| 1. |
Karr EA,
Sattley WM,
Jung DO,
Madigan MT,
Achenbach LA,
( 2003 ) Remarkable diversity of phototrophic purple bacteria in a permanently frozen Antarctic lake. PMID : 12902286 : DOI : 10.1128/aem.69.8.4910-4914.2003 PMC : PMC169089 Abstract >>
Although anoxygenic photosynthesis is thought to play an important role in the primary productivity of permanently frozen lakes in the Antarctic dry valleys, the bacterial communities responsible for this metabolism remain uncharacterized. Here we report the composition and activity of phototrophic purple bacteria in Lake Fryxell, Antarctica, as determined by analysis of a photosynthesis-specific gene, pufM. The results revealed an extensive diversity and highly stratified distribution of purple nonsulfur bacteria in Lake Fryxell and showed which phylotypes produced pufM transcripts in situ. Enrichment cultures for purple bacteria yielded two morphotypes, each with a pufM signature identical to signatures detected by environmental screening. The isolates also contained gas vesicles, buoyancy structures previously unknown in purple nonsulfur bacteria, that may be necessary for these organisms to position themselves at specific depths within the nearly freezing water column.
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2. |
McGuirl MA,
Lee JC,
Lyubovitsky JG,
Thanyakoop C,
Richards JH,
Gray HB,
Winkler JR,
( 2003 ) Cloning, heterologous expression, and characterization of recombinant class II cytochromes c from Rhodopseudomonas palustris. PMID : 12495812 : DOI : 10.1016/s0304-4165(02)00437-3 Abstract >>
The cytochrome (cyt) c', cyt c(556), and cyt c(2) genes from Rhodopseudomonas palustris have been cloned; recombinant cyt c' and cyt c(556) have been expressed, purified, and characterized. Unlike mitochondrial cyt c, these two proteins are structurally similar to cyt b(562), in which the heme is embedded in a four-helix bundle. The hemes in both recombinant proteins form covalent thioether links to two Cys residues. UV/vis spectra of the Fe(II) and Fe(III) states of the recombinant cyts are identical with those of the corresponding native proteins. Equilibrium unfolding measurements in guanidine hydrochloride solutions confirm that native Fe(II)-cyt c(556) is more stable than the corresponding state of Fe(III)-cyt c(556) (DeltaDeltaG(f)(o) =22 kJ/mol).
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3. |
Inui M,
Puskás LG,
( 2000 ) Cloning of genes participating in aerobic biodegradation of p-cumate from Rhodopseudomonas palustris. PMID : 10902905 : Abstract >>
Rhodopseudomonas palustris utilizes p-cumate as a carbon source both under anaerobic light and aerobic dark conditions. A gene cluster was isolated whose sequence showed high homology to genes which have been implicated the degradation of p-cumate in Pseudomonas pitida. Seven structural genes coding for dioxygenase-reductase, dihydroxy-dihydro dehydrogenase, and ring cleavage oxygenases were identified. A putative regulator and its possible recognition site was suggested on the basis of homology data. Mutant cells in which a kanamycin cassette was inserted into the dihydroxy-dihydro dehydrogenase gene could not grow aerobically on p-cumate. The mutation had no effect on growth using the para substituted benzoate derivatives 4-hydroxycinnamate, ferulate, protocatechuate, and 2,3,4-trihydroxybenzoate as sole carbon source. Moreover, mutant cells showed a growth pattern similar to wild type cells grown on these compounds under photoheterotrophic anaerobic conditions. These data suggest that genes of this operon are involved specifically in aerobic dissimilation of p-cumate. Intermediate products of p-cumate degradation could be detected from extracts of Escherichia coli heterologously expressing the first 5 genes responsible for the first two steps of p-cumate degradation in R. palustris. Primer extension analysis revealed the transcription regulation of the gene cluster which could be induced with para methyl-, ethyl- and isopropyl (cumate) benzoates. This is the first report on genes involved in aerobic degradation of these compounds in photosynthetic bacteria.
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4. |
Puskás LG,
Inui M,
Zahn K,
Yukawa H,
( 2000 ) A periplasmic, alpha-type carbonic anhydrase from Rhodopseudomonas palustris is essential for bicarbonate uptake. PMID : 11065374 : DOI : 10.1099/00221287-146-11-2957 Abstract >>
Intact cells of the purple non-sulfur bacterium Rhodopseudomonas palustris growing anaerobically, but not aerobically, contain carbonic anhydrase (CA) activity. The native enzyme was purified >2000-fold to apparent homogeneity and found to be a dimer with an estimated molecular mass of 54 kDa and a subunit molecular mass of 27 kDa. The CA gene (acaP) was cloned and its sequence revealed that it was homologous to alpha-type CAs. The upstream region of acaP was fused to the lacZ gene and beta-galactosidase activity was measured under different growth conditions. Acetazolamide inhibited purified CA with an IC(50) in the range of 10(-8) M, and in the culture media concentrations as low as 30 microM inhibited phototrophic growth under anaerobic, light conditions when bicarbonate was used. An acaP::KAN:(r) mutant strain was constructed by insertion of a kanamycin-resistance cassette and showed a growth pattern similar to wild-type cells grown in the presence of CA inhibitor. CO(2) gas supplied as an inorganic carbon source reversed the effect of mutation or acetazolamide. CA activity measurements, fusion and Western blot experiments confirmed that CA is expressed under different anaerobic conditions independently of bicarbonate or CO(2) and that there is no expression under aerobic conditions.
