Contributions of the Rcs Phosphorelay and FlhD4C2 to Swarming Behavior in Proteus mirabilis Open Access

Howery, Kristen (2016)

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Proteus mirabilis is a Gram-negative enteric bacterium known for its ability to utilize a specialized form of motility known as swarming. Swarming is the coordinated movement of a bacterial population across a solid or semi-solid surface. Upon surface contact, a subset of the initial population of vegetative, short-rod shaped cells undergoes differentiation into highly flagellated, elongated, and multinucleate swarmer cells. Swarmer cells then move outward from the point of inoculation and migrate for a few hours before de-differentiating back to short, vegetative rods. This work focuses on the role of two transcriptional regulators, RcsB and FlhD4C2, in the swarming process. The RcsB response regulator directly represses the flhDC operon, encoding the master flagellar activator FlhD4C2. Under conditions non-permissive for swarming, rcsB mutants differentiate and form swarmer cells indicating RcsB influences the expression of genes important for differentiation. These RcsB regulated genes were subsequently identified using RNA-Seq. One RcsB-activated locus, the minCDE cell division inhibition system, was found to be important for swarmer cell differentiation. Furthermore, RcsB was shown to regulate the expression of genes involved in biofilm formation and virulence. The bifurcation of P. mirabilis populations when cells are grown on agar surfaces was also examined. Following the initiation of swarming, only cells at the exterior of a colony were capable of differentiating into swarmer cells while cells in the interior remained short vegetative rods. To understand the mechanism regulating the spatial control of differentiation, expression of the flagellar master operon, flhDC, was investigated in single cells. The flhDC operon was found to exhibit bistable expression mediated by a positive feedback loop. A previously characterized mutant, SS-P, containing a transposon insertion in the non-coding region 325 bp upstream of flhDC transcriptional start site differentiated immediately upon surface contact and maintained swarmer cells throughout colony expansion. Deletion analysis of the region upstream of flhDC demonstrated that sequences from -322 to -1000 were required for spatial control of flhDC expression within the colony interior. Lastly, using DNA affinity chromatography, proteins bound to the flhDC upstream region were identified. The construction of in-frame deletions in the corresponding genes identified novel regulators required for swarmer cell differentiation.

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Table of Contents

List of Tables and Figures

Chapter 1: Introduction

Chapter 2: Regulation of the Min Cell Division Inhibition Complex by the Rcs Phosphorelay in Proteus mirabilis

Chapter 3: The Rcs Regulon in Proteus mirabilis: Implications for Motility, Biofilm Formation, and Virulence

Chapter 4: Expression of the Master Operon for Flagellar Synthesis flhDC in a Population of Swarming Proteus mirabilis Involves a Bistable Switch

Chapter 5: Discussion

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