UBC Theses and Dissertations
Studies on inter-species expression of photosynthesis genes in Rhodobacter capsulatus Zilsel, Joanna
The primary amino acid sequences of the L, M, and H photosynthetic reaction center peptide subunits from a number of purple non-sulfur bacteria, including Rhodopseudomonas viridis, Rhodobacter sphaeroides, and Rhodobacter capsulatus have been previously shown to be highly homologous, and detailed X-ray crystallographic analyses of reaction centers from two species of purple non-sulfur bacteria, Rps. viridis and R. sphaeroides have shown that all recognized structural and functional features are conserved. Experiments were undertaken to determine whether genes encoding reaction center and light harvesting peptide subunits from one species could be functionally expressed in other species. Plasmid-borne copies of R sphaeroides and Rps. viridis pigment binding-peptide genes were independently introduced into a photosynthetically incompetent R. capsulatus mutant host strain, deficient in all known pigment-binding peptide genes. The R. sphaeroides puf operon, which encodes the L and M subunits of the reaction center as well as both peptide subunits of light harvesting complex I, was shown to be capable of complementing the mutant R. capsulatus host. Hybrid reaction centers, comprised of R. sphaeroides-encoded L and M subunits and an R. capsulatus-encoded H subunit, were formed in addition to the R. sphaeroides-encoded LHI complexes. These hybrid cells were capable of photosynthetic growth, but their slower growth rates under low light conditions and their higher fluorescence emission levels relative to cells containing native complexes, indicated an impairment in energy transduction. The Rps. viridis puf operon was found to be incapable of functional expression in the R. capsulatus mutant host. Introduction of a plasmid-borne copy of the Rps. viridis puhA gene, which encodes the H subunit of the reaction center, into host cells already containing the Rps. viridis puf operon, such that all structural peptides of the Rps. viridis reaction center were present, still did not permit stable assembly of Rps. viridis photosynthetic complexes. RNA blot analysis demonstrated that the barrier to functional expression was not at the level of transcription. Differences between Rps. viridis and R. sphaeroides that may account for their differing abilities to complement the R. capsulatus mutant host strain are discussed.