Construction of a genetically engineered microorganism for CO2 fixation using a Rhodopseudomonas/Escherichia coli shuttle vector

The CO2 fixation ability of Rhodopseudomonas palustris DH was enhanced by introducing the recombinant plasmid pMG-CBBM containing the form II ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) gene (cbbM) isolated from Rps. palustris NO. 7. Sequencing of a 3.0-kb PstI fragment containing the cbbM gene revealed an open reading frame encoding 461 amino acids, homologous to known cbbM genes, with a ribosome binding site upstream of cbbM and a terminator downstream of cbbM, without promoter. pMG-CBBM, a Rhodopseudomonas/Escherichia coli shuttle expression plasmid, was derived from the Rhodopseudomonas/E. coli shuttle cloning vector pMG105, by inserting the promoter of the pckA gene and the cbbM gene into its multiple cloning site. Plasmid pMG-CBBM was transformed into Rps. palustris DH by electroporation, and was stably maintained when transformants were grown either photoheterotrophically or photolithoautotrophically in the absence of antibiotics. This is the first report of an expression plasmid containing a Rps. palustris-specific promoter that allows stable expression of a foreign gene in the absence of antibiotic selection.     

Optimization of wastewater feeding for single-cell protein production in an anaerobic wastewater treatment process utilizing purple non-sulfur bacteria in mixed culture condition

Impacts of operation timing of feeding and withdrawal on anaerobic wastewater treatment utilizing purple non-sulfur bacteria have been investigated in mixed culture condition with acidogenic bacteria. Simulated wastewater containing glucose was treated in a laboratory-scale chemostat reactor, changing the timing of wastewater feeding and withdrawal. Rhodopseudomonas palustris, which does not utilize glucose as a substrate, was inoculated in the reactor. Rps. palustris was detected by a fluorescent in situ hybridization (FISH) technique using the specific Rpal686 probe. As a result, population ratios of Rps. palustris were over 20% through the operation. Rps. palustris could grow by utilizing metabolites of acidogenic bacteria that coexisted in the reactor. A morning feed was effective for a good growth of purple non-sulfur bacteria. A protein content of cultured bacteria was the highest when wastewater was fed in the morning. Dissolved organic carbon (DOC) removal was 94% independent of the timing control. Consequently, feeding in the morning is the optimum feed-timing control from the aspects of growth of purple non-sulfur bacteria and single-cell protein production.     

Characterization of the different peripheral light-harvesting complexes from high- and low-light grown cells from Rhodopseudomonas palustris

In this paper we demonstrate that the spectroscopically different peripheral light-harvesting complexes from Rhodopseudomonas palustris, strain 2.6.1, isolated from high- and low-light grown cells have widely differing bacteriochlorophyll a (BChl a) resonance Raman spectra in the high-frequency carbonyl region (1550-1750 cm-1). Complexes synthesized in low-light grown cells exhibit Raman spectra characteristic of B800-850 and B800-820 complexes, depending on the excitation conditions. The in vivo strategy for low-light adaptation in this bacterium is thus somewhat different from that generally encountered in the Rhodospirillaceae. In these bacteria, as typified by Rps. acidophila and Rps. cryptolactis, low-light conditions induce the synthesis of B800-820 only complexes in which the hydrogen bonds between the acetyl carbonyl and the B850 binding pocket are broken, inducing changes in the absorption properties of the monomeric bacteriochlorophylls. In the case of Rps. palustris, additional spectral effects occur due to the coupling of the electronic levels of the differently interacting dimers. The extensive use of differential alpha/beta-polypeptide expression [Tadros et al. (1993) Eur. J. Biochem. 217, 867-875] thus allows Rps. palustris to alter its BChl a binding site environments causing the observed spread of BChl a Qy transitions, ranging from 801 to 856 nm.     

Hydrogen production by photoreactive nanoporous latex coatings of nongrowing Rhodopseudomonas palustris CGA009

Nonuniform light distribution is a fundamental limitation to biological hydrogen production by phototrophic bacteria. Numerous light distribution designs and culture conditions have been developed to reduce self-shading and nonuniform reactivity within bioreactors. In this study, highly concentrated (2.0 x 108 CFU/muL formulation) nongrowing Rhodopseudomonas palustris CGA009 were immobilized in thin, nanoporous, latex coatings. The coatings were used to study hydrogen production in an argon atmosphere as a function of coating composition, thickness, and light intensity. These coatings can be generated aerobically or anaerobically and are more reactive than an equivalent number of suspended or settled cells. Rhodopseudomonas palustris latex coatings remained active after hydrated storage for greater than 3 months in the dark and over 1 year when stored at -80 degrees C. The initial hydrogen production rate of the microphotobioreactors containing 6.25 cm2, 58.4 mum thick Rps. palustris latex coatings illuminated by 34.1 PAR mumol photons m-2 s-1 was 6.3 mmol H2 m-2 h-1 and had a final yield of 0.55 mol H2 m-2 in 120 h. A dispersible latex blend has been developed for direct comparison of the specific activity of settled, suspended, and immobilized Rps. palustris.     

