The Global Regulatory Role of the RNA Binding Protein CsrA Open Access
Edwards, Adrianne Nehrling (2010)
Abstract
CsrA influences several important cellular processes by binding
to target mRNAs and
altering their translation and/or stability. CsrA activity is
antagonized by two sRNAs,
CsrB and CsrC. To elucidate the global role of CsrA, we combined a
biochemical
method with high throughput cDNA sequencing to determine the
identities of mRNAs
that directly bound to Escherichia coli CsrA in vivo. Among
the 721 transcripts
discovered were relA, spoT and dksA, which
encode proteins that mediate the stringent
response. RelA and SpoT are involved in guanosine tetraphosphate
(ppGpp) metabolism,
while DksA typically functions synergistically with ppGpp to
potentiate the stringent
response. CsrA directly repressed expression of relA and
indirectly repressed spoT
transcript levels, suggesting that CsrA may affect cellular ppGpp
levels in certain
conditions. CsrA directly activated dksA expression, but
this effect was masked by DksA
negative autoregulation. In turn, DksA and ppGpp activated
csrA, csrB and csrC
transcription. CsrA and DksA were both required for full expression
of csrB and csrC,
which required the response regulator UvrY. These complex direct
and indirect
regulatory interactions between the Csr and stringent response
systems suggest that these
systems function to finely tune gene expression in response to
carbon metabolism and
nutrient availability. In addition, a screen designed to find novel
CsrA targets in E. coli
using a microarray-based approach revealed that CsrA affected the
expression of several
GGDEF and EAL domain proteins. GGDEF and EAL domains are associated
with c-di-
GMP metabolism, a second messenger that positively influences
biofilm formation and
represses motility. CsrA directly repressed the expression of two
GGDEF domain
proteins in a post-transcriptional manner while modestly
influencing the expression of
five additional GGDEF/EAL domain proteins through unknown
mechanisms. Cellular c-
di-GMP concentrations were increased modestly in the csrA
mutant, signifying that CsrA
globally affects c-di-GMP synthesis. Finally, analysis of effects
of CsrA on
GGDEF/EAL domain proteins in Salmonella Typhimurium
demonstrated that CsrA has
both direct and indirect effects on regulating the switch between
motility and biofilm
formation. These studies have expanded the regulatory role of CsrA
and suggest that
CsrA governs cellular physiology and behavior on a global
scale.
Table of Contents
Chapter 1. Introduction....................................................................................................1
Csr/Rsm regulatory system......................................................................................1 CsrA mechanism and regulation..................................................................2 Other components of the Csr regulatory system..........................................5 CsrA regulated genes in E. coli K-12..........................................................8 The Csr/Rsm system in other organisms....................................................11 Stringent Response.................................................................................................16 Metabolism and regulation of ppGpp........................................................17 DksA, a cofactor for ppGpp regulation.....................................................23 Mechanism of ppGpp and DksA................................................................25 Regulatory targets of ppGpp and DksA.....................................................29 Regulation of biofilm formation and c-di-GMP....................................................32 Metabolism of c-di-GMP...........................................................................33 Regulation by c-di-GMP............................................................................40 Global regulatory networks and global regulons in prokaryotes...........................44 Conclusion.............................................................................................................46 References..............................................................................................................48 Chapter 2. Circuitry linking the Csr system and stringent response global regulatory systems...........................................................................................................96 Abstract..................................................................................................................97 Introduction............................................................................................................97 Results..................................................................................................................101 Discussion............................................................................................................112 Materials and Methods.........................................................................................117 References............................................................................................................124 Chapter 3. The RNA binding protein CsrA controls cyclic di-GMP metabolism by directly regulating the expression of GGDEF proteins..............................................153 Abstract................................................................................................................154 Introduction..........................................................................................................154 Results..................................................................................................................157 Discussion............................................................................................................170 Materials and Methods.........................................................................................175 References............................................................................................................183 Chapter 4. Complex regulatory network encompassing the Csr, c-di-GMP and motility systems of Salmonella Typhimurium.............................................................213 Abstract................................................................................................................214 Introduction..........................................................................................................214 Results..................................................................................................................217 Discussion............................................................................................................227 Materials and Methods.........................................................................................232 References............................................................................................................238 Chapter 5. Discussion....................................................................................................261 Global role of CsrA in Escherichia coli..............................................................261 Potential role of CsrA in regulation of secondary messenger metabolism..........265 Complex interconnections between global regulatory networks.........................267 Outlook................................................................................................................271 References............................................................................................................272 Appendix. Supporting Information for Chapter 2.....................................................277About this Dissertation
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