20Sterols are essential components of eukaryotic cells whose biosynthesis and function in 21 eukaryotes has been studied extensively. Sterols are also recognized as the diagenetic precursors 22 of steranes preserved in sedimentary rocks where they can function as geological proxies for 23 eukaryotic organisms and/or aerobic metabolisms and environments. However, production of 24 these lipids is not restricted to the eukaryotic domain as a few bacterial species also synthesize 25 sterols. Phylogenomic studies have identified genes encoding homologs of sterol biosynthesis 26 proteins in the genomes of several additional species, indicating that sterol production may be 27 more widespread in the bacterial domain than previously thought. Although the occurrence of 28 sterol synthesis genes in a genome indicates the potential for sterol production, it provides 29 neither conclusive evidence of sterol synthesis nor information about the composition and 30 abundance of basic and modified sterols that are actually being produced. Here, we coupled 31 bioinformatics with lipid analyses to investigate the scope of bacterial sterol production. We 32 identified oxidosqualene cyclase (Osc), which catalyzes the initial cyclization of oxidosqualene 33 to the basic sterol structure, in 34 bacterial genomes from 5 phyla (Bacteroidetes, Cyanobacteria,
34Planctomycetes, Proteobacteria and Verrucomicrobia) and in 176 metagenomes. Our data indicate that 35 bacterial sterol synthesis likely occurs in diverse organisms and environments and also provides 36 evidence that there are as yet uncultured groups of bacterial sterol producers. Phylogenetic 37 analysis of bacterial and eukaryotic Osc sequences revealed two potential lineages of the sterol 38 pathway in bacteria indicating a complex evolutionary history of sterol synthesis in this domain. We 39 characterized the lipids produced by Osc-containing bacteria and found that we could generally 40 predict the ability to synthesize sterols. However, predicting the final modified sterol based on our current 41 knowledge of bacterial sterol synthesis was difficult. Some bacteria produced demethylated and 42 saturated sterol products even though they lacked homologs of the eukaryotic proteins required 43 3 for these modifications emphasizing that several aspects of bacterial sterol synthesis are still completely 44 unknown. It is possible that bacteria have evolved distinct proteins for catalyzing sterol 45 modifications and this could have significant implications for our understanding of the 46 evolutionary history of this ancient biosynthetic pathway. 47 48 Sterols are tetracyclic triterpenoid lipids that are required by all eukaryotes for critical cellular 49 functions including maintaining membrane fluidity, phagocytosis, stress tolerance and cell 50 signaling (Bloch, 1991;Swan and Watson, 1998;Castoreno et al., 2005;Xu et al., 2005;Riobo, 51 2012). Studies on the biosynthesis of sterols in eukaryotes have revealed a variety of novel 52 biochemical reactions while molecular and cell bio...