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Access Type

WSU Access

Date of Award

January 2019

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

Penelope I. Higgs

Abstract

Bacteria were long thought of as simple, unicellular organisms that lived independently of other cells in the environment, but It is now well-known, however, that bacteria are capable of numerous multicellular behaviors and often live in multicellular communities. In bacteria, multicellular behaviors can range from relatively simple cooperation between cells to the formation of specialized microbial tissues. While formation of microbial tissues is relatively simple compared to tissue formation in many higher eukaryotic organisms, many of the same basic principles are conserved in both systems. Identifying and characterizing the basic regulatory mechanisms that control multicellular behaviors in bacteria would provide valuable insight into the fundamental requirements for multicellularity in both prokaryotes and eukaryotes, which would significantly impact our understanding of multicellular tissue formation.

For examining multicellular behavior, the ubiquitous soil bacteria, Myxococcus xanthus, is an ideally model system. Upon nutrient limitation, M. xanthus enters multiphase developmental program in which cells form multicellular structures (fruiting bodies) and undergo differentiation into distinct cell types. This complex program is regulated by an extensive genetic regulatory network (GRN) that has been largely defined. MrpC is a CRP/Fnr family transcription factor that is one of the central regulators of the developmental GRN. and is subject to an extensive amount of transcriptional and post-transcriptional regulation. Proper regulation of MrpC is required for coordination of multicellular development because populations that have perturbed MrpC regulation display highly uncoordinated fruiting body structures compared to the wild type. In this thesis, I set out to examine how different recurring regulatory patterns (network motifs) influence MrpC regulation and affect coordinated multicellular behavior. Using fluorescent transcriptional reporters and electrophoretic mobility shift assays, I determined that MrpC functioned as a negative autoregulator by competing with MrpB, the activator for mrpC expression. I then set out to examine the role of MrpC negative autoregulation (NAR) in development by expressing mrpC from a mutant promoter. Using a high-resolution developmental assay, I observed that perturbation of NAR resulted in asynchronous progression through development. Fluorescence reporter data indicated that MrpC NAR limits cell-cell variability in mrpC expression, which likely promotes synchronize progression through development. Finally, I designed and performed a transposon-based forward genetic screen to identify factors that post-transcriptionally regulate MrpC.

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