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41995-G2
Aerobic and Anaerobic Pathways of Lipid Biosynthesis in an Evolutionary Context
In this project we sought to examine common assumptions about the prevalence and diversity of hopanoid producers in modern samples. Interpretation of hopanoids has traditionally been guided by several principles: hopanoid biosynthesis occurs in 50% of bacteria; it does not require oxygen; and paradoxically, hopanoid biosynthesis is found only in aerobic bacteria. Each of these assumptions remains under-explored, and many of these “universalities” have known exceptions. Here we examined both the whole genomes of microorganisms and the diversity of complex (natural) microbial assemblages in order to describe more fully the distribution of these pathways. We initially examined culturable species that have had their whole genomes sequenced to catalog the occurrence of the squalene-hopene cyclase (sqhC) gene. Subsequently, the same gene was amplified from several environmental samples representing anaerobic and aerobic environments. The diversity of these environmental sequences was extraordinary from several perspectives. First, most environmental sequences were only distantly related to their nearest relatives from culture collections. Second, we were unable to find any sequences that were related to Cyanobacteria, even though these bacteria have been believed to contribute a major fraction of the hopanoids found in some sedimentary systems. Third, we showed that hopanoid biosynthesis occurs among < 10% of bacterial species, not the previous estimate of 50%. This could suggest a specialized physiological function for hopanoids and certainly will prompt more work on this problem in the future. The results of this project have significant impact on our ability to understand hydrocarbons in ancient, geological deposits. Personnel Contributions: The work carried out throughout the two-year project was done by Prof. Ann Pearson, research technician Sarah Flood Page, post-doctoral investigator Dr. Kimberly Kraunz, graduate student Woodward Fischer, research technician-turned-graduate student William Leavitt, and undergraduate Lauren Wolchok. All of these individuals obtained their salary support from other sources. PRF funds have been used exclusively to support laboratory supplies, extensive DNA sequencing, and costs associated with traveling to conferences. Two papers have been published and two are in preparation for submission. This work has contributed to the career advancement of four female scientists: the PI, a post-doc, a technician who later became a graduate student, and an undergraduate. The undergraduate won the Hoopes Prize for excellence in research and graduated summa cum laude. Results: 1) We found that despite the widespread preservation of hopanoids in the geologic record, their biosynthesis is very sparsely distributed among the bacteria (Fischer and Pearson, 2007). This contrasts with the ubiquity of steroids in eukaryotes. Data from metagenome sequencing projects agreed: fewer than 10% of uncultured species contain triterpenoid cyclase genes. We proposed a model for the evolution of steroids, hopanoids, and other triterpenoids such as isoarborinol from a common precursor compound. We then suggested that this compound might be malabaricatriene, a known natural product found in sulfur-rich anaerobic systems. 2) We developed a set of degenerate PCR primers that target conserved sections of the squalene-hopene cyclase (sqhC) gene. These primers were used to amplify DNA from two aerobic, aquatic environments: the surface water of a highly stratified lake and from the oligotrophic central Pacific Ocean. We published a paper describing the diversity of sqhC genes obtained from these samples (Pearson et al., 2007). We applied six different methods, three of which were designed to favor the amplification of known cyanobacterial sequences and three of which targeted Proteobacteria. However, none of the sequences could be assigned definitively to any genus within these groups and none at all were affiliated with the Cyanobacteria even at the phylum level. The sequences exhibited tremendous phylogenetic diversity (Figure attached), and the results suggest that the dominant microbial sources of these important lipid biomarkers remain undiscovered. 3) Work on anaerobic environments failed to generate any sqhC sequences. It remains unknown if this was a problem with the particular sample used, or if in general the abundance of sqhC gene template in anaerobic systems is so low that it cannot be detected. This subject requires further work to answer important questions about synthesis of hopanoids in anaerobic environments. 4) We traveled to the Bahamas in 2007 to take samples across a terrestrial-estuarine-offshore transect in this carbonate-rich environment. We chose this location because if cyanobacterial sqhC sequences are to be found in any marine setting, it is likely that they will be in association with tropical carbonate platforms. Preliminary sequencing work using the primers and protocols above has yielded > 50 new sqhC sequences, representing three different sampling environments (soil, estuarine, and open marine). We are in the process of analyzing these data and preparing a publication (Leavitt and Pearson, 2007). 5) An undergraduate student thesis was supported by this grant. Lauren Wolchok explored the diversity of hopanoids produced by a bacterium, Gemmata obscuriglobus, that also produces sterols. Her thesis will be turned into a publication (Wolchok et al., 2007).
