Graduate Studies

 

First Advisor

Kurt H. Piepenbrink

Degree Name

Doctor of Philosophy (Ph.D.)

Committee Members

Donald Becker, Jennifer Auchtung, Limei Zhang, Mark Wilson

Department

Biochemistry

Date of this Version

5-2025

Document Type

Dissertation

Citation

A dissertation presented to the faculty of the Graduate College at the University of Nebraska in partial fulfillment of requirements for the degree of Doctor of Philosophy

Major: Biochemistry

Under the supervision of Professor Kurt H. Piepenbrink

Lincoln, Nebraska, May 2025

Comments

Copyright 2025, Yafan Yu. Used by permission

Abstract

Horizontal gene transfer (HGT) via natural competence allows bacteria to incorporate extracellular DNA (eDNA) into their genomes, facilitating genetic diversity and adaptation. In Acinetobacter species, natural competence depends on DNA binding and uptake mediated by type IV pili (T4P). T4P are dynamic extracellular filaments composed primarily of the major pilin subunit PilA, along with a few minor subunits. T4P drive a range of cellular functions including twitching motility, biofilm formation, and DNA uptake, with some functions dependent on pilus retraction and others not. However, how Acinetobacter T4P bind DNA and how T4P/eDNA interactions impact biofilm formation remain unclear. Here, I present my results probing 1) the mechanisms by which Acinetobacter T4P bind DNA and 2) the structural basis for differences in DNA uptake between naturally-occurring clonal groups of Acinetobacter baumannii.

To identify which pilus subunits are eDNA receptors in Acinetobacter, we recombinantly expressed Acinetobacter T4P subunits and measured binding to DNA as well as T4P functions in A. baumannii transposon mutants of T4P subunits. We found that FimT is not required for pilus assembly or twitching motility but promotes biofilm formation. Flow cytometry further showed that a fimT mutant is defective in DNA uptake. Although FimT does not contain known DNA-binding motifs, computational structure prediction suggests possible mechanisms for direct interactions with dsDNA.

Acinetobacter bacteria, including International Clone I (IC-I) and International Clone II (IC-II) strains, show variable phenotypes in assays of type IV pilus-dependent functions. Here we show that this variation is the result of variable efficiency in pilus retraction between pilus subtypes and from that, a differential balance between retraction-dependent and retraction-independent functions. We define type IV pilus subtypes based on the sequence of the major subunit, PilA. In both naturally-occurring pilA variants from the IC-I and IC-II groups and isogenic strains complemented with IC-I or IC-II pilA, the IC-I pilus subtype promotes greater twitching motility and DNA-uptake while the IC-II pilus subtype promotes biofilm formation while showing reduced capacity for DNA-uptake and twitching motility, similar to a retraction-deficient mutant and consistent with the hypothesis that pilus retraction of the IC-II pilus is naturally deficient.

Advisor: Kurt H. Piepenbrink

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