Denitrification of PVA-immobilized denitrifying photosynthetic bacterium,Rhodobacter sphaeroides

A newly isolated denitrifying strain, Rhodobacter sphaeroides NII2 was immobilized in polyvinyl alcohol (PVA) gel, and the properties of the cells in the gel were examined. The immobilized cells had low or almost no denitrification activity, but the cells were activated by incubation in light with culture medium for denitrification containing 0.5% nitrate and no other nitrogen source. Cells grown in the dark were activated by incubation at an earlier stage and to a higher rate than the light-grown cells. The activation was markedly enhanced in the PVA gel with a low cell concentration. The immobilized cells consumed nitrate with a temporary accumulation of NO₂ and evolved nitrogen gas. The immobilized cells could use various organic compounds as electron donors for denitrification. Thus, the immobilized cells were applied to a continuous treatment of synthetic wastewater using an aparatus devised by this laboratory. The results showed an efficient removal of NO₃-N from the test water.     

Immobilization of the purple non-sulfur bacterium Rhodobacter sphaeroides on glass surfaces

Summary A method for improving the adsorption of bacteria on glass surfaces was developed. The modification of a glass surface by LS-2480 greatly increased the number of bacteria that were immobilized. The conditions for bacteria immobilization on the modified glass surface were optimized.     

Rhythmic gene expression in a purple photosynthetic bacterium, Rhodobacter sphaeroides

Circadian rhythms are known to exist in all groups of eukaryotic organisms as well as oxygenic photosynthetic bacteria, cyanobacteria. However, little information is available regarding the existence of rhythmic behaviors in prokaryotes other than cyanobacteria. Here we report biological rhythms of gene expression in a purple bacterium Rhodobacter sphaeroides by using a luciferase reporter gene system. Self-bioluminescent strains of Rb. sphaeroides were constructed, which produced a bacterial luciferase and its substrate, a long chain fatty aldehyde, to sustain the luminescence reaction. After being subjected to a temperature or light entrainment regime, the reporter strains with the luciferase genes driven by an upstream endogenous promoter expressed self-sustained rhythmicity in the constant free-running period. The rhythms were controlled by oxygen and exhibited a circadian period of 20.5 h under aerobic conditions and an ultradian period of 10.6-12.7 h under anaerobic conditions. The data suggest a novel endogenous oscillation mechanism in purple photosynthetic bacteria. Elucidation of the clock-like behavior in purple bacteria has implications in understanding the origin and evolution of circadian rhythms.     

Osmoregulated periplasmic glucans of the free-living photosynthetic bacterium Rhodobacter sphaeroides.

The osmoregulated periplasmic glucans (OPGs) produced by Rhodobacter sphaeroides, a free-living organism, were isolated by trichloracetic acid treatment and gel permeation chromatography. Compounds obtained were characterized by compositional analysis, matrix-assisted laser desorption ionization mass spectrometry and nuclear magnetic resonance. R. sphaeroides predominantly synthesizes a cyclic glucan containing 18 glucose residues that can be substituted by one to seven succinyl esters residues at the C6 position of some of the glucose residues, and by one or two acetyl residues. The glucans were subjected to a mild alkaline treatment in order to remove the succinyl and acetyl substituents, analyzed by MALDI mass spectrometry and purified by high-performance anion-exchange chromatography. Methylation analysis revealed that this glucan is linked by 17 1,2 glycosidic bonds and one 1,6 glycosidic bond. Homonuclear and (1)H/(13)C heteronuclear NMR experiments revealed the presence of a single alpha-1,6 glycosidic linkage, whereas all other glucose residues are beta-1,2 linked. The different anomeric proton signals allowed a complete sequence-specific assignment of the glucan. The structural characteristics of this glucan are very similar to the previously described OPGs of Ralstonia solanacearum and Xanthomonas campestris, except for its different size and the presence of substituents. Therefore, similar OPGs are synthesized by phytopathogenic as well as free-living bacteria, suggesting these compounds are intrinsic components of the Gram-negative bacterial envelope.     

