Growth characteristics of Rhodopseudomonas palustris cultured outdoors, in an underwater tubular photobioreactor, and investigation on photosynthetic efficiency

The underwater tubular photobioreactor is a fully controlled outdoor system to study photosynthetic bacteria. Before growing bacteria cells outdoors, two modified van Niel medium (vN-A, vN-B) were tested under artificial light. During exponential growth, the specific growth rates were 0.0416 and 0.0434 h⁻¹, respectively; vN-B was chosen for outdoor experiments. The growth behavior of Rhodopseudomonas palustris was investigated under a natural light–dark cycle (sunrise–sunset, 15L/9D) and a forced light–dark cycle (9:00–19:00, 10L/14D). Relationships between solar radiations, daily growth rates, and biomass output rates were also investigated. After determining the elemental biomass molar composition and its combustion heat, some trends of photosynthetic efficiency (PE) were obtained over daylight. The PE trends were always of the oscillatory type, with the exception of that achieved at low biomass concentration. Under a natural light/dark cycle, the maximum PE (11.2%) was attained at sunset, while under a forced light/dark cycle, the highest PE (8.5%) was achieved in the morning. Three initial biomass concentrations were investigated (0.65, 1.01, and 1.54 g l⁻¹). The stoichiometric equation for bacteria cells indicated that 87.7% of the carbon of acetic acid was converted to biomass and only 12.3% was lost as CO₂.     

Bacterial Decolorization of Azo Dyes by Rhodopseudomonas palustris

Summary The ability of Rhodopseudomonas palustris AS1.2352 possessing azoreductase activity to decolorize azo dyes was investigated. It was demonstrated that anaerobic conditions were necessary for bacterial decolorization, and the optimal pH and temperature were pH 8 and 30–35 °C, respectively. Decolorization of dyes with different molecular structures was performed to compare their degradability. The strain could decolorize azo dye up to 1250 mg l⁻¹, and the correlation between the specific decolorization rate and dye concentration could be described by Michaelis–Menten kinetics. Long-term repeated operations showed that the strain was stable and efficient during five runs. Cell extracts from the strain demonstrated oxygen-insensitive azoreductase activity in vitro.     

Cytochrome P450 enzymes from the metabolically diverse bacterium Rhodopseudomonas palustris

Four (CYP195A2, CYP199A2, CYP203A1, and CYP153A5) of the seven P450 enzymes, and palustrisredoxin A, a ferredoxin associated with CYP199A2, from the metabolically diverse bacterium Rhodopseudomonas palustris have been expressed and purified. A range of substituted benzenes, phenols, benzaldehydes, and benzoic acids was shown to bind to the four P450 enzymes. Monooxygenase activity of CYP199A2 was reconstituted with palustrisredoxin A and putidaredoxin reductase of the P450cam system from Pseudomonas putida. We found that 4-ethylbenzoate and 4-methoxybenzoate were oxidized to single products, and 4-methoxybenzoate was demethylated to form 4-hydroxybenzoate. Crystals of substrate-free CYP199A2 which diffracted to approximately 2.0A have been obtained.     

Sulfoacetate generated by Rhodopseudomonas palustris from taurine

Genes thought to encode (a) the regulator of taurine catabolism under carbon-limiting or nitrogen-limiting conditions and (b) taurine dehydrogenase were found in the genome of Rhodopseudomonas palustris. The organism utilized taurine quantitatively as a sole source of nitrogen (but not of carbon) for aerobic and photoheterotrophic growth. No sulfate was released, and the C-sulfonate bond was recovered stoichiometrically as sulfoacetate, which was identified by mass spectrometry. An inducible sulfoacetaldehyde dehydrogenase was detected. R. palustris thus contains a pathway to generate a natural product that was previously believed to be formed solely from sulfoquinovose.The senior author (AMC) would like to express his thanks for the rewarding experience of doing postdoctoral research in the laboratory of Prof. H.-G. Schlegel.     

