Thebc 1 complexes ofRhodobacter sphaeroides andRhodobacter capsulatus

Photosynthetic bacteria offer excellent experimental opportunities to explore both the structure and function of the ubiquinol-cytochromec oxidoreductase (bc ₁ complex). In bothRhodobacter sphaeroides andRhodobacter capsulatus, thebc ₁ complex functions in both the aerobic respiratory chain and as an essential component of the photosynthetic electron transport chain. Because thebc ₁ complex in these organisms can be functionally coupled to the photosynthetic reaction center, flash photolysis can be used to study electron flow through the enzyme and to examine the effects of various amino acid substitutions. During the past several years, numerous mutations have been generated in the cytochromeb subunit, in the Rieske iron-sulfur subunit, and in the cytochromec ₁ subunit. Both site-directed and random mutagenesis procedures have been utilized. Studies of these mutations have identified amino acid residues that are metal ligands, as well as those residues that are at or near either the quinol oxidase (Q_o) site or the quinol reductase (Q_i) site. The postulate that these two Q-sites are located on opposite sides of the membrane is supported by these studies. Current research is directed at exploring the details of the catalytic mechanism, the nature of the subunit interactions, and the assembly of this enzyme.     

DNA repair systems in the phototrophic bacterium Rhodobacter capsulatus

UV irradiation and mitomycin C exposure trigger a protease-activity-dependent inhibition of cell division in Rhodobacter capsulatus, which begins about 2 h after the treatment is applied. UV irradiation also induces a dose-dependent mutagenesis with a maximal rate between 5 and 10 J m-2, with increased synthesis of a protein of Mr approximately 30,000 between 2 and 3 h after UV irradiation. In addition, R. capsulatus has an efficient photoreactivation system that reverses the lethal effects of UV irradiation in the presence of intense visible light.     

Degradation of p-nitrophenol by the phototrophic bacterium Rhodobacter capsulatus

The phototrophic bacterium Rhodobacter capsulatus detoxified p-nitrophenol and 4-nitrocatechol. The bacterium tolerated moderate concentrations of p-nitrophenol (up to 0.5 mM) and degraded it under light at an optimal O₂ pressure of 20 kPa. The bacterium did not metabolize the xenobiotic in the dark or under strictly anoxic conditions or high O₂ pressure. Bacterial growth with acetate in the presence of p-nitrophenol took place with the simultaneous release of nonstoichiometric amounts of 4-nitrocatechol, which can also be degraded by the bacterium. Crude extracts from R. capsulatus produced 4-nitrocatechol from p-nitrophenol upon the addition of NAD(P)H, although at a very low rate. A constitutive catechol 1,2-dioxygenase activity yielding cis,cis-muconate was also detected in crude extracts of R. capsulatus. Further degradation of 4-nitrocatechol included both nitrite- and CO₂-releasing steps since: (1) a strain of R. capsulatus (B10) unable to assimilate nitrate and nitrite released nitrite into the medium when grown with p-nitrophenol or 4-nitrocatechol, and the nitrite concentration was stoichiometric with the 4-nitrocatechol degraded, and (2) cultures of R. capsulatus growing microaerobically produced low amounts of ¹⁴CO₂ from radiolabeled p-nitrophenol. The radioactivity was also incorporated into cellular compounds from cells grown with uniformly labeled ¹⁴C-p-nitrophenol. From these results we concluded that the xenobiotic is used as a carbon source by R. capsulatus, but that only the strain able to assimilate nitrite (E1F1) can use p-nitrophenol as a nitrogen source. Received: 30 December 1996 / Accepted: 3 September 1997     

Induction of stress proteins in the phototrophic bacterium Rhodobacter sphaeroides

The stress response of the phototrophic bacterium Rhodobacter sphaeroides was investigated by two-dimensional gel electrophoresis. Oxygen, 0.5% and 4% ethanol, UV radiation, heat shock at 42°C and 0.01% phenanthrene were tested as stress factors. The protein pattern on two-dimensional gels of stressed as compared to control cells revealed that all stress factors applied caused modifications in the protein pattern of R. sphaeroides. The intensity of particular spots increased or decreased as a consequence of altered protein synthesis. Specific and general stress responses were observed.     

