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The role of the pufX gene product of Rhodobacter capsulatus Lilburn, Timothy George


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.

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