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 cytochromes and ferredoxins from the marine purple phototrophic bacterium, Rhodopseudomonas marina

Four soluble c-type cytochromes, the high redox potential 4-Fe-S ferredoxin known as HiPIP, a large molecular weight 2-Fe-S ferredoxin and a 4-Fe-S 'bacterial' ferredoxin, were isolated from extracts of two strains of Rps. marina. Cytochrome c-550, cytochrome c' and cytochrome c-549 were previously described, and we have extended their characterization. Cytochrome c-558, which has not previously been observed in Rps. marina, appears to be a low-spin isozyme of the more commonly observed high-spin cytochrome c'. HiPIP, which was not observed in previous work, was found to be abundant in Rps. marina. The 2-Fe-S ferredoxin, which has previously been observed only in Rps. palustris, has a native size greater than 100 kDa and a subunit size of 17 kDa. The 'bacterial' ferredoxin appears to have only a single four-iron-sulfur cluster. We examined photosynthetic membranes by difference spectroscopy and found abundant c-type cytochromes. Approximately one-quarter of the heme can be reduced by ascorbate and the remainder by dithionite. There is 2 nm difference between the high-potential heme (554 nm) and the low (552 nm). These characteristics resemble those of the tetraheme reaction center cytochrome of Rps. viridis. In addition to the electron transfer components, we found small amounts of a fluorescent yellow protein which has spectral resemblance to a photoactive yellow protein from Ec. halophila.     

Electron transfer proteins of the purple phototrophic bacterium, Rhodopseudomonas rutila.

The soluble electron transfer protein content of Rhodopseudomonas rutila was found to consist of two basic cytochromes and a (4Fe-4S) ferredoxin. Cytochrome c' was easily identified by its characteristic high spin absorption spectra. The native molecular weight is 29,000 and the subunit is 14,000. Cytochrome c-550 has low spin absorption spectra and a high redox potential (376 mV) typical of cytochromes c2. The molecular weight is about 14,000. The ferredoxin is apparently a dimer (43,000) of approximately 18,000 Da subunits. There are 1.3 to 1.5 iron-sulfur clusters per monomer of 18- to 21-kDa protein. The N-terminal amino acid sequence is like the (7Fe-8S) ferredoxins of Rhodobacter capsulatus and Azotobacter vinelandii. Remarkably, there are only 2 or 3 out of 25 amino acid substitutions. Difference absorption spectra of Rps. rutila membranes indicate that there is not tetraheme reaction center cytochrome c, such as is characteristic of Rps. viridis. However, there are a high potential cytochrome c and a low potential cytochrome b in the membrane, which are suggestive of a cytochrome bc1 complex. Rps. rutila is most similar to Rps. palustris in microbiological properties, yet it does not have the cytochromes c-556, c-554, and c-551 in addition to c2 and c', which are characteristic of Rps. palustris. Furthermore, the Rps. rutila cytochrome c' is dimeric, whereas the same protein from Rps. palustris is the only one known to be monomeric. The cytochrome pattern is more like that of Rhodospirillum rubrum and Rb. capsulatus, which are apparently only able to make cytochromes c2 and c'.     

Location of fatty acids in lipid A obtained from lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023

Monophosphoryl lipid A (MLA) obtained from the lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023 was initially purified by silicic acid column chromatography to yield a single major pentaacyl MLA fraction. This fraction was methylated and further purified by reverse-phase high performance liquid chromatography to yield three prominent peak fractions. Laser desorption mass spectrometry of these three fractions allowed us to complete the important structural analysis of lipid A from this source. Three structurally distinct forms of dimethyl MLA were identified where Mr = 1447, 1449, and 1451 atomic mass units. These forms differed only by the presence or absence of unsaturation and keto group in the fatty acids. We established that the acyloxyacyl group (either delta 7-tetradecenoyloxytetradecanoate or tetradecanoyloxytetradecanoate) and the 3-ketotetradecanoate or hydroxytetradecanoate occupied the 2'- and 2-positions of the glucosamine disaccharide, respectively. Analysis of several minor fractions suggests that there is considerable structural heterogeneity in the MLA. With this new knowledge, the study of the structure-to-function relationship of the reported lack of toxicity of lipopolysaccharide from R. sphaeroides can be completed.     

