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PubMed1991

Nucleotide sequence of the replication region of the marine Rhodobacter plasmid pRD31.

The minimum region required for replication of the marine Rhodobacter endogenous plasmid pRD31 has been sequenced. This region is located on a 1367-bp HincII-PstI restriction fragment and is 62% rich in G-C base pairs. A region homologous to the bacteriophage P1 repA promoter, which overlaps two inverted repeats, has been identified. Plasmids with mutations in this 1367-bp region could not replicate in marine Rhodobacter hosts. This is the first identified replication origin of a photosynthetic bacterium.

Amino Acid Sequence↗

PubMed1990

Stark effect in wild-type and heterodimer-containing reaction centers from Rhodobacter capsulatus.

The effect of an external electric field on the optical absorption spectra of wild-type Rhodobacter capsulatus and two Rb. capsulatus reaction centers that have been genetically modified through site-directed mutagenesis (HisM200----LeuM200 and HisM200----PheM200) was measured at 77 K. The two genetically modified reaction centers replace histidine M200, the axial ligand to the M-side bacteriochlorophyll of the special pair, with either leucine or phenylalanine. These substitutions result in the replacement of the M-side bacteriochlorophyll with bacteriopheophytin, forming a bacteriochlorophyll-bacteriopheophytin heterodimer. The magnitude of the change in dipole moment from the ground to excited state (delta mu app) and the angle delta between the Qy transition moment and the direction of delta mu app were measured for the special pair absorption band for all three reaction centers. The values for delta mu app and delta obtained for wild-type Rb. capsulatus (delta mu app = 6.7 +/- 1.0 D, delta = 38 +/- 3 degrees) were the same within experimental error as those of Rhodobacter sphaeroides and Rhodopseudomonas viridis. The values for delta mu app and delta obtained for the red-most Stark band of both heterodimers were the same, but delta mu was substantially different from that of wild-type reaction centers (HisM200----LeuM200, delta mu app greater than or equal to 14.1 D and delta = 33 +/- 3 degrees; HisM200----PheM200, delta mu app greater than or equal to 15.7 D and delta = 31 +/- 4 degrees).(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗

PubMed1988

Stark effect spectroscopy of Rhodobacter sphaeroides and Rhodopseudomonas viridis reaction centers.

The nature of the initially excited state of the primary electron donor or special pair has been investigated by Stark effect spectroscopy for reaction centers from the photosynthetic bacteria Rhodopseudomonas viridis and Rhodobacter sphaeroides at 77 K. The data provide values for the magnitude of the difference in permanent dipole moment between the ground and excited state, [unk]Deltamu[unk], and the angle [unk] between Deltamu and the transition dipole moment for the electronic transition. [unk]Deltamu[unk] and [unk] for the lowest-energy singlet electronic transition associated with the special pair primary electron donor were found to be very similar for the two species. [unk]Deltamu[unk] for this transition is substantially larger than for the Q(y) transitions of the monomeric pigments in the reaction center or for pure monomeric bacteriochlorophylls, for which Stark data are also reported. We conclude that the excited state of the special pair has substantial charge-transfer character, and we suggest that charge separation in bacterial photosynthesis is initiated immediately upon photoexcitation of the special pair. Data for Rhodobacter sphaeroides between 340 and 1340 nm are presented and discussed in the context of the detection of charge-transfer states by Stark effect spectroscopy.

Journal Article↗

PubMed1996

Preliminary crystallographic studies of dimethylsulfoxide reductase from Rhodobacter capsulatus.

Dimethylsulfoxide reductase from the photosynthetic bacterium Rhodobacter capsulatus has been crystallized in two similar forms which are suitable for X-ray structure determination. Both crystals forms belong to space group P4(1)22 or P4(3)22, with cell dimensions a = b = 80.81, c = 229.75 A (type I crystals) or a = b = 89.30, c = 230.05 A (type II crystals) and one molecule in the asymmetric unit. Diffraction has been observed to at least 2.0 A in type I crystals and to 2.6 A in type II crystals. Dimethylsulfoxide reductase from Rhodobacter is the simplest molybdenum oxotransferase known and this makes it an ideal model to study the structure and function of this class of enzymes.