Complete assimilation of cysteine by a newly isolated non-sulfur purple bacterium resembling Rhodovulum sulfidophilum (Rhodobacter sulfidophilus)

A rod-shaped, motile, phototrophic bacterium, strain SiCys, was enriched and isolated from a marine microbial mat, with cysteine as sole substrate. During phototrophic anaerobic growth with cysteine, sulfide was produced as an intermediate, which was subsequently oxidized to sulfate. The molar growth yield with cysteine was 103 g mol^–1, in accordance with complete assimilation of electrons from the carbon and the sulfur moiety into cell material. Growth yields with alanine and serine were proportionally lower. Thiosulfate, sulfide, hydrogen, and several organic compounds were used as electron donors in the light, whereas cystine, sulfite, or elemental sulfur did not support phototrophic anaerobic growth. Aerobic growth in the dark was possible with fructose as substrate. Cultures of strain SiCys were yellowish-brown in color and contained bacteriochlorophyll a, spheroidene, spheroidenone, and OH-spheroidene as major photosynthetic pigments. Taking the morphology, photosynthetic pigments, aerobic growth in the dark, and utilization of sulfide for phototrophic growth into account, strain SiCys was assigned to the genus Rhodovulum (formerly Rhodobacter) and tentatively classified as a strain of R. sulfidophilum. In cell-free extracts in the presence of pyridoxal phosphate, cysteine was converted to pyruvate and sulfide, which is characteristic for cysteine desulfhydrase activity (l-cystathionine γ-lyase, EC 4.4.1.1). Received: 15 December 1995 / Accepted: 1 April 1996     

Inhibitor-complexed structures of the cytochrome bc1 from the photosynthetic bacterium Rhodobacter sphaeroides

The cytochrome bc(1) complex (bc(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(1) complexes from the photosynthetic purple bacterium Rhodobacter sphaeroides (Rsbc(1)), stabilized with the quinol oxidation (Q(P)) site inhibitor stigmatellin alone or in combination with the quinone reduction (Q(N)) site inhibitor antimycin, were determined. The high quality electron density permitted assignments of a new metal-binding site to the cytochrome c(1) subunit and a number of lipid and detergent molecules. Structural differences between Rsbc(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(1) complex are derived from analyses of 10 independent molecules in various crystal forms and from comparisons with mitochondrial complexes.     

Regulation of nitrogenase in the photosynthetic bacterium Rhodobacter sphaeroides containing draTG and nifHDK genes from Rhodobacter capsulatus

The photosynthetic bacteria Rhodobacter capsulatus and Rhodospirillum rubrum regulate their nitrogenase activity by the reversible ADP-ribosylation of nitrogenase Fe-protein in response to ammonium addition or darkness. This regulation is mediated by two enzymes, dinitrogenase reductase ADP-ribosyl transferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG). Recently, we demonstrated that another photosynthetic bacterium, Rhodobacter sphaeroides, appears to have no draTG genes, and no evidence of Fe-protein ADP-ribosylation was found in this bacterium under a variety of growth and incubation conditions. Here we show that four different strains of Rba. sphaeroides are incapable of modifying Fe-protein, whereas four out of five Rba. capsulatus strains possess this ability. Introduction of Rba. capsulatus draTG and nifHDK (structural genes for nitrogenase proteins) into Rba. sphaeroides had no effect on in vivo nitrogenase activity and on nitrogenase switch-off by ammonium. However, transfer of draTG from Rba. capsulatus was sufficient to confer on Rba. sphaeroides the ability to reversibly modify the nitrogenase Fe-protein in response to either ammonium addition or darkness. These data suggest that Rba. sphaeroides, which lacks DRAT and DRAG, possesses all the elements necessary for the transduction of signals generated by ammonium or darkness to these proteins.     

