Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Rhodobacter”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 109 records · Page 6Linked to original sources
PubMed2001

Cloning, overproduction and characterization of cytochrome c peroxidase from the purple phototrophic bacterium Rhodobacter capsulatus.

The bacterial cytochrome c peroxidase (BCCP) from Rhodobacter capsulatus was purified as a recombinant protein from an Escherichia coli clone over-expressing the BCCP structural gene. BCCP from Rb. capsulatus oxidizes the Rhodobacter cytochrome c2 and reduces hydrogen peroxide, probably functioning as a detoxification mechanism. The enzyme binds two haem c groups covalently. The gene encoding BCCP from Rb. capsulatus was cloned through the construction of a 7-kb subgenomic clone. In comparison with the protein sequence, the sequence deduced from the gene has a 21-amino-acid N-terminal extension with the characteristics of a signal peptide. The purified recombinant enzyme showed the same physico-chemical properties as the native enzyme. Spectrophotometric titration established the presence of a high-potential (Em=+270 mV) and a low-potential haem (between -190 mV and -310 mV) as found in other BCCPs. The enzyme was isolated in the fully oxidized but inactive form. It binds calcium tightly and EGTA treatment of the enzyme was necessary to show calcium activation of the mixed valence enzyme. This activation is associated with the formation of a high-spin state at the low-potential haem. BCCP oxidizes horse ferrocytochrome c better than the native electron donor, cytochrome c2; the catalytic activities ('turnover number') are 85 800 min(-1) and 63 600 min(-1), respectively. These activities are the highest ever found for a BCCP.

Amino Acid Sequence↗
PubMed1999

Activation of the cycA P2 promoter for the Rhodobacter sphaeroides cytochrome c2 gene by the photosynthesis response regulator.

The Rhodobacter sphaeroides photosynthesis response regulator, PrrA, positively regulates cycA P2 expression. Deletion analysis has identified sequences within 73 bp upstream of the transcription initiation site that are required for the activation of cycA P2 by PrrA. A mutant form of the Rhodobacter capsulatus PrrA homologue, whose activity is independent of phosphorylation (RegA*), protects an approximately 26 bp region of cycA P2 that is centred at approximately -50 from DNase digestion, and activates transcription of a mutant -14T promoter with increased activity when using either R. sphaeroides RNA polymerase or Escherichia coli Esigma70. A 4 bp target site mutation that eliminated DNA binding and transcription activation by RegA* in vitro also abolished PrrA activation of cycA P2 transcription in vivo, indicating that this region contains a PrrA binding site. By analysing the behaviour of the -14T mutant cycA P2 promoter in vivo, we also found that PrrA uses the same target site to activate expression in both the presence and the absence of O2. However, the extent of transcription activation by PrrA at cycA P2 in vivo is greater under anaerobic conditions.

Bacterial Proteins↗
PubMed2002

A single flavoprotein, AppA, integrates both redox and light signals in Rhodobacter sphaeroides.

Anoxygenic photosynthetic proteobacteria exhibit various light responses, including changing levels of expression of photosynthesis genes. However, the underlying mechanisms are largely unknown. We show that expression of the puf and puc operons encoding structural proteins of the photosynthetic complexes is strongly repressed by blue light under semi-aerobic growth in Rhodobacter sphaeroides but not in the related species Rhodobacter capsulatus. At very low oxygen tension, puf and puc expression is independent of blue light in both species. Photosynthetic electron transport does not mediate the blue light repression, implying the existence of specific photoreceptors. Here, we show that the flavoprotein AppA is likely to act as the photoreceptor for blue light-dependent repression during continuous illumination. The FAD cofactor of AppA is essential for the blue light-dependent sensory transduction of this response. AppA, which is present in R. sphaeroides but not in R. capsulatus, is known to participate in the redox-dependent control of photosynthesis gene expression. Thus, AppA is the first example of a protein with dual sensing capabilities that integrates both redox and light signals.

Bacterial Proteins↗
PubMed2002

The opgGIH and opgC genes of Rhodobacter sphaeroides form an operon that controls backbone synthesis and succinylation of osmoregulated periplasmic glucans.

