Sites of phospholipid biosynthesis during induction of intracytoplasmic membrane formation in Rhodopseudomonas sphaeroides

A rapid, gratuitous and cell-division uncoupled induction of intracytoplasmic photosynthetic membrane formation was demonstrated in low-aeration suspensions of chemotrophically grown Rhodopseudomonas sphaeroides. Despite a nearly 2-fold increase in phospholipid levels, no significant increases were detected in the specific activities of CDP-1,2-diacyl-sn-glycerol:sn-glycerol-3-phosphate phosphatidyltransferase (phosphatidylglycerophosphate synthase, EC 2.7.8.5) and CDP-1,2-diacyl-sn-glycerol:L-serine O-phosphatidyltransferase (phosphatidylserine synthase, EC 2.7.8.8), the first committed enzymes of anionic and zwitterionic phospholipid biosyntheses, respectively. The distribution of phosphatidylglycerophosphate and phosphatidylserine synthase activities after rate-zone sedimentation of cell-free extracts indicated that intracytoplasmic membrane phospholipids were synthesized mainly within distinct domains of the conserved cytoplasmic membrane. Labeling studies with ³²P_i and L-[³H]phenylalanine suggested that preexisting phospholipid was utilized initially as the matrix for insertion of intracytoplasmic membrane protein that was synthesized and assembled de novo during induction.Abbreviations BChl bacteriochlorophyll a - B800-850, B875 peripheral and core light-harvesting BChl-protein complexes, respectively, identified by near-IR absorption maxima This paper is dedicated to Professor Gerhart Drews on the occasion of his sixtieth birthday     

Light-mediated regulation of phospholipid synthesis in Rhodopseudomonas sphaeroides.

The relationship between the culture levels of guanosine-5'-diphosphate-3'-diphosphate (ppGpp) and the rates of synthesis and accumulation of cellular phospholipids was examined in cultures of Rhodopseudomonas sphaeroides that had been subjected to immediate decreases in incident light intensity. After a high-to-low light transition of high-light-adapted cells, an immediate inhibition of total cellular phospholipid production occurred coincident with a rapid accumulation of culture ppGpp. The inhibition of phospholipid accumulation occurred at the level of phospholipid synthesis rather than turnover, and both the extent of ppGpp accumulation and the degree of inhibition of phospholipid synthesis were directly dependent upon the magnitude of the light transition. Maximum inhibition (greater than 90%) of the rate of cellular phospholipid synthesis occurred after transitions from 5,350 to 268 1x and lower, including transitions to the dark, with comparable inhibition being exerted upon the rates of synthesis of individual species of phospholipids. Reinitiation of culture phospholipid accumulation in cultures shifted from 5,350 to 1,070 1x and lower occurred 65 to 70 min subsequent to the downshift in light intensity, apparently irrespective of the culture level of ppGpp.     

In vivo intermembrane transfer of phospholipids in the photosynthetic bacterium Rhodopseudomonas sphaeroides.

The kinetics of accumulation of phospholipids into the intracytoplasmic membrane of Rhodopseudomonas sphaeroides have been examined. We have previously demonstrated that accumulation of phospholipids in the intracytoplasmic membrane is discontinuous with respect to the cell cycle. In this study we demonstrated a sevenfold increase in the rate of phospholipid incorporation into the intracytoplasmic membrane concurrent with the onset of cell division. Pulse-chase labeling studies revealed that the increase in the rate of phospholipid accumulation into the intracytoplasmic membrane results from the transfer of phospholipid from a site other than the intracytoplasmic membrane, and that the transfer of phospholipid, rather than synthesis of phospholipid, is most likely subject to cell cycle-specific regulation. The rates of synthesis of the individual phospholipid species (phosphatidylethanolamine, phosphatidyglycerol, and an unknown phospholipid) remained constant with respect to one another throughout the cell cycle. Similarly, each of these phospholipid species appeared to be transferred simultaneously to the intracytoplasmic membrane. We also present preliminary kinetic evidence which suggested that phosphatidylethanolamine may be converted to phosphatidycholine within the intracytoplasmic membrane.     

