The ‘light’ (L) and the ‘medium’ (M) subunits of the photosynthetic reaction centre from Rhodopseudomonas viridis were isolated and their amino‐terminal sequences, as well as the sequences of several chymotryptic peptides, determined. Rps. viridis DNA was cloned in the Escherichia coli plasmid pBR322. Mixed oligonucleotide probes derived from the amino acid sequences were synthesised and utilised to isolate one clone which contained the genes for the L and M subunits of the reaction centre as well as the α and β subunits of the light‐harvesting complex and part of the gene for the reaction centre cytochrome. The nucleotide sequences of the L and M subunit genes and the derived amino acid sequences are presented. The L subunit consists of 273 amino acids and has a mol. wt of 30 571. The M subunit consists of 323 amino acids and has a mol. wt of 35 902. The primary structure is discussed in the light of the recently published secondary and tertiary structure which has shown that both subunits contain five membrane‐spanning helices.
The puf operon in Rhodobacter sphaeroides is composed of the genes for the photosynthetic reaction center L and M subunits, light-harvesting antenna complex I, and one other open reading frame termed puJX.Complementation of a reaction center-deficient, photosynthetically incompetent pufLMX deletion strain in trans with a fragment containing the entire puf operon, including puJX and an additional 1,100 base pairs of DNA downstream of puJX, restored the reaction center and the photosynthesis-positive phenotype. Complementation of the same strain with puJBALM restores the reaction center to the level seen with the entire puf operon but not the photosynthesis-positive phenotype. Northern (RNA) blot analysis revealed that oxygen regulated transcription was not blocked in the absence of puJX and the downstream region. Spectroscopic and protein analyses indicated that the pigment-binding protein complexes, including the reaction center, were expressed and showed normal absorption characteristics. A 20% reduction in the amount of light-harvesting antenna complex II and a corresponding increase in the amount of light-harvesting antenna complex I were observed in the deletion strain harboring the plasmid with the puf insert lacking the puJX gene and the downstream region compared with those complemented with the entire puf operon and an additional downstream 1,100 base pairs.The purple nonsulfur bacterium Rhodobacter sphaeroides produces an extensive system of intracytoplasmic membranes (ICM) when grown photosynthetically or under conditions of low oxygen partial pressure (21). The ICM is physically continuous with the cytoplasmic membrane but functionally differentiated. The components for aerobic respiration are located in the cytoplasmic membrane, while the reaction center (RC) and two light-harvesting antenna complexes responsible for photosynthetic growth are located in the ICM (30, 31).The two light-harvesting complexes serve as antennae which capture and transfer light energy to the RC. Lightharvesting antenna complex I (LHI), characterized by a single infrared absorption maximum at 875 nm (at room temperature; 888 nm at 77K), is composed of two polypeptides, two bacteriochlorophyll (Bchl) a molecules, and two carotenoids (8). Light-harvesting antenna complex II (LHII), with two infrared absorption maxima at 800 and 850 nm, also consists of two polypeptides but contains three molecules of Bchl a and a single carotenoid (47).The RC is composed of three subunits termed H (heavy), M (medium), and L (light) on the basis of their migration properties in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The three subunits are present in a 1:1:1 stoichiometry and contain a Bchl a dimer, two monomeric molecules of Bchl a, two monomeric molecules of bacteriopheophytin a, two ubiquinones, and a nonheme iron (for a review, see reference 18). The RC undergoes reversible bleaching upon light excitation that can be measured at 860 or 600 nm. These two maxima have been assigned to the Qy and Qx transition ba...
The complete nucleotide sequence of the gene encoding the cytochrome subunit of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis, and the derived amino acid sequence are presented. The nucleotide sequence of the gene reveals the existence of a typical bacterial signal peptide of 20 amino acid residues which is not found in the mature cytochrome subunit. The gene encoding the cytochrome subunit is preceded by the gene encoding the M subunit. Both genes overlap by 1 bp. The mature cytochrome subunit consists of 336 amino acid residues; 73% of its amino acid sequence was confirmed by protein sequencing work. The mol. wt of the cytochrome subunit including the covalently bound fatty acids and the bound heme groups is 40 500. The internal sequence homology is low, despite the symmetric structure of the cytochrome subunit previously shown by X‐ray crystallographic analysis of the intact photosynthetic reaction centre. Sequence homologies to other cytochromes were not found.
A DNA probe to the signaling domain of a halobacterial transducer for phototaxis (HtrI) was used to clone and sequence four members of a new family of transducer proteins (Htps) in Halobacterium salinarium potentially involved in chemo- or phototactic signal transduction. The signaling domains in these proteins have 31-43% identity when compared with each other or with their bacterial analogs, the methyl-accepting chemotaxis proteins. An additional region of homology found in three of the Htps has 31-43% identity with HtrI. The Htps contain from 0 to 3 transmembrane helices and Western blotting showed that HtpIII is soluble. The arrangement of the domains in these Htps suggests a modular architecture in their construction.
The gene coding for the ‘heavy’ subunit of the photosynthetic reaction centre from Rhodopseudomonas viridis was isolated in an expression vector. Expression of the heavy subunit in Escherichia coli was detected with antibodies raised against crystalline reaction centres. The entire subunit, and not a fusion protein, was expressed in E. coli. The protein coding region of the gene was sequenced and the amino acid sequence derived. Part of the amino acid sequence was confirmed by chemical sequence analysis of the protein. The heavy subunit consists of 258 amino acids and its mol. wt. is 28 345. It possesses one membrane‐spanning α‐helical segment, as was revealed by the concomitant X‐ray structure analysis.
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