Immunochemical relationship of the major outer membrane protein of Rhodopseudomonas sphaeroides 2.4.1 to proteins of other photosynthetic bacteria.

Immunoblots of sodium dodecyl sulfate-polyacrylamide gels derived from outer membrane preparations of various strains of Rhodopseudomonas sphaeroides revealed polypeptides which cross-reacted with antibody directed against the major outer membrane protein of R. sphaeroides 2.4.1. Immunochemical quantitation of the major outer membrane protein of strain 2.4.1 showed approximately 5.5 x 10(4) molecules per cell whether cells were grown chemoheterotrophically or photoheterotrophically. Rhodospirillum rubrum outer membranes contained a cross-reactive protein, whereas the outer membranes derived from Rhodopseudomonas capsulata and Paracoccus denitrificans showed no cross-reaction with the antibody prepared against the major outer membrane protein from R. sphaeroides 2.4.1.     

Kinetic analysis of N-acylphosphatidylserine accumulation and implications for membrane assembly in Rhodopseudomonas sphaeroides.

The accumulation of N-acylphosphatidylserine (NAPS) in response to the inclusion of Tris in the growth medium of Rhodopseudomonas sphaeroides strain M29-5 has been examined. In the accompanying paper (Donohue et al., J. Bacteriol. 152:000--000, 1982), we show that in response to Tris, NAPS accumulated to as much as 40% of the total cellular phospholipid content. NAPS accumulation began immediately upon addition of Tris and was reflected as an abrupt 12-fold increase in the apparent rate of NAPS accumulation. We suggest that Tris altered the flow of metabolites through a preexisting and previously unknown metabolic pathway. NAPS accumulation ceased immediately upon the removal of Tris; however, accumulated NAPS remained largely metabolically stable. Importantly, under conditions in which NAPS was not accumulated, the intracytoplasmic membrane was shown to be virtually devoid of newly synthesized NAPS. The significance of this observation is discussed in terms of its physiological implications on phospholipid transfer and membrane biogenesis in R. sphaeroides.     

In vivo metabolic intermediates of phospholipid biosynthesis in Rhodopseudomonas sphaeroides

The in vivo metabolic pathways of phospholipid biosynthesis in Rhodopseudomonas sphaeroides have been investigated. Rapid pulse-chase-labeling studies indicated that phosphatidylethanolamine and phosphatidylglycerol were synthesized as in other eubacteria. The labeling pattern observed for N-acylphosphatidylserine (NAPS) was inconsistent with the synthesis of this phospholipid occurring by direct acylation of phosphatidylserine (PS). Rather, NAPS appeared to be kinetically derived from an earlier intermediate such as phosphatidic acid or more likely CDP-diglyceride. Tris-induced NAPS accumulation specifically reduced the synthesis of PS. Treatment of cells with a bacteriostatic concentration of hydroxylamine (10 mM) greatly reduced total cellular phospholipid synthesis, resulted in accumulation of PS, and stimulated the phosphatidylglycerol branch of phospholipid metabolism relative to the PS branch of the pathway. When the cells were treated with a lower hydroxylamine dosage (50 microM), total phospholipid synthesis lagged as PS accumulated, however, phospholipid synthesis resumed coincident with a reversal of PS accumulation. Hydroxylamine alone was not sufficient to promote NAPS accumulation but this compound allowed continued NAPS accumulation when cells were grown in medium containing Tris. The significance of these observations is discussed in terms of NAPS biosynthesis being representative of a previously undescribed branch of the phospholipid biosynthetic sequence.     

Characterization of a TOL-like plasmid from Alcaligenes eutrophus that controls expression of a chromosomally encoded p-cresol pathway.