Electron transfer proteins of the purple phototrophic bacterium, Rhodopseudomonas rutila.

The soluble electron transfer protein content of Rhodopseudomonas rutila was found to consist of two basic cytochromes and a (4Fe-4S) ferredoxin. Cytochrome c' was easily identified by its characteristic high spin absorption spectra. The native molecular weight is 29,000 and the subunit is 14,000. Cytochrome c-550 has low spin absorption spectra and a high redox potential (376 mV) typical of cytochromes c2. The molecular weight is about 14,000. The ferredoxin is apparently a dimer (43,000) of approximately 18,000 Da subunits. There are 1.3 to 1.5 iron-sulfur clusters per monomer of 18- to 21-kDa protein. The N-terminal amino acid sequence is like the (7Fe-8S) ferredoxins of Rhodobacter capsulatus and Azotobacter vinelandii. Remarkably, there are only 2 or 3 out of 25 amino acid substitutions. Difference absorption spectra of Rps. rutila membranes indicate that there is not tetraheme reaction center cytochrome c, such as is characteristic of Rps. viridis. However, there are a high potential cytochrome c and a low potential cytochrome b in the membrane, which are suggestive of a cytochrome bc1 complex. Rps. rutila is most similar to Rps. palustris in microbiological properties, yet it does not have the cytochromes c-556, c-554, and c-551 in addition to c2 and c', which are characteristic of Rps. palustris. Furthermore, the Rps. rutila cytochrome c' is dimeric, whereas the same protein from Rps. palustris is the only one known to be monomeric. The cytochrome pattern is more like that of Rhodospirillum rubrum and Rb. capsulatus, which are apparently only able to make cytochromes c2 and c'.     

Multiple copies of the coding regions for the light-harvesting B800-850 alpha- and beta-polypeptides are present in the Rhodopseudomonas palustris genome.

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.     

Aeration conditions affecting growth of purple nonsulfur bacteria in an organic wastewater treatment process

Effects of aeration on purple nonsulfur bacteria (PnSB) were studied in photobioreactors. Bacterial community changes were analyzed by denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH). DGGE band pattern change was small and only few prominent bands were obtained at non-aeration condition. Sequencing results of the prominent DGGE bands obtained at this condition revealed that they represented mainly the PnSB, Rhodobacter sphaeroides and Rhodopseudomonas palustris. On the other hand, under aerated condition, some prominent bands originated from heterotrophs appeared but no proliferation of PnSB was observed. FISH was applied to detect PnSB and their population was quantified. Maximum PnSB ratio (up to 80%) was obtained both at non-aeration condition and at constant ORPs less than -200 mV. In the presence of DO, Rps. palustris was more competitive to chemoheterotrophs than Rb. sphaeroides.     

Genotypic and phenotypic diversity within species of purple nonsulfur bacteria isolated from aquatic sediments

To assess the extent of genotypic and phenotypic diversity within species of purple nonsulfur bacteria found in aquatic sediments, a total of 128 strains were directly isolated from agar plates that had been inoculated with sediment samples from Haren and De Biesbosch in The Netherlands. All isolates were initially characterized by BOX-PCR genomic DNA fingerprinting, and 60 distinct genotypes were identified. Analyses of 16S rRNA gene sequences of representatives of each genotype showed that five and eight different phylotypes of purple nonsulfur bacteria were obtained from the Haren and De Biesbosch sites, respectively. At the Haren site, 80.5% of the clones were Rhodopseudomonas palustris, whereas Rhodoferax fermentans and Rhodopseudomonas palustris were numerically dominant at the De Biesbosch site and constituted 45.9 and 34.4% of the isolates obtained, respectively. BOX-PCR genomic fingerprints showed that there was a high level of genotypic diversity within each of these species. The genomic fingerprints of Rhodopseudomonas palustris isolates were significantly different for isolates from the two sampling sites, suggesting that certain strains may be endemic to each sampling site. Not all Rhodopseudomonas palustris isolates could degrade benzoate, a feature that has previously been thought to be characteristic of the species. There were differences in the BOX-PCR genomic fingerprints and restriction fragment length polymorphisms of benzoate-coenzyme A ligase genes and form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes between benzoate-degrading and non-benzoate-degrading genotypes. The ability to distinguish these two Rhodopseudomonas palustris groups based on multiple genetic differences may reflect an incipient speciation event resulting from adaptive evolution to local environmental conditions.     

