Characterization of metabolism in human gut Coriobacteriia using a newly developed genetic toolkit

Abstract

The human gastrointestinal tract is colonized by trillions of microorganisms that greatly impact health and disease. Among these organisms are Coriobacteriia, a class of prevalent human gut Actinobacteria implicated in drug and dietary phytochemical metabolism and associated with multiple human diseases. Gaining a mechanistic understanding of Coriobacteriia metabolic activities and their regulation could better inform efforts to modulate gut microbial activities to improve human health. However, the whole Coriobacteriia taxon, including Eggerthella lenta, is currently genetically intractable. This thesis describes our efforts to develop a comprehensive genetic toolkit for Coriobacteriia and our application of these tools to characterize biochemical activities of human gut Coriobacteriia and their genetic regulation. Chapter 2 describes our efforts to develop a genetic toolkit for Coriobacteriia. We construct shuttle vectors and develop methods to transform E. lenta, Gordonibacter urolithinfaciens, and other Coriobacteriia. With these tools, we characterize endogenous E. lenta constitutive and inducible promoters using a reporter system and construct inducible expression systems, enabling tunable gene regulation. We also achieve genome editing by harnessing an endogenous type I-C CRISPR-Cas system. We further create a transposon mutagenesis library for E. lenta and G. urolithinfaciens by engineering a native transposable element. By greatly expanding our ability to study and engineer gut Coriobacteriia, these tools will reveal mechanistic details of host-microbe interactions and provide a roadmap for genetic manipulation of other understudied human gut bacteria. Chapter 3 details our work characterizing Coriobacteriia enzymes involved in polyphenol metabolism. Polyphenols are an important group of phytochemicals known for their antioxidant and anti-inflammatory properties. These dietary compounds are greatly impacted by gut bacterial metabolism, which changes their bioactivity and bioavailability. A prominent reaction in polyphenol metabolism is the removal of para-hydroxyl groups from catechols by molybdenum-dependent catechol dehydroxylases encoded in Coriobacteriia. However, the substrates of most putative catechol dehydroxylases remain unidentified due to the challenges of obtaining these enzymes from standard heterologous expression systems. To solve this problem, we establish G. urolithinfaciens as a versatile bacterial host to express active catechol dehydroxylases. The heterologous expression system allows us to streamline the catechol dehydroxylase discovery process and rapidly deorphanize twelve previously uncharacterized gut bacterial catechol dehydroxylases that selectively dehydroxylate intermediates in the gut bacterial metabolism of plant-derived catechins and lignans. Unexpectedly, we discover multiple instances of distinct catechol dehydroxylases that selectively metabolize individual substrate enantiomers, setting the stage for future efforts to elucidate the mechanisms and evolution of these enantiocomplementary dehydroxylases. Altogether, these findings greatly increase our knowledge of these metalloenzymes and provide a more comprehensive understanding of phytochemical metabolism relevant to human health. Chapter 4 illustrates our work to elucidate the function and mechanism of a unique class of transmembrane transcriptional regulators in Coriobacteriia. Aiming to address the molecular details underlying the regulation of catechol dehydroxylase expression, we identify a previously unappreciated family of transcriptional regulators comprised of a 12-transmebrane helix domain and a LuxR-type DNA-binding domain, which are referred to here as 12-TM LuxR. Bioinformatic analyses show their high diversification and wide distribution in Coriobacteriia. We confirm that 12-TM LuxRs sense specific compounds and upregulate cognate metabolic enzymes. We further combine genetic and biochemical approaches to characterize the mechanism underlying 12-TM LuxR regulation. We show that 12-TM LuxRs are one-component systems that directly bind to their inducers. The 12-TM domains structurally resemble major facilitator superfamily (MFS) transporters, and we show these domains determine inducer specificity. Lastly, we show that inducer binding likely promotes 12-TM LuxR dimerization/oligomerization, which activates the regulator. Our findings suggest that Coriobacteriia evolved MFS-like domains for metabolic regulation, representing a new mechanism for bacterial nutrient sensing and signal transduction.