Strains of all 18 species of the family Rhodospirillaceae (nonsulfur photosynthetic bacteria) were studied for their comparative nitrogen-fixing abilities. All species, with the exception of Rhodocyclus purpureus, were capable of growth with N2 as the sole nitrogen source under photosynthetic (anaerobic) conditions. Most rapid growth on N2 was observed in strains of Rhodopseudomonas capsulata. Within the genus Rhodopseudomonas, the species R. capsulata, R. sphaeroides, R. viridis, R. gelatinosa, and R. blastica consistently showed the highest in vivo nitrogenase rates (with the acetylene reduction technique); nitrogenase rates in other species of Rhodopseudomonas and in most species of Rhodospirillum were notably lower. Chemotrophic (dark microaerobic) nitrogen fixation occurred in all species with the exception of one strain of Rhodospirillum fulvum; oxygen requirements for dark N2 fixation varied considerably among species and even within strains of the same species. We conclude that the capacity to fix molecular nitrogen is virtually universal among members of the Rhodospirillaceae but that the efficacy of the process varies considerably among species.     

Succinate dehydrogenase in Rhodopseudomonas sphaeroides: subunit composition and immunocross-reactivity with other related bacteria.

Antibodies were raised against the succinate dehydrogenase (SDH) present in the chromatophores of phototrophically grown Rhodopseudomonas sphaeroides. Crossed immunoelectrophoresis experiments indicated that the SDH present in the cytoplasmic membranes of heterotrophically grown R. sphaeroides is probably the same enzyme observed in the chromatophores. The enzyme was extracted by Triton X-100 in a form which consisted of only two subunits (molecular weight, 68,000 and 30,000) and was not associated with a cytochrome b. The antibodies directed against SDH from R. sphaeroides showed no immunocross-reactivity with SDH from phylogenetically related bacterial species, including Rhodopseudomonas capsulata, Paracoccus denitrificans, Rhodopseudomonas palustris, Rhodospirillum rubrum, and Rhodospirillum fulvum.     

Expression of the transposable lac operon Tn951 in Rhodopseudomonas sphaeroides

The transposon Tn951 (lac) was introduced into the photosynthetic bacterium Rhodopseudomonas sphaeroides 2.4.1, which is normally Lac-, via the P-group plasmid RP1. beta-Galactosidase was produced constitutively in both chemotrophically and phototrophically grown cells, and the levels were found to be the same but low. Mutants were isolated, however, that were able to grow on lactose minimal medium and which expressed different levels of beta-galactosidase when grown chemotrophically or phototrophically. The beta-galactosidase levels found in all R. sphaeroides strains were much less than those found in Escherichia coli.     

Influence of pH, O2, and temperature on the absorption properties of the secondary light-harvesting antenna in members of the family Rhodospirillaceae

In some Rhodospirillaceae, the primary light-harvesting (LH I) antenna absorbs near-infrared light around 870 nm, whereas LH II (holochrome B800-860) has a major absorption band between 850 and 860 nm (B860) and a minor absorbancy around 800 nm (B800). Results show that, unlike LH I, holochrome B800-860 (LH II) exhibits unstable light absorption properties in whole cells. This was observed in Rhodopseudomonas capsulata grown anaerobically in light in weakly buffered carbohydrate medium; cultures lost both carotenoid-dependent brown-yellow pigmentation and LH II absorbancy. The whole cell spectrophotometric changes were attributed to mild acid conditions generated during sugar metabolism. LH II absorbancy was also destroyed in both R. capsulata and Rhodopseudomonas gelatinosa when cultures growing at neutral pH were acidified to a pH value around 5.0 with HCl. In contrast, during the same time period of exposure to pH 5.0, only a 50% decrease in Rhodopseudomonas sphaeroides LH II B800 absorbancy was measured. At neutral pH, LH II absorbancy in suspensions of nongrowing Rhodopseudomonas spp. was also sensitive to O2 exposure and to incubation at 30 to 40 degrees C. During treatment with O2, the rate of LH II B800 absorption decrease in R. gelatinosa and R. sphaeroides was 60 and 40% per h, respectively, compared with their absorbancy maximum around 860 nm. Both 860-nm absorbancy and the total bacteriochlorophyll content of the cells remained unchanged. On the other hand, no significant decrease in B800 if LH II in R. capsulata occurred during O2 exposure, but a 20% absorption decay rate per h of B800 was observed in cells incubated anaerobically at 40 degrees C. These B800 LH II spectral changes Rhodopseudomonas spp. were prevented by maintaining cells at neutral pH and at 10 degrees C. The near-infrared absorption spectrum of Rhodospirillum rubrum, which does not form LH II, was not significantly influenced by these different pH, aerobic, or temperature conditions.     

