Artificial DNA-mediated genetic transformation of the photosynthetic nitrogen-fixing bacterium Rhodospirillum rubrum

Methods were identified for the introduction of plasmid DNA into Rhodospirillum rubrum, including freeze-thaw and CaCl₂-based techniques.Abbreviations cfu colony forming units - DMSO dimethyl sulfoxide - DTT dithiothreitol - O.D.₆₈₀ optical density at 680 nm     

Occurrence of oscillations in ATP synthesis and hydrolysis in chromatophores of Rhodospirillum rubrum

The conditions under which an oscillatory behaviour is observed during net hydrolysis or synthesis of ATP in chromatophores of Rhodospirillum rubrum FR1 are described. In the case of ATPase the oscillations are observed at low temperature (ca. 11°C) in the dark after an initial transient behaviour. These oscillations are attenuated or disappear by the addition of an uncoupler.Oscillations are also observed during ATP synthesis. At 3°C the oscillations appear spontaneously if photophosphorylation is measured during a sufficiently long time. At 30°C the mere intercalation of a dark period also at 30°C is sufficient to trigger the oscillations in the following light period.Abbreviations Bchl Bacteriochlorophyll - FCCP carbonyl cyanide p-trifluoromethoxyphenyl hydrazone - PMS phenazine methosulfate - TMPD, N,N,N,N tetramethyl-1,4-phenylenediamine Dedicated to Prof. Dr. Gerhart Drews as a homage for his permanent example as hard worker and careful scientist and also for his remarkable human quality     

Temperature dependency of the Mg-ATPase and photophosphorylation activities in Rhodospirillum rubrum chromatophores

Arrhenius plots for ATP synthesis, coupled to endogenous and Phenazine methosulfate or N,N,N,N,-Tetramenthyl-1,4-Phenylene diamine-mediated photosynthetic election transport and for ATP hydrolysis were studied in Rhodospirillum rubrum chromatophores.Coupled or uncoupler induced Mg-ATPase show no discontinuity in the range tested (30°C-5°C) and they also have the same activation energy. Phenazine methosulfatecatalyzed photophosphorylation has also a single activation energy where as the endogenous reaction shows complex and ageing dependent behaviour, alternating temperature ranges having high (45.2 to 144,4 kJ·mol^-1) and very low (ca 0.0 to 3.3 kJ·mol^-1) activation energy.Abbreviations Bchl Bacteriochlorophyll - Ea Activation energy - FCCP Carbonyl Cyanide p. Trifluoromethoxy henyl Hydrazone - PMS phenazine methosulfate - TMPD N,N,N,N-Tetramethyl-1,4-Phenylene diamine - R Rhodospirillum     

A serendipic legacy: Erwin Esmarch's isolation of the first photosynthetic bacterium in pure culture

During the 1880's, Erwin von Esmarch was a junior associate (Assistent) of Robert Koch studying bacteria of medical significance. In 1887, he isolated the first example of spiral-shaped bacteria in pure culture, from the dry residue of a dead mouse that he had suspended sometime earlier in Berlin tap-water. Under certain conditions, colonies of the organism were the color of red wine, and this led Esmarch to name the bacterium Spirillum rubrum. Twenty years later, Hans Molisch demonstrated that S. rubrum, an apparent heterotroph, was in fact a non-oxygenic purple photosynthetic bacterium, and it was renamed Rhodospirillum rubrum. Esmarch was a careful investigator and his classic paper of 1887 details the serendipitous isolation and general characteristics of the first pure culture of an anoxyphototroph, which later played a prominent role as an experimental system for study of basic aspects of bacterial photosynthesis. This report includes an English translation of his original paper (in German), a commentary on the historical significance of Esmarch's spirillum, and a summary of Esmarch's career.     