Regulatory properties of the nitrogenase fromRhodopseudomonas palustris

Ammonium salts, glutamine, asparagine, and urea cause an immediate inactivation (switch-off) of light-dependent acetylene reduction in intact cells of the photosynthetic bacteriumRhodopseudomonas palustris. This effect is reversible showing the same kinetic pattern of inactivation and reactivation with all effector compounds. Its duration depends on the amount of effector added to the cells. Both nitrogenase components are found catalytically active in a cell-free preparation after enzyme switch-off in vivo. Involvement of the ammonia assimilating system in this regulatory mechanism is indicated by the following observations: ammonia uptake during the switch-off period, resumption of acetylene reduction after disappearance of ammonia from the outer medium, and persistence of enzyme switch-off with dihydrogen and thiosulfate as electron donors in the absence of an additional carbon source. Nitrogenase activity in crude extracts is non-linear with time and is stimulated by manganese ions. After resolution of nitrogenase into its MoFe-protein and Fe-protein these properties are lost, indicating the presence of an activating factor. Nitrogenase ofR. palustris cross reacts reciprocally with the complementary proteins ofAzotobacter vinelandii, but not with those ofClostridium pasteurianum.Abbreviations CCCP m-chlorocarbonyl cyanide phenyl hydrazone - DNP 2,4-dinitrophenol - EPR electron paramagnetic resonance - HEPES N-2-hydroxyethylpiperazine--2-ethane sulfonic acid - NOQNO 2-n-nonyl-4-hydroxyquinoline-N-oxide - TES N-tris[hydroxymethyl]methyl-2-aminoethane sulfonic acid     

The close phylogenetic relationship of Nitrobacter and Rhodopseudomonas palustris

The phylogenetic position of Nitrobacter winogradskyi and two other nitrite-oxidizing bacteria was elucidated comparing oligonucleotides of the 16S ribosomal RNA. Nitrobacter winogradskyi and the Nitrobacter isolate Yukatan are genetically nearly identical; Nitrobacter isolate X14 is more distantly related. Phylogenetically, Nitrobacter is a member of a group of purple non-sulfur bacteria that is defined by various species of Rhodopseudomonas, Rhodomicrobium vannielii, Rhodospirillum rubum and their non-phototrophic relatives. Nitrobacter shares a high sequence similarity to Rhodopseudomonas palustris. These findings are in accord with several common taxonomic characteristics, and in addition support the conversion hypothesis for the origin of this group of chemolithotrophic bacteria.     

ATPase activity of the polar organelle demonstrated by cytochemical reaction in whole unstained cells of Rhodopseudomonas palustris

The polar organelle, a structure associated with the flaggelar apparatus of bacteria, has been demonstrated in whole unstained cells of the photosynthetic bacterium Rhodopseudomonas palustris. It is subpolarly located close to the surface of the bacterial cell and has a round or ellipsoidal shap. It shows a strong ATPase activity which enables its cytochemical electron microscopical visualization.     

Growth of Rhodopseudomonas palustris under phototrophic and non-phototrophic conditions and its CO-dependent H2 production

A new hydrogen producing bacterium, Rhodopseudomonas palustris P4, originally isolated under an anaerobic/phototrophic condition, grew well under aerobic/chemoheterotrophic or anaerobic/chemoheterotrophic conditions and showed CO-dependent, H₂ production activity when transferred to anaerobic conditions. Cell growth was best under an aerobic/chemoheterotrophic condition as the doubling time of 1 h, while the H₂ production activity was highest in the cells grown under an aerobic/chemoheterotrophic condition at 20 mmol g^–1 cell^–1 h^–1.     

The enzymatic system thiosulfate: Cytochrome c oxidoreductase from photolithoautotrophically grown Rhodopseudomonas palustris

Rhodopseudomonas palustris cells, characterized by a lamellar type intracytoplasmic chromatophore membrane system after phototrophic growth, yielded a crude supernatant cell-free fraction (S-144) after ultracentrifugation which retained the contents of both the cell compartments. After thiosulfate-dependent growth, a protein system was isolated from S-144 which catalyzed the thiosulfate-linked reduction of an endogenous c-type cytochrome. — The colorless oxidoreductase protein, after purification to homogeneity, revealed a molecular weight of 93,000 and, after SDS treatment, a particle weight of 48,000. It was focused at an average pI of 5.45. Apparent K _m values for several substrates were in the M range. The electron acceptor for thiosulfate oxidation was found to be a cytochrome c from S-144. The homogeneous acceptor protein, at liquid nitrogen temperature, exhibited absorption maxima at 549.0, 518.5 and 418.0 nm, and shoulders at 525.5, 512.0 and 508.0 nm. Its molecular weight was found to be 17,000 (gel filtration) and 16,000 (SDS gel electrophoresis). It was characterized by a pI of 10.0. Its midpoint redox potential of E _m,7.0=+228 mV was determined by redox titrations and the value of +205 mV by spectrophotometric calculations.Abbreviations BSA bovine serum albumin - HiPIP high potential nonheme iron protein - IEF isoclectric focusing - SDS dodecylsulfate, sodium salt - Temed N,N,N,N-tetramethylethylenediamine     

