Rhodospirillum rubrum possesses a variant of the bchP gene, encoding geranylgeranyl-bacteriopheophytin reductase

Abstract

The bchP gene product of Rhodobacter sphaeroides is responsible for the reduction of the isoprenoid moiety of bacteriochlorophyll (Bchl) from geranylgeraniol (GG) to phytol; here, we show that this enzyme also catalyzes the reduction of the isoprenoid moiety of bacteriopheophytin (Bphe). In contrast, we demonstrate that a newly identified homolog of this gene in Rhodospirillum rubrum encodes an enzyme, GG-Bphe reductase, capable of reducing the isoprenoid moiety of Bphe only. We propose that Rhodospirillum rubrum is a naturally occurring bchP mutant and that an insertion mutation may have been the initial cause of a partial loss of function. Normal BchP function can be restored to Rhodospirillum rubrum, creating a new transconjugant strain possessing Bchl esterified with phytol. We speculate on the requirement of Rhodospirillum rubrum for phytylated Bphe and on a potential link between the absence of LH2 and of phytylated Bchl from the wild-type bacterium. The identification of a second role for the fully functional BchP in catalyzing the synthesis of phytylated Bphe strongly suggests that homologs of this enzyme may be similarly responsible for the synthesis of phytylated pheophytin in organisms possessing photosystem 2. In addition to bchP, other members of a photosynthesis gene cluster were identified in Rhodospirillum rubrum, including a bchG gene, demonstrated to encode a functional Bchl synthetase by complementation of a Rhodobacter sphaeroides mutant.

FIG. 1.

Proposed pathway for reduction of BpheaGG in Rhodospirillum rubrum. The sequence of the hydrogenation reactions is based on that which occurs during Chl biosynthesis in greening plants (33). Reduction proceeds via dihydro-GG-esterified (BpheaDHGG) and tetrahydro-GG-esterified (BpheaTHGG) intermediates to the final product, phytylated Bphe (BpheaP). R represents the tetrapyrrole-derived moiety of Bphe.

FIG. 2.

(A) Physical map of part of a Rhodospirillum rubrum PGC. Arrows indicate the direction of transcription of the labeled genes. (B) Deduced amino acid sequence alignment between Rhodospirillum rubrum bchG (top) and Rhodobacter sphaeroides bchG (bottom). (C) Deduced amino acid sequence alignment between Rhodospirillum rubrum bchP (top) and Rhodobacter sphaeroides bchP (bottom). Asterisks above the alignment mark the positions of significant amino acids within a putative ADP-binding βαβ fold (37).

FIG. 3.

(A) Partial alignment of the Rhodospirillum rubrum bchP deduced amino acid sequence (Rs. rubrum) with sequences of known and putative GG-(b)chl reductases from the following species (sequence accession numbers in parentheses): Rubrivivax gelatinosus (EMBL AB034704); Rhodobacter sphaeroides (EMBL AJ010302); Rhodobacter capsulatus (Swissprot P26172); Synechocystis sp. strain PCC 6803 (Swissprot Q55087); Oryza sativa (EMBL AP001080); Mesembryanthemum crystallinum (PIR T12299); Glycine max (EMBL AF068686); Arabidopsis thaliana (EMBL Y14044); and Zantedeschia aethiopica (EMBL AF055296). The numbers above the alignment are amino acid positions within Rhodospirillum rubrum BchP. The sequences labeled Δx, P_R/P_S, P_S/P_R, and P_S/P_S represent the predicted products of the modified and hybrid bchP genes present in plasmids pSK1RRbchPΔx, pSK1bchP_R/P_S, pSK1bchP_S/P_R, and pSK1bchP_S/P_S, respectively. (B) Codon usage analysis for the multialigned region of Rhodospirillum rubrum BchP. Each of the three forward reading frames is presented, with frame i being the true reading frame. Codon frequencies add up to 1.0 for each amino acid species. Average codon frequency for the entire bchP gene (0.497) is indicated by a dashed line. It can be seen that the codon frequency drops substantially for the extra amino acids in the correct frame, but that it is more typical in frame iii in this region.

FIG. 4.

HPLC traces of acetone-methanol extracts of Rhodobacter sphaeroides and Rhodospirillum rubrum strains. (A) Functional analysis of Rhodospirillum rubrum bchP. (i) BpheaGG standard; (ii) Rhodobacter sphaeroides bchP mutant DBCΩ:T6G5; (iii) DBCΩ:T6G5[pSK1RRbchP], the Rhodobacter sphaeroides bchP mutant complemented with Rhodospirillum rubrum bchP; (iv) DBCΩ:T6G5[pSK1bchP], the Rhodobacter sphaeroides bchP mutant complemented with Rhodobacter sphaeroides bchP; (v) BpheaP standard. (B) Functional analysis of modified versions of Rhodospirillum rubrum bchP via complementation of Rhodobacter sphaeroides bchP mutant DBCΩ:T6G5. (i) DBCΩ:T6G5[pSK1RRbchPΔx]; (ii) DBCΩ:T6G5[pSK1bchP_R/P_S]; (iii) DBCΩ:T6G5[pSK1bchP_S/P_R]; and (iv) DBCΩ:T6G5[pSK1bchP_S/P_S]. (C) Functional analysis of Rhodospirillum rubrum bchG. (i) Rhodobacter sphaeroides bchG mutant T6G1[pRKSK1]; (ii) T6G1[pSK1RRbchG], the Rhodobacter sphaeroides bchG mutant complemented with Rhodospirillum rubrum bchG; (iii) T6G1[pEB bchG], the Rhodobacter sphaeroides bchG mutant complemented with Rhodobacter sphaeroides bchG. (D) Analysis of a novel mutant strain of Rhodospirillum rubrum. (i) Wild-type Rhodospirillum rubrum[pRKSK1]; (ii) Rhodospirillum rubrum[pSK1bchP], the new, BchlaP-containing strain; (iii) Rhodobacter sphaeroides wild-type strain DBCΩ. The retention times of the four principal pigments are indicated by vertical dashed lines: 1, BchlaGG; 2, BchlaP; 3, BpheaGG; and 4, BpheaP. Other known peaks are individually labeled: a, dihydro-GG-esterified Bchla; b, tetrahydro-GG-esterified Bchla. Traces are normalized according to the maximum Bchl peak, with the exception of the trace for strain T6G1[pRKSK1], displayed at the same scale as that of strain T6G1[pSK1RRbchG]. The Bphe region of most traces is expanded vertically sixfold; this is indicated by a break in the trace at a retention time of 21.5 min.

FIG. 5.

Absorbance spectra of intracytoplasmic membranes from (A) wild-type Rhodospirillum rubrum[pRKSK1] and (B) Rhodospirillum rubrum complemented with bchP in plasmid pSK1bchP. Spectra are normalized at their LH1 absorbance maxima. It can be seen that the 880-nm peak is slightly blue-shifted in Rhodospirillum rubrum[pSK1bchP].