Effect of aerobic growth conditions on the soluble cytochrome content of the purple phototrophic bacterium Rhodobacter sphaeroides: induction of cytochrome c554

When grown anaerobically in the light, Rhodobacter sphaeroides contains appreciable quantities of cytochromes c2 and c', but smaller amounts of other soluble cytochromes such as cytochrome c551.5, cytochrome c554, and an oxygen-binding heme protein. When R. sphaeroides is mass cultured aerobically in the dark to stationary phase, the content of cytochrome c2 does not change appreciably, whereas cytochrome c554 is approximately 8-fold more abundant, cytochrome c' is at least 10-fold less abundant, and cytochrome c551.5 is fivefold lower than in the phototrophically grown cells. These observations confirm previous literature reports that in this organism a cytochrome c553 (or c554 in our experience) is more abundant when cells are grown aerobically. Furthermore, the aerobic cytochrome c554 is positively identified with the previously characterized minor cytochrome c554 component of anaerobic photosynthetic cells. Preliminary sequence results show that cytochrome c554 is a member of the cytochrome c' structural family, but differs from normal cytochromes c' in having a methionine sixth ligand to the heme. The levels of electron carrier proteins of low redox potential had previously been reported to be less in aerobic than in photoheterotrophic cells and we have verified that observation for the specific examples of cytochromes c' and c551.5. The oxygen binding heme protein, SHP, is not induced by aerobic growth.     

Shotgun proteomic analysis of a chromatophore-enriched preparation from the purple phototrophic bacterium Rhodopseudomonas palustris

A proteomics approach was evaluated for analysis of photosyntheis-related proteins that are characteristic of chromatophores, particles derived from purple phototrophic bacterial intracytoplasmic membranes. Proteins of purified chromatophores from Rhodopseudomonas palustris were solubilized and digested with trypsin, to create a collection of peptides that were fractionated by liquid chromatography. Peptide sequences were determined and assigned to specific proteins by analysis of tandem mass spectra of peptides, and comparison to a library derived from the recently determined R. palustris genome sequence. A total of 300 proteins were detected with a probability value >/=0.9, and the number of proteins detected increased to 345 when the minimum probability value was reduced to 0.5. Membrane-integral proteins of the reaction center, cytochrome b/c (1), light-harvesting and ATPase complexes were used as controls to assess how well this approach performs with hydrophobic proteins. New genes were identified, and tentatively designated as encoding photosynthesis-related proteins. We conclude that this approach is a powerful method to evaluate the possible existence of new photosynthesis-related proteins (and genes), although alternative methods are needed to evaluate the exact functions of newly discovered genes.     

Isolation and purification of malate dehydrogenase isoforms from phototrophic purple bacteria Rhodobacter sphaeroides and Rhodopseudomonas palustris

A five-step procedure was used to obtain electrophoretically pure preparations of malate dehydrogenase (EC 1.1.1.37) from Rhodobacter sphaeroides and Rhodopseudomonas palustris. The procedure included extraction, ammonium sulfate fractionation, gel filtration, and ion exchange and gel permeation chromatography. The enzyme was found to exist in two isoforms, dimeric and tetrameric, formed by the oligomerization of identical subunits. The isoforms are assumed to be involved in different metabolic processes.     

The amino acid sequence of the cytochrome c2 from the phototrophic bacterium Rhodopseudomonas globiformis.

The amino acid sequence of the principal soluble cytochrome c from the phototrophic acidophilic bacterium Rhodopseudomonas (or Rhodopila) globiformis was determined. By the criteria of percentage sequence identity and fewness of internal insertions and deletions it is more similar in sequence to some mitochondrial cytochromes c than to any known bacterial cytochrome. The organism does not have any properties that commend it as being particularly similar to postulated prokaryotic precursors of the mitochondrion. We consider that the relatively high degree of sequence similarity is an instance of convergence, and is an example of the limitations that are imposed on attempts to deduce distant evolutionary relationships from sequence information. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50136 (12 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment [see Biochem. J. (1987) 241, 5].     

