A denitrifying strain of Rhodobacter capsulatus

Abstract Repeated subculturing of Rhodobacter capsulatus strain BK5 under phototrophic conditions on a medium containing butyrate and nitrate led to the appearance of cultures that, unlike the original, produced gas. Isolation of a pure culture of the gas-forming organism revealed that it was a derivative of R. capsulatus BK5 that by virtue of expressing a nitrite reductase can catalyse the complete sequence of the denitrification reactions. The nitrite reductase is of the type that contains copper rather than haem.     

The interaction between dimethylsulfoxide and nitrate reducing pathways in Rhodobacter capsulatus

Abstract The interaction between nitrate- and dimethyl-sulphoxide (DMSO)-reducing pathways was demonstrated in intact cells of Rhodobacter capsulatus AD2 removed from cultures grown under different conditions. The results provide evidence of competition between the DMSO and nitrate reductases for a common electron pool. Furthermore, strong inhibition was observed of the anaerobic dark DMSO-dependent growth of R. capsulatus by nitrate in the growth medium. This phenomenon is also discussed.     

The periplasmic nitrate reductase of Rhodobacter capsulatus; purification,characterisation and distinction from a single reductase for trimethylamine-N-oxide,dimethylsulphoxide and chlorate

The periplasmic dissimilatory nitrate reductase from Rhodobacter capsulatus N22DNAR⁺ has been purified. It comprises a single type of polypeptide chain with subunit molecular weight 90,000 and does not contain heme. Chlorate is not an alternative substrate. A molybdenum cofactor, of the pterin type found in both nitrate reductases and molybdoenzymes from various sources, is present in nitrate reductase from R. capsulatus at an approximate stoichiometry of 1 molecule per polypeptide chain. This is the first report of the occurrence of the cofactor in a periplasmic enzyme. Trimethylamine-N-oxide reductase activity was fractionated by ion exchange chromatography of periplasmic proteins. The fractionated material was active towards dimethylsulphoxide, chlorate and methionine sulphoxide, but not nitrate. A catalytic polypeptide of molecular weight 46,000 was identified by staining for trimethylamine-N-oxide reductase activity after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The same polypeptide also stained for dimethylsulphoxide reductase activity which indicates that trimethylamine-N-oxide and dimethylsulphoxide share a common reductase.Abbreviations DMSO dimethylsulphoxide - LDS lithium dodecyl sulphate - MVH reduced methylviologen - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - TMAO trimethylamine-N-oxide     

Phenotypic characterisation and genetic complementation of dimethylsulfoxide respiratory mutants of Rhodobacter sphaeroides and Rhodobacter capsulatus

Abstract Two chlorate resistant mutants of Rhodobacter sphaeroides were isolated which were deficient in dimethylsulfoxide reductase activity. Immunoblotting experiments showed that the phenotype of these mutants and that of Rhodobacter capsulatus strain DK9, a mutant unable to reduce dimethylsulfoxide, was correlated with low or undetectable levels of the dimethylsulfoxide reductase apoprotein. All three mutants were complemented by a cosmid from a library of Rhodobacter sphaeroides genomic DNA. Further genetic complementation analysis revealed that functions required for restoration of dimethylsulfoxide reductase activity in the Rhodobacter sphaeroides mutants were encoded on an 9 kb EcoR1 DNA fragment derived from this cosmid. Expression of this 9 kb DNA fragment in Escherichia coli showed that it encoded the dimethylsulfoxide reductase structural gene of Rhodobacter sphaeroides .     

Derivatives of Rhodobacter capsulatus strain AD2 cured of their endogenous plasmid are unable to utilize nitrate

Abstract Derivatives of Rhodobacter capsulatus AD2 unable to grow with nitrate as sole N source were isolated after conjugation with a plasmid containing a cloned 3.4 kb Hin dIII fragment from the endogenous plasmid of this strain. These derivatives lacked the M _r 74 × 10⁶ plasmid found in the wild-type, and failed to revert to growth on nitrate. Cultures of the plasmid-cured strains also lacked dissimilatory nitrate reductase activity, suggesting that genes required for both assimilatory and dissimilatory nitrate reduction are located on the endogenous plasmid.     

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.     

