Physical mapping of bchG, orf427, and orf177 in the photosynthesis gene cluster of Rhodobacter sphaeroides: Functional assignment of the bacteriochlorophyll synthetase gene

The purple photosynthetic bacterium Rhodobacter sphaeroides has within its genome a cluster of photosynthesis-related genes approximately 41 kb in len gth. In an attempt to identify genes involved in the terminal esterificatio n stage of bacteriochlorophyll biosynthesis, a previously uncharacterized 5 -kb region of this cluster was sequenced. Four open reading frames (ORFs) w ere identified, and each was analyzed by transposon mutagenesis. The produc t of one of these ORFs, bchG, shows close homologies with (bacterio)chlorop hyll synthetases, and mutants in this gene were found to accumulate bacteri opheophorbide, the metal-free derivative of the bacteriochlorophyll precurs or bacteriochlorophyllide, suggesting that bchG is responsible for the este rification of bacteriochlorophyllide with an alcohol moiety. This assignmen t of function to bchG was verified by the performance of assays demonstrati ng the ability of BchG protein, heterologously synthesized in Escherichia c oli, to esterify bacteriochlorophyllide with geranylgeranyl pyrophosphate i n vitro, thereby generating bacteriochlorophyll. This step is pivotal to th e assembly of a functional photosystem in R. sphaeroides, a model organism for the study of structure-function relationships in photosynthesis. A seco nd gene, orf177, is a member of a large family of isopentenyl diphosphate i somerases, while sequence homologies suggest that a third gene, orf427, may encode an assembly factor for photosynthetic complexes. The function of th e remaining ORF, bchP, is the subject of a separate paper (H, Addlesee and C. N. Hunter, J. Bacteriol. 181:7248-7255, 1999), An operonal arrangement o f the genes is proposed.