A SYSTEM FOR SITE-SPECIFIC MUTAGENESIS OF THE PHOTOSYNTHETIC REACTION CENTER IN RHODOPSEUDOMONAS-VIRIDIS

The purple non-sulfur bacterium Rhodopseudomonas viridis contains a photosynthetic reaction center which has been structurally resolved to 2.3 angstrom providing a unique basis for the study of biological electron transfer processes by the method of site-specific mutagenesis. Here we report the construction of a puf operon deleted mutant strain incapable of photosynthetic growth. The deletion was introduced with the help of a newly constructed suicide vector by electroporation which is with conjugation another gene transfer system for R. viridis. The deletion strain was complemented by conjugational gene transfer with wild-type (WT) and mutated LM genes of the puf operon. The complemented WT and mutations YL162F and HL153F grew photosynthetically, expressed and assembled the four subunits L, M, H and Cyt c of the reaction center correctly. These first mutations already demonstrate the value of the R. viridis system for a detailed structure-function analysis of photosynthetic electron transfer.