Directed mutational analysis of the Rhodobacter capsulatus puha gene and downstream open reading frames

Two mutant strains of Rhodobacter capsulatus which lacked the heavy (H) subunit of the reaction center (RC) were constructed. One strain, DW5, contains a translationally in-frame deletion of the puhA gene, which encodes the RC H subunit. The other strain, DW1, is a puhA polar mutation strain containing the same deletion of the puhA gene coupled with the insertion of a streptomydn/spectinomycin resistance omega (Ω) cartridge. Both strains were unable to grow under photosynthetic conditions (PS ). Absorption spectroscopy and SDS-PAGE analysis showed that these two strains have a reduction in the amount of the light-harvesting I (LHI) complex. In DW5, both photosynthetic growth and LHI complex level were restored by complementation in trans with plasmid pPUHA, which contains the puhA gene. However, when pPUHA was introduced into DW1, photosynthetic growth was only partially restored, and the LHI complex level was not restored to the wild type phenotype. When DW1 was doubly complemented with pPUHA and pORF214/162b (which contains orf214 and orfl62b, two open reading frames [orfs] downstream of puhA), photosynthetic growth was restored to the wild type level. However, LHI complex level was not restored in this doubly complemented strain. In a third mutant strain, DW2, orf214 was directly mutated with an insertion of the Ω. cartridge. DW2 also showed a PS" phenotype and a reduction in LHI complex level. Interestingly, SDS-PAGE analysis showed that the RC H subunit was missing in the intracytoplasmic membrane of DW2. Photosynthetic growth, but not LHI complex level, was restored when DW2 was complemented with pORFF214/162b. The roles of the RC H subunit and the gene products of the orfs downstream of puhA in photosynthesis are discussed.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat