Sphaeroides Strain Research Articles

Time-resolved and steady-state spectroscopic analysis of membrane-bound reaction centers from Rhodobacter sphaeroides: comparisons with detergent-solubilized complexes.

The spectroscopic analysis of the antenna-deficient Rhodobacter sphaeroides strain RCO1 has been extended to an investigation of the kinetics and spectroscopy of primary charge separation. Global analysis of time-resolved difference spectra demonstrated that the rate of charge separation in membrane-bound reaction centers is slightly slower than in detergent-solubilized reaction centers from the same strain. A kinetic analysis of the decay of the primary donor excited state at single wavelengths was carried out using a high repetition rate laser system, with the reaction centers being maintained in the open state using a combination of phenazine methosulfate and horse heart cytochrome c. The kinetics of primary charge separation in both membrane-bound and solubilized reaction centers were found to be non-monoexponential, with two exponential decay components required for a satisfactory description of the decay of the primary donor excited state. The overall rate of charge separation in membrane-bound reaction centers was slowed if the primary acceptor quinone was reduced using sodium ascorbate. This slowing was caused, in part, by an increase in the relative amplitude of the slower of the two exponential components. The acceleration in the rate of charge separation observed on removal of the reaction center from the membrane did not appear to be caused by a significant change in the electrochemical properties of the primary donor. The influence of the environment of the reaction center on primary charge separation is discussed together with the origins of the non-monoexponential decay of the primary donor excited state.

Polyol metabolism of Rhodobacter sphaeroides: biochemical characterization of a short-chain sorbitol dehydrogenase.

A sorbitol dehydrogenase (SDH; L-iditol:NAD+ 2-oxidoreductase; EC 1.1.1.14) was isolated from the phototrophic bacterium Rhodobacter sphaeroides strain M22, a transposon mutant of R. sphaeroides Si4 with the transposon inserted in the mannitol dehydrogenase (MDH) gene. SDH was purified 470-fold to apparent homogeneity by ammonium sulfate precipitation, chromatography on Phenyl-Sepharose, Q-Sepharose and Matrex Gel Red-A, and by gel filtration on Superdex 200. The relative molecular mass (M(r)) of the native SDH was 61000 as calculated from its Stokes' radius (rs = 3.5 nm) and sedimentation coefficient (S20,w = 4.23S). SDS-PAGE resulted in one single band representing a polypeptide with a M(r) of 29,000, indicating that the native protein is a dimer. The isoelectric point of SDH was determined to be pH 4.8. The enzyme was specific for NAD+ and catalysed the oxidation of D-glucitol (sorbitol) to D-fructose, galactitol to D-tagatose and of L-iditol. The apparent Km values were NAD+, 0.06 mM; D-glucitol, 6.2 mM; galactitol, 1.5 mM; NADH, 0.13 mM; D-fructose, 160 mM; and D-tagatose, 13 mM. The pH-optimum of substrate oxidation was 11.0 and that of substrate reduction 6.0-7.2. It was demonstrated that SDH is expressed in the wild-type strain R. sphaeroides Si4 together with MDH during growth on D-glucitol. Forty-four amino acids of the SDH N terminus were sequenced. This sequence exhibited 45-55% identity to the N-terminal sequence of 10 enzymes belonging to the short-chain alcohol dehydrogenase family.

Accumulation of poly-(hydroxybutyrate) by a non-sulfur photosynthetic bacterium, Rhodobacter sphaeroides RV at different pH

Effect of pH of culture media on intracellular accumulation of poly-(hydroxybutyrate) (PHB) by a non-sulfur photosynthetic bacterium, Rhodobacter sphaeroides strain RV was studied in pH-stat cultures. Sub-optimal pH for growth, 8.0 or 8.5 gave the higher content of PHB rather than optimal pH 7.5 for growth. These results show that growth and PHB accumulation of the bacteria can be controlled by pH of culture media.

Stabilization of reduced primary quinone by proton uptake in reaction centers of Rhodobacter sphaeroides.

Proton binding stoichiometry and kinetics of charge recombination were measured after single flash excitation in reaction centers from the purple photosynthetic bacterium, Rhodobacter sphaeroides strain R-26, where the native ubiquinone in the primary quinone acceptor site QA was removed and replaced by (benzo-, naphtho-, and anthra-) quinones of various structures and redox midpoint potentials. The observed proton binding stoichiometry was small (0.2-0.4 H+/QA-) and not specific to quinones in the acidic and neutral pH ranges. Above pH 9, however, significant differences were detected; reaction centers reconstituted by menadione failed to take up protons above pH 9.5. The pH dependence of the free energy change (stabilization) of the semiquinone was determined by integration of the proton uptake stoichiometry as a function of pH. Ubiquinone had the largest (100 meV at pH 5) and menadione the smallest (49 meV at pH 5) stabilization energy compared to those at very high (> 11) pH. In the case of the anthraquinone-reconstituted reaction center, acceptable agreement was obtained above pH 9 for the stabilization energies derived from energetic parameters of the thermally activated electron transfer (back reaction) and from proton binding stoichiometries. The stabilization at high pH could be attributed to a single protonatable amino acid, which might be either in the QB (secondary quinone) pocket (Glu L212) or in the vicinity of the QA binding domain (Tyr H40). It was shown that this residue had a negligible energy of interaction with bacteriopheophytin and that its coupling to the semiquinone was sensitive to the structure and physicochemical properties of QA.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and regulation of Bradyrhizobium japonicum and Xanthobacter flavus CO2 fixation genes in a photosynthetic bacterial host.

Calvin cycle carbon dioxide fixation genes encoded on DNA fragments from two nonphotosynthetic, chemolithoautotrophic bacteria, Bradyrhizobium japonicum and Xanthobacter flavus, were found to complement and support photosynthetic growth of a ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion mutant of the purple nonsulfur bacterium Rhodobacter sphaeroides. The regulation of RubisCO expression was analyzed in the complemented R. sphaeroides RubisCO deletion mutant. Distinct differences in the regulation of RubisCO synthesis were revealed when the complemented R. sphaeroides strains were cultured under photolithoautotrophic and photoheterotrophic growth conditions, e.g., a reversal in the normal pattern of RubisCO gene expression. These studies suggest that sequences and molecular signals which regulate the expression of diverse RubisCO genes may be probed by using the R. sphaeroides complementation system.

Effects of phospholipase A2 digestion on the carotenoid and bacteriochlorophyll components of the light-harvesting complexes in Rhodobacter sphaeroides chromatophores

The instantaneous electrochromic response of carotenoids associated with the B800-850 light-harvesting complex of Rhodobacter sphaeroides has been used widely as an intrinsic probe of membrane potential. In the present study, the structural basis for this phenomenon was examined by phospholipase A2 digestion of chromatophores from R. sphaeroides strain NF57G, containing B800-850 as the sole pigment-protein complex. The major phospholipase-induced alterations of the overall carotenoid absorption spectrum were characterized by an absorbance loss and a blue shift that were accompanied by a decrease in absorbance at 800 nm and a red shift in the B850 absorbance band. In wild-type chromatophores, the electrochromic carotenoid response induced by both flash illumination and a K+ diffusion potential was diminished by approximately 60% after 1 h of digestion. The initial loss of the carotenoid response was correlated specifically to the hydrolysis of phosphatidylethanolamine, and was shown to arise from effects exerted directly upon the electrochromically active carotenoid pool, possibly by alterations in the spatial relationship between the field-sensitive carotenoids and the polarizing permanent field. In phospholipase A2-digested NF57G preparations in which the B800 band was diminished by nearly half and the carotenoid response was abolished, no significant changes in the efficiency of energy transfer from carotenoids to bacteriochlorophyll were detected at 77 K, suggesting that the electrochromically active carotenoids are not energetically linked to B800 bacteriochlorophyll.

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

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.

Chromosome transfer in Rhodobacter sphaeroides: Hfr formation and genetic evidence for two unique circular chromosomes

A 600-bp oriT-containing DNA fragment from the Rhodobacter sphaeroides 2.4.1 S factor (oriTs) (A. Suwanto and S. Kaplan, J. Bacteriol. 174:1124-1134, 1992) was shown to promote polarized chromosomal transfer when provided in cis. A Kmr-oriTs-sacR-sacB (KTS) DNA cassette was constructed by inserting oriTs-sacR-sacB into a pUTmini-Tn5 Km1 derivative. With this delivery system, KTS appeared to be randomly inserted into the genome of R. sphaeroides, generating mutant strains which also gained the ability to act as Hfr donors. An AseI site in the Kmr cartridge (from Tn903) and DraI and SnaBI sites in sacR-sacB (the levansucrase gene from Bacillus subtilis) were employed to localize the KTS insertion definitively by pulsed-field gel electrophoresis. The orientation of oriTs at the site of insertion was determined by Southern hybridization analysis. Interrupted mating experiments performed with some of the Hfr strains exhibited a gradient of marker transfer and further provided genetic evidence for the circularity and presence of two chromosomal linkage groups in this bacterium. The genetic and environmental conditions for optimized mating between R. sphaeroides strains were also defined. The results presented here and our physical map of the R. sphaeroides 2.4.1 genome are discussed in light of the presence of two chromosomes.

