The bchU gene of Chlorobium tepidum encodes the c-20 methyltransferase in bacteriochlorophyll c biosynthesis

Abstract

Bacteriochlorophylls (BChls) c and d, two of the major light-harvesting pigments in photosynthetic green sulfur bacteria, differ only by the presence of a methyl group at the C-20 methine bridge position in BChl c. A gene potentially encoding the C-20 methyltransferase, bchU, was identified by comparative analysis of the Chlorobium tepidum and Chloroflexus aurantiacus genome sequences. Homologs of this gene were amplified and sequenced from Chlorobium phaeobacteroides strain 1549, Chlorobium vibrioforme strain 8327d, and C. vibrioforme strain 8327c, which produce BChls e, d, and c, respectively. A single nucleotide insertion in the bchU gene of C. vibrioforme strain 8327d was found to cause a premature, in-frame stop codon and thus the formation of a truncated, nonfunctional gene product. The spontaneous mutant of this strain that produces BChl c (strain 8327c) has a second frameshift mutation that restores the correct reading frame in bchU. The bchU gene was inactivated in C. tepidum, a BChl c-producing species, and the resulting mutant produced only BChl d. Growth rate measurements showed that BChl c- and d-producing strains of the same organism (C. tepidum or C. vibrioforme) have similar growth rates at high and intermediate light intensities but that strains producing BChl c grow faster than those with BChl d at low light intensities. Thus, the bchU gene encodes the C-20 methyltransferase for BChl c biosynthesis in Chlorobium species, and methylation at the C-20 position to produce BChl c rather than BChl d confers a significant competitive advantage to green sulfur bacteria living at limiting red and near-infrared light intensities.

FIG. 1.

Proposed reaction catalyzed by the bchU gene product. It should be noted that the substrate specificity of the BchU methyltransferase has not yet been established, so other related substrates may be methylated by an analogous reaction. Compounds: 1, [3-vinyl, 8-ethyl, 12-methyl] bacteriochlorophyllide d; 2, [3-vinyl, 8-ethyl, 12-methyl] bacteriochlorophyllide c; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine. R₁, ethyl, n-propyl, iso-butyl, or neo-pentyl; R₂, methyl or ethyl.

FIG. 2.

Methyltransferases involved in biosynthesis of bacteriochlorophylls and carotenoids. (A) Gene organization of an apparent photosynthesis-related operon in Chloroflexus aurantiacus. Genes in black are required for BChl c biosynthesis, and genes in gray encode chlorosome envelope proteins. (B) Phylogenetic relationships between translated sequences of BchU (encoding the C-20 methyltransferase) in green bacteria and CrtF (encoding neurosporene O-methyltransferases) in purple bacteria. Amino acid sequences were aligned by using the ClustalW program and the phylogenetic tree was generated with MacVector, version 7.1.1. UbiE, an O-methyltransferase involved in ubiquinone biosynthesis in the nonphotosynthetic plant pathogen Rickettsia coronii, was used as the outgroup sequence.

FIG. 3.

Amino acid and nucleotide sequence alignments of a portion of the BchU sequences from BChl c-, d-, and e-producing species. The type of BChl produced by each species is indicated in parentheses. Arrows indicate the insertion of a single nucleotide in the BchU gene of C. vibrioforme 8327d, which causes a frameshift mutation that results in a premature stop codon (*). The deletion of an adenine residue in the same region in the BChl c-producing revertant C. vibrioforme 8327c returns the gene to the correct reading frame.

FIG. 4.

Insertional inactivation of the bchU gene in C. tepidum. (A) The aadA gene, conferring resistance to streptomycin and spectinomycin, was inserted in bchU between KpnI sites that were 231 bp apart. (B) PCR confirmation of gene interruption. The bchU gene was amplified from genomic DNA extracted from the wild type (lane 1) and a mutant culture (lane 2) of C. tepidum, using primers CT0028 F1 and CT0028 B1. The fragment amplified from wild-type C. tepidum is 1.0 kbp; that amplified from the mutant is 1.9 kbp. Lane M, size markers; the sizes of bands (in kilobases) are indicated at the left.

FIG. 5.

Elution profiles of BChl c from wild-type C. tepidum (A) and BChl d from the bchU mutant (B). The numbers beside each peak indicate molecular masses (in daltons) of the compounds eluting in each peak, as determined by mass spectrometry. The four peaks in the upper panel correspond to [8-ethyl, 12-methyl] BChl c (792 Da), [8-ethyl, 12-ethyl] BChl c (806 Da), [8-propyl, 12-ethyl] BChl c (820 Da), and [8-isobutyl, 12-ethyl] BChl c (834 Da). Each BChl d peak is 14 mass units lighter than its corresponding BChl c counterpart; this difference reflects the loss of one methyl group (-CH₃) at the C-20 position.

FIG. 6.

Absorption profiles for BChls c and d. (A) Absorption spectra of BChl c (solid line) and BChl d (dashed line) in methanol from chlorosomes from C. tepidum. Pigments were separated on a reverse-phase C₁₈ HPLC column; spectra of the eluates were recorded with an online diode array detector. Spectra have been normalized to the molar extinction coefficients of the Q_y peaks. (B) Molar extinction coefficients of BChl c (solid line)- and d (dashed line)-containing chlorosomes in cell-harvesting buffer (10 mM KH₂PO₄, 50 mM NaCl, pH 7.0).

FIG. 7.

Growth rate measurements of the wild type (circles) and a bchU mutant (triangles) of C. tepidum at 146 μmol of photons m⁻² s⁻¹ (closed symbols) and 8 μmol of photons m⁻² s⁻¹ (open symbols). Culture conditions are described in the text. For the determination of growth rates, the first point in the growth curve and the first point of the linear portion of the curve were used. The nonlinear “bulge” areas of the curves are due to the transient appearance of polysulfide and sulfur globules in the growth medium.

FIG. 8.

Direct growth competition between BChl c- and d-producing strains of C. tepidum. Relative concentrations of BChls c (dark boxes) and d (light boxes), reported as fractions of the total BChl c plus BChl d concentration, were calculated as described in Materials and Methods. The percentages of BChl c plus BChl d composition at 0 generations and after 12 generations of growth at 8 μmol of photons m⁻² s⁻¹ (A) or 20 generations of growth at 146 μmol of photons m⁻² s⁻¹ (B) are indicated for each culture.