Photoresponsive flagellum-independent motility of the purple phototrophic bacterium Rhodobacter capsulatus

We report the discovery of photoresponsive, flagellum-independent motility of the alpha-proteobacterium Rhodobacter capsulatus, a nonsulfur purple phototrophic bacterium. This motility takes place in the 1.5% agar-glass interface of petri plates but not in soft agar, and cells move toward a light source. The appearances of motility assay plates inoculated with wild-type or flagellum-deficient mutants indicate differential contributions from flagellar and flagellum-independent mechanisms. Electron microscopy confirmed the absence of flagella in flagellar mutants and revealed the presence of pilus-like structures at one pole of wild-type and mutant cells. We suggest that R. capsulatus utilizes a flagellum-independent, photoresponsive mechanism that resembles twitching motility to move in a line away from the point of inoculation toward a light source.     

Periplasmic location of dissimilatory nitrate and nitrite reductases in a denitrifying phototrophic bacterium, Rhodopseudomonas sphaeroides forma sp. denitrificans

The localization of dissimilatory nitrate and nitrite reductasesof a denitrifying phototrophic bacterium, Rhodopseudomonas sphaeroidesforma sp. denitrificans, was investigated. Nitrate and nitritereductases were located in the periplasmic space of the bacteriumgrown anaerobically in the presence of nitrate either in lightor in darkness. Chromatophores showed nitrate and nitrite reductaseactivities when dithionite-reduced benzyl viologen was an electrondonor; this suggests that the enzymes were trapped inside thevesicles. ¹Present address: Japanese Red Cross Central Blood Center, Hiroo4-1-31, Shibuyaku, Tokyo 150, Japan. ²Present address: Plant Growth Laboratory, University of California,Davis, California 95616, U.S.A. (Received November 7, 1979; )     

Inhibition of nitrate reduction by light and oxygen in Rhodobacter sphaeroides forma sp. denitrificans

Light inhibited each step of the denitrification process in whole cells of Rhodobacter sphaeroides forma sp. denitrificans. This inhibition by light was prevented in the presence of exogenous electron donors like N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) plus ascorbate or in the presence of an uncoupler (carbonyl cyanide m-chlorophenylhydrazone). Addition of myxothiazol restored the inhibition by light in uncoupled cells. Measurements of light-induced absorbance changes under these conditions showed that this inhibition is due, for the steps of reduction of nitrite to dinitrogen, to the photooxidation of cytochromes c ₁ plus c ₂ and not due to the photoinduced membrane potential. Moreover, the presence of oxygen inhibited almost all of the reduction of nitrate and nitrous oxide but only 70% of the reduction of nitrite to nitrous oxide. These inhibitions were overcome in the presence of TMPD plus ascorbate. This implies that the inhibition in presence of oxygen was due to a diversion of the reducing power from the denitrifying chain to the respiratory chain. It was concluded from this series of experiments that the reduction of nitrate to nitrite is inhibited when the ubiquinone pool is partly oxidized and that nitrite and nitrous oxide reductions are inhibited when cytochromes c ₁ plus c ₂ are oxidized by photosynthesis or respiration.Abbreviations R Rhodobacter - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - HOQNO 2-n-heptyl-4-hydroxyquinoline N-oxide - CCCP carbonyl cyanide m-chlorophenylhydrazone - cytochrome c ₁ cytochrome c ₂ plus cytochrome c ₁     

Metabolism of the phase variants of the phototrophic bacterium Rhodobacter sphaeroides

Growth, bacteriochlorophyll a content, electron transport chain (ETC), and activities of the tricarboxylic acid (TCA) cycle enzymes were studied in R and M phase variants of Rhodobacter sphaeroides cells grown anaerobically in the light and aerobically in the dark. Under all cultivation conditions tested, bacteriochlorophyll a content was 2–3 times lower in the cells of the M variant compared to the R variant, which therefore was predominant in the cultures grown in the light. In both variants, activity of all TCA cycle enzymes was higher for the cells grown in the dark under aerobic conditions. When grown aerobically in the dark, the R variant, unlike the M variant, did not contain cytochrome aa ₃, acting as cytochrome c oxidase, in its ETC. An additional point of coupling the electron transfer to the generation of the proton gradient at the cytochrome aa ₃ level provided for more efficient oxidation of organic substrates, resulting in predominance of the M variant in the cultures grown in the dark under aerobic conditions.     