Time resolved ADMR applied to the triplet state of the primary donor of bacterial photosynthetic reaction centers

The population and depopulation kinetics of the triplet sublevels of reaction centers ofRhodobacter (Rb.) sphaeroides strains R26 and GA were determined at 9 K by time resolved absorption detected magnetic resonance (ADMR) using the RF pulse method. The comparison of the saturation and pulse methods of time resolved ADMR proofs the latter to be superior. The kinetic behavior of the 2|E| signal was investigated for the first time. It exhibits an unexpected slow decaying component with a rate comparable to thez-level decay. Using a simple kinetic 4-level model for the triplet dynamics we conclude that this slow component as well as the sign of the ADMR signal of the 2|E| transition can be explained by a selective spin lattice relaxation channel connecting the triplet sublevelsx andz.

Towards the understanding of the function of Rb sphaeroides Y wild type reaction center: gene cloning, protein and detergent structures in the three-dimensional crystals

We report various experiments aimed at the resolution of the 3-dimensional structure of the photosynthetic reaction center from wild type Y Rhodobacter sphaeroides. The genes encoding the L and M polypeptides have been cloned and sequenced. They bear 2 mutations each when compared to those already sequenced in another Rb sphaeroides strain (2.4.1). In the L gene, these codon changes are silent. In the M gene, one is silent and the other one leads to a Leu- Met substitution at position 140. At the present stage of the refinement of the X-ray data (0.3 nm resolution) the structure of the Y reaction center is shown to be highly similar to that of the Rhodopseudomonas viridis reaction center. The binding of spheroidene on the M side of the Y reaction center is shown to be determined by hydrophobic interactions with neighboring amino acids and by steric factors. Preliminary results concerning the localization of the detergent (β-octylglucoside) in the unit cell are presented. This method combines low angle neutron scattering at different contrasts in H 2O/D 2O with X-ray crystallographic data.

Light-dependent porphyrin production by suspended and immobilized cells of Rhodobacter sphaeroides

This study was undertaken to determine optimum conditions for porphyrin production by suspended and immobilized cells of Rhodobacter sphaeroides. Cell suspensions of R. sphaeroides strain NR3 previously grown photosynthetically excreted large amounts of porphyrins with coproporphyrin III as the major component under anaerobic-light conditions. The most pronounced excretion was found in the presence of 10 mM glycine, 10 mM succinate, and 0.1 mM Mg 2+ (or Ca 2+) and at pH between 7.5 and 8.0 and a high light intensity ( ≧ 15,000 lx ). The amount of porphyrins produced after 6 d of incubation by cell suspensions with an initial cell dry weight of 0.54 g was 170 μmol· l −1. Extracellular porphyrin formation was severely inhibited by levulinate, 2, 2′-dipyridyl, or chloramphenicol. Immobilization of cells with agar or carrageenan resulted in a marked decrease in the formation rate. However, intermittent treatment with a growth medium restored the ability of the immobilized cells to produce porphyrins effectively. Agar-immobilized cells in a repeated batch process including the intermittent treatment stage for re-activation produced higher levels of porphyrins than did cell suspension.

Expression of the meta-cleavage pathway operon of the TOL plasmid of Pseudomonas putida in the phototrophic bacterium Rhodobacter sphaeroides

Plasmid pPL392, a pBR322 derivative containing a 16.4 kb fragment harboring the whole meta-cleavage pathway operon genes of the TOL plasmid pWWO as well as its regulatory gene xylS, was inserted into the HindIII site of the broad-host-range plasmid pRK404 to yield the plasmid pUA58. This pUA58 plasmid was mobilized into the phototrophic bacteria Rhodobacter sphaeroides using the mobilizing plasmid pRK2013. Meta-cleavage pathway genes, measured by the catechol 2,3-dioxygenase (C230) enzyme activity, were constitutively expressed at a high level in the R. sphaeroides (pUA58) strain, since the inducing compound m-toluate did not increase the level of the C230 enzyme. This behaviour of R. sphaeroides (pUA58) strain was not due to any modification of the pUA58 plasmid in this bacterium, because synthesis of the C230 enzyme in both E. coli and P. putida cells containing this plasmid were inducible by m-toluate. Nevertheless, and despite the high level of expression of meta-cleavage genes, R. sphaeroides (pUA58) cells were unable to grow using m-toluate as the sole carbon source. By subcloning some of the whole meta-cleavage operon genes, it has been shown that the growth inability of R. sphaeroides (pUA58) using m-toluate must be attributed to an inhibitory effect of one of the intermediate metabolites of the m-toluate degradation over the R. sphaeroides cells.