Journal Article↗

PubMed1997

Structure of cytochrome c' from Rhodobacter capsulatus strain St Louis: an unusual molecular association induced by bridging Zn ions.

Rhodobacter capsulatus strain St Louis cytochrome c' (RCCP-SL) has been crystallized and the structure solved by molecular replacement. It was refined at 2.1 A resolution to an R value of 18.4%, and compared with Rhodobacter capsulatus strain M110 cytochrome c' (RCCP-M110). Although these two proteins are very similar in sequence and structure, the intermolecular interaction is largely different. In RCCP-M110, the molecules dimerize through interaction of helix B to form an antiparallel arrangement. When crystallized in the presence of Zn ions, molecules of RCCP-SL were found to be arranged as linear polymers connected by the bridging Zn ions. The changes in conformation of the side chains induced by binding of the Zn ions, by the substitution of Glu90 for Asp90, and by the different arrangement of the molecules, are discussed in detail.

Journal Article↗

PubMed1989

Electron transport pathways to nitrous oxide in Rhodobacter species.

1. Electron transport components involved in nitrous oxide reduction in several strains of Rhodobacter capsulatus and in the denitrifying strain of Rhodobacter sphaeroides (f. sp. denitrificans) have been investigated. Detailed titrations with antimycin A and myxothiazol, inhibitors of the cytochrome bc1 complex, show that part of the electron flow to nitrous oxide passes through this complex. The sensitivity to myxothiazol varies between strains and growth conditions of R. capsulatus; the higher rates of nitrous oxide reduction correlate with the higher sensitivities. Partial inhibition of the nitrous oxide reductase enzyme with azide decreased the sensitivity to myxothiazol of the strains that had the highest nitrous oxide reductase activity. 2. Inhibition of nitrous oxide reduction in cells of R. capsulatus by myxothiazol could be restored under dark conditions by addition of N,N,N',N'-tetramethyl-p-phenylene diamine. The highest activities observed after addition of this electron carrier were found in the strains that had the highest sensitivity to myxothiazol, consistent with the premise that this inhibitor is more effective at the higher flux rates to nitrous oxide. 3. Addition of nitrous oxide to cells of R. capsulatus strain N22DNAR+ under darkness caused oxidation of both b- and c-type cytochromes. The oxidation of b cytochromes was less pronounced in the presence of myxothiazol, consistent with a role for the cytochrome bc1 complex in the electron pathway to nitrous oxide. Ferricyanide, in the absence of myxothiazol, caused a similar extent of oxidation of b cytochromes, but a greater oxidation of c-type, suggesting that there was a pool of c-type cytochrome that was not oxidisable by nitrous oxide. The time course showed that both the b- and c-type cytochromes were oxidised within a few seconds of the addition of nitrous oxide. During the following seconds there was a partial re-reduction of the cytochromes such that after approximately 1 min a lower steady-state of oxidation was attained and this persisted until the nitrous oxide was exhausted. 4. A mutant, MTCBC1, of R. capsulatus that specifically lacked a functional cytochrome bc1 complex reduced nitrous oxide, albeit at 30% of the rate shown by the parent strain MT1131. A reduced minus nitrous-oxide-oxidised difference spectrum for MTCBC1 in the absence of myxothiazol was similar to the corresponding difference spectrum observed for strain N22DNAR+ in the presence of myxothiazol. It is suggested that these difference spectra identify the cytochrome components, including a b-type, involved in a pathway that is alternative to, and independent of, the cytochrome bc1 complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Anaerobiosis↗

PubMed1988

Properties of a Tn5 insertion mutant defective in the structural gene (fruA) of the fructose-specific phosphotransferase system of Rhodobacter capsulatus and cloning of the fru regulon.