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5. |
Roh JH,
Inui M,
( 2000 ) Sequence analysis of the cryptic plasmid pMG101 from Rhodopseudomonas palustris and construction of stable cloning vectors. PMID : 10618203 : DOI : 10.1128/aem.66.1.54-63.2000 PMC : PMC91785 Abstract >>
A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of alpha-proteobacteria.
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6. |
Egland PG,
( 2000 ) HbaR, a 4-hydroxybenzoate sensor and FNR-CRP superfamily member, regulates anaerobic 4-hydroxybenzoate degradation by Rhodopseudomonas palustris. PMID : 10613868 : DOI : 10.1128/jb.182.1.100-106.2000 PMC : PMC94245 Abstract >>
Under anaerobic conditions, structurally diverse aromatic compounds are catabolized by bacteria to form benzoyl-coenzyme A (benzoyl-CoA), the starting compound for a central reductive pathway for aromatic ring degradation. The structural genes required for the conversion of 4-hydroxybenzoate (4-HBA) to benzoyl-CoA by Rhodopseudomonas palustris have been identified. Here we describe a regulatory gene, hbaR, that is part of the 4-HBA degradation gene cluster. An hbaR mutant that was constructed was unable to grow anaerobically on 4-HBA. However, the mutant retained the ability to grow aerobically on 4-HBA by an oxygen-requiring pathway distinct from the anaerobic route of 4-HBA degradation. The effect of the HbaR protein on expression of hbaA encoding 4-HBA-CoA ligase, the first enzyme for 4-HBA degradation, was investigated by using hbaA::'lacZ transcriptional fusions. HbaR was required for a 20-fold induction of beta-galactosidase activity that was observed with a chromosomal hbaA::'lacZ fusion when cells grown anaerobically on succinate were switched to anaerobic growth on succinate and 4-HBA. HbaR also activated expression from a plasmid-borne hbaA-'lacZ fusion when it was expressed in aerobically grown Pseudomonas aeruginosa cells, indicating that the activity of this regulator is not sensitive to oxygen. The deduced amino acid sequence of HbaR indicates that it is a member of the FNR-CRP superfamily of regulatory proteins. It is most closely related to transcriptional activators that are involved in regulating nitrate reduction. Previously, it has been shown that R. palustris has an FNR homologue, called AadR, that is also required for 4-HBA degradation. Our evidence indicates that AadR activates expression of hbaR in response to anaerobiosis and that HbaR, in turn, activates expression of 4-HBA degradation in response to 4-HBA as an effector molecule.
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7. |
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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8. |
Oda Y,
Meijer WG,
Gibson JL,
Gottschal JC,
Forney LJ,
( 2004 ) Analysis of diversity among 3-chlorobenzoate-degrading strains of Rhodopseudomonas palustris. PMID : 15259271 : DOI : 10.1007/s00248-003-1028-5 Abstract >>
The phenotypic and genetic characteristics of 14 strains of the purple nonsulfur bacterium Rhodopseudomonas palustris were studied to assess diversity within this species. While all strains had certain phenotypic characteristics in common, including the ability to metabolize benzoate and degrade 2- and 3-chlorobenzoate, there were also significant differences among the strains such as the rate of growth in media containing benzoate as a carbon source. Genetic characterization of the strains revealed there were three divergent lineages in the species. Based on 16S rRNA gene sequences, the 14 strains could be grouped into three distinct clusters (A, B, and C), and this clustering was congruent with that based on gene sequences of form II ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). Although BOX-PCR genomic DNA fingerprints of all 14 strains exhibited differences, analysis of the fingerprint images and UPGMA/product-moment analysis of similarities showed there were three groupings that were entirely consistent with clusters based on other characteristics of the strains. Thus, regardless of the method of analysis used, strains in groups A and B consistently clustered together and were separate from those of group C. These results suggest that strains in groups A-B and C represent phylogenetically related clones that have diverged from one another. This indicates that at least three lineages of Rhodopseudomonas palustris exist among the strains included in this study, and that each may be particularly well adapted to a distinct ecological niche.