Osmoregulated periplasmic glucans (OPGs) of Rhodobacter sphaeroides are anionic cyclic molecules that accumulate in large amounts in the periplasmic space in response to low osmolarity of the medium. Their anionic character is provided by the substitution of the glucosidic backbone by succinyl residues. A wild-type strain was subject to transposon mutagenesis, and putative mutant clones were screened for changes in OPGs by thin layer chromatography. One mutant deficient in succinyl substitution of the OPGs was obtained and the gene inactivated in this mutant was characterized and named opgC. opgC is located downstream of three ORFs, opgGIH, two of which are similar to the Escherichia coli operon, mdoGH, governing OPG backbone synthesis. Inactivation of opgG, opgI or opgH abolished OPG production and complementation analysis indicated that the three genes are necessary for backbone synthesis. In contrast, inactivation of a gene similar to ndvB, encoding the OPG-glucosyl transferase in Sinorhizobium meliloti, had no consequence on OPG synthesis in Rhodobacter sphaeroides. Cassette insertions in opgH had a polar effect on glucan substitution, indicating that opgC is in the same transcription unit. Expression of opgIHC in E. coli mdoB/mdoC and mdoH mutants allowed the production of slightly anionic and abnormally long linear glucans.

Bacterial Proteins↗
PubMed1998

Characterization of cytochrome c-556 from the purple phototrophic bacterium Rhodobacter capsulatus as a cytochrome-c peroxidase.

A cytochrome c-556 was purified from Rhodobacter capsulatus and the complete amino acid sequence was determined. It contains 328 amino acid residues and two typical heme-binding sites at cysteine residues 54 and 57 and at residues 200 and 203. It is homologous to the family of bacterial cytochrome c peroxidases (BCCP) with 69% identity to Paracoccus denitrificans BCCP and 60% identity to Pseudomonas aeruginosa BCCP for which there is a three-dimensional structure. There is lesser similarity to the mauG gene products from methylotrophic bacteria which are thought to be involved in biosynthesis of the quinone cofactor of methylamine dehydrogenase. Translated genes from Escherichia coli and Helicobacter pylori are also related to the bacterial cytochrome c peroxidases. The divergence of this family of proteins is reflected in the fact that the reported sixth heme ligands are not conserved, except in Pseudomonas, Rhodobacter and Paracoccus. This suggests that homologs of BCCP may fold differently and/or may not have the same enzymatic activity as the prototypic protein from Ps. aeruginosa. We found that the Rb. capsulatus BCCP is active with both Rb. capsulatus cytochrome c2 and with horse cytochrome c as substrates (Km values 60 microm and 6 microm, respectively). The turnover number was 40 s(-1) and the Km for peroxide was 33 microm. We have thus confirmed that the Rb. capsulatus protein is a cytochrome c peroxidase.

Amino Acid Sequence↗
PubMed1995

Alignment of a 1.2-Mb chromosomal region from three strains of Rhodobacter capsulatus reveals a significantly mosaic structure.

High-resolution physical maps of the genomes of three Rhodobacter capsulatus strains, derived from ordered cosmid libraries, were aligned. The 1.2-Mb segment of the SB1003 genome studied here is adjacent to a 1-Mb region analyzed previously [Fonstein, M., Nikolskaya, T. & Haselkorn, H. (1995) J. Bacteriol. 177, 2368-2372]. Probes derived from the ordered cosmid set of R. capsulatus SB1003 were used to link cosmids from the St. Louis and 2.3.1 strain libraries. Cosmids selected this way did not merge into a single contig but formed several unlinked groups. EcoRV restriction maps of the ordered cosmids were then constructed using lambda terminase and fused to derive fragments of the chromosomal map. In order to link these fragments, their ends were transcribed to produce secondary probes for hybridization to gridded cosmid libraries of the same strains. This linking reduced the number of subcontigs to three for the St. Louis strain and one for the 2.3.1 strain. Hybridization of the same probes back to the ordered cosmid set of SB1003 positioned the subcontigs on the high-resolution physical map of SB1003. The final alignment of the restriction maps shows numerous large and small translocations in this 1.2-Mb chromosomal region of the three Rhodobacter strains. In addition, the chromosomes of the three strains, whose fine-structure maps can now be compared over 2.2 Mb, are seen to contain regions of 15-80 kb in which restriction sites are highly polymorphic, interspersed among regions in which the positions of restriction sites are highly conserved.

Chromosomes, Bacterial↗
PubMed1995

Reconstitution of the bacterial core light-harvesting complexes of Rhodobacter sphaeroides and Rhodospirillum rubrum with isolated alpha- and beta-polypeptides, bacteriochlorophyll alpha, and carotenoid.

