Date of Award

12-2025

Document Type

Thesis

Degree Name

Master of Science in Biology

Department

Biology

First Reader/Committee Chair

Daniel Nickerson

Abstract

Variovorax paradoxus is a Gram-negative aerobic bacterium present in both soil and the human oral microbiome. This species contributes to biotransformation of xenobiotics and promotes plant growth. Interestingly, although the genome of strain EPS lacks the flagellum operon annotated in strain S110, the microbe remains motile, and flagella can be observed microscopically. To investigate this unexpected phenotype, motility mutants were enriched from a Tn5 mutant library using a liquid culture settling strategy. A total of 122 isolates were screened for motility defects, and 57 exhibited significantly reduced motility compared to the wild type. Rescue cloning revealed that over 60% of these mutants contained disruptions in genes within the tight adherence (tad) locus. Based on previous studies, these genes are predicted to function in a single pilus biogenesis system, although their roles in swarming or planktonic motility is unclear.

Mutant N10 was found to contain a disruption in a pseudogene, Varpa_5149, located within the tad locus and hypothesized to encode a prepilin peptidase, a component documented in other bacterial tad systems. The prepilin peptidase, a core gene present in other tad loci, was missing from V. paradoxus EPS. Complementation of Varpa_5148 alone, which encodes the Flp/Fap prepilin, did not restore motility, whereas complementation with Varpa 5148-5150, which includes the putative prepilin peptidase, restored motility to that of the wild type. To examine the biochemical role of the pseudogene, a tagged flp/fap prepilin fusion protein was constructed, overexpressed with arabinose and purified. The purified protein was incubated with lysates from wild type V. paradoxus, mutant N10, and complemented N10. Cleavage of the Flp/Fap prepilin protein was expected in the wild type and complemented N10 strain, but absent in N10; however, cleavage activity was also detected in N10 lysates, suggesting potential nonspecific peptidase activity. These findings indicate that the experimental approach needs refinement and the role of the putative prepilin peptidase in V. paradoxus warrants further investigation.

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