Date of Award

Fall 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology

First Advisor

Groisman, Eduardo

Abstract

The mammalian gut is densely populated with trillions of microbes, collectively termed the gut microbiota. The gut microbiota is critical to the health of the host. Therefore, it is critical to understand how beneficial microbes persist in the gut. This dissertation presents molecular mechanisms by which conserved genetic factors promote gut colonization by the abundant human gut symbiotic bacteria from the Bacteroides genus. Bacteroides species have an extraordinary ability to utilize various complex polysaccharides available in the gut. I have identified the global regulon of a transcriptional regulator, BT4338, necessary for the utilization of polysaccharides and intestinal colonization by B. thetaiotaomicron. BT4338 regulates hundreds of genes, including several involved in carbohydrate utilization, central metabolism, and energy generation, as well as a gene specifying a translation factor homolog, EF-G2, that is dispensable for carbohydrate utilization but necessary for fitness in the gut. EF-G2 specifically accumulates under carbon starvation conditions, as a result of direct transcription activation of its encoding gene by BT4338. I established that EF-G2 is a novel translation factor that promotes ribosome translocation and protein synthesis, albeit more slowly than the ubiquitous canonical EF-G, without consuming energy. This is in contrast to the canonical EF-G which consumes energy for every amino acid incorporated into a protein. Therefore, EF-G2 enables B. thetaiotaomicron to save energy during the most energy-demanding step of protein synthesis. I also determined that the ability of EF-G2 to sustain protein synthesis is essential for the fitness of B. thetaiotaomicron in the murine gut. Critically, EF-G2 outnumbers the canonical EF-G more than 10-fold in bacteria harvested from the murine cecum. Additionally, I discovered a novel transcription anti-termination system that links transcription anti-termination at a specific site to the abundance of translation factors. EF-G2, or artificially over-expressed EF-G1, increases the abundance of mRNA of a second gene (BT0901) in a two-gene operon (BT0902-01) by promoting transcription read-through at an intrinsic transcription terminator in the intergenic region. This effect depends on an upstream cis-acting element located distantly from the terminator and is independent of the ability of EF-G2 or EF-G1 in protein synthesis. Lastly, I identified genes that have genetic interactions with the master regulator encoding BT4338 or the EF-G2 encoding BT2167 during murine gut colonization by B. thetaiotaomicron. Collectively, a master regulator in Bacteroides coordinates expression of genes involved in carbohydrate utilization and energy generation, as well as a gene specifying an alternative translation factor that mediates energy-efficient slow protein synthesis. These factors enable Bacteroides to calibrate both metabolism and protein synthesis to the availability of nutrient and energy resources, thereby advancing bacterial fitness in the gut.

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