Bioconversion of synthesis gas to hydrogen using a light-dependent photosynthetic bacterium,Rhodospirillum rubrum

Biological hydrogen production from synthesis gas was carried out in batch culture. The phototrophic anaerobic bacterium, Rhodospirillum rubrum was used to oxidize CO and water to CO₂ and hydrogen. The bacteria were grown under anaerobic conditions in liquid medium; also acetate was used as carbon source in presence of synthesis gas. Biological hydrogen production was catalysed by R. rubrum via the water–gas shift reaction. A light-dependent cell growth modelled with a desired rate of hydrogen production and CO uptake was determined. The effect of light intensity on microbial cell growth was also studied at 500, 1,000 and 1,500 m.cd. A complete conversion of CO to hydrogen and maximum light efficiency were obtained with an acetate concentration of 1 g/l and light intensity of 500 m.cd. Utilization of the carbon monoxide from the gas phase was often considered as a mass transfer limited process, which needed to diffuse through the gas–liquid interface and then further diffuse into liquid medium prior to reaction. The results from this study showed that maximum cell propagation and hydrogen production were achieved with a limited light intensity of 1,000 m.cd. It was also found that high-light intensity may interfere with cell metabolism. In low-light intensity and substrate concentration, no inhibition was observed, however at extreme conditions, non-competitive inhibition was identified. The adverse effect of high-light intensity was shown at 5,000 m.cd, where the CO conversion drastically dropped to as low as 21%. Maximum CO conversion of 98% and maximum yield of 86% with an acetate concentration of 1.5 g/l and a light intensity of 1,000 m.cd were achieved.     

The early formation of the photosynthetic apparatus in Rhodospirillum rubrum

The time dependent assembly of the photosynthetic apparatus was studied in Rhodospirillum rubrum after transfer of cells growing aerobically in the dark to low aeration. While bacteriochlorophyll (Bchl) cellular levels increase continuously levels of soluble cytochrome c ₂do not change significantly. Absorption spectra of membranes isolated at different times after transfer reveal that incorporation of carotenoids lags behind incorporation of Bchl. However, a carotenoid fraction exhibiting spectral properties of spirilloxanthin isomers was isolated apart from membranes. This carotenoid fraction even was present in homogenates from Bchl-free, aerobically grown cells. Incorporation of U-¹⁴C-proteinhydrolyzate into membrane proteins showed that proteins are mainly formed which are specific for photosynthetic membranes. Although the proportion of reaction center (RC) Bchl per light harvesting (LH) Bchl does not change the proportions of membrane proteins present in RC and LH preparations change initially. But later on the proportions of the different proteins also reach constant values. Concerning proteins characteristic for cytoplasmic membranes a differential incorporation of label can be observed. The data indicate that the photosynthetic apparatus in Rhodospirillum rubrum is assembled through a sequential mechanism.Abbreviations Bchl bacteriochlorophyll - LH light harvesting - RC reaction center - R. Rhodospirillum - R. Rhodopseudomonas     

Evidence for an ammonium transport system in the N2-fixing phototrophic bacteriumRhodospirillum rubrum

An ammonium transport system in the phototrophic N₂-fixing bacteriumRhodospirillum rubrum was characterized by using the uptake of¹⁴C-methylamine as a probe.Uptake showed saturation kinetics with an apparentK _m =110 M. It was competitively inhibited by ammonium (K _i =7 M). Uptake exhibited a narrow pH maximum around pH 7.0.Up to 200-fold gradients across the membrane were formed within 40–60 min. Gradient formation was inhibited by carbon starvation, azide or cyanide. Pre-accumulated methylamine was released by ammonium pulses to more than 80%, indicating only minor metabolization.The synthesis of the transport system was repressed by ammonium in high concentrations.     

Freeze fracture studies of reaction centers from Rhodospirillum rubrum in chromatophores and liposomes

In freeze-fractures of chromatophores of Rhodospirillum rubrum the reaction centers are seen as hexagonal arranged particles of 13 nm diameter with a density of around 5,500 particles per m². Similar regions on the cytoplasmic membrane suggest that these parts are the prospective invagination sites.Isolated reaction centers are easily incorporated into liposomes. In freeze fractures of liposomes particles similar in shape and size, although less dense as in chromatophores are observed. In negative staining much smaller units of only 5 nm in diameter are found indicating that reaction centers occur in the membrane as tri- or tetramers. There is a strong correlation between particle density in chromatophores and titratable reaction centers remaining in these membranes after extraction of reaction centers by detergents; both values are in good agreement with the yield of reaction centers at a given detergent concentration.Abbreviations LDAO Lauryldimethylamine oxide - PF protoplasmic fracture face - EF exoplasmic fracture face     

The lipopolysaccharides of Rhodospirillum rubrum,Rhodospirillum molischianum,and Rhodopila globiformis

