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Journal of Bacteriology, June 2000, p. 3175-3182, Vol. 182, No. 11
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Physical Mapping of bchG, orf427, and orf177 in the Photosynthesis Gene Cluster of Rhodobacter sphaeroides: Functional Assignment of the Bacteriochlorophyll Synthetase Gene

Hugh A. Addlesee,1,* Leszek Fiedor,2 and C. Neil Hunter1

Robert Hill Institute for Photosynthesis and Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom,1 and Institute of Molecular Biology, Jagiellonian University, 31-120 Cracow, Poland2

Received 12 November 1999/Accepted 10 March 2000

The purple photosynthetic bacterium Rhodobacter sphaeroides has within its genome a cluster of photosynthesis-related genes approximately 41 kb in length. In an attempt to identify genes involved in the terminal esterification stage of bacteriochlorophyll biosynthesis, a previously uncharacterized 5-kb region of this cluster was sequenced. Four open reading frames (ORFs) were identified, and each was analyzed by transposon mutagenesis. The product of one of these ORFs, bchG, shows close homologies with (bacterio)chlorophyll synthetases, and mutants in this gene were found to accumulate bacteriopheophorbide, the metal-free derivative of the bacteriochlorophyll precursor bacteriochlorophyllide, suggesting that bchG is responsible for the esterification of bacteriochlorophyllide with an alcohol moiety. This assignment of function to bchG was verified by the performance of assays demonstrating the ability of BchG protein, heterologously synthesized in Escherichia coli, to esterify bacteriochlorophyllide with geranylgeranyl pyrophosphate in vitro, thereby generating bacteriochlorophyll. This step is pivotal to the assembly of a functional photosystem in R. sphaeroides, a model organism for the study of structure-function relationships in photosynthesis. A second gene, orf177, is a member of a large family of isopentenyl diphosphate isomerases, while sequence homologies suggest that a third gene, orf427, may encode an assembly factor for photosynthetic complexes. The function of the 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 of the genes is proposed.

* Corresponding author. Mailing address: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom. Phone: (0114) 222 2000, ext. 24240. Fax: (0114) 272 8697. E-mail: h.a.addlesee{at}sheffield.ac.uk.

Journal of Bacteriology, June 2000, p. 3175-3182, Vol. 182, No. 11
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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