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9. |
Cantera JJ,
Kawasaki H,
Seki T,
( 2004 ) The nitrogen-fixing gene (nifH) of Rhodopseudomonas palustris: a case of lateral gene transfer? PMID : 15256566 : DOI : 10.1099/mic.0.26940-0 Abstract >>
Nitrogen fixation is catalysed by some photosynthetic bacteria. This paper presents a phylogenetic comparison of a nitrogen fixation gene (nifH) with the aim of elucidating the processes underlying the evolutionary history of Rhodopseudomonas palustris. In the NifH phylogeny, strains of Rps. palustris were placed in close association with Rhodobacter spp. and other phototrophic purple non-sulfur bacteria belonging to the alpha-Proteobacteria, separated from its close relatives Bradyrhizobium japonicum and the phototrophic rhizobia (Bradyrhizobium spp. IRBG 2, IRBG 228, IRBG 230 and BTAi 1) as deduced from the 16S rRNA phylogeny. The close association of the strains of Rps. palustris with those of Rhodobacter and Rhodovulum, as well as Rhodospirillum rubrum, was supported by the mol% G+C of their nifH gene and by the signature sequences found in the sequence alignment. In contrast, comparison of a number of informational and operational genes common to Rps. palustris CGA009, B. japonicum USDA 110 and Rhodobacter sphaeroides 2.4.1 suggested that the genome of Rps. palustris is more related to that of B. japonicum than to the Rba. sphaeroides genome. These results strongly suggest that the nifH of Rps. palustris is highly related to those of the phototrophic purple non-sulfur bacteria included in this study, and might have come from an ancestral gene common to these phototrophic species through lateral gene transfer. Although this finding complicates the use of nifH to infer the phylogenetic relationships among the phototrophic bacteria in molecular diversity studies, it establishes a framework to resolve the origins and diversification of nitrogen fixation among the phototrophic bacteria in the alpha-Proteobacteria.
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10. |
Choi HP,
Hong JW,
Rhee KH,
Sung HC,
( 2004 ) Cloning, expression, and characterization of 5-aminolevulinic acid synthase from Rhodopseudomonas palustris KUGB306. PMID : 15251194 : DOI : 10.1016/j.femsle.2004.05.048 Abstract >>
The hemA gene encoding 5-aminolevulinic acid synthase (ALAS) was cloned from the genomic DNA of photosynthetic bacterium Rhodopseudomonas palustris KUGB306. The deduced protein (ALAS) of this gene contained 409 amino acids. The hemA gene was subcloned into an expression vector pGEX-KG and the encoded protein was overexpressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli BL21. The recombinant ALAS was purified and isolated free of the fusion partner (GST) by affinity purification on glutathione-Sepharose 4B resin and cleavage of the purified fusion protein by thrombin protease. The optimum pH and temperature of the recombinant ALAS was found to be at pH 7.5-8.0 and 35-40 degrees C, respectively. The Km value of the enzyme was 2.01 mM for glycine and 49.55 microM for succinyl-CoA. The enzyme activity was strongly inhibited by Pb2+, Fe2+, Co2+, Cu2+, and Zn2+ at 1 mM, but slightly affected by Mg2+ and K+. The recombinant ALAS required pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor led to complete loss of the activity. Ultraviolet-visible spectroscopy with the ALAS suggested the presence of an aldimine linkage between the enzyme and PLP.
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11. |
Dispensa M,
Thomas CT,
Kim MK,
Perrotta JA,
Gibson J,
Harwood CS,
( 1992 ) Anaerobic growth of Rhodopseudomonas palustris on 4-hydroxybenzoate is dependent on AadR, a member of the cyclic AMP receptor protein family of transcriptional regulators. PMID : 1522059 : DOI : 10.1128/jb.174.18.5803-5813.1992 PMC : PMC207109 Abstract >>
The purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris converts structurally diverse aromatic carboxylic acids, including lignin monomers, to benzoate and 4-hydroxybenzoate under anaerobic conditions. These compounds are then further degraded via aromatic ring-fission pathways. A gene termed aadR, for anaerobic aromatic degradation regulator, was identified by complementation of mutants unable to grow anaerobically on 4-hydroxybenzoate. The deduced amino acid sequence of the aadR product is similar to a family of transcriptional regulators which includes Escherichia coli Fnr and Crp, Pseudomonas aeruginosa Anr, and rhizobial FixK and FixK-like proteins. A mutant with a deletion in aadR failed to grow on 4-hydroxybenzoate under anaerobic conditions and grew very slowly on benzoate. It also did not express aromatic acid-coenzyme A ligase II, an enzyme that catalyzes the first step of 4-hydroxybenzoate degradation, and it was defective in 4-hydroxybenzoate-induced expression of benzoate-coenzyme A ligase. The aadR deletion mutant was unaffected in other aspects of anaerobic growth. It grew normally on nonaromatic carbon sources and also under nitrogen-fixing conditions. In addition, aerobic growth on 4-hydroxybenzoate was indistinguishable from that of the wild type. These results indicate that AadR functions as a transcriptional activator of anaerobic aromatic acid degradation.
