||Historically, new microorganisms have been discovered either by setting up enrichment cultures in the laboratory that favor particular types of physiologies or by sequencing the amplified 16S ribosomal RNA gene from DNA extracted from environmental samples. These approaches have been successful in exposing the large variety of physiological capabilities (enrichment cultures) and vast phylogenetic diversity (16S rRNA surveys) of the microbial world. Yet they pose severe limitations to furthering our understanding of microbial diversity and to the discovery of microorganisms with novel physiologies. Another approach, metagenomics, has emerged as a powerful tool to study community composition, to define physiological capabilities of microbial communities and to discover novel microorganisms. A particularly fascinating group of microorganisms to study are the chlorophototrophs. Chlorophototrophs use membrane-embedded chlorophyll-protein complexes called reaction centers to harvest light energy and transform it into chemical energy. Prior to the work presented here, there were five known bacterial phyla that contained chlorophototrophs, including the Cyanobacteria, Chlorobi, Chloroflexi, Firmicutes and Proteobacteria. These distinct groups vary in their reaction centers, accessory pigments and carbon metabolism among other properties. Microbial mats in alkaline hot springs of Yellowstone National Park are dominated by chlorophototrophic microorganisms and provide an ideal setting for studying them. The mats from two particular springs, Octopus and Mushroom Springs, have been the subject of over three decades of research, and much is known about their geochemistry and microbial composition. Two phyla of chlorophototrophs, the Cyanobacteria and the Chloroflexi reside in these low-carbonate and low-sulfide mats. A metagenomic study conducted on these mats revealed the presence of a third type of chlorophototroph that had escaped detection by other approaches and that belongs to the phylum Acidobacteria, a phylum previously not known to contain chlorophototrophs. An analysis of the metagenome predicted this new chlorophototroph, Candidatus Chloracidobacterium thermophilum, to have Type-1 reaction centers, the Fenna Matthew Olson protein and chlorosomes as antenna structures, properties also found in the chlorophototrophic Chlorobi. Unlike the Chlorobiales, however, Cab. thermophilum was predicted to be an aerobe. The present work describes the isolation of this new chlorophototroph in culture and its initial characterization. An enriched culture of Cab. thermophilum has been generated from a cyanobacterial enrichment culture cultivated from the mats of Octopus Spring that contained Cab. thermophilum as a minor component. Like other Acidobacteria, Cab. thermophilum is difficult to culture and has long generation times and fastidious growth requirements. In addition, the culture contains heterotrophic microorganisms that seem to be providing unidentified, essential growth factor(s). Physiological studies of this culture have confirmed the metagenomic predictions that Cab. thermophilum is an aerobic chlorophototroph that synthesizes chlorosomes containing bacteriochlorophyll c as antenna pigments. In order to understand the physiology of Cab. thermophilum further, its genome has been completely sequenced. It consists of two chromosomes, both of which harbor essential genes. The genome contains all of the genes required for phototrophy with chlorosomes as antenna structures but lacks key genes of all known carbon fixation pathways, as well as genes for assimilatory nitrate and sulfate reduction. In addition, it lacks the biosynthetic pathways for the synthesis of the amino acids valine, isoleucine and leucine. These genomic analyses clearly define Cab. thermophilum as a chlorophotoheterotroph that is dependent on other members of the mat community for essential nutrients. The structure of the chlorosome antenna complex of Cab. thermophilum has been investigated and its protein, lipid, quinone and pigment composition elucidated. Although synthesized by an aerobe, the chlorosomes resemble those of other green bacteria in their general shape and by the presence of CsmA and CsmI-like proteins. However, they contain additional unique proteins and lipids. Moreover, the presence of the xanthophylls echinenone and canthaxanthin reflects the aerobic environment from which Cab. thermophilum was isolated. Like the chlorophototrophic Chlorobi, Cab. thermophilum synthesizes three types of chlorophylls, bacteriochlorophyll (BChl) c, chlorophyll (Chl) a and BChl a. High performance liquid chromatography analyses combined with mass spectrometry have revealed that Cab. thermophilum methylates its BChl c at the C-8 and C-12 positions and that its C-17 propionic group is esterified with a variety of isoprenoid and straight alkane moieties. The most abundant BChl c species, especially at high light intensities, has been found to be [8-iBu, 12-Et]-BChl c esterified with the unbranched C-18 alcohol, octadecanol. Interestingly, although Cab. thermophilum is an aerobe, its chlorosomes exhibit redox-dependent quenching of fluorescence emission. Lastly, carotenoid biosynthesis in Cab. thermophilum has been investigated. Three genes predicted to code for a lycopene cyclase, a ketolase, and a hydroxylase respectively have been expressed heterologously and their enzymatic activities confirmed.
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