In photosynthetic bacteria such as members of the genera Rhodospirillum, Rhodopseudomonas, and Rhodobacter a single sugar, fructose, is transported by the phosphotransferase system-catalyzed group translocation mechanism. Previous studies indicated that syntheses of the three fructose catabolic enzymes, the integral membrane enzyme II, the peripheral membrane enzyme I, and the soluble fructose-1-phosphate kinase, are coordinately induced. To characterize the genetic apparatus encoding these enzymes, a Tn5 insertion mutation specifically resulting in a fructose-negative, glucose-positive phenotype was isolated in Rhodobacter capsulatus. The mutant was totally lacking in fructose fermentation, fructose uptake in vivo, phosphoenolpyruvate-dependent fructose phosphorylation in vitro, and fructose 1-phosphate-dependent fructose transphosphorylation in vitro. Extraction of the membrane fraction of wild-type cells with butanol and urea resulted in the preparation of active enzyme II free of contaminating enzyme I activity. This preparation was used to show that the activity of enzyme I was entirely membrane associated in the parent but largely soluble in the mutant, suggesting the presence of an enzyme I-enzyme II complex in the membranes of wild-type cells. The uninduced mutant exhibited measurable activities of both enzyme I and fructose-1-phosphate kinase, which were increased threefold when it was grown in the presence of fructose. Both activities were about 100-fold inducible in the parental strain. Although the Tn5 insertion mutation was polar on enzyme I expression, fructose-1-phosphate kinase activity was enhanced, relative to the parental strain. ATP-dependent fructokinase activity was low, but twofold inducible and comparable in the two strains. A second fru::Tn5 mutant and a chemically induced mutant selected on the basis of xylitol resistance showed pleiotropic loss of enzyme I, enzyme II, and fructose-1-phosphate kinase. These mutants were used to clone the fru regulon by complementing the negative phenotype with a wild-type cosmid bank.

Biological Transport↗

PubMed1995

Structural model of the photosynthetic reaction center of Rhodobacter capsulatus.

The reaction center (RC) from the photosynthetic bacterium Rhodobacter (Rb.) capsulatus has been the subject of a considerable amount of molecular biological and spectroscopic work aimed at improving our understanding of the primary steps of photosynthesis. However, no three-dimensional structure is available for this protein. We present here a model obtained by combining information from the structure of the highly homologous RC from Rhodopseudomonas (Rps.) viridis with molecular mechanics and simulated annealing calculations. In the Rb. capsulatus model the orientations of the bacteriochlorophyll monomer and the bacteriopheophytin on the branch inactive in electron transfer differ significantly from those in the RCs of Rps. viridis and Rb. sphaeroides. The bacteriopheophytin orientational difference is in good accord with previous linear dichroism measurements. A comparison is made of interactions between the pigments and the protein environment that may be of functional significance in Rps. viridis, Rb. sphaeroides, and Rb. capsulatus.

Amino Acids↗

PubMed1993

Electron-transfer kinetics and electrostatic properties of the Rhodobacter sphaeroides reaction center and soluble c-cytochromes.

