Ferric iron reductase of Rhodopseudomonas sphaeroides.

Partially digested chromosomal DNA of Bacillus brevis ATCC 9999, a producer of the cyclic peptide antibiotic gramicidin S, was ligated into the BamHI site of the Escherichia coli expression vector pUR2-Bam. The ligated molecules were used to transfer E. coli to ampicillin resistance. Of 5 X 10(3) colonies tested by in situ immunoassay for a cross-reaction with antibodies against the gramicidin S synthetase 2, 6 colonies were found to be immunoreactive. A clone designated MK2, which had a 3.9-kilobase insert of B. brevis DNA, directed in E. coli under the lac promoter control the synthesis of polypeptides that were cross-reactive with the antibody to the gramicidin S synthetase 2. Partial purification of the gene products by gel filtration revealed a major fraction with an approximate molecular weight of 140,000 and with specific ornithine-dependent ATP-32PPi and 2'-dATP-32PPi exchange activities. These unique activities of the gramicidin S synthetase 2 were not detected in the E. coli strain harboring the vector.     

Porin from Rhodopseudomonas sphaeroides

A protein homooligomer was purified from both the cell envelope fractions and the saline extracts of Rhodopseudomonas sphaeroides cells. This oligomer exhibited strong porin activity when reconstituted into proteoliposomes with egg phosphatidylcholine. In the saline extracts of both chemotrophically and phototrophically grown cells, the porin oligomer was the most predominant polypeptide, which produced pores whose behavior toward various sugars could be approximated by hollow cylinders of 0.62 nm in radius. The oligomer was dissociated, in the presence of EDTA, into monomers that migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as though their molecular weight was about 47,000. The monomer was active in the reconstitution assay and produced pores with sizes comparable to those produced by the oligomer. Circular dichroism spectra indicated the predominance of beta-sheet structure in both the oligomeric and EDTA-dissociated monomeric forms. Drastic conditions, for example, precipitation with 10% trichloroacetic acid or heating for a few hours at 100 degrees C in sodium dodecyl sulfate, were necessary to denature the protein into a form with a reduced content of beta-sheet structure.     

Nontoxic lipopolysaccharide from Rhodopseudomonas sphaeroides ATCC 17023.

Chemical analysis of the lipopolysaccharide from Rhodopseudomonas sphaeroides ATCC 17023, isolated by the phenol-chloroform-petroleum ether method, revealed the presence of glucuronic acid, 2-keto-3-deoxyoctonate, threonine, and phosphorus in the polysaccharide moiety. The lipid A component contained glucosamine, glucosamine phosphate, amide-bound 3-oxotetradecanoic acid and 3-hydroxytetradecanoic acid, and ester-bound 3-hydroxydecanoic acid and 7-tetradecenoic acid. Structural similarity of the lipid A from R. sphaeroides ATCC 17023 to enterobacterial lipid A is indicated by the existence of a serological cross-reaction occurring between the lipid A from R. sphaeroides ATCC 17023 and that from Salmonella minnesota R595. The lipopolysaccharide and lipid A of R. sphaeroides, however, were found to be neither toxic in mice nor pyrogenic in rabbits.     

Soluble Dissimilatory Nitrate Reductase Containing Cytochrome c from a Photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

Dissimilatory nitrate reductase [nitrite: (acceptor) oxidoreductase.EC 1.7.99.4 [EC] ] from a denitrifying photosynthetic bacterium, Rhodopseudomonassphaeroides forma sp. denitrificans proved to be a soluble enzymethat could be purified 47-fold. It was labile, and containedcytochrome c, based on the results of specific staining forheme on polyacrylamide gel electrophoresis and on its absorptionspectrum. Its physiological molecular weight was determinedto be 112k, although heterogeneous molecular weights of 112k,100k, 73k and 60k were found for different preparations. Theoptimum for enzyme activity was about pH 6, and the K_m for thenitrate was 1.6 mM. As an electron donor, benzyl viologen wasvery good; but NADH, NADPH, FAD, FMN, cytochromes b₂ and c₂,dichlorophenolindophenol and phenazine methosulfate were noteffective. Bathophenanthroline and thiocyanate inhibited enzymaticactivity. The addition of 1 mM tungstate to the growing culturein place of molybdate decreased the nitrate reductase in thecells, but a further addition of 1 mM molybdate stopped it.This nitrate reductase is believed to be a molybdo-iron proteinsimilar to the enzymes from other bacteria with a nitrate respiratingability. (Received February 29, 1980; Accepted January 29, 1981)     

Delayed fluorescence from Rhodopseudomonas sphaeroides following single flashes.

Delayed fluorescence from Rhodopseudomonas sphaeroides chromatophores was studied with the use of short flashes for excitation. Although the delayed fluorescence probably arises from a back-reaction between the oxidized reaction center bacteriochlorophyll complex (P+) and the reduced electron acceptor (X-), the decay of delayed fluorescence after a flash is much faster (tau1/2 approximately 120 mus) than the decay of P+X-. The rapid decay of delayed fluorescence is not due to the uptake of a proton from the solution, nor to a change in membrane potential. It correlates with small optical absorbance changes at 450 and 770 nm which could reflect a change in the state of X-. The intensity of the delayed fluorescence is 11-18-fold greater if the excitation flashes are spaced 2 s apart than it is if they are 30 s apart. The enhancement of delayed fluorescence at high flash repetition rates occurs only at redox potentials which are low enough (less than +240 mV) so that electron donors are available to reduce P+X- to PX- in part of the reaction center population. The enhancement decays between flashes as PX- is reoxidized to PX, as measured by the recovery of photochemical activity. Evidently, the reduction of P+X- to PX- leads to the storage of free energy that can be used on a subsequent flash to promote delayed fluorescence. The reduction of P+X- also is associated with a carotenoid spectral shift which decays as PX- is reoxidized to PX. Although this suggests that the free energy which supports the delayed fluorescence might be stored as a membrane potential, the ionophore gramicidin D only partially inhibits the enhancement of delayed fluorescence. With widely separated flashes, gramicidin has no effect on delayed fluorescence. At redox potentials low enough to keep X fully reduced, delayed fluorescence of the type described above does not occur, but one can detect weak luminescence which probably is due to phosphorescence of a protoporphyrin.     

Glycerol dissimilation in Rhodopseudomonas sphaeroides.

Rhodopseudomonas sphaeroides followed a diauxic growth curve when grown on a malate-glycerol medium, the first phase of growth being supported by malate and the second by glycerol. A soluble glycerokinase and a particulate, pyridine nucleotide-independent glycerophosphate dehydrogenase, were induced by the presence of glycerol in the medium, but neither was fully expressed nor functional until all malate had been consumed.     

Fusion of chromatophores derived from Rhodopseudomonas sphaeroides

Fusion of chromatophores, the photosynthetic membrane vesicles isolated from the intracytoplasmic membranes of Rhodopseudomonas sphaeroides, was achieved by the use of poly(ethylene glycol) 6000 as fusogen. Ultracentrifugation, electron microscopy, intrinsic density and isotope labeling were used to demonstrate chromatophore fusion. Although studies of the flash-induced shift in the carotenoid absorbance spectrum indicated that the membrane was rendered leaky to ions by either the fusion procedure or the increased size of the fused products, the orientation and integrity of fused chromatophores were otherwise demonstrated to be identical to control chromatophores by freeze-fracture electron microscopy, proteolytic enzyme digestion, enzymatic radioiodination, and transfer of chromatophore phospholipids mediated by phospholipid exchange protein extracted from Rps. sphaeroides.     

Crossed immunoelectrophoretic analysis of chromatophore membranes from Rhodopseudomonas sphaeroides.

Triton extracts of intracytoplasmic photosynthetic membranes (chromatophores) purified from Rhodopseudomonas sphaeroides were subjected to crossed immunoelectrophoresis with antiserum raised in rabbits to purified chromatophores. A total of 31 immunoprecipitates was visualized; 2 of the immunoprecipitates were identified as reduced nicotinamide adenine dinucleotide (EC 1.6.99.3) and L-lactate dehydrogenases by enzyme staining techniques. Reaction with a monospecific antiserum identified the photochemical reaction center. Photopigments were associated with a major precipitate in the pattern which was identified on the basis of immunological identity as light-harvesting bacteriochlorophyll a . protein complex. These results provide the basis for a detailed structural and functional analysis of the chromatophore membrane by crossed immunoelectrophoresis.     

Characterization of a site-specific restriction endonuclease from Rhodopseudomonas sphaeroides.

