Defining the MtrR Regulon Beyond the mtrCDE Efflux Pump Operon Open Access
Johnson, Paul JT (2010)
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
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