed CooA binding site located upstream of the cowN transcription start site abolished cowN expression, indicating that cowN regulation by CooA is direct. In conclusion, a transcription factor-based network controls cowN expression to protect Mo-nitrogenase (but not Fe-nitrogenase) under appropriate conditions. PMID:25070737

  2. Cooperation between two periplasmic copper chaperones is required for full activity of the cbb3-type cytochrome c oxidase and copper homeostasis in Rhodobacter capsulatus

    DOE PAGES

    Trasnea, Petru -Iulian; Utz, Marcel; Khalfaoui-Hassani, Bahia; ...

    2016-02-28

    Copper (Cu) is an essential micronutrient that functions as a cofactor in several important enzymes, like respiratory heme-copper oxygen reductases. Yet, Cu is also toxic and therefore cells engage a highly coordinated Cu uptake and delivery system to prevent the accumulation of toxic Cu concentrations. In the current work we analyzed Cu delivery to the cbb3-type cytochrome c oxidase (cbb3-Cox) of Rhodobacter capsulatus. We identified the PCuAC-like periplasmic chaperone PccA and analyzed its contribution to cbb3-Cox assembly. Our data demonstrate that PccA is a Cu-binding protein with a preference for Cu(I), which is required for efficient cbb3-Cox assembly, in particularmore » at low Cu concentrations. By using in vivo and in vitro crosslinking we show that PccA forms a complex with the Sco1-homologue SenC. This complex is stabilized in the absence of the cbb3-Cox specific assembly factors CcoGHIS. In cells lacking SenC, the cytoplasmic Cu content is significantly increased, but the simultaneous absence of PccA prevents this Cu accumulation. Lastly, these data demonstrate that the interplay between PccA and SenC is not only required for Cu delivery during cbb3-Cox assembly, but that it also regulates Cu homeostasis in R. capsulatus.« less

  3. Synergistic dark and photo-fermentation continuous system for hydrogen production from molasses by Clostridium acetobutylicum ATCC 824 and Rhodobacter capsulatus DSM 1710.

    PubMed

    Morsy, Fatthy Mohamed

    2017-04-01

    This study investigated synergistic dark and photo-fermentation using continuous fermentation system (CFS). The system relies on connecting several fermenters from bottom of one to top culture level of the next in a manner that allows for delaying movement of the substrate and thus for its full consumption. While H2 was collected, CFS allowed for moving liquid byproducts toward the outlet and hence continuous productivity. CFS could be efficiently used for: (1) Continuous dark and photo-fermentation H2 production by Clostridium acetobutylicum and Rhodobacter capsulatus producing 5.65moleH2mole(-1) hexose; (2) Continuous dark-fermentation synergistic H2, acetone, butanol and ethanol (ABE) production by C. acetobutylicum which produced per mole hexose, 2.43mol H2 along with 73.08g ABE (3) Continuous H2 and methane production by C. acetobutylicum and bacterial sludge producing, per mole hexose, 1.64mol pure H2 and 2.56mol CH4 mixed with 0.37mol H2·The hydraulic retention time (HRT) for whole system was short where organic acids produced in dark-fermentation in first fermenter were synergistically utilized for H2 production by R. capsulatus in subsequent fermenters. CFS is suitable for fast-digestible sugars but not lignocelluloses or other hard-digestible organics, requiring prolonged HRT, unless such polymeric organics were hydrolyzed prior to fermentation.

  4. Purification of the integration host factor homolog of Rhodobacter capsulatus: cloning and sequencing of the hip gene, which encodes the beta subunit.

    PubMed

    Toussaint, B; Delic-Attree, I; De Sury D'Aspremont, R; David, L; Vinçon, M; Vignais, P M

    1993-10-01

    We describe a method for rapid purification of the integration host factor (IHF) homolog of Rhodobacter capsulatus that has allowed us to obtain microgram quantities of highly purified protein. R. capsulatus IHF is an alpha beta heterodimer similar to IHF of Escherichia coli. We have cloned and sequenced the hip gene, which encodes the beta subunit. The deduced amino acid sequence (10.7 kDa) has 46% identity with the beta subunit of IHF from E. coli. In gel electrophoretic mobility shift DNA binding assays, R. capsulatus IHF was able to form a stable complex in a site-specific manner with a DNA fragment isolated from the promoter of the structural hupSL operon, which contains the IHF-binding site. The mutated IHF protein isolated from the Hup- mutant IR4, which is mutated in the himA gene (coding for the alpha subunit), gave a shifted band of greater mobility, and DNase I footprinting analysis has shown that the mutated IHF interacts with the DNA fragment from the hupSL promoter region differently from the way that the wild-type IHF does.

  5. Optimization of the hydrogen yield from single-stage photofermentation of glucose by Rhodobacter capsulatus JP91 using response surface methodology.

    PubMed

    Ghosh, Dipankar; Sobro, Irma Flore; Hallenbeck, Patrick C

    2012-11-01

    Hydrogen production from glucose via single-stage photofermentation was examined with the photosynthetic bacterium Rhodobacter capsulatus JP91 (hup-). Response surface methodology with Box-Behnken design was used to optimize the independent experimental variables of glucose concentration, glutamate concentration and light intensity, as well as examining their interactive effects for maximization of molar hydrogen yield. Under optimal condition with a light intensity of 175W/m(2), 35mM glucose, and 4.5mM glutamate, a maximum hydrogen yield of 5.5 (±0.15)molH(2)/molglucose, and a maximum nitrogenase activity of 246 (±3.5)nmolC(2)H(4)/ml/min were obtained. Densitometric analysis of nitrogenase Fe-protein expression under different conditions showed significant variation in Fe-protein expression with a maximum at the optimized central point. Even under optimum conditions for hydrogen production, a significant fraction of the Fe-protein was found in the ADP-ribosylated state, suggesting that further improvement in yields might be possible.

  6. Bacteriochlorophyll-dependent expression of genes for pigment-binding proteins in Rhodobacter capsulatus involves the RegB/RegA two-component system.

    PubMed

    Abada, E M; Balzer, A; Jäger, A; Klug, G

    2002-04-01

    Expression of the puf and puc operons, which encode proteins of the photosynthetic apparatus of Rhodobacter capsulatus, is regulated by oxygen. A drop in the oxygen tension in the environment leads to an increase in the levels of puf and puc mRNAs. In strains lacking bacteriochlorophyll (Bchl) due to mutations in bch genes, the rise in puf and puc mRNA levels observed on reduction of oxygen tension is much less pronounced than in wild-type cells, indicating co-regulation of the syntheses of pigments and pigment-binding proteins. Here we show that Bchl synthesis also affects the expression of the bchC gene, which codes for a subunit of bacteriochlorophyll synthase, suggesting an autoregulatory mechanism for the Bchl biosynthetic pathway. Furthermore, our data provide evidence that the RegB/RegA two-component system, which is known to play a central role in oxygen-controlled expression of photosynthesis genes, is also involved in the Bchl-dependent regulation. Mutant strains which do not synthesize RegB or RegA show similar oxygen-dependent puf and puc expression in the presence and absence of Bchl. Our results support the view that the RegB/RegA system can directly or indirectly sense whether Bchl synthesis takes place or not.

  7. The ccoNOQP gene cluster codes for a cb-type cytochrome oxidase that functions in aerobic respiration of Rhodobacter capsulatus.

    PubMed

    Thöny-Meyer, L; Beck, C; Preisig, O; Hennecke, H

    1994-11-01

    The genes for a new type of a haem-copper cytochrome oxidase were cloned from Rhodobacter capsulatus strain 37b4, using the Bradyrhizobium japonicum fixNOQP gene region as a hybridizing probe. Four genes, probably organized in an operon (ccoNOQP), were identified; their products share extensive amino acid sequence similarity with the FixN, O, Q and P proteins that have recently been shown to be the subunits of a cb-type oxidase. CcoN is a b-type cytochrome, CcoO and CcoP are membrane-bound mono- and dihaem c-type cytochromes and CcoQ is a small membrane protein of unknown function. Genes for a similar oxidase are also present in other non-rhizobial bacterial species such as Azotobacter vinelandii, Agrobacterium tumefaciens and Pseudomonas aeruginosa, as revealed by polymerase chain reaction analysis. A ccoN mutant was constructed whose phenotype, in combination with the structural information on the gene products, provides evidence that the CcoNOQP oxidase is a cytochrome c oxidase of the cb type, which supports aerobic respiration in R. capsulatus and which is probably identical to the cbb3-type oxidase that was recently purified from a different strain of the same species. Mutant analysis also showed that this oxidase has no influence on photosynthetic growth and nitrogen-fixation activity.

  8. Photofermentative production of hydrogen and poly-β-hydroxybutyrate from dark fermentation products.

    PubMed

    Luongo, Vincenzo; Ghimire, Anish; Frunzo, Luigi; Fabbricino, Massimiliano; d'Antonio, Giuseppe; Pirozzi, Francesco; Esposito, Giovanni

    2017-03-01

    The aim of this work is to investigate the hydrogen and poly-β-hydroxybutyrate (PHB) production during the photofermentative treatment of the effluent from a dark fermentation reactor fed with the organic fraction of municipal solid waste. Two different inocula, an adapted culture of Rhodobacter sphaeroides AV1b and a mixed consortium of purple non sulphur bacteria have been investigated under the same operational conditions. Different hydrogen productivities of 364 and 559NmL H2 L(-1) were observed for the Rhodobacter sphaeroides and the mixed culture consortium tests, respectively: the consortium of PNSB resulted 1.5-fold more productive than the pure culture. On the other hand, Rhodobacter sphaeroides culture showed a higher PHB productivity (155mg PHB g COD(-1)) than the mixed culture (55mg PHB g COD(-1)). In all the tests, the concomitant H2 and PHB production was associated to a dissolved COD removal higher than 80%.

  9. Herbicide-quinone competition in the acceptor complex of photosynthetic reaction centers from Rhodopseudomonas sphaeroides: a bacterial model for PS-II-herbicide activity in plants.

    PubMed

    Stein, R R; Castellvi, A L; Bogacz, J P; Wraight, C A

    1984-01-01

    A select group of herbicides that inhibit photosystem II also act at the acceptor side of the reaction center (RC) from the photosynthetic bacterium Rhodopseudomonas sphaeroides, with much the same relative specificity as in plants. These include the triazines and some phenolic compounds. The proposal that herbicides inhibit the electron transfer from the primary quinone (QA) to the secondary quinone (QB) by competing for the secondary quinone binding site--the B-site--[5], is tested here with terbutryn, the most potent of the triazines. Competition between terbutryn and ubiquinone (Q-10) was observed using the kinetics of the back-reaction as a measure of inhibition. The model includes binding equilibria before and after flash activation. The binding constants for the preflash (dark) equilibria, for reaction centers in 0.14% lauryl dimethylamine-N-oxide (LDAO), were KDi = 0.8 microM terbutryn, KDq = 2 microM Q-10; both are detergent-concentration dependent. After flash activation, binding equilibrium is not fully restored on the time scale of the back-reaction because terbutryn unbinds slowly. This gives rise to biphasic decay kinetics from which koff for terbutryn was estimated to be 3 sec-1. Titrations of the rate of the slow back reaction indicated that the post-flash equilibrium is less sensitive to inhibitor, in a manner that is independent of the much stronger binding of the semiquinone, Q-B, and indicative of a direct effect of the redox state of QA on the affinity of the B-site for ligands. However, the effects on KLi and KDq could not be separated: either KLi greater than KDi or KLq less than KDq. Some triazine-resistant mutants have been isolated and are described. All appear to be herbicide binding site mutants. Whole cells and photosynthetic membrane vesicles (chromatophores) exhibit a 10-50-fold increase in resistance to triazines due, in large part, to an increase in the rate of unbinding (koff). The modifications of the binding site appear to

  10. Production of lipases by four anoxygenic purple non-sulphur phototrophic bacteria.

    PubMed

    Munjam, Srinivas; Girisham, S; Reddy, S M

    Production of lipases by Rhodopseudomonas palustris, Rhodobacter sphaeroides, Rhodocyclus gelatinosus and Rhodocyclus tenuis in different synthetic media was investigated. Rc. gelatinosus followed by Rb. sphaeroides were good producers of lipases, while Rps. palustris and Rc. tenuis were poor in lipase secretion. Lipase secretion by Rc. gelatinosus was adaptive in nature, while other three bacterial behavior was inconsistent. No positive correlation could be observed between growth and lipase production.