Intermembrane phospholipid transfer mediated by cell-free extracts of Rhodopseudomonas sphaeroides.

The transfer of phospholipids between two membrane substrates catalyzed by a soluble protein fraction from Rhodopseudomonas sphaeroides has been demonstrated. The assay employs purified intracytoplasmic membrane (ICM) vesicles derived from cells of R. sphaeroides grown on [3H]acetate as the phospholipid donor substrate and phosphatidylcholine (70%)/phosphatidylethanolamine (30%) unilamellar liposomes containing [14C]triolein, a nontransferable marker, as the acceptor substrate for transferred phospholipids. Incubation of these two membrane substrates with a 40 to 80% (NH4)2SO4 protein fraction from R. sphaeroides results in the transfer of tritium-labeled ICM phospholipids to the acceptor membrane substrate. Upon completion of the incubation period, the donor ICM vesicles are quantitatively separated from the acceptor liposomes by precipitation with antibody prepared against whole, purified ICM vesicles. Phospholipid transfer is linear with respect to time and protein concentration, is inhibited by trypsin and heat, and shows an absolute dependence upon the presence of acceptor liposomes and the 40 to 80% (NH4)2SO4 protein fraction. Control experiments indicate that no fusion of the donor and acceptor membrane occurs during the incubation period and that, following prolonged incubation there is no detectable degradation of the labeled lipid components. Preliminary data on the phospholipid specificity of the transfer reaction is also presented.     

Localization of phospholipid biosynthetic enzyme activities in cell-free fractions derived from Rhodopseudomonas sphaeroides

The phospholipid biosynthetic enzyme activities: CDP-diglyceride synthetase, phosphatidylglycerophosphate synthetase, PGP phosphatase, phosphatidylserine (PS) synthase, PS decarboxylase, and S-adenosyl-L-methionine:phosphatidylethanolamine (AdoMet:PE) N-methyltransferase were detected in crude cell-free extracts of Rhodopseudomonas sphaeroides. CDP-diglyceride synthetase and phosphatidylglycerophosphate synthetase co-enriched with penicillin-binding protein activity, a known cytoplasmic membrane marker, throughout fractionation of cell-free extracts of both chemoheterotrophically and photoheterotrophically grown cells. PS decarboxylase also co-enriched with the cytoplasmic membranes in fractions derived from chemoheterotrophically and photoheterotrophically grown cells, but substantially greater quantities of PS decarboxylase activity was found in the chromatophores derived from photoheterotrophically grown cells than could be accounted for by cytoplasmic membrane contamination of this sample. PS synthase (60% of the recovered activity) and S-adenosyl-L-methionine:phosphatidylethanolamine N-methyltransferase (90% of the recovered activity) were found in the supernatant fraction after high speed centrifugation of crude cell lysates, suggesting that these enzyme activities were not tightly membrane associated. The localization of phospholipid biosynthetic enzyme activity in R. sphaeroides is discussed in terms of the biosynthesis of the photosynthetic membranes.     

Lipid biosynthesis in synchronized cultures of the photosynthetic bacterium Rhodopseudomonas sphaeroides.

Lipid biosynthesis has been studied in photosynthetic cultures of Rhodopseudomonas sphaeroides that had been synchronized by stationary-phase cycling or by a centrifugation selection procedure. Synchrony index values in the range 0.70-0.80 were obtained for the first cell cycle with both synchronization methods. The major membrane lipids phosphatidylethanolamine and phosphatidylglycerol were accumulated discontinuously during the cell cycle, their mass doubling immediately before cell division. This accumulation of lipid corresponded to peaks in incorporation of radioactivity from either [1-14C]acetate or [2-3H]glycerol into individual acyl lipids as measured in individual portions of bacteria. For phosphatidylglycerol an additional peak of incorporation of radioactivity from [2-3H]glycerol was found midway through the cell cycle. In spite of their rather similar endogenous fatty acid compositions, the individual phosphoacylglycerols showed distinctive patterns of incorporation of radioactivity from [1-14C]acetate into their acyl moieties. The discontinuous synthesis of acyl lipids observed in cultures of Rhodopseudomonas sphaeroides synchronized by either stationary-phase cycling or centrifugation selection procedures contrasted with the accumulation of chlorophyll-protein complexes whose amounts were found to increase throughout the cell cycle. The implications of these findings for the control of lipid synthesis in bacterial photosynthetic membranes are discussed.     