Cloning, overproduction and characterization of cytochrome c peroxidase from the purple phototrophic bacterium Rhodobacter capsulatus.

The bacterial cytochrome c peroxidase (BCCP) from Rhodobacter capsulatus was purified as a recombinant protein from an Escherichia coli clone over-expressing the BCCP structural gene. BCCP from Rb. capsulatus oxidizes the Rhodobacter cytochrome c2 and reduces hydrogen peroxide, probably functioning as a detoxification mechanism. The enzyme binds two haem c groups covalently. The gene encoding BCCP from Rb. capsulatus was cloned through the construction of a 7-kb subgenomic clone. In comparison with the protein sequence, the sequence deduced from the gene has a 21-amino-acid N-terminal extension with the characteristics of a signal peptide. The purified recombinant enzyme showed the same physico-chemical properties as the native enzyme. Spectrophotometric titration established the presence of a high-potential (Em=+270 mV) and a low-potential haem (between -190 mV and -310 mV) as found in other BCCPs. The enzyme was isolated in the fully oxidized but inactive form. It binds calcium tightly and EGTA treatment of the enzyme was necessary to show calcium activation of the mixed valence enzyme. This activation is associated with the formation of a high-spin state at the low-potential haem. BCCP oxidizes horse ferrocytochrome c better than the native electron donor, cytochrome c2; the catalytic activities ('turnover number') are 85 800 min(-1) and 63 600 min(-1), respectively. These activities are the highest ever found for a BCCP.     

Plasmid rearrangements in the photosynthetic bacterium Rhodopseudomonas sphaeroides

Mu d1(Ap lac) was introduced into the photosynthetic bacterium Rhodopseudomonas sphaeroides 2.4.1. via the R-plasmid R751 in an attempt to isolate fusion derivatives involving photosynthetic operons. The selection system is potentially very powerful since R. sphaeroides is normally Lac negative. Among the exconjugants, photosynthesis-deficient mutants were recovered, some of which had elevated beta-galactosidase levels. Among the mutants examined, beta-galactosidase expression was linked exclusively to R751 . Many of the photosynthesis-deficient mutants were found to have alterations in their indigenous plasmids which apparently involved the exchange of DNA from one plasmid to another. Southern blot analysis revealed that there are extensive DNA sequences which are shared by the two plasmids that are involved in the rearrangements and that no exogenous DNA sequences appear to be involved. It was further discovered that plasmid rearrangement is a general phenomenon which can occur spontaneously in R. sphaeroides 2.4.1 and shows a high correlation with a photosynthesis minus phenotype.     

The lactose carrier of Escherichia coli functionally incorporated in Rhodopseudomonas sphaeroides obeys the regulatory conditions of the phototrophic bacterium

Rhodopseudomonas sphaeroides was provided with the ability to transport lactose via conjugation with a strain of Escherichia coli bearing a plasmid containing the lactose operon (including the lac Y gene, coding for the lactose carrier or M protein) and subsequent expression of the lac operon in Rps. sphaeroides (Nano, F.E. and Kaplan, S. submitted). The initial rate of lactose transport in Rps. sphaeroides was studied as a function of the light intensity and the magnitude of the proton-motive force. The results demonstrate that lactose transport is regulated by the rate of cyclic electron transfer in the same way as the endogenous transport systems.     