Spectroscopic characterization of reaction centers of the (M)Y210W mutant of the photosynthetic bacterium Rhodobacter sphaeroides

The tyrosine-(M)210 of the reaction center of Rhodobacter sphaeroides 2.4.1 has been changed to a tryptophan using site-directed mutagenesis. The reaction center of this mutant has been characterized by low-temperature absorption and fluorescence spectroscopy, time-resolved sub-picosecond spectroscopy, and magnetic resonance spectroscopy. The charge separation process showed bi-exponential kinetics at room temperature, with a main time constant of 36 ps and an additional fast time constant of 5.1 ps. Temperature dependent fluorescence measurements predict that the lifetime of P^* becomes 4–5 times slower at cryogenic temperatures. From EPR and absorbance-detected magnetic resonance (ADMR, LD-ADMR) we conclude that the dimeric structure of P is not significantly changed upon mutation. In contrast, the interaction of the accessory bacteriochlorophyll B_A with its environment appears to be altered, possibly because of a change in its position.Abbreviations ADMR - absorbance-detected magnetic resonance - LDAO - N, N dimethyl dodecyl amine-N-oxide - RC - reaction center - LD-ADMR - linear-dichroic absorbance-detected magnetic resonance - P - primary donor - B - accessory bacteriochlorophyll - - bacteriopheophytin     

Photochemical trapping of a bacteriopheophytin anion in site-specific reaction-center mutants from the photosynthetic bacterium Rhodobacter sphaeroides.

The mutant YY in the reaction center of Rhodobacter sphaeroides, in which Phe181 on the L chain has been replaced by Tyr, and the double mutant FY, with Tyr210 on the M chain replaced by Phe and Phe181 on the L chain replaced by Tyr, have been constructed by site-directed mutagenesis. The studies described here were performed to complement a previous mutational analysis of mutant FF with Tyr210 replaced by Phe. Both new strains grow photoheterotrophically. The optical absorption spectra of reaction centers isolated from these mutants have band shifts attributable to the monomer bacteriochlorophylls in the vicinity of the substitutions. Photochemical trapping of the bacteriopheophytin anion (I-) indicates that the bacteriopheophytin on the B branch is reduced to a much greater extent in FF and FY as compared to YY and wild-type YF. Low temperature (77 K) absorption spectra clearly show that in the wild-type (YF) and YY reaction centers only the 545-nm-absorbing bacteriopheophytin is reduced while in the FF and FY reaction centers both the 535-nm and 545-nm-absorbing bacteriopheophytins are reduced. A simple kinetic analysis is used to explain these results. This analysis suggests that, in order for the observed trapping results to occur, a decrease in the 'cycling' time must take place, that is changes in the rate(s) of charge recombination must accompany the already known decrease in the forward electron transfer rate.     

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

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.     

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

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 Co²⁺ and CrO₄ ^2? is reported and changes in membrane lipid profiles were rationalised.     

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

Photosynthesis Research - Chromatophores of purple non-sulfur bacteria (PNSB) are invaginations of the cytoplasmic membrane that contain a relatively simple system of light-harvesting...     

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

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.     

Hydrogen release by recombinant strains of Rhodobacter sphaeroides using a modified photosynthetic apparatus

Hydrogen release by recombinant strains of Rhodobacter sphaeroides pRK puf DD13 without a peripheral light-harvesting antenna complex and pRK puf deltaLM1 which is able to synthesize both antenna complexes, both of which were grown in conditions of nitrogen limitation, has been studied. The velocity of hydrogen release depended on light intensity. At high cell concentration (0.91 g l(-1)) of pRK puf DD 13, velocity was maximal at 2270 W m(-2) and was equal to 144.7 ml l(-1) h(-1) that evidences to an opportunity to increase the volume velocity of hydrogen release by application of the strains with low content of pigments.     

Amino acid sequence determination and three-dimensional modelling of thioredoxin from the photosynthetic bacterium Rhodobacter sphaeroides Y

The complete primary structure of thioredoxin from Rhodobacter sphaeroides Y has been determined by analysis of peptides after cleavage with cyanogen bromide, chymotrypsin and trypsin. Peptides were separated by HPLC and analyzed by liquid-phase and gas-phase sequencer degradations. The protein consists of 105 residues (Mr = 11,180); its amino acid sequence shows a clear homology to the five known thioredoxins from plant or bacterial sources, with 40-56% residue identity when the proteins are aligned at the active-site disulfide. Not only the active-site regions are conserved, but also residues which belong to the hydrophobic surface suggested to be important for binding of procaryote thioredoxins in redox interactions with other proteins (residues 75-76; 91-93 in Escherichia coli). A three-dimensional model of Rb. sphaeroides thioredoxin has been derived from the E. coli crystallographic structure with computer graphics. This model indicates that the overall structures as well as the active sites are closely similar; however, the residue substitutions allow both proteins to adopt different local folding as shown in the hydrophobic core.     

