Tools for Manipulation of Microbial Communities through Bacterial Conjugation

Abstract

Microbial communities are ubiquitous across the planet and play essential roles in numerous aspects of society including human health, agriculture, food production, and biodegradation. Controlled manipulation of natural microbial communities by introduction of biological agents is a promising option for precise, in situ microbiome editing. Bacterial conjugation is a well-studied mechanism for gene exchange (horizontal gene transfer) between bacteria in physical proximity to one another. Conjugation can be repurposed as a gene delivery technique into microbial communities and holds potential for addressing problems of environmental and clinical relevance, including the biodegradation of pollutants such as microplastics and delivery of precision antimicrobials. This thesis begins with a proof-of-concept study in which recombinant DNA is delivered to bacteria in wastewater via conjugation to enable the degradation of polyethylene terephthalate (PET), a major component of global plastic pollution. Using a broad-host-range conjugative plasmid, we enabled expression of FAST-PETase in various bacterial species from municipal wastewater, achieving substantial degradation of both commercial PET film and post-consumer PET products under laboratory conditions. Next, the thesis builds computational tools and methodologies to support model-based design of in situ gene delivery into microbial communities through conjugation. Prior to starting model development, a review of practices for calibrating spatio-temporal models to microscopy data was conducted, which demonstrated the need for a more formal process for generating predictive models. Drawing on practices from ecology, new strategies for systematic model validation were proposed. Some of these strategies were then implemented into a model calibration pipeline based on Pattern-Oriented-Modelling and Bayesian parameter inference, and the pipeline was demonstrated by fitting biophysical parameters in an agent-based model to time-lapse microscopy data. The calibrated model was able to reproduce several patterns in microcolony formation that were observed experimentally, but did not fully replicate patterns associated with colony shape. Finally, a single-cell-based approach to characterizing conjugation in microfluidic environments was developed to investigate spread of the previously developed conjugative plasmid that enabled expression of FAST-PETase. Although the plasmid could spread through conjugation, cells bearing the conjugative plasmid tended to get outcompeted for space by the faster-growinghealthier recipient population. The tendency of cells to self-orient in the direction towards the exit of the microfluidic traps through biomechanical processes also reduced conjugation efficiency. The previously developed model calibration pipeline was then applied to calibrate an agent-based model for conjugation to the microscopy data collected. Afterwards, the calibrated model was used to characterize how initial conditions and spatial factors influenced spread of conjugative plasmids in enclosed microenvironments. Collectively, these studies enhance understanding of engineering microbial communities through conjugation, offering novel solutions for plastic waste degradation and advancing model-based design of gene delivery into microbial communities.