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12. |
Filonov GS,
Piatkevich KD,
Ting LM,
Zhang J,
Kim K,
Verkhusha VV,
( 2011 ) Bright and stable near-infrared fluorescent protein for in vivo imaging. PMID : 21765402 : DOI : 10.1038/nbt.1918 PMC : PMC3152693 Abstract >>
Imaging biological processes in mammalian tissues will be facilitated by fluorescent probes with excitation and emission bands within the near-infrared optical window of high transparency. Here we report a phytochrome-based near-infrared fluorescent protein (iRFP) with excitation and emission maxima at 690 nm and 713 nm, respectively. iRFP does not require an exogenous supply of the chromophore biliverdin and has higher effective brightness, intracellular stability and photostability than earlier phytochrome-derived fluorescent probes. Compared with far-red GFP-like proteins, iRFP has a substantially higher signal-to-background ratio in a mouse model due to its infrared-shifted spectra.
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13. |
Okamura K,
Takata K,
Hiraishi A,
( 2009 ) Intrageneric relationships of members of the genus Rhodopseudomonas. PMID : 20118611 : Abstract >>
The intrageneric structure of the genus Rhodopseudomonas was evaluated by studying sequence information on 16S rRNA genes, 16S-23S rRNA gene internal transcribed spacer (ITS) regions, and puf genes using 33 test strains. The topology of phylogenetic trees based on these sequences was similar to those of every other independent method for tree construction. These phylogenetic data indicated that the test strains were grouped into at least 7 clusters possibly at the species level. This was supported by genomic DNA-DNA similarities among 12 representative test strains selected from these clusters. Our molecular data confirmed that the currently available strains of Rhodopseudomonas (Rps.) palustris are genetically quite heterogeneous within the genus. For example, Rps. palustris strains DSM 123(T) and ATCC 17001(T) are different from each other at the species level despite their status as the type strain of the species. Rps. palustris strain ATCC 17005 and the full genome-sequenced strains BisA53, BisB18, BisB5, and HaA2 should be re-classified into different species from Rps. palustris or as novel species of the genus Rhodopseudomonas.
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14. |
Sato I,
Yoshikawa J,
Furusawa A,
Chiku K,
Amachi S,
Fujii T,
( 2010 ) Isolation and properties of malic enzyme and its gene in Rhodopseudomonas palustris No. 7. PMID : 20057150 : DOI : 10.1271/bbb.90566 Abstract >>
Malic enzyme (ME) was purified as an electrophoretically homogenous protein from Rhodopseudomonas palustris No. 7. The molecular weight of ME was estimated to be 650 kDa and that of its subunit, 86 kDa. ME activity was remarkably enhanced by di- and mono-valent cations, and the K(a) values for Mg(2+) and NH(4)(+) were 0.26 and 0.56 mM respectively. Purified ME used both NAD(+) and NADP(+) as electron acceptors, with K(m) values of 0.11 and 1.8 mM. The K(m) value for L-malate was 1.7 mM using NAD(+) as electron acceptor. Gene cloning of the ME indicated that the ME from R. palustris strain No. 7 was composed of 774 amino acids encompassing the ME and phosphotransacetylase domains, although purified ME displayed no phosphotransacetylase activity. ME activity was inhibited by acetyl-CoA, oxaloacetate, and fructose-6-phosphate. These results suggest that ME plays an important role in the metabolic regulation of R. palustris No. 7 under photoheterotrophic conditions.
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15. |
Vuillet L,
Kojadinovic M,
Zappa S,
Jaubert M,
Adriano JM,
Fardoux J,
Hannibal L,
Pignol D,
Verméglio A,
Giraud E,
( 2007 ) Evolution of a bacteriophytochrome from light to redox sensor. PMID : 17581629 : DOI : 10.1038/sj.emboj.7601770 PMC : PMC1933401 Abstract >>
Bacteriophytochromes are red/far-red photoreceptors that bacteria use to mediate sensory responses to their light environment. Here, we show that the photosynthetic bacterium Rhodopseudomonas palustris has two distinct types of bacteriophytochrome-related protein (RpBphP4) depending upon the strain considered. The first type binds the chromophore biliverdin and acts as a light-sensitive kinase, thus behaving as a bona fide bacteriophytochrome. However, in most strains, RpBphP4 does not to bind this chromophore. This loss of light sensing is replaced by a redox-sensing ability coupled to kinase activity. Phylogenetic analysis is consistent with an evolutionary scenario, where a bacteriophytochrome ancestor has adapted from light to redox sensing. Both types of RpBphP4 regulate the synthesis of light harvesting (LH2) complexes according to the light or redox conditions, respectively. They modulate the affinity of a transcription factor binding to the promoter regions of LH2 complex genes by controlling its phosphorylation status. This is the first complete description of a bacteriophytochrome signal transduction pathway involving a two-component system.