  11. Temperature dependence of protein fluorescence in Rb. sphaeroides reaction centers frozen to 80 K in the dark or on the actinic light as the indicator of protein conformational dynamics.

    PubMed

    Knox, P P; Korvatovsky, B N; Krasilnikov, P M; Paschenko, V Z; Seifullina, N H; Grishanova, N P; Rubin, A B

    2016-03-01

    The differences in the average fluorescence lifetime (τav) of tryptophanyls in photosynthetic reaction center (RC) of the purple bacteria Rb. sphaeroides frozen to 80 K in the dark or on the actinic light was found. This difference disappeared during subsequent heating at the temperatures above 250 K. The computer-based calculation of vibration spectra of the tryptophan molecule was performed. As a result, the normal vibrational modes associated with deformational vibrations of the aromatic ring of the tryptophan molecule were found. These deformational vibrations may be active during the nonradiative transition of the molecule from the excited to the ground state. We assume that the differences in τav may be associated with the change in the activity of these vibration modes due to local variations in the microenvironment of tryptophanyls during the light activation.

  12. One for All or All for One: Heterogeneous Expression and Host Cell Lysis Are Key to Gene Transfer Agent Activity in Rhodobacter capsulatus

    PubMed Central

    Fogg, Paul C. M.; Westbye, Alexander B.; Beatty, J. Thomas

    2012-01-01

    The gene transfer agent (RcGTA) of Rhodobacter capsulatus is the model for a family of novel bacteriophage-related genetic elements that carry out lateral transfer of essentially random host DNA. Genuine and putative gene transfer agents have been discovered in diverse genera and are becoming recognized as potentially an important source of genetic exchange and microbial evolution in the oceans. Despite being discovered over 30 years ago, little is known about many essential aspects of RcGTA biology. Here, we validate the use of direct fluorescence reporter constructs, which express the red fluorescent protein mCherry in R. capsulatus. A construct containing the RcGTA promoter fused to mCherry was used to examine the single-cell expression profiles of wild type and RcGTA overproducer R. capsulatus populations, under different growth conditions and growth phases. The majority of RcGTA production clearly arises from a small, distinct sub-set of the population in the wild type strain and a larger sub-set in the overproducer. The most likely RcGTA release mechanism concomitant with this expression pattern is host cell lysis and we present direct evidence for the release of an intracellular enzyme accompanying RcGTA release. RcGTA ORF s is annotated as a ‘cell wall peptidase’ but we rule out a role in host lysis and propose an alternative function as a key contributor to RcGTA invasion of a target cell during infection. PMID:22916305

  13. Sulfite stimulates the ATP hydrolysis activity of but not proton translocation by the ATP synthase of Rhodobacter capsulatus and interferes with its activation by delta muH+.

    PubMed

    Cappellini, P; Turina, P; Fregni, V; Melandri, B A

    1997-09-01

    Sulfite stimulates the rate of ATP hydrolysis by the ATP synthase in chromatophores of Rhodobacter capsulatus. The stimulated activity is inhibited by oligomycin. The activation takes place also in uncoupled chromatophores. The activation consists in an increase of about 12-15-fold of the Vmax for the ATP hydrolysis reaction, while the Km for MgATP is unaffected at 0.16+/-0.03 mM. The dependence of Vmax on the sulfite concentration follows a hyperbolic pattern with half maximum effect at 12 mM. Sulfite affects the ability of the enzyme in translocating protons. Concomitant measurements of the rate of ATP hydrolysis and of ATP-induced protonic flows demonstrate that at sulfite concentrations of greater than 10 mM the hydrolytic reaction becomes progressively uncoupled from the process of proton translocation. This is accompanied by an inhibition of ATP synthesis, either driven by light or by artificially induced ionic gradients. ATP synthesis is totally inhibited at concentrations of at least 80 mM. Sulfite interferes with the mechanism of activation by delta muH+. Low concentrations of this anion (< or = 2 mM) prevent the activation by delta muH+. At higher concentrations a marked stimulation of the activity prevails, regardless of the occurrence of a delta muH+ across the membrane. Phosphate at millimolar concentrations can reverse the inhibition by sulfite. These experimental results can be simulated by a model assuming multiple and competitive equilibria for phosphate or sulfite binding with two binding sites for the two ligands (for sulfite K1S = 0.26 and K2S = 37 mM, and for phosphate K1P = 0.06 and K2P = 4.22 mM), and in which the state bound only to one sulfite molecule is totally inactive in hydrolysis. The competition between phosphate and sulfite is consistent with the molecular structures of the two ligands and of the enzyme.

  14. The puhE gene of Rhodobacter capsulatus is needed for optimal transition from aerobic to photosynthetic growth and encodes a putative negative modulator of bacteriochlorophyll production.

    PubMed

    Aklujkar, Muktak; Prince, Roger C; Beatty, J Thomas

    2005-05-15

    A conserved orf of previously unknown function (herein designated as puhE) is located 3' of the reaction centre H (puhA) gene in purple photosynthetic bacteria, in the order puhABCE in Rhodobacter capsulatus. Disruptions of R. capsulatus puhE resulted in a long lag in the growth of photosynthetic cultures inoculated with cells grown under high aeration, and increased the level of the peripheral antenna, light-harvesting complex 2 (LH2). The amount of the photosynthetic reaction centre (RC) and its core antenna, light-harvesting complex 1 (LH1), was reduced; however, there was no decrease in expression of a lacZ reporter fused to the puf (RC and LH1) promoter, in RC assembly in the absence of LH1, or in LH1 assembly in the absence of the RC. In strains that lack LH2, disruption of puhE increased the in vivo absorption at 780 nm, which we attribute to excess bacteriochlorophyll a (BChl) pigment production. This effect was seen in the presence and absence of PufQ, a protein that stimulates BChl biosynthesis. Expression of puhE from a plasmid reduced A(780) production in puhE mutants. We suggest that PuhE modulates BChl biosynthesis independently of PufQ, and that the presence of excess BChl in PuhE(-)LH2(+) strains results in excess LH2 assembly and also interferes with the adaptation of cells during the transition from aerobic respiratory to anaerobic photosynthetic growth.

  15. Coordinated Expression of fdxD and Molybdenum Nitrogenase Genes Promotes Nitrogen Fixation by Rhodobacter capsulatus in the Presence of Oxygen

    PubMed Central

    Hoffmann, Marie-Christine; Müller, Alexandra; Fehringer, Maria; Pfänder, Yvonne; Narberhaus, Franz

    2014-01-01

    Rhodobacter capsulatus is able to grow with N2 as the sole nitrogen source using either a molybdenum-dependent or a molybdenum-free iron-only nitrogenase whose expression is strictly inhibited by ammonium. Disruption of the fdxD gene, which is located directly upstream of the Mo-nitrogenase genes, nifHDK, abolished diazotrophic growth via Mo-nitrogenase at oxygen concentrations still tolerated by the wild type, thus demonstrating the importance of FdxD under semiaerobic conditions. In contrast, FdxD was not beneficial for diazotrophic growth depending on Fe-nitrogenase. These findings suggest that the 2Fe2S ferredoxin FdxD specifically supports the Mo-nitrogenase system, probably by protecting Mo-nitrogenase against oxygen, as previously shown for its Azotobacter vinelandii counterpart, FeSII. Expression of fdxD occurred under nitrogen-fixing conditions, but not in the presence of ammonium. Expression of fdxD strictly required NifA1 and NifA2, the transcriptional activators of the Mo-nitrogenase genes, but not AnfA, the transcriptional activator of the Fe-nitrogenase genes. Expression of the fdxD and nifH genes, as well as the FdxD and NifH protein levels, increased with increasing molybdate concentrations. Molybdate induction of fdxD was independent of the molybdate-sensing regulators MopA and MopB, which repress anfA transcription at micromolar molybdate concentrations. In this report, we demonstrate the physiological relevance of an fesII-like gene, fdxD, and show that the cellular nitrogen and molybdenum statuses are integrated to control its expression. PMID:24272776

  16. 1-Deoxy-d-Xylulose 5-Phosphate Synthase, the Gene Product of Open Reading Frame (ORF) 2816 and ORF 2895 in Rhodobacter capsulatus

    PubMed Central

    Hahn, Frederick M.; Eubanks, Lisa M.; Testa, Charles A.; Blagg, Brian S. J.; Baker, Jonathan A.; Poulter, C. Dale

    2001-01-01

    In eubacteria, green algae, and plant chloroplasts, isopentenyl diphosphate, a key intermediate in the biosynthesis of isoprenoids, is synthesized by the methylerythritol phosphate pathway. The five carbons of the basic isoprenoid unit are assembled by joining pyruvate and d-glyceraldehyde 3-phosphate. The reaction is catalyzed by the thiamine diphosphate-dependent enzyme 1-deoxy-d-xylulose 5-phosphate synthase. In Rhodobacter capsulatus, two open reading frames (ORFs) carry the genes that encode 1-deoxy-d-xylulose 5-phosphate synthase. ORF 2816 is located in the photosynthesis-related gene cluster, along with most of the genes required for synthesis of the photosynthetic machinery of the bacterium, whereas ORF 2895 is located elsewhere in the genome. The proteins encoded by ORF 2816 and ORF 2895, 1-deoxy-d-xylulose 5-phosphate synthase A and B, containing a His6 tag, were synthesized in Escherichia coli and purified to greater than 95% homogeneity in two steps. 1-Deoxy-d-xylulose 5-phosphate synthase A appears to be a homodimer with 68 kDa subunits. A new assay was developed, and the following steady-state kinetic constants were determined for 1-deoxy-d-xylulose 5-phosphate synthase A and B: Kmpyruvate = 0.61 and 3.0 mM, Kmd-glyceraldehyde 3-phosphate = 150 and 120 μM, and Vmax = 1.9 and 1.4 μmol/min/mg in 200 mM sodium citrate (pH 7.4). The ORF encoding 1-deoxy-d-xylulose 5-phosphate synthase B complemented the disrupted essential dxs gene in E. coli strain FH11. PMID:11114895

  17. Variation in composition and relative content of accumulated photopigments in a newly isolated Rhodobacter capsulatus strain XJ-1 in response to arsenic.