Biogeography of the purple nonsulfur bacterium Rhodopseudomonas palustris

The biogeography of the purple nonsulfur bacterium Rhodopseudomonas palustris on a local scale was investigated. Thirty clones of phototrophic bacteria were isolated from each of five unevenly spaced sampling locations in freshwater marsh sediments along a linear 10-m transect, and a total of 150 clones were characterized by BOX-PCR genomic DNA fingerprinting. Cluster analysis of 150 genomic fingerprints yielded 26 distinct genotypes, and 106 clones constituted four major genotypes that were repeatedly isolated. Representatives of these four major genotypes were tentatively identified as R. palustris based on phylogentic analyses of 16S rRNA gene sequences. The differences in the genomic fingerprint patterns among the four major genotypes were accompanied by differences in phenotypic characteristics. These phenotypic differences included differences in the kinetics of carbon source use, suggesting that there may be functional differences with possible ecological significance among these clonal linages. Morisita-Horn similarity coefficients (C(MH)), which were used to compare the numbers of common genotypes found at pairs of sampling locations, showed that there was substantial similarity between locations that were 1 cm apart (C(MH), >/=0.95) but there was almost no similarity between locations that were >/=9 m apart (C(MH), 相似文献   

Isolation and characterization of cytochrome C1 from photosynthetic bacterium Rhodopseudomonas sphaeroides R-26

Cytochrome c1 of photosynthetic bacterium R. sphaeroides R-26 has been purified from isolated cytochrome b-c1 complex to a single polypeptide, using a procedure involving Triton X-100 and urea solubilization, calcium phosphate column chromatography and ammonium sulfate fractionation. The purified protein contains 30 nmoles heme per mg protein and has an apparent molecular weight of 30,000, as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis. Bacterial cytochrome c1 is soluble in aqueous solution in the absence of detergent and has spectral characteristics similar to mammalian cytochrome c1. The amino acid compositions of these two proteins, however, are not comparable.     

Soluble cytochromes and a photoactive yellow protein isolated from the moderately halophilic purple phototrophic bacterium, Rhodospirillum salexigens

Three soluble cytochromes were found in two strains of the halophilic non-sulfur purple bacterium Rhodospirillum salexigens. These are cytochromes C2, C and c-551. Cytochrome C2 was recognized by the presence of positive charge at the site of electron transfer (measured by laser flash photolysis), although the protein has an overall negative charge (pI = 4.7). Cytochrome C2 has a high redox potential (300 mV) and is monomeric (13 kDa). Cytochrome c was recognized from its characteristic absorption spectrum. It has a redox potential of 95 mV, an isoelectric point of 4.3, and is isolated as a dimer (33 kDa) of identical subunits (14 kDa), a property which is typical of this family of proteins. R. salexigens cytochrome c-551 has an absorption spectrum similar to the low redox potential Rb. sphaeroides cytochrome c-551.5. It also has a low redox potential (-170 mV), is very acidic (pI = 4.5), and is monomeric (9 kDa), apparently containing 1 heme per protein. The existence of abundant membrane-bound cytochromes c-558 and c-551 which are approximately half reduced by ascorbate and completely reduced by dithionite suggests the presence of a tetraheme reaction center cytochrome in R. salexigens, although reaction centers purified in a previous study (Wacker et al., Biochim. Biophys. Acta (1988) 933, 299-305) did not contain a cytochrome. The most interesting observation is that R. salexigens contains a photoactive yellow protein (PYP), previously observed only in the extremely halophilic purple sulfur bacterium Ectothiorhodospira halophila. The R. salexigens PYP appears to be slightly larger than that of Ec. halophila (16 kDa vs. 14 kDa). Otherwise, these two yellow proteins have similar absorption spectra, chromatographic properties and kinetics of photobleaching and recovery.     

Periplasmic location of dissimilatory nitrate and nitrite reductases in a denitrifying phototrophic bacterium, Rhodopseudomonas sphaeroides forma sp. denitrificans

The localization of dissimilatory nitrate and nitrite reductasesof a denitrifying phototrophic bacterium, Rhodopseudomonas sphaeroidesforma sp. denitrificans, was investigated. Nitrate and nitritereductases were located in the periplasmic space of the bacteriumgrown anaerobically in the presence of nitrate either in lightor in darkness. Chromatophores showed nitrate and nitrite reductaseactivities when dithionite-reduced benzyl viologen was an electrondonor; this suggests that the enzymes were trapped inside thevesicles. ¹Present address: Japanese Red Cross Central Blood Center, Hiroo4-1-31, Shibuyaku, Tokyo 150, Japan. ²Present address: Plant Growth Laboratory, University of California,Davis, California 95616, U.S.A. (Received November 7, 1979; )     

