The Rhodobacter sphaeroides structural gene (puhA) for the reaction center H polypeptide has been identified and cloned by using restriction fragements specific for the analogous Rhodobacter capsulatus gene as a heterologous hybridization probe. The presence of puhA on a 1.45-kilobase BamHI restriction fragment was confirmed by partial DNA sequence analysis and by the synthesis of an immunoreactive Mr-28,000 reaction center H polypeptide in an R. sphaeroides coupled transcription-translation system. Approximately 450 base pairs of DNA upstream of the puhA gene were sufficient for expression of this protein in vitro. Northern RNA-DNA blot analysis with an internal puhA-specific probe identified at least two, apparently monocistronic, transcripts present at different cellular levels under physiological conditions known to affect the cellular content of both reaction center complexes and photosynthetic membrane. Northern blot analysis with specific upstream restriction fragment probes revealed that the 1,400-nucleotide puhA-specific mRNA had a 5' terminus upstream of the 1,130-nucleotide transcript. Both puhA-specific mRNA and immunoreactive reaction center H protein were detectable in chemoheterotrophically grown cells which lacked detectable bacteriochlorophyll and photosynthetic membrane.

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The reaction center of Rhodopseudomonas sphaeroides is an integral membrane protein complex responsible for primary photochemical charge separation in photosynthesis. We report the synthesis of two of the three subunits of the photosynthetic reaction center using a DNA-directed in vitro transcription-translation system prepared from R. sphaeroides. The in vitro-synthesized polypeptides, as resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, had apparent Mrs of 24,000 and 21,000 and were shown to be synthesized in equimolar amounts. This corresponds precisely to the in vivo reaction center subunits M and L, respectively. The in vitro-synthesized polypeptides were immunoprecipitated with antibody prepared against whole native reaction centers. In addition, the identity of the in vitro-synthesized polypeptides as L and M was verified by comparing the protease digestion products of in vivo-with in vitro-synthesized reaction center subunits. Both of the in vitro-synthesized polypeptides were also found to partition with the particulate material in the transcription-translation system and to associate with added membranes.When Rhodopseudomonas sphaeroides is grown chemoheterotrophically in the presence of oxygen, it is a typical gram-negative bacterial cell; when the oxygen tension is reduced below threshold levels, the cell responds by synthesizing an extensive intracytoplasmic membrane system (13). This new membrane houses the numerous components of the photosynthetic apparatus. In addition to regulation by oxygen tension, the synthesis of the components necessary for photophosphorylation is also modulated by light intensity (19). Given these attributes, the photosynthetic bacteria, and R. sphaeroides in particular, have been employed as a model system for the study of membrane biosynthesis and photosynthesis at the molecular level (20).One of the key complexes within the intracytoplasmic membrane is the photosynthetic reaction center (RC). The RC used light energy in the formation of high-energy electrons, and it is these electrons which are the driving force of photosynthetic electron transport. Studies have compared R. sphaeroides RC with the photosystem II RC of chloroplasts (17). Three integral membrane protein subunits, designated H, M, and L, make up the R. sphaeroides RC. In addition to the three polypeptides, there are four bacteriochlorophylls, two bacteriopheophytins, two quinones, and one iron in a functional RC (27). Polypeptides H, M, and L are extremely hydrophobic and have apparent Mrs of 28,000, 24,000, and 21,000, respectively, based on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, although these are not the true molecular weights (27). The actual role of each subunit is unclear, but dissociation of H from L and M leaves the L-M complex with spectral and functional properties similar to those of the whole RC (10).Despite extensive biochemical analysis of the photosynthetic membranes and RCs, very little is known about the assembly of these proteins or the regul...

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