Characterization of urease from the phototrophic bacteriumRhodobacter capsulatus E1F1

Rhodobacter capsulatus E1F1 showed high cytosolic urease activity when growing on urea, purines, and purine metabolites as nitrogen source. Molecular mass ofR. capsulatus enzyme is similar to that of other bacteria and greatly differs from that of jack bean. Kinetic parameters of partially purifiedR. capsulatus enzyme resemble those described in other bacterial ureases. The activity was inhibited by metal-chelating agents and by mercurials. Urease fromR. capsulatus E1F1 was negligible in nitrogen-starved cells or in cells cultured with nitrate, ammonium, or amino acids. Moreover, ammonium inhibited both the urea uptake and the urease activity expression inR. capsulatus cells.     

Involvement in denitrification of the napKEFDABC genes encoding the periplasmic nitrate reductase system in the denitrifying phototrophic bacterium Rhodobacter sphaeroides f. sp. denitrificans

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.     

Sulfide-quinone and sulfide-cytochrome reduction in Rhodobacter capsulatus

The reduction by sulfide of exogenous ubiquinone is compared to the reduction of cytochromes in chromatophores of Rhodobacter capsulatus. From titrations with sulfide values for V_max of 300 and 10 moles reduced/mg bacteriochlorophyll a·h, and for K_m of 5 and 3 M were estimated, for decyl-ubiquinone-and cytochrome c-reduction, respectively. Both reactions are sensitive to KCN, as has been found for sulfide-quinone reductase (SQR) in Oscillatoria limnetica, which is a flavoprotein. Effects of inhibitors interfering with quinone binding sites suggest that at least part of the electron transport from sulfide in R. capsulatus employs the cytochrome bc ₁-complex via the ubiquinone pool.Abbreviations BChl a bacteriochlorophyll a - DAD diaminodurene - decyl-UQ decyl-ubiquinone - LED light emitting diode - NQNO 2-n-nonyl-4-hydroxyquinoline-N-oxide - PQ-1 plastoquinone 1 - SQR sulfide-quinone reductase (E.C. 1.8.5.'.) - UQ ubiquinone 10 - Q_c the quinone reduction site on the cytochrome b ₆ f/bc ₁, complex (also termed Q_i or Q_r or Q_n) - Q_s the quinone reduction site on SQR - Q_z quinol oxidation site on the b ₆ f/bc ₁, complex (also termed Q_o or Q_p)     

Intracytoplasmic membrane vesiculation in light-harvesting mutants of Rhodobacter sphaeroides and Rhodobacter capsulatus

Abstract In Chlamydomonas reinhardtii there are three glutamate dehydrogenase isozymes which can use both NADH and NADPH as cofactors and respond differently to different nitrogen sources and several stress conditions. From data of induction of isozymes in different metabolic situations, we propose a possible physiological role for each of them in algal carbon and nitrogen metabolism.     

Purification and properties of a polyol dehydrogenase from the phototrophic bacterium Rhodobacter sphaeroides

A polyol dehydrogenase was detected in cell extracts of the facultative phototrophic bacterium Rhodobacter sphaeroides strain Si 4 grown on D-glucitol (sorbitol) as the sole carbon source. The enzyme was purified 150-fold to apparent homogeneity by steps involving fractionated (NH4)2SO4 precipitation, chromatography on Q-Sepharose and phenyl-Sepharose, and FPLC on Superose 12. The relative molecular mass (Mr) of the native polyol dehydrogenase was 47,200 as calculated from its Stokes' radius (rs = 2.76 nm) and sedimentation coefficient (s20, w = 4.15 S). SDS/PAGE resulted in one single band representing a polypeptide with a Mr of 52,200, indicating that the native protein is a monomer. The isoelectric point of the polyol dehydrogenase was determined to be pH 4.3. The enzyme was specific for NAD+ and oxidized both D-glucitol and D-mannitol to D-fructose, as well as D-arabinitol to D-ribulose. The pH optimum of substrate oxidation was pH 9.0 in 0.1 M Tris/HCl and that of substrate reduction was pH 6.5 in 0.1 M potassium phosphate. The reactions exhibited normal Michaelis-Menten kinetics allowing the estimation of KM values for NAD+ (0.18 mM) in the presence of D-glucitol, and for D-glucitol (31.8 mM), D-mannitol (0.29 mM) and D-arabinitol (1.8 mM), respectively. The KM value for D-fructose was 16.3 mM and that for NADH 0.02 mM. The equilibrium constants determined for the conversion of D-mannitol, D-glucitol and D-arabinitol were 4.5 nM, 0.58 nM and 80 pM, respectively. Based on the catalytic preference of the polyol dehydrogenase for D-mannitol, an enzymatic assay for D-mannitol was elaborated.     

