Elucidation of the bacteriochlorophyll c biosynthesis pathway in green sulfur bacterium Chlorobium tepidum

by Chew, Aline Gomez

Green sulfur bacteria (GSB) are a unique group of strictly anaerobic organisms. They have been isolated from microbial mats, from below the chemocline of stratified lakes, from sediments and even from the surroundings of deep-sea hydrothermal vents. The anaerobic environments in which GSB usually thrive are normally very light-limited. GSB have developed a large light-harvesting antenna, the chlorosome, in order to survive in such environments. Chlorosomes are unlike any other known light-harvesting antenna, because they do not require a protein scaffold for assembly. Structures formed from the self-aggregation of BChls c, d or e form the bulk of the chlorosome. The antenna BChls share unique characteristics that allow the formation of the chlorosome aggregates. These BChls have a hydroxyl moiety at the C-31 position that chelates the Mg of a neighboring molecule, which allows the formation of the higher order aggregates of the chlorosome. These chlorophylls also lack the C-132 methyl-carboxyl moiety found in other (B)Chls, which would otherwise interfere with aggregate formation. GSB also methylate these antenna BChls at the C-20, C-82 and C-121 positions. In addition to the antenna BChls, GSB produce Chl a and BChl a. Although the pathways for the biosynthesis of Chl a and BChl a have been elucidated in plants and algae and purple bacteria, respectively, very little was known about the biosynthesis of the chlorosome antenna BChls. By using the wealth of data available from the sequencing and annotation of the genome of the GSB Chlorobium tepidum, genes possibly involved in (B)Chl biosynthesis were identified and targeted for inactivation. Mutants of C. tepidum, affected in (B)Chl biosynthesis were constructed and characterized. The characterization of the mutants enabled the identification of previously unknown enzymes responsible for the C- 8 vinyl reduction (BciA), and C-82 (BchQ) and C-121 (BchR) methylations. Several orange mutants of C. tepidum, impaired in their ability to synthesize wild-type levels of BChl c, were also characterized. These mutants were impaired at different points in the BChl c biosynthetic pathway. A mutant of bchS, a paralog of the gene encoding the large Mg-chelatase subunit BchH, produced only 10% the BChl c of the wild type and excreted iv large amounts of protoporphyrin-IX into the growth medium. A strain carrying a mutation in bchJ, a gene that had been incorrectly assumed to encode the C-8 vinyl reductase, produced 9% the wild-type levels of BChl c and excreted large amounts of divinyl-protochlorophyllide into the media. Finally, a bchU bchQ bchR triple mutant of C. tepidum, which is unable to methylate the BChl c molecule, produced less than 30% of the BChl c of the wild type. Based on these results, a pathway for the biosynthesis of (B)Chls in C. tepidum was proposed. The accumulation of divinyl-protochlorophyllide in the bchJ mutant, helped place the branching of the three (B)Chl biosynthetic pathways of C. tepidum at the level of Chlide a. The only step remaining to be identified in BChl c biosynthesis is the removal of the C-13 2 methyl-carboxyl moiety of Chlide a. The results from this work not only have implications for the biosynthesis of (B)Chls in green sulfur bacteria but for the synthesis of all Chls and BChls. v