Defining the MtrR Regulon Beyond the mtrCDE Efflux Pump Operon Open Access

Johnson, Paul JT (2010)

Permanent URL: https://etd.library.emory.edu/concern/etds/dz010q790?locale=en
Published

Abstract

Neisseria gonorrhoeae, the causative agent of gonorrhea, is an important public health problem as it causes ~95 million cases of this sexually transmitted disease each year. As with other bacterial infections, previously successful antibiotic treatments have become less effective over time in treating gonorrhea. One way in which N. gonorrhoeae resists these antimicrobial agents is through the expression of efflux pump systems such as the MtrC-MtrD-MtrE efflux pump. Additionally, this pump and its regulators have recently been recognized as being important pathogenic determinants. Specifically, mutations that occur in the regulators of the MtrC-MtrD-MtrE efflux pump system alter the ability of the gonococcus to establish infections in in-vivo mouse studies. Loss of the repressor of this system, MtrR, results in an early fitness advantage followed by a loss of advantage in later stages of infection. Therefore, this work was designed to determine the MtrR regulon in order to further understand its role in gene regulation of the gonococcus given its central importance in efflux and pathogenesis. It was determined that MtrR is a global regulatory protein of significant importance in the gonococcus as it regulates at least 70 genes, including genes involved in pathogenesis, antimicrobial efflux, transport, stress response, and biosynthetic pathways. Given the breadth of these systems, these studies focused on the regulation of the gene encoding the alternative sigma factor RpoH, and select members of its regulon, as well as the glutamine biosynthetic pathway, glnE (glutamine synthetase adenylyltransferase) and glnA (glutamine synthetase). This work demonstrated that MtrR was responsible for repressing rpoH expression, and subsequently RpoH- activated genes, while inducible expression of MtrR could affect resistance to H2O2 via its affects on rpoH. Further, MtrR was found to repress glnA by its effects upon the DNA binding of a second transcriptional regulator, FarR, upstream of glnA as well as by its repression of farR expression. Additionally, MtrR was found to activate glnE. The disparate regulatory actions of MtrR identified in this research provide new insights regarding the contributions of this regulatory protein with respect to the physiology and pathogenic mechanisms of the gonococcus.

Table of Contents



Table of Contents

Abstract

Acknowledgements

List of Tables and Figures

Chapter 1: Introduction………………………………………………………………1


Chapter 2: MtrR Modulates rpoH Expression and Levels of Antimicrobial Resistance in
Neisseria gonorrhoeae………………………………………………………………92


Chapter 3: Differential Regulation of Glutamine Biosynthesis Genes glnA and glnE in
Neisseria gonorrhoeae by MtrR……………………………………………………134

Chapter 4: Unpublished Results…………………………………………………….178


Chapter 5: Summary and Discussion………………………………………………..206




















List of Figures and Tables

Chapter 1

Figure 1: Antimicrobial Usage Timeline for Neisseria gonorrhoeae

Figure 2: Major Efflux Pumps of Neisseria gonorrhoeae

Figure 3: MtrR Binding and Regulation of Directly Controlled Genes

Chapter 2

Table 1: Gonococcal strains, plasmids used

Table 2: Oligonucleotides used

Table 3: MtrR-regulated genes in Neisseria gonorrhoeae

Figure 1: Chromosomal map position of MtrR-regulated genes

Figure 2: Nucleotide sequence upstream of rpoH and identification of the MtrR-

binding site

Figure 3: MtrR regulation of rpoH expression

Figure 4: MtrR regulation of the RpoH-regulated grpE gene

Figure 5: H202 induction of rpoH expression

Figure 6: Identification of the MtrR-binding site within the rpoH promoter

Figure 7: Inducible production of MtrR represses rpoH expression and modulates

antimicrobial susceptibility levels in gonococci

Chapter 3

Table 1:Gonococcal strains and plasmids used in this study

Table 2:Oligonucleotides used in this study

Figure 1: The nucleotide sequence upstream of glnA and identification of the

MtrR and FarR-binding sites

Figure 2: MtrR and FarR regulation of glnA expression

Figure 3: Identification of the MtrR-binding site upstream of the glnA promoter

Figure 4: Identification of the FarR-binding site upstream of the glnA promoter

Figure 5: MtrR regulation of glnA expression is dependent on the MtrR binding

Site

Figure 6: MtrR influences FarR::DNA complexes

Figure 7: The nucleotide sequence upstream of glnE and MtrR-binding sites

Figure 8: Identification of the MtrR-binding site in the glnE upstream DNA

Chapter 4

Figure 1: Genes differentially regulated by MtrR during the late-log phase of

growth

Figure 2: Genes differentially regulated by MtrA during the mid-log phase of

growth

Figure 3: Genes differentially regulated by MtrA during the mid-log phase of

growth

Figure 4: Genes differentially regulated by MpeR during the mid-log phase of

growth

Figure 5: Genes differentially regulated by MpeR during the late-log phase of

growth

Figure 6: Growth profile of strain FA19, FA19 glnA::kan, and FA19

glnA::kan/glnA*

Table 1: Antimicrobial susceptibility of glnA::kan and glnA::kan/glnA* (pGCC3

glnA) compared to parental strain FA19 and ΔmtrR strain JF1

Chapter 5

Figure 1: Schematic of genes belonging to the MtrR regulon whose regulation

has been investigated in detail during this work, and previous work in

our laboratory

About this Dissertation

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
School
Department
Subfield / Discipline
Degree
Submission
Language
  • English
Research field
Keyword
Committee Chair / Thesis Advisor
Committee Members
Last modified

Primary PDF

Supplemental Files