Lipopolysaccharides of two strains of the phototrophic bacterium Rhodopseudomonas capsulata

The lipopolysaccharides of Rhodopseudomonas capsulata strains St. Louis (ATCC 23782) and Sp 11 both contain L-acofriose, rhamnose, glucose and glucosamine as the main sugar constituents. 2-Keto-3-deoxyoctonate and neuraminic acid were tentatively identified. The fatty acid spectrum found with both strains comprises 3-OH-C10 and C12:1 (ester-linked) and 3-oxo-C14 (amide-linked). Isolated lipid A from strain Sp 11 contains glucosamine, glucosamine-phosphate and the total of the fatty acids of the lipopolysaccharide. Methylation analysis of the degraded polysaccharide of this lipopolysaccharide shows L-acofriose in both terminal and 1 leads to 2 chain-linked positions in a 1:4 molar ratio. Rhamnose is exclusively chain-linked (1 leads to 2), glucose is both terminally and chain-linked (1 leads to 6) in a 1:1 molar ratio. The serological activity of the lipopolysaccharide of both the R. capsulata strains is low in antisera against living or heat-killed cells when tested by passive hemagglutination, Ouchterlony immunoprecipitation or gel-immunoelectrophoresis. No crossreaction was observed among the lipopolysaccharides of R. capsulata strains St. Louis, Sp 11 and 37b4 in immunoprecipitation. Lipopolysaccharide of strain Sp 11 was found to lack lethal toxicity in galactosamine-sensitized mice.     

Factors influencing the production of hydrogen by the purple non‐sulphur phototrophic bacterium Rhodopseudomonas acidophila KU001

Rhodopseudomonas acidophila KU001 was isolated from leather industry effluents and the effect of different cultural conditions on hydrogen production was studied. Anaerobic light induced more hydrogen production than anaerobic dark conditions. Growing cells produced more amounts of hydrogen between 96 and 144 h of incubation. Resting and growing cells preferred a pH of 6.0 ± 0.24 for hydrogen production. Succinate was the most preferred carbon source for the production of hydrogen while citrate was a poor source of carbon. Acetate and malate were also good carbon sources for hydrogen production under anaerobic light. Among the nitrogen sources, R. acidophila preferred ammonium chloride followed by urea for production of hydrogen_. L‐tyrosine was the least preferred nitrogen source by both growing and resting cells.     

Induction of stress proteins in the phototrophic bacterium Rhodobacter sphaeroides

The stress response of the phototrophic bacterium Rhodobacter sphaeroides was investigated by two-dimensional gel electrophoresis. Oxygen, 0.5% and 4% ethanol, UV radiation, heat shock at 42°C and 0.01% phenanthrene were tested as stress factors. The protein pattern on two-dimensional gels of stressed as compared to control cells revealed that all stress factors applied caused modifications in the protein pattern of R. sphaeroides. The intensity of particular spots increased or decreased as a consequence of altered protein synthesis. Specific and general stress responses were observed.     

Metabolism of the phase variants of the phototrophic bacterium Rhodobacter sphaeroides

Growth, bacteriochlorophyll a content, electron transport chain (ETC), and activities of the tricarboxylic acid (TCA) cycle enzymes were studied in R and M phase variants of Rhodobacter sphaeroides cells grown anaerobically in the light and aerobically in the dark. Under all cultivation conditions tested, bacteriochlorophyll a content was 2–3 times lower in the cells of the M variant compared to the R variant, which therefore was predominant in the cultures grown in the light. In both variants, activity of all TCA cycle enzymes was higher for the cells grown in the dark under aerobic conditions. When grown aerobically in the dark, the R variant, unlike the M variant, did not contain cytochrome aa ₃, acting as cytochrome c oxidase, in its ETC. An additional point of coupling the electron transfer to the generation of the proton gradient at the cytochrome aa ₃ level provided for more efficient oxidation of organic substrates, resulting in predominance of the M variant in the cultures grown in the dark under aerobic conditions.