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Sterol Side-Chain Methylation in Uncultured Bacteria.

Record Type:	Language materials, manuscript : Monograph/item
Title/Author:	Sterol Side-Chain Methylation in Uncultured Bacteria./
Author:	Brown, Malory Onessa.
Description:	1 online resource (155 pages)
Notes:	Source: Dissertations Abstracts International, Volume: 85-06, Section: B.
Contained By:	Dissertations Abstracts International85-06B.
Subject:	DNA methylation. -
Online resource:	click for full text (PQDT)
ISBN:	9798381021264

Sterol Side-Chain Methylation in Uncultured Bacteria.
Brown, Malory Onessa.

Sterol Side-Chain Methylation in Uncultured Bacteria. - 1 online resource (155 pages)

Source: Dissertations Abstracts International, Volume: 85-06, Section: B.

Thesis (Ph.D.)--Stanford University, 2023.

Includes bibliographical references

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.

Electronic reproduction.
Ann Arbor, Mich. :
ProQuest,
2024

Mode of access: World Wide Web

ISBN: 9798381021264Subjects--Topical Terms:

1468904
DNA methylation.
Index Terms--Genre/Form:

554714
Electronic books.

Sterol Side-Chain Methylation in Uncultured Bacteria.
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245 1 0 $a Sterol Side-Chain Methylation in Uncultured Bacteria.
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300 $a 1 online resource (155 pages)
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500 $a Source: Dissertations Abstracts International, Volume: 85-06, Section: B.
500 $a Advisor: Francis, Christopher;Sperling, Erik;Welander, Paula.
502 $a Thesis (Ph.D.)--Stanford University, 2023.
504 $a Includes bibliographical references
520 $a 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.
533 $a Electronic reproduction. $b Ann Arbor, Mich. : $c ProQuest, $d 2024
538 $a Mode of access: World Wide Web
650 4 $a DNA methylation. $3 1468904
650 4 $a Fungi. $3 580161
650 4 $a Bioinformatics. $3 583857
650 4 $a Eukaryotes. $3 1468905
650 4 $a Biosynthesis. $3 674536
650 4 $a Bacteria. $3 600016
650 4 $a Labeling. $3 1466860
650 4 $a Cholesterol. $3 1226662
650 4 $a Amino acids. $3 1066910
650 4 $a Sterols. $3 582399
650 4 $a Genomes. $3 600075
650 4 $a Drinking water. $3 712720
650 4 $a E coli. $3 1413565
650 4 $a Chromatography. $3 783938
650 4 $a Lipids. $3 556565
650 4 $a Creeks & streams. $3 1465326
650 4 $a Algae. $3 632686
650 4 $a Cloning. $3 572584
650 4 $a Steroids. $3 1026991
650 4 $a Biomarkers. $3 1155103
650 4 $a Enzymes. $3 557408
650 4 $a Aquatic sciences. $3 1178821
650 4 $a Biochemistry. $3 582831
650 4 $a Genetics. $3 578972
650 4 $a Microbiology. $3 591510
650 4 $a Water resources management. $3 1178858
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