Photoheterotrophic utilization of acetate by the wild type and an acetate-adapted mutant of Rhodopseudomonas capsulata

Rhodopseudomonas capsulata strain St. Louis can grow anaerobically in the light-with acetate as the carbon source. The organism is sensitive to acetate, however, initial concentrations exceeding 25 mM resulting in an extensive growth lag. Bicarbonate is not required for growth of this strain on acetate, but addition of bicarbonate shortens the lag phase in media with high initial acetate concentration. A spontaneous mutant which exhibited a minimal lag phase and rapid growth rates on acetate media was derived from strain St. Louis. This mutant possessed elevated levels of the glyoxylate cycle enzyme, isocitrate lyase.     

The influence of acetate on the oxidation of sulfide by Rhodopseudomonas capsulata

The capacity to oxidize sulfide and the influence of the simultaneous presence of acetate in heterotrophically (acetate) and autotrophically (sulfide/CO₂) grown Rhodopseudomonas capsulata was investigated.Sulfide oxidation of acetate-limited cultures was found inversely related to the specific growth rate. Upon acetate deprevation (metering pump stopped) increased rates of sulfide oxidation were observed. This points to the existence of a constitutive acceptor for the electrons from sulfide. It is suggested that a carrier functional in the light-induced cyclic electron flow operates as such. The rate of sulfide oxidation, however, is low when compared to autotrophically-grown cells. This is probably due to the low levels of Calvin cycle enzymes present in the acetate-grown cells.In cells growing on sulfide/CO₂, the addition of acetate resulted in less sulfide being oxidized. Upon depletion of the acetate, the rate of sulfide oxidation again increased, however, insufficiently to maintain the accelerated growth rate. This indicates that under mixotrophic conditions the enzymes of the Calvin cycle are being synthesized to a far lesser extent.Non-Standurd Abbreviations PHB poly--hydroxybutyric acid - D dilution rate - TCA Tri carboxylic acid cycle - RubPcase ribulose 1,5-bisphosphate carboxylase - RP reducing power     

A pleiotropic mutant of Rhodopseudomonas capsulata defective in nitrogen metabolism

Wild type strains of Rhodopseudomonas capsulata typically can use N₂, NH ₄ ⁺ , or various nitrogenous organic compounds as N sources for photosynthetic growth. One class of mutants selected for inability to grow on N₂ (Nif^–) also shows simultaneous loss of capacity to obtain N from numerous organic substrates. When supplied at relatively high concentrations, ammonia can be used as the sole N source for growth of such strains. Enzymatic analysis of one mutant (W11) indicates that the pleiotropic effect on N nutrition is neither due to detectable alteration in the activities of nitrogenase or the initial enzymes responsible for bulk assimilation of ammonia (glutamine synthetase and glutamate synthase) nor to absence of systems required for catabolism of organic N sources. The phenotype of W11 (Nit^–; defective in N metabolism) appears to result from loss of ability to grow using low concentrations of ammonia (supplied externally or generated in vivo).     

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     

Characterization of Rhodopseudomonas capsulata

Thirty-three strains of Rhodopseudomonas capsulata have been studied in order to develop a more comprehensive characterization of the species. On the basis of morphological, nutritional, physiological and other properties, the characteristics of an ideal biotype have been defined, which can be used to distinguish Rps. capsulata from similar purple bacteria. In this connection, two properties of Rps. capsulata are of particular note: a) sensitivity to penicillin G is 10³–10⁵ times greater than that shown by closely related species, and b) all strains examined are susceptible to lysis by one or more strains of host species-specific virulent bacteriophages. It appears that members of the species Rps. capsulata form a stringent taxonomic grouping.     

Ammonium uptake in Rhodopseudomonas capsulata

Investigations of the uptake of ammonium (NH ₄ ⁺ ) by Rhodopseudomonas capsulata B100 supported the presence of an NH ₄ ⁺ transport system. Experimentally NH ₄ ⁺ was determined by electrode or indophenol assay and saturation kinetics were observed with two apparent K _m's of 1.7 M and 11.1 M (pH 6.8, 30°) and a V _max at saturation of 50–60 nmol/min·mg protein. The optimum pH and temperature were 7.0 and 33° C, respectively. The Q₁₀ quotient was calculated to be 1.9 at 100 M NH ₄ ⁺ , indicating enzymatic involvement. In contrast to the wild type, B100, excretion of NH ₄ ⁺ , not uptake, was observed in a glutamine auxotroph, R. capsulata G29, which is derepressed for nitrogenase and lacks glutamine synthetase activity. G29R1, a revertant of G29, also took up NH ₄ ⁺ at the same rate as wild type and had fully restored glutamine synthetase activity. Partially restored derivatives, G29R5 and G29R6, grew more slowly than wild type on NH ₄ ⁺ as the nitrogen source, remained derepressed for nitrogenase in the presence of NH ₄ ⁺ , and displayed rates of NH ₄ ⁺ uptake in proportion to their glutamine synthetase activity. Ammonium uptake and glutamine synthetase activity were also restored in R. capsulata G29 exconjugants which had received the plasmid pPS25, containing the R. capsulata glutamine synthetase structural gene. These data suggest that NH ₄ ⁺ transport is tightly coupled to assimilation.Abbreviations used CHES cyclohexylaminoethanesulfonic acid - GS glutamine synthetase - SDS sodium dodecylsulfate     

