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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?
Microbial stromatolite signatures in a sedimentary rock


Lipids like sterols and hopanoids retain their chemical structure as deposited sediment becomes rock.

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.


Our friends in the Summons lab at MIT performed molecular clock analyses on sterol biosynthesis enzymes to date the earliest appearance of eukaryotes. Image credit: MIT


​Bacterial synthesis of the biomarker tetrahymanol (in red) ​ ​



C-4 sterol demethylation by SdmA and SdmB in Methylococcus capsulatus ​
The demosponge lanthella flabelliformis, PC: Clay Bryce
Lanthella flabelliformis, a Demosponge
Photo by Clay Bryce, image copyright WA Museum



2) Synthesis of the sponge biomarker 24-isopropylcholesterol (24-ipc). Our exploration of sterol synthesis genes in bacterial genomes and metagenomes revealed a potential role for bacteria in the synthesis of the 24-ipc sponge biomarker. We have identified proteins required for the addition of the unique C-24 isopropyl group, C-24 sterol methyltransferases (SMTs), in bacterial symbiont genomes associated with sponges. We developed a heterologous expression system that allowed us to express these putative bacterial SMTs in E. coli and demonstrate that these proteins are true sterol methyltransferases. These are the first bacterial sterol SMTs identified and confirmed to have biochemical activity. We are currently investigating the biochemical characteristics of these bacterial SMTs, determining if any free-living bacteria synthesize sterols methylated at C-24, and also exploring the evolutionary ancestry of bacterial and eukaryotic SMTs through phylogenetic analyses.

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.

Cyclic triterpenols found in E. adriatica
Marisa and Amy sampling hot springs at Lassen Volcanic National Park
Taking samples at Lassen Volcanic National Park

4) Environmental distribution of lipid biomarkers. In order to better understand the distribution of lipid biomarkers in the environment, we study the community composition and lipid profiles in an acidic, sulfidic hot spring at Lassen Volcanic National Park. During this survey, we found a biomarker used to indicate an oxic environment, 3-methylhopanoids, in a community of anoxygenic phototrophs. Since little is known about lipid biomarker production in living microbial mats and biofilms in this natural environment, we are currently comparing seasonal factors' effect on 3-methylhopanoid production. Modern hydrothermal systems, like the hot springs at Lassen, are thought to be analogs of habitats present on early Earth and may even provide insights into the origin and evolution of photosynthetic systems.

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. 

Two models for cyclase evolution
Proposed GDGT synthesis pathway (B) based on lipid profiles of WT & constructed archaea (A & C) ​

6) Lipid biosynthesis in archaea. Glycerol dialkyl glycerol tetraethers (GDGTs) are unique archaeal membrane lipids that can function not just as biomarkers for archaea but can also be used as paleotemperature proxies. However, the pathway for GDGT synthesis has not been fully characterized and we have been attempting to identify the missing synthesis proteins in the thermoacidophile Sulfolobus acidocaldarius. Thus far, we have identified a novel protein, calditol synthase (Cds), necessary for modifying the glycosylated membrane head groups of Sulfolobus (Zeng et al., 2018, PNAS). We have also identified two novel proteins, GDGT-ring synthases, required for introducing the cyclopentane rings within the core GDGT structure (Zeng et al., 2019, PNAS). We are currently characterizing these GDGT biosynthesis proteins and continue to search for other unknown tetraether lipid biosynthesis proteins in archaea and bacteria.