Alterations in the phospholipid composition of Rhodopseudomonas sphaeroides and other bacteria induced by Tris.

Alterations in the phospholipid head group composition of most strains of Rhodopseudomonas sphaeroides, as well as Rhodopseudomonas capsulata and Paracoccus denitrificans, occurred when cells were grown in medium supplemented with Tris. Growth of R. sphaeroides M29-5 in Tris-supplemented medium resulted in the accumulation of N-acylphosphatidylserine (NAPS) to as much as 40% of the total whole-cell phospholipid, whereas NAPS represented approximately 28 an 33% of the total phospholipid when R. capsulata and P. denitrificans respectively, were grown in medium containing 20 mM Tris. The accumulation of NAPS occurred primarily at the expense of phosphatidylethanolamine in both whole cells and isolated membranes of R. sphaeroides and had no detectable effect on cell growth under either chemoheterotrophic or photoheterotrophic conditions. Yeast extract (0.1%) and Casamino Acids (1.0%) were found to be antagonistic to the Tris-induced (20 mM) alteration in the phospholipid composition of R. sphaeroides. The wild-type strains R. sphaeroides 2.4.1 and RS2 showed no alteration in their phospholipid composition when they were grown in medium supplemented with Tris. In all strains of Rhodospirillaceae tested, as well as in P. denitrificans, NAPS represented between 1.0 and 2.0% of the total phospholipid when cells were grown in the absence of Tris. [32P]orthophosphoric acid entered NAPS rapidly in strains of R. sphaeroides that do (strain M29-5) and do not (strain 2.4.1) accumulate this phospholipid in response to Tris. Our data indicate that the phospholipid head group composition of many Rhodospirillaceae strains, as well as P. denitrificans, is easily manipulated; thus, these bacteria may provide good model systems for studying the effects of these modifications on membrane structure and function in a relatively unperturbed physiological system.     

Rhodopseudomonas sphaeroides membranes: alterations in phospholipid composition in aerobically and phototrophically grown cells.

The effects of growth conditions on phospholipid composition in Rhodopseudomonas sphaeroides have been reexamined. The levels of phosphatidylethanolamine (27 to 28%), phosphatidylglycerol (23 to 24%), and phosphatidylcholine (11 to 18%) were very similar in cells grown aerobically or phototrophically at a high light intensity, consistent with findings for another member of Rhodospirillaceae. In addition, an unknown phospholipid species was detected which comprised 20 to 30% of the total phospholipid in these cells. In cells growing phototrophically at low-intensity illumination, the level of phosphatidylethanolamine increased by about 1.6-fold and that of the unknown phospholipid markedly decreased. Although the synthesis of photosynthetic pigments, light-harvesting protein, and intracytoplasmic photosynthetic membranes also increased markedly, the ratios of individual phospholipid species were essentially identical in photosynthetic membrane and cell wall fractions purified from these cells. Since a significant exchange of lipids apparently did not occur during the isolation of these fractions, it was suggested that the changes in cellular phospholipid accumulation were not due to a unique composition within the photosynthetic membrane. Instead, these phosphoglyceride changes were found to be related to overall phospholipid metabolism and could be accounted for principally by differences in biosynthetic rates. These results, together with studies in nutrient-restricted aerobic cells, suggested that the mechanism by which phospholipid levels are regulated may be related to radiant energy flux rather than cellular energy limitation.     

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.     