    PubMed

    Lin, Hua Z; Yue, Ying H; Lü, Jiang C; Zhao, Gui C; Yang, Ping S

    2014-01-01

    This study aimed to isolate and characterize a new arsenic (As)-tolerant bacterial strain (XJ-1) from the Halosol soil, to evaluate its As tolerance, and to examine the variation in composition and relative content of accumulated photosynthetic pigments in response to As. The experiments were performed with high-performance liquid chromatography (HPLC), inductively-coupled plasma mass spectrometry (ICP-MS), liquid chromatography/mass spectrometry (LC/MS), thin-layer chromatography (TLC) and grayscale intensity image analysis using Gel-Pro analyzer software. Strain XJ-1 was identified as Rhodobacter (R.) capsulatus based on 16S rRNA gene sequencing and physiological characteristics. Strain XJ-1 was able to grow when exposed to arsenite [As(III)] and arsenate [As(V)] under anaerobic-light conditions. The median effective concentrations (EC50) of As(III) and As(V) were 0.61 mM and 2.03 mM, respectively. Strain XJ-1 could reduce As(V) to As(III), but As(III) could not be transformed back to As(V) or other organic As compounds. Accumulation of bacteriochlorophylls and carotenoids in strain XJ-1 varied in the presence of 0.2-1.2 mM As(III) and 0-2.5 mM As(V). As exposure resulted in pronounced variation in compositions and contents of photosynthetic pigments, especially hydroxyspheroidene, bacteriophaeophytin, the ratio of tetrahydrogeranylgeranyl to phytylated BChl a, and the ratio of spheroidene to spheroidenone. This research highlights the adaptative response of R. capsulatus strain XJ-1 photosystems to environmental As, and demonstrates the potential of utilizing the sensitivity of its photosynthetic pigments to As(III) and As(V) for the biodetection of As in the environment.

  18. Heterologous expression of the Rhodobacter capsulatus BchI, -D, and -H genes that encode magnesium chelatase subunits and characterization of the reconstituted enzyme.

    PubMed

    Willows, R D; Beale, S I

    1998-12-18

    Magnesium chelatase inserts Mg2+ into protoporphyrin IX in the chlorophyll and bacteriochlorophyll biosynthetic pathways. In photosynthetic bacteria, the products of three genes, bchI, bchD, and bchH, are required for magnesium chelatase activity. These genes from Rhodobacter capsulatus were cloned separately into expression plasmids pET3a and pET15b. The pET15b constructs produced NH2-terminally His6-tagged proteins. All proteins were highly expressed and were purified to near homogeneity. The BchI and BchH proteins were soluble. BchD proteins were insoluble, inactive inclusion bodies that were renatured by rapid dilution from 6 M urea. The presence of BchI in the solution into which the urea solution of BchD was diluted increased the yield of active BchD. A molar ratio of 1 BchI:1 BchD was sufficient for maximum renaturation of BchD. All of the proteins were active in the magnesium chelatase assay except His-tagged BchI, which was inactive and inhibited in incubations containing non-His-tagged BchI. Expressed BchH proteins contained tightly bound protoporphyrin IX, and they were susceptible to inactivation by light. Maximum magnesium chelatase activity per mol of BchD occurred at a stoichiometry of 4 BchI:1 BchD. The optimum reaction pH was 8.0. The reaction exhibited Michaelis-Menten kinetics with respect to protoporphyrin IX and BchH.

  19. Expression of Uptake Hydrogenase and Molybdenum Nitrogenase in Rhodobacter capsulatus Is Coregulated by the RegB-RegA Two-Component Regulatory System

    PubMed Central

    Elsen, Sylvie; Dischert, Wanda; Colbeau, Annette; Bauer, Carl E.

    2000-01-01

    Purple photosynthetic bacteria are capable of generating cellular energy from several sources, including photosynthesis, respiration, and H2 oxidation. Under nutrient-limiting conditions, cellular energy can be used to assimilate carbon and nitrogen. This study provides the first evidence of a molecular link for the coregulation of nitrogenase and hydrogenase biosynthesis in an anoxygenic photosynthetic bacterium. We demonstrated that molybdenum nitrogenase biosynthesis is under the control of the RegB-RegA two-component regulatory system in Rhodobacter capsulatus. Footprint analyses and in vivo transcription studies showed that RegA indirectly activates nitrogenase synthesis by binding to and activating the expression of nifA2, which encodes one of the two functional copies of the nif-specific transcriptional activator, NifA. Expression of nifA2 but not nifA1 is reduced in the reg mutants up to eightfold under derepressing conditions and is also reduced under repressing conditions. Thus, although NtrC is absolutely required for nifA2 expression, RegA acts as a coactivator of nifA2. We also demonstrated that in reg mutants, [NiFe]hydrogenase synthesis and activity are increased up to sixfold. RegA binds to the promoter of the hydrogenase gene operon and therefore directly represses its expression. Thus, the RegB-RegA system controls such diverse processes as energy-generating photosynthesis and H2 oxidation, as well as the energy-demanding processes of N2 fixation and CO2 assimilation. PMID:10781552

  20. Yeast Two-Hybrid Studies on Interaction of Proteins Involved in Regulation of Nitrogen Fixation in the Phototrophic Bacterium Rhodobacter capsulatus

    PubMed Central

    Pawlowski, Alice; Riedel, Kai-Uwe; Klipp, Werner; Dreiskemper, Petra; Groß, Silke; Bierhoff, Holger; Drepper, Thomas; Masepohl, Bernd

    2003-01-01

    Rhodobacter capsulatus contains two PII-like proteins, GlnB and GlnK, which play central roles in controlling the synthesis and activity of nitrogenase in response to ammonium availability. Here we used the yeast two-hybrid system to probe interactions between these PII-like proteins and proteins known to be involved in regulating nitrogen fixation. Analysis of defined protein pairs demonstrated the following interactions: GlnB-NtrB, GlnB-NifA1, GlnB-NifA2, GlnB-DraT, GlnK-NifA1, GlnK-NifA2, and GlnK-DraT. These results corroborate earlier genetic data and in addition show that PII-dependent ammonium regulation of nitrogen fixation in R. capsulatus does not require additional proteins, like NifL in Klebsiella pneumoniae. In addition, we found interactions for the protein pairs GlnB-GlnB, GlnB-GlnK, NifA1-NifA1, NifA2-NifA2, and NifA1-NifA2, suggesting that fine tuning of the nitrogen fixation process in R. capsulatus may involve the formation of GlnB-GlnK heterotrimers as well as NifA1-NifA2 heterodimers. In order to identify new proteins that interact with GlnB and GlnK, we constructed an R. capsulatus genomic library for use in yeast two-hybrid studies. Screening of this library identified the ATP-dependent helicase PcrA as a new putative protein that interacts with GlnB and the Ras-like protein Era as a new protein that interacts with GlnK. PMID:12923097

  1. Interaction between the H2 Sensor HupUV and the Histidine Kinase HupT Controls HupSL Hydrogenase Synthesis in Rhodobacter capsulatus

    PubMed Central

    Elsen, Sylvie; Duché, Ophélie; Colbeau, Annette

    2003-01-01

    The photosynthetic bacterium Rhodobacter capsulatus contains two [NiFe]hydrogenases: an energy-generating hydrogenase, HupSL, and a regulatory hydrogenase, HupUV. The synthesis of HupSL is specifically activated by H2 through a signal transduction cascade comprising three proteins: the H2-sensing HupUV protein, the histidine kinase HupT, and the transcriptional regulator HupR. Whereas a phosphotransfer between HupT and HupR was previously demonstrated, interaction between HupUV and HupT was only hypothesized based on in vivo analyses of mutant phenotypes. To visualize the in vitro interaction between HupUV and HupT proteins, a six-His (His6)-HupU fusion protein and the HupV protein were coproduced by using a homologous expression system. The two proteins copurified as a His6-HupUHupV complex present in dimeric and tetrameric forms, both of which had H2 uptake activity. We demonstrated that HupT and HupUV interact and form stable complexes that could be separated on a native gel. Interaction was also monitored with surface plasmon resonance technology and was shown to be insensitive to salt concentration and pH changes, suggesting that the interactions involve hydrophobic residues. As expected, H2 affects the interaction between HupUV and HupT, leading to a weakening of the interaction, which is independent of the phosphate status of HupT. Several forms of HupT were tested for their ability to interact with HupUV and to complement hupT mutants. Strong interaction with HupUV was obtained with the isolated PAS domain of HupT and with inactive HupT mutated in the phosphorylable histidine residue, but only the wild-type HupT protein was able to restore normal H2 regulation. PMID:14645270

  2. bchFNBH bacteriochlorophyll synthesis genes of Rhodobacter capsulatus and identification of the third subunit of light-independent protochlorophyllide reductase in bacteria and plants.

    PubMed

    Burke, D H; Alberti, M; Hearst, J E

    1993-04-01

    We present the nucleotide and deduced amino acid sequences of four contiguous bacteriochlorophyll synthesis genes from Rhodobacter capsulatus. Three of these genes code for enzymes which catalyze reactions common to the chlorophyll synthesis pathway and therefore are likely to be found in plants and cyanobacteria as well. The pigments accumulated in strains with physically mapped transposon insertion mutations are analyzed by absorbance and fluorescence spectroscopy, allowing us to assign the genes as bchF, bchN, bchB, and bchH, in that order. bchF encodes a bacteriochlorophyll alpha-specific enzyme that adds water across the 2-vinyl group. The other three genes are required for portions of the pathway that are shared with chlorophyll synthesis, and they were expected to be common to both pathways. bchN and bchB are required for protochlorophyllide reduction in the dark (along with bchL), a reaction that has been observed in all major groups of photosynthetic organisms except angiosperms, where only the light-dependent reaction has been clearly established. The purple bacterial and plant enzymes show 35% identity between the amino acids coded by bchN and chlN (gidA) and 49% identity between the amino acids coded by bchL and chlL (frxC). Furthermore, bchB is 33% identical to ORF513 from the Marchantia polymorpha chloroplast. We present arguments in favor of the probable role of ORF513 (chlB) in protochlorophyllide reduction in the dark. The further similarities of all three subunits of protochlorophyllide reductase and the three subunits of chlorin reductase in bacteriochlorophyll synthesis suggest that the two reductase systems are derived from a common ancestor.

  3. Enzymic systems proposed to be involved in the dissimilatory reduction of selenite in the purple non-sulfur bacteria Rhodospirillum rubrum and Rhodobacter capsulatus.

    PubMed

    Kessi, Janine

    2006-03-01

    Various enzymic systems, such as nitrite reductase, sulfite reductase and glutathione reductase, have been proposed for, or suspected to be involved in, the reduction of selenite in bacteria. As alphaproteobacteria have been shown to be highly tolerant to transition metal oxyanions, it seemed interesting to investigate the hypothetical involvement of these different enzymes in the reduction of selenite in the purple non-sulfur bacteria Rhodospirillum rubrum and Rhodobacter capsulatus. The hypothetical involvement of nitrite reductase and sulfite reductase in the reduction of selenite in these bacteria was investigated by analysing the effects of nitrite and sulfite amendments on the growth and kinetics of selenite reduction. The reduction of selenite was not concomitant with that of either sulfite or nitrite in Rs. rubrum, suggesting that the reduction pathways operate independently. In Rb. capsulatus, strong interactions were observed between the nitrite reduction and selenite reduction pathways. However, in both organisms, selenite reduction took place during both the growth phase and the stationary phase, indicating that selenite metabolism is constitutively expressed. In contrast, neither nitrite nor sulfite was transformed during stationary phase, suggesting that the metabolism of both ions is induced, which implies that identical reduction pathways for selenite and nitrite or selenite and sulfite are excluded. Buthionine sulfoximine (BSO, S-n-butyl homocysteine sulfoximine), a specific inhibitor of glutathione synthesis, was used to depress the intracellular glutathione level. In stationary-phase cultures of both Rs. rubrum and Rb. capsulatus amended with BSO, the rate of reduction of selenite was slowed, indicating that glutathione may be involved in the dissimilatory reduction of selenite in these organisms. The analysis of the headspace gases of the cultures indicated that the synthesis of methylated selenium compounds was prevented in the presence of 3

  4. Coupling of proton flow to ATP synthesis in Rhodobacter capsulatus: F(0)F(1)-ATP synthase is absent from about half of chromatophores.