Conjugational transfer systems of Rhodopseudomonas capsulata mediated by R plasmids

Plasmids RP1, R68.45 and RP4::Mu cts 61 were transferred into Rhodopseudomonas capsulata from Escherichia coli. The frequency of intraspecies transfer of these plasmids in R. capsulata was 10^-4–10^-5 per donor. The plasmids also mobilized chromosomal genes at a low frequency. Phototrophic recombinants from matings between recipient strains defective in the photosynthetic-apparatus and wild type donors were obtained at a frequency of 10^-7–10^-8 per donor.     

Modulation by fumarate of a P i -insensitive pyruvate kinase from Rhodopseudomonas capsulata

Cold lability was found to be responsible for the initial failure to detect pyruvate kinase activity in extracts of the facultative phototroph, Rhodopseudomonas capsulata. Taking advantage of the reversal of cold inactivation by high concentrations of monovalent cations, the enzyme could be partially purified by (NH₄)₂SO₄-precipitation and gelfiltration. In contrast to the enzyme from Rhodospirillum rubrum, the pyruvate kinase from R. capsulata is nearly insensitive to inorganic phosphate. Instead, it is susceptible to allosteric inhibition by fumarate. Adenosinemonophosphate and sugar phosphates as activators prevent the inhibitory action of fumarate.     

Characterization of the plasmid DNAs of Rhodopseudomonas capsulata

Presence of extrachromosomal DNa in Rhodopseudomonas capsulata strain BH9 was shown by the appearance of a satellite band in a dye-buoyant density gradient. Radioactively labelled DNA was prepared from this satellite band and examined on a 5–20% sucrose gradient. Three radioactive peaks with sedimentation coefficients of 100 S, 94 S, and 58–64 S, respectively, were consistently observed. Analysis of these sedimentation coefficients suggested that there are two species of plasmid DNA with molecular sizes of 94×10⁶ daltons (named pBH91) and 74×10⁶ daltons (named pBH92). The 58–64 S peak is attributed to open circular molecules. DNAs from each peak of the sucrose gradient were examined by electronmicroscopy, and the results agree closely with those of the sucrose gradient analysis. Reassociation kinetics of the plasmid DNA was also followed. Addition of total DNA of strain BH9 increased the renaturation rate of the plasmid DNA. It was calculated from the magnitude of the increase that approximately 10% of the BH9 total DNA may hybridize with the plasmid sequences. DNA prepared from the gene transfer agent (GTA) produced by R. capsulata increases the renaturation rate of the plasmid to the same extent as total DNA isolated from the GTA producing strain, Y262.     

Diazotrophic growth of Rhodopseudomonas acidophila and Rhodopseudomonas capsulata under microaerobic conditions in the dark

Diazotrophy of Rhodopseudomonas acidophila and Rhodopseudomonas capsulata was not obligatorily linked to photosynthesis. In the dark R. acidophila grew with dinitrogen as sole nitrogen source at a dissolved oxygen tension of 15 Torr (= 2.0 kPa); the doubling time was 8 h. Acetylene reduction by whole cells was more sensitive to oxygen in the light than in the dark. 16.5 mg N₂ were fixed per g lactic acid consumed. R. capsulata synthesized nitrogenase and fixed dinitrogen in the dark at a dissolved oxygen tension of less than one Torr (= 0.13 kPa). The doubling time of this bacterium was 16 h and 10.5 mg N₂ were fixed per g lactic acid consumed.Abbreviation kPa kilopascal     

Respiratory deficient mutants of Rhodopseudomonas capsulata

The facultative photosynthetic bacterium Rhodopseudomonas capsulata was mutagenized by transfer of the plasmid pSUP201::Tn5 from Escherichia coli to R. capsulata. Mutants defective in cytochrome oxidase and other respiratory functions were selected by replica plating, NADI-reaction and immunological methods. Among 20,000 mutants no clone was detected, which lacks the 65,000-protein of the cytochrome oxidase, but many mutants have been isolated which were cytochrome oxidase deficient (or inactive). Other mutants excrete heme and cytochrome c into the medium and lack cytochrome c ₂.Abbreviations Ap ampicillin - CIE crossed immunoelectrophoresis - cyt cytochrome - Cm chloramphenicol - Km kanamycin - SDS sodium dodecylsulfate - Tc tetracycline     