The kinetics of electron transfer between the Rhodobacter sphaeroides R-26 reaction center and nine soluble c-cytochromes have been analyzed and compared to the patterns of the surface electrostatic potentials for each of the proteins. Characteristic first-order electron-transfer rates for 1:1 complexes formed at low ionic strength between the reaction center and the different c-cytochromes were identified and found to vary by a factor of almost 100, while second-order rates were found to differ by greater than 10(6). A correlation was found between the location of likely electrostatic interaction domains on each cytochrome and its characteristic rate of electron transfer. The interaction domains were identified by mapping electrostatic potentials, calculated from the Poisson-Boltzmann equation, onto simulated "encounter surfaces" for each of the cytochromes and the reaction center. For the reaction center, the c-cytochrome binding domain was found to have almost exclusively net negative potential (< -3 kT) and to be shifted slightly toward the M-subunit side of the reaction center. The location of interaction domains of complementary, positive potential (> 3 kT) differed for each cytochrome. The correspondence between electrostatic, structural, and kinetic properties of 1:1 reaction center-cytochrome complexes leads to a proposed mechanism for formation of reaction center-cytochrome electron-transfer complexes that is primarily driven by the juxtaposition of regions of delocalized complementary potential. In this mechanism the clustering of charged residues is of primary importance and not the location of specific residues. A consequence of this mechanism is that many different sets of charge distributions are predicted to be capable of stabilizing a specific configuration for a reaction center-cytochrome complex. This mechanism for reaction center association with water-soluble c-cytochromes fits molecular recognition mechanisms proposed for c-cytochromes in nonphotosynthetic systems. In general, the kinetic scheme for reaction center driven cytochrome oxidation was found to vary between a simple two-state model, involving cytochrome in free and reaction center bound states, and a three-state model, that includes cytochrome binding in kinetically competent ("proximal") and incompetent ("distal") modes. The kinetically incompetent mode of cytochrome binding is suggested not to be an intrinsic feature of the reaction center-cytochrome association but is likely to be due to variation in the physical state of the reaction center.

Animals↗

PubMed2004

CIRCE is not involved in heat-dependent transcription of groESL but in stabilization of the mRNA 5'-end in Rhodobacter capsulatus.

The CIRCE element, an inverted DNA repeat, is known to be involved in the temperature-dependent regulation of genes for heat shock proteins in a variety of organisms. The CIRCE element was identified as the target for the HrcA protein, which represses transcription of heat shock genes under normal growth temperature. Our data reveal that the CIRCE element is not involved in the temperature-dependent transcription of the groESL genes in Rhodobacter capsulatus. Apparently, R.capsulatus does not harbour an HrcA protein. The mechanisms of heat shock regulation of the groESL genes in R.capsulatus therefore diverge significantly from the regulatory pathway identified in other organisms. A structural analysis of the CIRCE RNA element revealed a stem of 11 nt pairs and a loop of only 5 nt. This folding differs from a structure with a 9 nt loop suggested previously on the basis of computer analysis. The RNA structure leads to a slight stabilization of the groESL mRNA that is more pronounced at normal growth temperature than under heat shock conditions.

5' Untranslated Regions↗

PubMed1994

Cloning and nucleotide sequence of regA, a putative response regulator gene of Rhodobacter sphaeroides.

A 0.9 kb DNA fragment carrying the Rhodobacter capsulatus regA gene, which encodes an oxygen-dependent, positively-acting response regulator of photosynthetic gene expression, was used as a probe in Southern hybridisation experiments to determine whether a similar gene occurs in R. sphaeroides. A strongly hybridising DNA fragment isolated from a R. sphaeroides plasmid gene bank was isolated, sequenced and found to contain an open reading frame which exhibits 75% identity with the R. capsulatus regA gene. The deduced amino acid sequence of 184 residues shows 81% identity and 89% similarity with the R. capsulatus RegA protein, and significant similarities with other response regulators of the two component sensor-regulator type. Introduction of the R. sphaeroides gene into a R. capsulatus regA mutant, which exhibits abnormally low levels of membrane-bound photosynthetic complexes, resulted in a 22-33-fold increase in these complexes to approximately 62-65% of wild-type levels. This is the first study to identify a putative response regulator in R. sphaeroides and to complement a regulatory mutation in R. capsulatus with a gene from another species. Further studies of associated genes may identify the different mechanisms by which the regulation of photosynthesis complex formation occurs in response to environmental stimuli in R. sphaeroides and R. capsulatus.

Amino Acid Sequence↗

PubMed1999

Genomic complexity among strains of the facultative photoheterotrophic bacterium Rhodobacter sphaeroides.

Pulsed-field gel electrophoresis following the use of rare cutting restriction endonucleases together with Southern hybridization, using markers distributed on chromosomes I and II of Rhodobacter sphaeroides 2.4.1, has been used to examine approximately 25 strains of R. sphaeroides in an effort to assess the occurrence of genome complexity in these strains. The results suggest that genome complexity is widespread and is accompanied by substantial genomic heterogeneity.