Soluble cytochrome composition of the purple phototrophic bacterium, Rhodopseudomonas sphaeroides ATCC 17023

A detailed study of the soluble cytochrome composition of Rhodopseudomonas sphaeroides (ATCC 17023) indicates that there are five c-type cytochromes and one b-type cytochrome present. The molecular weights, heme contents, amino acid compositions, isoelectric points, and oxidation-reduction potentials were determined and the proteins were compared with those from other bacterial sources. Cytochromes c2 and c' have previously been well characterized. Cytochrome c-551.5 is a diheme protein which has a very low redox potential, similar to certain purple bacterial and algal cytochromes. Cytochrome c-554 is an oligomer, which is spectrally similar to the low-spin isozyme of cytochrome c' found in other purple bacteria (e.g., Rhodopseudomonas palustris cytochrome c-556). An unusual high-spin c-type heme protein has also been isolated. It is spectrally distinguishable from cytochrome c' and binds a variety of heme ligands including oxygen. A large molecular-weight cytochrome b-558 is also present which appears related to a similar protein from Rhodospirillum rubrum, and the bacterioferritin from Escherichia coli. None of the soluble proteins appear to be related to the abundant membrane-bound c-type cytochrome in Rps. sphaeroides which has a larger subunit molecular weight similar to mitochondrial cytochrome c1 and chloroplast cytochrome f.     

Photobiodegradation of pyridine by Rhodopseudomonas palustris JA1

A purple non-sulfur bacterium isolated from dairy effluent was identified as Rps. palustris JA1. This organism was able to grow on pyridine as sole source of carbon in a light dependent anaerobic process with a doubling time of 30 h. Intermediates of pyridine photobiodegradation were identified as glycine and malonate, produced in stoichiometric molar ratios with simultaneous utilization, yielding biomass.     

Isolation and Characterization of a Temperate Bacteriophage Specific for Rhodopseudomonas spheroides

The isolation of a temperature phage specific for the photosynthetic bacterium Rhodopseudomonas spheroides is reported. This phage, Rphi-1, establishes a state of lysogeny and can be induced from the prophage state by exposure to mitomycin C or UV irradiation. Mutants of Rphi-1 which grow on a standard laboratory strain (2.4.1) of Rhodopseudomonas spheroides were isolated. Although the original Rphi-1 isolated was chloroform sensitive, the mutant which plates on strain 2.4.1 is chloroform resistant. Rphi-1 does not grow on closely related bacteria, such as Rhodopseudomonas palustris or Rhodopseudomonas capsulata. Rphi-1 mutants forms plaques with the same efficiency whether the plates are incubated under aerobic conditions in the dark or under anaerobic conditions in the light (phototropic conditions).     

Structural characterization of high 800 nm-absorbing light-harvesting complexes from Rhodospirillales from their resonance Raman spectra

Resonance Raman spectroscopy provided evidence that high 800 nm-absorbing antennae from Rhodopseudomonas (Rps.) acidophila and Rps. palustris have similar structures around their dweller bacteriochlorophylls. These host-site structures are different from those of B 850-800 complexes from Chromatiaceae, which also exhibit a high absorbance at 800 nm. As also shown by previous biochemical data, these complexes might be stoichiometrically different from other antenna complexes, having one more BChl per minimal size unit of protein. A new classification of B 850-800 complexes is proposed, on the basis of resonance Raman and biochemical data: this classification distinguishes a class of B 850-800 S (involving the B 850-800 complexes from sulfur purple bacteria), two classes of B 850-800 NS (involving the B 850-800 complexes from non sulfur purple bacteria) and a class of H 800 complexes (involving the B 850-800 complexes from non sulfur purple bacteria exhibiting a high absorbance at 800 nm).     