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16. |
Tadros MH,
Waterkamp K,
( 1989 ) Multiple copies of the coding regions for the light-harvesting B800-850 alpha- and beta-polypeptides are present in the Rhodopseudomonas palustris genome. PMID : 2670551 : PMC : PMC400955 Abstract >>
A reverse-phase HPLC System for isolation of the water insoluble alpha- and beta-polypeptides of the light-harvesting complex II (LH II) of Rhodopseudomonas (Rps.) palustris without employment of any detergent was developed. The material obtained was of high purity and suitable for direct microsequence analysis. Chromatographic analysis could resolve at least two major beta-polypeptides, beta a and beta b, two major alpha-polypeptides, alpha a and alpha b, and two additional minor polypeptides. N-terminal amino acid sequencing shows that the resolved peaks correspond to different polypeptide species and that the minor species have an N-terminal sequence identical to that of the alpha b polypeptide. An oligonucleotide derived from the amino terminal sequence of the alpha a polypeptide was utilized to screen a genomic library from Rps.palustris. Several independent clones have been characterized by Southern blot and nucleotide sequence analysis. We show that Rps.palustris contains at least four different clusters of beta and alpha genes. Two clones contain sequences potentially coding for beta a-alpha a and beta b-alpha b polypeptides; and two additional clones potentially coding for beta and alpha peptides which we named beta c-alpha c and beta d-alpha d, which did not correspond to the major purified polypeptides. In addition to the protein chemistry data, the conservation at the amino acid level and the presence of canonical ribosomal binding sites upstream of each of the identified genes strongly suggest that all four coding regions are expressed.
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17. |
Thornburg CK,
Wortas-Strom S,
Nosrati M,
Geiger JH,
Walker KD,
( 2015 ) Kinetically and Crystallographically Guided Mutations of a Benzoate CoA Ligase (BadA) Elucidate Mechanism and Expand Substrate Permissivity. PMID : 26378464 : DOI : 10.1021/acs.biochem.5b00899 Abstract >>
A benzoate CoA ligase (BadA), isolated from the bacterium Rhodopseudomonas palustris, catalyzes the conversion of benzoate to benzoyl CoA on the catabolic pathway of aromatic carboxylic acids. Herein, apparent Michaelis constants K(app)cat and K(app)M were determined for an expanded array of 31 substrates chosen to systematically probe the active site architecture of the enzyme and provide a baseline for expansion of wild-type substrate specificity. Acyl CoA products were observed for 25 of the 31 substrates; in general, BadA converted ortho-substituted substrates better than the corresponding meta and para regioisomers, and the turnover number was more affected by steric rather than electronic effects. The kinetic data are interpreted in relation to six crystal structures of BadA in complex with several substrates and a benzoyl-AMP reaction intermediate. In contrast to other known natural substrate-bound benzoate ligase structures, all substrate-bound BadA structures adopted the thiolation conformation instead of the adenylation conformation. We also observed all the aryl carboxylates to be uniquely oriented within the active site, relative to other structures. Together, the kinetics and structural data suggested a mechanism that involves substrate binding in the thiolation conformation, followed by substrate rotation to an active orientation upon the transition to the adenylation conformation. On the basis of this hypothesis and the structural data, sterically demanding active site residues were mutated, and the substrate specificity was expanded substantially versus that of BadA. Novel activities were seen for substrates with larger substituents, including phenyl acetate. Additionally, the mutant Lys427Ala identified this nonconserved residue as essential for the thiolation step of BadA, but not adenylation. These variously acylated CoAs can serve as novel substrates of acyl CoA-dependent acyltransferases in coupled enzyme assays to produce analogues of bioactive natural products.
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18. |
Wong WT,
Tseng CH,
Hsu SH,
Lur HS,
Mo CW,
Huang CN,
Hsu SC,
Lee KT,
Liu CT,
( 2014 ) Promoting effects of a single Rhodopseudomonas palustris inoculant on plant growth by Brassica rapa chinensis under low fertilizer input. PMID : 25130882 : DOI : 10.1264/jsme2.me14056 PMC : PMC4159042 Abstract >>
Several Rhodopseudomonas palustris strains have been isolated from rice paddy fields in Taiwan by combining the Winogradsky column method and molecular marker detection. These isolates were initially screened by employing seed germination and seedling vigor assays to evaluate their potential as inoculants. To fulfill the demand in the present farming system for reducing the application of chemical fertilizers, we assessed the plant growth-promoting effects of the R. palustris YSC3, YSC4, and PS3 inoculants on Brassica rapa chinensis (Chinese cabbage) cultivated under a half quantity of fertilizer. The results obtained showed that supplementation with approximately 4.0��10(6) CFU g(-1) soil of the PS3 inoculant at half the amount of fertilizer consistently produced the same plant growth potential as 100% fertility, and also increased the nitrogen use efficiency of the applied fertilizer nutrients. Furthermore, we noted that the plant growth-promotion rate elicited by PS3 was markedly higher with old seeds than with new seeds, suggesting it has the potential to boost the development of seedlings that were germinated from carry-over seeds of poor quality. These beneficial traits suggest that the PS3 isolate may serve as a potential PGPR inoculant for integrated nutrient management in agriculture.