Mechanism of nitrogenase switch-off by oxygen.

Oxygen caused a reversible inhibition (switch-off) of nitrogenase activity in whole cells of four strains of diazotrophs, the facultative anaerobe Klebsiella pneumoniae and three strains of photosynthetic bacteria (Rhodopseudomonas sphaeroides f. sp. denitrificans and Rhodopseudomonas capsulata strains AD2 and BK5). In K. pneumoniae 50% inhibition of acetylene reduction was attained at an O2 concentration of 0.37 microM. Cyanide (90 microM), which did not affect acetylene reduction but inhibited whole-cell respiration by 60 to 70%, shifted the O2 concentration that caused 50% inhibition of nitrogenase activity to 2.9 microM. A mutant strain of K. pneumoniae, strain AH11, has a respiration rate that is 65 to 75% higher than that of the wild type, but its nitrogenase activity is similar to wild-type activity. Acetylene reduction by whole cells of this mutant was inhibited 50% by 0.20 microM O2. Inhibition by CN- of 40 to 50% of the O2 uptake in the mutant shifted the O2 concentration that caused 50% inhibition of nitrogenase to 1.58 microM. Thus, when the respiration rates were lower, higher oxygen concentrations were required to inhibit nitrogenase. Reversible inhibition of nitrogenase activity in vivo was caused under anaerobic conditions by other electron acceptors. Addition of 2 mM sulfite to cell suspensions of R. capsulata B10 and R. sphaeroides inhibited nitrogenase activity. Nitrite also inhibited acetylene reduction in whole cells of the photodenitrifier R. sphaeroides but not in R. capsulata B10, which is not capable of enzymatic reduction of NO2-. Lower concentrations of NO2- were required to inhibit the activity in NO3- -grown cells, which have higher activities of nitrite reductase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasmid distribution and analyses in Rhodopseudomonas sphaeroides

Ten strains of Rhodopseudomonas sphaeroides were analyzed by agarose gel electrophoresis for plasmid DNA content and, by filter-hybridizations, for their molecular relationships. All strains examined contained at least one plasmid. Several strains carried as many as six different plasmid species with sizes ranging from 42 to 140 kilobases (kb). Those larger than 89 kb showed extensive homology with each other; the 42-kb plasmid of R. sphaeroides strain 2.4.1 was homologous to the smaller plasmid DNA of three other strains. A partial map of the 42-kb plasmid derived from R. sphaeroides 2.4.1 was prepared by analysis of restriction endonuclease digests. Cross-hybridization among the large plasmids indicated that those present in any one strain of R. sphaeroides showed homology to one or more of the large plasmids detected in strains L and 2.4.1.     

Nitrous oxide reduction by members of the family Rhodospirillaceae and the nitrous oxide reductase of Rhodopseudomonas capsulata.

After growth in the absence of nitrogenous oxides under anaerobic phototrophic conditions, several strains of Rhodopseudomonas capsulata were shown to possess a nitrous oxide reductase activity. The enzyme responsible for this activity had a periplasmic location and resembled a nitrous oxide reductase purified from Pseudomonas perfectomarinus. Electron flow to nitrous oxide reductase was coupled to generation of a membrane potential and inhibited by rotenone but not antimycin. It is suggested that electron flow to nitrous oxide reductase branches at the level of ubiquinone from the previously characterized electron transfer components of R. capsulata. This pathway of electron transport could include cytochrome c', a component hitherto without a recognized function. R. capsulata grew under dark anaerobic conditions in the presence of malate as carbon source and nitrous oxide as electron acceptor. This confirms that nitrous oxide respiration is linked to ATP synthesis. Phototrophically and anaerobically grown cultures of nondenitrifying strains of Rhodopseudomonas sphaeroides, Rhodopseudomonas palustris, and Rhodospirillum rubrum also possessed nitrous oxide reductase activity.     

