Research

Earth’s history is marked by atmospheric and climatic fluctuations that have shaped life and its evolution. Floral and faunal fossils have revealed that these ancient events profoundly changed the abundance and diversity of macroscopic organisms, yet much less is known about how microbial communities responded to these dramatic environmental changes. This is one of the challenges in geomicrobiology - how do we study microorganisms in the context of Earth’s distant past?

While microbes do not readily leave diagnostic morphological fossils, subtle microbial signatures are preserved in sedimentary rocks for billions of years. One such group of biosignatures are well-preserved lipid compounds with specific biological origins, which can be used as biomarkers or "molecular fossils" for the presence of certain microbes or environmental conditions at the time of deposition.

Despite the significant implications biomarker studies have on our interpretation of microbial evolution and Earth’s ancient environment, our understanding of the phylogenetic distribution and physiological function of these molecules in modern bacteria is quite limited. In our lab, we combine techniques from bioinformatics, genetics, physiology and biochemistry to address three general questions that can be applied to any biomarker:

• What is its phylogenetic distribution in modern bacteria?
• What are its physiological roles in modern bacteria?
• What is the evolutionary history of its biosynthetic pathway?

Current Projects

1) Bacterial production of eukaryotic biomarkers. Eukaryotic biomarkers are specific lipid molecules that are considered diagnostic for certain eukaryotic organisms – from multicellular organisms like sponges to unicellular eukaryotes such as protists. However, some bacterial species have been shown to produce these “eukaryotic” lipids, and there are several open questions regarding how bacteria synthesize and utilize these lipids.

Tetrahymanol synthesis by bacteria. Tetrahymanol is primarily produced by ciliated protists commonly found in aquatic environments and is recognized as the diagenetic precursor to gammacerane, a polycyclic hydrocarbon detected in sedimentary rocks dating as far back as the late Proterozoic. A few bacterial species are also capable of tetrahymanol production but the biochemical mechanisms for producing this lipid in bacteria have remained a mystery. Using a combination of comparative genomics, gene deletion, and lipid analyses, we identified a novel bacterial protein, Ths, required for the synthesis of tetrahymanol in methane-consuming bacteria (Banta et al., 2015, PNAS). We demonstrated that Ths is found in other tetrahymanol producing bacteria including anoxygenic phototrophs and sulfate-reducing bacteria and is mechanistically distinct from eukaryotic synthesis of tetrahymanol. Bioinformatics analyses of Ths also revealed that bacterial tetrahymanol production is more prevalent in freshwater and terrestrial environments than marine systems. We are currently trying to better understand the mechanistic and structural characteristics of Ths as well as the evolutionary history of this bacterial pathway. 

Sterol synthesis by bacteria. Sterol lipids, such as cholesterol, are ubiquitous and essential components of eukaryotic cells whose diagenetic products, steranes, are utilized as general biomarkers for eukaryotes. However, sterol production has been observed in a few bacterial species and very little is known about the biosynthesis and function of sterols in these organisms. To better understand sterol-production in the bacterial domain, we searched bacterial genomes and metagenomes for one essential sterol synthesis protein, oxidosqualene cyclase. These analyses demonstrated that sterol production is more widespread in the bacterial domain than previously thought (Wei et al., 2016, Front Microbiol). In addition, we have discovered a set of novel bacterial proteins, SdmA and SdmB, required for demethylating sterols at the C-4 position - a modification that is essential for proper sterol function in eukaryotes (Lee et al., 2018, PNAS). SdmA and SdmB are phylogenetically and mechanistically distinct from the C-4 demethylase enzymes in eukaryotes and, like bacterial tetrahymanol synthesis discussed above, is an example of convergent evolution in lipid synthesis.

We are currently exploring the mechanistic details and functional significance of sterol demethylation, and sterols more broadly, in a variety of bacteria. These studies have led to the discovery of more complex sterol production by a marine δ-Proteobacterium, Enhygromyxa salina. In addition, we are utilizing the aerobic methanotroph Methylococcus capsulstus as a model system to study how sterols are transported from the cytoplasmic membrane to the outer membrane and whether sterol demethylation functions as an indicator of hypoxic conditions in these organisms.

3) Identification of orphan biomarker sources. Orphan biomarkers are lipids found in ancient or modern sediments for which there are no extant sources or the extant sources are not consistent with their occurrence in a specific environment or time period. One example is isoarborinol, an unusual pentacyclic triterpenol whose only known extant sources are certain flowering plants. Through our work, we have identified two novel arborinol lipids structurally similar to isoarborinol, which we named eudoraenol and adriaticol, in the marine bacterium Eudoraea adriatica (Banta et al., 2017, PNAS). We are currently investigating the phylogenetic distribution of eudoraneol cyclase homologs in environmental metagenomes and are using an E. coli heterologous expression system developed in our lab to express these cyclases.

5) Terpene cyclase evolution and the emergence of eukaryotes. Cyclic triterpenoids are a diverse class of lipids synthesized by enzymes called cyclases. The phylogenetic distribution of different types of cyclases in modern bacteria suggests two different evolutionary models. In one model, squalene-hopene cyclases (SHCs), which make hopanoids, share a common protein ancestor with oxidosqualene cyclases (OSCs) that make sterols and arborinols. However, another model suggests that SHCs are ancestral and that several critical residue changes in an SHC could give rise to an OSC. As sterols are essential in all eukaryotes yet rarely produced in bacteria, we are particularly interested in how cyclase evolution contributed to the emergence of eukaryotic life. We are currently expressing SHCs and OSCs from environmental metagenomes to elucidate phylogenetic relationships between different types of cyclases. In addition, we are exploring the cyclase activity of putative archaeal cyclases identified in metagenomes assembled genomes (MAGs). To date, no archaeon has been shown to produce cyclic triterpenoids. 

Friends & Collaborators

Roger Summons, MIT

José Giner, SUNY-ESF

Dianne Newman, Caltech

Zhirui (Ray) Zeng, Southern University of Science and Technology

Amy Banta, University of Wisconsin-Madison

Laura Meredith, University of Arizona

Laura Dassama, Department of Chemistry at Stanford

Linda Jahnke, NASA Ames Research Center

M. Niki Parenteau, NASA Ames Research Center

Michael T. Madigan, Southern Illinois University

William (Bill) Metcalf, University of Illinois Urbana-Champaign