Role of Rhodobacter sp. strain PS9, a purple non-sulfur photosynthetic bacterium isolated from an anaerobic swine waste lagoon,in odor remediation

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.     

Purification and characterization of a neutral serine protease from non-sulfur purple photosynthetic bacterium

A neutral serine protease was purified as a homogeneous protein from the culture broth of photosynthetic bacterium T-20 by sequential chromatographies on columns of DEAE-cellulose, Toyopearl HW 55F, hydroxyapatite, and CM-cellulose. The molecular weight was estimated to be approximately 44,000 by SDS-PAGE, while the value of approximately 80,000 was obtained when the Hedrick-Smith method was used; this suggested that the enzyme consists of two identical subunits. The isoelectric point was determined to be 6.3 by isoelectric focusing. The enzyme had a pH optimum at 7.8. Maximal enzyme activity was detected at 50°C, and the activity was stable up to 50°C for 5 min at pH 7.0–7.2. The substrate specificity of the protease was investigated with a series of synthetic peptidyl-p-nitroanilide. The best substrate examined was Suc-Ala-Ala-Pro-Phe-pNA. The protease activity was inhibited by various inhibitors of serine protease such as chymostatin, PMSF, and DFP. EDTA, which is an inhibitor of metal protease, also inhibited the protease activity, whereas inhibitors of thiol and aspartic proteases had no significant effect.     

The road to the crystal structure of the cytochrome bc 1 complex from the anoxigenic, photosynthetic bacterium Rhodobacter sphaeroides

The advantages of using bacterial systems to study the mechanism and function of cytochrome bc (1) complexes do not extend readily to their structural investigations. High quality crystals of bacterial complexes have been difficult to obtain despite the enzymes' smaller sizes and simpler subunit compositions compared to their mitochondrial counterparts. In the course of the structure determination of the bc (1) complex from R. sphaeroides, we observed that the growth of only low quality crystals correlated with low activity and stability of the purified complex, which was mitigated in part by introducing a double mutations to the enzyme. The S287R(cyt b)/V135S(ISP) mutant shows 40% increase in electron transfer activity and displays a 4.3 degrees C increase in thermal stability over wild-type enzyme. The amino acid histidine was found important in maintaining structural integrity of the bacterial complex, while the respiratory inhibitors such as stigmatellin are required for immobilization of the iron-sulfur protein extrinsic domain. Crystal quality of the R. sphaeroides bc (1) complex can be improved further by the presence of strontium ions yielding crystals that diffracted X-rays to better than 2.3 A resolution. The improved crystal quality can be understood in terms of participation of strontium ions in molecular packing arrangement in crystal.     

Expression of the gene encoding cytochrome c3 from the sulfate-reducing bacterium Desulfovibrio vulgaris in the purple photosynthetic bacterium Rhodobacter sphaeroides.

The gene encoding cytochrome c3 (cyc-gene) from Desulfovibrio vulgaris (Hildenborough) was cloned by G. Voordouw and S. Brenner (1986, Eur. J. Biochem. 159, 347-351). The gene was expressed in Escherichia coli but only the apoprotein was observed (W. Pollock, P. Chemerika, M. Forrest, J. Beatty, and G. Voordouw, 1989, J. Gen. Microbiol. 135, 2319-2328). In this study, the cyc-gene was cloned into the broad host range vector pRK404 and then introduced into the purple photosynthetic bacterium Rhodobacter sphaeroides. Cells grown anaerobically produced a significant amount of recombinant cytochrome c3. The purified protein contains four hemes and the N-terminal protein sequence is identical to the published sequence of the native cytochrome c3. Thus, R. sphaeroides was able to produce the mature cytochrome c3 by combining the four steps of protein synthesis, exporting the protein across the membrane, cleaving the signal peptide, and inserting four hemes. It appears that the D. vulgaris promoter is not very efficiently used by R. sphaeroides. However, replacement of the promoter with a R. sphaeroides promoter should result in cytochrome c3 overproduction.