Search

The NH2 termini of light-harvesting complex I (LHI) polypeptides alpha and beta of Rhodobacter capsulatus are thought to be involved in the assembly of the LHI complex. For a more detailed study of the role of the NH2-terminal segment of the LHI alpha protein in insertion into the intracytoplasmic membrane (ICM) of R. capsulatus, amino acids 6 to 8, 9 to 11, 12 and 13, or 14 and 15 of the LHI alpha protein were deleted. Additionally, the hydrophobic stretch of the amino acids 7 to 11 was lengthened by insertion of hydrophobic or hydrophilic amino acids. All mutations abolished the ability of the mutant strains to form a functional LHI antenna complex. All changes introduced into the LHI alpha protein strongly reduced the stability of its LHI beta partner protein in the ICM. The effects on the mutated protein itself, however, were different. Deletion of amino acids 6 to 8, 9 to 11, or 14 and 15 drastically reduced the amount of the LHI alpha protein inserted into the membrane or prevented its insertion. Deletion of amino acids 12 and 13 and lengthening of the stretch of amino acids 7 to 11 reduced the half-life of the mutated LHI alpha protein in the ICM in comparison with the wild-type LHI alpha protein. Under the selective pressure of low light, revertants which regained a functional LHI antenna complex were identified only for the mutant strain deleted of amino acids 9 to 11 of the LHI alpha polypeptide [U43 (pTPR15)]. The restoration of the LHI+ phenotype was due to an in-frame duplication of 9 bp in the pufA gene directly upstream of the site of deletion present in strain U43(pTPR15). The duplicated nucleotides code for the amino acids Lys, Ile, and Trp. Membranes purified from the revertants were different from that of the reaction center-positive LHI+ LHII- control strain U43(pTX35) in doubling of the carotenoid content and increase of the size of the photosynthetic unit. By separating the reaction center and LHI complexes of the revertants by native preparative gel electrophoresis, we confirmed that the higher amount of carotenoids was associated with the LHI proteins.

The transverse orientation of photosynthetic polypeptides in the membrane of Rhodopseudomonas capsulata was investigated using surface radioiodination of chromatophores and spheroplasts with the reagent 1,3,4,6-tetrachloro-3α, 6α-diphenylglycoluril. Native pigment-protein complexes were isolated by Triton X-100-polyacrylamide gel electrophoresis to identify and distinguish polypeptides not separated by sodium dodecylsulphate-polyacrylamide gel electrophoresis. The two antenna light-harvesting complexes were found to be asymmetrically oriented with polypeptides 14 kDa and 8 kDa of complex B800-850 and 12 kDa of B870 being exposed on either membrane surface. Subunits H and M of the photochemical reaction centre, and probably L, span the membrane.

The B800-850 light-harvesting complex of Rhodopseudomonas capsulata was crystallized in the presence of detergents. The crystals were obtained by a vapour diffusion technique, using polyethylene glycol as a precipitant. Crystals grew to a size of 0.5 × 0.5 × 0.3 mm within two weeks. Two different crystal forms were obtained; one is supposed to be triclinic, space group PI, the other orthorhombic, space group C2221. Both crystal forms diffract to approximately 1.0 nm. Absorption spectra and polyacrylamide gel electrophoresis demonstrate that all expected components, i.e. three polypeptides of apparent Mr,8000, 10000 and 14000, bacteriochlorophyll a and carotenoids, are present and in native conformation.

The larger (α) of the two bacteriochlorophyll-binding polypeptides of the antenna complex B870 of the photosynthetic apparatus of Rhodopseudomonas; capsulata was extracted by chloroform-methanol-ammonium acetate from the intracytoplasmic membrane and purified by column chromatography on LH60. The complete amino acid sequence has been determined. The Mr of the polypeptide is 6588. The protein consists of 58 amino acids, having a polarity of 28%.

The synthesis of the photosynthetic apparatus was induced in chemotrophically grown cultures of the wild type strain 37b4 of Rhodopseudomonas capsulata by lowering of oxygen partial pressure. In these induced but growth-limited cultures the amount of total RNA per cell increased. Using specific DNA-probes for genes of the light-harvesting complex B800–850 and for ribosomal RNA it was shown that the levels of mRNA for the proteins of the light-harvesting complex B800-850 and for ribosomal RNA increased. In the mutant strain G1pho+, missing the B800–850 complex, but synthesizing small amounts of the Mr 10000 polypeptide of the complex, the B800–850-specific mRNA was not increased during induction. It is concluded that the synthesis of the B800–850 complex is under transcriptional control and that, under these conditions, the level of rRNA also increased.

