Identification of a Key Step in the Biosynthetic Pathway of Bacteriochlorophyll c and Its Implications for Other Known and Unknown Green Sulfur Bacteria

The enzyme responsible for the methylation at the C-20 methine position of the bacteriochlorophylls c and e found in green sulfur photosynthetic bacteria has been identiﬁed by genomics and knockout mutagenesis. The distribution of this enzyme in other green sulfur bacteria is surprising.

cus. Fortunately, genes that code for chlorosome components are clustered in the FAP bacteria, while they are not in the green sulfur bacteria, and this clustering suggested a methyltransferase gene as a good candidate for the C-20 methylase. A knockout mutation in Chlorobium tepidum had the expected phenotype of containing bacteriochlorophyll d, which lacks the C-20 methyl group, instead of bacteriochlorophyll c. This established the identity of the gene, which was named bchU (4).

A SURPRISE FINDING AND SOME PREDICTIONS
Other groups of green photosynthetic bacteria contain functional chlorosomes that contain only bacteriochlorophyll d. Maresca et al. (4) examined some of these strains. A surprise in their findings is that at least one "wild-type" bacteriochlorophyll d-containing organism also contains a bchU gene, but with a frameshift mutation that leads to premature termination and an inactive enzyme. Certain bacteriochlorophyll d-containing strains have long been known to be prone to reversion to making bacteriochlorophyll c when grown for extended periods at low light intensities (2). In some of these strains, Maresca et al. (4) found that a second mutation restores the original reading frame and results in an active methyltransferase enzyme. Interesting questions remain about the observed distribution of these bacteriochlorophyll d-containing organisms in nature and how the C-20 methyl group affects the efficiency of light collection. In laboratory growth competition experiments, Maresca et al. (4) found that the bacteriochlorophyll d-containing Chlorobium tepidum bchU mutant cells did not grow as rapidly as the bacteriochlorophyll c-containing wild type under low light conditions but grew at the same rate at higher light intensity.
Can it be that all bacteriochlorophyll d-containing strains (which are often dominant isolates at somewhat higher positions in the water column and therefore higher light intensities) really contain a bchU gene that is inactivated by a frameshift and susceptible to reversion? This seems unlikely, but this question could be easily resolved by analysis of a series of bacteriochlorophyll d-containing strains. It may be that some of the common laboratory bacteriochlorophyll d-containing strains have arisen in the laboratory by selective pressure due to culturing at higher light intensities and that newly isolated bacteriochlorophyll d-containing strains will lack the gene entirely.
There is one additional pigment in the series of pigments that comprise the chlorobium chlorophylls, bacteriochlorophyll f (Fig. 1). This pigment has never been observed in nature, but it is the logical final member of this series of pigments, in that it contains hydrogen at C-20 and formyl at C-7. According to the progression of in vivo absorption maxima, in which bacteriochlorophyll c typically absorbs at 750 to 760 nm, bacteriochlorophyll d at 725 to 735 nm, and bacteriochlorophyll e at 710 to 720 nm, bacteriochlorophyll f should absorb at about 690 to 710 nm. It is a bit of a mystery why bacteriochlorophyll f-containing organisms have never been found, as both of the enzymes that make the two functional groups are clearly present in closely related species. The predicted 690-to 710-nm spectral window would appear to be a niche that is not well exploited, as it is just to the red of the chlorophyll a absorption band that is usually dominant. The only other known organisms that absorb in this spectral region are the chlorophyll d-containing cyanobacteria, which are not widely distributed (5).
It should be possible to produce an organism that contains bacteriochlorophyll f, simply by knocking out the C-20 meth-yltransferase enzyme in a bacteriochlorophyll e-containing strain. While none of the bacteriochlorophyll e-containing strains have genetic systems yet available, this should still be relatively straightforward.
A final puzzle yet to be solved is the identification of the enzyme that makes the formyl group at the C-7 position in bacteriochlorophyll e. This is the same position and functional group that is found in chlorophyll b. However, the enzymes are almost certainly not homologous, as the enzyme that makes chlorophyll b is a mixed-function oxidase that relies on O 2 as a substrate (9) and the bacteriochlorophyll e-containing bacteria are strict anaerobes. This is almost certainly another in a growing group of cases of gene replacement, in which the same biosynthetic steps are carried out by entirely different enzymes in anaerobic and aerobic organisms, with only the aerobic enzymes using O 2 as a substrate. Other examples include the coproporphyrinogen oxidase involved in heme and chlorophyll biosynthesis (HemN versus HemF), the oxidative cyclase that makes the isocyclic ring in chlorophylls (BchE versus AcsF), and ribonucleotide reductase (NrdG versus NrdB) (6). The anaerobic versions are probably the more ancient enzymes, dating to a time more than 2.2 billion years ago when the earth was largely anaerobic, and the more efficient aerobic versions that use the powerful oxidant O 2 have replaced the older ones whenever possible.