Tricarboxylic acid and glyoxylate cycle enzyme activities in Thiobacillus versutus,an isocitrate lyase negative organism

An analysis was made of the specific enzyme activities of the TCA and glyoxylate cycle in Thiobacillus versutus cells grown in a thiosulphate- or acetate-limited chemostat. Activities of all enzymes of the TCA cycle were detected, irrespective of the growth substrate and they were invariably lower in the thiosulphate-grown cells. Of the glyoxylate cycle enzymes, isocitrate lyase was absent but malate synthase activity was increased from 15 nmol·min^-1·mg^-1 protein in thiosulphate-grown cells to 58 nmol·min^-1·mg^-1 protein in acetate-grown cells. Suspensions of cells grown on thiosulphate were able to oxidize acetate, although the rate was 3 times lower than that observed with acetate-grown cells. The respiration of acetate was completely inhibited by 10 mM fluoroacetate or 5 mM arsenite. Partially purified citrate synthase from both thiosulphate- and acetate-grown cells was completely inhibited by 0.5 mM NADH and was insensitive to inhibition by 1 mM 2-oxoglutarate or 1 mM ATP. The specific enzyme activities of the TCA and glyoxylate cycle in T. versutus were compared with those of Pseudomonas fluorescens, an isocitrate lyase positive organism, after growth in a chemostat limited by acetate, glutarate, succinate or glutamate. The response of the various enzyme activities to a change in substrate was similar in both organisms, with the exception of isocitrate lyase.Abbreviations TCA tricarboxylic acid - DNTB 2,2-dinitro-5,5-dithiobenzoic acid - APAD acetylpyridine adenine dinucleotide - PMS phenazine methosulphate - DCPIP 2,6-dichlorophenol-indophenol - DOC dissolved organic carbon     

On insertion of pigment-associated polypeptides during membrane biogenesis in Rhodopseudomonas capsulata

Early stages in the formation of membranes and photosynthetic units were studied under growth-limiting phototrophic and chemotrophic conditions in cells of Rhodopseudomonas capsulata. The incorporation of polypeptides, forming bacteriochlorophyll-carotinoid-protein complexes in the membrane, was followed by use of pulse-labeling and immunoprecipitation techniques. The newly synthesized polypeptides were inserted into two distinct membrane fractions at both different rates and proportions. The two membrane fractions differed in sedimentation behavior, absorption spectra and activities of the respiratory chain. The individual pigment-associated proteins did not exhibit precursor-product relationship between the two membrane fractions. The data suggest that newly synthesized polypeptides were integrated both into cytoplasmic and pre-existing intracytoplasmic membranes, where the proteins and pigments were assembled to form reaction centers and light-harvesting pigment-protein complexes.Abbreviations Bchl bacteriochlorophyll - cpm counts per minute - M _r relative molecular mass - P 100 pellet of 100,000xg, 60 min - P300 pellet of 300,000xg, 90 min - pO₂ oxygen partial pressure - R Rhodopseudomonas - dodecyl sulfate sodium dodecyl sulfate. International standard units - Bq Becquerel (s^-1) - Pa Pascal (N/m²; 1 Torr=133,3 Pa)     

Contribution of dissolved dinitrogen in culture solutions to growth of Rhodopseudomonas capsulata with various sources of combined nitrogen

Fixation of dissolved dinitrogen in culture solutions by the photosynthetic bacterium Rhodopseudomonas capsulata, strain B10, reduced the lag phase associated with growth with glutamate. A comparable effect was not observed with ammonium chloride. This strain assimilated nitrate but nitrogen fixation was depressed during early growth on nitrate. It is shown that nitrite, the first product of nitrate assimilation, inhibits nitrogen fixation during the early stages of cell growth.     

Depression of the synthesis of the intermediate and large forms of ribulose-1,5-bisphosphate carboxylase/oxygenase in Rhodopseudomonas capsulata

Rhodopseudomonas capsulata produces both an intermediate (I) and a large (L) form of ribulose-1,5-bisphosphate carboxylase/oxygenase. Both forms are derepressed under CO₂-limiting conditions. The L-form of the enzyme is completely repressed when the culture is grown either photoautotrophically or photoheterotrophically with malate as the electron donor. The L-form is derepressed in the late logarithmic phase of growth when cells are grown photoheterotrophically with butyrate as the electron donor and the NaHCO₃ supplement is 0.01%. The level of the I-form is increased about fivefold under latter growth conditions when compared to malate-grown cells. Analytical ultracentrifugation revealed the molecular masses of the I-and L-forms to be 300,000 and 542,000, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the I-form to be composed of only one type subunit with a molecular weight of 64,000. The L-form possessed both large and small subunits with molecular weights of 58,000 and 10,000.     