The cell wall lipopolysaccharides from three phototrophic species of the alpha₁-group of Proteobacteria, Rhodospirillum rubrum, Rhodospirillum molischianum, and Rhodopila globiformis were isolated and chemically characterized. Sodium deoxycholate polyacrylamide gel electrophoresis patterns revealed that the lipopolysaccharides of all three species possess O-chains. They are composed of repeating units only in R. molischianum and R. globiformis. The presence of l-glycero-d-mannoheptose and 2-keto-3-deoxyoctonate indicated core structures in all three lipopolysaccharides. Glucosamine was found as backbone amino sugar in lipid A of R. molischianum and R. rubrum, while R. globiformis has 2,3-diaminoglucose as backbone amino sugar. The latter species also differed from the two former ones in its content of hydroxy fatty acids (3-OH-14:0, 3-OH-16:0 in R. rubrum and R. molischianum and 3-OH-14:0, 3-OH-18:0 and 3-OH-19:0 (possibly iso- or anteisobranched) in R. globiformis).Abbreviations DOC-PAGE sodium deoxycholate polyacrylamide gel electrophoresis - GC/MS combined gas-liquid chromatography/mass spectrometry - KDO 2-keto-3-deoxyoctonate     

Unidirectional inhibition of phosphoenolpyruvate carboxykinase from Rhodospirillum rubrum by ATP

The kinetic and regulatory properties of partially purified phosphoenolpyruvate (PEP) carboxykinase (EC 4.1.1.32) from Rhodospirillum rubrum were studied. The enzyme was active with guanosine-and inosinephosphates and must thus be classified as GTP (ITP): oxaloacetate carboxylyase (transphosphorylating). In the direction of oxaloacetate-formation, the enzyme was strongly inhibited by ATP (K_i=0.03 mM). ITP, UTP, CTP and GTP were less inhibitory. The inhibition was competitive with respect to GDP or IDP, but not with respect to PEP. In the direction of PEP-synthesis, the enzyme was not inhibited, but rather activated by ATP.     

Interaction of horse cytochromec with the photosynthetic reaction center ofRhodospirillum rubrum

Mitochondrial cytochromec (horse), which is a very efficient electron donor to bacterial photosynthetic reaction centersin vitro, binds to the reaction center ofRhodospirillum rubrum with an approximate dissociation constant of 0.3–0.5 µM at pH 8.2 and low ionic strength. The binding site for the reaction center is on the frontside of cytochromec which is the side with the exposed heme edge, as revealed by differential chemical acetylation of lysines of free and reaction-center-bound cytochromec. In contrast, bacterial cytochromec ₂ was found previously to bind to the detergent-solubilized reaction center through its backside, i.e., the side opposite to the heme cleft [Rieder, R., Wiemken, V., Bachofen, R., and Bosshard, H. R. (1985).Biochem. Biophys. Res. Commun. 128, 120–126]. Binding of mitochondrial cytochromec but not of mitochondrial cytochromec ₂ is strongly inhibited by low concentrations of poly-l-lysine. The results are difficult to reconcile with the existence of an electron transfer site on the backside of cytochromec ₂.     

Use of 8-analino-1-naphthalene sulfonate as a monitor for possible phase transition involving water at low temperatures in photoreaction center from Rhodospirillum rubrum

The increase in the rate of the primary back reaction on cooling the photoreaction center from Rhodospirillum rubrum was interpreted in terms of a model in which the peculiar temperature dependence of the rate results from a phase transition involving water. The primary back reaction is defined as the return of the electron from the reduced primary ubiquinone to the oxidized bacteriochlorophyll molecules following illumination. The dye 8-anilino-1-naphthalene sulfonate was used to detect the state of the water solvent as it transforms on cooling from a liquid to a solid glass. We inferred from studies with air-dried films of photoreaction center that the water which may be responsible for the unusual temperature dependence of the rate of the primary back reaction is not on the surface but is bound within the photoreaction center protein.     

