A puhA gene deletion and plasmid complementation system for facile site directed mutagenesis studies of the reaction center H protein of Rhodobacter Sphaeroides

The development of a Rhodobacter sphaeroides deletion/plasmid complementation system of the puhA gene (which encodes the reaction center [RC] heavy [H] subunit) for expression of site directed mutants of the RC H protein is described. The mutant strain ΔPUHA was constructed by introduction of a translationally in-frame deleted puhA allele at the chromosomal puhA gene site, and evaluated in plasmid complementation experiments. Strain ΔPUHA was unable to grow under photosynthetic conditions. Absorption spectroscopy showed this strain has a reduction in the amount of the light-harvesting I (LHI) complex. SDS-PAGE analysis of chromatophore proteins of strain ΔPUHA confirmed the absence of the RC H protein band. When ΔPUHA was complemented in trans with the wild type puhA gene in plasmids, photosynthetic growth and the RC H protein band in SDS-PAGE were restored. The results of comparisons of the properties of strains with different types of chromosomal puhA gene disruptions in complementation experiments are consistent with the idea that expression of one or more genes located 3' of puhA is required for optimal RC levels and photosynthetic growth. Since the ΔPUHA translationally in-frame deletion does not seem to interfere with transcription through and beyond the residual puhA sequences, this strain allows facile evaluation of the consequences of plasmid-borne RC H mutations in an otherwise wild type genetic background. The role of the RC H subunit in photosynthesis is discussed.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat