| 1. |
Cao J,
Hosler J,
Shapleigh J,
Revzin A,
Ferguson-Miller S,
( 1992 ) Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase. The coxII/coxIII operon codes for structural and assembly proteins homologous to those in yeast. PMID : 1332950 : Abstract >>
The coxII/coxIII operon of Rhodobacter sphaeroides cytochrome c oxidase has been sequenced and characterized by insertional inactivation/complementation analysis. The organization of the genes in this locus (coxII.orf1.orf3.coxIII) is the same as that of the equivalent operon of Paracoccus denitrificans (ctaC.ctaB.ctaG.ctaE), but unlike that of other bacteria whose cytochrome oxidase genes have been characterized so far. The predicted amino acid sequence homology with eukaryotic oxidases is also higher for Rb. sphaeroides (and P. denitrificans) than for other bacterial versions of the enzyme. The inactivation of coxII results in loss of the characteristic cytochrome oxidase spectrum from membranes of the mutant strain. Full recovery requires introduction into the bacterium of the complete operon containing coxII.orf1.orf3.coxIII; partial complementation yielding a spectrally altered enzyme is achieved with a plasmid containing coxII or coxII.orf1.orf3. These results indicate that the peptides ORF1, ORF3, and COXIII are all required for assembly of native cytochrome c oxidase, suggesting an oxidase-specific assembly or chaperonin function for the ORFs in Rb. sphaeroides similar to that observed for the homologous gene products in yeast, COX10 and COX11.
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2. |
Benning C,
Somerville CR,
( 1992 ) Identification of an operon involved in sulfolipid biosynthesis in Rhodobacter sphaeroides. PMID : 1400200 : DOI : 10.1128/jb.174.20.6479-6487.1992 PMC : PMC207608 Abstract >>
Two new mutants of Rhodobacter sphaeroides deficient in sulfolipid accumulation were isolated by directly screening mutagenized cell lines for polar lipid composition by thin-layer chromatography of lipid extracts. A genomic clone which complemented the mutations in these two lines, but not the previously described sulfolipid-deficient sqdA mutant, was identified. Sequence analysis of the relevant region of the clone revealed three, in tandem open reading frames, designated sqdB, ORF2, and sqdC. One of the mutants was complemented by the sqdB gene, and the other was complemented by the sqdC gene. Insertional inactivation of sqdB also inactivated sqdC, indicating that sqdB and sqdC are cotranscribed. The N-terminal region of the 46-kDa putative protein encoded by the sqdB gene showed slight homology to UDP-glucose epimerase from various organisms. The 30-kDa putative protein encoded by ORF2 showed very striking homology to rabbit muscle glycogenin, a UDP-glucose utilizing, autoglycosylating glycosyltransferase. The 26-kDa putative protein encoded by the sqdC gene was not homologous to any protein of known function.
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3. |
Meijer WG,
Tabita FR,
( 1992 ) Isolation and characterization of the nifUSVW-rpoN gene cluster from Rhodobacter sphaeroides. PMID : 1317839 : DOI : 10.1128/jb.174.12.3855-3866.1992 PMC : PMC206092 Abstract >>
The rpoN gene from Rhodobacter sphaeroides was isolated from a genomic library via complementation of a Rhodobacter capsulatus rpoN mutant. The rpoN gene was located on a 7.5-kb HindIII-EcoRI fragment. A Tn5 insertion analysis of this DNA fragment showed that a minimal DNA fragment of 5.3 kb was required for complementation. Nucleotide sequencing of the complementing region revealed the presence of nifUSVW genes upstream from rpoN. The rpoN gene was mutagenized via insertion of a gene encoding kanamycin resistance. The resulting rpoN mutant was not impaired in diazotrophic growth and was in all respects indistinguishable from the wild-type strain. Southern hybridizations using the cloned rpoN gene as a probe indicated the presence of a second rpoN gene. Deletion of the nifUS genes resulted in strongly reduced diazotrophic growth. Two conserved regions were identified in a NifV LeuA amino acid sequence alignment. Similar regions were found in pyruvate carboxylase and oxaloacetate decarboxylase. It is proposed that these conserved regions represent keto acid-binding sites.
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4. |
Shapleigh JP,
Gennis RB,
( 1992 ) Cloning, sequencing and deletion from the chromosome of the gene encoding subunit I of the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides. PMID : 1313140 : DOI : 10.1111/j.1365-2958.1992.tb01511.x Abstract >>
The ctaD gene encoding subunit I of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides has been cloned. The gene encodes a polypeptide of 565 residues which is highly homologous to the sequences of subunit I from other prokaryotic and eukaryotic sources, e.g. 51% identity with that from bovine, and 75% identity with that from Paracoccus denitrificans. The ctaD gene was deleted from the chromosome of R. sphaeroides, resulting in a strain that spectroscopically lacks cytochrome a. This strain maintains about 50% of the cytochrome c oxidase activity of the wild-type strain owing to the presence of an alternate o-type cytochrome c oxidase. The aa3-type oxidase was restored by complementing the chromosomal deletion with a plasmid-borne copy of the ctaD gene. This system is well suited for site-directed mutagenesis probing of the structure and function of cytochrome c oxidase.
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5. |
Oh JI,
Ko IJ,
Kaplan S,
( 2003 ) Digging deeper: uncovering genetic loci which modulate photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1. PMID : 12686637 : DOI : 10.1099/mic.0.26010-0 Abstract >>
A new genetic locus was identified in Rhodobacter sphaeroides which is required for optimal synthesis of the light-harvesting spectral complexes as well as for optimal growth under anaerobic conditions with dimethyl sulfoxide (DMSO) as a terminal electron acceptor. The primary structure of the deduced osp gene product shows significant homology to the receiver domain of known response regulators common to bacterial two-component systems. However, site-directed mutagenesis revealed that the Osp protein appears not to be involved in a phospho-relay signal transduction pathway. Paradoxically, the effect of the Osp protein upon spectral complex levels is exerted at the transcriptional level of photosynthesis gene expression. The absence of the Osp protein does not appear to have a general effect on house-keeping metabolism. In cells lacking Osp, the levels of DMSO reductase appear to be normal. The quaternary structure of the Osp protein was determined to be a homodimer and it was directly demonstrated that Osp does not bind to the promoter region of photosynthesis genes as judged by mobility-shift experiments and primary structure analysis.
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6. |
Cantera JJ,
Kawasaki H,
Seki T,
( 2002 ) Farnesyl diphosphate synthase gene of three phototrophic bacteria and its use as a phylogenetic marker. PMID : 12508853 : DOI : 10.1099/00207713-52-6-1953 Abstract >>
Farnesyl diphosphate (FPP) synthase is essential not only for phototrophic bacteria in carotenoid biosynthesis, but also for non-phototrophic bacteria in the biosynthesis of physiologically important compounds. The gene encoding FPP synthase was assessed as a molecular marker to investigate the intermingled relationship between the phototropic and non-phototropic bacteria in the alpha-Proteobacteria based on 16S rRNA analysis. The FPP synthase amino acid sequences from three phototropic bacteria, Rhodobacter sphaeroides ATCC 11167(T), Rhodobacter capsulatus ATCC 11166(T) and Rhodovulum sulfidophilum W4(T), were determined and used in conjunction with sequences of other representative members of the alpha-, gamma- and epsilon-Proteobacteria and the low-G+C Gram-positive bacteria for phylogenetic analyses by the neighbour-joining and maximum-likelihood methods. The overall topology of the FPP synthase gene tree is consistent with that of the 16S rRNA tree, producing a distinct cluster of the three phototropic bacteria. A minor discordance between the two trees was observed in the cluster of the non-phototrophic Bradyrhizobiumjaponicum USDA 110 and Mesorhizobium loti MAFF 303099; the FPP synthase genes of these two rhizobial species are highly homologous as compared with their respective 16S rRNA. The results suggest that the FPP synthase and 16S rRNA genes have the same evolutionary pattern, evolving vertically from each common ancestral gene; the FPP synthase gene, therefore, could possibly be used for further study on the molecular systematics of photosynthetic bacteria.
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7. |
Rios-Velazquez C,
Coller R,
Donohue TJ,
( 2003 ) Features of Rhodobacter sphaeroides CcmFH. PMID : 12511487 : DOI : 10.1128/jb.185.2.422-431.2003 PMC : PMC145331 Abstract >>
In this study, the in vivo function and properties of two cytochrome c maturation proteins, CcmF and CcmH from Rhodobacter sphaeroides, were analyzed. Strains lacking CcmH or both CcmF and CcmH are unable to grow under anaerobic conditions where c-type cytochromes are required, demonstrating their critical role in the assembly of these electron carriers. Consistent with this observation, strains lacking both CcmF and CcmH are deficient in c-type cytochromes when assayed under permissive growth conditions. In contrast, under permissive growth conditions, strains lacking only CcmH contain several soluble and membrane-bound c-type cytochromes, albeit at reduced levels, suggesting that this bacterium has a CcmH-independent route for their maturation. In addition, the function of CcmH that is needed to support anaerobic growth can be replaced by adding cysteine or cystine to growth media. The ability of exogenous thiol compounds to replace CcmH provides the first physiological evidence for a role of this protein in thiol chemistry during c-type cytochrome maturation. The properties of R. sphaeroides cells containing translational fusions between CcmF and CcmH and either Escherichia coli alkaline phosphatase or beta-galactosidase suggest that they are each integral cytoplasmic membrane proteins with their presumed catalytic domains facing the periplasm. Analysis of CcmH shows that it is synthesized as a higher-molecular-weight precursor protein with an N-terminal signal sequence.
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8. |
González-Pedrajo B,
de la Mora J,
Ballado T,
Camarena L,
Dreyfus G,
( 2002 ) Characterization of the flgG operon of Rhodobacter sphaeroides WS8 and its role in flagellum biosynthesis. PMID : 12401220 : DOI : 10.1016/s0167-4781(02)00504-3 Abstract >>
In this work, we show evidence regarding the functionality of a large cluster of flagellar genes in Rhodobacter sphaeroides. The genes of this cluster, flgGHIJKL and orf-1, are mainly involved in the formation of the basal body, and flgK and flgL encode the hook-associated proteins HAP1 and HAP3. In general, these genes showed a good similarity as compared with those reported for Salmonella enterica. However, flgJ and flgK showed particular features that make them unique among the flagellar sequences already reported. flgJ is only a third of the size reported for flgJ from Salmonella; whereas flgK is about three times larger than any other flgK sequence previously known. Our results indicate that both genes are functional, and their products are essential for flagellar assembly. In contrast, the interruption of orf-1, did not affect motility suggesting that this sequence, if functional, is not indispensable for flagellar assembly. Finally, we present genetic evidence suggesting that the flgGHIJKL genes are expressed as a single transcriptional unit depending on the sigma-54 factor.
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9. |
Porter SL,
Warren AV,
Martin AC,
Armitage JP,
( 2002 ) The third chemotaxis locus of Rhodobacter sphaeroides is essential for chemotaxis. PMID : 12421313 : DOI : 10.1046/j.1365-2958.2002.03218.x Abstract >>
The purple photosynthetic bacterium Rhodobacter sphaeroides has three loci encoding multiple homologues of the bacterial chemosensory proteins: 13 putative chemoreceptors, four CheW, four CheA, six CheY, two CheB and three CheR. Previously, studies have shown that, although deletion of cheOp1 led to only minor changes in behaviour, deletion of cheOp2 led to a loss of taxis. The third locus encodes two CheA, one CheR, one CheB, one CheW, one CheY, a putative cytoplasmic chemoreceptor (TlpT) and a protein showing homology to the chromosomal partitioning factor Soj (designated Slp). Here, we show that every protein encoded by this locus is essential for normal chemotaxis. Phototaxis is also dependent upon all the components of this locus, except CheB2 and Slp. The two putative CheA proteins encoded in this locus are unusual. CheA3 has only the P1 domain and the P5 regulatory domain linked by a large internal domain, whereas CheA4 lacks the P1 and P2 domains required for phosphorylation and response regulator binding. These data indicate that the minimal set of proteins required for normal chemotaxis in R. sphaeroides is all the proteins encoded by cheOp2 and the third chemotaxis locus, and that the multiple chemosensory protein homologues found in R. sphaeroides are not redundant.
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10. |
Svensson-Ek M,
Abramson J,
Larsson G,
Törnroth S,
Brzezinski P,
Iwata S,
( 2002 ) The X-ray crystal structures of wild-type and EQ(I-286) mutant cytochrome c oxidases from Rhodobacter sphaeroides. PMID : 12144789 : DOI : 10.1016/s0022-2836(02)00619-8 DOI : 10.1016/s0022-2836(02)00619-8 Abstract >>
The structure of cytochrome c oxidase from Rhodobacter sphaeroides has been solved at 2.3/2.8A (anisotropic resolution). This high-resolution structure revealed atomic details of a bacterial terminal oxidase including water molecule positions and a potential oxygen pathway, which has not been reported in other oxidase structures. A comparative study of the wild-type and the EQ(I-286) mutant enzyme revealed structural rearrangements around E(I-286) that could be crucial for proton transfer in this enzyme. In the structure of the mutant enzyme, EQ(I-286), which cannot transfer protons during oxygen reduction, the side-chain of Q(I-286) does not have the hydrogen bond to the carbonyl oxygen of M(I-107) that is seen in the wild-type structure. Furthermore, the Q(I-286) mutant has a different arrangement of water molecules and residues in the vicinity of the Q side-chain. These differences between the structures could reflect conformational changes that take place upon deprotonation of E(I-286) during turnover of the wild-type enzyme, which could be part of the proton-pumping machinery of the enzyme.
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11. |
Lu T,
Wu YQ,
Song HY,
( 1997 ) The Nucleotide Sequence of gltD Gene Encoding the Small Subunit of Rhodobacter sphaeroides Glutamate Synthase. PMID : 12219208 : Abstract >>
We have determined the complete nucleotide sequence of a 2 387 bp chromosomal SalI-EcoRI fragment, which contains the structural gene (gltD) for the small subunit of Rhodobacter sphaeroides glutamate synthase, as well as the 5'- and 3'-flanking regions. An open reading frame of 1 242 base pairs was identified as the R. sphaeroides gltD gene. The MW of the small subunit, as deduced from the nucleotide sequence, was estimated to be 44 kD. A comparison of the nucleotide sequence revealed a high similarity among gltD genes of R. sphaeroides, Azospirillum brasilense and Escherichia coli. The deduced amino acid sequence of R. sphaeroides GltD showed a high similarity with that of A. brasilense GltD. The analysis of the binding domains of R. sphaeroides GltD was also carried out. The gltD-lacZ fusion was observed in the presence of 15 mM leucine.
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12. |
Hickman JW,
Barber RD,
Skaar EP,
Donohue TJ,
( 2002 ) Link between the membrane-bound pyridine nucleotide transhydrogenase and glutathione-dependent processes in Rhodobacter sphaeroides. PMID : 11751816 : DOI : 10.1128/jb.184.2.400-409.2002 PMC : PMC139586 Abstract >>
The presence of a glutathione-dependent pathway for formaldehyde oxidation in the facultative phototroph Rhodobacter sphaeroides has allowed the identification of gene products that contribute to formaldehyde metabolism. Mutants lacking the glutathione-dependent formaldehyde dehydrogenase (GSH-FDH) are sensitive to metabolic sources of formaldehyde, like methanol. This growth phenotype is correlated with a defect in formaldehyde oxidation. Additional methanol-sensitive mutants were isolated that contained Tn5 insertions in pntA, which encodes the alpha subunit of the membrane-bound pyridine nucleotide transhydrogenase. Mutants lacking transhydrogenase activity have phenotypic and physiological characteristics that are different from those that lack GSH-FDH activity. For example, cells lacking transhydrogenase activity can utilize methanol as a sole carbon source in the absence of oxygen and do not display a formaldehyde oxidation defect, as determined by whole-cell (13)C-nuclear magnetic resonance. Since transhydrogenase can be a major source of NADPH, loss of this enzyme could result in a requirement for another source for this compound. Evidence supporting this hypothesis includes increased specific activities of other NADPH-producing enzymes and the finding that glucose utilization by the Entner-Doudoroff pathway restores aerobic methanol resistance to cells lacking transhydrogenase activity. Mutants lacking transhydrogenase activity also have higher levels of glutathione disulfide under aerobic conditions, so it is consistent that this strain has increased sensitivity to oxidative stress agents like diamide, which are known to alter the oxidation reduction state of the glutathione pool. A model will be presented to explain the role of transhydrogenase under aerobic conditions when cells need glutathione both for GSH-FDH activity and to repair oxidatively damaged proteins.
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13. |
Pignol D,
Adriano JM,
Fontecilla-Camps JC,
Sabaty M,
( 2001 ) Crystallization and preliminary X-ray analysis of the periplasmic nitrate reductase (NapA-NapB complex) from Rhodobacter sphaeroides f. sp. denitrificans. PMID : 11717511 : DOI : 10.1107/s0907444901015852 Abstract >>
The periplasmic nitrate reductase of Rhodobacter sphaeroides f. sp. denitrificans is a heterodimer responsible for the first step of reduction in the denitrification process by the conversion of nitrate to nitrite. It consists of a 91 kDa molybdenum-containing catalytic subunit (NapA) and a 17 kDa dihaem cytochrome c (NapB). Crystals of the NapA-NapB complex were obtained by the vapour-diffusion method using ammonium sulfate as precipitant. They belong to the P6(1)22 space group, with unit-cell parameters a = b = 151.9, c = 255.8 A, and contain a single complex in the asymmetric unit. A complete native data set was collected at a synchrotron source to 3.1 A resolution.
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14. |
Martin AC,
Wadhams GH,
Shah DS,
Porter SL,
Mantotta JC,
Craig TJ,
Verdult PH,
Jones H,
Armitage JP,
( 2001 ) CheR- and CheB-dependent chemosensory adaptation system of Rhodobacter sphaeroides. PMID : 11717272 : DOI : 10.1128/JB.183.24.7135-7144.2001 PMC : PMC95562 Abstract >>
Rhodobacter sphaeroides has multiple homologues of most of the Escherichia coli chemotaxis genes, organized in three major operons and other, unlinked, loci. These include cheA(1) and cheR(1) (che Op(1)) and cheA(2), cheR(2), and cheB(1) (che Op(2)). In-frame deletions of these cheR and cheB homologues were constructed and the chemosensory behaviour of the resultant mutants examined on swarm plates and in tethered cell assays. Under the conditions tested, CheR(2) and CheB(1) were essential for normal chemotaxis, whereas CheR(1) was not. cheR(2) and cheB(1), but not cheR(1), were also able to complement the equivalent E. coli mutants. However, none of the proteins were required for the correct polar localization of the chemoreceptor McpG in R. sphaeroides. In E. coli, CheR binds to the NWETF motif on the high-abundance receptors, allowing methylation of both high- and low-abundance receptors. This motif is not contained on any R. sphaeroides chemoreceptors thus far identified, although 2 of the 13 putative chemoreceptors, McpA and TlpT, do have similar sequences. This suggests that CheR(2) either interacts with the NWETF motif of E. coli methyl-accepting chemotaxis proteins (MCPs), even though its native motif may be slightly different, or with another conserved region of the MCPs. Methanol release measurements show that R. sphaeroides has an adaptation system that is different from that of Bacillus subtilis and E. coli, with methanol release measurable on the addition of attractant but not on its removal. Intriguingly, CheA(2), but not CheA(1), is able to phosphorylate CheB(1), suggesting that signaling through CheA(1) cannot initiate feedback receptor adaptation via CheB(1)-P.
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15. |
Jain R,
Shapleigh JP,
( 2001 ) Characterization of nirV and a gene encoding a novel pseudoazurin in Rhodobacter sphaeroides 2.4.3. PMID : 11535790 : DOI : 10.1099/00221287-147-9-2505 Abstract >>
Sequencing of the region flanking nirK, the gene encoding the copper-containing nitrite reductase in Rhodobacter sphaeroides 2.4.3, has identified two genes whose products could potentially be involved in nitrite reductase expression and activity. One of the genes has been designated nirV. Putative nirV orthologues are found in other denitrifiers, where they are also located downstream of the structural gene for nitrite reductase. The nirV in 2.4.3 is apparently cotranscribed with nirK. Inactivation of nirV had no effect on cell growth, or on nitrite reductase expression or activity. Downstream of nirV and divergently transcribed is a gene, designated ppaZ, encoding a protein with significant similarity to pseudoazurins from other denitrifiers. However, three of the four residues required for binding of the type I copper centre are not conserved in the deduced sequence of the protein in 2.4.3. ppaZ is expressed only when oxygen becomes limiting. ppaZ expression is dependent on both FnrL and NnrR, and a putative binding site for these proteins has been identified. Expression of ppaZ is also dependent on the two-component PrrB/PrrA system. Inactivation of ppaZ had no significant effect on cell growth or on nitrite reductase expression or activity. Expression of a maltose-binding protein-PpaZ fusion indicated that the protein could not bind copper. Examination of the genome of the related bacterium R. sphaeroides 2.4.1 revealed that it encodes ppaZ but not nirV and evidence is presented suggesting that a common ancestor of 2.4.3 and 2.4.1 had both nitrite and nitric oxide reductase activity but as the strains diverged 2.4.1 lost nirK and nirV, making it incapable of nitrite reduction.
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16. |
Bartnikas TB,
Wang Y,
Bobo T,
Veselov A,
Scholes CP,
Shapleigh JP,
( 2002 ) Characterization of a member of the NnrR regulon in Rhodobacter sphaeroides 2.4.3 encoding a haem-copper protein. PMID : 11882718 : DOI : 10.1099/00221287-148-3-825 Abstract >>
Upstream of the nor and nnrR cluster in Rhodobacter sphaeroides 2.4.3 is a previously uncharacterized gene that has been designated nnrS. nnrS is only expressed when 2.4.3 is grown under denitrifying conditions. Expression of nnrS is dependent on the transcriptional regulator NnrR, which also regulates expression of genes required for the reduction of nitrite to nitrous oxide, including nirK and nor. Deletion analysis indicated the sequence 5'-TTGCG(N4)CACAA-3', which is similar to sequences found in nirK and nor, is required for nnrS expression. Mutation of this sequence to the consensus Fnr-binding sequence by changing two bases in each half site caused nnrS expression to become nitrate independent. Inactivation of nnrS did not affect nitric oxide metabolism, nor did it affect expression of any of the genes involved in nitric oxide metabolism. However, taxis towards nitrate and nitrite was affected by nnrS inactivation. Purification of a histidine-tagged NnrS demonstrated that NnrS is a haem- and copper-containing membrane protein. Genes encoding putative orthologues of NnrS are sometimes but not always found in bacteria encoding nitrite and/or nitric oxide reductase.
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17. |
Klug RM,
Benning C,
( 2001 ) Two enzymes of diacylglyceryl-O-4'-(N,N,N,-trimethyl)homoserine biosynthesis are encoded by btaA and btaB in the purple bacterium Rhodobacter sphaeroides. PMID : 11331765 : DOI : 10.1073/pnas.101037998 PMC : PMC33312 Abstract >>
Betaine lipids are ether-linked, nonphosphorous glycerolipids that resemble the more commonly known phosphatidylcholine in overall structure. Betaine lipids are abundant in many eukaryotes such as nonseed plants, algae, fungi, and amoeba. Some of these organisms are entirely devoid of phosphatidylcholine and, instead, contain a betaine lipid such as diacylglyceryl-O-4'-(N,N,N,-trimethyl)homoserine. Recently, this lipid also was discovered in the photosynthetic purple bacterium Rhodobacter sphaeroides where it seems to replace phosphatidylcholine under phosphate-limiting growth conditions. This discovery provided the opportunity to study the biosynthesis of betaine lipids in a bacterial model system. Mutants of R. sphaeroides deficient in the biosynthesis of the betaine lipid were isolated, and two genes essential for this process, btaA and btaB, were identified. It is proposed that btaA encodes an S-adenosylmethionine:diacylglycerol 3-amino-3-carboxypropyl transferase and btaB an S-adenosylmethionine-dependent N-methyltransferase. Both enzymatic activities can account for all reactions of betaine lipid head group biosynthesis. Because the equivalent reactions have been proposed for different eukaryotes, it seems likely that orthologs of btaA/btaB may be present in other betaine lipid-containing organisms.
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18. |
Ballado T,
Camarena L,
González-Pedrajo B,
Silva-Herzog E,
Dreyfus G,
( 2001 ) The hook gene (flgE) is expressed from the flgBCDEF operon in Rhodobacter sphaeroides: study of an flgE mutant. PMID : 11160099 : DOI : 10.1128/JB.183.5.1680-1687.2001 PMC : PMC95053 Abstract >>
In this work we identified the flgE gene encoding the flagellar hook protein from Rhodobacter sphaeroides. Our results show that this gene is part of a flagellar cluster that includes the genes flgB, flgC, flgD, flgE, and flgF. Two different types of mutants in the flgE gene were isolated, and both showed a Fla(-) phenotype, indicating the functionality of this sequence. Complementation studies of these mutant strains suggest that flgE is included in a single transcriptional unit that starts in flgB and ends in flgF. In agreement with this possibility, a specific transcript of approximately 3.5 kb was identified by Northern blot. This mRNA is large enough to represent the complete flgBCDEF operon. FlgE showed a relatively high proline content; in particular, a region of 12 amino acids near the N terminus, in which four prolines were identified. Cells expressing a mutant FlgE protein lacking this region showed abnormal swimming behavior, and their hooks were curved. These results suggest that this region is involved in the characteristic quaternary structure of the hook of R. sphaeroides and also imply that a straight hook, or perhaps the rigidity associated with this feature, is important for an efficient swimming behavior in this bacterium.
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19. |
Fales L,
Kryszak L,
Zeilstra-Ryalls J,
( 2001 ) Control of hemA expression in Rhodobacter sphaeroides 2.4.1: effect of a transposon insertion in the hbdA gene. PMID : 11160087 : DOI : 10.1128/JB.183.5.1568-1576.2001 PMC : PMC95041 Abstract >>
The common precursor to all tetrapyrroles is 5-aminolevulinic acid (ALA), and in Rhodobacter sphaeroides its formation occurs via the Shemin pathway. ALA synthase activity is encoded by two differentially regulated genes in R. sphaeroides 2.4.1: hemA and hemT. In our investigations of hemA regulation, we applied transposon mutagenesis under aerobic conditions, followed by a selection that identified transposon insertion mutants in which hemA expression is elevated. One of these mutants has been characterized previously (J. Zeilstra-Ryalls and S. Kaplan, J. Bacteriol. 178:985-993, 1996), and here we describe our analysis of a second mutant strain. The transposon inserted into the coding sequences of hbdA, coding for S-(+)-beta-hydroxybutyryl-coenzyme A dehydrogenase and catalyzing an NAD-dependent reaction. We provide evidence that the hbdA gene product participates in polyhydroxybutyrate (PHB) metabolism and, based on our findings, we discuss possibilities as to how defective PHB metabolism might alter the level of hemA expression.
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20. |
Parker SD,
Duggan PS,
( 2000 ) Characterisation of a Rrhodobacter sphaeroides gene that encodes a product resembling Eescherichia coli cytochrome b(561) and R. sphaeroides cytochrome b(562). PMID : 10930745 : DOI : 10.1111/j.1574-6968.2000.tb09237.x Abstract >>
Analysis of the photoactive yellow protein (pyp) gene region of Rhodobacter sphaeroides has revealed the presence of an additional open reading frame, orfD, that had not previously been identified. Here we report the location of this new gene and the predicted amino acid sequence of the encoded protein. The translation product resembles a group of small cytochrome b-like proteins, including Escherichia coli cytochrome b(561), R. sphaeroides cytochrome b(562), and two new cytochrome b(561)-like proteins identified using the E. coli genome sequence, for which functions have not yet been established. To determine OrfD function in R. sphaeroides, an orfD mutant was constructed. The OrfD mutant exhibited growth rates and yields very similar to those of the wild-type strain when grown under a variety of growth conditions. Respiration rates, reduced-minus-oxidised spectra and levels of photosynthetic complexes were also very similar in the two strains. Although the role of OrfD was therefore not determined here, we demonstrate that the orfD gene is expressed in R. sphaeroides under aerobic, semi-aerobic and photosynthetic growth conditions.
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21. |
Roh JH,
( 2000 ) Genetic and phenotypic analyses of the rdx locus of Rhodobacter sphaeroides 2.4.1. PMID : 10852880 : DOI : 10.1128/jb.182.12.3475-3481.2000 PMC : PMC101935 Abstract >>
Previously, we reported that rdxB, encoding a likely membrane-bound two [4Fe-4S]-containing center, is involved in the aerobic regulation of photosystem gene expression in Rhodobacter sphaeroides 2.4.1. To further investigate the role of rdxB as well as other genes of the rdxBHIS operon on photosystem gene expression, we constructed a series of nonpolar, in-frame deletion mutations in each of the rdx genes. Using both puc and puf operon lacZ fusions to monitor photosystem gene expression, under aerobic conditions, in each of the mutant strains revealed significant increased photosynthesis gene expression. In the case of mutations in either rdxH, rdxI, or rdxS, the aerobic induction of photosystem gene expression is believed to be indirect by virtue of a posttranscriptional effect on cbb(3) cytochrome oxidase structure and integrity. For RdxB, we suggest that this redox protein has a more direct effect on photosystem gene expression by virtue of its interaction with the cbb(3) oxidase. An associated phenotype, involving the enhanced conversion of the carotenoid spheroidene to spheroidenone, is also observed in the RdxB, -H, -I, and -S mutant strains. This phenotype is also suggested to be the result of the role of the rdxBHIS locus in cbb(3) oxidase activity and/or structure. RdxI is suggested to be a new class of metal transporter of the CPx-type ATPases.