A type II restriction endonuclease, RshI, has been partially purified from photoheterotrophically grown Rhodopseudomonas sphaeroides strain 2.4.1. The enzyme preparation, after a single DE-52 column fractionation, is free of 5' exonuclease and phosphatase activities but contains a trace of 3' exonuclease activity. Based upon deoxyribonucleic acid (DNA) sequencing data in the vicinity of the enzyme-promoted cleavage of pBR322 DNA, we have concluded that RshI probably recognizes the palinodromic hexanucleotide sequence 5'-CGATCG-3' and cleaves between the T and C. lambda cI857 DNA contains three RshI sites, two of which lie in the replaceable region. The plasmid pBR322, which carries resistances to ampicillin and tetracycline, contains a single RshI site in the ampicillin resistance determinant. Insertion of DNA into the RshI site of pBR322 results in loss of ampicillin resistance but retention of tetracycline resistance, thereby providing a convenient screening procedure for recombinant plasmids.     

Electron Transport Coupled to Nitrate Reduction in a Photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

The electron transport system involved in nitrate reductionand its relationship to photosynthetic cyclic electron transportin a photodenitrifier, Rhodopseudomonas sphaeroides forma sp.denitrificans, were studied. Nitrate oxidized only b-type cytochromein the presence of cyanide, which inhibits nitrite reductase.Heptylhydroxyquinoline-N-oxide (HOQNO) inhibited the oxidationof b-type cytochrome by nitrate, but not the oxidation of b-and c-type cytochrome by nitrite. The inhibition by HOQNO wasovercome by phenazine methosulfate (PMS). Absorption changesof b-type cytochrome induced by illumination were in just theopposite directions for oxygen- and nitrate-oxidized cells;the cytochrome was reduced in oxygen-oxidized cells and oxidizedin nitrate-oxidized cells. Antimycin enhanced the reductionand inhibited the oxidation, but had no inhibitory effect onthe oxidation of b-type cytochrome by nitrate. Dithionite-reducedminus ferricyanide-oxidized difference spectra of cells at 77?Kshowed two b-type cytochrome components with a bands at 556.5and 562 nm. The proportion of the b-562 component decreasedin cells grown under denitrifying conditions. It was concludedthat a b-type cytochrome is involved in the nitrate reduction.The b-type cytochrome was presumed to be an alternative to thecytochrome b in the photosynthetic cyclic electron transport. ¹ Present address: Japanese Red Cross Tokyo-to Komagome BloodCenter, Komagome 2-2-2, Toshima-ku, Tokyo 170, Japan. (Received August 13, 1981; Accepted December 5, 1981)     

Regulatory properties of the ADPglucose pyrophosphorylase from Rhodopseudomonas sphaeroides and from Rhodopseudomonas gelatinosa

Highly purified fractions of chlorosomes and cytoplasmic membranes were isolated from Chloroflexus aurantiacus Ok-70-fl and Chlorobium limicola 6230. These fractions were comparatively analyzed for their pigmentation, phospholipid, glycolipid, and cytochrome c content as well as for their specific activities of succinate dehydrogenase and NADH-oxidase. The data showed that there are some differences in pigmentation and phospholipid content between the isolated fractions of Chloroflexus and Chlorobium. Chlorosomes of Chloroflexus contained a specific BChl a-complex with a characteristic absorption maximum at about 790 nm. This BChl a-complex could not be detected in spectra of chlorosomes from Chlorobium. The near infrared region of the spectra of the isolated cytoplasmic membranes of both organisms revealed considerable differences: The BChl a-complexes of Chloroflexus membranes exhibited peaks at 806 and 868 nm whereas the membranes of Chlorobium had a single BChl a-peak at 710 nm. In contrast to the findings with Chlorobium the chlorosomes of Chloroflexus contained at least twice as much phospholipids as did the cytoplasmic membranes. In Chlorobium the phospholipid content of cytoplasmic membranes is three times that of their chlorosomes. The distribution of all other components (carotenoid composition, enzyme activities, cytochrome c content, and glycolipids) was about the same in both strains. From the data it was concluded that differences in the organization of the photosynthetic apparatus are mainly based on differences of the organization of the photosynthetic units in the cytoplasmic membrane and probably the kind of linkage of the light harvesting system in the chlorosomes with the reaction center in the cytoplasmic membranes.Abbreviations BChl c bacteriochlorophyll c - BChl a bacteriochlorophyll a - DSM Deutsche Sammlung von Mikrorganismen     

Rhodopseudomonas sphaeroides forma sp. denitrificans,a denitrifying strain as a subspecies of Rhodopseudomonas sphaeroides

From polluted water of a lagoon pond a new type of denitrifying photosynthetic purple bacteria was isolated. With respect to morphology, fine structure, photopigments, requirement for growth factors, the range of utilization of organic substrates for phototrophic growth and DNA base ratio, the denitrifying strains show the closest resemblance to Rhodopseudomonas sphaeroides and were therefore described as a subspecies named R. sphaeroides forma sp. denitrificans. The new isolates grow well with nitrate anaerobically in the dark accompanying the evolution of nitrogen gas. They cannot assimilate nitrate as the nitrogen source for growth.     