A comparison of electron transport and photophosphorylation systems of Rhodopseudomonas capsulata and Rhodospirillum rubrum

The photophosphorylation systems of Rhodopseudomonas capsulata and Rhodospirillum rubrum chromatophores have been compared in respect to the effects of artificial electron carries [N-methyl-phenazonium methosulfate (PMS) and diaminodurene], reducing agents (ascorbate in particular), and various quinones in the absence and presence of the electron transport inhibitors antimycin A and dibromothymoquinone (DBMIB). In addition, the effects of both inhibitors on photosynthetic electron transport through cytochromes b and c has been followed. From the results obtained, it appears that in both organisms: a) ubiquinone functions as an electron carrier between the cytochromes, and b) both antimycin A and DBMIB inhibit cyclic electron flow in the segment ... cytochrome bubiquinone»cytochrome c ..., but at different sites. The systems apparently differ mainly in respect to the nature of the electron flow by-pass shunt that is evoked in the presence of PMS; thus, in R. rubrum, PMS catalyzes a shunt that by-passes both cytochrome b and ubiquinone, whereas in Rps. capsulata the PMS shunt seems to circumvent only ubiquinone.Abbreviations BChl bacteriochlorophyll - DAD diaminodurene=2,3,5,6-tetramethyl-p-phenylenediamine - DBMIB dibromothymoquinone=2,5-dibromo-6-isopropyl-3-methylbenzoquinone - HOQNO heptylhydroxyquinoline-N-oxide - PMS N-methylphenazonium methosulfate     

The effect of dibromothymoquinone on respiratory and photosynthetic electron transport in Rhodopseudomonas capsulata chromatophores

Dibromothymoquinone has been shown to inhibit light-induced cytochrome b reduction, and oxidation of succinate and NADH by chromatophores of Rhodopseudomonas capsulata. The half-inhibitory concentration of light-induced reactions and NADH oxidation is 2.5 M, but of succinate oxidation is 16 M. Hexane extraction inhibited oxidation of NADH and succinate equally. The results are interpreted to suggest that ubiquinone is concerned in all three processes described, but that the pools associated with NADH and succinate oxidation are not equally accessible to dibromothymoquinone.Abbreviations DBMIB Dibromothymoquinone - NADH Reduced nicotinamide adenine dinucleotide - Bchl Bacteriochlorophyll     

Amino acid sequence of ferredoxin II from the phototroph Rhodospirillum rubrum: Characteristics of a 7Fe ferredoxin

The complete sequence of amino acids of ferredoxin II (FdII) from Rhodospirillum rubrum was determined by repetitive Edman degradation using pyridylethylated-ferredoxin and oxidized, denatured ferredoxin. Peptides derived from trypsin, pepsin, Glu-C endoproteinase, Arg-C endoproteinase, tryptophan specific cleavage and partial acid hydrolysis and C-terminal sequence from carboxypeptidase digestion were used to construct the total sequence. RrFdII is a polypeptide of 104 amino acids having a calculated molecular weight of 11556 excluding the iron and sulfur atoms. The complete amino acid sequence was: PYVVTENCIKCKYQDCVEVCPVDCFYEGENFLVINPDECIDCGVCNPECPAEAIAGKWLEINRKFADLWPNITRKGPAL ADADDWKDKPDKTGLLSENPGKGTV. Sequence comparisons, EPR characteristics and iron analyses indicate that RrFdII has structural features in common with ferredoxins containing [3Fe-4S], [4Fe-4S] centers. Of 104 amino acids, 60 (58%) including all 9 cysteines, are found in identical locations in the 7Fe ferredoxin prototype, Azotobacter vinelandii FdI.The protein sequence data reported in this paper will appear in the SWISS-PROT database and EMBL Data Library under the accession number P80448.     

The antenna system of Rhodospirillum rubrum. Detection of macromolecular constituents not stainable by Coomassie brilliant blue in solubilized preparations of the B880 complex

A solubilized preparation of the major Rhodospirillum rubrum antenna complex (B880) was obtained by a described procedure and its polypeptide composition was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Only two polypeptides of molecular weights close to 7000 were detected after staining the gels with Coomassie brilliant blue. However, several other constituents could be visualized by silver staining or by an immunochemical method. When the preparation was chromatographed on Sephacryl, some of the resulting fractions exhibited the characteristic B880 absorption spectrum and contained only the two proteins that were detectable with Coomassie brilliant blue. In those fractions the A ₂₈₀/A ₈₈₀ratio was 0.4, which indicated a significant improvement of the bacteriochlorophyll to protein ratio over the unchromatographed preparation (A ₂₈₀/A ₈₈₀=0.7). Other chromatography fractions lacked bacteriochlorophyll and contained a carotenoid which seemed to be bound to protein. The macromolecular constituents present in these latter fractions differed from those associated to the purified B880 complex in their electrophoretic moblities and/or in their staining properties. That suggested the possible existence of a carotenoprotein that did not result from the B880 complex upon loss of bacteriochlorophyll.     

