Proteus mirabilis Swarming: O-antigen, Surface Sensing, and the Rcs System Público
Morgenstein, Randy M (2011)
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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