Edward J. Bylina^*, Kenneth A. Ohgi, Torrie Nute, Vikki Cerniglia, Scott O’Neal and Paul Weaver

KAIROS Scientific Inc. ^*

The high resolution crystal structure of the photosynthetic reaction center from the purple non-sulfur bacterium Blastochloris (formerly Rhodopseudomonas) viridis provides an excellent basis for structure-function studies of biological electron transfer processes. However, the inability to express and isolate mutagenized reaction centers from strains that cannot grow photosynthetically has limited the scope of such studies in this organism. Here we describe the characterization of Blastochloris viridis strain RA3, which is capable of expressing reaction centers under non-photosynthetic growth conditions. Low- aeration growth conditions have been established to produce dark-grown cultures containing a highly induced photosynthetic apparatus. A puf operon deletion strain incapable of photosynthetic growth has been constructed in RA3. This deletion strain can be complemented by the puf operon from B. viridis DSM133 using a sacB-containing suicide vector, resulting in a hybrid reaction center containing the H subunit of RA3 and the L, M and cytochrome subunits of DSM133. These hybrid reaction centers have been purified and characterized. Unique restriction sites have been introduced throughout the DSM133 puf operon to facilitate mutagenesis studies of the reaction center. Use of this strain will allow investigation of bacteriochlorophyll b biosynthesis, mutagenesis of the B. viridis light harvesting complex, and structure-function studies of the B. viridis reaction center.

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