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22. |
Mouncey NJ,
Gak E,
Choudhary M,
Oh J,
Kaplan S,
( 2000 ) Respiratory pathways of Rhodobacter sphaeroides 2.4.1(T): identification and characterization of genes encoding quinol oxidases. PMID : 11064196 : DOI : 10.1111/j.1574-6968.2000.tb09383.x Abstract >>
Rhodobacter sphaeroides 2.4.1(T) respires aerobically via a branched respiratory chain consisting of both cytochrome c oxidases and quinol oxidases. Here, genes from chromosome II encoding two distinct quinol oxidases have been characterized. The qoxBA genes encode a putative heme-copper quinol oxidase, whereas the qxtAB genes encode a quinol oxidase homologous to the cyanide-insensitive oxidase of Pseudomonas aeruginosa. No phenotype was observed for mutations in either oxidase in the wild-type background. A strain containing a qxtA mutation in a cytochrome bc(1) complex mutant background was unable to grow aerobically. No role was found for the Qox oxidase, nor was a qoxB::lacZ transcriptional fusion expressed under a variety of conditions. These are the first molecular studies to characterize the quinol oxidases of R. sphaeroides 2.4.1(T).
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23. |
Scavetta RD,
Thomas CB,
Walsh MA,
Szegedi S,
Joachimiak A,
Gumport RI,
Churchill ME,
( 2000 ) Structure of RsrI methyltransferase, a member of the N6-adenine beta class of DNA methyltransferases. PMID : 11024175 : DOI : 10.1093/nar/28.20.3950 PMC : PMC110776 Abstract >>
DNA methylation is important in cellular, developmental and disease processes, as well as in bacterial restriction-modification systems. Methylation of DNA at the amino groups of cytosine and adenine is a common mode of protection against restriction endonucleases afforded by the bacterial methyltransferases. The first structure of an N:6-adenine methyltransferase belonging to the beta class of bacterial methyltransferases is described here. The structure of M. RSR:I from Rhodobacter sphaeroides, which methylates the second adenine of the GAATTC sequence, was determined to 1.75 A resolution using X-ray crystallography. Like other methyltransferases, the enzyme contains the methylase fold and has well-defined substrate binding pockets. The catalytic core most closely resembles the PVU:II methyltransferase, a cytosine amino methyltransferase of the same beta group. The larger nucleotide binding pocket observed in M. RSR:I is expected because it methylates adenine. However, the most striking difference between the RSR:I methyltransferase and the other bacterial enzymes is the structure of the putative DNA target recognition domain, which is formed in part by two helices on an extended arm of the protein on the face of the enzyme opposite the active site. This observation suggests that a dramatic conformational change or oligomerization may take place during DNA binding and methylation.
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24. |
Poggio S,
Aguilar C,
Osorio A,
González-Pedrajo B,
Dreyfus G,
Camarena L,
( 2000 ) sigma(54) Promoters control expression of genes encoding the hook and basal body complex in Rhodobacter sphaeroides. PMID : 11004178 : DOI : 10.1128/jb.182.20.5787-5792.2000 PMC : PMC94701 Abstract >>
Gene expression of the flagellar system is tightly controlled by external stimuli or intracellular signals. A general picture of this regulation has been obtained from studies of Salmonella enterica serovar Typhimurium. However, these regulatory mechanisms do not apply to all bacterial groups. In this study, we have investigated regulation of the flagellar genetic system in Rhodobacter sphaeroides. Deletion analysis, site-directed mutagenesis, and 5'-end mapping were conducted in order to identify the fliO promoter. Our results indicate that this promoter is recognized by the factor sigma(54). Additionally, 5'-end mapping of the flgB and fliK transcripts suggests that these mRNAs are also transcribed from sigma(54) promoters. Finally, we showed evidence that suggests that fliC transcription is not entirely dependent on the presence of a complete basal body-hook structure. Our results are discussed in the context of a possible regulatory hierarchy controlling flagellar gene expression in R. sphaeroides.
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25. |
Shah DS,
Porter SL,
Martin AC,
Hamblin PA,
Armitage JP,
( 2000 ) Fine tuning bacterial chemotaxis: analysis of Rhodobacter sphaeroides behaviour under aerobic and anaerobic conditions by mutation of the major chemotaxis operons and cheY genes. PMID : 10970853 : DOI : 10.1093/emboj/19.17.4601 PMC : PMC302075 Abstract >>
Rhodobacter sphaeroides chemotaxis is significantly more complex than that of enteric bacteria. Rhodobacter sphaeroides has multiple copies of chemotaxis genes (two cheA, one cheB, two cheR, three cheW, five cheY but no cheZ), controlling a single 'stop-start' flagellum. The growth environment controls the level of expression of different groups of genes. Tethered cell analysis of mutants suggests that CheY(4) and CheY(5) are the motor-binding response regulators. The histidine protein kinase CheA(2) mediates an attractant ('normal') response via CheY(4), while CheA(1) and CheY(5) appear to mediate a repellent ('inverted') response. CheY(3) facilitates signal termination, possibly acting as a phosphate sink, although CheY(1) and CheY(2) can substitute. The normal and inverted responses may be initiated by separate sets of chemoreceptors with their relative strength dependent on growth conditions. Rhodobacter sphaeroides may use antagonistic responses through two chemosensory pathways, expressed at different levels in different environments, to maintain their position in a currently optimum environment. Complex chemotaxis systems are increasingly being identified and the strategy adopted by R.sphaeroides may be common in the bacterial kingdom.
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26. |
Shah DS,
Perehinec T,
Stevens SM,
Aizawa SI,
Sockett RE,
( 2000 ) The flagellar filament of Rhodobacter sphaeroides: pH-induced polymorphic transitions and analysis of the fliC gene. PMID : 10960108 : DOI : 10.1128/jb.182.18.5218-5224.2000 PMC : PMC94672 Abstract >>
Flagellar motility in Rhodobacter sphaeroides is notably different from that in other bacteria. R. sphaeroides moves in a series of runs and stops produced by the intermittent rotation of the flagellar motor. R. sphaeroides has a single, plain filament whose conformation changes according to flagellar motor activity. Conformations adopted during swimming include coiled, helical, and apparently straight forms. This range of morphological transitions is larger than that in other bacteria, where filaments alternate between left- and right-handed helical forms. The polymorphic ability of isolated R. sphaeroides filaments was tested in vitro by varying pH and ionic strength. The isolated filaments could form open-coiled, straight, normal, or curly conformations. The range of transitions made by the R. sphaeroides filament differs from that reported for Salmonella enterica serovar Typhimurium. The sequence of the R. sphaeroides fliC gene, which encodes the flagellin protein, was determined. The gene appears to be controlled by a sigma(28)-dependent promoter. It encodes a predicted peptide of 493 amino acids. Serovar Typhimurium mutants with altered polymorphic ability usually have amino acid changes at the terminal portions of flagellin or a deletion in the central region. There are no obvious major differences in the central regions to explain the difference in polymorphic ability. In serovar Typhimurium filaments, the termini of flagellin monomers have a coiled-coil conformation. The termini of R. sphaeroides flagellin are predicted to have a lower probability of coiled coils than are those of serovar Typhimurium flagellin. This may be one reason for the differences in polymorphic ability between the two filaments.
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27. |
Horne IM,
Lee HJ,
Pemberton JM,
Gomelsky M,
( 2000 ) Domain structure, oligomeric state, and mutational analysis of PpsR, the Rhodobacter sphaeroides repressor of photosystem gene expression. PMID : 10735869 : DOI : 10.1128/jb.182.8.2253-2261.2000 PMC : PMC111275 Abstract >>
The transcription factor PpsR from the facultative photoheterotroph Rhodobacter sphaeroides is involved in repression of photosystem gene expression under aerobic growth conditions. We have isolated a number of spontaneous mutations as well as constructed directed mutations and deletions in ppsR. Repressor activities and the oligomeric state of the wild-type and mutant proteins were assayed. Our results suggest that the wild-type PpsR exists in cell extracts as a tetramer. Analysis of the PpsR mutants confirmed that the carboxy-terminal region of PpsR (residues 400 to 464) is involved in DNA binding. The central region of the protein (residues 150 to 400) was found to contain two PAS domains (residues 161 to 259 and 279 to 367). PAS domains are ubiquitous protein modules involved in sensory transduction as well as in protein-protein interactions. All spontaneously isolated mutations, which significantly impaired repressor activity and which mapped outside the DNA binding region, were positioned in the PAS domains. None of these, however, affected the overall oligomeric state. This implies that the conformation of the PAS domains within the tetramer is critical for repressor activity. Upstream of the first PAS domain resides a putative glutamine-rich hinge (residues 127 to 136) that connects the first PAS domain to the amino-terminal region (residues 1 to 135). The role of the amino terminus of PpsR is not obvious; however, extended deletions within this region abolish repressor activity, thus suggesting that the amino terminus is essential for structural integrity of the protein. We present a model of the domain architecture of the PpsR protein according to which PpsR is comprised of three regions: the carboxy terminus responsible for DNA binding, the central region primarily involved in protein oligomerization and possibly signal sensing, and the amino terminus of unknown function. This model may prove useful for determining the mode of PpsR action.
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28. |
Parkes-Loach PS,
Loach PA,
Westerhuis WH,
Conroy MJ,
( 2000 ) The solution structure of Rhodobacter sphaeroides LH1beta reveals two helical domains separated by a more flexible region: structural consequences for the LH1 complex. PMID : 10756106 : DOI : 10.1006/jmbi.2000.3649 Abstract >>
Here, the solution structure of the Rhodobacter sphaeroides core light-harvesting complex beta polypeptide solubilised in chloroform:methanol is presented. The structure, determined by homonuclear NMR spectroscopy and distance geometry, comprises two alpha helical regions (residue -34 to -15 and -11 to +6, using the numbering system in which the conserved histidine residue is numbered zero) joined by a more flexible four amino acid residue linker. The C-terminal helix forms the membrane spanning region in the intact LH1 complex, whilst the N-terminal helix must lie in the lipid head groups or in the cytoplasm, and form the basis of interaction with the alpha polypeptide. The structure of a mutant beta polypeptide W(+9)F was also determined. This mutant, which is deficient in a hydrogen bond donor to the bacteriochlorophyll, showed an identical structure to the wild-type, implying that observed differences in interaction with other LH1 polypeptides must arise from cofactor binding. Using these structures we propose a modification to existing models of the intact LH1 complex by replacing the continuous helix of the beta polypeptide with two helices, one of which lies at an acute angle to the membrane plane. We suggest that a key difference between LH1 and LH2 is that the beta subunit is more bent in LH1. This modification puts the N terminus of LH1beta close to the reaction centre H subunit, and provides a rationale for the different ring sizes of LH1 and LH2 complexes.
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29. |
Yeliseev AA,
( 2000 ) TspO of rhodobacter sphaeroides. A structural and functional model for the mammalian peripheral benzodiazepine receptor. PMID : 10681549 : DOI : 10.1074/jbc.275.8.5657 Abstract >>
The function and specific structural aspects of the tryptophan-rich sensory protein (TspO) of Rhodobacter sphaeroides 2.4.1 were studied using site-directed mutagenesis involving some 17 different amino acids. The choice of these amino acids changes was dictated from an analysis of the TspO family of proteins derived from the data bases. These studies demonstrated the importance of several highly conserved tryptophan residues in the sensory transduction pathway involving TspO through the proposed binding of an intermediate(s) in the tetrapyrrole biosynthesis pathway. These studies also revealed that the substitution of one or several of the amino acid residues dramatically affected, either directly or indirectly, the levels of TspO in the membranes of R. sphaeroides. Mounting evidence is presented suggesting that TspO normally forms a dimer within the bacterial outer membrane, and the dimer form of TspO may be the active form for TspO function. Because our earlier studies provided us with a functional framework within which to view these amino acid substitutions, we are able to suggest a preliminary model for TspO structure-function. Not only do these studies tell us more about TspO, but they also shed light on the TspO homologue, the drug-binding component of the mitochondrial peripheral benzodiazepine receptor. Mounting evidence draws numerous parallelism between these proteins and supports the significance of using TspO as a model for the structure and function of the mitochondrial protein.
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30. |
Choudhary M,
( 2000 ) DNA sequence analysis of the photosynthesis region of Rhodobacter sphaeroides 2.4.1. PMID : 10648776 : DOI : 10.1093/nar/28.4.862 PMC : PMC102589 Abstract >>
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.
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31. |
Di Valentin M,
Hiser L,
( 2000 ) Cox11p is required for stable formation of the Cu(B) and magnesium centers of cytochrome c oxidase. PMID : 10617659 : DOI : 10.1074/jbc.275.1.619 Abstract >>
Assembly of the core subunits of the aa(3)-type cytochrome c oxidase in mitochondria and aerobic bacteria such as Rhodobacter sphaeroides requires the association of three subunits and the formation of five to seven metal centers. Several assembly proteins are required for the late stages of oxidase assembly in eukaryotes; some of these are also present in Rb. sphaeroides. To investigate the role of one of these proteins, Cox11p, the mitochondrial-like oxidase of Rb. sphaeroides was overexpressed and purified from cells that lacked cox11, the gene for Cox11p. The oxidase that assembled in the absence of Cox11p lacked Cu(B) at the active site and contained greatly reduced amounts of metal at the magnesium/manganese-binding site between subunits I and II. This inactive oxidase, however, did contain hemes a and a(3), Cu(A), and all three subunits. These results indicate that Cox11p is required at a late, perhaps final, step in the assembly of cytochrome oxidase, most likely the insertion of Cu(B). Oxidase which assembled in a strain with a low copy number of cox11 appeared nearly wild type, suggesting that Cox11p is required in substoichiometric amounts for its role in oxidase assembly.
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32. |
Kaplan S,
( 1999 ) A novel mechanism for the regulation of photosynthesis gene expression by the TspO outer membrane protein of Rhodobacter sphaeroides 2.4.1. PMID : 10409680 : DOI : 10.1074/jbc.274.30.21234 Abstract >>
A bacterial homolog of the mammalian mitochondrial benzodiazepine receptor, the tryptophan-rich sensory protein (TspO) has been previously demonstrated to negatively affect the transcriptional expression of several photosynthesis genes of Rhodobacter sphaeroides. To identify components of the signal transduction pathway from the outer membrane-localized TspO to the DNA-active transcription factor(s), we examined the involvement of TspO in the regulation of tetrapyrrole metabolism in R. sphaeroides. By analyzing the tetrapyrrole pigments accumulated by resting cell suspensions of R. sphaeroides, we demonstrated that TspO negatively regulates the activity of coproporphyrinogen III oxidase in this bacterium. hemN, encoding one of the isoenzymes of coproporphyrinogen III oxidase of R. sphaeroides, provided in trans to the wild type strain, produced a TSPO1 mutant phenotype by abolishing the negative effect of TspO on the transcription of the photosynthesis genes, crtI and puc. It is proposed that TspO, by regulating the exit of certain tetrapyrrole intermediates of the heme/bacteriochlorophyll biosynthetic pathways in R. sphaeroides in response to the availability of molecular oxygen, causes the accumulation of a biosynthetic intermediate that serves as a corepressor for both specific pigment gene transcription and the puc operon. The relationship between the bacterial TspO and the mitochondrial peripheral benzodiazepine receptor is discussed.
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33. |
Simmons AE,
Mackenzie C,
( 1999 ) Multiple chromosomes in bacteria. The yin and yang of trp gene localization in Rhodobacter sphaeroides 2.4.1. PMID : 10511537 : PMC : PMC1460784 Abstract >>
The existence of multiple chromosomes in bacteria has been known for some time. Yet the extent of functional solidarity between different chromosomes remains unknown. To examine this question, we have surveyed the well-described genes of the tryptophan biosynthetic pathway in the multichromosomal photosynthetic eubacterium Rhodobacter sphaeroides 2.4.1. The genome of this organism was mutagenized using Tn5, and strains that were auxotrophic for tryptophan (Trp(-)) were isolated. Pulsed-field gel mapping indicated that Tn5 insertions in both the large (3 Mb CI) and the small (0.9 Mb CII) chromosomes created a Trp(-) phenotype. Sequencing the DNA flanking the sites of the Tn5 insertions indicated that the genes trpE-yibQ-trpGDC were at a locus on CI, while genes trpF-aroR-trpB were at locus on CII. Unexpectedly, trpA was not found downstream of trpB. Instead, it was placed on the CI physical map at a locus 1.23 Mb away from trpE-yibQ-trpGDC. To relate the context of the R. sphaeroides trp genes to those of other bacteria, the DNA regions surrounding the trp genes on both chromosomes were sequenced. Of particular significance was the finding that rpsA1, which encodes ribosomal protein S1, and cmkA, which encodes cytidylate monophosphate kinase, were on CII. These genes are considered essential for translation and chromosome replication, respectively. Southern blotting suggested that the trp genes and rpsA1 exist in single copy within the genome. To date, this topological organization of the trp "operon" is unique within a bacterial genome. When taken with the finding that CII encodes essential housekeeping functions, the overall impression is one of close regulatory and functional integration between these chromosomes.
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34. |
Schwintner C,
Cahors S,
Sabaty M,
( 1999 ) Nitrite and nitrous oxide reductase regulation by nitrogen oxides in Rhodobacter sphaeroides f. sp. denitrificans IL106. PMID : 10498715 : PMC : PMC103630 Abstract >>
We have cloned the nap locus encoding the periplasmic nitrate reductase in Rhodobacter sphaeroides f. sp. denitrificans IL106. A mutant with this enzyme deleted is unable to grow under denitrifying conditions. Biochemical analysis of this mutant shows that in contrast to the wild-type strain, the level of synthesis of the nitrite and N(2)O reductases is not increased by the addition of nitrate. Growth under denitrifying conditions and induction of N oxide reductase synthesis are both restored by the presence of a plasmid containing the genes encoding the nitrate reductase. This demonstrates that R. sphaeroides f. sp. denitrificans IL106 does not possess an efficient membrane-bound nitrate reductase and that nitrate is not the direct inducer for the nitrite and N(2)O reductases in this species. In contrast, we show that nitrite induces the synthesis of the nitrate reductase.
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35. |
Liu HP,
Yamamoto I,
Satoh T,
( 1999 ) Involvement in denitrification of the napKEFDABC genes encoding the periplasmic nitrate reductase system in the denitrifying phototrophic bacterium Rhodobacter sphaeroides f. sp. denitrificans. PMID : 10227138 : DOI : 10.1271/bbb.63.530 Abstract >>
Seven genes, napKEFDABC, encoding the periplasmic nitrate reductase system were cloned from the denitrifying phototrophic bacterium Rhodobacter sphaeroides f. sp. denitrificans IL106. Two transmembrane proteins, NapK and NapE, an iron-sulfur protein NapF, a soluble protein NapD, a catalytic subunit of nitrate reductase precursor NapA, a soluble c-type diheme cytochrome precursor NapB, and a membrane-anchored c-type tetraheme cytochrome NapC were deduced as the gene products. Every mutant in which each nap gene was disrupted by omega-cassette insertion lost nitrate reductase activity as well as the ability of cells to grow with nitrate under anaerobic-dark conditions. A transconjugant of the napD-disrupted mutant with a plasmid bearing the napKEFDABC genes recovered both nitrate reductase activity and nitrate-dependent anaerobic-dark growth of cells. Denitrification activity, which was not observed in the napD mutant, was also restored by the conjugation. These results indicate that the periplasmic nitrate reductase encoded by the napKEFDABC genes is the enzyme responsible for denitrification in this phototroph, although the presence of a membrane-bound nitrate reductase has been reported in the same strain.
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36. |
Zannoni D,
Daldal F,
( 1999 ) The membrane-attached electron carrier cytochrome cy from Rhodobacter sphaeroides is functional in respiratory but not in photosynthetic electron transfer. PMID : 10200265 : DOI : 10.1073/pnas.96.8.4348 PMC : PMC16335 Abstract >>
Rhodobacter species are useful model organisms for studying the structure and function of c type cytochromes (Cyt c), which are ubiquitous electron carriers with essential functions in cellular energy and signal transduction. Among these species, Rhodobacter capsulatus has a periplasmic Cyt c2Rc and a membrane-bound bipartite Cyt cyRc. These electron carriers participate in both respiratory and photosynthetic electron-transfer chains. On the other hand, until recently, Rhodobacter sphaeroides was thought to have only one of these two cytochromes, the soluble Cyt c2Rs. Recent work indicated that this species has a gene, cycYRs, that is highly homologous to cycYRc, and in the work presented here, functional properties of its gene product (Cyt cyRs) are defined. It was found that Cyt cyRs is unable to participate in photosynthetic electron transfer, although it is active in respiratory electron transfer, unlike its R. capsulatus counterpart, Cyt cyRc. Chimeric constructs have shown that the photosynthetic incapability of Cyt cyRs is caused, at least in part, by its redox active subdomain, which carries the covalently bound heme. It, therefore, seems that this domain interacts differently with distinct redox partners, like the photochemical reaction center and the Cyt c oxidase, and allows the bacteria to funnel electrons efficiently to various destinations under different growth conditions. These findings raise an intriguing evolutionary issue in regard to cellular apoptosis: why do the mitochondria of higher organisms, unlike their bacterial ancestors, use only one soluble electron carrier in their respiratory electron-transport chains?
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37. |
Shimada H,
Ishida K,
Machiya Y,
Takamiya K,
( 2007 ) Isolation of SIP, a protein that interacts with SPB, a possible transcriptional regulatory factor in Rhodobacter sphaeroides. PMID : 17720716 : DOI : 10.1093/pcp/pcm114 Abstract >>
SPB is a transcriptional factor in Rhodobacter sphaeroides that represses expression of the puf operon under aerobic or semi-aerobic light conditions. Here, we identified a 17,500 Da protein designated SIP (SPB interaction protein) that interacts with SPB, as determined by binding to an SPB-His(x6) fusion protein-Ni column. The SPB-SIP interaction in vivo was confirmed by an immunoprecipitation assay. The level of transcripts and protein of SIP did not differ for all growth conditions tested, indicating that regulation of the SIP-SPB interaction, if any, is not through modulation of sip or spb expression but rather by modification of the proteins.
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38. |
Hunter CN,
McGlynn P,
Ashby MK,
Burgess JG,
Olsen JD,
( 1991 ) DNA sequencing and complementation/deletion analysis of the bchA-puf operon region of Rhodobacter sphaeroides: in vivo mapping of the oxygen-regulated puf promoter. PMID : 1779756 : DOI : 10.1111/j.1365-2958.1991.tb01974.x Abstract >>
Within the photosynthetic gene cluster of Rhodobacter sphaeroides the genes encoding light-harvesting LHI and reaction-centre complexes are transcriptionally linked in the order pufBALMX. The region stretching 1.6 kb upstream of pufB has been examined by DNA sequencing and by complementation/deletion analysis. These studies demonstrate that three open reading frames are located upstream of pufB. One open reading frame, designated bchA, terminates just inside pufQ, which is located proximal to pufB. BchA contains a 37 bp region that functions as the oxygen-regulated promoter for pufQ, and probably for the puf operon as a whole. We also demonstrate that the protein encoded by pufQ appears to play a role in bacteriochlorophyll biosynthesis.
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39. |
Wang ZY,
Suzuki H,
Kobayashi M,
Nozawa T,
( 2007 ) Solution structure of the Rhodobacter sphaeroides PufX membrane protein: implications for the quinone exchange and protein-protein interactions. PMID : 17335288 : DOI : 10.1021/bi0618060 Abstract >>
PufX membrane protein is found in Rhodobacter species of purple photosynthetic bacteria and has been known to play an essential role in ubiquinone/ubiquinol exchange between the reaction center and cytochrome bc1 complex and also contribute to the dimerization of the reaction center-light-harvesting core complex. We have determined the solution structure of the Rhodobacter sphaeroides PufX using multidimensional NMR spectroscopy. The PufX, functionally expressed in Escherichia coli, forms a stable alpha helix consisting of 21 residues over the central transmembrane domain. The overall structure of the PufX is very similar to those of the LH1 alpha- and beta-polypeptides from Rhodospirillum rubrum and LH2 polypeptides. A short segment (Lys28-Gly35) rich in Gly and Ala residues revealed a relatively fast exchange between the backbone amide protons and deuteriums in the hydroxyl groups of the solvent, indicating that the backbone of this segment is more easily accessible to the surrounding solvent molecules compared to those of its neighboring portions. The Gly- and Ala-rich segment is located in the middle of the central helix and forms an extensive groove-like conformation on the surface with the neighboring residues, where the residues with large side chains are aligned on one side of the helix, and small residues are aligned on the other face. Such a structural motif may serve as a functional site responsible for ubiquinol transport from the core complex to the membrane phase and for sequence-specific helix-helix interactions with the neighboring polypeptides.
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40. |
Tunnicliffe RB,
Ratcliffe EC,
Hunter CN,
Williamson MP,
( 2006 ) The solution structure of the PufX polypeptide from Rhodobacter sphaeroides. PMID : 17161397 : DOI : 10.1016/j.febslet.2006.11.065 Abstract >>
PufX organizes the photosynthetic reaction centre-light harvesting complex 1 (RC-LH1) core complex of Rhodobacter sphaeroides and facilitates quinol/quinone exchange between the RC and cytochrome bc(1) complexes. The structure of PufX in organic solvent reveals two hydrophobic helices flanked by unstructured termini and connected by a helical bend. The proposed location of basic residues and tryptophans at the membrane interface orients the C-terminal helix along the membrane normal, with the GXXXG motifs in positions unsuitable as direct drivers of dimerisation of the RC-LH1 complex. The N-terminal helix is predicted to extend approximately 40 Anggstrom along the membrane interface.
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41. |
Terlesky KC,
Tabita FR,
( 1991 ) Purification and characterization of the chaperonin 10 and chaperonin 60 proteins from Rhodobacter sphaeroides. PMID : 1678280 : DOI : 10.1021/bi00247a013 Abstract >>
Two heat-shock proteins that show high identity with the Escherichia coli chaperonin 60 (groEL) and chaperonin 10 (groES) chaperonin proteins were purified and characterized from photolithoautotrophically grown Rhodobacter sphaeroides. The proteins were purified by using sucrose density gradient centrifugation and Mono-Q anion-exchange chromatography. In the presence of 1 mM ATP, the chaperonin 10 and chaperonin 60 proteins bound to each other and comigrated as a large complex during sucrose density gradient centrifugation. The native molecular weights of each protein as determined by gel filtration chromatography were 889,200 for chaperonin 60 and 60,000 for chaperonin 10. Chaperonin 60 is comprised of monomers with a molecular weight of 61,000 and chaperonin 10 is comprised of monomers with a molecular weight of 12,700 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chaperonin 60 was 9.3% of the total soluble cell protein during photolithoautotrophic growth which increased to 28.5% following heat-shock treatment. When cells were grown photoheterotrophically or chemoheterotrophically, chaperonin 60 was reduced to 6.7% and 3.5%, respectively, of the total soluble protein. The N-terminal amino acid sequence of each protein was determined; chaperonin 60 of R. sphaeroides showed 72% identity to E. coli chaperonin 60 protein, and R. sphaeroides chaperonin 10 showed 45% identity with E. coli chaperonin 10. R. sphaeroides chaperonin 60 catalyzed ATP hydrolysis with a specific activity of 134 nmol min-1 mg-1 (kcat = 0.13 s-1) and was inhibited by R. sphaeroides chaperonin 10, but not E. coli chaperonin 10. The E. coli chaperonin 60 ATPase activity was inhibited by chaperonin 10 from both R. sphaeroides and E. coli.
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42. |
Yoshida Y,
Takai M,
Satoh T,
Takami S,
( 1991 ) Molybdenum requirement for translocation of dimethyl sulfoxide reductase to the periplasmic space in a photodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans. PMID : 1710616 : DOI : 10.1128/jb.173.11.3277-3281.1991 PMC : PMC207938 Abstract >>
Translocation of dimethyl sulfoxide (DMSO) reductase to the periplasmic space was studied in vivo with a photodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans, using immunoblotting analysis and radioactive labeling. A polypeptide with an apparent molecular mass about 2,000 Da higher than that of DMSO reductase accumulated during induction of the reductase with DMSO. An uncoupler, carbonyl cyanide-m-chlorophenylhydrazone, inhibited the processing of the polypeptide after cells had been radioactively pulse-labeled with [35S]methionine. These results indicated that the higher-molecular-mass polypeptide was the precursor form of DMSO reductase. The precursor form accumulated in either the cytoplasm or the membrane, whereas the mature form accumulated in the periplasmic space. The membrane-bound precursor was sensitive to proteinase K treatment from both the cytoplasmic and periplasmic sides of the membrane, indicating that the polypeptide binds to the membrane, exposing it to both the outer and inner surfaces of the cytoplasmic membrane. Processing of the precursor was hampered by removal of molybdate from the medium and was restored by its readdition. It was also inhibited by the addition of tungstate in the medium.
