A novel genetically engineered pathway for synthesis of poly(hydroxyalkanoic acids) in Escherichia coli. (1/6)

A new pathway to synthesize poly(hydroxyalkanoic acids) (PHA) was constructed by simultaneously expressing butyrate kinase (Buk) and phosphotransbutyrylase (Ptb) genes of Clostridium acetobutylicum and the two PHA synthase genes (phaE and phaC) of Thiocapsa pfennigii in Escherichia coli. The four genes were cloned into the BamHI and EcoRI sites of pBR322, and the resulting hybrid plasmid, pBPP1, conferred activities of all three enzymes to E. coli JM109. Cells of this recombinant strain accumulated PHAs when hydroxyfatty acids were provided as carbon sources. Homopolyesters of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB), or 4-hydroxyvalerate (4HV) were obtained from each of the corresponding hydroxyfatty acids. Various copolyesters of those hydroxyfatty acids were also obtained when two of these hydroxyfatty acids were fed at equal amounts: cells fed with 3HB and 4HB accumulated a copolyester consisting of 88 mol% 3HB and 12 mol% 4HB and contributing to 68.7% of the cell dry weight. Cells fed with 3HB and 4HV accumulated a copolyester consisting of 94 mol% 3HB and 6 mol% 4HV and contributing to 64.0% of the cell dry weight. Cells fed with 3HB, 4HB, and 4HV accumulated a terpolyester consisting of 85 mol% 3HB, 13 mol% 4HB, and 2 mol% 4HV and contributing to 68.4% of the cell dry weight.  (+info)

Thiocapsa litoralis sp. nov., a new purple sulfur bacterium from microbial mats from the White Sea. (2/6)

A new phototrophic purple sulfur bacterium, isolated from benthic microbial mats from the White Sea littoral zone, is described. Individual cells were spherical, non-motile and lacked gas vesicles. In pure cultures cells appeared in regular platelet-like arrangements of four, eight or sixteen cells. Cell division occurred inside a common envelope, surrounded by a thick capsule. Internal photosynthetic membranes were of the vesicular type. The colour of cell suspensions was pink to rose-red. Bacteriochlorophyll a and carotenoids of the spirilloxanthin series were found as photosynthetic pigments. Under anoxic conditions in the light, photolithoautotrophic growth occurred with sulfide, thiosulfate, sulfite and elemental sulfur as electron donors. Sulfur globules were stored as an intermediary oxidation product and were visible microscopically inside the cells. In the presence of sulfide and bicarbonate, photomixotrophic growth occurred with a number of organic substrates. Sulfate could serve as sole assimilatory source of sulfur. Chemolithoautotrophic growth in the dark was possible with sulfide and thiosulfate as electron donors. Optimum growth occurred in the presence of 1% NaCl, at pH 6.5 and at 30 degrees C. The DNA base composition of the type strain, BM5T, was 64.0 mol% G+C. According to 16S rDNA sequence information and DNA-DNA hybridization, the new isolate clearly belongs to the genus Thiocapsa, but is sufficiently different from other recognized Thiocapsa species to be described as a new species of this genus for which the name Thiocapsa litoralis sp. nov. is proposed. The type strain is BM5T (= ATCC 700894).  (+info)

Accessory proteins functioning selectively and pleiotropically in the biosynthesis of [NiFe] hydrogenases in Thiocapsa roseopersicina. (3/6)

There are at least two membrane-bound (HynSL and HupSL) and one soluble (HoxEFUYH) [NiFe] hydrogenases in Thiocapsa roseopersicina BBS, a purple sulfur photosynthetic bacterium. Genes coding for accessory proteins that participate in the biosynthesis and maturation of hydrogenases seem to be scattered along the chromosome. Transposon-based mutagenesis was used to locate the hydrogenase accessory genes. Molecular analysis of strains showing mutant phenotypes led to the identification of hupK (hoxV ), hypC1, hypC2, hypD, hypE, and hynD genes. The roles of hynD, hupK and the two hypC genes were investigated in detail. The putative HynD was found to be a hydrogenase-specific endoprotease type protein, participating in the maturation of the HynSL enzyme. HupK plays an important role in the formation of the functionally active membrane-bound [NiFe] hydrogenases, but not in the biosynthesis of the soluble enzyme. In-frame deletion mutagenesis showed that HypC proteins were not specific for the maturation of either hydrogenase enzyme. The lack of either HypC protein drastically reduced the activity of every hydrogenase. Hence both HypCs might participate in the maturation of [NiFe] hydrogenases. Homologous complementation with the appropriate genes substantiated the physiological roles of the corresponding gene products in the H2 metabolism of T. roseopersicina.  (+info)

Thiocapsa marina sp. nov., a novel, okenone-containing, purple sulfur bacterium isolated from brackish coastal and marine environments. (4/6)

Four marine, phototrophic, purple sulfur bacteria (strains 5811T, 5812, BM-3 and BS-1) were isolated in pure culture from different brackish to marine sediments in the Mediterranean Sea, the White Sea and the Black Sea. Single cells of these strains were coccus-shaped, non-motile and did not contain gas vesicles. The colour of cell suspensions that were grown in the light was purple-red. Bacteriochlorophyll a and carotenoids of the okenone series were present as photosynthetic pigments. Photosynthetic membrane systems were of the vesicular type. Hydrogen sulfide, thiosulfate, elemental sulfur and molecular hydrogen were used as electron donors during photolithotrophic growth under anoxic conditions; carbon dioxide was utilized as the carbon source. During growth on sulfide, elemental sulfur globules were stored inside the cells. In the presence of hydrogen sulfide, several organic substances could be photoassimilated. Comparative 16S rDNA sequence analysis revealed an affiliation of these four strains to the genus Thiocapsa. Both phylogenetic analysis and the results of DNA-DNA hybridization studies revealed that these strains formed a separate cluster within the genus Thiocapsa. Thus, according to phenotypic characteristics and mainly the carotenoid composition, 16S rDNA sequence analysis and DNA-DNA hybridization data, it is proposed that these strains should be classified as a novel species, Thiocapsa marina sp. nov., with strain 5811T (=DSM 5653T=ATCC 43172T) as the type strain.  (+info)

Cross-crystallization method used for the crystallization and preliminary diffraction analysis of a novel di-haem cytochrome c4. (5/6)

The newly discovered di-haem cytochrome c4 from the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina is the first cytochrome c4 to be crystallized from an anaerobic organism. It was crystallized using the addition of metal-ion salts to the standard vapour-diffusion method. Coloured well shaped three-dimensional crystals with dimensions of approximately 0.6 x 0.05 x 0.02 mm grew within 3-4 d at pH 5 and diffracted to 1.72 angstroms without radiation damage. Cytochrome c4 crystallized in space group P4(1)2(1)2 as a primitive tetragonal system with unit-cell parameters a = b = 75.29, c = 37.12 angstroms, alpha = beta = gamma = 90 degrees.  (+info)

Anaerobic phototrophic nitrite oxidation by Thiocapsa sp. strain KS1 and Rhodopseudomonas sp. strain LQ17. (6/6)

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