Photosynthetic Bacterium Rhodospirillum Rubrum Research Articles

Nucleotide sequence of cDNA clones encoding the beta subunit of mitochondrial ATP synthase from the green alga Chlamydomonas reinhardtii: the precursor protein encoded by the cDNA contains both an N-terminal presequence and a C-terminal extension.

cDNA and genomic clones encoding the beta subunit of mitochondrial ATP synthase from Chlamydomonas reinhardtii have been isolated using heterologous DNA probes from the photosynthetic bacterium Rhodospirillum rubrum. The protein encoded by the cDNA is 79-83% identical to corresponding proteins from higher-plant and mammalian mitochondria, and 75% identical to the R. rubrum protein. It contains both an N-terminal presequence and a unique C-terminal extension. The presequence, which is the first mitochondrial presequence determined in C. reinhardtii, is similar in structure to mitochondrial presequences from other organisms. As chloroplast presequences from C. reinhardtii also share features with mitochondrial presequences from other organisms (L.-G. Franzén et al., FEBS Lett 260 (1990) 165-168), this raises interesting questions about protein targeting to chloroplasts and mitochondria in C. reinhardtii. The possibility that the C-terminal extension is involved in targeting the protein to the mitochondrion is discussed. Southern blot analysis indicates that the protein is encoded by a single-copy gene.

Picosecond fluorescence spectroscope of light-harvesting antenna complexes from Rhodospirillum rubrum in the 300-4 K temperature range. Comparison with the data on chromatophores

Time- and spectrally-resolved picosecond fluorescence of light-harvesting antenna complexes B880, isolated from the membranes of the photosynthetic purple bacterium Rhodospirillum rubrum were studied over a wide temperature range from room temperature down to 4 K. At low temperatures, the kinetics are dependent on the fluorescence recording wavelength, analogous with the observation for chromatophores. The kinetics presumably reflect excitation transfer between different antenna complexes in the spectrally inhomogeneous antenna. An excitation lifetime in an antenna complex of the order of 10 ps, which is insensitive to temperature, was measured. This time seems to be characteristic of the “macroscopic” light excitation transfer in a broader class of photosynthetic systems.

Photosynthesis and photorespiration in a mutant of the cyanobacterium Synechocystis PCC 6803 lacking carboxysomes

A mutant of the cyanobacterium Synechocystis PCC 6803 was obtained by replacing the gene of the carboxylation enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) with that of the photosynthetic bacterium Rhodospirillum rubrum. This mutant consequently lacks carboxysomes - the protein complexes in which the original enzyme is packed. It is incapable of growing at atmospheric CO2 levels and has an apparent photosynthetic affinity for inorganic carbon (Ci) which is 1000 times lower than that of the wild type, yet it accumulates more Ci than the wild type. The mutant appears to be defective in its ability to utilize the intracellular Ci pool for photosynthesis. Unlike the carboxysomal carboxylase activity of Rubisco, which is almost insensitive to inhibition by O2 in vitro, the soluble enzyme is competitively inhibited by O2. The photosynthetic rate and Ci compensation point of the wild type were hardly affected by low O2 levels. Above 100 μM O2, however, both parameters became inhibited. The CO2 compensation point of the mutant was linearly dependent on O2 concentration. The higher sensitivity of the mutant to O2 inhibition than that expected from in-vitro kinetics parameters of Rubisco, indicates a low capacity to recycle photorespiratory metabolites to Calvin-cycle intermediates.

Mutation of asparagine 111 of rubisco from Rhodospirillum rubrum alters the carboxylase/oxygenase specificity

The conserved asparagine 111 of ribulose-1,5-bisphosphate carboxylase/oxygenase from the photosynthetic bacteria Rhodospirillum rubrum was identified as a candidate for a side-chain that might be involved in the carboxylase/oxygenase specificity. It was replaced by site-directed mutagenesis with aspartic acid, leucine, glutamine or glycine residues. The mutant enzymes exhibit a very low carboxylase activity compared with the wild-type enzyme. The values of K m(RuBp) and k cat for Asn111 → Gly, the most active mutant, are 420 μ m and 0.034 s −1, compared with 13 μ m and 3.0 s −1 for wild-type. The mutation of Asn111 → Gly causes a more than tenfold decrease in the CO 2 O 2 specificity factor, τ, τ Asn111 → Gly = 0.56 and τ wlld-type = 6.7. This is the first reported change in rubisco specificity by a single site-directed mutation alone and suggests a target for future protein engineering studies.