Generic interrelations of purple bacteria in the genus Rhodopseudomonas

The technique of DNA--DNA hybridization was used to study relations offween purple nonsulfur bacteria (the family Rhodospirillaceae). The level of homologies with Rhodopseudomonas sphaeroides 8259 was nearly the same for different species (8-17%) in the genus Rhodopseudomonas under the conditions optimal for hybridization. The same level of homologies was found for the DNA of Rhodospirillum rubrum, a species belonging to another genus of purple nonsulfur bacteria (13%). Rhodomicrobium vannielli was most remote from R. sphaeroides 8259 (3%). Similar results were obtained under other conditions of hybridization. The intraspecial heterogeneity of R. sphaeroides was studied in this work. The thermal stability of hybrid duplexes was analysed. The results are indicative of a considerable divergence of different R. sphaeroides strains (delta T50 = 2.1-11.6).     

Fermentation of pyruvate by 7 species of phototrophic purple bacteria]

The dark, anaerobic fermentation of pyruvate under growth conditions was examined with the following species of phototrophic purple bacteria: Rhodospirillum rubrum strains Ha and S1, Rhodopseudomonas gelatinosa strain 2150, Rhodopseudomonas acidophila strain 7050, Rhodopseudomonas palustris strain ATCC 17001, Rhodopseudomonas capsulata strains Kb1 and 6950, Rhodopseudomonas sphaeroides strain ATCC 17023, and Chromatium vinosum strain D. Fermentation balances were established for all experiments. Under fermentative conditions cell protein and dry weight increased only slightly, if at all. The species differed considerably in their fermentative activity; R. rubrum and R. gelatinosa exhibited the highest rates (2-8 mumoles pyruvate/mg protein-h). R. acidophila and R. capsulata showed an intermediate fermentation rate (0.4--2.0 mumoles pyruvate/mg protein-h), while the other strains tested fermented at quite low rates (0.2-0.4 mumoles pyruvate/mg protein-h). The extremes of fermentation times were from 30-380 hours. Based on the products of fermentation which were formed in addition to acetate, formate, and CO2, the species can be grouped as follows: a) R. rubrum, R. gelatinosa, and R. sphaeroides additionally form propionate. b) R. gelatinosa, R. palustris, R. capsulata, R. sphaeroides, and C. vinosum additionally form lactate. R. palustris also produces butyrate. c) R. acidophila and R. capsulata additionally form much 2,3-butanediol, acetoin, and diacetyl. Small amounts of acetoin were formed by the rest of the strains. A comparison of the fermentation of pyruvate by normal and starved cells (4 days in the light without a carbon source) of R. rubrum and R. gelatinosa shows that the latter ferment more slowly and produce less acetate and formate, but more propionate or lactate. The fermentation of pyruvate by R. rubrum was also studied in cultures in which the pH fell (7.2--6.6). Compared with the fermentation at neutral pH (7.3, 7.4), the following differences were found: a slower fermentation rate, an increased production of dry weight, an increased formation of propionate, but a reduced formation of acetate and a very low production of formate.     

Molecular evolution of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)

Abstract The evolutionary relationship of the RuBisCO large subunit gene(s) ( rbcL ) of several prokaryotes was examined using the technique of heterologous DNA hybridization. Restriction fragments of cloned rbcL from Anacystis nidulans 6301, Chlamydomonas reinhardtii, Rhodospirillum rubrum , and maize were nick-translated and used as probes. The C. reinhardtii and maize probes hybridized with restriction fragment(s) only from cyanobacteria: Agmenellum quadruplicatum, Fremyella diplosiphon , and Mastigocladus laminosus . In addition, the A. nidulans probe hybridized with restriction fragment(s) from Alcaligenes eutrophus, Chromatium vinosum, Nitrobacter hamburgensis, Paracoccus denitrificans, Pseudomonas oxalaticus, Rhodomicrobium vannielii, Rhodopseudomonas capsulata, Rhodopseudomonas palustris, Rhodopseudomonas sphaeroides, Thiobacillus intermedius, Thiobacillus neapolitanus , and Thiothrix nivea . The elucidated fragment of Rhodopseudomonas species is presumably for the Form I RuBisCO LSU of these organisms. The R. rubrum probe hybridized only to a restriction fragment(s) from R. capsulata, R. palustris, R. sphaeroides, T. neapolitanus , and T. nivea . The fragment(s) of Rhodopseudomonas species is the Form II rbcL of these organisms. The restriction fragments of T. neapolitanus and T. nivea were also different from those elucidated by the A. nidulans probe, suggesting the presence of a second (different) rbcL in these organisms. Positive hybridization was not obtained using any of the probes with DNA from Beggiatoa alba, Chlorobium vibrioforme or Chloroflexus aurantiacus . It appears that all rbcL have evolved from a common ancestor. Our data are consistent with and supportive of the evolutionary scheme for RuBisCO proposed by Akazawa, Takabe, and Kobayashi [1].     