Methodology has been developed to reconstitute carotenoids and bacteriochlorophyll alpha with isolated light-harvesting complex I (LHI) polypeptides of both Rhodobacter sphaeroides and Rhodospirillum rubrum. Reconstitution techniques first developed in this laboratory using the LHI polypeptides of R. rubrum, R. sphaeroides, and Rhodobacter capsulatus reproduced bacteriochlorophyll alpha spectral properties characteristic of LHI complexes lacking carotenoids. In this study, carotenoids are supplied either as organic-solvent extracts of chromatophores or as thin-layer chromatography or high performance liquid chromatography-purified species. The resulting LHI complexes exhibit carotenoid and bacteriochlorophyll a spectral properties characteristic of native LHI complexes of carotenoid-containing bacteria. Absorption and circular dichroism spectra support the attainment of a native-like carotenoid environment in the reconstituted LHI complexes. For both R. sphaeroides- and R. rubrum-reconstituted systems, fluorescence excitation spectra reveal appropriate carotenoid to bacteriochlorophyll alpha energy-transfer efficiencies based on comparisons with the in vivo systems. In the case of R. rubrum reconstitutions, carotenoids afford protection from photodynamic degradation. Thus, carotenoids reconstituted into LHI exhibit spectral and functional characteristics associated with native pigments. Heterologous reconstitutions demonstrate the applicability of the developed assay in dissecting the molecular environment of carotenoids in light-harvesting complexes.

Bacteriochlorophylls↗
PubMed1995

Accumulation of UDP-sulfoquinovose in a sulfolipid-deficient mutant of Rhodobacter sphaeroides.

The sulfolipid 6-sulfo-alpha-D-quinovosyl diacylglycerol is found in the photosynthetic membranes of all plants and most photosynthetic bacteria. Progress toward the elucidation of the pathway for sulfolipid biosynthesis has been slow in the past. However, the recent isolation of three genes of the photosynthetic bacterium Rhodobacter sphaeroides known to be involved in sulfolipid biosynthesis provides promising new opportunities. Two of the genes flank an open reading Rhodobacter sphaeroides known to be involved in sulfolipid biosynthesis provides promising new opportunities. Two of the genes flank an open reading frame predicted to encode a protein with amino acid sequence similarity to sugar nucleotide-dependent glycosyltransferases. The UDP-sulfoquinovose:diacylglcerol sulfoquinovosyltransferase thought to catalyze the last step of sulfolipid biosynthesis belongs to this group of glycosyltransferases. To test whether this open reading frame encodes the sulfoquinovosyltransferase of R. sphaeroides, it was inactivated by gene replacement avoiding polar mutagenesis. The resulting sulfolipid-deficient mutant defines a new gene, designated sqdD. Mutant cells grown in the presence of [35S]sulfate accumulate a water-soluble 35S-labeled compound. The purified compound was tentatively identified by co-chromatography with standards and enzymatic conversion as UDP-sulfoquinovose, the final precursor of sulfolipid biosynthesis. This result strongly suggests that the inactivation of sqdD causes a metabolic block in the last step of sulfolipid biosynthesis.

Bacterial Proteins↗
PubMed2001

Alteration of product specificity of Rhodobacter sphaeroides phytoene desaturase by directed evolution.

Phytoene desaturases occurring in nature convert phytoene to either neurosporene or lycopene in most eubacteria. Approximately 10% of known phytoene desaturases, as in Rhodobacter, produce neurosporene, whereas the rest produce lycopene. These two types of enzymes, although similar in function, have relatively low similarity (below 60%) in terms of nucleotide or amino acid sequence. The mechanism controlling the product specificity of these enzymes is unclear. Here we used directed evolution to change the product of Rhodobacter sphaeroides phytoene desaturase (crtI gene product), a neurosporene-producing enzyme, to lycopene. Two generations of random mutagenesis were performed, from which three positive mutants were isolated and sequenced. We then used site-directed mutagenesis to determine the effect of each amino acid change. Gathering information from random mutagenesis, we further recombined the beneficial mutations by site-directed mutagenesis and increased the percent of lycopene production to 90%.

Base Sequence↗
PubMed1998

Different cleavage specificities of RNases III from Rhodobacter capsulatus and Escherichia coli.