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43. |
Wadsten P,
Wöhri AB,
Snijder A,
Katona G,
Gardiner AT,
Cogdell RJ,
Neutze R,
Engström S,
( 2006 ) Lipidic sponge phase crystallization of membrane proteins. PMID : 17005199 : DOI : 10.1016/j.jmb.2006.06.043 DOI : 10.1016/j.jmb.2006.06.043 Abstract >>
Bicontinuous lipidic cubic phases can be used as a host for growing crystals of membrane proteins. Since the cubic phase is stiff, handling is difficult and time-consuming. Moreover, the conventional cubic phase may interfere with the hydrophilic domains of membrane proteins due to the limited size of the aqueous pores. Here, we introduce a new crystallization method that makes use of a liquid analogue of the cubic phase, the sponge phase. This phase facilitates a considerable increase in the allowed size of aqueous domains of membrane proteins, and is easily generalised to a conventional vapour diffusion crystallisation experiment, including the use of nanoliter drop crystallization robots. The appearance of the sponge phase was confirmed by visual inspection, small-angle X-ray scattering and NMR spectroscopy. Crystals of the reaction centre from Rhodobacter sphaeroides were obtained by a conventional hanging-drop experiment, were harvested directly without the addition of lipase or cryoprotectant, and the structure was refined to 2.2 Angstroms resolution. In contrast to our earlier lipidic cubic phase reaction centre structure, the mobile ubiquinone could be built and refined. The practical advantages of the sponge phase make it a potent tool for crystallization of membrane proteins.
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44. |
Zahiri HS,
Noghabi KA,
Shin YC,
( 2006 ) Biochemical characterization of the decaprenyl diphosphate synthase of Rhodobacter sphaeroides for coenzyme Q10 production. PMID : 16896603 : DOI : 10.1007/s00253-006-0524-1 Abstract >>
Coenzyme Q(10) (CoQ(10)), like other CoQs of various organisms, plays indispensable roles not only in energy generation but also in several other processes required for cells' survival. In this study, a gene encoding for a decaprenyl diphosphate synthase (Rsdds) was cloned from Rhodobacter sphaeroides in Escherichia coli. The in vivo catalytic activity and product specificity of Rsdds were compared with those of a counterpart enzyme from Agrobacterium tumefaciens (Atdds) in E. coli as a heterologous host. In contrast with Atdds, Rsdds showed lower catalytic activity but higher product specificity for CoQ(10) production, as indicated by the amount of CoQ(9) formation. The higher product specificity of Rsdds was also confirmed by utilizing both Rsdds and Atdds for in vitro synthesis of polyprenyl diphosphates. Thin layer chromatography indicated that the Rsdds enzyme resulted in relatively much less solanesyl diphosphate formation. The purified Rsdds catalyzed the addition of isopentenyl diphosphate to dimethyl allyl diphosphate, geranyl diphosphate, omega,E,E-farnesyl diphosphate (FPP), and omega,E,E,E-geranylgeranyl diphosphate as priming substrates. The kinetic parameters of V (max) (pmol/min), K (M) (microM), k (cat) (1/min), and k (cat) /K (M) of the enzyme using FPP as the most appropriate substrate were determined to be 264.6, 13.1, 8.8, and 0.67, respectively.
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45. |
Williams JC,
Steiner LA,
Ogden RC,
Simon MI,
Feher G,
( 1983 ) Primary structure of the M subunit of the reaction center from Rhodopseudomonas sphaeroides. PMID : 16593385 : DOI : 10.1073/pnas.80.21.6505 PMC : PMC390381 Abstract >>
The reaction center is a membrane-bound bacteriochlorophyll-protein complex that mediates the primary photochemical events in the photosynthetic bacterium Rhodopseudomonas sphaeroides. The previously determined amino-terminal sequences of the three subunits of the reaction center protein were used to design synthetic mixed oligonucleotide probes for the structural genes encoding the subunits. One of these probes was used to isolate and clone a fragment of DNA from R. sphaeroides that contained the gene encoding the M subunit. The nucleotide sequence of this gene was determined by the dideoxy method. In addition, a number of tryptic and chymotryptic peptides from the M protein were isolated and subjected to sequence analysis, and the sequence of the carboxyl terminus was determined. Together with the amino-terminal sequence, the data establish the primary structure of the M protein. The distribution of hydrophobic residues in the amino acid sequence suggests the presence of five membrane-spanning segments. A significant homology was found between the amino acid sequence of the M subunit and a thylakoid membrane protein (M(r) 32,000) from spinach that has been implicated in herbicide and quinone binding.
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46. |
Yun CH,
Van Doren SR,
Crofts AR,
Gennis RB,
( 1991 ) The use of gene fusions to examine the membrane topology of the L-subunit of the photosynthetic reaction center and of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides. PMID : 1645718 : Abstract >>
The topology of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides has been examined by generating gene fusions with alkaline phosphatase. Gene fusions were generated at random locations within the fbcB gene encoding the cytochrome b subunit. These fusion products were expressed in Escherichia coli and were screened for alkaline phosphatase activity on chromogenic plates. 33 in-frame fusions which showed activity were further characterized. The fusion junctions of all those fusions which had a high specific activity were clustered in three regions of the cytochrome b polypeptide, and thus these regions were tentatively assigned as being near the periplasmic surface. The data are consistent with a model containing eight transmembrane helices. In order to explore the validity of the gene fusion approach for a protein not normally expressed in E. coli, the topology of the L-subunit of the photosynthetic reaction center from R. sphaeroides was also explored using phoA gene fusions. A similar protocol was used as with the cytochrome b subunit. The gene fusions with high specific activity were shown to be in regions of the L-subunit polypeptide known to be at or near the periplasmic surface, as defined by the high resolution structure determined by X-ray crystallography. These data demonstrate the utility of this approach for determining membrane protein topology and extend potential applications to include at least some proteins not normally expressed in E. coli.
|
47. |
Usui S,
Yu L,
( 1991 ) Subunit IV (Mr = 14,384) of the cytochrome b-c1 complex from Rhodobacter sphaeroides. Cloning, DNA sequencing, and ubiquinone binding domain. PMID : 1651916 : Abstract >>
The Rhodobacter sphaeroides gene encoding subunit IV of the cytochrome b-c1 complex (fbcQ) was cloned and sequenced. The fbcQ cistron is 372 base pairs long and encodes 124 amino acid residues. The molecular mass of subunit IV, deduced from the nucleotide sequence, is 14,384 Da. A hydropathy plot of the predicted amino acid sequence revealed only one transmembrane helix; it is near the C-terminal end. The 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinone ([3H]azido-Q)-labeled subunit IV was isolated from the [3H]-azido-Q-treated cytochrome b-c1 complex. A ubiquinone-binding peptide was obtained by digesting the labeled subunit IV with V8 protease followed by high performance liquid chromatography separation. Amino acid analysis and partial N-terminal sequencing of this ubiquinone-binding peptide revealed that it corresponded to residues 77-124 of subunit IV. Based on the hydropathy profile and predicted tendency to form alpha-helices and beta-sheets, we propose a structural model for subunit IV. In this model the ubiquinone-binding domain is located near the surface of the membrane.
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48. |
van Berkum P,
Leibold JM,
Eardly BD,
( 2006 ) Proposal for combining Bradyrhizobium spp. (Aeschynomene indica) with Blastobacter denitrificans and to transfer Blastobacter denitrificans (Hirsch and Muller, 1985) to the genus Bradyrhizobium as Bradyrhizobium denitrificans (comb. nov.). PMID : 16564957 : DOI : 10.1016/j.syapm.2005.07.014 Abstract >>
The symbiotic bradyrhizobia of Aeschynomene indica and the aquatic budding bacterium Blastobacter denitrificans have much in common and this study broadens the characters that are shared between the two. The 23S rRNA gene sequences of the bradyrhizobial isolates were most similar to each other and to the sequence of Bl. denitrificans. Evidence for the presence of photosynthetic genes in the genome of Bl. denitrificans was obtained by PCR using primers to the conserved M subunit (pufM) of the photosynthetic reaction center present in purple sulfur and purple nonsulfur bacteria. The deduced amino acid sequences of the partial PufM protein of Bl. denitrificans and the corresponding sequences obtained from the bradyrhizobial isolates were identical. Both the bradyrhizobial isolates and the type strain of Bl. denitrificans shared the ability to propagate by budding, demonstrated by electron microscopy. Even though many interspecific characters were shared among the bradyrhizobial isolates including Bl. denitrificans, it was evident from Amplified Fragment Length Polymorphism (AFLP) analysis that genomic variation existed among the collection that was examined. Variation among bradyrhizobial isolates and Bl. denitrificans also was established in carbon and nitrogen source utilization and the ability to grow at elevated temperature. Based on these results and previously reported evidence it is suggested that the type strain for Bl. denitrificans and the bradyrhizobial isolates from nodules of A. indica belong to a common group of bacteria. Therefore, it is proposed that they be combined into the genus Bradyrhizobium and that LMG 8443 be transferred to this genus as the type strain for B. denitrificans.
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49. |
Cao J,
Shapleigh J,
Gennis R,
Revzin A,
Ferguson-Miller S,
( 1991 ) The gene encoding cytochrome c oxidase subunit II from Rhodobacter sphaeroides; comparison of the deduced amino acid sequence with sequences of corresponding peptides from other species. PMID : 1648008 : DOI : 10.1016/0378-1119(91)90235-4 Abstract >>
The gene (coxII) encoding subunit II of Rhodobacter sphaeroides cytochrome c oxidase (cytochrome aa3) has been isolated by screening a genomic DNA library in phage lambda with a probe derived from coxII of Paracoccus denitrificans. A 2-kb fragment containing coxII DNA was subcloned into the phage M13mp18 and the sequence determined. The 2-kb insert contains the entire coding region for coxII gene, including the ATG start codon and a TGA stop codon. The deduced amino acid (aa) sequence of subunit II of R. sphaeroides shows regions of substantial homology to the corresponding subunit of the bovine mitochondrial oxidase (63% overall) and P. denitrificans oxidase (68% overall). The postulated redox-active copper ion (CuA) binding site involving two Cys and two His residues (as well as an alternative Met residue) is conserved among these species, along with four invariant acidic aa residues (two Asp and two Glu) that may be involved in interactions with cytochrome c, and a region of aromatic residues (Tyr-Gln-Trp-Tyr-Trp-Gly-Tyr-Glu-Tyr) which is postulated to play a role in electron transfer. Hydropathy profile analysis suggests that while the bovine COXII secondary structure contains two transmembrane helices, the R. sphaeroides subunit II has a third such helix that may function as part of a signal sequence, as suggested for P. denitrificans.
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50. |
Slovak PM,
Porter SL,
Armitage JP,
( 2006 ) Differential localization of Mre proteins with PBP2 in Rhodobacter sphaeroides. PMID : 16484180 : DOI : 10.1128/JB.188.5.1691-1700.2006 PMC : PMC1426539 Abstract >>
In Rhodobacter sphaeroides, MreB, MreC, MreD, PBP2, and RodA are encoded at the same locus. The localizations of PBP2, MreB, and MreC, which have all been implicated in the synthesis of the peptidoglycan layer, were investigated under different growth conditions to gain insight into the relationships between these proteins. Immunofluorescence microscopy showed that PBP2 localized to specific sites at the midcell of elongating cells under both aerobic and photoheterotrophic conditions. Visualizing PBP2 at different stages of the cell cycle showed that in elongating cells, PBP2 was found predominately at the midcell, with asymmetric foci and bands across the cell. PBP2 remained at midcell until the start of septation, after which it moved to midcell of the daughter cells. Deconvolution and three-dimensional reconstructions suggested that PBP2 forms a partial ring at the midcell of newly divided cells and elongated cells, while in septating cells, partial PBP2 rings were present at one-quarter and three-quarter positions. Due to the diffraction limits of light microscopy, these partial rings could represent unresolved helices. Colocalization studies showed that MreC always colocalized with PBP2, while MreB colocalized with PBP2 only during elongation; during septation, MreB remained at the septation site, whereas PBP2 relocalized to the one-quarter and three-quarter positions. These results suggest that PBP2 and MreC are involved in peptidoglycan synthesis during elongation and that this occurs at specific sites close to midcell in R. sphaeroides.
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51. |
Anderson S,
Dragnea V,
Masuda S,
Ybe J,
Moffat K,
Bauer C,
( 2005 ) Structure of a novel photoreceptor, the BLUF domain of AppA from Rhodobacter sphaeroides. PMID : 15924418 : DOI : 10.1021/bi0502691 PMC : PMC2774740 Abstract >>
The flavin-binding BLUF domain of AppA represents a new class of blue light photoreceptors that are present in a number of bacterial and algal species. The dark state X-ray structure of this domain was determined at 2.3 A resolution. The domain demonstrates a new function for the common ferredoxin-like fold; two long alpha-helices flank the flavin, which is bound with its isoalloxazine ring perpendicular to a five-stranded beta-sheet. The hydrogen bond network and the overall protein topology of the BLUF domain (but not its sequence) bear some resemblance to LOV domains, a subset of PAS domains widely involved in signaling. Nearly all residues conserved in BLUF domains surround the flavin chromophore, many of which are involved in an intricate hydrogen bond network. Photoactivation may induce a rearrangement in this network via reorientation of the Gln63 side chain to form a new hydrogen bond to the flavin O4 position. This shift would also break a hydrogen bond to the Trp104 side chain, which may be critical in induction of global structural change in AppA.
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52. |
Grinstead JS,
Hsu ST,
Laan W,
Bonvin AM,
Hellingwerf KJ,
Boelens R,
Kaptein R,
( 2006 ) The solution structure of the AppA BLUF domain: insight into the mechanism of light-induced signaling. PMID : 16323221 : DOI : 10.1002/cbic.200500270 Abstract >>
The transcriptional antirepressor AppA from the photosynthetic bacterium Rhodobacter sphaeroides senses both the light climate and the intracellular redox state. Under aerobic conditions in the dark, AppA binds to and thereby blocks the function of PpsR, a transcriptional repressor. Absorption of a blue photon dissociates AppA from PpsR and allows the latter to repress photosynthesis gene expression. The N terminus of AppA contains sequence homology to flavin-containing photoreceptors that belong to the BLUF family. Structural and chemical aspects of signal transduction mediated by AppA are still largely unknown. Here we present NMR studies of the N-terminal flavin-binding BLUF domain of AppA. Its solution structure adopts an alpha/beta-sandwich fold with a beta alpha beta beta alpha beta beta topology, which represents a new flavin-binding fold. Considerable disorder is observed for residues near the chromophore due to conformational exchange. This disorder is observed both in the dark and in the light-induced signaling state of AppA. Furthermore, we detect light-induced structural changes in a patch of surface residues that provide a structural link between light absorption and signal-transduction events.
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53. |
Philippsen A,
Schirmer T,
Stein MA,
Giffhorn F,
Stetefeld J,
( 2005 ) Structure of zinc-independent sorbitol dehydrogenase from Rhodobacter sphaeroides at 2.4 A resolution. PMID : 15805591 : DOI : 10.1107/S0907444904034390 Abstract >>
Recombinant sorbitol dehydrogenase (SDH) from Rhodobacter sphaeroides has been crystallized in the absence of the cofactor NAD(H) and its structure determined to 2.4 A resolution using molecular replacement (refined R and R free factors of 18.8 and 23.8%, respectively). As expected from the sequence and shown by the conserved fold, SDH can be assigned to the short-chain dehydrogenase/reductase protein family. The cofactor NAD and the substrate sorbitol have been modelled into the structure and the active-site architecture, which displays the highly conserved catalytic tetrad of Asn-Ser-Tyr-Lys residues, is discussed in relation to the enzyme mechanism. This is the first structure of a bacterial SDH belonging to the SDR family.
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54. |
Smith D,
Gray J,
Mitchell L,
Antholine WE,
Hosler JP,
( 2005 ) Assembly of cytochrome-c oxidase in the absence of assembly protein Surf1p leads to loss of the active site heme. PMID : 15764605 : DOI : 10.1074/jbc.C500061200 Abstract >>
Surf1p is a protein of the inner membrane of mitochondria that functions in the assembly of cytochrome-c oxidase. The specifics of the role of Surf1p have remained unresolved. Numerous mutations in human Surf1p lead to severe mitochondrial disease. A homolog of human Surf1p is encoded by the genome of the alpha-proteobacterium Rhodobacter sphaeroides, which synthesizes a mitochondrial-like aa(3)-type cytochrome-c oxidase. The gene for Surf1p was deleted from the genome of R. sphaeroides. The resulting aa(3)-type oxidase was purified and analyzed by biochemical methods plus optical and EPR spectroscopy. The oxidase that assembled in the absence of Surf1p was composed of three subpopulations with structurally distinct heme a(3)-Cu active sites. 50% of the oxidase lacked heme a(3), 10-15% contained heme a(3) but lacked Cu(BB), and 35-40% had a normal heme a(3) -Cu(B) active site with normal activity. Cu(A) assembly was unaffected. All of the oxidase contained low-spin heme a, but the environment of the heme a center was slightly altered in the 50% of the enzyme that lacked heme a(3). Introduction of a normal copy of the gene for Surf1p on an exogenous plasmid resulted in a single population of normally assembled, highly active enzyme. The data indicate that Surf1p plays a role in facilitating the insertion of heme a(3) into the active site of cytochrome-c oxidase. The results suggest that maturation of the heme a(3)-Cu(B) center is a step that limits the association of subunits I and II in the assembly of mitochondrial cytochrome oxidase.
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55. |
Tabata A,
Yamamoto I,
Matsuzaki M,
Satoh T,
( 2005 ) Differential regulation of periplasmic nitrate reductase gene (napKEFDABC) expression between aerobiosis and anaerobiosis with nitrate in a denitrifying phototroph Rhodobacter sphaeroides f. sp. denitrificans. PMID : 16136296 : DOI : 10.1007/s00203-005-0029-9 Abstract >>
A denitrifying phototroph, Rhodobacter sphaeroides f. sp. denitrificans, has the ability to denitrify by respiring nitrate. The periplasmic respiratory nitrate reductase (Nap) catalyses the first step in denitrification and is encoded by the genes, napKEFDABC. By assaying the ss-galactosidase activity of napKEFD-lacZ fusions in wild type and nap mutant cells grown under various growth conditions, the environmental signal for inducing nap expression was examined. Under anoxic conditions with nitrate, nap genes expression in the wild-type strain was highest in the dark, and somewhat lowered by incident light, but that of the napA, napB, and napC mutant strains was low, showing that nap expression is dependent on nitrate respiration. Under oxic conditions, both the wild type and nap mutant cells showed high ss-galactosidase activities, comparable to the wild-type grown under anoxic conditions with nitrate. Myxothiazol, a specific inhibitor of the cytochrome bc (1) complex, did not affect the beta-galactosidase activity in the wild-type cells grown aerobically, suggesting that the redox state of the quinone pool was not a candidate for the activation signal for aerobic nap expression. These results suggested that the trans-acting regulatory signals for nap expression differ between anoxic and oxic conditions. Deletion analysis showed that the nucleotide sequence from -135 to -88 with respect to the translational start point is essential for nap expression either under anoxic or oxic conditions, suggesting that the same cis-acting element is involved in regulating nap expression under either anoxic with nitrate or oxic conditions.
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56. |
Franchi E,
Tosi C,
Scolla G,
Penna GD,
Rodriguez F,
Pedroni PM,
( N/A ) Metabolically engineered Rhodobacter sphaeroides RV strains for improved biohydrogen photoproduction combined with disposal of food wastes. PMID : 15645340 : DOI : 10.1007/s10126-004-1007-y Abstract >>
Three differently metabolically engineered strains, 2 single PHA- and Hup- mutants and one double PHA-/Hup- mutant, of the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides RV, were constructed to improve a light-driven biohydrogen production process combined with the disposal of solid food wastes. These phenotypes were designed to abolish, singly or in combination, the competition of H2 photoproduction with polyhydroxyalkanoate (PHA) accumulation by inactivating PHA synthase activity, and with H2 recycling by abolishing the uptake hydrogenase enzyme. The performance of these mutants was compared with that of the wild-type strain in laboratory tests carried out in continuously fed photobioreactors using as substrates both synthetic media containing lactic acid and media from the acidogenic fermentation of actual fruit and vegetable wastes, containing mainly lactic acid, smaller amounts of acetic acia, and traces of higher volatile acids. With the lactic acid-based synthetic medium, the single Hup- and the double PHA-/Hup- mutants, but not the single PHA- mutant, exhibited increased rates of H2 photoproduction, about one third higher than that of the wild-type strain. With the food-waste-derived growth medium, only the single Hup- mutant showed higher rates of H2 production, but all 3 mutants sustained a longer-term H2 photoproduction phase than the wild-type strain, with the double mutant exhibiting overall the largest amount of H2 evolved. This work demonstrates the feasibility of single and multiple gene engineering of microorganisms to redirect their metabolism for improving H2 photoproduction using actual waste-derived substrates.
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57. |
Slovak PM,
Wadhams GH,
Armitage JP,
( 2005 ) Localization of MreB in Rhodobacter sphaeroides under conditions causing changes in cell shape and membrane structure. PMID : 15601688 : DOI : 10.1128/JB.187.1.54-64.2005 PMC : PMC538805 Abstract >>
MreB is thought to be a bacterial actin homolog that defines the morphology of rod-shaped bacteria. Rhodobacter sphaeroides changes shape, from a rod to coccobacillus, and undergoes extensive cytoplasmic membrane invagination when it switches from aerobic to photoheterotrophic growth. The role of MreB in defining R. sphaeroides shape was therefore investigated. Attempts at deleting or insertionally inactivating mreB were unsuccessful under all growth conditions. Immunofluorescence microscopy showed MreB localized to mid-cell in elongating cells under both aerobic and photoheterotrophic conditions. Three-dimensional reconstruction showed that MreB formed a ring at mid-cell. MreB remained at mid-cell as septation began but localized to new sites in the daughter cells before the completion of septation. MreB localized to putative septation sites in cephalexin-treated filamentous cells. Genomic single-copy mreB was replaced with gfp-mreB, and green fluorescent protein (GFP)-MreB localized in the same pattern, as seen with immunofluorescence microscopy. Some of the cells expressing GFP-MreB were abnormal, principally displaying an increase in cell width, suggesting that the fusion was not fully functional in all cells. GFP-MreB localized to swellings at mid-cell in cells treated with the penicillin-binding protein 2 inhibitor amdinocillin. These data suggest that MreB is essential in R. sphaeroides, performing a role at mid-cell in elongating cells, and in early septation, putatively in the cytoplasmic control of the peptidoglycan synthetic complexes.
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58. |
Benning C,
Somerville CR,
( 1992 ) Isolation and genetic complementation of a sulfolipid-deficient mutant of Rhodobacter sphaeroides. PMID : 1551852 : DOI : 10.1128/jb.174.7.2352-2360.1992 PMC : PMC205858 Abstract >>
All photosynthetic organisms are thought to contain the sulfolipid 6-sulfo-alpha-D-quinovosyl diacylglycerol. However, the pathway of sulfolipid biosynthesis has not been elucidated, and the functional or structural significance of this lipid is not known. Mutants of Rhodobacter sphaeroides deficient in sulfolipid accumulation were isolated by directly screening for altered sulfolipid content. The mutants had no apparent phenotype except for the sulfolipid deficiency. A gene, designated sqdA, which complemented one of the mutations was isolated and characterized. The putative sqdA gene product is a protein with a molecular mass of 33.6 kDa that has no sequence similarity to any enzyme of known function.
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59. |
Spiridonova EM,
Berg IA,
Kolganova TV,
Ivanovskiĭ RN,
Kuznetsov BB,
Turova TP,
( N/A ) [An oligonucleotide primer system for amplification of the ribulose-1,5-bisphosphate carboxylase/oxygenase genes of bacteria of various taxonomic groups]. PMID : 15315232 : Abstract >>
Based on the analysis of GenBank nucleotide sequences of the cbbL and cbbM genes, coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPC), the key enzyme of the Calvin cycle, a primer system was designed that allows about 800-bp-long fragments of these genes to be PCR-ampliflied in various photo- and chemotrophic bacteria. The efficiency of the designed primer system in detection of RuBPC genes was demonstrated in PCR with DNA of taxonomically diverse bacteria possessing RuBPC genes with a known primary structure. Nucleotide sequences of RuBPC gene fragments of bacteria belonging to the genera Acidithiobacillus. Ectothiorhodospira, Magnetospirillum, Methylocapsa, Thioalkalispira, Rhodobacter, and Rhodospirillum were determined to be deposited with GenBank and to be translated into amino acid sequences and subjected to phylogenetic analysis.
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60. |
Axelrod HL,
Feher G,
Allen JP,
Chirino AJ,
Day MW,
Hsu BT,
Rees DC,
( 1994 ) Crystallization and X-ray structure determination of cytochrome c2 from Rhodobacter sphaeroides in three crystal forms. PMID : 15299423 : DOI : 10.1107/S0907444994001319 Abstract >>
Cytochrome c(2) serves as the secondary electron donor that reduces the photo-oxidized bacteriochlorophyll dimer in photosynthetic bacteria. Cytochrome c(2) from Rhodobacter sphaeroides has been crystallized in three different forms. At high ionic strength, crystals of a hexagonal space group (P6(1)22) were obtained, while at low ionic strength, triclinic (P1) and tetragonal (P4(1)2(1)2) crystals were formed. The three-dimensional structures of the cytochrome in all three crystal forms have been determined by X-ray diffraction at resolutions of 2.20 A (hexagonal), 1.95 A, (triclinic) and 1.53 A (tetragonal). The most significant difference observed was the binding of an imidazole molecule to the iron atom of the heme group in the hexagonal structure. This binding displaces the sulfur atom of Met l00, which forms the axial ligand in the triclinic and tetragonal structures.
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61. |
Steinbüchel A,
Hustede E,
Liebergesell M,
Pieper U,
Timm A,
Valentin H,
( 1992 ) Molecular basis for biosynthesis and accumulation of polyhydroxyalkanoic acids in bacteria. PMID : 1476773 : DOI : 10.1111/j.1574-6968.1992.tb05841.x Abstract >>
The current knowledge on the structure and on the organization of polyhydroxyalkanoic acid (PHA)-biosynthetic genes from a wide range of different bacteria, which rely on different pathways for biosynthesis of this storage polyesters, is provided. Molecular data will be shown for genes of Alcaligenes eutrophus, purple non-sulfur bacteria, such as Rhodospirillum rubrum, purple sulfur bacteria, such as Chromatium vinosum, pseudomonads belonging to rRNA homology group I, such as Pseudomonas aeruginosa, Methylobacterium extorquens, and for the Gram-positive bacterium Rhodococcus ruber. Three different types of PHA synthases can be distinguished with respect to their substrate specificity and structure. Strategies for the cloning of PHA synthase structural genes will be outlined which are based on the knowledge of conserved regions of PHA synthase structural genes and of the PHA-biosynthetic routes in bacteria as well as on the heterologous expression of these genes and on the availability of mutants impaired in the accumulation of PHA. In addition, a terminology for the designation of PHAs and of proteins and genes relevant for the metabolism of PHA is suggested.
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62. |
Cobbe N,
Heck MM,
( 2004 ) The evolution of SMC proteins: phylogenetic analysis and structural implications. PMID : 14660695 : DOI : 10.1093/molbev/msh023 Abstract >>
The SMC proteins are found in nearly all living organisms examined, where they play crucial roles in mitotic chromosome dynamics, regulation of gene expression, and DNA repair. We have explored the phylogenetic relationships of SMC proteins from prokaryotes and eukaryotes, as well as their relationship to similar ABC ATPases, using maximum-likelihood analyses. We have also investigated the coevolution of different domains of eukaryotic SMC proteins and attempted to account for the evolutionary patterns we have observed in terms of available structural data. Based on our analyses, we propose that each of the six eukaryotic SMC subfamilies originated through a series of ancient gene duplication events, with the condensins evolving more rapidly than the cohesins. In addition, we show that the SMC5 and SMC6 subfamily members have evolved comparatively rapidly and suggest that these proteins may perform redundant functions in higher eukaryotes. Finally, we propose a possible structure for the SMC5/SMC6 heterodimer based on patterns of coevolution.
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63. |
Purvis DJ,
Theiler R,
Niederman RA,
( 1990 ) Chromatographic and protein chemical analysis of the ubiquinol-cytochrome c2 oxidoreductase isolated from Rhodobacter sphaeroides. PMID : 2153104 : Abstract >>
The ubiquinol-cytochrome c2 oxidoreductase (cytochrome bc1 complex) purified from chromatophores of Rhodobacter sphaeroides consists of four polypeptide subunits corresponding to cytochrome b, c1, and the Rieske iron-sulfur protein, as well as a 14-kDa polypeptide of unknown function, respectively. In contrast, the complex isolated from Rhodospirillum rubrum by the same procedure lacked a polypeptide corresponding to the 14-kDa subunit. Gel-permeation chromatography of the R. sphaeroides cytochrome bc1 complex in the presence of 200 mM NaCl removed the iron-sulfur protein, while the 14-kDa polypeptide remained tightly bound to the cytochromes; this is consistent with the possibility that the latter protein is an authentic component of the complex rather than an artifact of the isolation procedure. The individual polypeptides of the R. sphaeroides complex were purified to homogeneity by gel-permeation chromatography in the presence of 50% aqueous formic acid and their amino acid compositions determined. The 14-kDa polypeptide was found to be rich in charged and polar residues. Edman degradation analysis indicated that its N terminus is blocked and not rendered accessible by de-blocking procedures. Cyanogen bromide cleavage gave rise to a blocked N-terminal fragment as well as a C-terminal peptide comprising more than one-third of the protein. Gas-phase sequence analysis of this peptide established a sequence of 48 residues and identified a putative trans-membrane segment near the C terminus. The blocked N-terminal fragment was cleaved at tryptophan with BNPS-skatole. The resulting peptides, together with tryptic fragments derived from the intact protein, yielded additional sequence information; however, none of the sequences exhibited significant homologies to any known proteins. Tryptic fragments were also used to generate sequence information for cytochrome c1.