Effect of selenite on growth and protein synthesis in the phototrophic bacterium Rhodobacter sphaeroides

The effect of selenite on the growth rate and protein synthesis has been investigated in Rhodobacter sphaeroides. This photosynthetic bacterium efficiently reduces selenite with intracellular accumulation under both dark aerobic and anaerobic photosynthetic conditions. Addition of 1 mM selenite under these two growth conditions does not affect the final cell density, although a marked slowdown in growth rate is observed under aerobic growth. The proteome analysis of selenite response by two-dimensional gel electrophoresis shows an enhanced synthesis of some chaperones, an elongation factor, and enzymes associated to oxidative stress. The induction of these antioxidant proteins confirms that the major toxic effect of selenite is the formation of reactive oxygen species during its metabolism. In addition, we show that one mutant unable to precipitate selenite, selected from a transposon library, is affected in the smoK gene. This encodes a constituent of a putative ABC transporter implicated in the uptake of polyols. This mutant is less sensitive to selenite and does not express stress proteins identified in the wild type in response to selenite. This suggests that the entry of selenite into the cytoplasm is mediated by a polyol transporter in R. sphaeroides.     

Identification and solubilization of a signal peptidase from the phototrophic bacterium Rhodobacter capsulatus.

In Gram-negative bacteria, exported proteins are synthesized with an amino-terminal signal sequence which is cleaved off by the signal peptidase during, or shortly after the translocation process. Here, we report the identification and solubilization of a signal peptidase from the phototrophic bacterium Rhodobacter capsulatus which cleaves homologous and heterologous precursor proteins at the authentic cleavage site. This signal peptidase is the first identified component of the R. capsulatus protein export machinery.     

Tactic responses to oxygen in the phototrophic bacterium Rhodobacter sphaeroides WS8N

The temporal and spatial behavior of a number of mutants of the photosynthetic, facultative anaerobe Rhodobacter sphaeroides to both step changes and to gradients of oxygen was analyzed. Wild-type cells, grown under a range of conditions, showed microaerophilic behavior, accumulating in a 1.3-mm band about 1.3 mm from the meniscus of capillaries. Evidence suggests this is the result of two signaling pathways. The strength of any response depended on the growth and incubation conditions. Deletion of either the complete chemosensory operons 1 and 2 plus the response regulator genes cheY(4) and cheY(5) or cheA(2) alone led to the loss of all aerotactic responses, although the cells still swam normally. The Prr system of R. sphaeroides responds to electron flow through the alternative high-affinity cytochrome oxidase, cbb(3), controlling expression of a wide range of metabolic pathways. Mutants with deletions of either the complete Prr operon or the histidine kinase, PrrB, accumulated up to the meniscus but still formed a thick band 1.3 mm from the aerobic interface. This indicates that the negative aerotactic response to high oxygen levels depends on PrrB, but the mutant cells still retain the positive response. Tethered PrrB(-) cells also showed no response to a step-down in oxygen concentration, although those with deletions of the whole operon showed some response. In gradients of oxygen where the concentration was reduced at 0.4 micro M/s, tethered wild-type cells showed two different phases of response, with an increase in stopping frequency when the oxygen concentration fell from 80 to 50% dissolved oxygen and a decrease in stopping at 50 to 20% dissolved oxygen, with cells returning to their normal stopping frequency in 0% oxygen. PrrB and CheA(2) mutants showed no response, while PrrCBA mutants still showed some response.     

Non-reciprocal regulation of Rhodobacter capsulatus and Rhodobacter sphaeroides recA genes expression

Abstract The Rhodobacter capsulatus recA gene has been isolated and sequenced. Its deduced amino acid sequence showed the closest identity with the Rhodobacter sphaeroides RecA protein (91% identity). However, the promoter regions of both R. capsulatus and R. sphaeroides recA genes are only 64% similar. An Escherichia coli -like LexA binding site was not present in the upstream region of the R. capsulatus recA gene. Nevertheless, the R. capsulatus recA gene is inducible by DNA damage in both hetero- and phototrophically growing conditions. The R. capsulatus recA gene is poorly induced when inserted into the chromosome of R. sphaeroides , indicating that the recA gene of both bacteria possess different control sequences despite their phylogenetically close relationship.