Characterization of the pet operon of Rhodospirillum rubrum

The three genes of the pet operon, coding, respectively, for the Rieske iron-sulfur protein, cytochrome b and cytochrome c 1 components of the cytochrome bc 1 complex in the photosynthetic bacterium Rhodospirillum rubrum have been sequenced. The amino acid sequences deduced for these three peptides from the nucleotide sequences of the genes have been confirmed, in part, by direct sequencing of portions of the three peptides separated from a sample of the purified, detergent-solubilized complex. These sequences show considerable homology with those previously obtained for the pet operons of other photosynthetic bacteria. Northern blots of R. rubrum mRNA have established that the operon is transcribed as a single polycistronic message, the start site of which has been determined by both primer extension and nuclease protection. Photosynthetic growth of R. rubrum was shown to be inhibited by antimycin A, a specific inhibitor of cytochrome bc 1 complexes, and antimycin A-resistant mutants of R. rubrum have been isolated. Preliminary results suggest that it may be possible to express the R. rubrum pet operon in a strain of the photosynthetic bacterium Rhodobacter capsulatus from which the native pet operon has been deleted.

Simple Determination of the CO2/O2 Specificity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by the Specific Radioactivity of [14C]Glycerate 3-Phosphate

A new method is presented for measurement of the CO(2)/O(2) specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The [(14)C]3-phosphoglycerate (PGA) from the Rubisco carboxylase reaction and its dilution by the Rubisco oxygenase reaction was monitored by directly measuring the specific radioactivity of PGA. (14)CO(2) fixation with Rubisco occurred under two reaction conditions: carboxylase with oxygenase with 40 micromolar CO(2) in O(2)-saturated water and carboxylase only with 160 micromolar CO(2) under N(2). Detection of the specific radioactivity used the amount of PGA as obtained from the peak area, which was determined by pulsed amperometry following separation by high-performance anion exchange chromatography and the radioactive counts of the [(14)C]PGA in the same peak. The specificity factor of Rubisco from spinach (Spinacia oleracea L.) (93 +/- 4), from the green alga Chlamydomonas reinhardtii (66 +/- 1), and from the photosynthetic bacterium Rhodospirillum rubrum (13) were comparable with the published values measured by different methods.

Electrostatic fields at the active site of ribulose-1,5-bisphosphate carboxylase.

A macroscopic approach has been employed to calculate the electrostatic potential field of nonactivated ribulose-1,5-bisphosphate carboxylase and of some complexes of the enzyme with activator and substrate. The overall electrostatic field of the L2-type enzyme from the photosynthetic bacterium Rhodospirillum rubrum shows that the core of the dimer, consisting of the two C-terminal domains, has a predominantly positive potential. These domains provide the binding sites for the negatively charged phosphate groups of the substrate. The two N-terminal domains have mainly negative potential. At the active site situated between the C-terminal domain of one subunit and the N-terminal domain of the second subunit, a large potential gradient at the substrate binding site is found. This might be important for polarization of chemical bonds of the substrate and the movement of protons during catalysis. The immediate surroundings of the activator lysine, K191, provide a positive potential area which might cause the pK value for this residue to be lowered. This observation suggests that the electrostatic field at the active site is responsible for the specific carbamylation of the epsilon-amino group of this lysine side chain during activation. Activation causes a shift in the electrostatic potential at the position of K166 to more positive values, which is reflected in the unusually low pK of K166 in the activated enzyme species. The overall shape of the electrostatic potential field in the L2 building block of the L8S8-type Rubisco from spinach is, despite only 30% amino acid homology for the L-chains, strikingly similar to that of the L2-type Rubisco from Rhodospirillum rubrum. A significant difference between the two species is that the potential is in general more positive in the higher plant Rubisco. In particular, the second phosphate binding site has a considerably more positive potential, which might be responsible for the higher affinity for the substrate of L8S8-type enzymes. The higher potential at this site might be due to two remote histidine residues, which are conserved in the plant enzymes.