Nitrogenase switch-off by ammonia in Rhodopseudomonas palustris: Loss under nitrogen deficiency and independence from the adenylylation state of glutamine synthetase

Nitrogenase activity in Rhodopseudomonas palustris is subject to a rapid switch-off in response to exogenous ammonia. When cells were grown on limiting nitrogen and eventually became nitrogen deficient, nitrogenase synthesis was fully derepressed but the enzyme was insensitive to ammonia. The transformation of ammonia-sensitive to ammonia-insensitive cells was a slow, but fully reversible process. The switch-off effect in ammonia-sensitive cells paralleled changes in the adenylylation state of glutamine synthetase. Ammonia-insensitive cells, however, showed similar changes in glutamine synthetase activity although nitrogenase activity was unaffected. We conclude that nitrogenase regulation and adenylylation of glutamine synthetase are independent processes, at least under conditions of nitrogen deficiency.     

A Comparative Study of the Isocitrate Dehydrogenases of Chlorobium limicola forma thiosulfatophilum and Rhodopseudomonas palustris

The carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle in the obligate photolithotroph Chlorobium limicola forma thiosulfatophilum and the oxidation of isocitrate in the tricarboxylic acid cycle in the photoheterotroph Rhodopseudomonas palustris are catalyzed by isocitrate dehydrogenases. A comparative study of these enzymes isolated from the two bacteria showed that they virtually do not differ in enzymatic and kinetic properties.     

Augmentation of H2 photoproduction in Rhodopseudomonas palustris by N-heterocyclic aromatic compounds

Increases of 23- (5.6 mmol acetylene reduced mg dry wt^–1) and 16- (4 mmol acetylene reduced mg dry wt^–1) fold in nitrogenase activity and 12- (671 l H₂ mg dry wt^–1 h^–1) and 6- (349 l mg dry wt^–1 h^–1) fold in H₂ photoproduction in Rhodopseudomonas palustris JA1 over 24 h were achieved with pyrazine 2-carboxylate (3 mM) and 3-picoline (3 mM), respectively, and were higher than earlier reports of enhancement (1.5 to 5- fold) in biological H₂ production using various alternative methods.     

Spectral and functional characterization of membrane fragments from the facultative photosynthetic bacterium Rhodopseudomonas blastica

The cytochromes of photosynthetically grown Rhodopseudomonas blastica have been thermodynamically characterized using the technique of redox titrations. Six cytochromes were present; two cytochromes c, E _m7= +295mV, E _m7=+345mV; and four cytochromes b, E _m7=+290mV, E _m7=+130mV, E _m7=+60mV, E _m7=-4mV. These cytochromes were tightly bound except for cytochrome c with E _m7 of+345mV which was mostly present in the soluble cell extracts.The effects of cyanide on both the cytochrome c oxidase activity and the NADH-dependent respiration, revealed the presence of a branched respiratory chain, one branch leading to a cyanide-resistant oxidase containing pathway and the other including the cyanide-sensitive cytochrome c-oxidase.The effects of antimycin A, myxothiazol and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) on the steadystate NADH-dependent respiration were also studied. Antimycin A and myxothiazol appeared to act at the level of the ubiquinol-cytochrome c oxidoreductase while UHDBT drastically affected both respiratory branches.Absorption spectra of chromatophore photopigments resulted to be similar to those reported in many species of facultative photosynthetic bacteria although carotenoid absorption maxima were blue-shifted by 5 nm.The light-induced oxygen reduction performed by chromatophores from R. blastica suggested a strict interaction between photosynthetic and respiratory apparatuses.     