    PubMed

    Feniouk, B A; Cherepanov, D A; Junge, W; Mulkidjanian, A Y

    2001-11-01

    F(0)F(1)-ATP synthase (H(+)-ATP synthase, F(0)F(1)) utilizes the transmembrane protonmotive force to catalyze the formation of ATP from ADP and inorganic phosphate (P(i)). Structurally the enzyme consists of a membrane-embedded proton-translocating F(0) portion and a protruding hydrophilic F(1) part that catalyzes the synthesis of ATP. In photosynthetic purple bacteria a single turnover of the photosynthetic reaction centers (driven by a short saturating flash of light) generates protonmotive force that is sufficiently large to drive ATP synthesis. Using isolated chromatophore vesicles of Rhodobacter capsulatus, we monitored the flash induced ATP synthesis (by chemoluminescence of luciferin/luciferase) in parallel to the transmembrane charge transfer through F(0)F(1) (by following the decay of electrochromic bandshifts of intrinsic carotenoids). With the help of specific inhibitors of F(1) (efrapeptin) and of F(0) (venturicidin), we decomposed the kinetics of the total proton flow through F(0)F(1) into (i) those coupled to the ATP synthesis and (ii) the de-coupled proton escape through F(0). Taking the coupled proton flow, we calculated the H(+)/ATP ratio; it was found to be 3.3+/-0.6 at a large driving force (after one saturating flash of light) but to increase up to 5.1+/-0.9 at a smaller driving force (after a half-saturating flash). From the results obtained, we conclude that our routine chromatophore preparations contained three subsets of chromatophore vesicles. Chromatophores with coupled F(0)F(1) dominated in fresh material. Freezing/thawing or pre-illumination in the absence of ADP and P(i) led to an increase in the fraction of chromatophores with at least one de-coupled F(0)(F(1)). The disclosed fraction of chromatophores that lacked proton-conducting F(0)(F(1)) (approx. 40% of the total amount) remained constant upon these treatments.

  5. Hydroxylamine assimilation by Rhodobacter capsulatus E1F1. requirement of the hcp gene (hybrid cluster protein) located in the nitrate assimilation nas gene region for hydroxylamine reduction.

    PubMed

    Cabello, Purificación; Pino, Carmen; Olmo-Mira, M Francisca; Castillo, Francisco; Roldán, M Dolores; Moreno-Vivián, Conrado

    2004-10-29

    Rhodobacter capsulatus E1F1 grows phototrophically with nitrate as nitrogen source. Using primers designed for conserved motifs in bacterial assimilatory nitrate reductases, a 450-bp DNA was amplified by PCR and used for the screening of a genomic library. A cosmid carrying an insert with four SalI fragments of 2.8, 4.1, 4.5, and 5.8 kb was isolated, and DNA sequencing revealed that it contains a nitrate assimilation (nas) gene region, including the hcp gene coding for a hybrid cluster protein (HCP). Expression of hcp is probably regulated by a nitrite-sensitive repressor encoded by the adjacent nsrR gene. A His(6)-HCP was overproduced in Escherichia coli and purified. HCP contained about 6 iron and 4 labile sulfide atoms per molecule, in agreement with the presence of both [2Fe-2S] and [4Fe-2S-2O] clusters, and showed hydroxylamine reductase activity, forming ammonia in vitro with methyl viologen as reductant. The apparent K(m) values for NH(2)OH and methyl viologen were 1 mM and 7 microM, respectively, at the pH and temperature optima (9.3 and 40 degrees C). The activity was oxygen-sensitive and was inhibited by sulfide and iron reagents. R. capsulatus E1F1 grew phototrophically, but not heterotrophically, with 1 mM NH(2)OH as nitrogen source, and up to 10 mM NH(2)OH was taken up by anaerobic resting cells. Ammonium was transiently accumulated in the media, and its assimilation was prevented by L-methionine-D,L-sulfoximine, a glutamine synthetase inhibitor. In addition, hydroxylamine- or nitrite-grown cells showed the higher hydroxylamine reductase activities. However, R. capsulatus B10S, a strain lacking the whole hcp-nas region, did not grow with 1 mM NH(2)OH. Also, E. coli cells overproducing HCP tolerate hydroxyl-amine better during anaerobic growth. These results suggest that HCP is involved in assimilation of NH(2)OH, a toxic product that could be formed during nitrate assimilation, probably in the nitrite reduction step.

  6. X-ray crystallographic and mass spectrometric structure determination and functional characterization of succinylated porin from Rhodobacter capsulatus: implications for ion selectivity and single-channel conductance.

    PubMed Central

    Przybylski, M.; Glocker, M. O.; Nestel, U.; Schnaible, V.; Blüggel, M.; Diederichs, K.; Weckesser, J.; Schad, M.; Schmid, A.; Welte, W.; Benz, R.

    1996-01-01

    The role of charges near the pore mouth has been discussed in theoretical work about ion channels. To introduce new negative charges in a channel protein, amino groups of porin from Rhodobacter capsulatus 37b4 were succinylated with succinic anhydride, and the precise extent and sites of succinylations and structures of the succinylporins determined by mass spectrometry and X-ray crystallography. Molecular weight and peptide mapping analyses using matrix-assisted laser desorption-ionization mass spectrometry identified selective succinylation of three lysine-epsilon-amino groups (Lys-46, Lys-298, Lys-300) and the N-terminal alpha-amino group. The structure of a tetra-succinylated porin (TS-porin) was determined to 2.4 A and was generally found unchanged in comparison to native porin to form a trimeric complex. All succinylated amino groups found in a mono/di-succinylated porin (MS-porin) and a TS-porin are localized at the inner channel surface and are solvent-accessible: Lys-46 is located at the channel constriction site, whereas Lys-298, Lys-300, and the N-terminus are all near the periplasmic entrance of the channel. The Lys-46 residue at the central constriction loop was modeled as succinyl-lysine from the electron density data and shown to bend toward the periplasmic pore mouth. The electrical properties of the MS-and TS-porins were determined by reconstitution into black lipid membranes, and showed a negative charge effect on ion transport and an increased cation selectivity through the porin channel. The properties of a typical general diffusion porin changed to those of a channel that contains point charges near the pore mouth. The single-channel conductance was no longer a linear function of the bulk aqueous salt concentration. The substantially higher cation selectivity of the succinylated porins compared with the native protein is consistent with the increase of negatively charged groups introduced. These results show tertiary structure

  7. The Effects of Rhodobacter capsulatus KCTC-2583 on Cholesterol Metabolism, Egg Production and Quality Parameters during the Late Laying Periods in Hens.

    PubMed

    Lokhande, Anushka; Ingale, S L; Lee, S H; Kim, J S; Lohakare, J D; Chae, B J; Kwon, I K

    2013-06-01

    An experiment was conducted to investigate the effects of dietary supplementation of Rhodobacter capsulatus KCTC-2583 on egg-yolk and serum cholesterol, egg production and quality parameters during the late laying periods in hens. A total of 160 Hy-Line Brown layers (54 wk-old) were randomly allotted to 4 treatment groups on the basis of laying performance. Each treatment had 4 replicates with 10 birds each (40 birds per treatment). Two hens were confined individually with cage size 35×35×40 cm and each 10 birds (5 cages) shared a common feed trough between them forming one experimental unit. Dietary treatments were; basal diet supplemented with 0 (control), 0.05, 0.10 and 0.15% R. capsulatus KCTC-2583. Experimental diets were fed in meal form for 56 d. Dietary supplementation of increasing levels of R. capsulatus KCTC-2583 reduced (linear, p<0.05) egg-yolk cholesterol and triglycerides (d 28, 42 and 56) concentrations. Also, serum cholesterol and triglycerides (d 21, 42 and 56) concentrations were linearly reduced (p<0.05) with increasing dietary R. capsulatus KCTC-2583. Laying hens fed a diet supplemented with increasing levels of R. capsulatus KCTC-2583 had increased (linear; p<0.05) overall egg production, egg weight, egg mass and feed efficiency. However, dietary treatments had no effect (linear or quadratic; p>0.05) on feed intake of laying hens. At d 28 and 56, breaking strength and yolk colour of eggs were linearly improved (p<0.05) in laying hens fed dietary increasing levels of R. capsulatus KCTC-2583. Dietary treatment had no effects (linear or quadratic; p>0.05) on albumin height, shell thickness and shell weight at any period of experiment. These results indicate that dietary supplementation of R. capsulatus KCTC-2583 has the potential to improve the laying hen performance and lead to the development of low cholesterol eggs during late laying period in Hy-Line Brown hens.

  8. Functional assignment of gene AAC16202.1 from Rhodobacter capsulatus SB1003: new insights into the bacterial SDR sorbitol dehydrogenases family.

    PubMed

    Sola-Carvajal, Agustín; García-García, María Inmaculada; Sánchez-Carrón, Guiomar; García-Carmona, Francisco; Sánchez-Ferrer, Alvaro

    2012-11-01

    Short-chain dehydrogenases/reductases (SDR) constitute one of the largest enzyme superfamilies with over 60,000 non-redundant sequences in the database, many of which need a correct functional assignment. Among them, the gene AAC16202.1 (NCBI) from Rhodobacter capsulatus SB1003 has been assigned in Uniprot both as a sorbitol dehydrogenase (#D5AUY1) and, as an N-acetyl-d-mannosamine dehydrogenase (#O66112), both enzymes being of biotechnological interest. When the gene was overexpressed in Escherichia coli Rosetta (DE3)pLys, the purified enzyme was not active toward N-acetyl-d-mannosamine, whereas it was active toward d-sorbitol and d-fructose. However, the relative activities toward xylitol and l-iditol (0.45 and 6.9%, respectively) were low compared with that toward d-sorbitol. Thus, the enzyme could be considered sorbitol dehydrogenase (SDH) with very low activity toward xylitol, which could increase its biotechnological interest for determining sorbitol without the unspecific cross-determination of added xylitol in food and pharma compositions. The tetrameric enzyme (120 kDa) showed similar catalytic efficiency (2.2 × 10(3) M(-1) s(-1)) to other sorbitol dehydrogenases for d-sorbitol, with an optimum pH of 9.0 and an optimum temperature of 37 °C. The enzyme was also more thermostable than other reported SDH, ammonium sulfate being the best stabilizer in this respect, increasing the melting temperature (T(m)) up to 52.9 °C. The enzyme can also be considered as a new member of the Zn(2+) independent SDH family since no effect on activity was detected in the presence of divalent cations or chelating agents. Finally, its in silico analysis enabled the specific conserved sequence blocks that are the fingerprints of bacterial sorbitol dehydrogenases and mainly located at C-terminal of the protein, to be determined for the first time. This knowledge will facilitate future data curation of present databases and a better functional assignment of newly described

  9. The Effects of Rhodobacter capsulatus KCTC-2583 on Cholesterol Metabolism, Egg Production and Quality Parameters during the Late Laying Periods in Hens

    PubMed Central

    Lokhande, Anushka; Ingale, S. L.; Lee, S. H.; Kim, J. S.; Lohakare, J. D.; Chae, B. J.; Kwon, I. K.

