Proteus mirabilis Swarming: O-antigen, Surface Sensing, and the Rcs System Público

Morgenstein, Randy M (2011)

Permanent URL: https://etd.library.emory.edu/concern/etds/2f75r8544?locale=es
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Abstract

Proteus mirabilis Swarming
O-antigen, Surface Sensing, and the Rcs System

Randy M. Morgenstein

Proteus mirabilis is a Gram-negative bacterium that exists as a short rod when
grown in liquid media. On surfaces, P. mirabilis undergoes a
distinct physical and biochemical change that culminates in the formation of a
swarmer cell. Swarmer cells are elongated, polyploid, and hyper-flagellated cells
that up-regulate virulence factors. How P. mirabilis senses a surface is not fully
understood; however, the inhibition of flagella rotation and accumulation of
putrescine have been proposed to be sensory mechanisms. Our lab has isolated
a transposon insertion in waaL, encoding O-antigen ligase (PM942), that results
in loss of swarming, but not swimming motility. Upon further examination, it was
shown that the swarming decfect in the waaL mutant stemmed from a failure to
activate flhDC, the class 1 activator of the flagellar cascade, when grown on solid
surfaces. The swarming defect could be returned to the waaL mutant by
overexpression of flhDC in trans or by making a mutation in the response
regulator rcsB. We propose that surface sensing is relayed by O-antigen, to the
Rcs phosphorelay, a known repressor of flhDC. In order to test this hypothesis,
mutations were made in rcsC, rcsB, rcsF, and umoB ( igaA), and umoD in wild-
type and waaL backgrounds. By comparing the swarming phenotypes of the
single and double mutants, along with overexpression strains, we have begun to
establish a working model for the role of O-antigen in surface sensing and the
Rcs pathway in P. mirabilis. We have shown that along with RcsF, UmoD acts
on the Rcs system, and that UmoD is activated by solid surfaces.

Table of Contents

Table of Contents

Abstract
Acknowledgements
Table of Contents

List of Tables and Figures
Chapter 1: Introduction…………………………………………………1

Chapter 2: Regulation of gene expression during swarmer cell differentiation in
Proteus mirabilis…………………………………………………………71

Chapter 3: Loss of the WaaL O-antigen ligase prevents surface activation of the
flagellar gene cascade in Proteus mirabilis…………………………..112

Chapter 4: Genetic Dissection of the Rcs Signaling Pathway and its Role in
Swarming Motility in Proteus mirabilis…………………………………155

Chapter 5: Discussion/Conclusions…………………………………….198

Figures and Tables

Chapter 2

Figure 1: Swarming phenotype of P. mirabilis

Figure 2: Key regulators of gene expression during swarming

Chapter 3

Figure 1: Kyte-Doolittle hydropathy profiles of WaaL proteins

Figure 2: WaaL is necessary for swarming, but not swimming

Figure 3: SDS-PAGE analysis of O-antigen production

Figure 4: A wzz mutation prevents swarming

Figure 5: Analysis of FlaA and flhDC expression in wild-type and waaL
mutant strains

Figure 6: Suppression of the swarming defect in a waaL mutant

Table 1: Strains and Plasmids

Table 2: Primers

Chapter 4

Figure 1: Differential effects of Rcs mutations on swarming in wild-type
and PM942 waaL::kmr cells

Figure 2: UmoB and UmoD effect swarming to varying degrees

Figure 3: UmoB is an input into the Rcs TCS

Figure 4: RcsF overexpression counters the phenotype of UmoB
overexpression, but not UmoD overexpression

Figure 5: rcsF is not needed for UmoB or UmoD activity in PM7002

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