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64. |
Gibson JL,
Chen JH,
Tower PA,
Tabita FR,
( 1990 ) The form II fructose 1,6-bisphosphatase and phosphoribulokinase genes form part of a large operon in Rhodobacter sphaeroides: primary structure and insertional mutagenesis analysis. PMID : 2175647 : DOI : 10.1021/bi00487a014 Abstract >>
Fructose 1,6-bisphosphatase (FBPase) and phosphoribulokinase (PRK) are two key enzymes of the reductive pentose phosphate pathway or Calvin cycle of photosynthetic carbon dioxide assimilation. Early studies had indicated that the properties of enzymes isolated from photosynthetic bacteria were clearly distinct from those of enzymes obtained from the chloroplasts of higher plants [for a review, see Tabita (1988)]. The eucaryotic enzymes, which are light activated by the thioredoxin/ferredoxin system (Buchanan, 1980), were each shown to contain a putative regulatory amino acid sequence (Marcus et al., 1988; Porter et al., 1988). The enzymes from photosynthetic bacteria are not controlled by the thioredoxin/ferredoxin system but exhibit complex kinetic properties and, in the case of PRK, there is an absolute requirement of NADH for activity. In the photosynthetic bacterium Rhodobacter sphaeroides, the structural genes of the Calvin cycle, including the genes that encode FBPase (fbp) and PRK (prk), are found in two distinct clusters, and the fbp and prk genes are closely associated in each cluster. In the present investigation, we have determined the nucleotide sequence of the fbpB and prkB genes of the form II cluster and have compared the deduced amino acid sequences to previously determined sequences of light-activated enzymes from higher plants and from other eucaryotic and procaryotic sources. In the case of FBPase, there are several regions that are conserved in the R. sphaeroides enzymes, including a protease-sensitive area located in a region equivalent to residues 51-71 of mammalian FBPase.(ABSTRACT TRUNCATED AT 250 WORDS)
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65. |
Platt MW,
Miller KJ,
Lane WS,
Kennedy EP,
( 1990 ) Isolation and characterization of the constitutive acyl carrier protein from Rhizobium meliloti. PMID : 2144277 : DOI : 10.1128/jb.172.9.5440-5444.1990 PMC : PMC213210 Abstract >>
Rhizobium species produce an inducible acyl carrier protein (ACP), encoded by the nodF gene, that somehow functions in an exchange of cell signals between bacteria and specific plant hosts, leading to nodulation of plant roots and symbiotic nitrogen fixation, as well as a constitutive ACP needed for the synthesis of essential cell lipids. The periplasmic cyclic glucans of Rhizobium spp. are also involved in specific rhizobium-plant interaction. These glucans are strongly similar to the periplasmic membrane-derived oligosaccharides (MDO) of Escherichia coli. E. coli ACP is an essential component of a membrane-bound transglucosylase needed for the biosynthesis of MDO, raising the possibility that either or both of the rhizobial ACPs might have a similar function. We have now isolated the constitutive ACP of R. meliloti and determined its primary structure. We have also examined its function, together with those of ACPs from E. coli, Rhodobacter sphaeroides, and spinach, in the MDO transglucosylase system and as substrate for the E. coli ACP acylase enzyme. All four ACPs act as acceptors of acyl residues, but only the E. coli ACP functions in the transglucosylase system.
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66. |
Yun CH,
Beci R,
Crofts AR,
Kaplan S,
Gennis RB,
( 1990 ) Cloning and DNA sequencing of the fbc operon encoding the cytochrome bc1 complex from Rhodobacter sphaeroides. Characterization of fbc deletion mutants and complementation by a site-specific mutational variant. PMID : 2176595 : DOI : 10.1111/j.1432-1033.1990.tb15633.x Abstract >>
The ubiquinol: cytochrome-c oxidoreductase (cytochrome bc1 complex) is a central component of the mitochondrial respiratory chain as well as the respiratory and/or photosynthetic systems of numerous prokaryotic organisms. In Rhodobacter sphaeroides, the bc1 complex has a dual function. When the cells are grown photosynthetically, the bc1 complex is present in the intracytoplasmic membrane and is a critical component of the cyclic electron transport system. When the cells are grown in the dark in the presence of oxygen, the same bc1 complex is a necessary component of the cytochrome-c2-dependent respiratory chain. The fact that the bc1 complex from R. sphaeroides has been extensively studied, plus the ability to manipulate this organism genetically, makes this an ideal system for using site-directed mutagenesis to address questions relating to the structure and function of the bc1 complex. In the current work, the cloning and complete sequence of the fbc operon from R. sphaeroides is reported. As in other bacteria, this operon contains three genes, encoding the Rieske 2Fe-2S subunit, the cytochrome b subunit, and the cytochrome c1 subunit. Recombination techniques were used to delete the entire fbc operon from the chromosome. The resulting strain cannot grow photosynthetically, but can grow aerobically utilizing a quinol oxidase. Photosynthetic growth is restored by providing fbc operon on a plasmid, and the reappearance of the protein subunits and the spectroscopic features due to the bc1 complex are also demonstrated. Finally, a mutation is introduced within the gene encoding the cytochrome b subunit which is predicted to confer resistance to the inhibitor myxothiazol. It is shown that the resulting strain contains a functional bc1 complex which, as expected, is resistant to the inhibitor. Hence, this system is suitable for the detailed characterization of the bc1 complex, combining site-directed mutagenesis with the biochemical and biophysical techniques which have been previously developed for the study of photosynthetic bacteria.
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67. |
Pille S,
Chuat JC,
Breton AM,
Clément-Métral JD,
Galibert F,
( 1990 ) Cloning, nucleotide sequence, and expression of the Rhodobacter sphaeroides Y thioredoxin gene. PMID : 2137818 : DOI : 10.1128/jb.172.3.1556-1561.1990 PMC : PMC208632 Abstract >>
Synthetic oligodeoxynucleotide probes based on the known amino acid sequence of Rhodobacter sphaeroides Y thioredoxin were used to identify, clone, and sequence the structural gene. The amino acid sequence derived from the DNA sequence of the R. sphaeroides gene was identical to the known amino acid sequence of R. sphaeroides thioredoxin. An NcoI site was created by directed mutagenesis at the beginning of the thioredoxin gene, inducing in the encoded protein the replacement of serine in position 2 by alanine. The 421-base-pair NcoI-PstI restriction fragment obtained was ligated in the pKK233-2 expression vector and the resulting hybrid plasmid was used to transform Escherichia coli strains lacking functional thioredoxin. Transformants that complemented mutations in the trxA gene were identified by increased colony size on rich medium, growth on minimal medium with methionine sulfoxide, and ability to support M13 growth and T7 replication; this latter phenotype implies correct interaction between R. sphaeroides thioredoxin and the product of T7 gene 5. The presence of R. sphaeroides thioredoxin was further confirmed by enzyme assay.
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68. |
Arnoux B,
Ducruix A,
Astier C,
Picaud M,
Roth M,
Reiss-Husson F,
( 1990 ) 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. PMID : 2126457 : DOI : 10.1016/0300-9084(90)90116-x Abstract >>
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 (beta-octylglucoside) in the unit cell are presented. This method combines low angle neutron scattering at different contrasts in H2O/D2O with X-ray crystallographic data.
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69. |
Korkhov VM,
Sachse C,
Short JM,
Tate CG,
( 2010 ) Three-dimensional structure of TspO by electron cryomicroscopy of helical crystals. PMID : 20541505 : DOI : 10.1016/j.str.2010.03.001 PMC : PMC2911597 Abstract >>
The 18 kDa TSPO protein is a polytopic mitochondrial outer membrane protein involved in a wide range of physiological functions and pathologies, including neurodegeneration and cancer. The pharmacology of TSPO has been extensively studied, but little is known about its biochemistry, oligomeric state, and structure. We have expressed, purified, and characterized a homologous protein, TspO from Rhodobacter sphaeroides, and reconstituted it as helical crystals. Using electron cryomicroscopy and single-particle helical reconstruction, we have determined a three-dimensional structure of TspO at 10 A resolution. The structure suggests that monomeric TspO comprises five transmembrane alpha helices that form a homodimer, which is consistent with the dimeric state observed in detergent solution. Furthermore, the arrangement of transmembrane domains of individual TspO subunits indicates a possibility of two substrate translocation pathways per dimer. The structure provides the first insight into the molecular architecture of TSPO/PBR protein family that will serve as a framework for future studies.
|
70. |
Carius Y,
Christian H,
Faust A,
Zander U,
Klink BU,
Kornberger P,
Kohring GW,
Giffhorn F,
Scheidig AJ,
( 2010 ) Structural insight into substrate differentiation of the sugar-metabolizing enzyme galactitol dehydrogenase from Rhodobacter sphaeroides D. PMID : 20410293 : DOI : 10.1074/jbc.M110.113738 PMC : PMC2888412 Abstract >>
Galactitol 2-dehydrogenase (GatDH) belongs to the protein superfamily of short-chain dehydrogenases. As an enzyme capable of the stereo- and regioselective modification of carbohydrates, it exhibits a high potential for application in biotechnology as a biocatalyst. We have determined the crystal structure of the binary form of GatDH in complex with its cofactor NAD(H) and of the ternary form in complex with NAD(H) and three different substrates. The active form of GatDH constitutes a homo-tetramer with two magnesium-ion binding sites each formed by two opposing C termini. The catalytic tetrad is formed by Asn(116), Ser(144), Tyr(159), and Lys(163). GatDH structurally aligns well with related members of the short-chain dehydrogenase family. The substrate binding pocket can be divided into two parts of different size and polarity. In the smaller part, the side chains of amino acids Ser(144), Ser(146), and Asn(151) are important determinants for the binding specificity and the orientation of (pro-) chiral compounds. The larger part of the pocket is elongated and flanked by polar and non-polar residues, enabling a rather broad substrate spectrum. The presented structures provide valuable information for a rational design of this enzyme to improve its stability against pH, temperature, or solvent concentration and to optimize product yield in bioreactors.
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71. |
Bell CH,
Porter SL,
Strawson A,
Stuart DI,
Armitage JP,
( 2010 ) Using structural information to change the phosphotransfer specificity of a two-component chemotaxis signalling complex. PMID : 20161720 : DOI : 10.1371/journal.pbio.1000306 PMC : PMC2817712 Abstract >>
Two-component signal transduction pathways comprising histidine protein kinases (HPKs) and their response regulators (RRs) are widely used to control bacterial responses to environmental challenges. Some bacteria have over 150 different two-component pathways, and the specificity of the phosphotransfer reactions within these systems is tightly controlled to prevent unwanted crosstalk. One of the best understood two-component signalling pathways is the chemotaxis pathway. Here, we present the 1.40 A crystal structure of the histidine-containing phosphotransfer domain of the chemotaxis HPK, CheA(3), in complex with its cognate RR, CheY(6). A methionine finger on CheY(6) that nestles in a hydrophobic pocket in CheA(3) was shown to be important for the interaction and was found to only occur in the cognate RRs of CheA(3), CheY(6), and CheB(2). Site-directed mutagenesis of this methionine in combination with two adjacent residues abolished binding, as shown by surface plasmon resonance studies, and phosphotransfer from CheA(3)-P to CheY(6). Introduction of this methionine and an adjacent alanine residue into a range of noncognate CheYs, dramatically changed their specificity, allowing protein interaction and rapid phosphotransfer from CheA(3)-P. The structure presented here has allowed us to identify specificity determinants for the CheA-CheY interaction and subsequently to successfully reengineer phosphotransfer signalling. In summary, our results provide valuable insight into how cells mediate specificity in one of the most abundant signalling pathways in biology, two-component signal transduction.
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72. |
Watson GM,
Mann NH,
MacDonald GA,
Dunbar B,
( 1990 ) Identification and characterization of a GroEL homologue in Rhodobacter sphaeroides. PMID : 1982105 : DOI : 10.1016/0378-1097(90)90330-s Abstract >>
A protein closely related to the Escherichia coli GroEL protein has been isolated from Rhodobacter sphaeroides. Native and SDS-polyacrylamide gel electrophoresis of this protein have shown that it is present in the cell as a multimeric complex of Mr 670,000 which is composed of a monomer of Mr 58,000. Antisera raised against the Mr 58,000 polypeptide have been shown to cross-react with GroEL and the alpha subunit of the pea plastid chaperonin. The N-terminal amino acid sequence of the Mr 58,000 polypeptide is identical to that of GroEL at 15 of 19 residues and is also closely related to the alpha subunit of the pea plastid chaperonin, though less so to the beta subunit.
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73. |
Kornberger P,
Gajdzik J,
Natter H,
Wenz G,
Giffhorn F,
Kohring GW,
Hempelmann R,
( 2009 ) Modification of galactitol dehydrogenase from Rhodobacter sphaeroides D for immobilization on polycrystalline gold surfaces. PMID : 19778027 : DOI : 10.1021/la9010168 Abstract >>
Galactitol dehydrogenase (GatDH) from Rhodobacter sphaeroides is a multifunctional enzyme that catalyzes in the presence of oxidized beta-nicotinamide adenine dinucleotide (NAD(+)) the interconversion of various multivalent aliphatic alcohols to the corresponding ketones. The recombinant GatDH was provided with an N-terminal His(6)-tag to which distally up to three cysteine residues were attached. This protein construct maintained nearly full enzymatic activity, and it could be covalently immobilized via thiol bonds onto the surface of a gold electrode. Binding of GatDH onto the gold electrode was verified by SPR measurements, and residual enzyme activity was measured by cyclic voltammetry using 1,2-hexanediol as substrate, the cofactor NAD(+) and the redox mediator CTFM (4-carboxy-2,5,7-trinitrofluorenyliden-malonnitrile) in solute form. The results demonstrate the possibility of a directed functional immobilization of proteins on gold surfaces, which represents a proof-of-concept for the development of reactors for electrochemical synthon preparation using dehydrogenases.
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74. |
Chen JH,
Gibson JL,
McCue LA,
Tabita FR,
( 1991 ) Identification, expression, and deduced primary structure of transketolase and other enzymes encoded within the form II CO2 fixation operon of Rhodobacter sphaeroides. PMID : 1939098 : Abstract >>
Previous studies had indicated that the form II or B cluster of CO2 fixation structural genes is part of a large operon in Rhodobacter sphaeroides (Gibson, J. L., Chen, J.-H., Tower, P. A., and Tabita, F. R. (1990) Biochemistry 29, 8085-8093). In this investigation, we have sequenced the DNA between the prkB and rbpL genes and provide evidence for three distinct open reading frames which encode additional structural genes of the Calvin reductive pentose phosphate pathway; these genes encode the enzymes transketolase, glyceraldehyde phosphate dehydrogenase, and aldolase. Noteworthy is transketolase, which may be expressed to high levels in Escherichia coli. This study thus represents the initial description of the primary structure of bacterial transketolase, a key enzyme of the reductive and the oxidative pentose phosphate pathways. Each of the genes are separated by short stretches of intergenic sequence, consistent with earlier evidence which suggested that these genes are cotranscribed and part of a large operon controlled by sequences upstream from fbpB.
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75. |
Zhang L,
Mu W,
Jiang B,
Zhang T,
( 2009 ) Characterization of D-tagatose-3-epimerase from Rhodobacter sphaeroides that converts D-fructose into D-psicose. PMID : 19205890 : DOI : 10.1007/s10529-009-9942-3 Abstract >>
A non-characterized gene, previously proposed as the D-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with D-fructose and decreased for other substrates in the order: D-tagatose, D-psicose, D-ribulose, D-xylulose and D-sorbose. Its activity was maximal at pH 9 and 40 degrees C while being enhanced by Mn(2+). At pH 9 and 40 degrees C, 118 g D-psicose l(-1) was produced from 700 g D-fructose l(-1) after 3 h.
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76. |
Gibson JL,
Falcone DL,
Tabita FR,
( 1991 ) Nucleotide sequence, transcriptional analysis, and expression of genes encoded within the form I CO2 fixation operon of Rhodobacter sphaeroides. PMID : 1907281 : Abstract >>
In Rhodobacter sphaeroides, many of the structural genes encoding enzymes of the Calvin cycle are duplicated and grouped within two separate clusters. In this study, the nucleotide sequence of a 5627-base pair region of DNA that contains the form I Calvin cycle gene cluster has been determined. The five open reading frames are arranged in the order, fbpA prkA cfxA rbcL rbcS and are tightly linked and oriented in the same direction. The results of insertional mutagenesis studies suggest the genes are organized within an operon. Consistent with this proposal, the cfxA gene has been tentatively identified as a gene encoding the Calvin cycle enzyme, aldolase. Measurement of the activities of various Calvin cycle enzymes in the insertion mutants showed that inactivation of genes within one CO2 fixation cluster affected expression of genes within the second cluster, revealing a complex regulatory network.
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77. |
Erb TJ,
Rétey J,
Fuchs G,
Alber BE,
( 2008 ) Ethylmalonyl-CoA mutase from Rhodobacter sphaeroides defines a new subclade of coenzyme B12-dependent acyl-CoA mutases. PMID : 18819910 : DOI : 10.1074/jbc.M805527200 Abstract >>
Coenzyme B(12)-dependent mutases are radical enzymes that catalyze reversible carbon skeleton rearrangement reactions. Here we describe Rhodobacter sphaeroides ethylmalonyl-CoA mutase (Ecm), a novel member of the family of coenzyme B(12)-dependent acyl-CoA mutases, that operates in the recently discovered ethylmalonyl-CoA pathway for acetate assimilation. Ecm is involved in the central reaction sequence of this novel pathway and catalyzes the transformation of ethylmalonyl-CoA to methylsuccinyl-CoA in combination with a second enzyme that was further identified as promiscuous ethylmalonyl-CoA/methylmalonyl-CoA epimerase. In contrast to the epimerase, Ecm is highly specific for its substrate, ethylmalonyl-CoA, and accepts methylmalonyl-CoA only at 0.2% relative activity. Sequence analysis revealed that Ecm is distinct from (2R)-methylmalonyl-CoA mutase as well as isobutyryl-CoA mutase and defines a new subfamily of coenzyme B(12)-dependent acyl-CoA mutases. In combination with molecular modeling, two signature sequences were identified that presumably contribute to the substrate specificity of these enzymes.
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78. |
Zarzycki J,
Schlichting A,
Strychalsky N,
Müller M,
Alber BE,
Fuchs G,
( 2008 ) Mesaconyl-coenzyme A hydratase, a new enzyme of two central carbon metabolic pathways in bacteria. PMID : 18065535 : DOI : 10.1128/JB.01621-07 PMC : PMC2238226 Abstract >>
The coenzyme A (CoA)-activated C5-dicarboxylic acids mesaconyl-CoA and beta-methylmalyl-CoA play roles in two as yet not completely resolved central carbon metabolic pathways in bacteria. First, these compounds are intermediates in the 3-hydroxypropionate cycle for autotrophic CO2 fixation in Chloroflexus aurantiacus, a phototrophic green nonsulfur bacterium. Second, mesaconyl-CoA and beta-methylmalyl-CoA are intermediates in the ethylmalonyl-CoA pathway for acetate assimilation in various bacteria, e.g., in Rhodobacter sphaeroides, Methylobacterium extorquens, and Streptomyces species. In both cases, mesaconyl-CoA hydratase was postulated to catalyze the interconversion of mesaconyl-CoA and beta-methylmalyl-CoA. The putative genes coding for this enzyme in C. aurantiacus and R. sphaeroides were cloned and heterologously expressed in Escherichia coli, and the proteins were purified and studied. The recombinant homodimeric 80-kDa proteins catalyzed the reversible dehydration of erythro-beta-methylmalyl-CoA to mesaconyl-CoA with rates of 1,300 micromol min(-1) mg protein(-1). Genes coding for similar enzymes with two (R)-enoyl-CoA hydratase domains are present in the genomes of Roseiflexus, Methylobacterium, Hyphomonas, Rhodospirillum, Xanthobacter, Caulobacter, Magnetospirillum, Jannaschia, Sagittula, Parvibaculum, Stappia, Oceanicola, Loktanella, Silicibacter, Roseobacter, Roseovarius, Dinoroseobacter, Sulfitobacter, Paracoccus, and Ralstonia species. A similar yet distinct class of enzymes containing only one hydratase domain was found in various other bacteria, such as Streptomyces species. The role of this widely distributed new enzyme is discussed.
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79. |
Liu J,
Hiser C,
Ferguson-Miller S,
( 2017 ) Role of conformational change and K-path ligands in controlling cytochrome c oxidase activity. PMID : 28842531 : DOI : 10.1042/BST20160138 PMC : PMC6103453 DOI : 10.1042/BST20160138 PMC : PMC6103453 Abstract >>
Given the central role of cytochrome c oxidase (CcO) in health and disease, it is an increasingly important question as to how the activity and efficiency of this key enzyme are regulated to respond to a variety of metabolic states. The present paper summarizes evidence for two modes of regulation of activity: first, by redox-induced conformational changes involving the K-proton uptake path; and secondly, by ligand binding to a conserved site immediately adjacent to the entrance of the K-path that leads to the active site. Both these phenomena highlight the importance of the K-path in control of CcO. The redox-induced structural changes are seen in both the two-subunit and a new four-subunit crystal structure of bacterial CcO and suggest a gating mechanism to control access of protons to the active site. A conserved ligand-binding site, first discovered as a bile salt/steroid site in bacterial and mammalian oxidases, is observed to bind an array of ligands, including nucleotides, detergents, and other amphipathic molecules. Highly variable effects on activity, seen for these ligands and mutations at the K-path entrance, can be explained by differing abilities to inhibit or stimulate K-path proton uptake by preventing or allowing water organization. A new mutant form in which the K-path is blocked by substituting the conserved carboxyl with a tryptophan clarifies the singularity of the K-path entrance site. Further study in eukaryotic systems will determine the physiological significance and pharmacological potential of ligand binding and conformational change in CcO.
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80. |
Aiken C,
Gumport RI,
( 1988 ) Restriction endonuclease RsrI from Rhodobacter sphaeroides, an isoschizomer of EcoRI: purification and properties. PMID : 2843805 : DOI : 10.1093/nar/16.16.7901 PMC : PMC338499 Abstract >>
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.
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81. |
Davidson E,
Daldal F,
( 1987 ) fbc operon, encoding the Rieske Fe-S protein cytochrome b, and cytochrome c1 apoproteins previously described from Rhodopseudomonas sphaeroides, is from Rhodopseudomonas capsulata. PMID : 2821272 : DOI : 10.1016/0022-2836(87)90324-x Abstract >>
Detailed comparison of the 'Rhodopseudomonas sphaeroides GA' strain used by Gabellini et al. (1985) with genuine R. sphaeroides and R. capsulata strains indicated that the previously reported fbc operon of R. sphaeroides (Gabellini and Sebald, 1986) encoding the structural genes for the Rieske Fe-S protein, cytochrome b and cytochrome c1 subunits of the ubiquinol:cytochrome c2 oxidoreductase, is not from R. sphaeroides, but is rather from a strain of R. capsulata. Consequently, the genuine bc1 genes from R. sphaeroides were cloned using corresponding R. capsulata genes as probes, and a partial nucleotide sequence for the Rieske Fe-S protein of R. sphaeroides was determined and compared with that of R. capsulata.
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82. |
Allen JP,
Feher G,
Yeates TO,
Komiya H,
Rees DC,
( 1987 ) Structure of the reaction center from Rhodobacter sphaeroides R-26: the protein subunits. PMID : 2819866 : DOI : 10.1073/pnas.84.17.6162 PMC : PMC299029 Abstract >>
The three-dimensional structure of the protein subunits of the reaction center (RC) of Rhodobacter sphaeroides has been determined by x-ray diffraction at a resolution of 2.8 A with an R factor of 26%. The L and M subunits each contain five transmembrane helices and several helices that do not span the membrane. The L and M subunits are related to each other by a 2-fold rotational symmetry axis that is approximately the same as that determined for the cofactors. The H subunit has one transmembrane helix and a globular domain on the cytoplasmic side, which contains a helix that does not span the membrane and several beta-sheets. The structural homology with RCs from other purple bacteria is discussed. A structure of the complex formed between the water soluble cytochrome c2 and the RC from Rb. sphaeroides is proposed.
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83. |
Burgess JG,
Ashby MK,
Hunter CN,
( 1989 ) Characterization and complementation of a mutant of Rhodobacter sphaeroides with a chromosomal deletion in the light-harvesting (LH2) genes. PMID : 2693605 : DOI : 10.1099/00221287-135-7-1809 Abstract >>
An LH2- strain of Rhodobacter sphaeroides, DBC1, has been constructed by deleting the puc operon, which encodes the LH2 alpha and beta polypeptides, from the chromosome and replacing it with a kanamycin resistance gene. Southern blot analysis indicates that the 950 bp BamHI restriction fragment which contains the puc operon has been lost and has been replaced by the 1.25 kb Km(R) cassette derived from Tn903. Strain DBC1 lacked the LH2 complex, as shown by loss of the characteristic absorbance bands at 800 and 850 nm. The LH2 polypeptides were also found to be absent after SDS-PAGE. The wild-type phenotype was restored to DBC1 by the transfer of a 3.8 kb BscI fragment containing the puc operon in plasmid pMA81. Transconjugants possessed a wild-type absorbance spectrum and LH2 polypeptides.
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84. |
Stephenson FH,
Greene PJ,
( 1989 ) Nucleotide sequence of the gene encoding the RsrI methyltransferase. PMID : 2602165 : DOI : 10.1093/nar/17.24.10503 PMC : PMC335328 Abstract >>
N/A
|
85. |
Stephenson FH,
Ballard BT,
Boyer HW,
Rosenberg JM,
Greene PJ,
( 1989 ) Comparison of the nucleotide and amino acid sequences of the RsrI and EcoRI restriction endonucleases. PMID : 2695392 : DOI : 10.1016/0378-1119(89)90458-7 Abstract >>
The RsrI endonuclease, a type-II restriction endonuclease (ENase) found in Rhodobacter sphaeroides, is an isoschizomer of the EcoRI ENase. A clone containing an 11-kb BamHI fragment was isolated from an R. sphaeroides genomic DNA library by hybridization with synthetic oligodeoxyribonucleotide probes based on the N-terminal amino acid (aa) sequence of RsrI. Extracts of E. coli containing a subclone of the 11-kb fragment display RsrI activity. Nucleotide sequence analysis reveals an 831-bp open reading frame encoding a polypeptide of 277 aa. A 50% identity exists within a 266-aa overlap between the deduced aa sequences of RsrI and EcoRI. Regions of 75-100% aa sequence identity correspond to key structural and functional regions of EcoRI. The type-II ENases have many common properties, and a common origin might have been expected. Nevertheless, this is the first demonstration of aa sequence similarity between ENases produced by different organisms.
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86. |
Bracher A,
Sharma A,
Starling-Windhof A,
Hartl FU,
Hayer-Hartl M,
( 2015 ) Degradation of potent Rubisco inhibitor by selective sugar phosphatase. PMID : 27246049 : DOI : 10.1038/nplants.2014.2 Abstract >>
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the conversion of atmospheric carbon dioxide into organic compounds in photosynthetic organisms. Alongside carboxylating the five-carbon sugar ribulose-1,5-bisphosphate (RuBP)(1-3), Rubisco produces a small amount of xylulose-1,5-bisphosphate (XuBP), a potent inhibitor of Rubisco(4). The AAA+ protein Rubisco activase removes XuBP from the active site of Rubisco in an ATP-dependent process(5,6). However, free XuBP rapidly rebinds to Rubisco, perpetuating its inhibitory effect. Here, we combine biochemical and structural analyses to show that the CbbY protein of the photosynthetic bacterium Rhodobacter sphaeroides and Arabidopsis thaliana is a highly selective XuBP phosphatase. We also show that CbbY converts XuBP to the non-inhibitory compound xylulose-5-phosphate, which is recycled back to RuBP. We solve the crystal structures of CbbY from R. sphaeroides and A. thaliana, and through mutational analysis show that the cap domain of the protein confers the selectivity for XuBP over RuBP. Finally, in vitro experiments with CbbY from R. sphaeroides reveal that CbbY cooperates with Rubisco activase to prevent a detrimental build-up of XuBP at the Rubisco active site. We suggest that CbbY, which is conserved in algae and plants, is an important component of the cellular machinery that has evolved to deal with the shortcomings of the ancient enzyme Rubisco.