Reduction of potassium tellurite to elemental tellurium and its effect on the plasma membrane redox components of the facultative phototroph Rhodobacter capsulatus

Summary.  Anaerobically light-grown cells of Rhodobacter capsulatus B100 are highly resistant to the toxic oxyanion tellurite (TeO₃ ²⁻; minimal inhibitory concentration, 250 μg/ml). This study examines, for the first time, some structural and biochemical features of cells and plasma membrane fragments of this facultative phototroph grown in the presence of 50μg of K₂TeO₃ per ml. Through the use of transmission microscopy and X-ray microanalysis we show that several “needlelike” shaped granules of elemental tellurium are accumulated into the cytosol near the intracytoplasmic membrane system. Flash-spectroscopy, oxygen consumption measurements, and difference spectra analysis indicated that membrane vesicles (chromatophores) isolated from tellurite-grown cells are able to catalyze both photosynthetic and respiratory electron transport activities, although they are characterized by a low c-type cytochrome content (mostly soluble cytochrome c ₂). This feature is paralleled by a low cytochrome c oxidase activity and with an NADH-dependent respiration which is catalyzed by a pathway leading to a quinol oxidase (Qox) inhibited by high (millimolar) concentrations of cyanide (CN⁻). Conversely, membranes from R. capsulatus B100 cells grown in the absence of tellurite are characterized by a branched respiratory chain in which the cytochrome c oxidase pathway (blocked by CN⁻ in the micromolar range) accounts for 35–40% of the total NADH-dependent oxygen consumption, while the remaining activity is catalyzed by the quinol oxidase pathway. These data have been interpreted to show that tellurite resistance of R. capsulatus B100 is characterized by the presence of a modified plasma-membrane-associated electron transport system. Received May 2, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Department of Biology, University of Bologna, Via Irnerio 42, 40126 Bologna, Italy.     

Mediated electrochemistry of dimethyl sulfoxide reductase from Rhodobacter capsulatus

Electrochemically driven catalysis of the bacterial enzyme dimethyl sulfoxide (DMSO) reductase (Rhodobacter capsulatus) has been studied using the macrocyclic complex (trans-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane-6,13-diamine)cobalt(III) as a mediator. In the presence of both DMSO and DMSO reductase, the normal transient Co^III/II voltammetric response of the complex is transformed into an amplified and sigmoidal (steady-state) waveform characteristic of a catalytic EC′ mechanism. At low concentrations of DMSO (approximately K _M) or high mediator concentrations (more than the concentration of DMSO reductase), the steady-state character of the voltammetric response disappears and is replaced by more complicated waveforms that are a convolution of transient and steady-state behavior as different steps within the catalytic cycle become rate limiting. Through digital simulation of cyclic voltammetry performed under conditions where the sweep rate, DMSO concentration, DMSO reductase concentration and mediator concentration were varied systematically, we were able to model all voltammograms with a single set of rate and equilibrium constants which provide new insights into the kinetics of the DMSO reductase catalytic mechanism that have hitherto been inaccessible from steady state or stopped flow kinetic studies.

Redox characteristics of the tungsten DMSO reductase of Rhodobacter capsulatus

The dimethylsulfoxide reductase (DMSOR) from Rhodobacter capsulatus is known to retain its three-dimensional structure and enzymatic activity upon substitution of molybdenum, the metal that occurs naturally at the active site, by tungsten. The redox properties of tungsten-substituted DMSOR (W-DMSOR) have been investigated by a dye-mediated reductive titration with the concentration of the W(V) state monitored by EPR spectroscopy. At pH 7.0, E(m)(W(VI)/W(V)) is -194 mV and E(m)(W(V)/W(IV)) is -134 mV. Each E(m) value of W-DMSOR is significantly lower (220 and 334 mV, respectively) than that of the corresponding couple of Mo-DMSOR. These redox potentials are consistent with the ability of Mo-DMSOR to catalyze both the reduction of DMSO to DMS and the back reaction, whereas W-DMSOR is very effective in catalyzing the forward reaction, but shows no ability to catalyze the oxidation of DMS to DMSO.     

Isolation, characterisation and expression of the bacterioferritin gene of Rhodobacter capsulatus

Abstract The nucleotide sequence of the Rhodobacter capsulatus bacterioferritin gene ( bfr ) was determined and found to encode a protein of 161 amino acids with a predicted molecular mass of 18 174 Da. The molecular mass of the purified protein was estimated to be 18 176.06 ± 0.80 Da by electrospray mass spectrometry. The bfr gene was introduced into an expression vector, and bacterioferritin was produced to a high level in Escherichia coli . The amino acids which are involved in haem ligation, and those which provide ligands in the binuclear metal centre in bacterioferritin from E. coli are conserved in the R. capsulatus protein. The sequences of bacterioferritins, ferritin-like proteins, and proteins similar to Dps of E. coli are compared, and membership of the bacterioferritin family re-evaluated.     

