The Regulation of Fitness Factors in a Commensal Gut Bacterium

Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology

First Advisor

Groisman, Eduardo

Abstract

The human gut microbiome is a dynamic environment with significant variation in available nutrients. Bacteroides thetaiotaomicron is able to colonize and persist in the gut microbiome due to its ability to adapt to utilizing different carbohydrate. Fitness factors are genes or proteins that are necessary to enable B. thetaiotaomicron’s colonization of the murine gut. These fitness factors are either diet-dependent or diet-independent. I identify different methods of regulation occurring via the fitness factor Cur as well as regulation of the fitness factors EF-G2 and Roc.I identify regulators of the elongation factor G2, EF-G2, a paralog to an essential translation factor. EF-G2 is required for wild type colonization in the murine gut and allows B. thetaiotaomicron to continue translocation without utilizing energy. EF-G2 is regulated through a master transcriptional regulator Cur and additionally by proteins found upstream of Cur, PfkA and BT3803. Cur also regulates Roc, regulator of colonization. Roc is required for wild type colonization in the murine gut on a diet-dependent basis. As Cur does not directly bind the roc promoter, I investigated how Cur regulates Roc protein amounts. I determined Cur regulates Roc via the roc leader sequence. This regulation is relieved when the roc leader is replaced with a different leader sequence. This exploration included utilizing a transposon mutagenesis screen and an RNA-RNA interaction prediction tool. Finally, I also identify a rut site in the roc transcript that results in a Rho-dependent terminated transcript through an in vitro transcription assay. I also investigated differentially regulated genes resulting from an RNA-seq experiment deficient for two predicted sRNAs. Both of these sRNAs are regulated by Cur in glucose and under carbon starvation conditions. In addition to identifying differentially regulated genes as a result of the absence of the two predicted sRNAs, I identified genes that are differentially regulated in a Cur-deficient background as well. Consequently, identifying genes that are differentially regulated by the two sRNAs downstream of Cur. Overall, I have identified different forms of regulation that occur on and from three fitness factors that are required for wild type colonization of B. thetaiotaomicron in the murine gut.

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