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87. |
Kaszubska W,
Aiken C,
O'Connor CD,
Gumport RI,
( 1989 ) Purification, cloning and sequence analysis of RsrI DNA methyltransferase: lack of homology between two enzymes, RsrI and EcoRI, that methylate the same nucleotide in identical recognition sequences. PMID : 2690017 : DOI : 10.1093/nar/17.24.10403 PMC : PMC335309 Abstract >>
RsrI DNA methyltransferase (M-RsrI) from Rhodobacter sphaeroides has been purified to homogeneity, and its gene cloned and sequenced. This enzyme catalyzes methylation of the same central adenine residue in the duplex recognition sequence d(GAATTC) as does M-EcoRI. The reduced and denatured molecular weight of the RsrI methyltransferase (MTase) is 33,600 Da. A fragment of R. sphaeroides chromosomal DNA exhibited M.RsrI activity in E. coli and was used to sequence the rsrIM gene. The deduced amino acid sequence of M.RsrI shows partial homology to those of the type II adenine MTases HinfI and DpnA and N4-cytosine MTases BamHI and PvuII, and to the type III adenine MTases EcoP1 and EcoP15. In contrast to their corresponding isoschizomeric endonucleases, the deduced amino acid sequences of the RsrI and EcoRI MTases show very little homology. Either the EcoRI and RsrI restriction-modification systems assembled independently from closely related endonuclease and more distantly related MTase genes, or the MTase genes diverged more than their partner endonuclease genes. The rsrIM gene sequence has also been determined by Stephenson and Greene (Nucl. Acids Res. (1989) 17, this issue).
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88. |
Bartsch RG,
Ambler RP,
Meyer TE,
Cusanovich MA,
( 1989 ) Effect of aerobic growth conditions on the soluble cytochrome content of the purple phototrophic bacterium Rhodobacter sphaeroides: induction of cytochrome c554. PMID : 2543295 : DOI : 10.1016/0003-9861(89)90293-2 Abstract >>
When grown anaerobically in the light, Rhodobacter sphaeroides contains appreciable quantities of cytochromes c2 and c', but smaller amounts of other soluble cytochromes such as cytochrome c551.5, cytochrome c554, and an oxygen-binding heme protein. When R. sphaeroides is mass cultured aerobically in the dark to stationary phase, the content of cytochrome c2 does not change appreciably, whereas cytochrome c554 is approximately 8-fold more abundant, cytochrome c' is at least 10-fold less abundant, and cytochrome c551.5 is fivefold lower than in the phototrophically grown cells. These observations confirm previous literature reports that in this organism a cytochrome c553 (or c554 in our experience) is more abundant when cells are grown aerobically. Furthermore, the aerobic cytochrome c554 is positively identified with the previously characterized minor cytochrome c554 component of anaerobic photosynthetic cells. Preliminary sequence results show that cytochrome c554 is a member of the cytochrome c' structural family, but differs from normal cytochromes c' in having a methionine sixth ligand to the heme. The levels of electron carrier proteins of low redox potential had previously been reported to be less in aerobic than in photoheterotrophic cells and we have verified that observation for the specific examples of cytochromes c' and c551.5. The oxygen binding heme protein, SHP, is not induced by aerobic growth.
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89. |
Li F,
Liu J,
Zheng Y,
Garavito RM,
Ferguson-Miller S,
( 2015 ) Protein structure. Crystal structures of translocator protein (TSPO) and mutant mimic of a human polymorphism. PMID : 25635101 : DOI : 10.1126/science.1260590 PMC : PMC5125025 Abstract >>
The 18-kilodalton translocator protein (TSPO), proposed to be a key player in cholesterol transport into mitochondria, is highly expressed in steroidogenic tissues, metastatic cancer, and inflammatory and neurological diseases such as Alzheimer's and Parkinson's. TSPO ligands, including benzodiazepine drugs, are implicated in regulating apoptosis and are extensively used in diagnostic imaging. We report crystal structures (at 1.8, 2.4, and 2.5 angstrom resolution) of TSPO from Rhodobacter sphaeroides and a mutant that mimics the human Ala(147)��Thr(147) polymorphism associated with psychiatric disorders and reduced pregnenolone production. Crystals obtained in the lipidic cubic phase reveal the binding site of an endogenous porphyrin ligand and conformational effects of the mutation. The three crystal structures show the same tightly interacting dimer and provide insights into the controversial physiological role of TSPO and how the mutation affects cholesterol binding.
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90. |
Yin L,
Dragnea V,
Feldman G,
Hammad LA,
Karty JA,
Dann CE,
Bauer CE,
( 2013 ) Redox and light control the heme-sensing activity of AppA. PMID : 23982072 : DOI : 10.1128/mBio.00563-13 PMC : PMC3760249 Abstract >>
The DNA binding activity of the photosystem-specific repressor PpsR is known to be repressed by the antirepressor AppA. AppA contains a blue-light-absorbing BLUF domain and a heme-binding SCHIC domain that controls the interaction of AppA with PpsR in response to light and heme availability. In this study, we have solved the structure of the SCHIC domain and identified the histidine residue that is critical for heme binding. We also demonstrate that dark-adapted AppA binds heme better than light-excited AppA does and that heme bound to the SCHIC domain significantly reduces the length of the BLUF photocycle. We further show that heme binding to the SCHIC domain is affected by the redox state of a disulfide bridge located in the Cys-rich carboxyl-terminal region. These results demonstrate that light, redox, and heme are integrated inputs that control AppA's ability to disrupt the DNA binding activity of PpsR. Photosynthetic bacteria must coordinate synthesis of the tetrapyrroles cobalamin, heme, and bacteriochlorophyll, as overproduction of the latter two is toxic to cells. A key regulator controlling tetrapyrrole biosynthesis is PpsR, and the activity of PpsR is controlled by the heme-binding and light-regulated antirepressor AppA. We show that AppA binds heme only under dark conditions and that heme binding significantly affects the length of the AppA photocycle. Since AppA interacts with PpsR only in the dark, bound heme thus stimulates the antirepressor activity of PpsR. This causes the redirection of tetrapyrrole biosynthesis away from heme into the bacteriochlorophyll branch.
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91. |
Winkler A,
Heintz U,
Lindner R,
Reinstein J,
Shoeman RL,
Schlichting I,
( 2013 ) A ternary AppA-PpsR-DNA complex mediates light regulation of photosynthesis-related gene expression. PMID : 23728293 : DOI : 10.1038/nsmb.2597 PMC : PMC3702404 Abstract >>
The anoxygenic phototrophic bacterium Rhodobacter sphaeroides uses different energy sources, depending on environmental conditions including aerobic respiration or, in the absence of oxygen, photosynthesis. Photosynthetic genes are repressed at high oxygen tension, but at intermediate levels their partial expression prepares the bacterium for using light energy. Illumination, however, enhances repression under semiaerobic conditions. Here, we describe molecular details of two proteins mediating oxygen and light control of photosynthesis-gene expression: the light-sensing antirepressor AppA and the transcriptional repressor PpsR. Our crystal structures of both proteins and their complex and hydrogen/deuterium-exchange data show that light activation of AppA-PpsR2 affects the PpsR effector region within the complex. DNA binding studies demonstrate the formation of a light-sensitive ternary AppA-PpsR-DNA complex. We discuss implications of these results for regulation by light and oxygen, highlighting new insights into blue light-mediated signal transduction.
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92. |
Li F,
Xia Y,
Meiler J,
Ferguson-Miller S,
( 2013 ) Characterization and modeling of the oligomeric state and ligand binding behavior of purified translocator protein 18 kDa from Rhodobacter sphaeroides. PMID : 23952237 : DOI : 10.1021/bi400431t PMC : PMC3756528 Abstract >>
Translocator Protein 18 kDa (TSPO), previously known as the peripheral-type benzodiazepine receptor (PBR), is a mitochondrial outer membrane protein that has been identified as a key player in cholesterol and porphyrin transport, apoptotic signaling, and cancer development, as well as neurological inflammation and disease. Despite a number of TSPO ligands whose effects have been studied with respect to these varied biological activities, the nature of their interactions with TSPO and the molecular mechanism of their effects remain controversial, in part because of the lack of an atomic-resolution structure. We expressed and purified the homologue of mammalian TSPO from Rhodobacter sphaeroides (RsTSPO), as well as a mutant form in a proposed drug binding loop, RsTSPOW38C. We characterized their binding behaviors with endogenous ligands and a series of compounds that affect apoptosis by using a sensitive tryptophan fluorescence quenching assay. Our results show that RsTSPO behaves as a dimer in the purified state and binds with low micromolar affinity to many of these ligands, including retinoic acid, curcumin, and a known Bcl-2 inhibitor, gossypol, suggesting a possible direct role for TSPO in their regulation of apoptosis. A computational model of the RsTSPO dimer is constructed using EM-Fold, Rosetta, and a cryo-electron microscopy density map. Binding behaviors of known ligands are discussed in the context of the model with respect to regions that may be involved in binding.
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93. |
( 1997 ) Nucleotide sequence and transcriptional analysis of the flanking region of the gene (spb) for the trans-acting factor that controls light-mediated expression of the puf operon in Rhodobacter sphaeroides. PMID : 9210332 : DOI : 10.1093/oxfordjournals.pcp.a029205 Abstract >>
We recently reported the existence of a trans-acting factor (SPB) in Rhodobacter sphaeroides that repressed the expression of the puf operon during illumination. SPB was somewhat homologous to HvrA of Rhodobacter capsulatus, but these proteins appear to have functionally different properties. We now report an analysis of the flanking region of spb in the genome of R.sphaeroides, and we show that spb is a positional counterpart of hvrA of R. capsulatus. The region directly downstream of spb was found to contain three genes, two of which were highly homologous to orf5 and ahcY in R. capsulatus. However, a gene corresponding to hvrB, which controls the expression of orf5 and ahcY in R. capsulatus, was absent in R. sphaeroides. The level of the transcript of ahcY did not change in cells grown under photosynthetic and by respiratory conditions. By contrast, orf5 was transcribed at a higher rate in photosynthetically grown cells under high-intensity light than under low-intensity light, indicating features of transcription different from those in R. capsulatus. A third gene, orf318, which was absent in the corresponding region of R. capsulatus, encoded an amino acid sequence that was significantly homologous to the consensus sequence of RfaI and RfaJ of E.coli, which are glycosyl transferases involved in the synthesis of lipopolysaccharide. orf318 was transcribed in the opposite direction to ahcY, and at only a low level, under all conditions tested.
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94. |
( 1997 ) A quorum-sensing system in the free-living photosynthetic bacterium Rhodobacter sphaeroides. PMID : 9393720 : DOI : 10.1128/jb.179.23.7530-7537.1997 PMC : PMC179706 Abstract >>
Rhodobacter sphaeroides is a free-living, photoheterotrophic bacterium known for its genomic and metabolic complexity. We have discovered that this purple photosynthetic organism possesses a quorum-sensing system. Quorum sensing occurs in a number of eukaryotic host-associated gram-negative bacteria. In these bacteria there are two genes required for quorum sensing, the luxR and luxI homologs, and there is an acylhomoserine lactone signal molecule synthesized by the product of the luxI homolog. In R. sphaeroides, synthesis of a novel homoserine lactone signal, 7,8-cis-N-(tetradecenoyl)homoserine lactone, is directed by a luxI homolog termed cerI. Two open reading frames immediately upstream of cerI are proposed to be components of the quorum-sensing system. The first of these is a luxR homolog termed cerR, and the second is a small open reading frame of 159 bp. Inactivation of cerI in R. sphaeroides results in mucoid colony formation on agar and formation of large aggregates of cells in liquid cultures. Clumping of CerI mutants in liquid culture is reversible upon addition of the acylhomoserine lactone signal and represents a phenotype unlike those controlled by quorum sensing in other bacteria.
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95. |
( 1997 ) Evidence for the role of redox carriers in photosynthesis gene expression and carotenoid biosynthesis in Rhodobacter sphaeroides 2.4.1. PMID : 9068641 : DOI : 10.1128/jb.179.6.1951-1961.1997 PMC : PMC178919 Abstract >>
Previous work from this laboratory revealed that alterations in the structure of the ccoNOQP operon of Rhodobacter sphaeroides 2.4.1 could lead to induction of the photosynthetic apparatus under aerobic growth conditions. Immediately downstream of the ccoNOQP operon is the rdxB gene, the first gene of the rdxBHIS cluster. The rdxB gene product is predicted to encode a membrane protein which can bind two [4Fe-4S] clusters. The ccoP gene product is a diheme cytochrome which is a component of the cbb3-type cytochrome oxidase. Under aerobic growth conditions, strains possessing ccoP and rdxB mutations both singly and in combination produced light-harvesting complexes, suggesting that normal functioning of these proteins is required to maintain repression of photosynthesis gene expression in the presence of oxygen. Analysis of the expression of puc::lacZ fusions under aerobic conditions revealed an approximately 12-fold increase in puc operon expression in the RDXB1 and CCOP1 mutant strains compared with that for wild-type 2.4.1. Similarly, puf::lacZ activity was observed to be elevated fourfold above wild-type levels. Further indication of the importance of the RdxB and CcoP proteins was derived from studies of mutant and wild-type cells grown under anoxygenic photosynthetic and nitrogen-fixing conditions. These mutant strains were observed to accumulate spheroidenone to approximately 50% or more of the total carotenoid. In wild-type cultures, spheroidenone normally accumulates to approximately 10 to 20% of the total carotenoid under the same growth conditions. This effect was most pronounced when both the rdxB and the ccoP mutations were present together in cells cultured under nitrogen-fixing photosynthetic growth conditions in which spheroidenone represented approximately 90% of the total carotenoid. We propose that mutations in the rdxB or ccoP gene may lead to changes in a membrane-generated redox signal or the accumulation of a critical redox intermediate in the mutant strains which results in increased photosynthesis gene expression under aerobic conditions by alteration of the activity of a transcriptional regulator(s) of photosynthesis gene expression. Mutations in these genes also appear to posttranscriptionally influence the terminal step of carotenoid biogenesis. Potential regulators interacting with an aberrant redox signal in the mutants and the possible nature of such a redox signal are discussed.
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96. |
( 1997 ) Characterization of the nitric oxide reductase-encoding region in Rhodobacter sphaeroides 2.4.3. PMID : 9171397 : DOI : 10.1128/jb.179.11.3534-3540.1997 PMC : PMC179145 Abstract >>
A gene cluster which includes genes required for the expression of nitric oxide reductase in Rhodobacter sphaeroides 2.4.3 has been isolated and characterized. Sequence analysis indicates that the two proximal genes in the cluster are the Nor structural genes. These two genes and four distal genes apparently constitute an operon. Mutational analysis indicates that the two structural genes, norC and norB, and the genes immediately downstream, norQ and norD, are required for expression of an active Nor complex. The remaining two genes, nnrT and nnrU, are required for expression of both Nir and Nor. The products of norCBQD have significant identity with products from other denitrifiers, whereas the predicted nnrT and nnrU gene products have no similarity with products corresponding to other sequences in the database. Mutational analysis and functional complementation studies indicate that the nnrT and nnrU genes can be expressed from an internal promoter. Deletion analysis of the regulatory region upstream of norC indicated that a sequence motif which has identity to a motif in the gene encoding nitrite reductase in strain 2.4.3 is critical for nor operon expression. Regulatory studies demonstrated that the first four genes, norCBQD, are expressed only when the oxygen concentration is low and nitrate is present but that the two distal genes, nnrTU, are expressed constitutively.
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97. |
( 1997 ) Cloning, nucleotide sequence, and overexpression of smoS, a component of a novel operon encoding an ABC transporter and polyol dehydrogenases of Rhodobacter sphaeroides Si4. PMID : 9335280 : DOI : 10.1128/jb.179.20.6335-6340.1997 PMC : PMC179547 Abstract >>
The gene coding for sorbitol dehydrogenase (SDH) of Rhodobacter sphaeroides Si4 was located 55 nucleotides upstream of the mannitol dehydrogenase gene (mtlK) within a previously unrecognized polyol operon. This operon probably consists of all the proteins necessary for transport and metabolization of various polyols. The gene encoding SDH (smoS) was cloned and sequenced. Analysis of the deduced amino acid sequence revealed homology to enzymes of the short-chain dehydrogenase/reductase protein family. For structure analysis of this unique bacterial enzyme, smoS was subcloned into the overexpression vector pET-24a(+) and then overproduced in Escherichia coli BL21(DE3), which yielded a specific activity of 24.8 U/mg of protein and a volumetric yield of 38,000 U/liter. Compared to values derived with the native host, R. sphaeroides, these values reflected a 270-fold increase in expression of SDH and a 971-fold increase in the volumetric yield. SDH was purified to homogeneity, with a recovery of 49%, on the basis of a three-step procedure. Upstream from smoS, another gene (smoK), which encoded a putative ATP-binding protein of an ABC transporter, was identified.
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98. |
( 1997 ) Structural and genetic analysis of a mutant of Rhodobacter sphaeroides WS8 deficient in hook length control. PMID : 9352903 : DOI : 10.1128/jb.179.21.6581-6588.1997 PMC : PMC179582 Abstract >>
Motility in the photosynthetic bacterium Rhodobacter sphaeroides is achieved by the unidirectional rotation of a single subpolar flagellum. In this study, transposon mutagenesis was used to obtain nonmotile flagellar mutants from this bacterium. We report here the isolation and characterization of a mutant that shows a polyhook phenotype. Morphological characterization of the mutant was done by electron microscopy. Polyhooks were obtained by shearing and were used to purify the hook protein monomer (FlgE). The apparent molecular mass of the hook protein was 50 kDa. N-terminal amino acid sequencing and comparisons with the hook proteins of other flagellated bacteria indicated that the Rhodobacter hook protein has consensus sequences common to axial flagellar components. A 25-kb fragment from an R. sphaeroides WS8 cosmid library restored wild-type flagellation and motility to the mutant. Using DNA adjacent to the inserted transposon as a probe, we identified a 4.6-kb SalI restriction fragment that contained the gene responsible for the polyhook phenotype. Nucleotide sequence analysis of this region revealed an open reading frame with a deduced amino acid sequence that was 23.4% identical to that of FliK of Salmonella typhimurium, the polypeptide responsible for hook length control in that enteric bacterium. The relevance of a gene homologous to fliK in the uniflagellated bacterium R. sphaeroides is discussed.
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99. |
( 1997 ) The dmsR gene encoding a dimethyl sulfoxide-responsive regulator for expression of dmsCBA (dimethyl sulfoxide respiration genes) in Rhodobacter sphaeroides f. sp. denitrificans. PMID : 9256068 : DOI : 10.1016/s0167-4781(97)00062-6 Abstract >>
Upstream of the dmsCBA genes encoding dimethyl sulfoxide (DMSO) reductase in the phototrophic bacterium Rhodobacter sphaeroides f. sp. denitrificans, there was found one gene (referred to dmsR) which encoded a protein composed of 232 amino acid residues and was divergently transcribed from the dmsCBA genes. The deduced amino acid sequence was homologous to the OmpR subfamily of response regulators in two-component systems for transcriptional regulation. The encoded protein DmsR was shown to bind to a DNA fragment containing the four direct repeats of a decameric nucleotide motif located between the dmsR and dmsCBA genes. The DNA-binding activity of DmsR was observed when the organism was grown anaerobically in the presence of DMSO. A dmsR-defected mutant strain showed no synthesis of DMSO reductase. These results indicate that the dmsR gene product acts as a positive regulator for expression of the dmsCBA genes in response to DMSO under anaerobic growth conditions.
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100. |
( 1997 ) Mutation in ntrC gene leading to the derepression of nitrogenase synthesis in Rhodobacter sphaeroides. PMID : 9037764 : DOI : 10.1111/j.1574-6968.1997.tb10220.x Abstract >>
The Rhodobacter sphaeroides mutants Drn12 and Drn21 derepressed for nitrogenase synthesis in the presence of ammonia and impaired in utilization of certain nitrogen sources have been analyzed. Both mutants show a low level of expression of the glnBA operon. The DNA fragment restoring the wild-type phenotype to these mutants contains the 3'-portion of ntrB gene and the entire ntrC gene. Sequence analysis showed that Drn12 bears a missense mutation in the ntrC gene. The mutation results in the replacement of a glycine residue by aspartate within the N-terminal domain of the NtrC protein. Pleiotropic phenotypes of Drn12 and Drn21 appear to be associated with an alteration in the regulation of glnBA expression.
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101. |
( 1993 ) Isolation and functional expression in Escherichia coli of a gene encoding phosphatidylethanolamine methyltransferase (EC 2.1.1.17) from Rhodobacter sphaeroides. PMID : 8340421 : Abstract >>
Phosphatidylcholine is a major component of membranes in most eukaryotes, but it is found only in a small number of bacteria, where it is synthesized by N-methylation of phosphatidylethanolamine. In yeast and other fungi the methylation of phosphatidylethanolamine to phosphatidylcholine proceeds in two steps: the methylation of phosphatidylethanolamine by phosphatidylethanolamine methyltransferase followed by the methylation of monomethylphosphatidylethanolamine by phospholipid methyltransferase. Here we describe the isolation of two allelic phosphatidylcholine-deficient mutants of Rhodobacter sphaeroides which are unable to methylate phosphatidylethanolamine, monomethylphosphatidylethanolamine, or dimethylphosphatidylethanolamine. A DNA fragment containing a gene designated pmtA, which encodes a 22.9-kDa protein, was found to complement both mutants. Expression of this gene in Escherichia coli, which normally lacks phosphatidylcholine or methylated derivatives of phosphatidylethanolamine, resulted in the formation of phosphatidylcholine. A protein extract derived from the E. coli strain expressing the pmtA gene was able to convert phosphatidylethanolamine, mono- and dimethylphosphatidylethanolamine into phosphatidylcholine. Based on these data we conclude that the product of the pmtA gene catalyzes a sequence of three chemically distinct, methylation reactions beginning with phosphatidylethanolamine and leading to the formation of phosphatidylcholine in R. sphaeroides.
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102. |
( 1996 ) Molecular cloning of dimethyl sulfoxide reductase from Rhodobacter sphaeroides. PMID : 8645727 : DOI : 10.1016/0167-4838(96)00015-5 Abstract >>
The dsrA gene encoding the molybdoenzyme dimethyl sulfoxide reductase was isolated by screening phagemid libraries containing restriction fragments of Rhodobacter sphaeroides f. sp. denitrificans genomic DNA with a pool of degenerate oligonucleotides. The encoded 822 amino-acid protein includes a 42 amino-acid periplasmic signal sequence that is cleaved during activation of the enzyme. Both forms of the protein were heterologously expressed in inactive states in E. coli and identified by Western blot analysis.
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103. |
( 1993 ) Cloning, nucleotide sequence and characterization of the mannitol dehydrogenase gene from Rhodobacter sphaeroides. PMID : 8254318 : DOI : 10.1099/00221287-139-10-2475 Abstract >>
Transposon mutagenesis and antibiotic enrichment were employed to isolate a mutant of Rhodobacter sphaeroides Si4 designated strain M22, that had lost the ability to grow on D-mannitol and to produce the enzyme mannitol dehydrogenase (MDH). DNA flanking the transposon in the mutant strain was used as a probe for the identification and cloning of the MDH gene (mtlK). A 5.5 kb EcoRI/BglII fragment from R. sphaeroides Si4 was isolated and shown to complement the mutation in R. sphaeroides M22. Successful complementation required that a promoter of the vector-plasmid pRK415 be present, suggesting that the mtlK gene is part of a larger operon. Using oligonucleotides derived from the N-terminal sequence of MDH as probes mtlK was located on the complementing fragment and the gene was sequenced. The mtlK open reading frame encodes a protein of 51,404 Da with an N-terminal sequence identical to that obtained from amino acid analysis of the purified MDH. The MDH of R. sphaeroides Si4 exhibits distant similarity to the mannitol-1-phosphate dehydrogenases from Escherichia coli and Enterococcus faecalis, with 28.1% and 26.3% identity, respectively. Mutant strains deficient in MtlK displayed substantial levels of sorbitol dehydrogenase activity, originally thought to be only a minor activity associated with the MDH enzyme. It is likely that we have uncovered an additional polyol dehydrogenase with activity for sorbitol. The mtlK gene can be used for overexpression of MDH in E. coli in order to obtain sufficient amounts of enzyme for further investigations and applications.
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104. |
( 1996 ) The Rhodobacter sphaeroides 2.4.1 rho gene: expression and genetic analysis of structure and function. PMID : 8606169 : DOI : 10.1128/jb.178.7.1946-1954.1996 PMC : PMC177890 Abstract >>
The gene which encodes transcription termination factor Rho from Rhodobacter sphaeroides 2.4.1, the gram-negative facultative photosynthetic bacterium, has been cloned and sequenced. The deduced protein shows a high level of sequence similarity to other bacterial Rho factors, especially those from proteobacteria. However, several amino acid substitutions in the conserved ATP-binding site have been identified. When expressed in Escherichia coli, the R. sphaeroides rho gene relieves Rho-dependent polarity of the trp operon, indicating interference with the transcription termination machinery of E. coli. A truncated version of R. sphaeroides Rho (Rho') is toxic to a bacterium related to R. sphaeroides, Paracoccus denitrificans, and is lethal to R. sphaeroides. We suggest that toxicity is due to the ability of Rho' to form inactive heteromers with the chromosomally encoded intact Rho. We localized a minimal amino acid sequence within Rho which appears to be critical for its toxic effect and which we believe may be involved in protein-protein interactions. This region was previously reported to be highly conserved and unique among various Rho proteins. The lethality of rho' in R. sphaeroides together with our inability to obtain a null mutation in rho suggests that Rho-dependent transcription termination is essential in R. sphaeroides. This is analogous to what is observed for gram-negative E. coli and contrasts with what is observed for gram-positive Bacillus subtilis. The genetic region surrounding the R. sphaeroides rho gene has been determined and found to be different compared with those of other bacterial species. rho is preceded by orf1, which encodes a putative integral membrane protein possibly involved in cytochrome formation or functioning. The gene downstream of rho is homologous to thdF, whose product is involved in thiophene and furan oxidation.
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105. |
( 1996 ) Sequential 1H and 15N NMR resonance assignment and secondary structure of ferrocytochrome c2 from Rhodobacter sphaeroides. PMID : 8827449 : DOI : 10.1093/oxfordjournals.jbchem.a021359 Abstract >>
Sequence-specific 1H and 15N assignments have been made for the amino acids of the ferrocytochrome c2 from Rhodobacter sphaeroides. Initial assignments were made by analysis of a series of homonuclear 2D COSY, TOCSY, and NOESY spectra obtained with the unlabeled protein. 2D and 3D 1H-15N correlated spectra obtained for a uniformly 15N-labeled ferrocytochrome c2 were used to confirm and extend the assignments. Partial 13C assignments have also been made by means of HSQC experiments on 13C at natural abundance, in particular for about two-thirds of the 13C alpha. Medium-range NOE connectivities, together with 3J(HC alpha NH) coupling constants, indicated the presence of five helices at positions 6-16, 60-68, 74-82, 84-91, and 109-120. No other regular secondary structure was observed. This folding is similar to that previously observed for the ferrocytochrome c2 of Rhodobacter capsulatus in solution, which exhibits approximately 50% sequence identity. Moreover, the rotation rates of the aromatic rings of phenylalanine or tyrosine, when conserved, were similar to those observed for R. capsulatus. Furthermore, C alpha H chemical shifts, which are sensitive to the secondary structure and ring current effects of the heme, appear to be very similar for the two proteins. Consequently, the solution structure of R. sphaeroides ferrocytochrome c2 appears to be very similar to that of R. capsulatus ferrocytochrome c2. These results are compared with the X-ray crystal structure of the R. sphaeroides ferrocytochrome c2.
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106. |
( 1996 ) 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. PMID : 8759915 : DOI : 10.1093/oxfordjournals.pcp.a028974 Abstract >>
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.
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107. |
( 1996 ) Cloning and characterization of nnrR, whose product is required for the expression of proteins involved in nitric oxide metabolism in Rhodobacter sphaeroides 2.4.3. PMID : 8759861 : DOI : 10.1128/jb.178.16.4958-4964.1996 PMC : PMC178280 Abstract >>
During denitrification, the production and consumption of nitric oxide (NO), an obligatory and freely diffusible intermediate, must be tightly regulated in order to prevent accumulation of this highly reactive nitrogen oxide. Sequencing upstream of norCB, the structural genes for NO reductase, in the denitrifying bacterium Rhodobacter sphaeroides 2.4.3, we have identified a gene, designated nnrR, which encodes a protein that is a member of the cyclic AMP receptor family of transcriptional regulators. Insertional inactivation of nnrR prevents growth on nitrite, as well as the reduction of nitrite and NO, but has no effect on reduction of nitrate or photosynthetic growth. By using nirK-lacZ and norB-lacZ fusions, we have shown that NnrR is a positive transcriptional regulator of these genes. nnrR is expressed at a low constitutive level throughout the growth of R. sphaeroides 2.4.3. These results show that NnrR is not a global regulator but is instead a regulator of genes whose products are directly responsible for production and reduction of NO. Evidence is also presented suggesting that an NnrR homolog may be present in the nondenitrifying bacterium R. sphaeroides 2.4.1. The likely effector of NnrR activity, as determined on the basis of work detailed in this paper and other studies, is discussed.
