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Plasmids, Immunity, and Phages of Gut Bacterium Eggerthella lenta

  • Author(s): Soto Perez, Paola Andrea
  • Advisor(s): Bondy-Denomy, Joseph
  • et al.
Abstract

The human gut microbiota encompasses all the microorganisms that reside in the gastrointestinal tract. Bacteria, its most abundant member, are known to have an impact on their human host. Bacteriophages (phages), also abundant within the microbiota, can potentially alter gut composition yet their interactions with bacteria remain poorly understood, particularly with respect to CRISPR-Cas immunity. Here, we focus on gut bacterium Eggerthella lenta: attempting to develop genetic tools, investigating its CRISPR-Cas system, and isolating phages against it. While developing a transformation protocol, we discovered endogenous plasmids and antibiotic resistance genes that we used for constructing shuttle vectors. We demonstrated that its CRISPR-Cas system is transcribed and sufficient for targeting of DNA. Furthermore, we assembled a database to identify protospacers that revealed matches for a majority of spacers and uncovered “hyper-targeted” phage and determined the mismatch tolerance of observed spacer/protospacer pairs. To study phage interactions with microbiome members and gut bacterial evasion of phages, we screened wastewater for phage against E. lenta. We isolated, sequenced, and annotated 4 novel and highly related phages. Using the phage to target a sensitive strain in gnotobiotic mice didn’t have an impact on bacterial abundance, despite their ability to lyse E. lenta in vitro. Our work lays a foundation for the development of tools for E. lenta, an understanding of its CRISPR-Cas system and characterization of novel phages.

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