23S rRNA in Rhodobacter capsulatus shows endoribonuclease III (RNase III)-dependent fragmentation in vivo at a unique extra stem-loop extending from position 1271 to 1331. RNase III is a double strand (ds)-specific endoribonuclease. This substrate preference is mediated by a double-stranded RNA binding domain (dsRBD) within the protein. Although a certain degree of double strandedness is a prerequisite, the question arises what structural features exactly make this extra stem-loop an RNase III cleavage site, distinguishing it from the plethora of stem-loops in 23S rRNA? We used RNase III purified from R.capsulatus and Escherichia coli, respectively, together with well known substrates for E.coli RNase III and RNA substrates derived from the special cleavage site in R.capsulatus 23S rRNA to study the interaction between the Rhodobacter enzyme and the fragmentation site. Although both enzymes are very similar in their amino acid sequence, they exhibit significant differences in binding and cleavage of these in vitro substrates.

Bacterial Proteins↗
PubMed2000

DNA sequence analysis of the photosynthesis region of Rhodobacter sphaeroides 2.4.1.

This paper describes the DNA sequence of the photosynthesis region of Rhodobacter sphaeroides 2.4.1 (T). The photosynthesis gene cluster is located within a approximately 73 kb Ase I genomic DNA fragment containing the puf, puhA, cycA and puc operons. A total of 65 open reading frames (ORFs) have been identified, of which 61 showed significant similarity to genes/proteins of other organisms while only four did not reveal any significant sequence similarity to any gene/protein sequences in the database. The data were compared with the corresponding genes/ORFs from a different strain of R.sphaeroides and Rhodobacter capsulatus, a close relative of R. sphaeroides. A detailed analysis of the gene organization in the photosynthesis region revealed a similar gene order in both species with some notable differences located to the pucBAC = cycA region. In addition, photosynthesis gene regulatory protein (PpsR, FNR, IHF) binding motifs in upstream sequences of a number of photosynthesis genes have been identified and shown to differ between these two species. The difference in gene organization relative to pucBAC and cycA suggests that this region originated independently of the photosynthesis gene cluster of R.sphaeroides.

Base Sequence↗
PubMed2003

Solution structure and DNA binding of the effector domain from the global regulator PrrA (RegA) from Rhodobacter sphaeroides: insights into DNA binding specificity.

Prr/RegA response regulator is a global transcription regulator in purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus, and is essential in controlling the metabolic changes between aerobic and anaerobic environments. We report here the structure determination by NMR of the C-terminal effector domain of PrrA, PrrAC. It forms a three-helix bundle containing a helix-turn-helix DNA binding motif. The fold is similar to FIS protein, but the domain architecture is different from previously characterised response regulator effector domains, as it is shorter than any characterised so far. Alignment of Prr/RegA DNA targets permitted a refinement of the consensus sequence, which contains two GCGNC inverted repeats with variable half-site spacings. NMR titrations of PrrAC with specific and non-specific DNA show which surfaces are involved in DNA binding and suggest residues important for binding specificity. A model of the PrrAC/DNA complex was constructed in which two PrrAC molecules are bound to DNA in a symmetrical manner.

Bacterial Proteins↗
PubMed1999

Anaerobic purification and characterization of nitrous oxide reductase from Rhodobacter sphaeroides f. sp. denitrificans IL106.

The nitrous oxide reductase from the photodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans IL106, has been purified under anaerobic conditions. The specific activity of the enzyme was 78 micromol nitrous oxide reduced per min per mg protein, which was approximately 80% higher than that of the aerobic form. The enzyme purified anaerobically retained most of its activity after aerobic storage at 4 degrees C for 2 months without any additives. Visible absorption spectra of the Rhodobacter nitrous oxide reductase resembled those of the enzymes from other origins. The enzyme retained its activity after reduction with sodium dithionite, and the enzyme activity could be determined using dithionite-reduced benzyl viologen. Turnover-dependent inactivation of the enzyme was suppressed by complete removal of oxygen from the reaction mixture, and promoted by zinc ions.

Chromatography, Gel↗
PubMed1996

A transcription factor with a leucine-zipper motif involved in light-dependent inhibition of expression of the puf operon in the photosynthetic bacterium Rhodobacter sphaeroides.

In the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides the synthesis of components of the photosystem is regulated in response to oxygen tension and light intensity. We have purified and cloned a trans-acting protein (SPB) that binds to the promoter region of the puf operon, which encodes the apoproteins of light-harvesting complex I and the reaction center. The SPB was composed of a single polypeptide with an apparent molecular mass of 15.0 kDa. The nucleotide sequence of the spb gene was determined. The gene encoded 104 amino acid residues, which correspond to a molecular mass of 11.5 kDa. SPB exhibited 53% homology to HvrA in Rhodobacter capsulatus. The deduced amino acid sequence indicated that SPB contained a region with homology to the leucine-zipper motif of c-JUN, a transcription factor in eukaryotes, and SPB also had a DNA-binding domain on the amino-terminal side of the leucine-zipper motif. The leucine-zipper motif of SPB might contribute to the formation of a dimer. Northern analysis indicated that spb was constitutively and monocistronically transcribed in R. sphaeroides, irrespective of growth conditions. Structural and functional differences between SPB and HvrA are discussed.

