Physical mapping and functional assignment of the geranylgeranyl-bacteriochlorophyll reductase gene, bchP, of Rhodobacter sphaeroides

Abstract

The bacteriochlorophyll of the purple photosynthetic bacterium Rhodobacter sphaeroides is esterified with phytol. The presence of this alcohol moiety is essential for the correct assembly of the photosynthetic apparatus. Despite this, and the fact that R. sphaeroides is widely used for the study of structure-function relationships in photosynthesis, the molecular genetics of the steps in which the alcohol is added and modified have not previously been investigated in this organism. Sequencing near the center of the photosynthesis gene cluster has now revealed the existence of an open reading frame encoding a putative 394-amino-acid polypeptide displaying strong homology with the products of a number of genes from other photosynthetic organisms, each proposed to be responsible for the reduction of the alcohol moiety of (bacterio)chlorophyll to phytol. An R. sphaeroides transposon mutant in this gene, bchP, possessed a structurally modified photosystem assembled with bacteriochlorophyll esterified with geranylgeraniol, rather than with phytol, implying that the product of this gene was geranylgeranyl-bacteriochlorophyll reductase. This identification was confirmed by the performance of in vitro assays using heterologously expressed protein, providing the first direct demonstration of the activity of a bchP gene product.

FIG. 1

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 chl biosynthesis in greening plants (30). The genes involved are in italics.

FIG. 2

Physical map of the R. sphaeroides photosynthesis gene cluster, adapted from reference , with an expanded map of the bchP locus indicating its direction of transcription and the position of Tn5 insertion T6G5. Structural genes, and those with putative regulatory or assembly functions, are shown in the darkest grey; the next darkest represents carotenoid biosynthesis genes; bacteriochlorophyll 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 longer horizontal bar immediately below the full map represents the insert of pSCN6-G, cloned in the pSUP202 vector. (B) Nucleotide and deduced amino acid sequences of the bchP gene. A potential ribosome-binding sequence is underlined.

FIG. 3

Partial alignment of R. sphaeroides bchP deduced amino acid sequence (Rsp_BchP) with sequences of other putative gene products. Fully conserved residues are in bold type. Sequences (and, in parentheses, accession numbers) are as follows: Rca_BchP, R. capsulatus BchP (P26172); Syn_ChlP, Synechocystis sp. strain PCC 6803 ChlP (X97972); Ath_CHLP, A. thaliana CHL P (Y14044); Mcr_CHLP, M. crystallinum CHL P (AF069318); Zae_CHLP, Z. aethiopica CHL P partial sequence (AF055296); Eco_YDIS, E. coli YDIS (P77337); PR00420, segments representing blocks of the flavoprotein monooxygenase signature. Asterisks below the alignment mark the positions of significant amino acids within a putative ADP-binding βαβ fold (36); residues which match the proposed fingerprint are underlined. Numbers above the alignment are amino acid positions within R. sphaeroides BchP.

FIG. 4

Spectral analyses of R. sphaeroides strains. (A) Whole-cell absorbance spectra balanced according to cell density; (B) HPLC traces of acetone-methanol extracts. R. sphaeroides strains: (ii), bchP mutant T6G5; (iii), T6G5[pSK1bchP]; (iv), wild-type. HPLC trace (i) represents a Rhodospirillum rubrum cell extract. Labeled peaks on HPLC traces: 1, bchlaGG; 2, bchlaDHGG; 3, bchlaTHGG; 4, bchlaP.

FIG. 5

HPLC analysis of geranylgeranyl-bchl reductase assays. Chromatograms are of acetone-methanol extracts of incubations containing porphyrin substrates and E. coli extracts as follows: bchlaGG plus pET9a control (trace A) and bchlaGG plus pET9a-bchP (trace B). Trace C represents a mixture of bchlidea esters. The breaks in traces A and B indicate a change in vertical scale: to the right of the breaks, the height of these traces is expanded by a factor of 4. Labeled peaks: 1, bchlaGG; 2, bchlaDHGG; 3, bchlaTHGG; 4, bchlaP.