The following projects combine bioinformatics with bacterial genetics and microbial physiology to get a better understanding of the biosynthesis and function of hopanoids and sterols in the aerobic methanotrophs. Our initial studies have been focused on utilizing the model organism Methylococcus capsulatus Bath. Originally isolated from a hot spring in the City of Bath, M. capsulatus is known to produce 3-methylhopanoids as well as sterols. We have expanded our studies to include M. capsulatus Texas, a close relative of the Bath strain, and Methylomicrobium alcaliphilum 20Z, an alkiliphilic, halotolerant methanotroph isolated from a soda lake in Siberia. Both Texas and 20Z are also sterol and 3-methylhopanoid producers. Therefore, these organisms are excellent models for studying the physiological function of both sterols and hopanoids in the bacterial domain. The genome sequence of each strain is publicly available and we have made significant progress in developing a set of genetic tools in these organisms. Furthermore, methanotrophs such as M. capsulatus are a critical component of the Earth’s ecosystem as their oxidation of biogenic methane plays a significant role in the global carbon cycle and in reducing the release of a potent greenhouse gas.
Biosynthesis of sterols and hopanoids in M. capsulatus. In addition to various amino functionalized and C-3 methylated hopanoids, M. capsulatus produces four different sterols, all of which contain one or two C-4 methyl groups. The 4-methyl sterols produced by M. capsulatus are considered biosynthetic intermediates in the formation of various sterols and these intermediates are rarely observed in eukaryotes. This project currently has three specific goals. The first is to construct sterol and hopanoid deletion mutants in M. capsulatus and other methanotrophs to determined if these two lipid classes share a common functional role. Second, is to identify proteins specifically involved in the conversion of 4-dimethyl sterols into 4-methyl sterols. Finally, we are pursuing studies to understand the localization of sterol molecules in methanotrophic membranes and how these lipids are transported throughout the cell.
Understanding 3-methylhopanoid function. One of the most characteristic hopanoids produced by obligate aerobic methanotrophs such as M. capsulatus are those methylated at the C-3 position. As such, 3-methylhopanes found in the rock record have been used as biomarkers not just for methanotrophy but also for evidence of aerobiosis on the ancient Earth. Yet nothing is known about the physiological significance of this methylation in extant methanotrophs. We have used bioinformatics and genetic deletion analysis to identify a C-3 methylase, HpnR, required for 3-methylhopanoid production in M. capsulatus. The main objective of this project is to identify the functional role of 3-methylhopanoids through further characterization of the C-3 methylase mutant in M. capsulatus and other organisms. Utilizing whole genome transcriptomics we are attempting to identify and understand the environmental and regulatory factors required for the expression of 3-methylhopanoids.
Tetrahymanol biosynthesis and function in the bacterial domain. Our studies on sterol and hopanoids in aerobic methanotrophs revealed the production of a third polycyclic lipid in M. alcaliphilum. This lipid is recognized as the biological precursor to gammacerane, a hydrocarbon biomarker identified in ancient sediments dating as far back as 1.6 billion years ago. Tetrahymanol was first identified in the eukaryotic ciliate, Tetrahymena pyroformis, where it is thought to function as a sterol surrogate under sterol-limiting conditions. While tetrahymanol has been observed in numerous ciliates since this initial discovery, its biosynthesis in bacteria was thought to be restricted to two species. Through comparative genomics and gene deletion analysis, we have identified a protein specifically required for tetrahymanol production in bacteria. Through bioinformatics analyses we have shown that tetrahymanol production is more widespread in the bacterial domain. Further, tetrahymanol biosynthesis occurs via distinct pathway not observed in tetrahymanol-producing ciliates. Our current goals for this project are to better understand the physiological function of tetrahymanol in aerobic methanotrophs, the biochemical mechanism of the newly identified bacterial tetrahymanol synthase and to identify any isotopic signatures that may distinguish bacterial tetrahymanol sources from eukarytoic ones.