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

We describe the development of a Rhodobacter sphaeroides RC H (puhA) gene deletion/plasmid complementation system for expression of site directed mutants of the RC H protein. The mutant strain Delta PUHA 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 Delta PUHA was not capable of photosynthetic growth, and SDS-PAGE chromatophore proteins confirmed the absence of the RC H protein band. When Delta PUHA was complemented 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 with plasmid-borne DNA fragments containing the wild type puhA gene 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 Delta PUHA 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. Two plasmid-borne site directed mutants of the puhA gene (Glu(H173) --> Gln and Glu(H173) --> Asp) exhibited different deficiencies in photosynthetic growth when present in Delta PUHA, indicating that a carboxylic acid amino acid side chain at the H173 position is important for RC function.