    2013-01-01

    An experiment was conducted to investigate the effects of dietary supplementation of Rhodobacter capsulatus KCTC-2583 on egg-yolk and serum cholesterol, egg production and quality parameters during the late laying periods in hens. A total of 160 Hy-Line Brown layers (54 wk-old) were randomly allotted to 4 treatment groups on the basis of laying performance. Each treatment had 4 replicates with 10 birds each (40 birds per treatment). Two hens were confined individually with cage size 35×35×40 cm and each 10 birds (5 cages) shared a common feed trough between them forming one experimental unit. Dietary treatments were; basal diet supplemented with 0 (control), 0.05, 0.10 and 0.15% R. capsulatus KCTC-2583. Experimental diets were fed in meal form for 56 d. Dietary supplementation of increasing levels of R. capsulatus KCTC-2583 reduced (linear, p<0.05) egg-yolk cholesterol and triglycerides (d 28, 42 and 56) concentrations. Also, serum cholesterol and triglycerides (d 21, 42 and 56) concentrations were linearly reduced (p<0.05) with increasing dietary R. capsulatus KCTC-2583. Laying hens fed a diet supplemented with increasing levels of R. capsulatus KCTC-2583 had increased (linear; p<0.05) overall egg production, egg weight, egg mass and feed efficiency. However, dietary treatments had no effect (linear or quadratic; p>0.05) on feed intake of laying hens. At d 28 and 56, breaking strength and yolk colour of eggs were linearly improved (p<0.05) in laying hens fed dietary increasing levels of R. capsulatus KCTC-2583. Dietary treatment had no effects (linear or quadratic; p>0.05) on albumin height, shell thickness and shell weight at any period of experiment. These results indicate that dietary supplementation of R. capsulatus KCTC-2583 has the potential to improve the laying hen performance and lead to the development of low cholesterol eggs during late laying period in Hy-Line Brown hens. PMID:25049857

  10. Biochemical, Molecular, and Genetic Analyses of the Acetone Carboxylases from Xanthobacter autotrophicus Strain Py2 and Rhodobacter capsulatus Strain B10

    PubMed Central

    Sluis, Miriam K.; Larsen, Rachel A.; Krum, Jonathan G.; Anderson, Ruth; Metcalf, William W.; Ensign, Scott A.

    2002-01-01

    Acetone carboxylase is the key enzyme of bacterial acetone metabolism, catalyzing the condensation of acetone and CO2 to form acetoacetate. In this study, the acetone carboxylase of the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus was purified to homogeneity and compared to that of Xanthobacter autotrophicus strain Py2, the only other organism from which an acetone carboxylase has been purified. The biochemical properties of the enzymes were virtually indistinguishable, with identical subunit compositions (α2β2γ2 multimers of 85-, 78-, and 20-kDa subunits), reaction stoichiometries (CH3COCH3 + CO2 + ATP→CH3COCH2COO− + H+ + AMP + 2Pi), and kinetic properties (Km for acetone, 8 μM; kcat = 45 min−1). Both enzymes were expressed to high levels (17 to 25% of soluble protein) in cells grown with acetone as the carbon source but were not present at detectable levels in cells grown with other carbon sources. The genes encoding the acetone carboxylase subunits were identified by transposon mutagenesis of X. autotrophicus and sequence analysis of the R. capsulatus genome and were found to be clustered in similar operons consisting of the genes acxA (β subunit), acxB (α subunit), and acxC (γ subunit). Transposon mutagenesis of X. autotrophicus revealed a requirement of σ54 and a σ54-dependent transcriptional activator (AcxR) for acetone-dependent growth and acetone carboxylase gene expression. A potential σ54-dependent promoter 122 bp upstream of X. autotrophicus acxABC was identified. An AcxR gene homolog was identified 127 bp upstream of acxA in R. capsulatus, but this activator lacked key features of σ54-dependent activators, and the associated acxABC lacked an apparent σ54-dependent promoter, suggesting that σ54 is not required for expression of acxABC in R. capsulatus. These studies reveal a conserved strategy of ATP-dependent acetone carboxylation and the involvement of transcriptional enhancers in acetone carboxylase gene

  11. Single amino acid substitutions in the B870 alpha and beta light-harvesting polypeptides of Rhodobacter capsulatus. Structural and spectral effects.

    PubMed

    Babst, M; Albrecht, H; Wegmann, I; Brunisholz, R; Zuber, H

    1991-12-05

    To obtain information on the structural and functional role of highly conserved amino acid residues in the B870 alpha and beta light-harvesting polypeptides of Rhodobacter capsulatus, site-directed mutagenesis was performed. 18 mutants with single amino acid substitutions at nine different positions in the B870 antenna polypeptides were prepared in a B800-850-lacking strain. The characterization of the resulting phenotypes was based on a quantification of the core-complex elements (reaction center, light-harvesting polypeptides, bacteriochlorophyll a and carotenoid) and the core-complex spectral characteristics (absorption maximum, absorption coefficient and fluorescence intensity). These data generally showed that strong structural effects were caused by the amino acid substitutions. Thus, the three tryptophan exchanges at the position alpha 8 resulted in either the absence of a core complex (alpha Trp8----Leu), the absence of the core antenna (alpha Trp8----Ala) or a reduction in the carotenoid content (alpha Trp8----Tyr). Likewise, the mutants alpha Pro13Gly (i.e. alpha Pro13----Gly), beta Gly10Val and alpha Phe23Ala demonstrated an abnormal protein/pigment ratio in the core antenna, while a drastically reduced antenna size resulted from the amino acid exchange beta Arg45Asp. In contrast to the structural effects, the absorption maxima and the fluorescence intensities of the mutant antennae differed only slightly from the wild type. The strongest blue shift of the bacteriochlorophyll a (8-11 nm) was induced by substitutions of the Trp at position alpha 43 (alpha Trp43----Ala, Leu or Tyr). Contrary to the other spectral effects, the absorption coefficient of bacteriochlorophyll a was strongly influenced by the amino acid substitutions and varied by 1.6-times less (beta Arg45Asp) and 1.3-times greater (alpha Phe25Ala) than normal. The antenna-free mutant, alpha Trp8Ala, yielded a high rate of B800-850 revertants during phototrophic growth, indicating a direct

  12. Characterization of a nif-regulated flavoprotein (FprA) from Rhodobacter capsulatus. Redox properties and molecular interaction with a [2Fe-2S] ferredoxin.

    PubMed

    Jouanneau, Y; Meyer, C; Asso, M; Guigliarelli, B; Willison, J C

    2000-02-01

    A flavoprotein from Rhodobacter capsulatus was purified as a recombinant (His)6-tag fusion from an Escherichia coli clone over-expressing the fprA structural gene. The FprA protein is a homodimer containing one molecule of FMN per 48-kDa monomer. Reduction of the flavoprotein by dithionite showed biphasic kinetics, starting with a fast step of semiquinone (SQ) formation, and followed by a slow reduction of the SQ. This SQ was in the anionic form as shown by EPR and optical spectroscopies. Spectrophotometric titration gave a midpoint redox potential for the oxidized/SQ couple of Em1 = +20 mV (pH 8.0), whereas the SQ/hydroquinone couple could not be titrated due to the thermodynamic instability of SQ associated with its slow reduction process. The inability to detect the intermediate form, SQ, upon oxidative titration confirmed this instability and led to an estimate of Em2 - Em1 of > 80 mV. The reduction of SQ by dithionite was significantly accelerated when the [2Fe-2S] ferredoxin FdIV was used as redox mediator. The midpoint redox potential of this ferredoxin was determined to be -275 +/- 2 mV at pH 7.5, consistent with FdIV serving as electron donor to FprA in vivo. FdIV and FprA were found to cross-react when incubated together with the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, giving a covalent complex with an Mr of approximately 60 000. Formation of this complex was unaffected by the redox states of the two proteins. Other [2Fe-2S] ferredoxins, including FdV and FdVI from R. capsulatus, were ineffective as electron carriers to FprA, and cross-reacted poorly with the flavoprotein. The possible function of FprA with regard to nitrogen fixation was investigated using an fprA-deleted mutant. Although nitrogenase activity was significantly reduced in the mutant compared with the wild-type strain, nitrogen fixation was apparently unaffected by the fprA deletion even under iron limitation or microaerobic conditions.

  13. CO2 reduction and organic compounds production by photosynthetic bacteria with surface displayed carbonic anhydrase and inducible expression of phosphoenolpyruvate carboxylase.

    PubMed

    Park, Ju-Yong; Kim, Yang-Hoon; Min, Jiho

    2017-01-01

    In Rhodobacter sphaeroides, carbonic anhydrase (CA; EC 4.2.1.1) is a zinc-containing metalloenzyme that catalyzes the reversible hydration of CO2 to HCO3(-) while phosphoenolpyruvate carboxylase (PEPC; 4.1.1.31), an enzyme involved in the carbon metabolism that catalyzed the fixation of CO2 to PEP, is a key factor for biological fixation of CO2 and enhances the production of organic compounds. In this study, the recombinant R. sphaeroides with highly-expressed CA was developed based on a surface displayed system of CA (pJY-OmpCA) on the outer membrane of R. sphaeroides using outer membrane protein (Omp) in R. sphaeroides, Finally, two more different recombinant R. sphaeroides were developed, which transformed with a two-vector system harboring cytosolic expressed CA (pJY-OmpCA-CA)or PEPC (pJY-OMPCA-PEPC) in R. sphaeroides with surface displayed CA on the outer membrane. In case of recombinant R. sphaeroides with the pJY-OmpCA-PEPC, it has shown the highest CO2 reduction efficiency and the production of several organic compounds (carotenoids, polyhydroxybutyrate, malic acid, succinic acid). It means that the surface displayed CA on the R. sphaeroides would accelerate the CO2-bicabonate conversion on the bacterial outer membrane. Moreover, inducible over-expression of PEPC with surface-displayed CA was successfully used to facilitate a rapider CO2 reduction and quicker production of organic compounds.

  14. Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H2 production

    SciTech Connect

    Wecker, Matt S. A.; Beaton, Stephen E.; Chado, Robert A.; Ghirardi, Maria L.

    2016-08-17

    The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H2 as a by-product of its nitrogenase-catalyzed nitrogen-fixing activity. Such H2 production, however, is expensive from a metabolic perspective, requiring nearly four times as many photons as the equivalent algal hydrogenase-based system. Here we report the insertion of a Clostridium acetobutylicum [FeFe]-hydrogenase and its three attendant hydrogenase assembly proteins into an R. capsulatus strain lacking its native uptake hydrogenase. Further, this strain is modified to fluoresce upon sensing H2. The resulting strain photoproduces H2 and self-reports its own H2 production through fluorescence. Furthermore, this model system represents a unique method of developing hydrogenase-based H2 production in R. capsulatus, may serve as a powerful system for in vivo directed evolution of hydrogenases and hydrogenase-associated genes, and provides a means of screening for increased metabolic production of H2.

  15. A point mutation in the ATP synthase of Rhodobacter capsulatus results in differential contributions of Delta(pH) and Delta(phi) in driving the ATP synthesis reaction.