Evidence of an essential carboxyl residue in membrane-bound pyrophosphatase ofRhodospirillum rubrum

Chemical modifications with water-soluble carbodiimides (EDC and CMC) were performed to elucidate whether some carboxyl residues are involved in the catalytic activity of membrane-bound pyrophosphatase ofRhodospirillum rubrum. EDC and CMC cause a loss of hydrolytic activity following pseudo-first-order kinetics up to 10 min of reaction. The enzyme was completely protected against EDC inhibition by PPi or Mg²⁺, whereas PPi or Mg²⁺ gave partial protection against CMC inactivation. Mg-PPi protected completely against the inhibition caused by both carbodiimides. These data suggest that the carboxyl moiety modified by EDC is at the active site. At longer times of inactivation with both carbodiimides, we could not observe a linear relationship in semilogarithmic plots of residual activity versus time, indicating that at least two carboxyls are involved in the inactivation, which correlates with the partial protection against CMC inactivation by PPi. We found that the activator site for Mg²⁺ is apparently at or near the active site of the enzyme. This is supported by the fact that PPi protects completely the activator effect of this divalent cation.     

Purification and properties of the carbonic anhydrase of Rhodospirillum rubrum

The carbonic anhydrase (EC 4.2.1.1) of Rhodospirillum rubrum has been purified to apparent homogeneity and some of its properties have been determined. The enzyme was cytoplasmic and was found only in photosynthetically grown cells. It had a molecular weight of about 28,000, and was apparently composed of two equal subunits. The amino acid composition was similar to that of other reported carbonic anhydrases except that the R. rubrum enzyme contained no arginine. The isoelectric point of the enzyme was 6.2 and the pH optimum was 7.5. It required Zn(II) for stability and enzymatic activity. The K _m(CO₂) was 80 mM. Typical carbonic anhydrase inhibition patterns were found with the R. rubrum enzyme. Strong acetazolamide and sulfanilamide inhibition confirmed the importance of Zn(II) for enzymatic activity as did the anionic inhibitors iodide, and azide. Other inhibitors indicated that histidine, sulfhydryl, lysine and serine residues were important for enzymatic activity.Abbreviation CA carbonic anhydrase In memory of R. Y. Stanier     

Regulation of divalent cations of the membrane-bound pyrophosphatase of Rhodospirillum rubrum,as shown by the hydrolysis of tripositive-pyrophosphate complexes

Tripositive-pyrophosphate [M(III)-PPi] complexes were used to investigate the role of free divalent cations on the membrane-bound pyrophosphatase. Divalent cations remain free and the M(III)-PPi complexes were employed as substrates. Formation of a La-PPi complex was studied by fluorescence, and the fact that Zn²⁺ and Mg²⁺ remain free in the solution was validated. Hydrolysis of La-PPi is stimulated by the presence of fixed concentrations of free Mg²⁺ or Zn²⁺ and this stimulation depends on the concentration of the cations when the La-PPi complex is fixed. The divalent cation stimulation order is Zn²⁺ > Co²⁺ > Mg²⁺ > Mn²⁺ > Ca²⁺ (at 0.5 mm of free cation). With different M(III)-PPi complexes, Zn²⁺ produces the same K _m, for all the complexes and Mg²⁺ stimulates with a different K _m. The results suggest that both Mg²⁺ and Zn²⁺ activate the membrane-bound pyrophosphatase but through different mechanisms.     

Studies of the adenylate and pyridine nucleotide pools during nitrogenase ‘switch-off’ inRhodospirillum rubrum

Summary When ammonium ions are added to a nitrogen fixing culture ofRhodospirillum rubrum, nitrogenase activity decreases due to inactivation of the Fe-protein. We have studied the adenylate and pyridine nucleotide pools during switch-off using the sensitive bioluminescence method. Immediately after the addition of ammonium ions there is a decrease in the ATP pool which is quickly reversed and no change is seen during the switch-off period. The pyridine nucleotide pools also do not change significantly during the switch-off. Consequently we conclude that changes in the pools studied were not the signal promoting inactivation of the Fe-protein.