Cloning, Molecular↗

PubMed1999

Reorientation of the acetyl group of the photoactive bacteriopheophytin in reaction centers of Rhodobacter sphaeroides: an ENDOR/TRIPLE resonance study.

The freeze-trapped bacteriopheophytin alpha radical anion phi(*)A- has been investigated by 1H-ENDOR/Special TRIPLE resonance spectroscopy in photosynthetic reaction centers of Rhodobacter sphaeroides, in which the Tyr at position M210 had been replaced by either Phe, Leu, His or Trp. In the wild type reaction center and the mutants YF(M210) and YW(M210) two distinct states of phi(*)A-, denoted I(*)1- and I(*)2-, can be stabilized below 200 K. The state I(*)1 is metastable and relaxes to I(*)2- as the temperature is raised from 135 K to 180 K. The difference in the electronic structure of phi(*)A- between the two states is interpreted in terms of a conformational change of phiA after freeze-trapping, involving a reorientation of the 3-acetyl group with respect to the macrocycle of the bacteriopheophytin. This interpretation is supported by the results of RHF-INDO/SP calculations. In the YH(M210) reaction center only one phiA- state is obtained that is distinct from I(*)1- and I(*)2, and the observed electronic structure indicates an almost in-plane orientation of the 3-acetyl group. This is consistent with the proposal that a hydrogen bond is formed between His M210 and the 3(1)-keto oxygen of phiA that impedes the reorientation of the acetyl group. Only one phi(*)A- state is observed in the YL(M210) reaction center, which is similar to the metastable state I(*)1 in the wild type complex. This result is interpreted in terms of a steric hindrance of the reorientation of the 3-acetyl group that is exerted by the side chain of Leu at position M210. Possible implications of these findings for the mechanism of electron transfer in bacterial reaction centers are discussed.

Electron Spin Resonance Spectroscopy↗

PubMedDate not supplied

Testing the photosynthetic bacterium Rhodobacter sphaeroides as heavy metal removal tool.

We present some preliminary results relevant to the ability of the purple non-sulphur bacterium Rhodobacter sphaeroides strain R26.1 to sequester heavy metals from contaminated growth media. The microorganism was chosen because of its significant tolerance to relatively high concentrations of the investigated ions Ni2+, Co2+, CrO4(2-), and MoO4(2-). In this paper the optimized conditions for the bacterial growth and the sample preparation used to infer the ability of the microorganism to cope with metal pollutants are presented. Elemental analysis has been performed by inductively coupled plasma atomic emission spectrometry previous mineralization of samples by a microwave system.

Environmental Pollutants↗

PubMed2004

NMR structural model of the interaction of herbicides with the photosynthetic reaction center from Rhodobacter sphaeroides.

The interaction of the herbicides acifluorfen and paraquat with the photosynthetic reaction center from Rhodobacter sphaeroides has been studied by NMR relaxation measurements. Interaction in aqueous solution has been demonstrated by evaluating motional features of the bound form through cross-relaxation terms of protons at fixed distances on the herbicides. Contributions to longitudinal nonselective relaxation rates different from the proton-proton dipolar relaxation were inferred, most probably due to paramagnetic effects originating from the high-spin nonheme Fe(II) ion in the reaction center. Paramagnetic contributions to proton relaxation rates were converted into distance constraints in order to build a model for the interaction. The models place paraquat in the QB site, where most herbicides interact, in agreement with docking calculations, whereas acifluorfen was placed between the metal and the QB site, as also demonstrated by the induced paramagnetic shifts. Acifluorfen could therefore act to break the electron-transfer pathway between the QA and QB sites.

Crystallography, X-Ray↗

PubMed1990

Initial characterization of site-directed mutants of tyrosine M210 in the reaction centre of Rhodobacter sphaeroides.