Purification and partial characterization of glutamate synthase from Rhodospirillum rubrum grown under nitrogen-fixing conditions

Glutamate synthase, a key enzyme in ammonia assimilation, has been purified from the photosynthetic bacterium Rhodospirillum rubrum. The purification procedure involves ion-exchange chromatography, affinity chromatography and gel filtration. The recovery in the procedure is high (62%) and the specific activity is 21 mumol of NADPH oxidized/min per mg. The enzyme is specific for its substrates, and no activity was demonstrated with NADH or NH4+ ions substituting for NADPH and glutamine respectively. The enzyme is composed of two dissimilar subunits with molecular masses of 53 and 152 kDa, and it is shown that Cl- ions have an effect on the aggregation of the enzyme. Km values for the substrates are: NADPH, 16 microM; 2-oxoglutarate, 10 microM; and glutamine, 65 microM. The enzyme is inhibited by amidotransferase inhibitors at micromolar concentrations. The role of the enzyme in the metabolic regulation of nitrogenase is discussed.

Purification and partial characterization of a pyruvate oxidoreductase from the photosynthetic bacterium Rhodospirillum rubrum grown under nitrogen-fixing conditions

A pyruvate oxidoreductase with the capacity to support pyruvate-dependent nitrogenase activity in vitro has been purified from the photosynthetic bacterium Rhodospirillum rubrum. The enzyme requires CoA for activity and is irreversibly inactivated by oxygen. The molecular properties and Km values for the substrates have been studied. In supporting nitrogenase activity addition of ferredoxin is required. Overall the enzyme is similar to the nif-specific pyruvate: flavodoxin oxidoreductase purified from Klebsiella pneumoniae.

Picosecond kinetics of light excitations in photosynthetic purple bacteria in the temperature range of 300-4 K

Comparative measurements of time- and spectrally resolved picosecond fluorescence of the photosynthetic purple bacterium Rhodospirillum rubrum in a wide temperature range from room temperature down to 4 K have been performed with different excitation wavelengths within the main infrared absorption band employed. Several kinetically different spectral components have been characterized in the fluorescence decay at low temperatures. This gives substantial support to the viewpoint that the B880 light-harvesting antenna band of R. rubrum (and probably the core antenna of other purple bacteria) consists of more than just two spectral forms, as has been suggested by several authors. The rates of the antenna singlet excitation quenching by reaction centres in different states have been determined. Although almost unchanged in the temperature range from 300 to 77 K, these rates decrease by 2 to 4 times on temperature lowering down to 4 K. It was found that at 4 K, the oxidized primary donor is a stronger fluorescence quencher than its triplet state.

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.

Exciton interactions in the light-harvesting antenna of photosynthetic bacteria studied with triplet-singlet spectroscopy and singlet-triplet annihilation on the B820 subunit form of Rhodospirillum rubrum

The pigment-protein complex, B820, isolated from the long-wavelength antenna (LH-1) of the photosynthetic purple bacterium Rhodospirillum rubrum was studied with polarized nanosecond laser spectroscopy. The polarized triplet (T)-singlet (S) spectrum was obtained at 77 K. The spectrum is significantly different from the T-S spectrum of monomeric BChl a , and can be explained by assuming that upon excitation the absorption band at 825 nm, which is due to a dimeric pair of BChl a molecules, disappears from the absorption spectrum, and is replaced by a monomer absorption band peaking at 809 nm. From the energy-dependence of the triplet yield, the high polarization of the bleaching, and the absence of singlet-triplet quenching we conclude that the B820 complex contains only one dimer of interacting BChl a molecules.

The requirement for Mn2+ and Ca2+ in nitrogen fixation by the photosynthetic bacterium Rhodospirillum rubrum

The role of Ca2+ and Mn2+ in Rhodospirillum rubrum grown under different conditions with respect to nitrogen source has been studied. The results show that this phototroph does not have an absolute requirement for these cations. In vitro studies of one of the enzymes operative in the metabolic regulation of nitrogenase in Rsp. rubrum have shown that Mn2+ or Fe2+ is required for activity. This investigation indicates that Mn2+ is not required in vivo for the function of this enzyme, suggesting that either Fe2+ is functional or that the enzyme has other properties when active in the cell.

