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

Abstract

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.

FIG. 1

Physical map of the R. sphaeroides photosynthesis gene cluster, with expanded physical and restriction maps of the newly sequenced region containing the bchG locus. Structural genes, and those with putative regulatory or assembly functions, are shown in the darkest grey; the next darkest represents carotenoid and other isoprenoid biosynthesis genes; Bchl biosynthesis genes are paler again, while very pale grey indicates genes of no known function. For further information on the photosynthesis gene cluster of R. sphaeroides, see reference . The horizontal bar immediately below the full map (A) represents the insert of pSCN6-G, cloned in the pSUP202 vector. Horizontal arrows on the expanded physical map (B) indicate the direction of transcription of the labeled genes, while vertical arrows below this map mark the positions of Tn5 insertions within these genes; stem-loops indicate potential transcription terminators.

FIG. 2

Proposed pathway for the phytylation of Bchlidea in R. sphaeroides and R. capsulatus. The sequence of the hydrogenation reactions is based on that which occurs during chlorophyll biosynthesis in greening plants (32). The genes involved are in italics. DHGG, dihydro-GG; THGG, tetrahydro-GG.

FIG. 3

Partial alignment of the R. sphaeroides bchG deduced amino acid sequence (Rsp_BchG) with sequences of other known and putative Bchl and Chl synthetases and of three polyprenyltransferases showing the four domains highly conserved between these sequences. Sequences (and, in parentheses, accession numbers): Rca_BchG, R. capsulatus BchG (Swissprot P26170); Cau_BchG, C. aurantiacus BchG (Swissprot P33326); Syn_ChlG, Synechocystis sp. strain PCC 6803 slr0056 gene product (DDBJ D64001); Ath_CHLG, Arabidopsis thaliana G4 (GenBank U19382); Cau_Orf2, C. aurantiacus orf2 gene product (EMBL Z34000). Polyprenyltransferase sequences and accession numbers: Sce_Coq2, Saccharomyces cerevisiae Coq2 (Swissprot P32378); Eco_UbiA, E. coli UbiA (Swissprot P32166); Eco_MenA, E. coli MenA (Swissprot P26601). Residues fully conserved within the synthetase enzymes are in bold type. A putative cation-binding DRXXD motif is marked by asterisks above the alignment (24). The figures above the alignment are amino acid positions within R. sphaeroides BchG.

FIG. 4

Spectral analyses of R. sphaeroides strains: (A) Whole-cell absorbance spectra of nonphotosynthetic cultures, balanced according to cell density; (B) HPLC traces of acetone-methanol extracts. Strains: (i), complemented bchG mutant T6G1[pEBbchG]; (ii), orf427 mutant T6G4; (iii), T6G4[pSK1bchP]; (iv), orf177 mutant T6G6; (v), wild-type R. sphaeroides. Labeled peaks on HPLC traces: 1, BchlaGG; 2, BchlaDHGG; 3, BchlaTHGG; 4, BchlaP.

FIG. 5

HPLC analysis of Bchl synthetase assays. Chromatograms are of acetone-methanol extracts of incubations containing porphyrin substrates and E. coli extracts as follows: Bchlidea plus pET9a control (trace A) and Bchlidea plus pET9a-bchG (trace B). Trace C represents a mixture of Bchlidea esters. Labeled peaks: 1, BchlaGG; 2, BchlaDHGG; 3, BchlaTHGG; 4, BchlaP. Peak 1a is likely to be 13²-hydroxy-BchlaGG.