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108. |
( 1993 ) Genetic analysis of the bchC and bchA genes of Rhodobacter sphaeroides. PMID : 8437569 : DOI : 10.1007/bf00277117 Abstract >>
This study has identified by sequence analysis a single gene in the bchC locus of Rhodobacter sphaeroides and three genes, designated bchX, Y and Z, in the bchA locus, which was previously thought to contain only a single gene. All four genes may reside within the same operon and are transcribed in the order bchC-X-Y-Z. Complementation analysis of eight transposon insertion mutants within these genes suggests that bchX, Y and Z are essential for the reduction of 2-devinyl-2-hydroxyethyl chlorophyllide a and that bchC encodes the 2-desacetyl-2-hydroxyethyl bacteriochlorophyllide a dehydrogenase. Similarity between the putative BchX protein and dinitrogenase reductase proteins suggests that BchX may also be a reductase, supplying electrons for reduction of 2-devinyl-2-hydroxyethyl chlorophyllide a.
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109. |
( 1996 ) Cloning of the fliI gene from Rhodobacter sphaeroides WS8 by analysis of a transposon mutant with impaired motility. PMID : 8759796 : DOI : 10.1111/j.1574-6968.1996.tb08416.x Abstract >>
A transposon mutant of Rhodobacter sphaeroides WS8 was isolated that showed reduced swarming on soft agar plates. Liquid cultures of this mutant (M18) showed a low percentage of motile swimming cells in mid-exponential phase and a low level of extracellular flagellin protein by Western blotting. M18 was complemented by a clone from a library of R. sphaeroides WS8 DNA, and restriction mapping of the site of TnphoA insertion in the mutant, coupled with DNA sequencing, showed that it had a defect in the fliI gene. To determine if a partly functional fliI gene was giving the low-motility phenotype of M18, a drug resistance omega cartridge was inserted into the gene to give a complete null mutant. This null strain also produced a low percentage of motile cells. Possible reasons for this apparent fliI-independent flagellar formation are discussed.
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110. |
( 1996 ) Cloning of the Rhodobacter sphaeroides hisL gene: unifunctionality of the encoded protein and lack of linkage to other his genes. PMID : 8760919 : DOI : 10.1099/13500872-142-8-2071 Abstract >>
The Rhodobacter sphaeroides 2.4.1 hisI gene, which encodes a phosphoribosyl-AMP-cyclohydrolase that catalyses the third step in the histidine biosynthetic pathway, has been isolated from a genomic library of this phototrophic bacterium by complementation of an Escherichia coli hisI mutant. Analysis of the nucleotide sequence of the R. sphaeroides hisI gene reveals that it encodes a deduced product of 119 aa with a predicted molecular mass of 13.4 kDa. In contrast to the situation in E. coli, the R. sphaeroides hisI gene encodes a unifunctional protein and it is not linked to the hisE gene. The absence of a single histidine operon like that of E. coli was confirmed by PFGE experiments and complementation analysis of a R. sphaeroides hisI mutant that was constructed by marker exchange. The location of hisI in the R. sphaeroides genome has been determined to be at map co-ordinate 2275 +/- 20 of chromosome l.
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( 1993 ) Nucleotide sequence and functional analysis of cbbR, a positive regulator of the Calvin cycle operons of Rhodobacter sphaeroides. PMID : 8376325 : DOI : 10.1128/jb.175.18.5778-5784.1993 PMC : PMC206655 Abstract >>
Structural genes encoding Calvin cycle enzymes in Rhodobacter sphaeroides are duplicated and organized within two physically distinct transcriptional units, the form I and form II cbb operons. Nucleotide sequence determination of the region upstream of the form I operon revealed a divergently transcribed open reading frame, cbbR, that showed significant similarity to the LysR family of transcriptional regulatory proteins. Mutants containing an insertionally inactivated cbbR gene were impaired in photoheterotrophic growth and completely unable to grow photolithoautotrophically with CO2 as the sole carbon source. In the cbbR strain, expression of genes within the form I operon was completely abolished and that of the form II operon was reduced to about 30% of the wild-type level. The cloned cbbR gene complemented the mutant for wild-type growth characteristics, and normal levels of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) were observed. However, rocket immunoelectrophoresis revealed that the wild-type level of RubisCO was due to overexpression of the form II enzyme, whereas expression of the form I RubisCO was 10% of that of the wild-type strain. The cbbR insertional inactivation did not appear to affect aerobic expression of either CO2 fixation operon, but preliminary evidence suggests that the constitutive expression of the form II operon observed in the cbbR strain may be subject to repression during aerobic growth.
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( 1996 ) Isolation of periplasmic nitrate reductase genes from Rhodobacter sphaeroides DSM 158: structural and functional differences among prokaryotic nitrate reductases. PMID : 8730872 : DOI : 10.1111/j.1365-2958.1996.tb02475.x Abstract >>
The phototrophic bacterium Rhodobacter sphaeroides DSM 158 has a periplasmic nitrate reductase which is induced by nitrate and it is not repressed by ammonium or oxygen. In a Tn5 mutant lacking nitrate reductase activity, transposon insertion is localized in a 1.2 kb EcoRI fragment. A 0.6 kb BamHI-EcoRI segment of this region was used as a probe to isolate, from the wild-type strain, a 6.8 kb PstI fragment carrying the putative genes coding for the periplasmic nitrate reductase. In vivo protein expression and DNA sequence analysis reveal the presence in this region of three genes, napABC, probably organized in an operon. These genes are required for nitrate reduction, as deduced by mutational and complementation studies. The napA gene codes for a protein with a high homology to the periplasmic nitrate reductase from Alcaligenes eutrophus and, to a lesser extent, to other prokaryotic nitrate reductases and molybdenum-containing enzymes. The napB gene product has two haem c-binding sites and shows a high homology with the cytochrome c-type subunit of the periplasmic nitrate reductase from A. eutrophus. NAPA and NAPB proteins appear to be translated with signal peptides of 29 and 24 residues, respectively, indicating that mature proteins are located in the periplasm. The napC gene codes for a 25 kDa protein with a transmembrane sequence of 17 hydrophobic residues. NAPC has four haem c-binding sites and is homologous to the membrane-bound c-type cytochromes encoded by Pseudomonas stutzeri nirT and Escherichia coli torC genes. The phenotypes of defined insertion mutants constructed for each gene also indicate that periplasmic nitrate reductase from R. sphaeroides DSM 158 is a dimeric complex of a 90 kDa catalytic subunit (NAPA) and a 15 kDa cytochrome c (NAPB), which receives electrons from a membrane-anchored tetrahaem protein (NAPC), thus allowing electron flow between membrane and periplasm. This nitrate-reducing system differs from the assimilatory and respiratory bacterial nitrate reductases at the level of cellular localization, regulatory properties, biochemical characteristics and gene organization.
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( 1997 ) Analysis of the cbbXYZ operon in Rhodobacter sphaeroides. PMID : 9006018 : DOI : 10.1128/jb.179.3.663-669.1997 PMC : PMC178745 Abstract >>
Three genes, cbbX, cbbY, and cbbZ were found downstream from the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes of Rhodobacter sphaeroides. As in chemoautotrophic bacteria, cbbZ was shown to encode phosphoglycolate phosphatase (PGP), whereas the identities of cbbX and cbbY are not known. To determine the physiological function of the cbbXYZ gene products, we constructed R. sphaeroides strains in which the genes were inactivated and characterized the resultant mutant strains according to growth phenotype and levels of RubisCO and PGP. Only a mutation in cbbX resulted in a discernible phenotype, namely, impaired photoautotrophic growth. No PGP activity was observed in any of the mutants, suggesting that the three genes are transcriptionally linked. Studies with a spontaneous chemoautotrophic competent derivative of the CBBX mutant suggested that the cbbXYZ gene products are not essential for chemoautotrophic growth. PGP activity determined in the wild-type strain grown under a variety of growth conditions, and in various strains containing mutations in Calvin-Benson-Bassham cycle structural and regulatory genes, indicated that transcription of the cbb(I) operon influenced expression of the downstream cbbXYZ operon.
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( 1997 ) Cloning and characterization of two groESL operons of Rhodobacter sphaeroides: transcriptional regulation of the heat-induced groESL operon. PMID : 8990302 : DOI : 10.1128/jb.179.2.487-495.1997 PMC : PMC178720 Abstract >>
The nonsulfur purple bacterium Rhodobacter sphaeroides was found to contain two groESL operons. The groESL1 heat shock operon was cloned from a genomic library, and a 2.8-kb DNA fragment was sequenced and found to contain the groES and groEL genes. The deduced amino acid sequences of GroEL1 (cpn60) and GroES1 (cpn10) were in agreement with N-terminal sequences previously obtained for the isolated proteins (K. C. Terlesky and F. R. Tabita, Biochemistry 30:8181-8186, 1991). These sequences show a high degree of similarity to groESL genes isolated from other bacteria. Northern analysis indicated that the groESL1 genes were expressed as part of a 2.2-kb polycistronic transcript that is induced 13-fold after heat shock. Transcript size was not affected by heat shock; however, the amount of transcript was induced to its greatest extent 15 to 30 min after a 40 degrees C heat shock, from an initial temperature of 28 degrees C, and remained elevated up to 120 min. The R. sphaeroides groESL1 operon contains a putative hairpin loop at the start of the transcript that is present in other bacterial heat shock genes. Primer extension of the message showed that the transcription start site is at the start of this conserved hairpin loop. In this region were also found putative -35 and -10 sequences that are conserved upstream from other bacterial heat shock genes. Transcription of the groESL1 genes was unexpectedly low under photoautotrophic growth conditions. Thus far, it has not been possible to construct a groESL1 deletion strain, perhaps indicating that these genes are essential for growth. A second operon (groESL2) was also cloned from R. sphaeroides, using a groEL1 gene fragment as a probe; however, no transcript was observed for this operon under several different growth conditions. A groESL2 deletion strain was constructed, but there was no detectable change in the phenotype of this strain compared to the parental strain.
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( 1997 ) Molecular systematic studies of eubacteria, using sigma70-type sigma factors of group 1 and group 2. PMID : 9045836 : DOI : 10.1128/jb.179.5.1734-1747.1997 PMC : PMC178889 Abstract >>
Sigma factors of the sigma70 family were used as a phylogenetic tool to compare evolutionary relationships among eubacteria. Several new sigma factor genes were cloned and sequenced to increase the variety of available sequences. Forty-two group 1 sigma factor sequences of various species were analyzed with the help of a distance matrix method to establish a phylogenetic tree. The tree derived by using sigma factors yielded subdivisions, including low-G+C and high-G+C gram-positive bacteria, cyanobacteria, and the alpha, beta, gamma, and delta subdivisions of proteobacteria, consistent with major bacterial groups found in trees derived from analyses with other molecules. However, some groupings (e.g., the chlamydiae, mycoplasmas, and green sulfur bacteria) are found in different positions than for trees obtained by using other molecular markers. A direct comparison to the most extensively used molecule in systematic studies, small-subunit rRNA, was made by deriving trees from essentially the same species set and using similar phylogenetic methods. Differences and similarities based on the two markers are discussed. Additionally, 31 group 2 sigma factors were analyzed in combination with the group 1 proteins in order to detect functional groupings of these alternative sigma factors. The data suggest that promoters recognized by the major vegetative sigma factors of eubacteria will contain sequence motifs and spacing very similar to those for the sigma70 sigma factors of Escherichia coli.
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( 1997 ) Thioredoxin is essential for Rhodobacter sphaeroides growth by aerobic and anaerobic respiration. PMID : 9025281 : DOI : 10.1099/00221287-143-1-83 Abstract >>
To investigate the biological role of thioredoxin in the facultative photosynthetic bacterium Rhodobacter sphaeroides, attempts were made to construct a thioredoxin-deficient mutant by site-specific mutagenesis, using the Tn903 kanamycin resistance gene for selection. In situ and Southern hybridization analyses have demonstrated that the TrxA- mutation is lethal for R. sphaeroides growth under anaerobic conditions with DMSO as terminal electron acceptor and under aerobic conditions. In addition, the DNA region upstream of the trxA initiation codon is essential for aerobic growth of R. sphaeroides. An ORF of unknown function was identified in this region and is suggested to encode a product essential for aerobic metabolism of R. sphaeroides. The mechanism of thioredoxin action was also analysed by using the procedure for gene replacement to introduce a Cys33 to Ser mutation into the trxA chromosomal copy. The strain carrying this mutation produced a thioredoxin impaired in its protein-disulfide reductase activity and was also not viable. These data suggest that the physiological function of R. sphaeroides thioredoxin is redox-dependent. Thioredoxin purified from R. sphaeroides was shown to have a glutathione-disulfide oxidoreductase activity typical of glutaredoxins. This unexpected finding suggests that R. sphaeroides thioredoxin, in contrast to Escherichia coli thioredoxin, has the potential to act in GSH-dependent processes. Thus, the fundamental role of R. sphaeroides thioredoxin in cell growth probably originates from the multiple functions it can serve in vivo.
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( 1996 ) Characterization of a glutathione-dependent formaldehyde dehydrogenase from Rhodobacter sphaeroides. PMID : 8631716 : DOI : 10.1128/jb.178.5.1386-1393.1996 PMC : PMC177813 Abstract >>
Glutathione-dependent formaldehyde dehydrogenases (GSH-FDH) represent a ubiquitous class of enzymes, found in both prokaryotes and eukaryotes. During the course of studying energy-generating pathways in the photosynthetic bacterium Rhodobacter sphaeroides, a gene (adhI) encoding a GSH-FDH homolog has been identified as part of an operon (adhI-cycI) that also encodes an isoform of the cytochrome c2 family of electron transport proteins (isocytochrome c2). Enzyme assays with crude Escherichia coli extracts expressing AdhI show that this protein has the characteristic substrate preference of a GSH-FDH. Ferguson plot analysis with zymograms suggests that the functional form of AdhI is a homodimer of approximately40-kDa subunits, analogous to other GSH-FDH enzymes. These properties of AdhI were used to show that mutations which increase or decrease adhI expression change the specific activity of GSH-FDH in R. sphaeroides extracts. In addition, expression of the presumed adhI-cycI operon appears to be transcriptionally regulated, since the abundance of the major adhI-specific primer extension product is increased by the trans-acting spd-7 mutation, which increases the level of both isocytochrome c2 and AdhI activity. While transcriptional linkage of adhI and cycI could suggest a function in a common metabolic pathway, isocytochrome c2 (periplasm) and AdhI (cytoplasm) are localized in separate compartments of R. sphaeroides. Potential roles for AdhI in carbon and energy generation and the possible relationship of GSH-FDH activity to isocytochrome c2 will be discussed based on the commonly accepted physiological functions of GSH-FDH enzymes in prokaryotes and eukaryotes.
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( 1996 ) Crystal structure of DMSO reductase: redox-linked changes in molybdopterin coordination. PMID : 8658134 : DOI : 10.1126/science.272.5268.1615 Abstract >>
The molybdoenzyme dimethylsulfoxide (DMSO) reductase contributes to the release of dimethylsulfide, a compound that has been implicated in cloud nucleation and global climate regulation. The crystal structure of DMSO reductase from Rhodobacter sphaeroides reveals a monooxo molybdenum cofactor containing two molybdopterin guanine dinucleotides that asymmetrically coordinate the molybdenum through their dithiolene groups. One of the pterins exhibits different coordination modes to the molybdenum between the oxidized and reduced states, whereas the side chain oxygen of Ser147 coordinates the metal in both states. The change in pterin coordination between the Mo(VI) and Mo(IV) forms suggests a mechanism for substrate binding and reduction by this enzyme. Sequence comparisons of DMSO reductase with a family of bacterial oxotransferases containing molybdopterin guanine dinucleotide indicate a similar polypeptide fold and active site with two molybdopterins within this family.
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( 1996 ) Nucleotide sequence of the genes, encoding the pentaheme cytochrome (dmsC) and the transmembrane protein (dmsB), involved in dimethyl sulfoxide respiration from Rhodobacter sphaeroides f. sp. denitrificans. PMID : 8950368 : DOI : 10.1016/s0005-2728(96)00101-6 Abstract >>
The nucleotide sequence of the genes encoding a pentaheme cytochrome (dmsC) and a transmembrane protein (dmsB) were determined upstream of the dmsA gene encoding dimethyl sulfoxide reductase from Rhodobacter sphaeroides f. sp. denitrificans. dmsC and dmsB encode proteins of 404 and 226 amino acid residues, which show 40% and 26% identity to the pentaheme cytochrome TorC and the transmembrane protein TorD, respectively, of the trimethylamine N-oxide reduction system in Escherichia coli.
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( 1996 ) Flagellar genes from Rhodobacter sphaeroides are homologous to genes of the fliF operon of Salmonella typhimurium and to the type-III secretion system. PMID : 8621091 : DOI : 10.1016/0378-1119(95)00855-1 Abstract >>
A flagellar region of the genome of Rhodobacter sphaeroides was cloned and sequenced. Three ORFs were identified and arranged in the same order as fliH, fliI and fliJ of Salmonella typhimurium (St). ORF2 is highly similar to FliI from St (49% similarity) showing Walker's A and B motifs. Similar scores were found with proteins of the type-III secretion system of virulence factors. ORF3 shows 16.4 and 11.1% similarity to FliJ from St and Bacillus subtilis, respectively. This work also shows that ORF3 is similar to HrpJ5 from Pseudomonas syringae (19.2% similarity). It was found that ORF2 and ORF3 start immediately downstream from the adjacent coding region, suggesting a single transcriptional unit.
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( 1996 ) Co-crystallization and characterization of the photosynthetic reaction center-cytochrome c2 complex from Rhodobacter sphaeroides. PMID : 8611557 : DOI : 10.1021/bi9522054 Abstract >>
The photosynthetic reaction center (RC) of Rhodobacter sphaeroides and cytochrome c2 (cyt c2), its physiological secondary electron donor, have been co-crystallized. The molar ratio of RC/cyt c2 was found by SDS-PAGE and optical absorbance changes in the co-crystals to be 4. The crystals diffracted X-rays to 3.5 angstroms. However, the resolution degraded during data collection. A data set, 82.5% complete, was collected to 4.5 angstroms. The crystals belong to the tetragonal space group P4(3)2(1)2, with unit cell dimensions of a = b = 142.7 angstroms and c = 254.8 angstroms. The positions of the RCs in the unit cell were determined by molecular replacement. A comparable search for the cyt c2 by this method was unsuccessful because of the small contribution of the cytochrome to the total scattering and because of its low occupancy. The cyt c2 was positioned manually into patches of difference electron density, adjacent to the periplasmic surface of the M polypeptide subunit of the RC. The difference electron density was not sufficient for precise positioning of the cyt c2, and its orientation was modeled by placing the exposed edge of the heme toward the primary donor of the reaction center D and by forming pairs for electrostatically interacting RC and cyt c2 amino acid residues. The RC-cyt c2 structure derived from the co-crystal data was supported by use of omit maps and structure refinement analyses. Cyt c2 reduces the photooxidized primary donor D+ in 0.9 +/- 0.1 micros in the co-crystals, which is the same as the fast electron transfer rate in vivo and in solution. This result provides strong evidence that the structure of the complex in the co-crystal is the same as in solution. Two additional methods were used to investigate the structure of the RC-cyt c2 complex: (i) Docking calculations based on interprotein electrostatic interactions identified possible binding positions of the cyt c2 on the RC. The cyt c2 position with the lowest electrostatic energy is very similar to that of the cyt c2 in the proposed co-crystal structure. (ii) Site-directed mutagenesis was used to modify two aspartic acid residues (M184 and L155) on the periplasmic surface of the RC. Cyt c2 binding affinity to these RCs and electron transfer rates to D+ in these RCs support the co-crystal structure of th RC-cyt c2 complex.
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( 1995 ) Analysis of the motA flagellar motor gene from Rhodobacter sphaeroides, a bacterium with a unidirectional, stop-start flagellum. PMID : 8596445 : DOI : 10.1111/j.1365-2958.1995.mmi_17050961.x Abstract >>
Rhodobacter sphaeroides swims by unidirectional rotation of a single medial flagellum, re-orienting randomly by Brownian motion when flagellar rotation stops and restarts. Previously we identified a mutant with a paralysed flagellum, which was complemented by a Rhodobacter gene that had homology to motB of Escherichia coli, a bacterium with bidirectional flagella. In the current work, interposon mutagenesis upstream of the Rhodobacter motB gene gave rise to another paralysed mutant, RED5. DNA sequence analysis of this upstream region showed one open reading frame, the predicted polypeptide sequence of which shows homology to the MotA protein of E. coli. MotA is thought to be a proton 'pore' involved in converting proton-motive force into flagellar rotation. Several potential proton-binding amino acids were conserved between putative membrane-spanning regions of R. sphaeroides and E. coli MotA sequences, along with a highly charged cytoplasmic linker region. Complementation studies with mutant RED5 showed the presence of an active promoter upstream from motA which was found to be necessary for expression of both motA and motB. Examination of the upstream DNA sequence showed only one putative promoter-like sequence which resembled a sigma 54-type promoter, including a potential enhancer binding site. The overall similarities between the R. sphaeroides MotA protein and those from other bacteria suggest that, despite the novel unidirectional rotation of the R. sphaeroides flagellum, the function of the MotA protein is similar to that in bacteria with bidirectional flagella.
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( 1995 ) Identification of a methyl-accepting chemotaxis protein in Rhodobacter sphaeroides. PMID : 8596451 : DOI : 10.1111/j.1365-2958.1995.mmi_18010115.x Abstract >>
Analysis of the DNA sequence directly upstream of the chemotaxis operon of Rhodobacter sphaeroides identified a single gene whose product has strong similarity to the methyl-accepting chemotaxis proteins (MCPs) found in enteric bacteria. The deduced protein had a highly conserved signalling sequence and only one very hydrophobic region at the N-terminus, in contrast to enteric MCPs. A possible cytoplasmic location of the majority of the protein was supported by Western blotting. The mcpA gene was insertionally inactivated and the resulting phenotype examined using swarm plate assays. The mutant lacking McpA lost chemotaxis to a wide range of attractant stimuli but only under aerobic conditions; it retained almost normal chemotaxis under anaerobic/photosynthetic conditions. The identification of a sensory protein which is active only under one set of growth conditions suggests that R. sphaeroides probably has several MCPs, which co-ordinately respond to changes in environmental conditions. Southern hybridization at relaxed stringency to the conserved sequence of the R. sphaeroides and Caulobacter crescentus mcp genes identified three possible additional mcp genes.
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( 1996 ) mgpS, a complex regulatory locus involved in the transcriptional control of the puc and puf operons in Rhodobacter sphaeroides 2.4.1. PMID : 8550440 : DOI : 10.1128/jb.178.1.35-45.1996 PMC : PMC177618 Abstract >>
A new method has been developed in order to select mutants showing decreased puc operon transcription in Rhodobacter sphaeroides 2.4.1. A transcriptional fusion of a promoterless fragment derived from the sacB gene, encoding the levansucrase from Bacillus subtilis, to the upstream regulatory region of the puc operon has been constructed. With appropriate levels of exogenous sucrose, survivors of a sucrose killing challenge have been isolated. Subsequent analysis revealed the presence of both cis- and trans-acting "down" mutations in relation to puc operon expression. One of the trans-acting regulatory mutations was chosen for further study. The original mutation showed less than 2% of the level of puc operon transcription compared with the wild type under aerobic conditions and an 86% reduction under dark dimethyl sulfoxide conditions. This mutation can be complemented by a 3.9-kb BamHI DNA fragment derived from a cosmid contained within a genomic cosmid bank. DNA sequence analysis of this fragment revealed the presence of a 2.8-kb open reading frame, designated mgpS, which would encode a 930-amino-acid protein. The N-terminal portion of the putative protein product presents homologies to proteins of the RNA helicase family. Disruption of the chromosomal mgpS resulted in decreased transcription of both puc and puf, while the presence of mgpS in multicopy in the wild type, 2.4.1., increased puc expression by a factor of 2 under aerobic conditions. Structural analysis of the mgpS locus revealed that expression of mgpS was likely to be complex. A smaller protein containing the 472 C-terminal amino acids of MgpS is able to act by itself as an activator of puc transcription and is expressed independently of the large open reading frame in which it is contained.
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( 1995 ) Cloning and nucleotide sequence of the gene encoding dimethyl sulfoxide reductase from Rhodobacter sphaeroides f. sp. denitrificans. PMID : 8534974 : Abstract >>
The gene encoding dimethyl sulfoxide (DMSO) reductase, which contains a molybdenum cofactor, of the phototrophic bacterium Rhodobacter sphaeroides f. sp. denitrificans was isolated using an oligonucleotide probe, which was synthesized based on a internal amino acid sequence of the purified enzyme. The DMSO reductase gene coded for 822 amino acids (2466 base pairs, M(r) = 89,206) as a precursor form having a signal peptide of 42 amino acids. The deduced amino acid sequence had high homology with those of some enzymes containing a molybdenum cofactor: trimethyl amine N-oxide reductase (48%), biotin sulfoxide reductase (44%), and DMSO reductase (29%) of Escherichia coli.
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( 1994 ) A novel cytochrome c oxidase from Rhodobacter sphaeroides that lacks CuA. PMID : 8130226 : DOI : 10.1021/bi00176a046 Abstract >>
Rhodobacter sphaeroides contains at least two different cytochrome c oxidases. When these bacteria are grown with high aeration, the traditional aa3-type cytochrome c oxidase is present at relatively high levels. However, under microaerophilic growth conditions or when the bacteria are grown photosynthetically, the amount of the aa3-type oxidase is greatly diminished and an alternate cytochrome c oxidase is evident. This alternate oxidase has been purified and characterized. The enzyme consists of three subunits by SDS-PAGE analysis (Mapp 45, 35, and 29 kDa). Two of the three subunits (Mapp 35 and 29 kDa) contain covalently bound heme C. Metal and heme analyses indicate that the oxidase contains heme C, heme B (protoheme IX), and Cu in a ratio of 3:2:1. Cryogenic Fourier transform infrared (FTIR) difference spectroscopy of the CO adduct of the reduced enzyme shows that the oxidase contains a heme-copper binuclear center and, thus, is a member of the heme-copper oxidase superfamily. In contrast to other members of this superfamily, however, this oxidase does not contain either heme O or heme A as a component of the binuclear center, but has heme B at this site. The single equivalent of Cu found in the oxidase is accounted for by the CuB component at the binuclear center. This suggests that this oxidase does not contain CuA, which is found in all other well-characterized cytochrome c oxidases. Both EPR and optical spectroscopic studies are consistent with this conclusion, also indicating that this oxidase does not contain CuA.(ABSTRACT TRUNCATED AT 250 WORDS)
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( 1996 ) A global signal transduction system regulates aerobic and anaerobic CO2 fixation in Rhodobacter sphaeroides. PMID : 8550404 : DOI : 10.1128/jb.178.1.12-18.1996 PMC : PMC177615 Abstract >>
Complementation of a mutant of Rhodobacter sphaeroides defective in photosynthetic CO2 reduction led to the identification of a gene which encodes a protein that is related to a class of sensor kinases involved in bacterial signal transduction. The nucleotide sequence and deduced amino acid sequence led to the finding that the gene which complemented the mutant is the regB (prrB) gene, previously isolated from both R. sphaeroides and Rhodobacter capsulatus and shown to regulate the anaerobic expression of structural genes required for the synthesis of the reaction center and light-harvesting systems of these organisms. The current investigation indicates that in addition to its role in the regulation of photosystem biosynthesis, regB (prrB) of R. sphaeroides is intimately involved in the positive regulation of the cbbI and cbbII Calvin cycle CO2 fixation operons. In addition to regulating the expression of structural genes encoding enzymes of the primary pathway for CO2 fixation in R. sphaeroides, regB was also found to be required for the expression of a gene(s) important for the putative alternative CO2 fixation pathway(s) of this organism. A mutation in regB also blocked expression of structural genes of the cbb regulon in a strain of R. sphaeroides capable of aerobic CO2-dependent growth in the dark. It is thus apparent that regB is part of a two-component system and encodes a sensor kinase involved in the global regulation of both anoxygenic light-dependent- and oxygenic light-independent CO2 fixation as well as anoxygenic photosystem biosynthesis.
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( 1993 ) Genetic evidence for the role of isocytochrome c2 in photosynthetic growth of Rhodobacter sphaeroides Spd mutants. PMID : 8380401 : DOI : 10.1128/jb.175.2.358-366.1993 PMC : PMC196149 Abstract >>
In Rhodobacter sphaeroides, cytochrome c2 (cyt c2)-deficient mutants are photosynthetically incompetent (PS-). However, mutations which suppress the photosynthetic deficiency (spd mutations) of cyt c2 mutants increase the levels of a cyt c2 isoform, isocyt c2. To determine whether isocyt c2 was required for photosynthetic growth of Spd mutants, we used Tn5 mutagenesis to generate a PS- mutant (TP39) that lacks both cyt c2 and isocyt c2. DNA sequence analysis of wild-type DNA that restores isocyt c2 production and photosynthetic growth to TP39 indicates that it encodes the isocyt c2 structural gene, cycI. The Tn5 insertion in TP39 is approximately 1.5 kb upstream of cycI, and our results show that it is polar onto cycI. The cycI gene has been physically mapped to a region of chromosome I that is approximately 700 kb from the R. sphaeroides photosynthetic gene cluster. Construction of a defined cycI null mutant and complementation of several mutants with the cycI gene under the control of the cyt c2 promoter region indicate that an increase in the levels of isocyt c2 alone is necessary and sufficient for photosynthetic growth in the absence of cyt c2. The data are discussed in terms of the obligate role of isocyt c2 in cyt c2-independent photosynthesis of R. sphaeroides.