Picosecond fluorescence of simple photosynthetic membranes: Evidence of spectral inhomogeneity and directed energy transfer

The picosecond time-domain singlet excitation transfer and trapping kinetics in photosynthetic membranes in case of low excitation intensities is studied by numerical integration of the appropriate master equation. The essential features of our two-dimensional-lattice random walk model are spectral heterogeneity of the light-harvesting antenna, inclusion of temperature effects, nonabsolute excitation trap, correlation between spectral and spatial parameters. A reasonably good agreement between theoretical and experimental fluorescence decay kinetics for purple photosynthetic bacterium Rhodospirillum rubrum is achieved only by assuming relatively large spectral inhomogeneity. From this comparison the average excitation lifetime on the lattice site is estimated to be 5–8 ps at the effective nearest neighbour lattice distance of 32 Å. If the model is correct, the relatively slow hopping rate determines that excitation transfer and trapping in R. rubrum at active photosynthesis conditions is a diffusion-limited process. The invariably present spectral disorder of photosynthetic systems promoting directed energy transfer serves for higher light-utilizing efficiency.

Cellular differentiation in the process of generation of the eukaryotic cell

Primitive atmosphere of the earth did not contain oxygen gas (O2) when the proto-cells were generated successfully as the result of chemical evolution and then evolved. Therefore, they first had acquired anaerobic energy metabolism, fermentation. The cellular metabolisms have often been formed by reorganizing to combine or recombinate between pre-existing metabolisms and newly born bioreactions. Photosynthetic metabolism in eukaryotic chloroplast consists of an electron-transfer photosystem and a fermentative reductive pentose phosphate cycle. On the other hand, O2-respiration of eukaryotic mitochondrion is made of Embden-Meyerhof (EM) pathway and tricarboxylic acid cycle, which originate from a connection of fermentative metabolisms, and an electron-transfer respiratory chain, which has been derived from the photosystem. These metabolisms already are completed in some evolved prokaryotes, for example the cyanobacterium Chlorogloea fritschii and aerobic photosynthetic bacteria Rhodospirillum rubrum and Erythrobacter sp. Therefore, it can be reasonably presumed that the eukaryotic chloroplast and mitochondrion have once been formed as the result of metabolic (and genetic) differentiations in most evolved cyanobacterium. Symbiotic theory has explained the origin of eukaryotic cell as that in which the mitochondrion and chloroplast have been derived from endosymbionts of aerobic bacterium and cyanobacterium, respectively, and has mentioned as one of the most potent supportive evidences that amino acid sequences of the photosynthetic and O2 -respiratory enzymes show similarities to corresponding prokaryotic enzymes. However, as will be shown in this discussion, many examples have shown currently that prokaryotic sequences of informative molecules are conserved well not only in those of the mitochondrial and chloroplast molecules but also in the nuclear molecules. In fact, the similarities in sequence of informative molecules are preserved well among the organisms not only in phylogenetically close relationships but also under highly selective pressure, that is under a physiological constraint for the species in their habitats. Therefore, the similarities in amino acid sequences of proteins between the prokaryotes and the organelles are not necessarily direct evidence for their phylogenetical closeness: it gives still less evidence for a symbiotic relationship between the prokaryotes and the organelles. The metabolic compartmentalization of the membranes is an important tendency in cellular evolution to guarantee high specificity and rate of the metabolisms. It is suggested from the data that the intracellular membranes are not static but undergo dynamic turnover. Furthermore, these facts strongly support the Membrane Evolution Theory which was proposed by one of the authors in 1975.

ATP-dependent and NAD-dependent modification of glutamine synthetase from Rhodospirillum rubrum in vitro.

Glutamine synthetase from the photosynthetic bacterium Rhodospirillum rubrum is the target of both ATP- and NAD-dependent modification. Incubation of R. rubrum cell supernatant with [alpha-32P]NAD results in the labeling of glutamine synthetase and two other unidentified proteins. Dinitrogenase reductase ADP-ribosyltransferase does not appear to be responsible for the modification of glutamine synthetase or the unidentified proteins. The [alpha-32P]ATP- and [alpha-32P] NAD-dependent modifications of R. rubrum glutamine synthetase appear to be exclusive and the two forms of modified glutamine synthetase are separable on two-dimensional gels. Loss of enzymatic activity by glutamine synthetase did not correlate with [alpha-32P]NAD labeling. This is in contrast to inactivation by nonphysiological ADP-ribosylation of other glutamine synthetases by an NAD:arginine ADP-ribosyltransferase from turkey erythrocytes (Moss, J., Watkins, P.A., Stanley, S.J., Purnell, M.R., and Kidwell, W.R. (1984) J. Biol. Chem. 259, 5100-5104). A 32P-labeled protein spot comigrates with the NAD-treated glutamine synthetase spot when glutamine synthetase purified from H3 32PO4-grown cells is analyzed on two-dimensional gels. The adenylylation site of R. rubrum glutamine synthetase has been determined to be Leu-(Asp)-Tyr-Leu-Pro-Pro-Glu-Glu-Leu-Met; the tyrosine residue is the site of modification.