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19. |
Ambler RP,
Daniel M,
Hermoso J,
Meyer TE,
Bartsch RG,
Kamen MD,
( 1979 ) Cytochrome c2 sequence variation among the recognised species of purple nonsulphur photosynthetic bacteria. PMID : 221822 : DOI : 10.1038/278659a0 Abstract >>
N/A
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20. |
( 1997 ) A cluster of bacterial genes for anaerobic benzene ring biodegradation. PMID : 9177244 : DOI : 10.1073/pnas.94.12.6484 PMC : PMC21076 Abstract >>
A reductive benzoate pathway is the central conduit for the anaerobic biodegradation of aromatic pollutants and lignin monomers. Benzene ring reduction requires a large input of energy and this metabolic capability has, so far, been reported only in bacteria. To determine the molecular basis for this environmentally important process, we cloned and analyzed genes required for the anaerobic degradation of benzoate and related compounds from the phototrophic bacterium, Rhodopseudomonas palustris. A cluster of 24 genes was identified that includes twelve genes likely to be involved in anaerobic benzoate degradation and additional genes that convert the related compounds 4-hydroxybenzoate and cyclohexanecarboxylate to benzoyl-CoA. Genes encoding benzoyl-CoA reductase, a novel enzyme able to overcome the resonance stability of the aromatic ring, were identified by directed mutagenesis. The gene encoding the ring-cleavage enzyme, 2-ketocyclohexanecarboxyl-CoA hydrolase, was identified by assaying the enzymatic activity of the protein expressed in Escherichia coli. Physiological data and DNA sequence analyses indicate that the benzoate pathway consists of unusual enzymes for ring reduction and cleavage interposed among enzymes homologous to those catalyzing fatty acid degradation. The cloned genes should be useful as probes to identify benzoate degradation genes from other metabolically distinct groups of anaerobic bacteria, such as denitrifying bacteria and sulfate-reducing bacteria.
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21. |
( 1997 ) 4-hydroxybenzoyl coenzyme A reductase (dehydroxylating) is required for anaerobic degradation of 4-hydroxybenzoate by Rhodopseudomonas palustris and shares features with molybdenum-containing hydroxylases. PMID : 9006014 : DOI : 10.1128/jb.179.3.634-642.1997 PMC : PMC178741 Abstract >>
The anaerobic degradation of 4-hydroxybenzoate is initiated by the formation of 4-hydroxybenzoyl coenzyme A, with the next step proposed to be a dehydroxylation to benzoyl coenzyme A, the starting compound for a central pathway of aromatic compound ring reduction and cleavage. Three open reading frames, divergently transcribed from the 4-hydroxybenzoate coenzyme A ligase gene, hbaA, were identified and sequenced from the phototrophic bacterium Rhodopseudomonas palustris. These genes, named hbaBCD, specify polypeptides of 17.5, 82.6, and 34.5 kDa, respectively. The deduced amino acid sequences show considerable similarities to a group of hydroxylating enzymes involved in CO, xanthine, and nicotine metabolism that have conserved binding sites for [2Fe-2S] clusters and a molybdenum cofactor. Cassette disruption of the hbaB gene yielded a mutant that was unable to grow anaerobically on 4-hydroxybenzoate but grew normally on benzoate. The hbaB mutant cells did not accumulate [14C]benzoyl coenzyme A during short-term uptake of [14C]4-hydroxybenzoate, but benzoyl coenzyme A was the major radioactive metabolite formed by the wild type. In addition, crude extracts of the mutant failed to convert 4-hydroxybenzoyl coenzyme A to benzoyl coenzyme A. This evidence indicates that the hbaBCD genes encode the subunits of a 4-hydroxybenzoyl coenzyme A reductase (dehydroxylating). The sizes of the specified polypeptides are similar to those reported for 4-hydroxybenzoyl coenzyme A reductase isolated from the denitrifying bacterium Thauera aromatica. The amino acid consensus sequence for a molybdenum cofactor binding site is in HbaC. This cofactor appears to be an essential component because anaerobic growth of R. palustris on 4-hydroxybenzoate, but not on benzoate, was retarded unless 0.1 microM molybdate was added to the medium. Neither tungstate nor vanadate replaced molybdate, and tungstate competitively inhibited growth stimulation by molybdate.
