ORCID

http://orcid.org/0000-0001-6165-4700

Date of Award

Winter 12-15-2022

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Computational & Systems Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The human experience is indelibly linked to microbial life. So much so, that the holobiont theory has been coined to define the assemblage of host and microbe as a discrete ecological unit. Perturbation of the commensal microbiome can create niche space for pathogens, which through the concomitant rise of antimicrobial resistance represent an ever-evolving danger. In Chapter 2 of this dissertation, I describe the results of an interventional study designed to directly perturb the healthy gut microbiome and observe the dynamics of taxonomic composition and functional recovery. I observe significant decreases after 5 days of antibiotic treatment in the gold standard metrics used to measure bacterial viability, yet see these same metrics recover to pre-treatment baseline levels within a few weeks. Recovery of species richness does not come without consequences, however, and results in significant functional enrichment of resistance in healthy volunteer microbiomes in three out of four treatment groups. An increase in compositional dissimilarity for taxonomic and resistome composition up to the end of the 6-month study window further confirms the entrenchment of a novel microbiome structure post-treatment. By looking past standard metrics of microbiome health and diversity, I observe both acute and long-term changes to the taxonomic assembly of commensal bacteria, the resulting consequences on the resistome of healthy volunteers, and identify individuals at greater risk of microbiome dysbiosis after treatment.

Beyond the bacteria residing on and within us are a slew of fascinating organisms which humanity coopts for another reason: bioproduction of chemicals essential for a functioning society. Escherichia coli is an excellent chassis for bioproduction of organic compounds due to its fast growth, genetic tractability, and well-understood metabolism. In chapter 3, four E. coli MG1655 (E.coli) strains are synthetically engineered to produce altered fatty acid (FA) compositions via the overexpression of novel biosynthesis pathways, resulting in new membrane phospholipid compositions. Two of which are not natively produced by WT E. coli. I observe that increased production of cyclopropane FA (CFA) and novel production of internally branched-chain FA (IBFA) results in largely similar growth rates and cell densities as WT. Production of double unsaturated FA (DUFA) results in reduced growth and metabolic output in multiple environmental conditions, as well as a highly perturbed transcriptomic state likely related to an increased need for maintaining iron homeostasis. Overall, I find the E. coli chassis tolerates altered or even novel phospholipid compositions while maintaining WT-like growth.On the opposite side of the spectrum, Rhodococcus opacus PD630 (R. opacus) is less genetically tractable, but it’s oleaginous nature and incredible metabolic potential have led to efforts to optimize R. opacus for degradation of recalcitrant carbon sources. Lignin is an underutilized resource produced from plant matter which R. opacus can degrade into the fuel precursor molecule triacylglyceride (TAG). Unfortunately, R. opacus only stores carbon as TAGs during nutrient shortage, which limits overall growth and production. In chapter 4 of this dissertation, we overexpress autologous transcription factors identified using a top-down transcriptome screen and demonstrate increased TAG production when grown in phenol, an aromatic compound commonly found in lignin breakdown products (LBPs). This is directly tied to increased expression of the aromatic catabolism genes of the β-ketoadipate pathway, and expression of the phenylacetic acid (paa) pathway repressor PaaX. Using genetic deletion experiments, we demonstrate the existence of a complex functional regulation mechanism for increased TAG production which requires the expression of the feaR activator of the phenylethylamine pathway in the +paaX background.

Finally, in chapter 5 we use R. opacus strains previously adapted to increasingly diverse mixtures of LBPs (MLBPS) to identify adaptive mechanisms for increased tolerance to aromatic compounds. Adapted strains exhibit increased growth rate in MLBPs, and significantly higher utilization of vanillic acid after adaptation. At high concentrations non-permissive to WT growth however, adapted strains exhibit catabolic repression, preferentially utilizing 4-hydroxybenzoate before other carbon sources. Compared to WT grown in a low concentration of MLBPs, adapted strains exhibit little shared differential expression or differential expression of the aromatic degradation clusters and catabolic pathways required for MLBP utilization. It is at high concentrations non-permissive to WT growth, when the effects of adaptation are strongest, that R. opacus exhibits divergent DE in the β-ketoadipate pathway. This led to the identification of a putative operon of 8 genes which are similarly divergently DE in all strains, and contain genes likely involved in aromatic catabolism and lipid biosynthesis. Through each chapter of the dissertation, I study the effect of perturbation on microbial systems at the community and cellular level, identifying in each case the emergent properties and mechanisms used for system resilience, and how this results in recovery, increased bioproduction, and tolerance.

Language

English (en)

Chair and Committee

Guatam Dantas

Committee Members

Juliane Bubeck-Wardenburg

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