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( 1994 ) Sequencing, chromosomal inactivation, and functional expression in Escherichia coli of ppsR, a gene which represses carotenoid and bacteriochlorophyll synthesis in Rhodobacter sphaeroides. PMID : 8188588 : DOI : 10.1128/jb.176.10.2869-2876.1994 PMC : PMC205441 Abstract >>
Sequencing of a DNA fragment that causes trans suppression of bacteriochlorophyll and carotenoid levels in Rhodobacter sphaeroides revealed two genes: orf-192 and ppsR. The ppsR gene alone is sufficient for photopigment suppression. Inactivation of the R. sphaeroides chromosomal copy of ppsR results in overproduction of both bacteriochlorophyll and carotenoid pigments. The deduced 464-amino-acid protein product of ppsR is homologous to the CrtJ protein of Rhodobacter capsulatus and contains a helix-turn-helix domain that is found in various DNA-binding proteins. Removal of the helix-turn-helix domain renders PpsR nonfunctional. The promoter of ppsR is located within the coding region of the upstream orf-192 gene. When this promoter is replaced by a lacZ promoter, ppsR is expressed in Escherichia coli. An R. sphaeroides DNA fragment carrying crtD', -E, and -F and bchC, -X, -Y, and -Z' exhibited putative promoter activity in E. coli. This putative promoter activity could be suppressed by PpsR in both E. coli and R. sphaeroides. These results suggest that PpsR is a transcriptional repressor. It could potentially act by binding to a putative regulatory palindrome found in the 5' flanking regions of a number of R. sphaeroides and R. capsulatus photosynthesis genes.
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Shah DS,
Armitage JP,
Sockett RE,
( 1995 ) Rhodobacter sphaeroides WS8 expresses a polypeptide that is similar to MotB of Escherichia coli. PMID : 7751310 : DOI : 10.1128/jb.177.10.2929-2932.1995 PMC : PMC176972 Abstract >>
A gene which complements a paralyzed flagellar mutant of Rhodobacter sphaeroides was sequenced. The derived protein sequence has similarity to MotB. R. sphaeroides MotB lacks the C-terminal peptidoglycan-binding motif of other MotB proteins. This divergence of sequence may reflect the unusual, unidirectional, stop-start action of the R. sphaeroides flagellar motor.
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( 1994 ) Positive and negative regulation of sequences upstream of the form II cbb CO2 fixation operon of Rhodobacter sphaeroides. PMID : 7961502 : DOI : 10.1128/jb.176.23.7299-7308.1994 PMC : PMC197119 Abstract >>
The unlinked form I and form II Calvin cycle CO2 fixation (cbb) operons of the photosynthetic bacterium Rhodobacter sphaeroides are located on different genetic elements, yet both operons are positively regulated by the transcription activator protein CbbR, the product of the cbbR gene located immediately upstream of the form I operon. By employing deletion mutagenesis, and a newly constructed promoter probe vector, the form II operon promoter (cbbFIIp) and three other promoters (Up, Vp, and Wp) were localized within 2.1 kb upstream of the form II operon. Mutations in both cbbR and the first gene of the form I operon (cbbFI) elicited both positive and negative responses when transcriptional fusions controlled by these four promoters were examined. With the exception of Wp, all these upstream promoters were repressed by oxygen. In addition, these promoters were associated with open reading frames of unknown function whose deduced amino acid sequences showed no significant relationship to proteins in current databases. The results of these experiments suggest that the promoter sequences and genes upstream of the form II cbb operon may be intimately involved with control of the cbb regulon of this photosynthetic organism.
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132. |
( 1996 ) Isolation and expression of the Rhodobacter sphaeroides gene (pgsA) encoding phosphatidylglycerophosphate synthase. PMID : 8576035 : DOI : 10.1128/jb.178.4.1030-1038.1996 PMC : PMC177762 Abstract >>
The Rhodobacter sphaeroides pgsA gene (pgsARs), encoding phosphatidylglycerophosphate synthase (PgsARs), was cloned, sequenced, and expressed in both R. sphaeroides and Escherichia coli. As in E. coli, pgsARs is located immediately downstream of the uvrC gene. Comparison of the deduced amino acid sequences revealed 41% identity and 69% similarity to the pgsA gene of E. coli, with similar homology to the products of the putative pgsA genes of several other bacteria. Comparison of the amino acid sequences of a number of enzymes involved in CDP-diacylglycerol-dependent phosphatidyltransfer identified a highly conserved region also found in PgsARs. The pgsARs gene carried on multicopy plasmids was expressed in R. sphaeroides under the direction of its own promoter, the R. sphaeroides rrnB promoter, and the E. coli lac promoter, and this resulted in significant overproduction of PgsARs activity. Expression of PgsARs activity in E. coli occurred only with the E. coli lac promoter. PgsARs could functionally replace the E. coli enzyme in both a point mutant and a null mutant of E. coli pgsA. Overexpression of PgsARs in either E. coli or R. sphaeroides did not have dramatic effects on the phospholipid composition of the cells, suggesting regulation of the activity of this enzyme in both organisms.
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133. |
( 1994 ) Nucleotide sequence and characterization of the Rhodobacter sphaeroides glnB and glnA genes. PMID : 7921264 : DOI : 10.1099/13500872-140-8-2143 Abstract >>
The glnA gene of Rhodobacter sphaeroides encoding glutamine synthetase (GS) has been cloned and sequenced. Molecular analysis revealed that there is a glnB gene upstream of glnA, in a single glnBA operon. A putative glnAp1-type promoter sequence, a consensus ntrC gene product binding site and a consensus upstream activator sequence were detected upstream of the glnB gene. The deduced amino acid sequences of the GS and GlnB proteins of R. sphaeroides showed strong homology with the same proteins from other Gram-negative bacteria. The sequence of the glnA gene isolated from glutamine auxotroph Gln83 was also determined. The glnA83 mutation was shown to result in premature termination of GS synthesis and formation of a 17 kDa C-truncated GS which could be complemented by a 5'-truncated glnA gene which encodes a 30 kDa N-truncated GS. This phenomenon is characteristic for interallelic complementation.
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134. |
( 1993 ) Expression of the Rhodobacter sphaeroides hemA and hemT genes, encoding two 5-aminolevulinic acid synthase isozymes. PMID : 8468290 : DOI : 10.1128/jb.175.8.2292-2303.1993 PMC : PMC204517 Abstract >>
The nucleotide sequences of the Rhodobacter sphaeroides hemA and hemT genes, encoding 5-aminolevulinic acid (ALA) synthase isozymes, were determined. ALA synthase catalyzes the condensation of glycine and succinyl coenzyme A, the first and rate-limiting step in tetrapyrrole biosynthesis. The hemA and hemT structural gene sequences were 65% identical to each other, and the deduced HemA and HemT polypeptide sequences were 53% identical, with an additional 16% of aligned amino acids being similar. HemA and HemT were homologous to all characterized ALA synthases, including two human ALA synthase isozymes. In addition, they were evolutionarily related to 7-keto-8-aminopelargonic acid synthetase (BioF) and 2-amino-3-ketobutyrate coenzyme A ligase (Kbl), enzymes which catalyze similar reactions. Two hemA transcripts were identified, both expressed under photosynthetic conditions at levels approximately three times higher than those found under aerobic conditions. A single transcriptional start point was identified for both transcripts, and a consensus sequence at this location indicated that an Fnr-like protein may be involved in the transcriptional regulation of hemA. Transcription of hemT was not detected in wild-type cells under the physiological growth conditions tested. In a mutant strain in which the hemA gene had been inactivated, however, hemT was expressed. In this mutant, hemT transcripts were characterized by Northern (RNA) hybridization, primer extension, and ribonuclease protection techniques. A small open reading frame of unknown function was identified upstream of, and transcribed in the same direction as, hemA.
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135. |
Theiler R,
Suter F,
Wiemken V,
Zuber H,
( 1984 ) The light-harvesting polypeptides of Rhodopseudomonas sphaeroides R-26.1. I. Isolation, purification and sequence analyses. PMID : 6384009 : Abstract >>
Four low-molecular-mass polypeptides were isolated and purified from chromatophore membranes of Rhodopseudomonas sphaeroides blue-green mutant R-26.1 by a combination of gel filtration and ion-exchange chromatography in organic solvents. On dodecyl sulfate polyacrylamide gels, the purified polypeptides comigrate with bands LH-1, LH-2 and LH-3 known to be related to the antenna-pigment-protein complexes. The complete primary structures were elucidated by automated Edman degradation of the intact polypeptides and of overlapping C-terminal fragments obtained after chemical cleavage at tryptophan and methionine residues. The C-termini were verified by hydrazinolysis and, in one case where an overlapping C-terminal fragment could not be obtained, by digestion with carboxypeptidase A. The four polypeptides show a tripartite structure: i.e. a polar N-terminal region is separated from a polar C-terminal region by a segment of about 21 predominantly hydrophobic amino-acid residues. All hydrophobic segments contain a characteristic conservative histidine residue. The C-terminal region is reduced to only a few amino acids in the two polypeptides which together form band LH-3, i.e. LH-3A and LH-3B. Their extended N-terminal region is rich in charged residues and contains an additional conserved histidine residue close to the beginning of the hydrophobic segment. These properties place LH-3A and LH-3B into subgroup (beta-polypeptides: B 870-beta and B 850-beta, respectively). LH-1 and LH-2 appear to form another subgroup (alpha-polypeptides: B 870-alpha and B 850-alpha, respectively) as suggested during a search for conservative elements within their sequences (structural basis for classification). N-Terminal analyses carried out with intact antenna-pigment-protein complexes revealed the following: (i) LH-1 and LH-3 are associated with the B 870 complex in Rp. sphaeroides 24.1 (wild type), (ii) the same polypeptides are almost exclusively present in chromatophore membranes of Rp. sphaeroides R-26, a blue-green mutant which absorbs at 870 nm, (iii) LH-2 and LH-3B are the constituent polypeptides of the B 800-850 complex of Rp. sphaeroides 2.4.1 and of the spectrally altered B 850 complex isolated from the blue-green mutant R-26.1 which absorbs at 860 nm. This mutant contains LH-2 and LH-3B along with LH-1 and LH-3A and apparently is able to form both types of antenna complexes.(ABSTRACT TRUNCATED AT 400 WORDS)
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136. |
Lang HP,
Cogdell RJ,
Takaichi S,
Hunter CN,
( 1995 ) Complete DNA sequence, specific Tn5 insertion map, and gene assignment of the carotenoid biosynthesis pathway of Rhodobacter sphaeroides. PMID : 7721699 : DOI : 10.1128/jb.177.8.2064-2073.1995 PMC : PMC176850 Abstract >>
The carotenoid biosynthesis genes form a cluster within the genome of Rhodobacter sphaeroides, lying in the middle of a larger cluster and 45 kb in length, which contains genes for bacteriochlorophyll biosynthesis and for the reaction center and light-harvesting apoproteins. The positions and approximate limits of the carotenoid genes were determined previously by localized transposon Tn5 mutagenesis and by comparison with the closely related Rhodobacter capsulatus carotenoid gene cluster. In this report, analysis of the DNA and deduced amino acid sequences of the carotenoid genes in R. sphaeroides are presented. Twenty-five Tn5 insertion mutants were used to produce a base-specific Tn5 insertion map of this region, and carotenoid gene assignment was supported by spectroscopic, ultrastructural, and high-pressure liquid chromatography analyses of these mutants. A region in the 3' end of crtD which affects bacteriochlorophyll biosynthesis was discovered, and CrtA was found to possess a proline-rich C-terminal region containing a repeated (Ala-Pro)n motif. CrtF also showed a high degree of sequence conservation with eukaryotic O-methyltransferases. This study provides gene sequences and assignments based upon a comprehensive structural, spectroscopic, and biochemical analysis of a range of carotenoid biosynthetic mutants; in each mutation, the point of Tn5 insertion is determined accurate to 1 bp on the gene cluster.
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137. |
Pollock VV,
Barber MJ,
( 1995 ) Molecular cloning and expression of biotin sulfoxide reductase from Rhodobacter sphaeroides forma sp. denitrificans. PMID : 7733660 : DOI : 10.1006/abbi.1995.1236 Abstract >>
Biotin sulfoxide reductase catalyzes the conversion of d-biotin d-sulfoxide (BSO) to d-biotin. Oligonucleotides directed against common sequences in Escherichia coli biotin sulfoxide reductase and in Rhodobacter sphaeroides f.sp. denitrificans dimethyl sulfoxide reductase have been utilized to amplify by PCR a 651-bp fragment from R. sphaeroides total genomic DNA that showed a high degree of sequence similarity with both E. coli biotin sulfoxide reductase and R. sphaeroides dimethyl sulfoxide reductase. Screening of a genomic cosmid library, prepared from R. sphaeroides genomic DNA, with this probe resulted in the isolation of a 7-kb EcoRI-EcoRI fragment that contained the complete coding region for R. sphaeroides BSO reductase which has been sequenced. The sequence data indicated a single open reading frame of 2231 nucleotides encoding a protein of 744 amino acid residues corresponding to a subunit molecular weight of 80,234 Da. The translated protein sequence contained the prokaryotic Mo-pterin signatures 2 and 3 (Mo-cofactor binding motifs) and a ATP/GTP-binding P-loop. The R. sphaeroides BSO reductase sequence showed 51% sequence similarity with the corresponding E. coli enzyme. In addition, there were only two conserved cysteines between the two BSO reductase sequences. The R. sphaeroides gene was demonstrated, by complementation, to rescue a mutant E. coli strain that was deficient in BSO reductase when grown on BSO as the sole source of biotin. When expressed from the FLAG*Shift 12c expression vector, in the presence of IPTG, the BSO reductase gene encoded a protein of approximately 80 kDa, which cross-reacted with the anti-FLAG monoclonal antibody and exhibited BSO reductase activity by the disk microbiological assay.
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138. |
Mackenzie C,
Chidambaram M,
Sodergren EJ,
Kaplan S,
Weinstock GM,
( 1995 ) DNA repair mutants of Rhodobacter sphaeroides. PMID : 7768798 : DOI : 10.1128/jb.177.11.3027-3035.1995 PMC : PMC176989 Abstract >>
The genome of the photosynthetic eubacterium Rhodobacter sphaeroides 2.4.1 comprises two chromosomes and five endogenous plasmids and has a 65% G+C base composition. Because of these characteristics of genome architecture, as well as the physiological advantages that allow this organism to live in sunlight when in an anaerobic environment, the sensitivity of R. sphaeroides to UV radiation was compared with that of the more extensively studied bacterium Escherichia coli. R. sphaeroides was found to be more resistant, being killed at about 60% of the rate of E. coli. To begin to analyze the basis for this increased resistance, a derivative of R. sphaeroides, strain 2.4.1 delta S, which lacks the 42-kb plasmid, was mutagenized with a derivative of Tn5, and the transposon insertion mutants were screened for increased UV sensitivity (UVs). Eight UVs strains were isolated, and the insertion sites were determined by contour-clamped homogeneous electric field pulsed-field gel electrophoresis. These mapped to at least five different locations in chromosome I. Preliminary analysis suggested that these mutants were deficient in the repair of DNA damage. This was confirmed for three loci by DNA sequence analysis, which showed the insertions to be within genes homologous to uvrA, uvrB, and uvrC, the subunits of the nuclease responsible for excising UV damage.
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139. |
Williams JC,
Steiner LA,
Feher G,
Simon MI,
( 1984 ) Primary structure of the L subunit of the reaction center from Rhodopseudomonas sphaeroides. PMID : 6095283 : DOI : 10.1073/pnas.81.23.7303 PMC : PMC392134 Abstract >>
The reaction center is an integral membrane protein that, together with several cofactors, mediates the primary photochemical events in bacterial photosynthesis. The amino-terminal sequences of the three subunits, L, M, and H, of the reaction center protein and the sequence of the structural gene encoding the M subunit have been reported previously. In the present study, we found that the 3' end of the structural gene encoding the L subunit overlaps by eight bases the 5' end of the gene encoding the M subunit. The primary structure of the L subunit has been determined from the nucleotide sequence of the gene and from analyses of the amino and carboxyl termini of the protein. The sequences of a number of tryptic and chymotryptic peptides were used to corroborate the nucleotide sequence. The L subunit was found to be composed of 281 amino acids (Mr 31,319) and to contain five hydrophobic segments. It is homologous to the M subunit and to a plant thylakoid protein referred to as the QB or Mr 32,000 protein.
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140. |
( 1994 ) The bacteriochlorophyll biosynthesis gene, bchM, of Rhodobacter sphaeroides encodes S-adenosyl-L-methionine: Mg protoporphyrin IX methyltransferase. PMID : 7925960 : DOI : 10.1016/0014-5793(94)00934-1 Abstract >>
The bchM gene of Rhodobacter sphaeroides has been sequenced and then overexpressed in E. coli producing a protein of M(r) approximately. 27,500. Cell-free extracts of the transformed E. coli strain are able to methylate added Mg protoporphyrin, resulting in the formation of Mg protoporphyrin monomethyl ester. The identity of this product was verified by HPLC. The bchM gene product is therefore assigned to the methyltransferase step in bacteriochlorophyll biosynthesis.
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141. |
Yeliseev AA,
Kaplan S,
( 1995 ) A sensory transducer homologous to the mammalian peripheral-type benzodiazepine receptor regulates photosynthetic membrane complex formation in Rhodobacter sphaeroides 2.4.1. PMID : 7673149 : DOI : 10.1074/jbc.270.36.21167 Abstract >>
The Rhodobacter sphaeroides 2.4.1 tryptophan-rich sensory protein gene, tspO (formerly crtK, ORF160) encodes a 17-kDa protein which has an unusually high content of aromatic amino acids in general and of L-tryptophan in particular. The TspO protein was localized to the outer membrane of aerobically grown R. sphaeroides 2.4.1 by use of a polyclonal antibody against the purified protein. This protein is present in severalfold higher levels in photosynthetic as opposed to aerobic grown cells. Although tspO lies within the crt gene cluster, null mutations have an intact carotenoid biosynthetic pathway. In the TSPO1 mutant there was an increased production of carotenoids and bacteriochlorophyll relative to the wild type, particularly when cells were grown aerobically or semiaerobically. When present in trans the tspO gene restored "normal" pigment production to TSPO1. The effect of the tspO gene on pigment production was shown to take place at the level of gene expression. Because the tspO gene product of R. sphaeroides 2.4.1 shows significant sequence homology and similarity to the peripheral-type benzodoazepine receptor from mammalian sources, TspO-specific antibodies when probed against liver and kidney mitochondrial protein showed strong cross-reactivity. The role of TspO in R. sphaeroides 2.4.1 and its relation to photosynthesis gene expression are discussed.
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142. |
Sabaty M,
Gagnon J,
Verméglio A,
( 1994 ) Induction by nitrate of cytoplasmic and periplasmic proteins in the photodenitrifier Rhodobacter sphaeroides forma sp. denitrificans under anaerobic or aerobic condition. PMID : 7857198 : Abstract >>
The synthesis of nitrate, nitrite, and nitrous oxide reductases is highly enhanced by the addition of nitrate during growth of Rhodobacter sphaeroides forma sp. denitrificans. Contrary to what is observed in many denitrifiers, the synthesis of these enzymes is not repressed by oxygen at concentrations as high as 37% air saturation. When oxygen concentration is increased up to 100% air saturation, the synthesis of nitrite and nitrous oxide reductases is repressed while the nitrate reductase is still synthesized. Two proteins, one periplasmic (35 kDa) and the other cytoplasmic (32 kDa), are also induced by nitrate, but not by trimethylamine-N-oxide or oxygen. Although their function is not yet known, these two proteins appear to be specifically linked to the denitrification pathway. The amino acid sequences of tryptic peptides and of the N-terminal ends of these proteins indicate no significant similarity with the sequences in the Swiss Prot Data Bank. However, a very good alignment is obtained between the amino acid sequences of the periplasmic nitrate reductase of Alcaligenes eutrophus H16 and those of various tryptic peptides of the nitrate reductase of R. sphaeroides forma sp. denitrificans.
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143. |
Gibson LC,
Willows RD,
Kannangara CG,
von Wettstein D,
Hunter CN,
( 1995 ) Magnesium-protoporphyrin chelatase of Rhodobacter sphaeroides: reconstitution of activity by combining the products of the bchH, -I, and -D genes expressed in Escherichia coli. PMID : 7892204 : DOI : 10.1073/pnas.92.6.1941 PMC : PMC42398 Abstract >>
Magnesium-protoporphyrin chelatase lies at the branch point of the heme and (bacterio)chlorophyll biosynthetic pathways. In this work, the photosynthetic bacterium Rhodobacter sphaeroides has been used as a model system for the study of this reaction. The bchH and the bchI and -D genes from R. sphaeroides were expressed in Escherichia coli. When cell-free extracts from strains expressing BchH, BchI, and BchD were combined, the mixture was able to catalyze the insertion of Mg into protoporphyrin IX in an ATP-dependent manner. This was possible only when all three genes were expressed. The bchH, -I, and -D gene products are therefore assigned to the Mg chelatase step in bacteriochlorophyll biosynthesis. The mechanism of the Mg chelation reaction and the implications for chlorophyll biosynthesis in plants are discussed.
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144. |
Gomelsky M,
Kaplan S,
( 1995 ) 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. PMID : 7551045 : DOI : 10.1099/13500872-141-8-1805 Abstract >>
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.
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145. |
Eraso JM,
Kaplan S,
( 1995 ) Oxygen-insensitive synthesis of the photosynthetic membranes of Rhodobacter sphaeroides: a mutant histidine kinase. PMID : 7751278 : DOI : 10.1128/jb.177.10.2695-2706.1995 PMC : PMC176939 Abstract >>
Two new loci, prrB and prrC, involved in the positive regulation of photosynthesis gene expression in response to anaerobiosis, have been identified in Rhodobacter sphaeroides. prrB encodes a sensor histidine kinase that is responsive to the removal of oxygen and functions through the response regulator PrrA. Inactivation of prrB results in a substantial reduction of photosynthetic spectral complexes as well as in the inability of cells to grow photosynthetically at low to medium light intensities. Together, prrB and prrA provide the major signal involved in synthesis of the specialized intracytoplasmic membrane (ICM), harboring components essential to the light reactions of photosynthesis. Previously, J. K. Lee and S. Kaplan (J. Bacteriol. 174:1158-1171, 1992) identified a mutant which resulted in high-level expression of the puc operon, encoding the apoproteins giving rise to the B800-850 spectral complex, in the presence of oxygen as well as in the synthesis of the ICM under conditions of high oxygenation. This mutation is shown to reside in prrB, resulting in a leucine-to-proline change at position 78 in mutant PrrB (PRRB78). Measurements of mRNA levels in cells containing the prrB78 mutation support the idea that prrB is a global regulator of photosynthesis gene expression. Two additional mutants, PRRB1 and PRRB2, which make two truncated forms of the PrrB protein, possess substantially reduced amounts of spectral complexes. Although the precise role of prrC remains to be determined, evidence suggests that it too is involved in the regulatory cascade involving prrB and prrA. The genetic organization of the photosynthesis response regulatory (PRR) region is discussed.
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146. |
Williams JC,
Steiner LA,
Feher G,
( 1986 ) Primary structure of the reaction center from Rhodopseudomonas sphaeroides. PMID : 3329732 : DOI : 10.1002/prot.340010405 Abstract >>
The reaction center is a pigment-protein complex that mediates the initial photochemical steps of photosynthesis. The amino-terminal sequences of the L, M, and H subunits and the nucleotide and derived amino acid sequences of the L and M structural genes from Rhodopseudomonas sphaeroides have previously been determined. We report here the sequence of the H subunit, completing the primary structure determination of the reaction center from R. sphaeroides. The nucleotide sequence of the gene encoding the H subunit was determined by the dideoxy method after subcloning fragments into single-stranded M13 phage vectors. This information was used to derive the amino acid sequence of the corresponding polypeptide. The termini of the primary structure of the H subunit were established by means of the amino and carboxy terminal sequences of the polypeptide. The data showed that the H subunit is composed of 260 residues, corresponding to a molecular weight of 28,003. A molecular weight of 100,858 for the reaction center was calculated from the primary structures of the subunits and the cofactors. Examination of the genes encoding the reaction center shows that the codon usage is strongly biased towards codons ending in G and C. Hydropathy analysis of the H subunit sequence reveals one stretch of hydrophobic residues near the amino terminus; the L and M subunits contain five such stretches. From a comparison of the sequences of homologous proteins found in bacterial reaction centers and photosystem II of plants, an evolutionary tree was constructed. The analysis of evolutionary relationships showed that the L and M subunits of reaction centers and the D1 and D2 proteins of photosystem II are descended from a common ancestor, and that the rate of change in these proteins was much higher in the first billion years after the divergence of the reaction center and photosystem II than in the subsequent billion years represented by the divergence of the species containing these proteins.
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147. |
Donohue TJ,
Hoger JH,
Kaplan S,
( 1986 ) Cloning and expression of the Rhodobacter sphaeroides reaction center H gene. PMID : 3023292 : DOI : 10.1128/jb.168.2.953-961.1986 PMC : PMC213577 Abstract >>
The Rhodobacter sphaeroides structural gene (puhA) for the reaction center H polypeptide has been identified and cloned by using restriction fragements specific for the analogous Rhodobacter capsulatus gene as a heterologous hybridization probe. The presence of puhA on a 1.45-kilobase BamHI restriction fragment was confirmed by partial DNA sequence analysis and by the synthesis of an immunoreactive Mr-28,000 reaction center H polypeptide in an R. sphaeroides coupled transcription-translation system. Approximately 450 base pairs of DNA upstream of the puhA gene were sufficient for expression of this protein in vitro. Northern RNA-DNA blot analysis with an internal puhA-specific probe identified at least two, apparently monocistronic, transcripts present at different cellular levels under physiological conditions known to affect the cellular content of both reaction center complexes and photosynthetic membrane. Northern blot analysis with specific upstream restriction fragment probes revealed that the 1,400-nucleotide puhA-specific mRNA had a 5' terminus upstream of the 1,130-nucleotide transcript. Both puhA-specific mRNA and immunoreactive reaction center H protein were detectable in chemoheterotrophically grown cells which lacked detectable bacteriochlorophyll and photosynthetic membrane.
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148. |
Barber MJ,
Van Valkenburgh H,
Trimboli AJ,
Pollock VV,
Neame PJ,
Bastian NR,
( 1995 ) The amino acid sequence of Rhodobacter sphaeroides dimethyl sulfoxide reductase. PMID : 7625833 : DOI : 10.1016/0003-9861(95)90009-8 Abstract >>
The complete amino acid sequence of the soluble, monomeric molybdenum-containing enzyme dimethyl sulfoxide reductase from Rhodobacter sphaeroides f sp. denitrificans has been determined using a combination of gas-phase Edman sequencing of isolated peptides and direct sequencing of PCR products generated from R. sphaeroides genomic DNA. The protein comprises 777 residues corresponding to an apoenzyme molecular weight of 84,748 Da. The amino acid sequence was rich in Ala and Gly residues which represented 21% of the protein's composition. The DNA sequence was 67% rich in G and C nucleotides. The amino acid sequence contained 10 cysteine residues which were relatively evenly distributed throughout the sequence and featured regions of sequence corresponding to the prokaryotic molybdopterin-binding signatures 2 and 3. While exhibiting limited sequence similarity to the corresponding membrane-bound molybdenum-containing subunit (DmsA) of Escherichia coli dimethyl sulfoxide reductase, the Rhodobacter sequence showed extensive sequence similarity to that of the E. coli molybdoprotein, trimethylamine N-oxide reductase (torA). Comparison with other related prokaryotic molybdenum-containing enzymes indicated the presence of two highly conserved cysteine residues (Cys-268 and Cys-616) which may function in molybdenum coordination.
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149. |
Gomelsky M,
Kaplan S,
( 1995 ) appA, a novel gene encoding a trans-acting factor involved in the regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1. PMID : 7642486 : DOI : 10.1128/jb.177.16.4609-4618.1995 PMC : PMC177224 Abstract >>
A new gene, the product of which is involved in the regulation of photosynthesis gene expression in the anoxygenic photosynthetic bacterium Rhodobacter sphaeroides 2.4.1, has been identified. The isolation of this gene, designated appA (activation of photopigment and puc expression), was based on its ability, when provided in extra copies, to partially suppress mutations in the two-component PrrB-PrrA regulatory system. The presence of extra copies of the appA gene in either prrB, prrA, or wild-type strains resulted in an activation of puc::lacZ expression under aerobic conditions. Constructed AppA null mutants did not grow photosynthetically and were impaired in the synthesis of both bacteriochlorophyll and carotenoids, as well as the structural proteins of the photosynthetic spectral complexes. When grown anaerobically in the dark, these mutants accumulated bacteriochlorophyll precursors. The expression of lacZ fusions to several photosynthesis genes and operons, including puc, puf, and bchF, was decreased in the AppA mutant strains in comparison with the wild type. To examine the role of AppA involvement in bacteriochlorophyll biosynthesis, we inactivated an early gene, bchE, of the bacteriochlorophyll pathway in both wild-type and AppA- mutant backgrounds. The double mutant, AppA- BchE-, was found to be severely impaired in photosynthesis gene expression, similar to the AppA- BchE+ mutant and in contrast to the AppA+ BchE- mutant. This result indicated that AppA is more likely involved in the regulation of expression of the bch genes than in the biosynthetic pathway per se. The appA gene was sequenced and appears to encode a protein of 450 amino acids with no obvious homology to known proteins.
