DIRECTED MUTAGENESIS OF THE RHODOBACTER-CAPSULATUS PUHA GENE AND ORF-214 - PLEIOTROPIC EFFECTS ON PHOTOSYNTHETIC REACTION-CENTER AND LIGHT-HARVESTING-1 COMPLEXES

Rhodobacter capsulatus puhA mutant strains containing either a nonpola r, translationally in-frame deletion or a polar insertion of an antibi otic resistance cartridge were constructed and evaluated for their pho tosynthetic growth properties, absorption spectroscopy profiles, and c hromatophore protein compositions. Both types of mutants were found to be incapable of photosynthetic growth and deficient in the reaction c enter (RC) and light-harvesting 1 (LH1) complexes. The translationally in-frame puhA deletion strains were restored to the parental strain p henotypes by complementation with a plasmid containing the puhA gene, whereas the polar puhA mutants were not. Analogous nonpolar and polar disruptions of orf 214 (located immediately 3' of the puhA gene) were made, and the resultant mutant strains were evaluated as described abo ve. The strain containing the nonpolar deletion of orf 214 exhibited s everely impaired photosynthetic growth properties and had greatly redu ced levels of the RC and LH1 complexes. Complementation of this strain with a plasmid that expressed orf 214 from the nifHDK promoter restor ed photosynthetic growth capability, as well as the RC and LH1 complex es. The polar disruption of orf 214 yielded cells that were incapable of photosynthetic growth and had even lower levels of the RC and LH1 c omplexes, and complementation in trans with orf 214 only marginally im proved these deficiencies. These results indicate that orf 214 and at least one additional gene located 3' of orf 214 are required to obtain the RC and LH1 complexes, and transcription read-through from the puh A superoperon is necessary for optimal expression of these new photosy nthesis genes.