Oxygen inhibition of the photoassimilation of CO2 in Rhodopseudomonas capsulata

The photoassimilation of ¹⁴CO₂ by washed cells of the photosynthetic bacterium Rhodopseudomonas capsulata was greatly inhibited in air. The inhibition was partially reversed by either sparging with argon or by adding inhibitors, e.g. CO [50% (v/v) in air] and NaN₃ (0.2 mM), which at these concentrations effectively restricted respiration. The effect of oxygen on the photoassimilation of ¹⁴CO₂ may be associated with a change in the redox state of the cells resulting in less reducing equivalents being available for this process.     

Evidence for an involvement of glutamine synthetase in regulation of nitrogenase activity in Rhodopseudomonas capsulata

In the presnet studies with whole cells and extracts of the photosynthetic bacterium Rhodopseudomonas capsulata the rapid inhibition of nitrogenase dependent activities (i.e. N₂-fixation acetylene reduction, or photoproduction of H₂) by ammonia was investigated. The results suggest, that the regulation of the nitrogenase activity by NH ₄ ⁺ in R. capsulata is mediated by glutamine synthetase (GS). (i) The glutamate analogue methionine sulfoximine (MSX) inhibited GS in situ and in vitro, and simultaneously prevented nitrogenase activity in vivo. (ii) When added to growing cultures ammonia caused rapid adenylylation of GS whereas MSX abolished the activity of both the adenylylated and unadenylylated form of the enzyme. (iii) Recommencement of H₂ production due to an exhaustion of ammonia coincided with the deadenylylation of GS. (iv) In extracts, the nitrogenase was found to be inactive only when NH ₄ ⁺ or MSX were added to intact cells. Subsequently the cells had to be treated with cetyltrimethylammonium bromide (CTAB). (v) In extracts the nitrogenase activity declined linearily with an increase of the ration of adenylylated vs. deadenylylated GS. A mechanism for inhibition of nitrogenase activity by ammonia and MSX is discussed.Abbreviations BSA bovin serum albumine - CTAB cetyltrimethylammonium bromide - GOGAT l-glutamine: 2-oxoglutarate amino transferase - GS glutamine synthetase - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MSX l-methionine-d,l-sulfoximine     

Characterization of three membrane fractions isolated from cells of Rhodopseudomonas capsulata adapting from chemotrophic to phototrophic conditions

By means of sucrose density centrifugation three membrane fractions, named light, medium and heavy have been isolated from cells of Rhodopseudomonas capsulata strain 37b4, adapting from chemotrophic to phototrophic growth conditions. Succinate dehydrogenase activity of aerobically grown cells was mainly confined to the heavy (chromatophore) fraction. Upon changing to phototrophic conditions the activity of the succinate dehydrogenase increased in the medium and light fraction. All fractions contain bacteriochlorophyll. NADH dehydrogenase of chemotrophically grown cells was enriched in the light and medium fraction but is increased in the heavy fraction under phototrophic growth conditions. The capacity of photophosphorylation is high in the light and heavy fraction. The results indicate a differentially incorporation of functional subunits into specific parts of the membrane system during membrane differentiation.Abbreviations Bchl bacteriochlorophyll - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DCCD N,N-dicyclohexyl carbodiimide     

The capsule and slime polysaccharides of the wild type and a phage resistant mutant ofRhodopseudomonas capsulata St. Louis

A rhamnose, galactose and pyruvic acid containing polysaccharide (capsule) together with the peptidoglycan was isolated fromRhodopseudomonas capsulata St. Louis as the insoluble sediment after sodium dodecyl sulfate extraction of cell envelope fractions. Treatment with pronase E separated the soluble polysaccharide from the insoluble peptidoglycan. After lysozyme-digestion, both the capsule polysaccharide and peptidoglycan were soluble.The capsule was also accumulated in the combined interphase/phenol-phase of hot phenol-water extracts of whole cells. Again, the capsule and peptidoglycan were sedimented together as long as no pronase E-treatment was performed. With the phage-resistant mutant (R. capsulata St. Louis RC1^-), no capsule polysaccharide was obtained in the combined interphase/phenol phase.An acidic polysaccharide (slime) different from the capsule in composition and serology was obtained by Cetavlon fractionation of hot phenol/water extracts of cells of both the wild-type and the mutant cells. It was shown to consist mainly of rhamnose, glucosamine and galacturonic acid.The use of O/K-antisera and of capsule polysaccharideantisera allowed a separate visualization of the capsule and slime layers.This paper is dedicated to Professor Hans G. Schoegel on the occasion of his 60th birthday