Competition between hydrogen peroxide and nitrate for electrons from the respiratory chains of Thiosphaera pantotropha and Rhodobacter capsulatus

Abstract Thiosphaera pantotropha and some strains of Rhodobacter capsulatus express both a periplasmic nitrate reductase and cytochrome c peroxidase when grown under aerobic conditions. Harvested cell suspensions of either species can respire nitrate in the presence of 200 μM O₂ (∼ 80% air saturation), at 70–80% of the anaerobic rate. Addition of hydrogen peroxide to such cells causes a 90% inhibition of nitrate reduction under anaerobic or aerobic conditions. The duration of the inhibition is proportional to the concentration of hydrogen peroxide added and can be ascribed to the expression of periplasmic peroxidases that compete with the nitrate reductase for electrons from the respiratory chain. The results reveal a hitherto unrecognised interaction between reactions of denitrification and the reduction of hydrogen peroxide by a periplasmic peroxidase that may have implications for the denitrification in microaerobic environments. The creation of aerobic conditions in bacterial cultures by addition of hydrogen peroxide, and relying on the generation of oxygen by endogenous catalase activity, is a commonly used technique for studying respiratory processes. The observations presented here demonstrate that results derived from such experiments should be interpreted with caution.     

Response of four rice varieties to Rhodobacter capsulatus at seedling stage

The phototrophic purple nonsulphur bacterium (PPNSB) Rhodobacter capsulatus was used to inoculate seedlings of four rice varieties Giza 159, Giza 171, Giza 176 and Giza 181, grown in hydroponic culture with or without nitrogen. After three weeks the seedling growth parameters were measured. Inoculation with R. capsulatus enhanced seedling growth of all rice varieties tested. The response to inoculation as compared to control plants (no nitrogen, no R. capsulatus) were 52% to 75% for shoot height, 47% to 100% for aerial part dry weight, 45% to 78% for aerial part N content, –37% to –9% for maximum root length, –4% to 8% for root system dry weight and 50% to 62% for root N content.     

Nitrate reductase activity in spheroplasts from Rhodobacter capsulatus E1F1 requires a periplasmic protein

Spheroplasts from Rhodobacter capsulatus E1F1 cells grown in nitrate maintained nitrate uptake and nitrate reductase activity only when they were illuminated under anaerobiosis in the presence of the periplasmic fraction and nitrate. The effects on nitrate uptake and nitrate reductase activity of spheroplasts were observed at low concentrations of periplasmic protein (about 50 x ml^-1). Periplasm from nitrate-grown cells was also required for nitrate reductase activity in spheroplasts isolated from ammonia-grown or diazotrophic cells which initially lacked this enzymatic activity. Both the maintenance of nitrate reductase in spheroplasts from nitrate-grown cells and the appearance of the activity in spheroplasts from diazotrophic cells were dependent on de novo protein synthesis. A periplasmic, 45-kDa protein which maintained the activity of nitrate reductase in spheroplasts was partially purified by gel filtration chromatography of periplasm obtained from nitrate-grown cells.Abbreviations NR nitrate reductase - CCCP carbonyl cyanide m-chlorophenylhydrazone - CAM chloramphenicol     

X-ray absorption spectroscopic studies of the methane monooxygenase hydroxylase component from Methylosinus trichosporium OB3b

The conversion from methane to methanol is catalyzed by methane monooxygenase (MMO) in methanotrophic bacteria. Earlier work on the crystal structures of the MMO hydroxylase component (MMOH) from Methylococcus capsulatus (Bath) at 4??°C and –160??°C has revealed two different core arrangements for the diiron active site. To ascertain the generality of these results, we have now carried out the first structural characterization on MMOH from Methylosinus trichosporium OB3b. Our X-ray absorption spectroscopic (XAS) analysis suggests the presence of two Fe-Fe distances of about 3?Å and 3.4?Å, which are proposed to reflect two populations of MMOH molecules with either a bis(μ-hydroxo)(μ-carboxylato)- or a (μ-hydroxo)(μ-carboxylato)diiron(III) core structure, respectively. The observation of these two different core structures, together with the crystallographic results of the MMOH from Methylococcus capsulatus (Bath), suggests the presence of an equilibrium that may reflect a core flexibility that is required to accommodate the various intermediates in the catalytic cycle of the enzyme. XAS studies on the binding of component B (MMOB) to the hydroxylase component show that MMOB does not perturb either this equilibrium or the gross structure of the oxidized diiron site in MMOH.     

Role of glutamine as a direct co-repressor of glutamine synthetase in Rhodobacter capsulatus E1F1

High performance liquid chromatography (HPLC) has been used to determine the internal levels of amino acids in Rhodobacter capsulatus E1F1 cells, subjected to different treatments and nutritional conditions. Glutamine synthetase activity and enzyme concentration correlated negatively with the level of glutamine, suggesting that glutamine per se acts as a co-repressor in the enzyme synthesis. Moreover, addition of the specific inhibitor L-methionine-D,L-sulfoximine, that produced an increase in enzyme concentration, specifically promoted a depletion of intracellular glutamine.     