DNA-DNA hybridization of Rhodopseudomonas capsulata,Rhodopseudomonas sphaeroides and Rhodopseudomonas sulfidophila strains

The genetic relatedness of 21 Rhodopseudomonas strains has been studied by means of DNA-DNA hybridization. All strains included in the study belonged to the subgroup of the genus Rhodopseudomonas which is characterized by a short-rod to coccus morphology, a vesicular intracytoplasmic membrane system and carotenoids of the spheroidene group. Mol percentages guanine + cytosine ranged from 64 to 73, most strains having values between 68 and 72. With few exceptions, the hybridization data obtained were in agreement with the subdivision in three (or possibly four) species on the basis of classical taxonomy. Strain SCJ, formerly considered to be a somewhat atypical R. capsulata strain, is most probably a R. sphaeroides strain and two out of seven strains that were received as R. sulfidophila did not fit in this species on the basis of the hybridization data. The results also showed that two undesignated strains that were previously thought to be related to R. capsulata (Hansen et al. 1975) cannot be assigned to this species and may be representatives of another species. The seven strains that required approximately 2.5% NaCl in the medium and that had been designated R. sulfidophila were found to synthesize far higher levels of bacteriochlorophyll during fully aerobic growth in the dark than the purple bacteria studied thus far.Abbreviations GC guanosine + cytosine - SSC standard saline citrate buffer     

Intracellular localization of phospholipid transfer activity in Rhodopseudomonas sphaeroides and a possible role in membrane biogenesis.

The cellular content of phospholipid transfer activity in Rhodopseudomonas sphaeroides was examined as a function of both oxygen partial pressure and light intensity used for growth. Cells grown under high light conditions (100 W/m2) had over two times the cellular level of phospholipid transfer activity when compared with cells grown under other conditions. Although cells grown under low light conditions (3 W/m2) had the lowest amount of total phospholipid transfer activity, they had the highest level (49%) of membrane-associated transfer activity. The soluble phospholipid transfer activity was further localized into periplasmic and cytoplasmic fractions. The distribution of phospholipid transfer activity in cells grown under medium light intensity (10 W/m2) was calculated as 15.1% membrane-associated, 32.4% in the periplasm, and 52.5% in the cytoplasm. The phospholipid transfer activities in the periplasmic and cytoplasmic fractions had distinctly different properties with respect to their molecular weights (56,000 versus 27,000) and specificities of transfer (phosphatidylethanolamine greater than phosphatidylglycerol versus phosphatidylglycerol greater than phosphatidylethanolamine).     

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.     

Flash-induced proton release in Rhodopseudomonas sphaeroides spheroplasts

Proton release by flash excitations was measured with right-side-out vesicles prepared from Rhodopseudomonas sphaeroides by lysozyme-EDTA treatment followed by hypotonic treatment. Absorbance change at 586 nm in the presence of bromcresol purple was measured to monitor the pH change. In the presence of horse heart cytochrome c, which catalyzes the electron transfer from the cytochrome b-c1 complex to the primary electron donor, the single-turnover flash elicited release of about two protons per primary electron donor, which was rereduced rapidly by the added cytochrome c. The halftime of the proton release was about 70 ms at pH 6.3 and at a redox potential of about 150 mV. The rate was considerably lower than that of the electron transfer from the cytochrome b-c1 complex to cytochrome c. However, multiple flashes with intervals of 60 ms caused release of the same amount of protons as that by flashes with longer intervals. This indicated that the proton release itself was rapid, but delocalization was slower. Antimycin A inhibited the proton release, and myxothiazol almost completely abolished it.     

Rhodopseudomonas sphaeroides mutants defective in nitrogen fixation

Mutants of phototrophic bacterium Rhodopseudomonas sphaeroides deficient in nitrogen fixation and unable to utilize alanine, proline, arganine and glutamic acid as nitrogen sources have been obtained as a result of nitrosomethylurea mutagenesis. The majority of the nif-mutants have no nitrogenase activity and aminotransferase activity of glutamine synthetase during their growth in glutamine containing medium is sharply lowered. The specific activity of glutamate synthase and alanine dehydrogenase in the mutants does not differ from that of the wild type strain. One of the mutants (NF-42) has higher glutamine synthetase activity in comparison with the wild type strain. The pleiotropic character of the changes obtained in the nif-mutants shows that the loss of nitrogen fixation ability is due to defects in regulation system of nitrogen metabolism.     

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.     

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