Author ORCID Identifier

https://orcid.org/0000-0001-7447-5218

Date of Award

Spring 6-9-2024

Document Type

Thesis (Ph.D.)

Department or Program

Microbiology and Immunology

First Advisor

George O'Toole

Abstract

Pseudomonas fluorescens is a gram-negative Gammaproteobacterium that survives and persists in diverse environments. This survivability is dependent on the bacterium’s ability to form a biofilm. Within Pseudomonas fluorescens, biofilm formation is a complex and dynamic process involving multiple protein- and polysaccharide-based components which each individually serve a specific purpose. Of these components, the repeats-in-toxin (RTX) adhesins LapA and MapA are critical for biofilm formation. Specifically, LapA is important for initial establishment of a biofilm and MapA is important in forming a fully mature biofilm. Deployment of the RTX adhesins during biofilm formation is a highly coordinated process involving multiple transcriptional, post-transcriptional, and post-translation mechanisms. For the transcriptional regulator FleQ, it has been previously shown that FleQ and the Gac/Rsm pathway both impact biofilm formation. However, it is currently unclear which biofilm components are regulated by these regulators.

In this thesis, I show that FleQ both transcriptionally and post-transcriptionally regulates the RTX adhesins. I provide evidence that FleQ directly regulates lapA and mapA RNA levels. Additionally, we determined that the regulation of LapA and MapA protein levels is mediated by the Gac/Rsm pathway. I further show that this regulation is specifically mediated by the small regulatory protein RsmE and the small regulatory RNA rsmZ.

While it was previously thought that Gac/Rsm pathway in the Pseudomonas fluorescens Pf0-1 strain was completely non-function due to a point mutation in GacA, I demonstrated through overexpression of the GacS histidine kinase that the Gac/Rsm pathway in the Pseudomonas fluorescens Pf0-1 strain is functional but attenuated compared to other lab strains. I additionally showed that this strain deploys the biosurfactant Gacamide A and swarms in an RsmA- and RsmE-dependent manner. Through mutagenic studies, I provide evidence that the proteins encoded by the pleABC operon secrete Gacamide A. I then demonstrate that multiple pathways regulate swarming motility by regulating flagellar function or biosurfactant deployment.

Collectively, the work in this thesis provides additional mechanistic insight into how FleQ and the Gac/Rsm pathway regulate biofilm formation and swarming motility.

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