Denitrification in Rhodopseudomonas sphaeroides f. denitrificans

Rhodopseudomonas sphaeroides f. denitrificans grown photosynthetically with NO ₃ ^- under anaerobic conditions accumulated NO ₂ ^- in the culture medium. In washed cells succinate, lactate, fumarate, citrate and malate, were effective electron donors for the reduction of NO ₃ ^- , NO ₂ ^- and N₂O to N₂ gas. Nitrate reductase was inhibited by amytal and potassium thocyanate. Nitrite reductase activity was severely restricted by potassium cyanide, sodium diethyldithiocarbamate, Amytal and 2-n-heptyl-4-hydroxyquinoline-N-oxide whereas N₂O reductase was inhibited by NaN₃, C₂H₂ and KCNS. Cells incubated with either K¹⁵NO₃ or K¹⁵NO₂ produced ¹⁵N₂O and ¹⁵N₂. A stoichiometry of 2:1 was recorded for the reduction of either NO ₃ ^- or NO ₂ ^- to N₂O and N₂ and for N₂O to N₂ it was 1:1.Abbreviations BVH reduced benzyl viologen - MVH reduced methyl viologen - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - CCCP carbonyl cyanide-m-chlorophenyl-hydrazone - DIECA diethyl dithiocarbamate - KCN potassium cyanide     

The carotenoid band shift in reaction centers from from Rhodopseudomonas sphaeroides.

A specific carotenoid associated with reaction centers purified from Rhodopseudomonas sphaeroides shows an optical absorbance change in response to photochemical activity, at temperatures down to 35 K. The change corresponds to a bathochromic shift of 1 nm of each absorption band. The same change is induced by either chemical oxidation or photo-oxidation of reaction center bacteriochlorophyll (P-870). Reduction of the electron acceptor of the reaction center, either chemically or photochemically, does not cause a carotenoid absorbance change or modify a change already induced by oxidation of P-870. The change of the carotenoid spectrum can therefore be correlated with the appearance of positive charge in the reaction center. In these studies we observed that at 35 K the absorption band of reaction center bacteriochlorophyll near 600 nm exhibits a shoulder at 605 nm. The resolution into two components is more pronounced in the light-dark difference spectrum. This observation is consistent with our earlier finding, that the "special pair" of bacteriochlorophyll molecules that acts as photochemical electron donor has a dimer-like absorption spectrum in the near infrared.     

Purification and properties of dimethylsulfoxide reductase containing a molybdenum cofactor from a photodenitrifier, Rhodopseudomonas sphaeroides f.s. denitrificans

Dimethylsulfoxide (DMSO) reductase was purified to electrophoretic homogeneity from the periplasmic fraction of a photodenitrifier, Rhodopseudomonas sphaeroides f.s. denitrificans. The enzyme had a molecular weight of 82,000 and had no subunit. It contained 1 mol of molybdenum per mol of enzyme, but iron and acid-labile sulfur were not present. The UV-visible spectrum showed only one absorption maximum at 280 nm. Denaturation of the enzyme released a molybdopterin cofactor, the fluorescence spectra of which were almost the same as those of a form B derivative of molybdopterin found in formate dehydrogenase. The Km value for DMSO was 15 microM, which was much lower than that for trimethylamin-N-oxide (TMAO), whereas Vmax with TMAO was larger than that with DMSO.     

RsaI: a new sequence-specific endonuclease activity from Rhodopseudomonas sphaeroides.

A new type II sequence-specific endonuclease, RsaI, has been identified from Rhodopseudomonas sphaeroides strain 28/5. An RsaI purification scheme that yields enzyme which is free of contaminating exonuclease and phosphatase activities after a single column fractionation has been developed. The enzyme recognized the tetranucleotide sequence 5'-GTAC-3' and cleaved between the T and A, thereby generating flush ends. RsaI should be extremely useful in deoxyribonucleic acid sequencing experiments.     

Bacteriochlorophyll a-types in chromatophore and subchromatophore preparations from Rhodopseudomonas sphaeroides.

