Roland Dierstein

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.

Nitrogen-limited continuous culture ofRhodopseudomonas capsulata growing photosynthetically or heterotrophically under low oxygen tensions

A continuous culture apparatus is described, which allows cultivation of photosynthetic bacteria anaerobically in the light and semiaerobically in the dark at constant oxygen tensions. The growth-parameters 1. substrate concentration at half maximum growth rate (Ks) and 2. yield (Y) forRhodopseudomonas capsulata are calculated.An automatic control system of the oxygen partial pressure in the culture medium is elaborated. It is shown, that the discontinuous regulation with a control unit in connection with two magnetic valves, which give short pulses of either oxygen-free gas or gas mixed with oxygen, is an economic, practicable and reliable method.The yield coefficientY during growth limited by ammonium sulfate is variable due to the synthesis of nitrogen independent metabolites, such as poly-β-hydroxybutyrate. The storage of poly-β-hydroxybutyrate in continuous culture is a function of both the actual concentration of ammonia and of theC/N ratio. At very low growth rates (1/6 µm) the poly-β-hydroxybutyrate content increases amounting to 33% of the dry weight.In semiaerobically dark grown cells (pO2: 5 mm Hg) the protein and bacteriochlorophyll content increased definitely on dry weight basis with increasing growth rates. In contrast, in anaerobic light cultures only a small increase of the bacteriochlorophyll level but no change of the protein content per dry weight of cells was observed at increasing growth rates.

Genetic transfer of the capacity to form bacteriochlorophyll-protein complexes in Rhodopseudomonas capsulata.

Reaction center (RC) and light harvesting (LH) complexes are major constituents of the bacterial photosynthetic apparatus [ 1,2] . RC of Rhodopseudomonas (R.) capsulata and of R. sphaeroides contain three proteins (molecular weights 28,24 and 21 kD) and RC bacteriochlorophyll (b&l) [3,4]. The LH-bchl of these species was proposed to be associated with one or more proteins of about 10 kD [5-lo]. It has been shown that the amounts of both RC bchl and LH-bchl correlate with their respective proteins, under various physiological conditions. The nature of co-regulation of the biosynthesis of the two non-covalently bound constituents of membrane subparticles is at present not understood. Recently a bacteriophage-like particle (GTA) with linear and double-stranded DNA (molecular weight 3.6 106) was discovered ( [ 131 and B. Marrs, personal communication), which efficiently transfers genes between various strains of R. capsulata [ 14,l S] . In this study the transfer of genes for bchl, proteins, and carotenoids, resulting in reconstitution of a functional photosynthetic apparatus, will be described.

Control of composition and activity of the photosynthetic apparatus of Rhodopseudomonas capsulata grown in ammonium-limited continuous culture

Rhodopseudomonas capsulata was grown either phototropically in the light or chemotrophically in the dark at oxygen tensions of 5 mm and 3 mm Hg in ammonium-limited continuous culture. During growth limitation bacteriochlorophyll content of cells and membranes varied dependent on growth rate drastically in chemotrophic cultures. Concomittantly, the ratio of membrane protein to total protein varied in the range of 30–41%. This dependence of membrane differentiation on growth rate was less evident in phototrophically grown cells.The incorporation of the bulk of bacteriochlorophyll was shown to be quantitatively correlated to the incorporation of 1–3 low molecular weight proteins with molecular weights in the range of 14 to less than 10 k daltons. Supported by similar findings of other authors it is proposed, that these proteins are to be attributed to the species of antenna bacteriochlorophyll and represent components of the photosynthetic apparatus. With decreasing growth rates the size of the photosynthetic unit with respect to the population of bacteriochlorophyll-and protein molecules was reduced subsequent to a reduction in the rate of incorporation of antenna-bacteriochlorophyll and the low molecular weight proteins, the reaction-center bacteriochlorophyll content of the membranes remaining constant. A parallel decrease in potential phosphorylating capacity was observed.It is concluded, that under these conditions, primary photochemical reactions in the reaction center were not the rate-limiting step in photophosphorylation. The interaction of growth limitation by an anabolic precursor (NH4+) and control of membrane differentiation by light intensity or oxygen tension is discussed.

Biosynthesis of pigment‐protein complex polypeptides in bacteriochlorophyll‐less mutant cells of Rhodopseudomonas capsulata YS

Bacteriochlorophyll‐less mutant cells of Rhodopseudomonas capsulata YS were capable of synthesizing pigment—protein complex polypeptides under conditions permitting the formation of the photosynthetic apparatus in the wild type. Individual polypeptides were identified by immunoprecipitation. Pulse‐chase experiments revealed that the polypeptides did not accumulate in the membranes but underwent rapid degradation. The data suggest that bacteriochlorophyll is needed to stabilize the polypeptides of pigment—protein complexes.