    PubMed

    Turina, Paola; Melandri, B Andrea

    2002-04-01

    The interface between the c-subunit oligomer and the a subunit in the F0 sector of the ATP synthase is believed to form the core of the rotating motor powered by the protonic flow. Besides the essential cAsp61 and aArg210 residues (Escherichia coli numbering), a few other residues at this interface, although nonessential, show a high degree of conservation, among these aGlu219. The homologous residue aGlu210 in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus has been substituted by a lysine. Inner membranes prepared from the mutant strain showed approximately half of the ATP synthesis activity when driven both by light and by acid-base transitions. As estimated with the ACMA assay, proton pumping rates in the inner membranes were also reduced to a similar extent in the mutant. The most striking impairment of ATP synthesis in the mutant, a decrease as low as 12 times as compared to the wild-type, was observed in the absence of a transmembrane electrical membrane potential (Delta(phi)) at low transmembrane pH difference (Delta(pH)). Therefore, the mutation seems to affect both the mechanism responsible for coupling F1 with proton translocation by F0, and the mechanism determining the relative contribution of Delta(pH) and Delta(phi) in driving ATP synthesis.

  16. Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H2 production

    SciTech Connect

    Wecker, Matt S. A.; Beaton, Stephen E.; Chado, Robert A.; Ghirardi, Maria L.

    2016-08-17

    The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H2 as a by-product of its nitrogenase-catalyzed nitrogen-fixing activity. Such H2 production, however, is expensive from a metabolic perspective, requiring nearly four times as many photons as the equivalent algal hydrogenase-based system. Here we report the insertion of a Clostridium acetobutylicum [FeFe]-hydrogenase and its three attendant hydrogenase assembly proteins into an R. capsulatus strain lacking its native uptake hydrogenase. Further, this strain is modified to fluoresce upon sensing H2. The resulting strain photoproduces H2 and self-reports its own H2 production through fluorescence. Furthermore, this model system represents a unique method of developing hydrogenase-based H2 production in R. capsulatus, may serve as a powerful system for in vivo directed evolution of hydrogenases and hydrogenase-associated genes, and provides a means of screening for increased metabolic production of H2.

  17. Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H 2 production: A Model System for Optimizing H 2 Production

    DOE PAGES

    Wecker, Matt S. A.; Beaton, Stephen E.; Chado, Robert A.; ...

    2016-08-23

    The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H2 as a by-product of its nitrogenase-catalyzed nitrogen-fixing activity. Such H2 production, however, is expensive from a metabolic perspective, requiring nearly four times as many photons as the equivalent algal hydrogenase-based system (Ghirardi et al. 2009). Here we report the insertion of a Clostridium acetobutylicum [FeFe]-hydrogenase and its three attendant hydrogenase assembly proteins into an R. capsulatus strain lacking its native uptake hydrogenase. Further, this strain is modified to fluoresce upon sensing H2. The resulting strain photoproduces H2 and self-reports its own H2 production through fluorescence. This model system represents a unique methodmore » of developing hydrogenase-based H2 production in R. capsulatus, may serve as a powerful system for in vivo directed evolution of hydrogenases and hydrogenase-associated genes, and provides a means of screening for increased metabolic production of H2.« less

  18. Open reading frame 5 (ORF5), encoding a ferredoxinlike protein, and nifQ are cotranscribed with nifE, nifN, nifX, and ORF4 in Rhodobacter capsulatus.

    PubMed Central

    Moreno-Vivian, C; Hennecke, S; Pühler, A; Klipp, W

    1989-01-01

    DNA sequence analysis of a 1,600-base-pair fragment located downstream of nifENX in nif region A of Rhodobacter capsulatus revealed two additional open reading frames (ORFs): ORF5, encoding a ferredoxinlike protein, and nifQ. The ferredoxinlike gene product contained two cysteine motifs, typical of ferredoxins coordinating two 4Fe-4S clusters, but the distance between these two motifs was unusual for low-molecular-weight ferredoxins. The R. capsulatus nifQ gene product shared a high degree of homology with Klebsiella pneumoniae and Azotobacter vinelandii NifQ, including a typical cysteine motif located in the C-terminal part. nifQ insertion mutants and also an ORF5-nifQ double deletion mutant showed normal diazotrophic growth only in the presence of high concentrations of molybdate. This demonstrated that the gene encoding the ferredoxinlike protein is not essential for nitrogen fixation. No NifA-activated consensus promoter could be found in the intergenic region between nifENX-ORF4 and ORF5-nifQ. Analyses of a nifQ-lacZYA fusion revealed that transcription of nifQ was initiated at a promoter in front of nifE. In contrast to other nitrogen-fixing organisms, R. capsulatus nifE, nifN, nifX, ORF4, ORF5, and nifQ were organized in one transcriptional unit. PMID:2708314

  19. Structure of the puf operon of the obligately aerobic, bacteriochlorophyll alpha-containing bacterium Roseobacter denitrificans OCh114 and its expression in a Rhodobacter capsulatus puf puc deletion mutant.

    PubMed Central

    Kortlüke, C; Breese, K; Gad'on, N; Labahn, A; Drews, G

    1997-01-01

    Roseobacter denitrificans (Erythrobacter species strain OCh114) synthesizes bacteriochlorophyll a (BChl) and the photosynthetic apparatus only in the presence of oxygen and is unable to carry out primary photosynthetic reactions and to grow photosynthetically under anoxic conditions. The puf operon of R. denitrificans has the same five genes in the same order as in many photosynthetic bacteria, i.e., pufBALMC. PufC, the tetraheme subunit of the reaction center (RC), consists of 352 amino acids (Mr, 39,043); 20 and 34% of the total amino acids are identical to those of PufC of Chloroflexus aurantiacus and Rubrivivax gelatinosus, respectively. The N-terminal hydrophobic domain is probably responsible for anchoring the subunit in the membrane. Four heme-binding domains are homologous to those of PufC in several purple bacteria. Sequences similar to pufQ and pufX of Rhodobacter capsulatus were not detected on the chromosome of R. denitrificans. The puf operon of R. denitrificans was expressed in trans in Escherichia coli, and all gene products were synthesized. The Roseobacter puf operon was also expressed in R. capsulatus CK11, a puf puc double-deletion mutant. For the first time, an RC/light-harvesting complex I core complex was heterologously synthesized. The strongest expression of the R. denitrificans puf operon was observed under the control of the R. capsulatus puf promoter, in the presence of pufQ and pufX and in the absence of pufC. Charge recombination between the primary donor P+ and the primary ubiquinone Q(A)- was observed in the transconjugant, showing that the M and L subunits of the RC were correctly assembled. The transconjugants did not grow photosynthetically under anoxic conditions. PMID:9286973

  20. Cooperation between two periplasmic copper chaperones is required for full activity of the cbb3-type cytochrome c oxidase and copper homeostasis in Rhodobacter capsulatus

    SciTech Connect

    Trasnea, Petru -Iulian; Utz, Marcel; Khalfaoui-Hassani, Bahia; Lagies, Simon; Daldal, Fevzi; Koch, Hans -Georg

    2016-02-28

    Copper (Cu) is an essential micronutrient that functions as a cofactor in several important enzymes, like respiratory heme-copper oxygen reductases. Yet, Cu is also toxic and therefore cells engage a highly coordinated Cu uptake and delivery system to prevent the accumulation of toxic Cu concentrations. In the current work we analyzed Cu delivery to the cbb3-type cytochrome c oxidase (cbb3-Cox) of Rhodobacter capsulatus. We identified the PCuAC-like periplasmic chaperone PccA and analyzed its contribution to cbb3-Cox assembly. Our data demonstrate that PccA is a Cu-binding protein with a preference for Cu(I), which is required for efficient cbb3-Cox assembly, in particular at low Cu concentrations. By using in vivo and in vitro crosslinking we show that PccA forms a complex with the Sco1-homologue SenC. This complex is stabilized in the absence of the cbb3-Cox specific assembly factors CcoGHIS. In cells lacking SenC, the cytoplasmic Cu content is significantly increased, but the simultaneous absence of PccA prevents this Cu accumulation. Lastly, these data demonstrate that the interplay between PccA and SenC is not only required for Cu delivery during cbb3-Cox assembly, but that it also regulates Cu homeostasis in R. capsulatus.

  1. The Thiol:Disulfide Oxidoreductase DsbB Mediates the Oxidizing Effects of the Toxic Metalloid Tellurite (TeO32−) on the Plasma Membrane Redox System of the Facultative Phototroph Rhodobacter capsulatus▿

    PubMed Central

    Borsetti, Francesca; Francia, Francesco; Turner, Raymond J.; Zannoni, Davide

    2007-01-01

    The highly toxic oxyanion tellurite (TeO32−) is a well known pro-oxidant in mammalian and bacterial cells. This work examines the effects of tellurite on the redox state of the electron transport chain of the facultative phototroph Rhodobacter capsulatus, in relation to the role of the thiol:disulfide oxidoreductase DsbB. Under steady-state respiration, the addition of tellurite (2.5 mM) to membrane fragments generated an extrareduction of the cytochrome pool (c- and b-type hemes); further, in plasma membranes exposed to tellurite (0.25 to 2.5 mM) and subjected to a series of flashes of light, the rate of the QH2:cytochrome c (Cyt c) oxidoreductase activity was enhanced. The effect of tellurite was blocked by the antibiotics antimycin A and/or myxothiazol, specific inhibitors of the QH2:Cyt c oxidoreductase, and, most interestingly, the membrane-associated thiol:disulfide oxidoreductase DsbB was required to mediate the redox unbalance produced by the oxyanion. Indeed, this phenomenon was absent from R. capsulatus MD22, a DsbB-deficient mutant, whereas the tellurite effect was present in membranes from MD22/pDsbBWT, in which the mutant gene was complemented to regain the wild-type DsbB phenotype. These findings were taken as evidence that the membrane-bound thiol:disulfide oxidoreductase DsbB acts as an “electron conduit” between the hydrophilic metalloid and the lipid-embedded Q pool, so that in habitats contaminated with subinhibitory amounts of TeIV, the metalloid is likely to function as a disposal for the excess reducing power at the Q-pool level of facultative phototrophic bacteria. PMID:17098900

  2. Activity of the Molybdopterin-Containing Xanthine Dehydrogenase of Rhodobacter capsulatus Can Be Restored by High Molybdenum Concentrations in a moeA Mutant Defective in Molybdenum Cofactor Biosynthesis

    PubMed Central

    Leimkühler, Silke; Angermüller, Sieglinde; Schwarz, Günter; Mendel, Ralf R.; Klipp, Werner

    1999-01-01

    During the screening for Rhodobacter capsulatus mutants defective in xanthine degradation, one Tn5 mutant which was able to grow with xanthine as a sole nitrogen source only in the presence of high molybdate concentrations (1 mM), a phenotype resembling Escherichia coli mogA mutants, was identified. Unexpectedly, the corresponding Tn5 insertion was located within the moeA gene. Partial DNA sequence analysis and interposon mutagenesis of regions flanking R. capsulatus moeA revealed that no further genes essential for molybdopterin biosynthesis are located in the vicinity of moeA and revealed that moeA forms a monocistronic transcriptional unit in R. capsulatus. Amino acid sequence alignments of R. capsulatus MoeA (414 amino acids [aa]) with E. coli MogA (195 aa) showed that MoeA contains an internal domain homologous to MogA, suggesting similar functions of these proteins in the biosynthesis of the molybdenum cofactor. Interposon mutants defective in moeA did not exhibit dimethyl sulfoxide reductase or nitrate reductase activity, which both require the molybdopterin guanine dinucleotide (MGD) cofactor, even after addition of 1 mM molybdate to the medium. In contrast, the activity of xanthine dehydrogenase, which binds the molybdopterin (MPT) cofactor, was restored to wild-type levels after the addition of 1 mM molybdate to the growth medium. Analysis of fluorescent derivatives of the molybdenum cofactor of purified xanthine dehydrogenase isolated from moeA and modA mutant strains, respectively, revealed that MPT is inserted into the enzyme only after molybdenum chelation, and both metal chelation and Mo-MPT insertion can occur only under high molybdate concentrations in the absence of MoeA. These data support a model for the biosynthesis of the molybdenum cofactor in which the biosynthesis of MPT and MGD are split at a stage when the molybdenum atom is added to MPT. PMID:10498704

  3. Molecular Regulation of Photosynthetic Carbon Dioxide Fixation in Nonsulfur Purple Bacteria

    SciTech Connect

    Tabita, Fred Robert

    2015-12-01

    The overall objective of this project is to determine the mechanism by which a transcriptional activator protein affects CO2 fixation (cbb) gene expression in nonsulfur purple photosynthetic bacteria, with special emphasis to Rhodobacter sphaeroides and with comparison to Rhodopseudomonas palustris. These studies culminated in several publications which indicated that additional regulators interact with the master regulator CbbR in both R. sphaeroides and R. palustris. In addition, the interactive control of the carbon and nitrogen assimilatory pathways was studied and unique regulatory signals were discovered.

  4. Temperature Dependence of the Rate Constants of Charge Recombination Reactions in Bacterial Reaction Centers

    NASA Astrophysics Data System (ADS)

    Thuy, T. T.; Yen, V. T. H.; Thao, T. T.; Viet, Nguyen Ai

    The bacterial reaction center couples light-induced electron transfer via a tightly bound ubiquinone (QA) to a mobile ubiquinone (QB). Based on the electron transfer theory by Marcus, we have investigated the rate of charge recombination reactions from Rhodopseudomonas viridis and Rhodobacter sphaeroides, by mean of finding an approximation formula. The results obtained are verified for not only at high and low temperature as the previous works but also at the medium temperature range.

  5. Electrocatalytic reduction of nitric oxide at electrodes modified with electropolymerized films of [Cr(v-tpy){sub 2}]{sup 3+} and their application to cellular NO determinations

    SciTech Connect

    Maskus, M.; Wu, Q.; Shapleigh, J.P.; Abruna, H.D.; Pariente, F.; Toffanin, A.

    1996-09-15

    Nitric oxide can be electrocatalytically reduced at electrodes modified with electropolymerized films of [Cr(v-tpy){sub 2}]{sup 3+}. Upon further modification with a thin film of Nafion (to prevent interferences from anions, especially nitrite), these electrodes can be employed as NO sensors in solution with submicromolar detection limits and fast response. We have carried out preliminary studies of cellular NO release from Rhodobacter sphaeroides bacterial cells with excellent results. 21 refs., 7 figs., 1 tab.

  6. Electrocatalytic reduction of nitric oxide at electrodes modified with electropolymerized films of [Cr(v-tpy)2]3+ and their application to cellular NO determinations.

    PubMed

    Maskus, M; Pariente, F; Wu, Q; Toffanin, A; Shapleigh, J P; Abruña, H D

    1996-09-15

    Nitric oxide can be electrocatalytically reduced at electrodes modified with electropolymerized films of [Cr(v-tpy)2]3+. Upon further modification with a thin film of Nafion (to prevent interferences from anions, especially nitrite), these electrodes can be employed as NO sensors in solution with submicromolar detection limits and fast response. We have carried out preliminary studies of cellular NO release from Rhodobacter sphaeroides bacterial cells with excellent results.

  7. The energies and kinetics of triplet carotenoids in the LH2 antenna complexes as determined by phosphorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Rondonuwu, Ferdy S.; Taguchi, Tokio; Fujii, Ritsuko; Yokoyama, Kyosuke; Koyama, Yasushi; Watanabe, Yasutaka

    2004-01-01

    The triplet (T 1) states of carotenoids (Cars) and bacteriochlorophyll a (BChl) in the LH2 antenna complexes from Rhodobacter sphaeroides G1C, Rba. sphaeroides 2.4.1 and Rhodospirillum molischianum, containing neurosporene, spheroidene and lycopene, respectively, were examined by stationary-state and time-resolved phosphorescence spectroscopy. The T 1 energies of Cars were determined, irrespective of the Car or BChl excitation, to be 7030 cm -1 (neurosporene), 6920 cm -1 (spheroidene) and 6870 cm -1 (lycopene), respectively, whereas that of BChl to be 7590 cm -1. In the Rba. sphaeroides G1C, the Car and BChl triplet states decayed in similar time constant as the BChl Q y state, a fact which indicates that the pair of triplet states decays through the triplet-triplet annihilation mechanism.

  8. Phylogenetic origins of the plant mitochondrion based on a comparative analysis of 5S ribosomal RNA sequences

    NASA Technical Reports Server (NTRS)

    Villanueva, E.; Delihas, N.; Luehrsen, K. R.; Fox, G. E.; Gibson, J.

    1985-01-01

    The complete nucleotide sequences of 5S ribosomal RNAs from Rhodocyclus gelatinosa, Rhodobacter sphaeroides, and Pseudomonas cepacia were determined. Comparisons of these 5S RNA sequences show that rather than being phylogenetically related to one another, the two photosynthetic bacterial 5S RNAs share more sequence and signature homology with the RNAs of two nonphotosynthetic strains. Rhodobacter sphaeroides is specifically related to Paracoccus denitrificans and Rc. gelatinosa is related to Ps. cepacia. These results support earlier 16S ribosomal RNA studies and add two important groups to the 5S RNA data base. Unique 5S RNA structural features previously found in P. denitrificans are present also in the 5S RNA of Rb. sphaeroides; these provide the basis for subdivisional signatures. The immediate consequence of obtaining these new sequences is that it is possible to clarify the phylogenetic origins of the plant mitochondrion. In particular, a close phylogenetic relationship is found between the plant mitochondria and members of the alpha subdivision of the purple photosynthetic bacteria, namely, Rb. sphaeroides, P. denitrificans, and Rhodospirillum rubrum.

  9. Phylogenetic origins of the plant mitochondrion based on a comparative analysis of 5S ribosomal RNA sequences

    NASA Technical Reports Server (NTRS)

    Villanueva, E.; Delihas, N.; Luehrsen, K. R.; Fox, G. E.; Gibson, J.

    1985-01-01

    The complete nucleotide sequences of 5S ribosomal RNAs from Rhodocyclus gelatinosa, Rhodobacter sphaeroides, and Pseudomonas cepacia were determined. Comparisons of these 5S RNA sequences show that rather than being phylogenetically related to one another, the two photosynthetic bacterial 5S RNAs share more sequence and signature homology with the RNAs of two nonphotosynthetic strains. Rhodobacter sphaeroides is specifically related to Paracoccus denitrificans and Rc. gelatinosa is related to Ps. cepacia. These results support earlier 16S ribosomal RNA studies and add two important groups to the 5S RNA data base. Unique 5S RNA structural features previously found in P. denitrificans are present also in the 5S RNA of Rb. sphaeroides; these provide the basis for subdivisional signatures. The immediate consequence of obtaining these new sequences is that it is possible to clarify the phylogenetic origins of the plant mitochondrion. In particular, a close phylogenetic relationship is found between the plant mitochondria and members of the alpha subdivision of the purple photosynthetic bacteria, namely, Rb. sphaeroides, P. denitrificans, and Rhodospirillum rubrum.

  10. Studies on Hydrogen Production by Photosynthetic Bacteria after Anaerobic Fermentation of Starch by a Hyperthermophile, Pyrococcus furiosus

    NASA Astrophysics Data System (ADS)

    Sugitate, Toshihiro; Fukatsu, Makoto; Ishimi, Katsuhiro; Kohno, Hideki; Wakayama, Tatsuki; Nakamura, Yoshihiro; Miyake, Jun; Asada, Yasuo

    In order to establish the sequential hydrogen production from waste starch using a hyperthermophile, Pyrococcus furiosus, and a photosynthetic bacterium, basic studies were done. P. furiosus produced hydrogen and acetate by anaerobic fermentation at 90°C. A photosynthetic bacterium, Rhodobacter sphaeroides RV, was able to produce hydrogen from acetate under anaerobic and light conditions at 30°C. However, Rb. sphaeroides RV was not able to produce hydrogen from acetate in the presence of sodium chloride that was essential for the growth and hydrogen production of P. furiosus although it produced hydrogen from lactate at a reduced rate with 1% sodium chloride. A newly isolated strain, CST-8, from natural environment was, however, able to produce hydrogen from acetate, especially with 3 mM L-alanine and in the presence of 1% sodium chloride. The sequential hydrogen production with P. furiosus and salt-tolerant photosynthetic bacteria could be probable at least in the laboratory experiment scale.

  11. Species differences in unlocking B-side electron transfer in bacterial reaction centers

    DOE PAGES

    Dylla, Nicholas P.; Faries, Kaitlyn M.; Wyllie, Ryan M.; ...

    2016-06-21

    The structure of the bacterial photosynthetic reaction center (RC) reveals symmetry-related electron transfer (ET) pathways, but only one path is used in native RCs. Analogous mutations have been made in two Rhodobacter (R.) species. A glutamic acid at position 133 in the M subunit increases transmembrane charge separation via the naturally inactive (B-side) path through impacts on primary ET in mutant R. sphaeroidesRCs. Prior work showed that the analogous substitution in the R. capsulatusRC also increases B-side activity, but mainly affects secondary ET. Finally, the overall yields of transmembrane ET are similar, but enabled in fundamentally different ways.

  12. A Conserved Steroid Binding Site in Cytochrome c Oxidase

    SciTech Connect

    Qin, Ling; Mills, Denise A.; Buhrow, Leann; Hiser, Carrie; Ferguson-Miller, Shelagh

    2010-09-02

    Micromolar concentrations of the bile salt deoxycholate are shown to rescue the activity of an inactive mutant, E101A, in the K proton pathway of Rhodobacter sphaeroides cytochrome c oxidase. A crystal structure of the wild-type enzyme reveals, as predicted, deoxycholate bound with its carboxyl group at the entrance of the K path. Since cholate is a known potent inhibitor of bovine oxidase and is seen in a similar position in the bovine structure, the crystallographically defined, conserved steroid binding site could reveal a regulatory site for steroids or structurally related molecules that act on the essential K proton path.

  13. A Computational Strategy to Analyze Label-Free Temporal Bottom-Up Proteomics Data§

    PubMed Central

    Du, Xiuxia; Callister, Stephen J.; Manes, Nathan P.; Adkins, Joshua N.; Alexandridis, Roxana A.; Zeng, Xiaohua; Roh, Jung Hyeob; Smith, William E.; Donohue, Timothy J.; Kaplan, Samuel; Smith, Richard D.; Lipton, Mary S.

    2008-01-01

    Biological systems are in a continual state of flux, which necessitates an understanding of the dynamic nature of protein abundances. The study of protein abundance dynamics has become feasible with recent improvements in mass spectrometry-based quantitative proteomics. However, a number of challenges still remain related to how best to extract biological information from dynamic proteomics data, for example, challenges related to extraneous variability, missing abundance values, and the identification of significant temporal patterns. This paper describes a strategy that addresses these issues and demonstrates its values for analyzing temporal bottom-up proteomics data using data from a Rhodobacter sphaeroides 2.4.1 time-course study. PMID:18442284

  14. Femtosecond processes and ultrafast biological electron transfer

    NASA Astrophysics Data System (ADS)

    Suzuki, Satoru; Sung, H. C.; Hayashi, M.; Lin, S. H.

    1995-02-01

    In this paper, we report the calculated femtosecond transient spectra of the primary electron transfer in the bacterial reaction center of Rhodobacter sphaeroides R26. The excitation dependence of the time-resolved spectra is simulated. The effects of vibrational coherence on the time-resolved spectra are discussed. The electron transfer accompanying vibrational relaxation is theoretically investigated using the single-vibronic level electron transfer rate constants. The effects of multi-modes and temperature on the electron transfer rate constants are also reported.

  15. Organization and evolution of the biological response to singlet oxygen stress.

    PubMed

    Dufour, Yann S; Landick, Robert; Donohue, Timothy J

    2008-11-14

    The appearance of atmospheric oxygen from photosynthetic activity led to the evolution of aerobic respiration and responses to the resulting reactive oxygen species. In Rhodobacter sphaeroides, a photosynthetic alpha-proteobacterium, a transcriptional response to the reactive oxygen species singlet oxygen ((1)O(2)) is controlled by the group IV sigma factor sigma(E) and the anti-sigma factor ChrR. In this study, we integrated various large datasets to identify genes within the (1)O(2) stress response that contain sigma(E)-dependent promoters both within R. sphaeroides and across the bacterial phylogeny. Transcript pattern clustering and a sigma(E)-binding sequence model were used to predict candidate promoters that respond to (1)O(2) stress in R. sphaeroides. These candidate promoters were experimentally validated to nine R. sphaeroides sigma(E)-dependent promoters that control the transcription of 15 (1)O(2)-activated genes. Knowledge of the R. sphaeroides response to (1)O(2) and its regulator sigma(E)-ChrR was combined with large-scale phylogenetic and sequence analyses to predict the existence of a core set of approximately eight conserved sigma(E)-dependent genes in alpha-proteobacteria and gamma-proteobacteria. The bacteria predicted to contain this conserved response to (1)O(2) include photosynthetic species, as well as free-living and symbiotic/pathogenic nonphotosynthetic species. Our analysis also predicts that the response to (1)O(2) evolved within the time frame of the accumulation of atmospheric molecular oxygen on this planet.

  16. Organization and Evolution of the Biological Response to Singlet Oxygen Stress

    PubMed Central

    Dufour, Yann S.; Landick, Robert; Donohue, Timothy J.

    2008-01-01

    The appearance of atmospheric oxygen from photosynthetic activity led to the evolution of aerobic respiration and responses to the resulting reactive oxygen species. In Rhodobacter sphaeroides, a photosynthetic α-proteobacterium, a transcriptional response to the reactive oxygen species singlet oxygen (1O2) is controlled by the group IV σ factor σE and the anti-σ factor ChrR. In this study, we integrated various large datasets to identify genes within the 1O2 stress response that contain σE-dependent promoters both within R. sphaeroides and across the bacterial phylogeny. Transcript pattern clustering and a σE-binding sequence model were used to predict candidate promoters that respond to 1O2 stress in R. sphaeroides. These candidate promoters were experimentally validated to nine R. sphaeroides σE-dependent promoters that control the transcription of 15 1O2-activated genes. Knowledge of the R. sphaeroides response to 1O2 and its regulator σE–ChrR was combined with large-scale phylogenetic and sequence analyses to predict the existence of a core set of approximately eight conserved σE-dependent genes in α-proteobacteria and γ-proteobacteria. The bacteria predicted to contain this conserved response to 1O2 include photosynthetic species, as well as free-living and symbiotic/pathogenic nonphotosynthetic species. Our analysis also predicts that the response to 1O2 evolved within the time frame of the accumulation of atmospheric molecular oxygen on this planet. PMID:18723027

  17. Heterologous carotenoid-biosynthetic enzymes: functional complementation and effects on carotenoid profiles in Escherichia coli.

    PubMed

    Song, Gyu Hyeon; Kim, Se Hyeuk; Choi, Bo Hyun; Han, Se Jong; Lee, Pyung Cheon

    2013-01-01

    A limited number of carotenoid pathway genes from microbial sources have been studied for analyzing the pathway complementation in the heterologous host Escherichia coli. In order to systematically investigate the functionality of carotenoid pathway enzymes in E. coli, the pathway genes of carotenogenic microorganisms (Brevibacterium linens, Corynebacterium glutamicum, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodopirellula baltica, and Pantoea ananatis) were modified to form synthetic expression modules and then were complemented with Pantoea agglomerans pathway enzymes (CrtE, CrtB, CrtI, CrtY, and CrtZ). The carotenogenic pathway enzymes in the synthetic modules showed unusual activities when complemented with E. coli. For example, the expression of heterologous CrtEs of B. linens, C. glutamicum, and R. baltica influenced P. agglomerans CrtI to convert its substrate phytoene into a rare product-3,4,3',4'-tetradehydrolycopene-along with lycopene, which was an expected product, indicating that CrtE, the first enzyme in the carotenoid biosynthesis pathway, can influence carotenoid profiles. In addition, CrtIs of R. sphaeroides and R. capsulatus converted phytoene into an unusual lycopene as well as into neurosporene. Thus, this study shows that the functional complementation of pathway enzymes from different sources is a useful methodology for diversifying biosynthesis as nature does.

  18. Heterologous Carotenoid-Biosynthetic Enzymes: Functional Complementation and Effects on Carotenoid Profiles in Escherichia coli

    PubMed Central

    Song, Gyu Hyeon; Kim, Se Hyeuk; Choi, Bo Hyun; Han, Se Jong

    2013-01-01

    A limited number of carotenoid pathway genes from microbial sources have been studied for analyzing the pathway complementation in the heterologous host Escherichia coli. In order to systematically investigate the functionality of carotenoid pathway enzymes in E. coli, the pathway genes of carotenogenic microorganisms (Brevibacterium linens, Corynebacterium glutamicum, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodopirellula baltica, and Pantoea ananatis) were modified to form synthetic expression modules and then were complemented with Pantoea agglomerans pathway enzymes (CrtE, CrtB, CrtI, CrtY, and CrtZ). The carotenogenic pathway enzymes in the synthetic modules showed unusual activities when complemented with E. coli. For example, the expression of heterologous CrtEs of B. linens, C. glutamicum, and R. baltica influenced P. agglomerans CrtI to convert its substrate phytoene into a rare product—3,4,3′,4′-tetradehydrolycopene—along with lycopene, which was an expected product, indicating that CrtE, the first enzyme in the carotenoid biosynthesis pathway, can influence carotenoid profiles. In addition, CrtIs of R. sphaeroides and R. capsulatus converted phytoene into an unusual lycopene as well as into neurosporene. Thus, this study shows that the functional complementation of pathway enzymes from different sources is a useful methodology for diversifying biosynthesis as nature does. PMID:23144136

  19. Effect of integral membrane proteins on the lateral mobility of plastoquinone in phosphatidylcholine proteoliposomes

    PubMed Central

    Blackwell, Mary F.; Whitmarsh, John

    1990-01-01

    Pyrene fluorescence quenching by plastoquinone was used to estimate the rate of plastoquinone lateral diffusion in soybean phosphatidylcholine proteoliposomes containing the following integral membrane proteins: gramicidin D, spinach cytochrome bf complex, spinach cytochrome f, reaction centers from Rhodobacter sphaeroides, beef heart mitochondrial cytochrome bc1, and beef heart mitochondrial cytochrome oxidase. The measured plastoquinone lateral diffusion coefficient varied between 1 and 3 · 10-7 cm2 s-1 in control liposomes that lacked protein. When proteins were added, these values decreased: a 10-fold decrease was observed when 16-26% of the membrane surface area was occupied by protein for all the proteins but gramicidin. The larger protein complexes (cytochrome bf, Rhodobacter sphaeroides reaction centers, cytochrome bc1, and cytochrome oxidase), whose hydrophobic volumes were 15-20 times as large as that of cytochrome f and the gramicidin transmembrane dimer, were 15-20 times as effective in decreasing the lateral-diffusion coefficient over the range of concentrations studied. These proteins had a much stronger effect than that observed for bacteriorhodopsin in fluorescence photobleaching recovery measurements. The effect of high-protein concentrations in gramicidin proteoliposomes was in close agreement with fluorescence photobleaching measurements. The results are compared with the predictions of several theoretical models of lateral mobility as a function of integral membrane concentration. PMID:19431774

  20. Production of ubiquinone-10 using bacteria.

    PubMed

    Yoshida, Hajime; Kotani, Yukinobu; Ochiai, Keiko; Araki, Kazumi

    1998-02-01

    Among the bacterial strains known to contain ubiquinone-10, three strains, Agrobacterium tumefaciens KY-3085 (ATCC4452), Paracoccus denitrificans KY-3940 (ATCC19367) and Rhodobacter sphaeroides KY-4113 (FERM-P4675), were selected as excellent producers of this ubiquinone. The ubiquinone-10 production by the Agrobacterium and Rhodobacter strains was affected by aeration. An ethionine-resistant mutant (M-37) derived from A. tumefaciens KY-3085 promoted increased production of ubiquinone-10 (20% higher than the parent). Another Agrobacterium mutant (AU-55), which was induced by the successive addition of four genetic markers, showed a tolerance to the suppression of ubiquinone-10 production caused by aeration, and the fermentation time for production was remarkably shortened. The amount of ubiquinone-10 produced by this Agrobacterium mutant reached 180 mg/l in a 58 h culture. A green mutant (carotenoid-deficient mutant, Co-22-11) derived from R. sphaeroides KY-4113 produced 350 mg/l of ubiquinone-10 under culturing conditions with a limited supply of air, the ubiquinone-10 content being 8.7 mg/g-dry cell. In this case, the amount and content corresponded to 2.8 and 3.6 times larger than those given by the wild-type strain, respectively. A multiple-layer structure of cell membrane was observed in the highly ubiquinone-10 accumulating cell of the green mutant by electron microscopy. The amount of ubiquinone-10 produced by P. denitrificans was much lower than those of the other two strains.

  1. Different Functions of Phylogenetically Distinct Bacterial Complex I Isozymes

    PubMed Central

    Spero, Melanie A.; Brickner, Joshua R.; Mollet, Jordan T.; Pisithkul, Tippapha; Amador-Noguez, Daniel

    2016-01-01

    ABSTRACT NADH:quinone oxidoreductase (complex I) is a bioenergetic enzyme that transfers electrons from NADH to quinone, conserving the energy of this reaction by contributing to the proton motive force. While the importance of NADH oxidation to mitochondrial aerobic respiration is well documented, the contribution of complex I to bacterial electron transport chains has been tested in only a few species. Here, we analyze the function of two phylogenetically distinct complex I isozymes in Rhodobacter sphaeroides, an alphaproteobacterium that contains well-characterized electron transport chains. We found that R. sphaeroides complex I activity is important for aerobic respiration and required for anaerobic dimethyl sulfoxide (DMSO) respiration (in the absence of light), photoautotrophic growth, and photoheterotrophic growth (in the absence of an external electron acceptor). Our data also provide insight into the functions of the phylogenetically distinct R. sphaeroides