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The 2.7 kilobase transcript of the puf operon of the photosynthetic bacterium Rhodobacter capsulatus has five open reading frames. The gene products of four of these open reading frames (pufB, A, L,and M) are well characterized as structural polypeptides of the reaction center (pufL and M) and the B870 light-harvesting antenna complex (pufB and A), The role of the pufX gene product has been unknown. By deleting the pufX gene from a plasmid carrying the puf operon and using this plasmid to reconstitute a strain of R. capsulatus which had the puf operon deleted, it was possible to characterize the pufX gene product. It was found that the pufX⁻ mutant was unable to grow photosynthetically until a secondary suppressor mutation had occurred. It appeared that either more than one type of suppressor mutation could occur or that one suppressor mutation could be accompanied by further mutations. To determine the nature of the lesion caused by the deletion of pufX, the structure of the photosynthetic unit and the ability of the subunits of the photosynthetic unit to accomplish energy and electron transfer of the mutant were compared to a pseudo-wild type. Spectrophotometric techniques, including fluorescence detection, reduced minus oxidized spectra, flash-induced absorbance change spectra, and ground state absorption spectra were used for these comparisons as well as biochemical assays. The biochemical assays measured the ability of chromatophores to transfer electrons from a quinone analog to horse-heart cytochrome c and to pump protons in response to light irradiation. The results of these comparisons indicated that the individual components of the photosynthetic unit functioned normally but that electron transfer between these components, specifically between the reaction center and the cytochrome b⁄c₁ complex, was impaired. It thus seemed likely that there was some structural defect in the photosynthetic unit. The structure of the photosynthetic units of pseudo-wild type and mutant strains was probed using sodium dodecyl sulfate-polyacrylamide gels, absorption spectroscopy, and electron microscopy. It was determined that the mutant had chromatophore vesicles that were about 50% larger than those of the pseudo-wild type and contained higher levels of reaction center and B870 light-harvesting antenna polypeptides. The suppressor mutants also had altered levels of polypeptides and showed differences in the way the expression of their B800-850 polypeptides was regulated. It was concluded that the pufX gene product plays a role in the correct assembly of the photosynthetic unit as a structural component of the unit and/or as a regulator of its assembly. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

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. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

This research was an attempt to characterize previously constructed mutants with a specifically altered psbO gene which encodes a 33 kDa protein active in photosynthesis. This polypeptide was believed to function in stabilization of manganese ions during photolysis of water at the photosystem II. The initial phase of this work was concerned with determining the manganese content of the genetically manipulated PS II particles of the photosynthetically active cyanobacteria.We found however, that the results of the isolation procedure for PS II particles of photosynthetically active cyanobacteria as described by Burnap et al. was not reproducible in our research organism. This prevented the chemical characterization of function of these particles as had been planned.In the second phase of the research sequencing of the mutated gene was to be performed for several clones in order to determine the kinds of specific alterations that had been made. The mutated genes had been cloned into both pUC1 20 and pPGV5 vectors which were transformed into Escherichia OR (EQQJi) and the cyanobacterium Synechococcus PCC 7942, respectively.Several attempts were mad o isolate plasmid DNA from both the transformed E QQJI and cyanobacterium. Isolation of pUC120 DNA was not achieved due to the toxicity of the 33 kDa protein product of the psbO gene in sgJj. The pPGV5 plasmid isolation was successful and PCR-sequencing was performed. However, the sequencing did not result in a readable sequence. Instead, banding patterns showed more than one nucleotide per lane. Since pPGV5 contains a strong constitutive promoter, a large amount of mutant protein was being produced. Our findings suggested that transformed cyanobacteria may have been under pressure to revert the altered gene to wild-type. Thus, upon growth of a single colony to a larger volume, a heterogeneous population of cells with different sizes of plasmids may have resulted. Restriction analysis of isolated plasmid DNA confirmed the presence of multiple-sized plasmid molecules. Therefore, this research has shown that the previously constructed mutants are not stable enough to characterize for alterations in manganese binding. / Department of Biology

The 33 kDa manganese stabilizing protein (MSP) has been proposed to provide ligands to stabilize Mn ions in the water lysis reaction of photosystem II of photosynthesis. In previous research site-directed mutagenesis had been performed on regions of the psbO gene encoding two aspartic acid residues of MSP which were thought to have the potential to form carboxyl bridges with Mn ions. The purpose of this research was to analyze these mutants. Plasmids pUC120-33 (#1,3,5,7,9,11,15) containing mutant psbO genes could not be isolated from E.coli because the expressed MSP was toxic to the cells. However, a psbO mutant gene carried in pPGV5-33 (#7) was isolated from E.coli and transformed into cyanobacterium Svnechococcus PCC 7942. Cyanobacterial cells carrying the MSP mutant showed a susceptibility to intensive light (100 footcandles) with a decrease of 30% in the growth rate within the first 100 hours after inoculation. This result suggested a possible function of the MSP in protecting the oxygen evolving complex from intensive light exposure. However, the mutant appeared to revert after this time probably due to homologous gene recombination with the wild type gene. In order to further analyze the function of mutants without recombination occurring, the construction of an MSP deletion was attempted using insertion of a kanamycin cartridge into the middle of the psbO gene. The inactivated psbO gene was transformed into E.coli and transformants were selected by kanamycin resistance. However, plasmid DNA carrying the interrupted genes could not be isolated, probably due to toxicity of the expression product in E.coli cells. Thus, future studies should be directed to reconstruction of a deletion mutant by direct transformation into cyanobacterial cells. Once a deletion mutant has been constructed analyses of the site-directed mutations could be performed in cyanobacteria. / Department of Biology