Comparison of absorption and circular dichroism (CD) spectra in the near infrared region was made with chromatophore and subchromatophore preparations obtained from Rhodopseudomonas sphaeroides. The 850 nm absorption band had a positive correlation with the 850 nm and 870 nm CD bands. The 800 nm and 870 nm absorption bands seemed not to correlate with any CD bands. Lipid contents in chromatophores and subchromatophores were measured. Lipids in membranes seemed to contribute to the appearance of the 870 nm absorption band, but not to that of the 800 nm and 850 nm absorption bands. The time courses of absorbance changes were compared at 800, 850, and 870 nm in detergent-treated chromatophores. Relative changes of absorbances differed from one another. The present results suggest that the three absorption bands are due to three different bacteriochlorophyll a-types and the 850 nm absorption band originates from exciton-coupling of bacteriochlorophyll a.     

Lipid-protein associations in chromatophores from the photosynthetic bacterium Rhodopseudomonas sphaeroides.

Lipid-protein interactions were examined in chromatophores isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides using lipid spin-labels. The chromatophores contain fluid bilayer and a significant amount of lipid immobilized by membrane proteins. For a typical preparation of cells grown under 600 ft-c illumination, 59% of the spin-labeled fatty acids were bound. Essentially the entire length of the 18-carbon fatty acid chain was immobilized, judging from results obtained with the spin-label at the 7, 12, and 16 positions. The amount immobilized varies directly with the bacteriochlorophyll content of the chromatophore material, suggesting that a significant fraction of the lipid spin-labels is immoblized on the hydrophobic surfaces of the chlorophyll-binding proteins. Changing the lipid spin-label head group from a negatively charged carboxyl group to a positively charged quarternary amine greatly decreased the amount of immobilized lipid. The changes in immobilized lipid with light level and polar head group suggest that the anntenna bacteriochlorophyll-binding proteins preferentially associate with negatively charged lipids.     

Purification and properties of the adenylate kinases from Rhodopseudomonas palustris,Rhodopseudomonas sphaeroides and Rhodopseudomonas rubrum

The adenylate kinases (EC 2.7.4.3) from photosynthetically grown Rhodopseudomonas palustris, Rhodopseudomonas sphaeroides and Rhodospirillum rubrum were purified to homogeneity by the same procedure. The purified enzymes showed optimal rates of activity with MgCl₂ at 25° C and pH 8.0. They were found to be heat labile and were characterized by pI-values of 4.5. Apparent molecular weights of 33 500 for R. palustris, 34 400 for R. sphaeroides and 32 100 for R. rubrum were determined by high performance liquid chromatography. No separation into subunits was observed by use of sodium dodecylsulfate polyacrylamide gel electrophoresis. The apparent K _m -values for ADP corresponded to 0.26 mM for R. palustris, 0.27 mM for R. sphaeroides and 0.24 mM for R. rubrum. ADP in excess had a strong inhibitory effect. Competitive product inhibition was found for AMP, with K _i-values of 0.017 mM for R. palustris, 0.018 mM for R. sphaeroides and 0.014 mM for R. rubrum. A competitive inhibitor likewise was P¹,P⁵-di(adenosine-5)pentaphosphate with K _i-values of 0.020 M for R. palustris and R. sphaeroides, and 0.017 M for R. rubrum. Sulfhydryl-reacting reagents like p-chloromercuribenzoate and iodoacetic acid were found to be non-inhibitory. All measurements of adenylate kinase activity were carried out with the stabilized and most sensitive luciferin-luciferase system.     

Protein composition of Rhodopseudomonas sphaeroides outer membrane.

The outer membrane polypeptide profile of Rhodopseudmonas sphaeroides was characterized. Solubilization of the outer membrane at 75 or 100 degrees C as opposed to room temperature resulted in the dissociation of 75-, 72-, and 68-kilodalton (kdal) polypeptide aggregates into 29-, 26.5-, and 21.5-kdal polypeptides, respectively, and a shared 47-kdal subunit. Similarly, an 88.5-kdal polypeptide dissociates into a 45-kdal monomeric form, and the electrophoretic mobility of a 58.5-kdal polypeptide was altered to 83 kdal.Lysozyme treatment of outer membrane fractions altered the 21.5-kdal polypeptide mobility to 23 kdal. The presence of lipid in both the 47-kdal polypeptide and an 8- to 10-kdal polypeptide was demonstrated by lipid staining and [14C]acetate incorporation. The lipid component of the 47-kdal polypeptide was neither lipopolysaccharide nor phospholipid. The 8- to 10-kdal polypeptide may be the equivalent of the Braun lipoprotein. Outer membrane fractions isolated from R. sphaeroides-specific phage RS1-resistant mutants were deficient in several of the high-molecular-weight aggregates involving the 47-kdal polypeptide.