Chromatophores (intracytoplasmic inside-out membrane vesicles) from the mutant strain Ala+ of Rhodopseudomonas capsulata were treated with proteinase K in order to determine the orientation of the pigment-binding polypeptides of the photochemical reaction center and of the light-harvesting complex B870. Nine amino acid residues of the B870α polypeptide were cleaved up to position Leu 9 -Val 10 of the N-terminus and 22 residues up to position Val 22 -Tyr 23 of the β-chain N-terminus. From the reaction center L-chain the N-terminus up to position 27 (Val) was split off. The C-termini of the B870α and β polypeptides including the hydrophobic α-helical transmembrane portion remained intact. It is concluded that the N-terminal region of the pigment-binding polypeptides α and β of the B870 light-harvesting complex and of the L-chain of the reaction center point to the cytoplasm while the C-termini of the B870α,β polypeptides are exposed or pointing toward the periplasmic membrane surface.

The B1020 antenna pigment—protein complex of Rhodopseudomonas viridis was isolated and found to contain two polypeptide subunits of apparent M r 8000 and 12000. Near-neighbour relationships between both antenna 8-kDa and 12-kDa polypeptides were revealed by reversible chemical cross-linking. Cross-linked homooligomers, probably up to the respective hexamer, were observed. Subunit M of the photochemical reaction center (RC) and the membrane-bound cytochrome c -553/558 were cross-linked in high yield at a 1:1 stoichiometry. The heavy subunit H of the RC was cross-linked to the 8-kDa polypeptide.

The kinetics of protein phosphorylation was studied in cells of phototrophic cultures and in dark-grown cells induced to form the photosynthetic apparatus by lowering of the oxygen tension. Cells of Rhodobacter capsulatus grown in a malate medium with 0.2 mM potassium phosphate were shifted to semiaerobic conditions, and 32 PO 4 3− or [ 35 S]Met was added 25 min after induction. The label of both radioactive precursors appeared in the membrane fraction about 20 min after addition. The maximum of 32 P was found after 1 h of labeling in the α polypeptide of the light-harvesting (LH) complex 1 (13870). The LHIα protein was phosphorylated after insertion into the membrane. Chloramphenicol inhibited the phosphorylation of LHI a but not of phospholipids. The steady-state level of phosphorylation was higher in anaerobic cultures grown at the low light intensity of 2000 lux than in cultures grown at high light intensity of 35000 lux. The phosphate label did not change significantly during a chase with unlabelled phosphate for 2 h. The phosphoamino acids in LHIα were detected with monoclonal antibodies and radioautography of labeled and hydrolyzed LHIα. Serine was shown to be the amino acid with the highest phosphate content; threonine and tyrosine were weakly phosphorylated. From the positions of these three amino acids in LHIα it was concluded that serine-2, which is exposed on the cytoplasmic side of the membrane, is the main phosphorylated amino acid. P-threonine and P-tyrosine are exposed on the periplasmic surface of the membrane.

Membranes active in oxidative phosphorylation but inactive in photophosphorylation were isolated from a carotenoid-less and bacteriochlorophyll-less mutant strain of Rhodopseudomonas capsulata . These membranes were uncoupled and recombined with coupling factor from the same strain and with reaction centers from a photosynthetically active revertant strain. The reconstituted system was able to form ATP from ADP and inorganic phosphate under anaerobic conditions in the light. Rates of 2 nmoles of ATP formed per min per μg of bacteriochlorophyll were obtained. A high incorporation of 32 P from inorganic phosphate into ADP in the light as well as in the dark was observed, the activity of which was associated with coupling factor. This incorporation occurs both aerobically and anaerobically and appears to be independent of photophosphorylation.

Electron micrographs of phenol-water-extracted lipopolysaccharide (LPS) of Rhodopseudomonas capsulata show filamentous and netlike aggregates. Treatment of the LPS with sodium deoxycholate resulted in a reversible splitting into subunits. The LPS represents a cell wall constituent with O-antigenic specificity. In passive hemagglutination tests, high titers were obtained when erythrocytes sensitized with untreated or heat-treated LPS were incubated with antisera obtained by immunization of rabbits with whole cells of R. capsulata . The alkali-treated LPS was not active in this test. Mouse lethality tests have shown that the LPS of R. capsulata is less toxic than LPS of Escherichia coli . Also, the X-ray protection efficacy and the phagocytic activity stimulation of LPS from R. capsulata in mice are small, as compared with LPS of E. coli . Incubation of living bacteria in saline (37 C) resulted in a solubilization of an LPS-protein-lipid complex from the outer layer of the cell wall. The isolated complex contained the components which were found in the LPS. In addition, 20% amino acids and a large amount of palmitic and stearic acids, which are typical phospholipid components, were present.