Amino Acid Sequence↗
PubMed1998

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

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.

Bacterial Proteins↗
PubMed2003

An abundant periplasmic protein of the denitrifying phototroph Rhodobacter sphaeroides f. sp. denitrificans is PstS, a component of an ABC phosphate transport system.

To understand a physiological role of an abundant 34-kDa periplasmic protein in the denitrifying phototroph Rhodobacter sphaeroides f. sp. denitrificans grown in a medium containing malate as the carbon source, the gene for the protein was isolated. The deduced amino acid sequence of the protein had a sequence similarity of 66.2% to that of PstS from Sinorhizobium meliloti. The downstream sequence of the Rhodobacter pstS contained five genes similar to pstCAB and phoUB, and its upstream sequence contained a putative regulatory sequence that is analogous to the Pho box involved in phosphate-limitation-induced gene expression in Escherichia coli. Both the amount of the PstS and the pstS promoter-driven expression of lacZ activity increased about two-fold in response to phosphate limitation. This is the first isolation of pst genes encoding proteins of an ABC phosphate transporter system from phototrophic bacteria.

ATP-Binding Cassette Transporters↗
PubMed1995

Isolation of regulatory mutants in photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1 and partial complementation of a PrrB mutant by the HupT histidine-kinase.

The photosynthetic bacterium Rhodobacter sphaeroides responds to the transition from aerobiosis to anaerobic photosynthesis by increasing the expression of the photosynthesis genes. Mutants have been isolated based on their inability, following such a transition, to increase transcription of the puc operon encoding the apoproteins of the light-harvesting complex II. Mutant D5, a representative of one mutant class, described here, although remaining photosynthetically competent, produced only low levels of the photosynthetic spectral complexes. Complementation analysis revealed that either the gene for the photosynthesis response regulator prrA or the gene encoding its cognate sensor kinase, prrB, was capable of rescuing this mutant. However, partial complementation of this mutant was achieved by placing in trans additional copies of other defined genes from the cosmid library of R. sphaeroides. We describe this effect in detail, attributable to the hupT gene, which has been proposed to encode a histidine-kinase for the hydrogen uptake system in Rhodobacter capsulatus. The effect of HupT on the expression of the photosynthesis genes was mediated through PrrA and independent of a functioning hydrogen uptake system. Thus, we raise the possibility that HupT can participate in phosphorylation of the heterologous response regulator PrrA by so-called cross-talk and therefore partially compensate for the defect in the mutant described. The observation of cross-talk, together with the complementation analysis, allowed us to assign the original mutation to the prrB gene; this was confirmed by DNA sequencing. Analysis of cross-talk in the wild-type, prrB and prrA genetic backgrounds suggested that besides kinase activity, PrrB may possess phosphatase activity toward PrrA. We also report the cloning, organization and structure of some of the hup genes from R. sphaeroides and construction of a Hup- strain.

Amino Acid Sequence↗
PubMed1999

Characterization of a molybdenum cofactor biosynthetic gene cluster in Rhodobacter capsulatus which is specific for the biogenesis of dimethylsulfoxide reductase.

The DMSO reductase of Rhodobacter capsulatus contains a pterin molybdenum cofactor (Moco) and is located in the periplasm. DNA sequence analysis identified four genes involved in the biosynthesis of the Moco (moaA, moaD, moeB and moaC) immediately downstream of the dor (DMSO respiratory) gene cluster. Rhodobacter capsulatus MoaA was expressed in Escherichia coli as a His6-tagged protein. Although, the expressed protein formed inclusion bodies, EPR spectroscopy showed that MoaA contains a [3Fe-4S] cluster. A moaA mutant was constructed and its phenotype indicates that the Moco biosynthetic gene cluster downstream of the dor operon is specific for the biogenesis of DMSO reductase. Two forms of DMSO reductase were purified by immunoaffinity chromatography from the moaA mutant. A mature form of DMSO reductase was located in the periplasm and a precursor form was found in the cytoplasm.

Amino Acid Sequence↗