A description of the properties of site-directed mutants of the reaction centre (RC) of Rhodobacter sphaeroides is presented. The residue tyrosine M210 (YM210) has been changed to phenylalanine (FM210) and leucine (LM210). Both mutants grew photoheterotrophically under conditions of high light but only the FM210 mutant grew under low light. Photobleaching spectra of chromatophores isolated from these mutants showed that the amount of functional RC was comparable to wild-type and that the spectral features were essentially unchanged. Shifts were observed in the absorption spectra in regions attributable to all the chromophores. An increase in intensity and a 3 nm red shift (from 803 to 806 nm) was observed in the Qy band of the monomer bacteriochlorophylls. A new extinction coefficient for the RC was determined at 806 nm (332 +/- 15 mM-1 cm-1). Linear dichroism (LD) spectra showed that there was no significant large scale change in the angles of the individual pigments relative to the C2 axis of symmetry. Cytochrome turnover assays were performed on isolated RC and light harvesting I complex (LH I)-RC (photosynthetic units, PSU) preparations. A turnover number of 120 cyt RC-1 s-1 was calculated for both the mutants while wild-type had a turnover number of 228 cyt RC-1 s-1. The cytochrome c2-mediated re-reduction kinetics of P+ were comparable to those observed in the wild-type. The half-time of charge recombination within the RC increased in the mutants to the wild-type (100 ms in the wild type, and 150 and 200 ms in FM210 and LM210, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗

PubMed1991

Analysis of the Rhodobacter capsulatus puc operon: the pucC gene plays a central role in the regulation of LHII (B800-850 complex) expression.

Formation of the light harvesting complex B800-850 (LHII) of Rhodobacter capsulatus requires the expression of more than the three known genes specific for that complex (pucA, pucB and pucE) encoding the alpha, beta and gamma subunits of LHII, respectively. In this work evidence is presented that the product of the gene pucC, which is located downstream from pucA, is essential for high-level transcription of the pucBACDE operon and formation of LHII. Plasmids were constructed containing deletions in one or several puc genes and transferred to a pucC::Tn5 mutant in which the puc operon is not expressed. It was found that the LHII- phenotype of the mutant was due to the missing PucC protein and that all known puc genes are located in one operon. To dissect the pucC, pucD and pucE genes from pucB and pucA and independently regulate them, they were placed under control of the nifHDK promoter. Only under nitrogen-fixing growth conditions was the LHII- pucC::Tn5 mutant complemented by this construction. It is concluded that expression of pucC is essential for formation of the LHII complex in R.capsulatus. Analysis of the pucD and pucE genes led to the conclusion that the products of these genes stabilize the B800-850 complex.

Blotting, Northern↗

PubMed1992

Studies on the expression of the pufX polypeptide and its requirement for photoheterotrophic growth in Rhodobacter sphaeroides.

The puf operon in Rhodobacter sphaeroides contains the genes for the light-harvesting antenna complex I (LHI), the reaction centre (RC) L and M subunits and an additional small open reading frame identified as pufX. It has been demonstrated before that a photosynthetically incompetent pufLMX deletion strain was not complemented by a plasmid-borne truncated puf operon version lacking only pufX, although expression of the pufL and pufM gene products was restored. We demonstrate here that the functional reinsertion of only the pufX open reading frame into the same construct is sufficient and necessary for complementation of the non-photosynthetic phenotype. We also demonstrate that the observed lack of photoheterotrophic growth in the absence of pufX is not the result of decreased light-harvesting ability, but rather the result of an impairment in light-driven cyclic electron transfer. Western blots using polyclonal antibodies against a synthetic peptide corresponding to a portion of the DNA-derived pufX amino acid sequence showed that the pufX open reading frame is expressed and that the gene product has an M(r) of 8-10,000 on SDS gels; a value close to the predicted mass of 9 kDa. The pufX polypeptide was localized to the intracytoplasmic membrane fraction and appeared to co-purify with the RC-LHI complex. It is suggested that the pufX polypeptide is associated with the RC-LHI complex and that it may play a critical role in facilitating the interaction between this complex and other components required for light-driven cyclic electron transfer.

Base Sequence↗