Identification of two new denitrifying strains of Rhodobacter sphaeroides

Abstract Highly specific polyclonal and antibodies against either nitrate, nitrite or nitrous oxide reductases from a photosynthetic denitrifying bacterium Rhodobacter sphaeroides f. sp. denitrificans were used to show the presence of immunologically reactive proteins in strains that Pellerin and Gest had shown to grow in the dark with nitrate as a terminal acceptor [9]. Two strains of this bacterium, namely 81-3 and 2.4.3 synthesized the three denitrifying enzymes and were capable of denitrification. Strains 81-1 and 2.4.1 (neotype) both expressed nitrate reductase activities but nitrite reductase was not detected since these strains did not reduce nitrite. They also did not grow in the dark with nitrate as a terminal acceptor. Each of strains 81-1, 81-3, 2.4.1 and 2.4.3 contain four plasmids. R. sphaeroides f. sp. denitrificans , however, contains only one large 108 kb plasmid, which is distinctly different in size from those detected in the other strains. This indicates that the 108 kb plasmid is not necessarily specific for denitrification.     

Changes in the acyl lipid composition of photosynthetic bacteria grown under photosynthetic and non-photosynthetic conditions

The acyl lipids and their constituent fatty acids were studied in the photosynthetic bacteria Rhodospirillum rubrum, Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides, which were grown under photosynthetic and non-photosynthetic conditions. The major lipids were found to be phosphatidylethanolamine, phosphatidylglycerol and cardiolipin in each bacterium. The two Rhodopseudomonas species also contained significant quantities of phosphatidylcholine. Other acyl lipids accounted for less than 10% of the total. On changing growth conditions from non-photosynthetic to photosynthetic a large increase in the relative proportion of phosphatidylglycerol was seen at the expense of phosphatidyl-ethanolamine. In Rhodospirillum rubrum the fatty acids of the major phospholipids showed an increase in the proportion of palmitate and stearate and a decrease in palmitoleate and vaccenate on changing growth conditions to photosynthetic. In contrast, the exceptionally high levels (>80%) of vaccenate in individual phospholipids of Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides were unaffected by changing growth conditions to photosynthetic. Analysis of the lipids of chromatophores, isolated from the three bacteria, showed that these preparations were enriched in phosphatidylglycerol. The large increase in this phospholipid, seen during growth under photosynthetic conditions, appeared, therefore, to be due to a proliferation of chromatophore membranes. Possible roles for acyl lipids in the formation and function of the photosynthetic apparatus of bacteria are discussed.     

Nitric oxide-dependent proton translocation in various denitrifiers.

Respiration of NO resulted in transient proton translocation in anaerobically grown cells of four physiologically diverse denitrifiers. Paracoccus denitrificans, Rhodopseudomonas sphaeroides subsp. denitrificans, "Achromobacter cycloclastes," and Rhizobium japonicum gave, respectively, H+/NO ratios of 3.65, 4.96, 1.94, and 1.12. Antimycin A completely inhibited NO-dependent proton translocation in P. denitrificans and severely restricted translocation in the R. sphaeroides strain. Proton uptake during NO respiration with antimycin A-inhibited cells supplied with an artificial electron source provided evidence for the periplasmic consumption of protons. Values obtained were consistent with the expected ratios of 0.5 mol of H+/mol of NO for reduction of NO to N2O and 1.0 mol of H+/mol of NO for reduction of NO to N2. These data are consistent with the presence of a unique NO reductase found only in anaerobically grown denitrifying cells.     

Isolation and preliminary characterization of two forms of ribulose 1,5-bisphosphate carboxylase from Rhodopseudomonas capsulata.

