Sterol side-chain methylation in uncultured bacteria
- Sterols are polycyclic triterpenoid lipids required by eukaryotes for many critical cellular functions. In addition, sterols are quite recalcitrant and are preserved as sterane molecular fossils, or biomarkers, in sedimentary rocks up to 1.6 billion years old. Given the persistence of these molecules in ancient rocks and their wide distribution amongst extant eukaryotes, geologists often interpret steranes broadly as biomarkers of past eukaryotic life, although diverse bacteria are also known to produce sterol lipids. Sterols with certain modifications to the side-chain, such as methylations at the C-24 position, can act as more specific biomarkers given their restriction to certain eukaryotic lineages and absence in cultured bacteria. One such side-chain methylated sterane biomarker, 24-isopropylcholestane (24-ipc), has historically been attributed to sea sponges of the Demospongiae class. 24-ipc first appears in the Cryogenian and may therefore represent the first evidence of animals on Earth. However, enzymes that produce sterols with the 24-isopropyl side-chain have not been identified in any extant organism. In this dissertation, I investigate the biochemical requirements for sterol side-chain propylation and explore the potentional for sterol side-chain methylation in yet-uncultured bacteria more broadly through a combination of laboratory experiments and bioinformatic analyses. In Chapter 1, I find that several bacterial sterol methyltransferases (SMTs) identified in metagenomes methylate the sterol side-chain in vitro and identify three bacterial SMTs that produce sterols with the 24-isopropyl side-chain of the 24-ipc biomarker. Several functional metagenomic SMTs occur in sterol biosynthesis gene clusters suggesting yet-uncultured bacteria have the genomic capacity to produce side-chain alkylated sterols de novo. In Chapter 2, I identify a necessary but not sufficient glycine residue conserved among propylating bacterial SMTs that occurs in the active site of SMT model structures. The presence of this glycine residue can therefore act as indicator of an SMT's ability to produce 24-isopropyl sterols. In Chapter 3, I find that bacterial SMTs are widely distributed in the environment but are particularly enriched in sponge metagenomes. Bacterial SMTs are actively transcribed in wastewater, soil, creeks, hot springs, and estuaries. Together, the results presented in this dissertation suggest bacteria may be an overlooked source of side-chain methylated sterols in the rock record, provide a framework for predicting SMT function directly from sequencing data, and highlight environments that can be targeted for future cultivation of bacteria that produce side-chain methylated sterols. Overall, this work demonstrates how techniques in molecular biology, biochemistry, and bioinformatics can be leveraged to improve the interpretation of molecular fossils.
|Type of resource
|electronic resource; remote; computer; online resource
|1 online resource.
|Brown, Malory Onessa
|Degree committee member
|Degree committee member
|Stanford Doerr School of Sustainability
|Stanford University, Department of Earth System Science
|Statement of responsibility
|Malory Onessa Brown.
|Submitted to the Department of Earth System Science.
|Thesis Ph.D. Stanford University 2023.
- © 2023 by Malory Onessa Brown
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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