SIGNAL PERCEPTION AND TRANSMISSION THROUGH THE VIRA/VIRG TWO-COMPONENT SYSTEM IN AGROBACTERIUM TUMEFACIENS Open Access

Fang, Fang (2007)

Permanent URL: https://etd.library.emory.edu/concern/etds/79407x36f?locale=en
Published

Abstract

Prokaryotes, fungi and plants sense and adjust to environmental change by two-component regulatory systems composed of HK (histidine kinase) and RR (response regulator) domains. Precise adaptation to complex environments often necessitates the integration of information from multiple cues. In Agrobacterium tumefaciens, the broad host range relies on the generality of signals and the integration of multiple input signals are regulated by the VirA/VirG two-component system. In this study, the VirA/VirG two-component system was used to explore the mechanism of signal perception and transmission and to reveal general strategies used for environmental sensing in prokaryotes.

VirA has been dissected into four domains, the periplasmic, linker, kinase and receiver domains. One striking feature is that VirA, as a hybrid kinase, carries an additional receiver at its C-terminus. The study of the role of the receiver domain in VirA/VirG activation indicated that the receiver domain encompasses both activating and inhibitory elements. With signal induction, the repression is relieved through dissociation of the linker/receiver interaction which makes the receiver to interact with the DNA binding domain of VirG for facilitating the phospho-transfer to the receiver domain of VirG. This may be a general mechanism for hybrid histidine kinases to integrate signals.

Another remarkable feature is that VirA, as a single receptor, detects multiple phenolic compounds. The short region aa288-293 in the VirA linker domain was found to regulate phenols specificity. Most importantly, single mutations such as Y293T in this region greatly reduce the range of known vir inducers. This short region therefore appears to be critical for the multi-host strategy developed by A. tumefaciens and has important practical consequences for the success of Agrobacterium-plant interaction.

Table of Contents

TABLE OF CONTENTS


LIST OF ILLUSTRATIONS
LIST OF TABLES
ABBREVIATIONS


1. The A. tumefaciens VirA/VirG Two-Component System and Signal Perception...1

Two-component paradigm
The VirA/VirG two-component system of Agrobacterium tumefaciens

2. Phenolic Activation of A. tumefaciens Virulence Genes: Exploiting Heterologous Hosts to Define Crucial Components...17

Introduction...17
Materials and methods...18
Results...24

VirA condition monitoring for vir induction both in A. tumefciens and in E. coli

VirA localization is not the factor for lower activity of VirA/VirG in E. coli
Probing specificity: Inhibitors ASBr and HDI

Discussion...29

3. Role of the Interaction between the Linker and the Receiver in the Phenolic Activation of A. tumefaciens Virulence Genes...32

Introduction...32
Materials and Methods...33
Results...42

Null mutants in the receiver domain are located proximal to D766N
Suppressor mutations of the LKR(285-829)(V765D, D766N and L770P) phenotypes

Requirements at aa293
Amino acid 293 and the receiver domain

Discussion...57

4. Genetic Evidence for Phenol Specificity Switch Caused by the Region aa288-aa293 of VirA Protein in Agrobacterium tumefaciens...62

Introduction...62
Materials and Methods...64
Results...76

LKR(285-829) and LKR(294-829) respond very differently to AS induction
Does aa285-aa293 affect VirA dimer formation?
Does aa285-aa293 regulate phenol specificity?
Are specific residues across the region aa285-293 responsible for the phenol specificity switch?

The presence of receiver domain of VirA is not involved in the specificity
Maximal activity change is the determinant for phenol specificity
Residues across aa285-293 control specificity for para position
Y293T dramatically narrows specificity

Discussion...95

5. The Receiver Domain of Agrobacterium tumefaciens VirA Histidine Kinase Has an Activating Role in vir Gene Expression...100

Introduction...100
Materials and Methods...104
Results...112

VirA receiver truncation alleles VirAΔ707-829 and VirAΔ712-829 have similar functions.

The VirA receiver domain has an activating function.
VirAΔR requires a higher concentration of VirG than wild type VirA in order to activate vir gene expression.

Point mutations in the VirA receiver domain can be corrected by overexpression of virG.

The DNA-binding domain of VirG interacts with the receiver domain of VirA.

Discussion...127

6. SUMMARY...131
REFERENCES...135

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