The presence of two distinct forms of ribulose 1,5-bisphosphate carboxylase has been demonstrated in extracts of Rhodopseudomonas capsulata, similar to the form I (peak I) and form II (peak II) carboxylases previously described from R. sphaeroides (J. Gibson and F. R. Tabita, J. Biol. Chem 252:943-949, 1977). The two activities, separated by diethylaminoethyl-cellulose chromatography, were shown to be of different molecular size after assay on polyacrylamide gels. The higher-molecular-weight carboxylase from R. capsulata was designated form I-C, whereas the smaller enzyme was designated form II-C. Catalytic studies revealed significant differences between the two enzymes in response to pH and the effector 6-phosphogluconate. Immunological studies with antisera directed against the carboxylases from R. sphaeroides demonstrated antigenic differences between the two R. capsulata enzymes; cross-reactivity was observed only between R. sphaeroides anti-form II serum and the corresponding R. capsulata enzyme, form II-C.     

Genetic transformation of Rhodopseudomonas sphaeroides by plasmid DNA.

A broad-host-range cloning vector, pUI81, was constructed in vitro from plasmids RSF1010 and pSL25 (a pBR322 derivative) and used to assay for transformation in Rhodopseudomonas sphaeroides. Washing cells with 500 mM Tris was an effective means of inducing competence for DNA uptake. Transformation frequencies as high as 10(-5) (transformants per viable cell) have been achieved by incubating Tris-treated cells with plasmid DNA, 100 mM CaCl2, and 20% polyethylene glycol 6000. Maximum frequencies were obtained when recipient cells were spread onto selective media after a 6.5-h outgrowth period in antibiotic-free medium. The structure (open circular versus closed, covalent circular), size, and concentration of plasmid DNA all significantly affected the transformation frequency. Four different plasmids, all small and suitable as cloning vectors, have been introduced by transformation into several different R. sphaeroides strains. Recombinant DNA carried on small, nonconjugative plasmids with broad host ranges can now be directly transferred to R. sphaeroides by this method.     

Identification of nitrogenase and carboxylase genes in the photosynthetic bacteria and cloning of a carboxylase gene from Rhodopseudomonas sphaeroides

The presumptive genes for the ribulose 1,5-bisphosphate carboxylase large subunit and for nitrogenase-specific components from Rhodopseudomonas sphaeroides and several other photosynthetic bacteria were identified and located by interspecific probing. Restriction digests of R. sphaeroides genomic DNA were hybridized under stringent conditions to cloned DNA from Rhodospirillum rubrum (plasmid pRR2119 carrying the carboxylase gene) and Klebsiella pneumoniae (pSA30 carrying the nitrogenase genes). The nitrogenase probe hybridized with different signal intensities to several distinct HindIII, BglII, EcoRI, BamHI and PvuII fragments of R. sphaeroides 2.4.1.DNA. The carboxylase probe hybridized to only single R. sphaeroides 2.4.1.DNA fragments produced with all five restriction enzymes. A 3000-bp EcoRI-BamHI R. sphaeroides 2.4.1.DNA fragment carrying the presumptive gene for the large subunit of ribulose 1,5-bisphosphate carboxylase was cloned into pBR322 and positively identified by probing with a 32P-labeled internal PstI fragment of the Rhodospirillum carboxylase gene.     

fbc operon, encoding the Rieske Fe-S protein cytochrome b, and cytochrome c1 apoproteins previously described from Rhodopseudomonas sphaeroides, is from Rhodopseudomonas capsulata

Detailed comparison of the 'Rhodopseudomonas sphaeroides GA' strain used by Gabellini et al. (1985) with genuine R. sphaeroides and R. capsulata strains indicated that the previously reported fbc operon of R. sphaeroides (Gabellini and Sebald, 1986) encoding the structural genes for the Rieske Fe-S protein, cytochrome b and cytochrome c1 subunits of the ubiquinol:cytochrome c2 oxidoreductase, is not from R. sphaeroides, but is rather from a strain of R. capsulata. Consequently, the genuine bc1 genes from R. sphaeroides were cloned using corresponding R. capsulata genes as probes, and a partial nucleotide sequence for the Rieske Fe-S protein of R. sphaeroides was determined and compared with that of R. capsulata.