几种重金属离子对光合细菌生长的抑制效应

光合细菌因其在自然生态系统中的意义与较大的应用价值 ,一直受到国内外科学工作者的重视 ,无论基础研究 ,还是应用研究 ,都取得了重大的进展。其应用研究主要为 3方面 :①用于高负荷有机废水处理 ;②用于水产养殖。它具有作为养殖水质净化剂、用于鱼虾贝幼体培育、作为动物性生物饵料的饵料、作为成鱼成虾的饲料添加剂、防治病等方面作用[6] ;③在农业上的应用。在光照嫌气条件下 ,大多数细菌能有效地固定Ｎ2 ,故在淹水的耕作土壤(如水稻田 )里 ,它能提高土壤Ｎ素水平 ,从而提高土壤肥力 ,光合细菌还能氧化或分解土壤中Ｈ2 Ｓ和胺等有毒化…     

Sites of phospholipid biosynthesis during induction of intracytoplasmic membrane formation in Rhodopseudomonas sphaeroides

A rapid, gratuitous and cell-division uncoupled induction of intracytoplasmic photosynthetic membrane formation was demonstrated in low-aeration suspensions of chemotrophically grown Rhodopseudomonas sphaeroides. Despite a nearly 2-fold increase in phospholipid levels, no significant increases were detected in the specific activities of CDP-1,2-diacyl-sn-glycerol:sn-glycerol-3-phosphate phosphatidyltransferase (phosphatidylglycerophosphate synthase, EC 2.7.8.5) and CDP-1,2-diacyl-sn-glycerol:L-serine O-phosphatidyltransferase (phosphatidylserine synthase, EC 2.7.8.8), the first committed enzymes of anionic and zwitterionic phospholipid biosyntheses, respectively. The distribution of phosphatidylglycerophosphate and phosphatidylserine synthase activities after rate-zone sedimentation of cell-free extracts indicated that intracytoplasmic membrane phospholipids were synthesized mainly within distinct domains of the conserved cytoplasmic membrane. Labeling studies with ³²P_i and L-[³H]phenylalanine suggested that preexisting phospholipid was utilized initially as the matrix for insertion of intracytoplasmic membrane protein that was synthesized and assembled de novo during induction.Abbreviations BChl bacteriochlorophyll a - B800-850, B875 peripheral and core light-harvesting BChl-protein complexes, respectively, identified by near-IR absorption maxima This paper is dedicated to Professor Gerhart Drews on the occasion of his sixtieth birthday     

Structural analysis of photosynthetic membranes by cryo-electron tomography of intact Rhodopseudomonas viridis cells

During the photosynthetic process, highly organized membranal assemblies convert light into biochemical energy with high efficiency. We have used whole-mount cryo-electron tomography to study the intracellular architecture of the photosynthetic membranes of the anaerobic purple photosynthetic bacterium Rhodopseudomonas viridis, as well as the organization of the photosynthetic units within the membranes. Three-dimensional reconstruction demonstrates a continuity of the plasma membrane with the photosynthetic membranes that form tunnel-like structures with an average diameter of 31 nm ± 8 nm at the connection sites. The spacing between the photosynthetic membranes at their cytoplasmic faces was found to be 11 nm, thus enforcing a highly close packaging of the photosynthetic membranes. Analysis of successive tomographic slices allowed for derivation of the spacing between adjacent photosynthetic core complexes from a single-layered photosynthetic membrane, in situ. This analysis suggests that most, if not all, photosynthetic membranes in R. viridis are characterized by a similar two-dimensional hexagonal lattice organization.     

Regulation of isocitrate lyase in a mutant of Rhodopseudomonas capsulata adapted to growth on acetate

Studies on acetate utilization by Rhodopseudomonas capsulata strain St. Louis indicated that the wild type grew poorly on acetate and made little if any of the glyoxylate cycle enzyme isocitrate lyase. A spontaneous mutant, Ac-l, capable of vigorous and immediate growth on acetate and exhibiting high levels of isocitrate lyase activity, was isolated in the course of those studies.Isocitrate lyase was not formed when the mutant was grown on malate. Addition of malate to cultures of Ac-l growing on acetate resulted in loss of the enzyme by dilution through growth.Starvation of acetate-grown Ac-l for acetate resulted in a rapid and complete loss of isocitrate lyase activity which was shown to be energy dependent. Readdition of acetate to a starved culture previously grown on acetate resulted in a rapid recovery of enzyme activity. The recovery required energy and was sensitive to chloramphenicol inhibition at any time during the recovery phase.     

Tributyl phosphate degradation by <Emphasis Type="Italic">Rhodopseudomonas palustris</Emphasis> and other photosynthetic bacteria

Tributyl phosphate (TBP) is widely used in nuclear fuel processing and other waste generating chemical industries. Although TBP is bacteriostatic, some microbes are resistant to it and may degrade it. Under dark aerobiosis, purple non-sulfur photosynthetic bacteria degraded up to 0.6 mM TBP, initially present at 2 mm, within 3 weeks and under photosynthetic conditions, Rhodopseudomonas palustris degraded 1.6 mM TBP within 3 weeks. The curing of the Rhodopseudomonas palustris endogenous plasmid demonstrated that the genes involved in the TBP degradation are chromosomal.     

Differentiation of the photosynthetic apparatus of Chloroflexus aurantiacus depending on growth with different amino acids

The phototrophic green bacterium Chloroflexus aurantiacus was grown anaerobically in batch culture with different amino acids at 56°C and constant illumination of 25 klx. The composition of the photosynthetic apparatus was measured by quantitation of the bacteriochlorophylls (Bchl) a and c (representing the membrane-bound and the chlorosomal moieties, respectively). Ser added at concentrations up to 15 mM stimulated protein formation and Bchl a and c syntheses. A comparable stimulation was found with Glu and Ala. Coproporphyrin accumulation approached saturation at 5 mM of Ala, Asp, Orn, and Ser, while with Glu and Arg saturating concentrations were above 5 mM. Protein and tetrapyrrole syntheses became saturated at 2.5 to 5 mM of Asp, Ile, and Val. However, with Arg and Orn Bchl c synthesis was stimulated up to 2.5 mM, growth and Bchl a synthesis up to 5 mM. At higher Arg or Orn concentrations these activities were inhibited. Coproporphyrin accumulation was highest with Arg or Orn, at concentrations which inhibited growth and Bchl formation. Stimulation of Bchl synthesis took place preferentially at the level of Bchl c, while Bchl a was more sensitive toward inhibition. In both cases however, the ratio of Bchl c to Bchl a increased with higher amino acid concentrations. Nevertheless, each amino acid induced a typical effect. To understand different effects exerted by different amino acids, chemostat cultures were grown limited by either Ser or Glu. With Ser, steady state protein levels and specific Bchl a contents decreased slightly when increasing the dilution rate (D). Concomitantly Bchl c and coproporphyrin levels as well as the ratio of Bchl a/Bchl c increased. With Glu as the limiting substrate, all of the above mentioned parameters decreased. Since all of the Ser was consumed and increasing amounts of Glu remained unutilized in the spent medium, it is concluded that differences in the formation of the three pyrrole derivatives tested are due to differences in the affinities of uptake systems for Ser and Glu.     

Differentiation of the intracytoplasmic membrane of Rhodopseudomonas palustris induced by variations of oxygen partial pressure or light intensity

The photosynthetic apparatus of Rhodopseudomonas palustris contains, in addition to reaction center bacteriochlorophyll (Bchl) two spectral forms of light harvesting (LH) Bchl, i.e. LH Bchl I, characterized by an infrared absorption maximum at 880 nm (890 nm at 77°K) and LH Bchl II absorbing at 805 and 855 nm (805 and 870 nm at 77°K). LH Bchl I seems to be associated with a single protein species of an apparent mol. wt. of 13000 whereas LH Bchl II is apparently associated with two proteins of mol. wts. of 9000 and 11000.Cells in anaerobic cultures adapt to changes of light intensity 1. by variation of the size of the photosynthetic unit, i.e. the molar ratio of LH Bchl II to reaction center Bchl, 2. by variation of the number of photosynthetic units per unit of membrane area, 3. by regulation of the size of the intracytoplasmic membrane system.During adaptation of changes of oxygen partial pressure cells are able to synthesize reaction center Bchl, LH Bchl and intracytoplasmic membranes at different rates. The synthesis of reaction center Bchl and LH Bchl I are, however, coordinated with each other, while the syntheses of LH Bchl II and reaction center Bchl proceed independently.List of Non-Standard Abbreviations Bchl bacteriochlorophyll - ICM mitracytoplasmic membrane - LDAO lauryldimethyl aminoxide - R Rhodopseudomonas - RC reaction center - SDS sodium dodecylsulfate