All cells are bound by a lipid membrane that separates the interior of the cell from the exterior environment, which is a universal characteristic of all known life on Earth. Some types of membrane lipids are found only in specific groups of organisms, and historically have been used as biomarkers for these groups. The carbon skeletons of branched or cyclic lipids are more resistant to degradation after cell death and can survive in the rock record for billions of years. Such 'geolipids' provide important fossil information and can help reconstruct the record of past microbial ecosystems on the early Earth, and potentially other planetary bodies. Previous lipid biomarker work has largely been directed at establishing taxonomic proxies by identifying compounds in pure cultures and environmental samples. But recent molecular work identifying the genes involved in the biosynthesis of geologically relevant lipids such as hopanoids has opened the door to surveying genomic and metagenomic data sets for the ability to synthesize these compounds. This work has revealed a much broader distribution of the genes than was previously realized via the pure culture studies. This discovery has necessitated a shift from taxonomic studies to investigating the cellular function of these compounds. The goal is to link the biological function of the lipid biomarkers to specific environmental conditions or bio(geo)chemical processes. Such a relationship will offer new criteria for interpreting the rock record. In keeping with the discovery of the wider distribution of certain lipid biomarkers, we have recently found that three purple non-sulfur (PNS) anoxygenic phototrophs isolated from marine, hypersaline, and hot spring environments synthesize 3-methylbacteriohopanepolyols (3-MeBHPs). These compounds were previously thought to be exclusively made by oxygen-requiring methanotrophic and acetic acid bacteria, and have long been considered a signal for methanotrophy in the rock record. However, our cultures are the first example of both the production of 3-MeBHPs by phototrophic bacteria and their production under anoxic conditions. We propose to investigate the cellular function of these compounds in PNS by utilizing a novel combination of molecular genetics, laboratory culture, and environmental studies. The goal is to link the laboratory and environmental experiments to ensure that the observations made with pure cultures reflect the biosynthesis of 3-MeBHPs in the environment. Such a relationship is necessary for properly interpreting the occurrence of these compounds in the sedimentary rock record. Preliminary PNS pure culture experiments have revealed that both total BHP and 3-MeBHP increased relative to total membrane fatty acid with increasing pH. We also observed a concomitant increase in cyclopropane fatty acid (cy19), indicating a membrane response to pH stress. We propose to investigate the role of hopanoids in pH homeostasis. This will be achieved by identifying the 3-MeBHP biosynthesis gene (hpnR) in these organisms, and creating deletion mutants of this gene for subsequent physiological studies. The aim of these studies will be to determine how 3-MeBHP production is regulated in response to membrane stress. We will also perform detailed compound-specific C isotopic studies, as preliminary analyses indicate that the PNS 3-MeBHPs will be distinguishable from methanotrophic 3-MeBHPs in the rock record. The proposed research is relevant to the Exobiology theme of Early Evolution of Life and the Biosphere, and objectives (ii) understand the phylogeny and physiology of microorganisms, including extremophiles, whose characteristics may reflect the nature of primitive environments; and vi) investigate the evolution of genes, pathways, and microbial species subject to long-term environmental change relevant to the origin of life on Earth and the search for life elsewhere.