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22. |
( 1995 ) Metabolism of cyclohexane carboxylic acid by the photosynthetic bacterium Rhodopseudomonas palustris. PMID : 8572887 : Abstract >>
Cyclohexane carboxylate supported relatively rapid growth (doubling times 7-8 h) of Rhodopseudomonas palustris under oxic or photosynthetic conditions, but did not serve as a substrate for either of the known aromatic CoA ligases. A CoA ligase that thioesterifies cyclohexane carboxylate was partially purified and did not cross react immunologically with the two CoA ligases purified previously from this bacterium. Crude extracts of R. palustris cells grown with a range of aromatic or alicyclic acids contained a dehydrogenase that reacted with cyclohexane carboxyl-CoA or cyclohex-1-ene carboxyl-CoA, using 2,6-dichlorophenolindophenol or ferricenium ion as electron carrier. This activity was not detected in extracts of adipate-, glutamate-, or succinate-grown cells. No oxidation or reduction of nonesterified cyclohexane carboxylate or cyclohexene carbocylate was detected in extracts of cells grown with aromatic or aliphatic substrates, neither aerobically nor anaerobically. A constitutively expressed thioesterase that hydrolyzed cyclohexane carboxyl-CoA and also some alicyclic and aliphatic CoA derivatives was purified and characterized. The enzyme had little or no activity on benzoyl-CoA or 4-hydroxybenzoyl-CoA. The presence of a thioesterase that effectively hydrolyzes cyclohexane carboxyl-CoA suggests that transient production of cyclohexane carboxylate is a physiological response to temporary excess of reductant during metabolism of aromatic compounds.
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23. |
Egland PG,
Gibson J,
Harwood CS,
( 1995 ) Benzoate-coenzyme A ligase, encoded by badA, is one of three ligases able to catalyze benzoyl-coenzyme A formation during anaerobic growth of Rhodopseudomonas palustris on benzoate. PMID : 7592432 : DOI : 10.1128/jb.177.22.6545-6551.1995 PMC : PMC177507 Abstract >>
The first step of anaerobic benzoate degradation is the formation of benzoyl-coenzyme A by benzoate-coenzyme A ligase. This enzyme, purified from Rhodopseudomonas palustris, is maximally active with 5 microM benzoate. To study the molecular basis for this reaction, the benzoate-coenzyme A ligase gene (badA) was cloned and sequenced. The deduced amino acid sequence of badA showed substantial similarity to other coenzyme A ligases, with the highest degree of similarity being that to 4-hydroxybenzoate-coenzyme A ligase (50% amino acid identity) from R. palustris. A badA mutant that was constructed had barely detectable levels of ligase activity when cell extracts were assayed at 10 microM benzoate. Despite this, the mutant grew at wild-type rates on benzoate under laboratory culture conditions (3 mM benzoate), and mutant cell extracts had high levels of ligase activity when assayed at a high concentration of benzoate (1 mM). This suggested that R. palustris expresses, in addition to BadA, a benzoate-activating enzyme(s) with a relatively low affinity for benzoate. A possible role of 4-hydroxybenzoate-coenzyme A ligase (encoded by hbaA) in this capacity was investigated by constructing a badA hbaA double mutant. Although the double mutant grew more slowly on benzoate than badA cells, growth rates were still significant, suggesting the involvement of a third enzyme in benzoate activation. Competition experiments involving the addition of a small amount of cyclohexanecarboxylate to ligase assay mixtures implicated cyclohexanecarboxylate-coenzyme A ligase as being this third enzyme. These results show that wild-type R. palustris cells synthesize at least three enzymes that can catalyze the initial step in anaerobic benzoate degradation during growth on benzoate. This observation supports previous suggestions that benzoyl-coenzyme A formation plays a central role in anaerobic aromatic compound biodegradation.
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24. |
Imhoff JF,
Rahn T,
Künzel S,
Neulinger SC,
( 2018 ) New insights into the metabolic potential of the phototrophic purple bacterium Rhodopila globiformis DSM 161T from its draft genome sequence and evidence for a vanadium-dependent nitrogenase. PMID : 29423563 : DOI : 10.1007/s00203-018-1489-z Abstract >>
Rhodopila globiformis: is the most acidophilic anaerobic anoxygenic phototrophic purple bacterium and was isolated from a warm acidic sulfur spring in Yellowstone Park. Its genome is larger than genomes of other phototrophic purple bacteria, containing 7248 Mb with a G + C content of 67.1% and 6749 protein coding and 53 RNA genes. The genome revealed some previously unknown properties such as the presence of two sets of structural genes pufLMC for the photosynthetic reaction center genes and two types of nitrogenases (Mo-Fe and V-Fe nitrogenase), capabilities of autotrophic carbon dioxide fixation and denitrification using nitrite. Rhodopila globiformis assimilates sulfate and utilizes the C1 carbon substrates CO and methanol and a number of organic compounds, in particular, sugars and aromatic compounds. It is among the few purple bacteria containing a large number of pyrroloquinoline quinone-dependent dehydrogenases. It has extended capacities to resist stress by heavy metals, demonstrates different resistance mechanisms to antibiotics, and employs several toxin/antitoxin systems.