Nontoxic and toxic oligopeptides with D-amino acids and unusual residues in Microcystis aeruginosa PCC 7806

Toxic and nontoxic peptides were isolated from the cyanobacterium Microcystis aeruginosa PCC 7806 by a procedure including extraction of cells with water-saturated 1-butanol, chromatography of the extract on silica gel plates and high performance liquid chromatography (HPLC) on Partisil-5. The toxin was shown to be only a minor constituent, being negatively charged and thus separable by electrophoresis, within the HPLC-purified fraction. It contained erythro-β-methyl-D-Asp, D-Glu, D-Ala, L-Leu, and L-Arg known to be part of the Microcystis peptide-toxin with Mr 994. The major part of the HPLC-purified fraction was assigned, however, to a nontoxic peptide with a Mr of 956. Partial hydrolysis studies of the nontoxic peptide(s) revealed amino acid sequences composed of D-Glu, N-methyl-Phe, and 3,4-dehydro-Pro, aside from the common L-amino acids. Cyclic linkage in the nontoxic peptide(s) appears likely.

Two closely related peptide toxins in axenically grown Microcystis aeruginosa PCC 7806.

Summary Two closely related peptide toxins from axenically grown Microcystis aeruginosa PCC 7806 were purified using molecular mass, charge and solubility as criteria. Water extracts of cells were applied to reversed phase C18 cartridges, and toxic fractions eluting between 20% and 100% methanol in water (vol/vol) were subjected to gel filtration. The fraction with an apparent M r of about 1 kDa was further separated by analytical paper electrophoresis and preparative ion exchange chromatography. Acidic, ninhydrin-negative toxic material was finally purified by reversed phase high performance liquid chromatography yielding two distinct toxic peptides. The LD 100 for mice was determined at 0.075 mg per kg body weight for each toxin. The major toxin (T17) with M r 994 was composed of equimolar amounts of erythro -β-methyl-D-aspartic acid, D-glutamic acid, D-alanine, L-leucine, L-arginine, N-methyl dehydroalanine and 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid (Adda). The other toxin (T16) with M r 980 had an identical composition except for a replacement of erythro -β-methyl-D-aspartic acid by D-aspartic acid. Mass spectrometric data suggested cyclic linkage for both toxins.

Isolation and characterization of two hydrocarbon-degrading Bacillus subtilis strains from oil contaminated soil of Kuwait.

Two strains of hydrocarbon-utilizing bacteria were isolated from soil samples of the Kuwait Burqan oil field at a temperature of 37 degrees C. The bacteria were motile endospore-forming rods with slight differences in their metabolic patterns and 16S rRNA sequence. Vegetative cells of the strains designated as AHI and AHII had an ultrastructure typical of gram-positive bacteria and showed gram-positive staining. The bacteria did not show pigmentation. Best growth was observed at 37 degrees C at neutral pH and NaCl concentrations in the range of 5-10 g per l. Both strains were obligatory aerobic and developed on synthetic media with either Diesel fuel, n-decan or naphthalene as the sole carbon and energy source. No specific growth factors were required. On the basis of their morphological, physiological and biochemical features, as well as their 16S rRNA analysis and electron microscope study, both strains were assigned to the species of Bacillus subtilis.

Isolation and complete amino acid sequence of the β‐ and α‐polypeptides from the peripheral light‐harvesting pigment‐protein complex II of Rhodobacter sulfidophilus

The peripheral light‐harvesting bacteriochlorophyll‐carotenoid‐protein complex B800‐850 (LHII) has been isolated from membranes of semi‐aerobic dark‐grown cells of Rhodobacter sulfidophilus strain W4. A reversed‐phase HPLC system resolved one β‐ and one α‐polypeptide in the ratio 1:1. The material obtained was of high purity and suitable for direct microsequence analysis. The primary structures of the β‐ and α‐polypeptides have been determined. The β‐polypeptide consists of 51 amino acid residues, yielding a molecular mass of 5512 Da and having 64.7% hydrophobicity. The α‐polypeptide consists of 52 amino acid residues, with a calculated molecular mass of 5661 Da and 75% hydrophobicity. The significance of uncommon structure motives with respect to the unusual spectroscopic characteristics of this light‐harvesting complex is discussed.

Inhibition of prodigiosin formation in Serratia marcescens by extracts of toxic cyanobacteria

Summary Inhibition of prodigiosin formation in growing cells of the chromogenic bacterium Serratia marcescens was studied with toxic strains of the cyanobacteria Microcystis aeruginosa and Aphanizomenon flosaquae . Increasing amounts of extracts added to cultures of Serratia marcescens caused a dose-dependent decrease in the spectral absorbance of prodigiosin in whole cells. Purified microcystin and saxitoxin were assayed as the representative toxins of Microcystis aeruginosa and Aphanizomenon flos-aquae , respectively. With Microcystis aeruginosa , pigmentation inhibition was not caused primarily by the characteristic peptide toxin but by a yet unknown low molecular weight component. By contrast, saxitoxin directly inhibited prodigiosin formation which was measured by a specific effect on the spectral absorbance. Thus, pigmentation inhibition in Serratia marcescens might be employed as a quantitative assay for the detection of toxic cyanobacteria.