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150. |
Zeilstra-Ryalls JH,
Kaplan S,
( 1995 ) Aerobic and anaerobic regulation in Rhodobacter sphaeroides 2.4.1: the role of the fnrL gene. PMID : 7592416 : DOI : 10.1128/jb.177.22.6422-6431.1995 PMC : PMC177491 Abstract >>
In Rhodobacter sphaeroides 2.4.1, the cellular requirements for 5-aminolevulinic acid (ALA) are in part regulated by the level of ALA synthase activity, which is encoded by the hemA and hemT genes. Under standard growth conditions, only the hemA gene is transcribed, and the level of ALA synthase activity varies in response to oxygen tension. The presence of an FNR consensus sequence upstream of hemA suggested that oxygen regulation of hemA expression could be mediated, in part, through a homolog of the fnr gene. Two independent studies, one detailed here, identified a region of the R. sphaeroides 2.4.1 genome containing extensive homology to the fix region of the symbiotic nitrogen-fixing bacteria Rhizobium meliloti and Bradyrhizobium japonicum. Within this region that maps to 443 kbp on chromsome I, we have identified an fnr homolog (fnrL), as well as a gene that codes for an anaerobic coproporphyrinogen III oxidase, the second such gene identified in this organism. We also present an analysis of the role of fnrL in the physiology of R. sphaeroides 2.4.1 through the construction and characterization of fnrL-null strains. Our results further show that fnrL is essential for both photosynthetic and anaerobic-dark growth with dimethyl sulfoxide. Analysis of hemA expression, with hemA::lacZ transcriptional fusions, suggests that FnrL is an activator of hemA under anaerobic conditions. On the other hand, the open reading frame immediately upstream of hemA appears to be an activator of hemA transcription regardless of either the presence or the absence of oxygen or FnrL. Given the lack of hemT expression under these conditions, we consider FnrL regulation of hemA expression to be a major factor in bringing about changes in the level of ALA synthase activity in response to changes in oxygen tension.
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151. |
Clement-Metral JD,
Holmgren A,
Cambillau C,
Jörnvall H,
Eklund H,
Thomas D,
Lederer F,
( 1988 ) Amino acid sequence determination and three-dimensional modelling of thioredoxin from the photosynthetic bacterium Rhodobacter sphaeroides Y. PMID : 3280308 : DOI : 10.1111/j.1432-1033.1988.tb13902.x Abstract >>
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.
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152. |
Allen JP,
Feher G,
Yeates TO,
Komiya H,
Rees DC,
( 1988 ) Structure of the reaction center from Rhodobacter sphaeroides R-26: protein-cofactor (quinones and Fe2+) interactions. PMID : 3054889 : DOI : 10.1073/pnas.85.22.8487 PMC : PMC282483 Abstract >>
The three-dimensional structure of the reaction center (RC) from Rhodobacter sphaeroides has been determined by x-ray diffraction to a resolution of 2.8 A with an R value of 24%. The interactions of the protein with the primary quinone, QA, secondary quinone, QB, and the nonheme iron are described and compared to those of RCs from Rhodopseudomonas viridis. Structural differences between the QA and QB environments that contribute to the function of the quinones (the electron transfer from QA- to QB and the charge recombination of QA-, QB- with the primary donor) are delineated. The protein residues that may be involved in the protonation of QB are identified. A pathway for the doubly reduced QB to dissociate from the RC is proposed. The interactions between QB and the residues that have been changed in herbicide-resistant mutants are described. The environment of the nonheme iron is compared to the environments of metal ions in other proteins.
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Ward MJ,
Bell AW,
Hamblin PA,
Packer HL,
Armitage JP,
( 1995 ) Identification of a chemotaxis operon with two cheY genes in Rhodobacter sphaeroides. PMID : 7494484 : DOI : 10.1111/j.1365-2958.1995.mmi_17020357.x Abstract >>
A large chemotaxis operon was identified in Rhodobacter sphaeroides WS8-N using a probe based on the 3' terminal portion of the Rhizobium meliloti cheA gene. Two genes homologous to the enteric cheY were identified in an operon also containing cheA, cheW, and cheR homologues. The deduced protein sequences of che gene products were aligned with those from Escherichia coli and shown to be highly conserved. A mutant with an interrupted copy of cheA showed normal patterns of swimming, unlike the equivalent mutants in E. coli which are smooth swimming. Tethered cheA mutant cells showed normal responses to changes in organic acids, but increased, inverted responses to sugars. The unusual behaviour of the cheA mutant and the identification of two homologues of cheY suggests that R. sphaeroides has at least two pathways controlling motor activity. To identify functional similarity between the newly identified R. sphaeroides Che pathway and the methyl-accepting chemotaxis protein (MCP)-dependent pathway in enteric bacteria, the R. sphaeroides cheW gene was expressed in a cheW mutant strain of E. coli and found to complement, causing a partial return to a swarming phenotype. In addition, expression of the R. sphaeroides gene in wild-type E. coli resulted in the same increased tumbling and reduced swarming as seen when the native gene is overexpressed in E. coli. The identification of che homologues in R. sphaeroides and complementation by cheW suggests the presence of MCPs in an organism previously considered to use only MCP-independent sensing. The MCP-dependent pathway, appears conserved.(ABSTRACT TRUNCATED AT 250 WORDS)
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154. |
Schauder S,
Schneider KH,
Giffhorn F,
( 1995 ) Polyol metabolism of Rhodobacter sphaeroides: biochemical characterization of a short-chain sorbitol dehydrogenase. PMID : 7551049 : DOI : 10.1099/13500872-141-8-1857 Abstract >>
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.
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155. |
Hallenbeck PL,
Kaplan S,
( 1987 ) Cloning of the gene for phosphoribulokinase activity from Rhodobacter sphaeroides and its expression in Escherichia coli. PMID : 3038847 : DOI : 10.1128/jb.169.8.3669-3678.1987 PMC : PMC212449 Abstract >>
A 3.4-kilobase EcoRI restriction endonuclease fragment has been cloned from the facultatively photoheterotrophic bacterium Rhodobacter sphaeroides and shown to contain the structural gene (prkA) for phosphoribulokinase (PRK) activity. The PRK activity was characterized in Escherichia coli, and the product of the reaction was identified. The prkA gene was localized to a 1,565-base-pair EcoRI-PstI restriction endonuclease fragment and gave rise to a 33-kilodalton polypeptide both in vivo and in vitro. The gene product produced in E. coli was shown to be identical to the gene product produced in R. sphaeroides. The amino acid sequence for the amino-terminal region deduced from the DNA sequence confirmed that derived for partially purified PRK derived from both E. coli and R. sphaeroides. In addition, the 3.4-kilobase EcoRI restriction endonuclease fragment coded for a 37-kilodalton polypeptide of unknown function, and preliminary evidence indicates that this DNA fragment is linked to genes coding for other activities significant in photosynthetic carbon assimilation. The genetic organization and proposed operon structure of this DNA fragment are discussed.
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156. |
Cooper CL,
Boyce SG,
Lueking DR,
( 1987 ) Purification and characterization of Rhodobacter sphaeroides acyl carrier protein. PMID : 3496918 : DOI : 10.1021/bi00384a013 Abstract >>
Acyl carrier protein (ACP) has been purified from the facultative phototrophic bacterium Rhodobacter sphaeroides. The ACP preparation was greater than 95% homogeneous as determined by native and disodium dodecyl sulfate (Na2DodSO4)-polyacrylamide gel electrophoreses and N-terminal amino acid analysis. Amino acid compositional analysis revealed that the protein contains approximately 75 amino acids, has a calculated minimum molecular weight of 8700, and lacks the amino acids tyrosine and tryptophan. The presence of the characteristic 4'-phosphopantetheine prosthetic group was indicated by the occurrence of equimolar quantities of beta-alanine and taurine in amino acid hydrolysates and was confirmed by independent chemical analysis. The protein displayed a pI of 3.8 and had a calculated partial specific volume of 0.732 mL/g. The primary structure of the protein has been determined for the first 46 amino acid residues from the N terminus of the molecule, and the region of the molecule encompassing the amino acids from residues 31 to 44 was found to have 100% homology with the identical residues in Escherichia coli ACP. In contrast to E. coli ACP, R. sphaeroides ACP migrated according to its molecular weight during Na2DodSO4 gel electrophoresis, was resistant to pH-induced denaturation, and comigrated with the cis-vaccenoyl-ACP derivative during native gel electrophoresis. It is proposed that the basis for these properties is the enhanced hydrophobic character of the protein.
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157. |
( 2013 ) Chemical catalysis by the translocator protein (18 kDa). PMID : 23651039 : DOI : 10.1021/bi400364z Abstract >>
Translocator proteins (18 kDa) (TSPOs) are conserved integral membrane proteins. In both eukaryotes and prokaryotes, TSPOs interact with porphyrins, precursors of heme, and photosynthetic pigments. Here we demonstrate that bacterial TSPOs catalyze rapid porphyrin degradation in a light- and oxygen-dependent manner. The reaction is inhibited by a synthetic TSPO ligand PK11195 and by mutations of conserved residues, which affect either porphyrin binding or catalytic activity. We hypothesize that TSPOs are ancient enzymes mediating porphyrin catabolism with the consumption of reactive oxygen species.
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( 1990 ) Molybdopterin guanine dinucleotide: a modified form of molybdopterin identified in the molybdenum cofactor of dimethyl sulfoxide reductase from Rhodobacter sphaeroides forma specialis denitrificans. PMID : 2326278 : DOI : 10.1073/pnas.87.8.3190 PMC : PMC53861 Abstract >>
The nature of molybdenum cofactor in the bacterial enzyme dimethyl sulfoxide reductase has been investigated by application of alkylation conditions that convert the molybdenum cofactor in chicken liver sulfite oxidase and milk xanthine oxidase to the stable, well-characterized derivative [di(carboxamidomethyl)]molybdopterin. The alkylated pterin obtained from dimethyl sulfoxide reductase was shown to be a modified form of alkylated molybdopterin with increased absorption in the 250-nm region of the spectrum and altered chromatographic behavior. The complex alkylated pterin was resolved into two components by treatment with nucleotide pyrophosphatase. These were identified as di(carboxamidomethyl)molybdopterin and GMP by their absorption spectra, coelution with standard compounds, and by further degradation by alkaline phosphatase to dephospho [di(carboxamidomethyl)]molybdopterin and guanosine. The GMP moiety was sensitive to periodate, identifying it as the 5' isomer. Chemical analysis of the intact alkylated pterin showed the presence of two phosphate residues per pterin. These results established that the pterin isolated from dimethyl sulfoxide reductase contains the phosphoric anhydride of molybdopterin and 5'-GMP, which is designated molybdopterin guanine dinucleotide.
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( 1998 ) The cbb3-type cytochrome c oxidase from Rhodobacter sphaeroides, a proton-pumping heme-copper oxidase. PMID : 9711295 : DOI : 10.1016/s0005-2728(98)00095-4 Abstract >>
Rhodobacter sphaeroides expresses a bb3-type quinol oxidase, and two cytochrome c oxidases: cytochrome aa3 and cytochrome cbb3. We report here the characterization of the genes encoding this latter oxidase. The ccoNOQP gene cluster of R. sphaeroides contains four open reading frames with high similarity to all ccoNOQP/fixNOQP gene clusters reported so far. CcoN has the six highly conserved histidines proposed to be involved in binding the low spin heme, and the binuclear center metals. ccoO and ccoP code for membrane bound mono- and diheme cytochromes c. ccoQ codes for a small hydrophobic protein of unknown function. Upstream from the cluster there is a conserved Fnr/FixK-like box which may regulate its expression. Analysis of a R. sphaeroides mutant in which the ccoNOQP gene cluster was inactivated confirms that this cluster encodes the cbb3-type oxidase previously purified. Analysis of proton translocation in several strains shows that cytochrome cbb3 is a proton pump. We also conclude that cytochromes cbb3 and aa3 are the only cytochrome c oxidases in the respiratory chain of R. sphaeroides.
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( 1998 ) Sequence, chromophore extraction and 3-D model of the photoactive yellow protein from Rhodobacter sphaeroides. PMID : 9630474 : DOI : 10.1016/s0167-4838(98)00050-8 Abstract >>
The photoactive yellow protein (pyp) gene has been isolated from Rhodobacter sphaeroides by probing with a homologous PCR-product. A sequence analysis shows that this pyp gene encodes a 124 AA protein with 48% identity to the three known PYPs. Downstream from pyp, a number of adjacent open reading frames were identified, including a gene encoding a CoA-ligase homologue (pCL). This latter protein is proposed to be involved in PYP chromophore activation, required for attachment to the apoprotein. We have demonstrated the presence of the chromophoric group, previously identified in PYP from Ectothiorhodospira halophila as trans 4-hydroxy cinnamic acid, in phototrophically cultured R. sphaeroides cells by capillary zone electrophoresis. The basic structure of the chromophore binding pocket in PYP has been conserved, as shown by a 3D model of R. sphaeroides PYP, constructed by homology-based molecular modelling. In addition, this model shows that R. sphaeroides PYP contains a characteristic, positively charged patch.
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161. |
( 1998 ) Identification of the Rhodobacter sphaeroides SOS box. PMID : 9663685 : DOI : 10.1046/j.1365-2958.1998.00860.x Abstract >>
Gel-mobility shift assays with crude cell extracts of Rhodobacter sphaeroides, which belongs to the alpha group of the proteobacteria, have shown that a protein binds to the promoter of its recA gene, resulting in two retardation bands. Analysis of the minimal region of the R. sphaeroides recA gene required for the formation of the DNA-protein complexes, revealed the presence of the motifs GTTCN7GATC and GAACN7GAAC, which are centred at positions -21 and +8 from the transcriptional starting point respectively. Using PCR mutagenesis, we have demonstrated that these two motifs are required for the formation of both DNA-protein complexes in vitro as well as for the DNA damage-mediated inducibility of the recA gene in vivo. Furthermore, the level of the recA gene expression in the constitutive mutants is the same as that achieved by the wild-type cells after DNA damage, indicating that the binding protein must be a repressor. The motif GTTCN7GTTC is also present upstream of the R. sphaeroides uvrA promoter, which in vitro specifically binds to a protein and whose expression is DNA damage inducible. Mutagenesis of this motif abolishes both the binding of this protein to the uvrA promoter and the DNA damage-mediated expression of this gene. The fact that the recA and uvrA wild-type promoters compete with each other for the retardation band formation, but not with their mutant derivatives in any of these motifs, indicates that the same repressor binds to the operator of both genes. All these results lead us to propose the sequence GTTCN7GTTC as the SOS box of R. sphaeroides. This is the first SOS box known whose sequence is a direct repeat and not a palindrome.
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( 1998 ) Characterisation of the mob locus from Rhodobacter sphaeroides required for molybdenum cofactor biosynthesis. PMID : 9473631 : DOI : 10.1016/s0167-4781(97)00145-0 Abstract >>
A clone carrying the mob locus from Rb. sphaeroides WS8 has been isolated from a cosmid library by Southern blotting with a probe covering the mob genes of Escherichia coli. The mob DNA has been subcloned and partially restores molybdoenzyme activities when transformed into E. coli mob strains. DNA sequence analysis of the subclone carrying the mob genes predicted at least 2 open reading frames. The mobA gene encodes protein FA whilst mobB encodes a nucleotide binding protein which has at least one extra domain relative to its E. coli counterpart.
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( 1999 ) Closely related form I ribulose bisphosphate carboxylase/oxygenase molecules that possess different CO2/O2 substrate specificities. PMID : 9882445 : DOI : 10.1006/abbi.1998.0979 Abstract >>
The deduced primary sequence (cbbL and cbbS) of form I ribulose 1, 5-bisphosphate carboxylase/oxygenase (rubisco) from Bradyrhizobium japonicum places this enzyme within the Type IC subgroup of red-like rubisco enzymes. In addition, B. japonicum appears to organize most of the structural genes of the Calvin-Benson-Bassham (CBB) pathway in at least one major operon. Functional expression and characterization of the B. japonicum and Xanthobacter flavus enzymes from this group revealed that these molecules exhibit diverse kinetic properties despite their relatively high degree of sequence relatedness. Of prime importance was the fact that these closely related enzymes exhibited CO2 and O2 substrate specificities that varied from relatively low values [tau = (VcKo)/(VoKc) = 45] to values that approximated those obtained for higher plants (tau = 75). These results, combined with the metabolic and genetic versatility of the organisms from which these enzymes were derived, suggest a potential rich resource for future biological selection and structure-function studies aimed at elucidating structural features that govern key enzymological properties of rubisco.
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( 1997 ) [Nucleotide sequence of gltB gene encoding the large subunit of Rhodobacter sphaeroides glutamate synthase]. PMID : 9863198 : Abstract >>
The complete nucleotide sequence of a 5.4-kb chromosomal EcoRI-SalI fragment was determined, which contains the structural gene (gltB) for the large subunit of Rhodobacter sphaeroides glutamate synthase, as well as the 5'- and 3'- flanking regions. A open reading frame of 4636 base pairs was identified as R. sphaeroides gltB gene. The MW of the large subunit, as deduced from the nucleotide sequence, was estimated as 164kD. Comparision of the nucleotide sequences revealed a high similarity among gltB genes of R. sphaeroides, Azospirillum brasilense and Escherichia coli. The deduced amino acid sequence of R. sphaeroides GltB showed a high similarity with that of A. brasilense GltB.
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( 1998 ) Expression of glnB and a glnB-like gene (glnK) in a ribulose bisphosphate carboxylase/oxygenase-deficient mutant of Rhodobacter sphaeroides. PMID : 9721307 : PMC : PMC107479 Abstract >>
In a ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO)-deficient mutant of Rhodobacter sphaeroides, strain 16PHC, nitrogenase activity was derepressed in the presence of ammonia under photoheterotrophic growth conditions. Previous studies also showed that reintroduction of a functional RubisCO and Calvin-Benson-Bassham (CBB) pathway suppressed the deregulation of nitrogenase synthesis in this strain. In this study, the derepression of nitrogenase synthesis in the presence of ammonia in strain 16PHC was further explored by using a glnB::lacZ fusion, since the product of the glnB gene is known to have a negative effect on ammonia-regulated nif control. It was found that glnB expression was repressed in strain 16PHC under photoheterotrophic growth conditions with either ammonia or glutamate as the nitrogen source; glutamine synthetase (GS) levels were also affected in this strain. However, when cells regained a functional CBB pathway by trans complementation of the deleted genes, wild-type levels of GS and glnB expression were restored. Furthermore, a glnB-like gene, glnK, was isolated from this organism, and its expression was found to be under tight nitrogen control in the wild type. Surprisingly, glnK expression was found to be derepressed in strain 16PHC under photoheterotrophic conditions in the presence of ammonia.
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( 1997 ) Identification and molecular genetic analysis of multiple loci contributing to high-level tellurite resistance in Rhodobacter sphaeroides 2.4.1. PMID : 9406390 : PMC : PMC168794 Abstract >>
The ability of the facultative photoheterotroph Rhodobacter sphaeroides to tolerate and reduce high levels of tellurite in addition to at least 10 other rare earth metal oxides and oxyanions has considerable potential for detoxification and bioremediation of contaminated environments. We report the identification and characterization of two loci involved in high-level tellurite resistance. The first locus contains four genes, two of which, trgAB, confer increased tellurite resistance when introduced into the related bacterium Paracoccus denitrificans. The trgAB-derived products display no significant homology to known proteins, but both are likely to be membrane-associated proteins. Immediately downstream of trgB, the cysK (cysteine synthase) and orf323 genes were identified. Disruption of the cysK gene resulted in decreased tellurite resistance in R. sphaeroides, confirming earlier observations on the importance of cysteine metabolism for high-level tellurite resistance. The second locus identified is represented by the telA gene, which is separated from trgAB by 115 kb. The telA gene product is 65% similar to the product of the klaB (telA) gene from the tellurite-resistance-encoding kilA operon from plasmid RK2. The genes immediately linked to the R. sphaeroides telA gene have no similarity to other components of the kilA operon. R. sphaeroides telA could not functionally substitute for the plasmid RK2 telA gene, indicating substantial functional divergence between the two gene products. However, inactivation of R. sphaeroides telA resulted in a significant decrease in tellurite resistance compared to the wild-type strain. Both cysK and telA null mutations readily gave rise to suppressors, suggesting that the phenomenon of high-level tellurite resistance in R. sphaeroides is complex and other, as yet uncharacterized, loci may be involved.
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( 1997 ) Evidence for two chemosensory pathways in Rhodobacter sphaeroides. PMID : 9426144 : DOI : 10.1046/j.1365-2958.1997.6502022.x Abstract >>
In contrast to enteric bacteria, chemotaxis in Rhodobacter sphaeroides requires transport and partial metabolism of chemoattractants. Although a chemotaxis operon has been identified containing homologues of the enteric cheA, cheW, cheR genes and two homologues of the cheY gene, deletion of the entire chemotaxis operon had only minor effects on chemotactic behaviour under the conditions tested. Responses to sugars were enhanced in tethered cells but in all other chemotaxis assays behaviour of the operon deletion mutant was wild type. The mutant also showed wild-type responses to weak organic acids such as acetate and propionate, the dominant chemoattractants for this organism, under all conditions. This is in direct contrast to the enterics in which CheA, CheW and CheY are absolutely essential for taxis to PTS sugars, oxygen and MCP-dependent chemoeffectors. The operon deletion mutant was subjected to Tn5 transposon mutagenesis and new mutants selected using a chemotaxis and phototaxis screen. One mutant, JPA203, was non-chemotactic on swarm plates and showed inverted responses when tethered or subjected to changes in light intensity. Characterization of the Tn5 insertion in JPA203 identified a second chemotaxis operon in R. sphaeroides that contains homologues of cheY, cheA and cheR, the first homologue of cheB and two homologues of cheW. The new genes were labelled orf10, cheY(III), cheA(II) cheW(II), cheW(III), cheR(II), cheB and tlpC. When introduced into a wild-type background, deletion of cheA(II) produced a chemotaxis minus phenotype in R. sphaeroides, suggesting that cheA(II) forms part of a dominant chemotactic pathway, whereas the earlier identified operon plays only a minor role under laboratory conditions. The data presented here support the existence of two chemosensory pathways in R. sphaeroides, a feature that so far is unique in bacterial chemotaxis.
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( 1998 ) The flagellar switch genes fliM and fliN of Rhodobacter sphaeroides are contained in a large flagellar gene cluster. PMID : 9683497 : PMC : PMC107384 Abstract >>
In this work, the genes that encode the FliM and FliN proteins of Rhodobacter sphaeroides were characterized. These genes are part of a large flagellar gene cluster in which six additional open reading frames encoding products homologous to FliL, FliO, FliP, FliQ, FliR, and FlhB proteins from other bacteria were identified. The inactivation of the fliM gene gave a nonflagellate phenotype (Fla-), suggesting that FliM is required for flagellar assembly. Complementation analysis of this fliM mutant indicated that fliM and fliN transcription starts beyond the 5' end of fliK and terminates after fliN.
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169. |
( 1998 ) Structural studies of wild-type and mutant reaction centers from an antenna-deficient strain of Rhodobacter sphaeroides: monitoring the optical properties of the complex from bacterial cell to crystal. PMID : 9537989 : DOI : 10.1021/bi971717a Abstract >>
Reaction centers have been crystallized from the antenna-deficient RCO2 strain of Rhodobacter sphaeroides, and a structural model has been constructed at 2.6 A resolution. The antenna-deficient strain allows assessment of the structural integrity of the reaction center at each stage in the purification-crystallization procedure. Spectroscopic evidence indicates that the properties of the reaction center bacteriopheophytins and the primary donor bacteriochlorophylls are modified somewhat on removal of the protein complex from the membrane and that these changes are carried through to the crystal form of the reaction center. The structure of a FM197R/YM177F mutant reaction center has also been determined to 2.55 A resolution. The mutant complex shows an unexpected change in structure, with a significant reorientation of the new arginine, the incorporation of a new water molecule into the structure, and rotation of the 2-acetyl carbonyl group of one of the primary donor bacteriochlorophylls to a more out-of-plane geometry. Changes in the optical spectrum of the FM197R/YM177F reaction center are discussed with respect to the altered structure of the complex.
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( 1998 ) Metabolic roles of a Rhodobacter sphaeroides member of the sigma32 family. PMID : 9422586 : PMC : PMC106842 Abstract >>
We report the role of a gene (rpoH) from the facultative phototroph Rhodobacter sphaeroides that encodes a protein (sigma37) similar to Escherichia coli sigma32 and other members of the heat shock family of eubacterial sigma factors. R. sphaeroides sigma37 controls genes that function during environmental stress, since an R. sphaeroides deltaRpoH mutant is approximately 30-fold more sensitive to the toxic oxyanion tellurite than wild-type cells. However, the deltaRpoH mutant lacks several phenotypes characteristic of E. coli cells lacking sigma32. For example, an R. sphaeroides deltaRpoH mutant is not generally defective in phage morphogenesis, since it plates the lytic virus RS1, as well as its wild-type parent. In characterizing the response of R. sphaeroides to heat, we found that its growth temperature profile is different when cells generate energy by aerobic respiration, anaerobic respiration, or photosynthesis. However, growth of the deltaRpoH mutant is comparable to that of a wild-type strain under each of these conditions. The deltaRpoH mutant mounted a heat shock response when aerobically grown cells were shifted from 30 to 42 degrees C, but it exhibited altered induction kinetics of approximately 120-, 85-, 75-, and 65-kDa proteins. There was also reduced accumulation of several presumed heat shock transcripts (rpoD P(HS), groESL1, etc.) when aerobically grown deltaRpoH cells were placed at 42 degrees C. Under aerobic conditions, it appears that another sigma factor enables the deltaRpoH mutant to mount a heat shock response, since either RNA polymerase preparations from an deltaRpoH mutant, reconstituted Esigma37, or a holoenzyme containing a 38-kDa protein (sigma38) each transcribed E. coli Esigma32-dependent promoters. The lower growth temperature profile of photosynthetic cells is correlated with a difference in heat-inducible gene expression, since neither wild-type cells or the deltaRpoH mutant mount a typical heat shock response after such cultures were shifted from 30 to 37 degrees C.
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( 1998 ) The crystal structure of phosphoribulokinase from Rhodobacter sphaeroides reveals a fold similar to that of adenylate kinase. PMID : 9548738 : DOI : 10.1021/bi972805y Abstract >>
The essential photosynthetic enzyme phosphoribulokinase (PRK) is responsible for the conversion of ribulose 5-phosphate (Ru5P) to ribulose 1,5-bisphosphate, the substrate for the CO2 fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). We have determined the structure of the octameric bacterial form of PRK to a resolution of 2.5 A. The protein is folded into a seven-member mixed beta-sheet surrounded by alpha-helices, giving the overall appearance of the nucleotide monophosphate family of kinases. Homology with the nucleotide monophosphate kinases suggests a number of amino acid residues that are likely to be important in catalysis and suggests the roles of some amino acid residues that have been mutated prior to the determination of the structure. Further, sequence identity across eukaryotic and prokaryotic species and a calculation of the buried surface area suggests the identity within the octamer of a dimer conserved throughout evolution. The width of the groove leading to the active site is consistent with an oriented molecule of thioredoxin controlling the oxidation state of two cysteines that regulate activity in the eukaryotic enzymes. Although neither Asp 42 nor Asp 169 can be definitively assigned as the catalytic base, the crystal structure suggests the location of a ribulose 5-phosphate binding site and suggests a role for several of the conserved basic residues.
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( 1997 ) Characterization of genes encoding dimethyl sulfoxide reductase of Rhodobacter sphaeroides 2.4.1T: an essential metabolic gene function encoded on chromosome II. PMID : 9401017 : DOI : 10.1128/jb.179.24.7617-7624.1997 PMC : PMC179721 Abstract >>
Rhodobacter sphaeroides 2.4.1T is a purple nonsulfur facultative phototrophic bacterium which exhibits remarkable metabolic diversity as well as genomic complexity. Under anoxic conditions, in the absence of light and the presence of dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO), R. sphaeroides 2.4.1T utilizes DMSO or TMAO as the terminal electron acceptor for anaerobic respiration, which is mediated by the molybdoenzyme DMSO reductase. Sequencing of a 13-kb region of chromosome II revealed the presence of 10 putative open reading frames, of which 5 possess homology to genes encoding the TMAO reductase (the tor system) of Escherichia coli. The dorS and dorR genes encode a sensor-regulator pair of the two-component sensory transduction protein family, homologous to the torS and torR gene products. The dorC gene was shown to encode a 44-kDa DMSO-inducible c-type cytochrome. The dorB gene encodes a membrane protein of unknown function homologous to the torD gene product. The dorA gene encodes DMSO reductase, containing the molybdopterin active site. Mutations were constructed in each of these dor genes, and the resulting mutants were shown to be impaired for DMSO-dependent anaerobic growth in the dark. The mutant strains exhibited negligible levels of DMSO reductase activity compared to the wild-type strain under similar growth conditions. Further, no DorA protein was detected in DorS and DorR mutant strains with anti-DorA antisera, suggesting that the products of these genes are required for the positive regulation of dor expression in response to DMSO. This characterization of the dor gene cluster is the first evidence that genes of chromosome CII encode metabolic functions which are essential under particular growth conditions.
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