Date of Award

Spring 5-15-2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Advisor(s)

Welch, Roy D

Second Advisor

Garza, Anthony G.

Keywords

antibiotics, genotype, knockout, Myxococcus, phenotype, xanthus

Subject Categories

Biology | Genetics | Genetics and Genomics | Life Sciences | Microbiology

Abstract

The question of how genotype affects phenotype has fascinated and puzzled scientists for generations. Regardless of organism, the correlation between observation and underlying condition is necessary for scientists to understand the world around us. Myxococcus xanthus, a fascinating organism with a large genome (7,314 genes), is known for complex social behaviors and is an excellent model system for the study of this relationship. Upon starvation, M. xanthus cells condense into large multicellular aggregates to await renewed nutrient availability. M. xanthus cells swarm together on surfaces as a predatory biofilm, lysing and killing prey as a singular unit. During predation, swarms display dynamic multicellular patterns called ripples. Individual cells also retain the ability to move independently, leaving behind a slime trail for other cells to follow. This creates complex flare structures at the edge of multicellular swarms. The first part of this thesis addresses the problem of genotype-to-phenotype accuracy in complex organisms. Using 50 single gene knockout M. xanthus strains, I performed the traditional motility phenotype assay as well as a novel motility phenotype assay under magnification. Comparison of these results demonstrates not only that genes not related to motility machinery can induce a motility phenotype, but also that motility phenotypes are dynamic and can change over time. Additionally, I demonstrate that motility phenotype can change significantly when swarms move over prey, as opposed to simple agar surfaces. This more in-depth analysis allows for phenotypic classification of knockout strains previously thought to be without phenotype. The complexity of the M. xanthus genome is not only responsible for its incredibly complex multicellular phenotype, but for its potential use as a model for the study of antibiotic resistance and production of novel antibiotic compounds. The production of secondary metabolites like antibiotics is not ubiquitous in bacteria. The unique resources or "building blocks" required for their manufacture as well as their complicated molecular scaffolds restricts their production to more complex organisms like M. xanthus. The laboratory-friendly and non-pathogenic nature of M. xanthus adds another category to its list of interesting attributes. By sharing many qualities with pathogenic bacteria while remaining a biosafety level one organism, M. xanthus represents a unique opportunity to study how antibiotic resistance is obtained, maintained, and compensated for in a complex genome. The second part of this thesis studies M. xanthus' usefulness in the fight against antibiotic resistance. I created strains of M. xanthus resistant to a wide variety of antibiotics. Some were exposed to initially low concentrations slowly building over time while others experienced higher antibiotic concentrations over a shorter period. The creation of these strains, simulating the manner in which pathogenic bacteria may gain resistance to antibiotics in vivo, allowed for a laboratory-friendly study of phenotypes and fitness costs associated with varying levels of resistance. I also supply preliminary proof of antibiotic-resistant M. xanthus' utility as a general heterologous host for novel antibiotics. Upon introduction of the oxytetracycline gene cluster, resistant strains were significantly more likely to retain the plasmid than WT. These results represent an important step in both understanding how and why antibiotic resistance arises in complex bacteria as well as a potential mechanism for novel antibiotic discovery.

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