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25. |
Bastard K,
Perret A,
Mariage A,
Bessonnet T,
Pinet-Turpault A,
Petit JL,
Darii E,
Bazire P,
Vergne-Vaxelaire C,
Brewee C,
Debard A,
Pellouin V,
Besnard-Gonnet M,
Artiguenave F,
Médigue C,
Vallenet D,
Danchin A,
Zaparucha A,
Weissenbach J,
Salanoubat M,
de Berardinis V,
( 2017 ) Parallel evolution of non-homologous isofunctional enzymes in methionine biosynthesis. PMID : 28581482 : DOI : 10.1038/nchembio.2397 Abstract >>
Experimental validation of enzyme function is crucial for genome interpretation, but it remains challenging because it cannot be scaled up to accommodate the constant accumulation of genome sequences. We tackled this issue for the MetA and MetX enzyme families, phylogenetically unrelated families of acyl-L-homoserine transferases involved in L-methionine biosynthesis. Members of these families are prone to incorrect annotation because MetX and MetA enzymes are assumed to always use acetyl-CoA and succinyl-CoA, respectively. We determined the enzymatic activities of 100 enzymes from diverse species, and interpreted the results by structural classification of active sites based on protein structure modeling. We predict that >60% of the 10,000 sequences from these families currently present in databases are incorrectly annotated, and suggest that acetyl-CoA was originally the sole substrate of these isofunctional enzymes, which evolved to use exclusively succinyl-CoA in the most recent bacteria. We also uncovered a divergent subgroup of MetX enzymes in fungi that participate only in L-cysteine biosynthesis as O-succinyl-L-serine transferases.
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26. |
( 2013 ) Cloning of two 5-aminolevulinic acid synthase isozymes HemA and HemO from Rhodopseudomonas palustris with favorable characteristics for 5-aminolevulinic acid production. PMID : 23338702 : DOI : 10.1007/s10529-013-1143-4 Abstract >>
5-Aminolevulinic acid (ALA) synthase (ALAS) HemA from non-sulfur photosynthetic bacteria has been used for the ALA bioproduction, whereas the isoenzyme HemT/HemO is less studied and not used for ALA production. Two ALAS-encoding genes, hemA and hemO from Rhodopseudomonas palustris were cloned, purified and characterized. The ALASs had very high specific activity, 3.6 and 2.7 U/mg, respectively, and strong affinity for one of its substrates, succinyl-CoA, K m with values of 11 and 4.4 �gM, respectively. HemO retained up to 60 % maximum activity within a broad range of concentrations of hemin, while HemA kept only 20 % at 10 �gM hemin. Escherichia coli overexpressing HemA or HemO produced 5.7 and 6.3 g ALA/l, respectively, in a 5 l bioreactor.
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27. |
( 1998 ) 2-Ketocyclohexanecarboxyl coenzyme A hydrolase, the ring cleavage enzyme required for anaerobic benzoate degradation by Rhodopseudomonas palustris. PMID : 9573182 : PMC : PMC107172 Abstract >>
2-Ketocyclohexanecarboxyl coenzyme A (2-ketochc-CoA) hydrolase has been proposed to catalyze an unusual hydrolytic ring cleavage reaction as the last unique step in the pathway of anaerobic benzoate degradation by bacteria. This enzyme was purified from the phototrophic bacterium Rhodopseudomonas palustris by sequential Q-Sepharose, phenyl-Sepharose, gel filtration, and hydroxyapatite chromatography. The sequence of the 25 N-terminal amino acids of the purified hydrolase was identical to the deduced amino acid sequence of the badI gene, which is located in a cluster of genes involved in anaerobic degradation of aromatic acids. The deduced amino acid sequence of badI indicates that 2-ketochc-CoA hydrolase is a member of the crotonase superfamily of proteins. Purified BadI had a molecular mass of 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a native molecular mass of 134 kDa as determined by gel filtration. This indicates that the native form of the enzyme is a homotetramer. The purified enzyme was insensitive to oxygen and catalyzed the hydration of 2-ketochc-CoA to yield pimelyl-CoA with a specific activity of 9.7 micromol min(-1) mg of protein(-1). Immunoblot analysis using polyclonal antiserum raised against the purified hydrolase showed that the synthesis of BadI is induced by growth on benzoate and other proposed benzoate pathway intermediates but not by growth on pimelate or succinate. An R. palustris mutant, carrying a chromosomal disruption of badI, did not grow with benzoate and other proposed benzoate pathway intermediates but had wild-type doubling times on pimelate and succinate. These data demonstrate that BadI, the 2-ketochc-CoA hydrolase, is essential for anaerobic benzoate metabolism by R. palustris.
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