PHOTOSYNTHETIC REACTION CENTER MUTAGENESIS VIA CHIMERIC RESCUE OF A NONFUNCTIONAL RHODOBACTER-CAPSULATUS PUF OPERON WITH SEQUENCES FROM RHODOBACTER-SPHAEROIDES

Photosynthetically active chimeric reaction centers which utilize gene tic information from both Rhodobacter capsulatus and Rb. sphaeroides p uf operons were isolated using a novel method termed chimeric rescue. This method involves in vivo recombination repair of a Rb. capsulatus host operon harboring a deletion in pufM with a non-expressed Rb. spha eroides donor puf operon. Following photosynthetic selection, three re vertant classes were recovered: 1) those which used Rb. sphaeroides do nor sequence to repair the Rb. capsulatus host operon without modifica tion of Rb. sphaeroides puf operon sequences (conversions), 2) those w hich exchanged sequence between the two operons (inversions), and 3) t hose which modified plasmid or genomic sequences allowing expression o f the Rb. sphaeroides donor operon. The distribution of recombination events across the Rb. capsulatus puf operon was decidedly non-random a nd could be the result of the intrinsic recombination systems or could be a reflection of some species-specific, functionally distinct chara cteristic(s). The minimum region required for chimeric rescue is the D -helix and half of the D/E-interhelix of M. When puf operon sequences 3' of nucleotide M882 are exchanged, significant impairment of excitat ion trapping is observed. This region includes both the 3' end of pufM and sequences past the end of pufM.