Purification and properties of a polyol dehydrogenase from the phototrophic bacterium Rhodobacter sphaeroides.

A polyol dehydrogenase was detected in cell extracts of the facultative phototrophic bacterium Rhodobacter sphaeroides strain Si 4 grown on D-glucitol (sorbitol) as the sole carbon source. The enzyme was purified 150-fold to apparent homogeneity by steps involving fractionated (NH4)2SO4 precipitation, chromatography on Q-Sepharose and phenyl-Sepharose, and FPLC on Superose 12. The relative molecular mass (Mr) of the native polyol dehydrogenase was 47,200 as calculated from its Stokes' radius (rs = 2.76 nm) and sedimentation coefficient (s20, w = 4.15 S). SDS/PAGE resulted in one single band representing a polypeptide with a Mr of 52,200, indicating that the native protein is a monomer. The isoelectric point of the polyol dehydrogenase was determined to be pH 4.3. The enzyme was specific for NAD+ and oxidized both D-glucitol and D-mannitol to D-fructose, as well as D-arabinitol to D-ribulose. The pH optimum of substrate oxidation was pH 9.0 in 0.1 M Tris/HCl and that of substrate reduction was pH 6.5 in 0.1 M potassium phosphate. The reactions exhibited normal Michaelis-Menten kinetics allowing the estimation of KM values for NAD+ (0.18 mM) in the presence of D-glucitol, and for D-glucitol (31.8 mM), D-mannitol (0.29 mM) and D-arabinitol (1.8 mM), respectively. The KM value for D-fructose was 16.3 mM and that for NADH 0.02 mM. The equilibrium constants determined for the conversion of D-mannitol, D-glucitol and D-arabinitol were 4.5 nM, 0.58 nM and 80 pM, respectively. Based on the catalytic preference of the polyol dehydrogenase for D-mannitol, an enzymatic assay for D-mannitol was elaborated.

Restriction endonuclease AfaI from Acidiphilium facilis, a new isoschizomer of RsaI: purification and properties

We have purified AfaI endonuclease, an isoschizomer of RsaI, from Acidiphilium facilis strain 28H. The enzyme is homogeneous as judged by polyacrylamide gel electrophoresis, and composed of a single polypeptide chain with a molecular weight of 30 000. AfaI endonuclease, like RsaI, recognizes the tetranucleotide sequence 5′-G-T-A-C-3′, and cleaves between the T and A to produce blunt-ended fragments. The yield of the enzyme is 50–100-times that of the RsaI, which is form a phototrophic bacterium, Rhodospseudomonas sphaeroides strain 28/5.

Isolation and Characterization of Two Endogenous Phages of Rhodobacter sphaeroides

Two new temperate bacteriophages for Rhodobacter sphaeroides, designated ϕRsA and ϕRsD, were isolated from a stock of phage ϕRsGl using R. sphaeroides strain DSM 159–2 as the indicator strain. Electron-microscopic examination showed that phage ϕRsA belonged to group B and phage ϕRsD belonged to group A of Bradley’s morphological classification. Phage ϕRsA had a polyhedral head (50 nm in diameter) and a long, flexible, non-contractile tail (250 by 11 nm). Phage ϕRsD also had a polyhedral head (56 nm in diameter) and a sheathed contractile tail (160 by 18 nm). Both phages contained double-stranded DNA with cohesive ends. The size of the ϕRsA genome was about 40 kb and the G + C content was 72·4 mol%. The size of the ϕRsD genome was about 56 kb and the G + C content was approximately 72·6 mol%. Biotinylated DNA probes of phage ϕRsA and of phage ϕRsD hybridized to genomic DNA from R. sphaeroides strains Y and Si 4 but not to DNA from other sources. The host ranges of the two phages were determined using 26 R. sphaeroides strains and six strains of Rhodobacter capsulatus. Phage ϕRsA formed plaques on R. sphaeroides DSM 159–2 only, while phage ϕRsD plaqued on another six strains of R. sphaeroides but not on R. capsulatus.