Photoinactivation of the detoxifying enzyme nitrophenol reductase from Rhodobacter capsulatus

The phototrophic bacterium Rhodobacter capsulatus E1F1 detoxifies 2,4-dinitrophenol by inducing an NAD(P)H-dependent iron flavoprotein that reduces this compound to the less toxic end product 2-amino-4-nitrophenol. This nitrophenol reductase was stable in crude extracts containing carotenes, but it became rapidly inactivated when purified protein was exposed to intense white light or moderate blue light intensities, especially in the presence of exogenous flavins. Red light irradiation had no effect on nitrophenol reductase activity. Photoinactivation of the enzyme was irreversible and increased under anoxic conditions. This photoinactivation was prevented by reductants such as NAD(P)H and EDTA and by the excited flavin quencher iodide. Addition of superoxide dismutase, catalase, tryptophan or histidine did not affect photoinactivation of nitrophenol reductase, thus excluding these reactive dioxygen species as the inactivating agent. Substantial protection by 2,4-dinitrophenol also took place when the enzyme was irradiated at a wavelength coinciding with one of the absorption peaks of this compound (365nm). These results suggest that the lability of nitrophenol reductase was due to the absorption of blue light by the flavin prosthetic group, thus producing an excited flavin that might irreversibly oxidize some functional group(s) necessary for enzyme catalysis. Nitrophenol reductase may be preserved in vivo from blue light photoinactivation by the high content of carotenes and excess of reducing equivalents in phototrophic growing cells.Abbreviations 2,4-DNP 2,4-dinitrophenol - ANP 2-amino-4-nitrophenol - EDTA ethylenediamine tetraacetic acid - MES 2-(N-Morpholino) ethanesulfonic acid - NPR nitrophenol reductase     

Effects of Cadmium Stress on Growth,Morphology, and Protein Expression in Rhodobacter capsulatus B10

The effects of cadmium stress on growth, morphology, and protein expression were investigated in Rhodobacter capsulatus B10 using two-dimensional polyacrylamide gel electrophoresis and a scanning electron microscope with an energy dispersive X-ray spectrometer. The bacterium grew in the presence of 150 μM CdCl₂ and highly induced heat-shock proteins (GroEL and Dnak), S-adenosylmethionine synthetase, ribosomal protein S1, aspartate aminotransferase, and phosphoglycerate kinase. Interestingly, the ribosomal protein S1 was proportionally expressed as the amount of cadmium in the medium, suggesting that S1 may be required for the repair of cadmium-mediated cellular damage. On the other hand, we identified five cadmium-binding proteins: 2-methylcitrate dehydratase, phosphate peripalsmic binding protein, inosine-5′-monophosphate dehydrogenase/guanosine-5′-monophosphate reductase, inositol monophosphatase, and lytic murein transglycosylase. The cadmium-treated cells had a filamentous structure and contained less phosphorous than the untreated cells. We propose that these characteristics of the cadmium-treated cells may be due to the inactivation of the phosphate peripalsmic binding protein and lytic murein transglycosylase by cadmium.     

Aerobic chemolithoautotrophic growth and RubisCO function in Rhodobacter capsulatus and a spontaneous gain of function mutant of Rhodobacter sphaeroides

Photosynthetic prokaryotes that assimilate CO₂ under anoxic conditions may also grow chemolithoautotrophically with O₂ as the electron acceptor. Among the nonsulfur purple bacteria, two species (Rhodobacter capsulatus and Rhodopseudomonas acidophilus), exhibit aerobic chemolithoautotrophic growth with hydrogen as the electron donor. Although wild-type strains of Rhodobacter sphaeroides grow poorly, if at all, with hydrogen plus oxygen in the dark, we report here the isolation of a spontaneous mutant (strain HR-CAC) of Rba. sphaeroides strain HR that is fully capable of this mode of growth. Rba. sphaeroides and Rba. capsulatus fix CO₂ via the reductive pentose phosphate pathway and synthesize two forms of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). RubisCO levels in the aerobic-chemolithoautotrophic-positive strain of Rba. sphaeroides were similar to those in wild-type strains of Rba. sphaeroides and Rba. capsulatus during photoheterotrophic and photolithoautotrophic growth. Moreover, RubisCO levels of Rba. sphaeroides strain HR-CAC approximated levels obtained in Rba. capsulatus when the organisms were grown as aerobic chemolithoautotrophs. Either form I or form II RubisCO was able to support aerobic chemolithoautotrophic growth of Rba. capsulatus strain SB 1003 and Rba. sphaeroides strain HR-CAC at a variety of CO₂ concentrations, although form II RubisCO began to lose the capacity to support aerobic CO₂ fixation at high O₂ to CO₂ ratios. The latter property and other facets of the physiology of this system suggest that Rba. sphaeroides and Rba. capsulatus strains may be effectively employed for the biological selection of RubisCO molecules of altered substrate specificity. Received: 8 August 1997 / Accepted: 26 December 1997