Large‐scale purification procedure of spinach leaf mitochondria —isolation and immunological studies of the F1–ATPase

A large–scale purification procedure for mitochondria from spinach (Spinacia oteracea L, cv Medania) leaves is described. It involves differential centrifugation and density gradient centrifugation on a self–generating gradient of Percoll, From 3 kg of spinach leaves, 150 mg mitochondrial protein are obtained. The thylakoid contamination is lower than 0.2% on a chlorophyll basis. The mitochondria oxidize malate and glycine with state 3 rates of 108 and 140 nmol (mg protein)‐1 min‐1, with respiratory control ratios of 2,7 and 3,8 and ADP/O ratios of 2,0 and 2.1, respectively. The present large–scale purification procedure will facilitate further biochemical and molecular biological studies of leaf mitochondrial proteins.A pure and active catalytic moiety of the F1–ATPase (EC 3,6,1,3) was purified from the isolated mitochondria. The yield was 5 mg of F1–ATPase from 150 mg mitochondria. The F1–ATPase contained five polypeptides of apparent molecular mass 54 kDa (α), 52 kDa (β), 33 kDa (γ), 22 kDa (ω) and 11 kDa (ɛ). An additional component at 24 kDa was present in variable amounts in some preparations and was therefore not ascribed to the ATPase complex. The enzyme catalyzed ATP hydrolysis at a rate of 12.5 nmol (mg protein)‐1 min‐1. Antibodies against the spinach mitochondrial F1–ATPase cross–reacted only with the a and β subunits of F1–ATPases of spinach chloroplasts, photosynthetic bacteria Rhodospirillum rubrum and beef heart mitochondria.

Molecular cloning, sequencing and expression of cytochrome c2 from Rhodospirillum rubrum

Cytochrome c2 (Mr 12,840) of the purple photosynthetic bacterium Rhodospirillum rubrum functions as a mobile electron carrier in the cyclic photosynthetic electron-transport system of this organism. It acts as the electron donor to photochemically oxidized reaction centres and is reduced in turn by electrons from the cytochrome bc1 complex. By using synthetic oligonucleotides based on the known amino acid sequence of the protein, the structural gene (cycA) has been identified and isolated. DNA sequence analysis indicates the presence of a typical prokaryotic 23-residue signal sequence, suggesting that the protein is synthesized as a precursor which is processed during its secretion into the periplasm. Evidence is presented for the production of assembled cytochrome c2 in Escherichia coli, but recombinants grow poorly and are unstable, suggesting toxicity of the gene product in this organism.

Physical map of the cryptic 55 kilobase plasmid from the photosynthetic bacterium Rhodospirillum rubrum.

Physical map of the cryptic 55 kilobase plasmid from the photosynthetic bacterium Rhodospirillum rubrum.

Preliminary studies on the mechanisms of action of phosphonic analogues of morphactins on plants and bacteria

Two classes of phosphonopeptides, those containing P-terminal 9-aminofluoren-9-ylphos-phonic acid and those of dialkyl 9-aminofluoren-9-ylphosphine oxides, influence plant growth according to different mechanisms. The effect of these compounds on the growth of several bacterial species, including the photosynthetic bacteriumRhodospirillum rubrum, as well as on the activity of photosystems 1 and 2 in isolatedPisum sativum andSpirodela oligorrhiza chloroplasts was studied. The peptides of free, unblocked 9-aminofluoren-9-ylphosphonic acid acted in a morphactin-like manner, whereas those of dialkyl 9-aminofluoren-9-ylphosphine oxides influenced photosynthesis indirectly.