Purification and characterization of the restriction endonuclease RsrI, an isoschizomer of EcoRI

Rhodobacter sphaeroides strain 630 produces restriction enzyme RsrI which is an isoschizomer of EcoRI. We have purified this enzyme and initiated a comparison with the EcoRI endonuclease. The properties of RsrI are consistent with a reaction mechanism similar to that of EcoRI: the position of cleavage within the -GAATTC-site is identical, the MgCl 2 optimum for the cleavage is identical, and the pH profile is similar. Methylation of the substrate sequence by the EcoRI methylase protects the site from cleavage by the RsrI endonuclease. RsrI cross-reacts strongly with anti- EcoRI serum indicating three-dimensional structural similarities. We have determined the sequence of 34 N terminal amino acids for RsrI and this sequence possesses significant similarity to the EcoRI N terminus.

Transposon mutagenesis and physiological analysis of strains containing inactivated form I and form II ribulose bisphosphate carboxylase/oxygenase genes in Rhodobacter sphaeroides.

Strains of Rhodobacter sphaeroides (Rhodopseudomonas sphaeroides) were constructed such that either the gene encoding form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC-O) or the gene encoding form II RuBPC-O was inactivated. Both strains were capable of photoheterotrophic growth with malate as the electron donor, with only slight differences in growth rate and overall carboxylase specific activity compared with the wild-type strain. Photolithotrophic growth with 1.5% CO2 in hydrogen was also possible for R. sphaeroides strains containing only one of the two RuBPC-O enzyme forms, although the differences in growth rates between wild-type and carboxylase mutant strains were greater under these conditions. These results indicate that the two forms of RuBPC-O are independently regulated. In addition, the regulatory system governing RuBPC-O synthesis may, in some cases, compensate for the lack of the missing enzyme.

The relation between electron transfer, protonmotive force and lactose transport in membrane vesicles from aerobically grown Rhodobacter sphaeroides 4P1

Membrane vesicles were isolated from Rhodobacter sphaeroides strain 4P1, grown aerobically in the dark. This strain is equipped with the lactose transport protein from Escherichia coli. In the membrane vesicles a branched electron-transfer chain is located; one branch contains cytochrome c oxidase and the other the so-called ‘alternative oxidase’. Cytochrome c oxidase is the major energy-coupling device in these membrane vesicles. Several electron donors (succinate, NADH, reduced cytochrome c, etc.) are oxidized via the electron-transfer chain with different contributions of the two branches of the terminal part of the electron-transfer chain. The capacity of these membrane vesicles to generate a protonmotive force correlates well with the activity of the cytochrome c oxidase-containing branch of the electron-transfer chain. This correlation was revealed by the use of electron-transfer chain inhibitors like antimycin A and KCN. The rate and maximal level of lactose accumulation correlated chemiosmotically with the magnitude of the protonmotive force, assuming 1 H +/lactose symport, without the need to invoke additional regulatory mechanisms. Electron donors that can directly reduce cytochrome c oxidase were most efficient in protonmotive force generation and in the energization of lactose transport.

Restriction endonuclease RsrI from Rhodobacter sphaeroides, an isoschizomer of EcoRI: purification and properties.

We have purified RsrI endonuclease (R.RsrI), an isoschizomer of EcoRI, from Rhodobacter sphaeroides strain 630. The enzyme is homogeneous as judged by polyacrylamide gel electrophoresis and size-exclusion high-performance liquid chromatography. RsrI endonuclease is a dimer over the concentration range of 0.05 to 1.4 mg/ml. The reduced and denatured molecular weight of the enzyme is 30,000 Da. R.RsrI, like R.EcoRI, catalyzes the cleavage of duplex DNA and oligodeoxyribonucleotides between the first two residues of the sequence GAATTC. R.RsrI exhibits a KM of 14 nM and a kcat of 6.5 min-1 when reacting with pBR322 DNA at 25 degrees C. R.RsrI differs from R.EcoRI in its N-terminal amino acid sequence, susceptibility to inhibition by antibodies, sensitivity to N-ethylmaleimide, isoelectric point, state of aggregation at high concentrations, temperature lability, and conditions for optimal reaction. R.RsrI displays a reduction of specificity ("star activity") under conditions that also relax the specificity of R.EcoRI.

Crystallization and preliminary X-ray and optical spectroscopic characterization of the photochemical reaction center from Rhodobacter sphaeroides strain 2.4.1

The photochemical reaction center from Rhodobacter sphaeroides 2.4.1 has been crystallized. The crystals were obtained in a solution of β-octylglucoside by the vapor diffusion technique using polyethylene glycol 4000 as the precipitant at 22 °C. The orthorhombic crystals (space group P2 12 12 1) have cell constants a = 142.5 A ̊ , b = 136.1 a ̊ A , c = 78.5 A ̊ , and diffract to 3.7 Å. The crystals display pronounced linear dichroism in the carotenoid absorption spectral region.