Cloning and expression of the Rhodobacter sphaeroides reaction center H gene

Abstract

The Rhodobacter sphaeroides structural gene (puhA) for the reaction center H polypeptide has been identified and cloned by using restriction fragements specific for the analogous Rhodobacter capsulatus gene as a heterologous hybridization probe. The presence of puhA on a 1.45-kilobase BamHI restriction fragment was confirmed by partial DNA sequence analysis and by the synthesis of an immunoreactive Mr-28,000 reaction center H polypeptide in an R. sphaeroides coupled transcription-translation system. Approximately 450 base pairs of DNA upstream of the puhA gene were sufficient for expression of this protein in vitro. Northern RNA-DNA blot analysis with an internal puhA-specific probe identified at least two, apparently monocistronic, transcripts present at different cellular levels under physiological conditions known to affect the cellular content of both reaction center complexes and photosynthetic membrane. Northern blot analysis with specific upstream restriction fragment probes revealed that the 1,400-nucleotide puhA-specific mRNA had a 5' terminus upstream of the 1,130-nucleotide transcript. Both puhA-specific mRNA and immunoreactive reaction center H protein were detectable in chemoheterotrophically grown cells which lacked detectable bacteriochlorophyll and photosynthetic membrane.