Anaerobic degradation of halogenated benzoic acids by photoheterotrophic bacteria

Abstract From light-exposed enrichment cultures containing benzoate and a mixture of chlorobenzoates, a pure culture was obtained able to grow with 3-chlorobenzoate (3-CBA) or 3-bromobenzoate (3-BrBA) as the sole growth substrate anaerobically in the light. The thus isolated organism is a photoheterotroph, designated isolate DCP3. It is preliminarily identified as a Rhodopseudomonas palustris strain. It differs from Rhodopseudomonas palustris WS17, the only other known photoheterotroph capable of using 3-CBA for growth, in its independence of benzoate for growth with 3-CBA and in its wider substrate range: if grown on 3-CBA, it can also use 2-CBA, 4-CBA or 3,5-CBA.     

Comparative study of B890 pigment-lipoprotein complexes from sulfur (Chromatium minutissimum) and non-sulfur (Rhodopseudomonas palustris) purple photosynthesizing bacteria]

Pigment-lipoprotein B890 complexes containing reaction center and "light-focusing" bacteriochlorophyll a were isolated from photosynthetic membranes of sulfur (Chromatium minutissimum) and non-sulfur (Rhodopseudomonas palustris) purple bacteria after the treatment with Triton X-100. The molecular weights of complexes were evaluated using several methods (200 000-300 000). By means of electron microscopy the sizes of complexes were found to be about 80 A. On the air-water interface hexagonal packing of complexes was observed. The chemical compositions of complexes are very similar except bacteriochlorophyll a whose specific content is somewhat higher in Chromatium minutissimum. The protein composition of complexes was studied and the molecular weights of proteins were estimated by SDS-gel electrophoresis. The results obtained show significant similarities in molecular organization of B890 complexes isolated from sulfur (Chromatium minutissimum) and non-sulfur (Rhodopseudomonas palustris) purple bacteria.     

Zur Taxonomie von Rhodopseudomonas palustris

Zusammenfassung Eine Reihe von Rhodopseudomonas palustris-Stämmen aus verschiedenen Herkünften wurden vergleichend unter Verwendung folgender Merkmale untersucht: Substratverwertung, in vivo-Absorptionsspektrum und Serologie der O-Antigene. Die gegen 2 Stämme gerichteten Antiseren zeigen hohe Spezifität. Die Verwendbarkeit der serologischen Kreuzreaktion für taxonomische Untersuchungen bei photosynthetischen Bakterien wird diskutiert.

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On the taxonomy of Rhodopseudomonas palustris

Summary Strains of Rhodopseudomonas palustris isolated from different habitats were compared with respect to their taxonomic features. All strains grew very well on formiate, acetate, propionate, butyrate, aspartate, inositol, ethanol, fructose, and p-amino-benzoate, respectively, as single carbon source. Most of the strains were able to use benzoic acid or glucose, too. But alanine was not found to be a good substrate. The maxima of the bacteriochlorophyll in-vivo-absorption spectra were estimated to be 376, 589, 802–805, and 858–875 nm. The shift of the infrared peak in the different strains is loosely correlated with the change of the carotenoid in vivo spectrum, the maxima of which were measured to be 470–480 nm (shoulder) 495–505 nm, and 520–545 nm (shoulder). Antisera were prepared against the strains 1e5 and 11/1. It was demonstrated that these antisera were directed against the lipopolysaccharides (O-antigen) of these bacteria. The antigen of 1e5 does not cross react with the antigen of 11/1. Strain 1e5 is the only one of 17 strains tested which is sensitive to the bacteriophage Rp1. The antigen of this strain cross reacted only with the antigen of strain K1. In contrast, the antigen of strain 11/1 cross reacted in some degree with most of the tested strains of Rps. palustris. No or very weak cross reaction was observed between the antigens of Rps. palustris (1e5, 11/1) and Rps. capsulata, Rps. spheroides, or R. rubrum, respectively. In contrast to 11/1 only heat-killed cells of strain 1e5 were agglutinated by anti-1e5.

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Im Text verwendete Abkürzungen LPS Lipopolysaccharid - R Rhodospirillum - Rps. Rhodopseudomonas - i.m. intramuskulär - s.c. subcutan - i.v. intravenös     

The photosynthetic apparatus of Rhodopseudomonas palustris: structures and organization

The structural analysis of the individual components of the photosynthetic apparatus of Rhodopseudomonas palustris, or those of related species, is almost complete. To shed light on the assembly and organization of this machinery, we have studied native membranes of Rps.palustris grown under different light conditions using atomic force microscopy (AFM). The organization of the complexes in the membranes is different from any previously observed: with areas of crystalline core-complexes, crystalline peripheral antennae, mixed domains, and apparently pure lipid membranes devoid of protein. Examination of antennae structure shows that chromatic adaptation is associated with modifications in absorption and size of the peripheral light harvesting complexes (LH2) as light intensity is reduced. The core-complex is observed to contain a reaction centre (RC) surrounded by an elliptical assembly of 15 LH1 subunits and a "gap" attributed to the W-subunit. The localization of the W-subunit is not restricted to the periapsis of the core-complex but randomly located with respect to the RC imposed axis.     

Substrate specificity of citrate lyase deacetylase of Rhodopseudomonas gelatinosa and Rhodopseudomonas palustris.

Citrate lyase (EC 4.1.3.6) isolated from Rhodopseudomonas palustris was investigated with regard to its kinetic properties and its subunit composition. This enzyme was inactivated by citrate lyase deacetylase (EC 3.1.2.-) of Rhodopseudomonas gelatinosa. A corresponding cross-reaction was measured with partially purified deacetylase of R. palustris and citrate lyase of R. gelatinosa. The three different subunit types (alpha, beta, and gamma) of citrate lyase from R. gelatinosa wee purified to homogeneity, and antibodies were prepared against each of the three subunits and against the native enzyme complex. In corresondence with the enzymatic interactions, immunological cross-reactions were found between anti-enzyme and anti-large subunit antibodies and citrate lyase from R. palustris. On the other hand, no immunological cross-reactions were detectable among each of the antibodies and citrate lyases from Enterobacter aerogenes, Streptococcus diacetilactis, and Clostridium sphenoides. Antibodies against the large subunit of citrate lyase inhibited the deacetylase, but antibodies against the middle and small subunits did not, indicating that the large subunits of citrate lyase are involved in binding the deacetylase.     

内蒙古碱湖中紫色非硫光合细菌的分离和鉴定

从内蒙古碱湖水样中分离得到一株紫色非硫光合细菌,命名为JH1-6.对该菌株进行了形态学观察、生理生化鉴定、活细胞吸收光谱以及16S rDNA序列分析.16S rDNA序列分析结果表明该菌株与沼泽红假单胞菌的16S rDNA序列同源性高达99%,结合形态特征和生理生化特性以及活细胞吸收光谱特征等,确定菌株JH1- 6在分类地位上属于沼泽红假单胞菌(Rhodopseudomonas palustris).     

Isolation and Identification of Photosynthetic Bacteria (Rhodospirillaceae) from Antarctic Marine and Freshwater Sediments

Sediment samples obtained from three freshwater lakes and off-shore coastal marine waters on Signy Island, South Orkney Islands, Antarctica have been inoculated into selective enrichment media for purple non-sulphur bacteria (Rhodospirillaceae). From the freshwater sediments strains of Rhodopseudomonas sphaeroides (1), Rhodopseudomonas palustris (1), Rhodospirillum fulvum (1), Rhodospirillum molischanum (1) and Rhodomicrobium vannielii (3) have been isolated. The only purple non-sulphur bacteria obtained from Lake 10 (Amos lake) were strains of Rhodomicrobium vannielii which were able to tolerate hydrogen sulphide (up to 0.04% w/v) found in this lake. Growth of all the other isolates is inhibited by the presence of hydrogen sulphide. Marine sediments yielded strains of Rhodopseudomonas palustris and Rhodomicrobium vannielii . All the isolates grow optimally at temperatures between 25 and 30 °C. mean generation times vary between 8 and 10.7 h depending on species. There is no evidence of cold adaptation in any of the strains studied.     

Phototrophic transformation of phenol to 4-hydroxyphenylacetate by <Emphasis Type="Italic">Rhodopseudomonas palustris</Emphasis>

Newly isolated and culture collection strains of Rhodopseudomonas palustris were able to transform phenol to 4-hydroxyphenylacetate under phototrophic conditions in the presence of acetate, malate, benzoate, or cinnamate as growth substrates. The reaction was examined with uniformly (14)C-labelled phenol and the product was identified by HPLC retention time, UV-scans, and (1)H- and (13)C-NMR analysis. The transformation reaction was detectable in cell-free extracts in the presence of NAD(+) and acetyl-CoA. For further degradation of 4-hydroxyphenylacetate by R. palustris, low partial pressures of oxygen were essential, presumably for aerobic aromatic ring fission reactions by mono- and di-oxygenases.