Regulatory mechanisms of MarA, the activator of multiple antibiotic resistance

Download Statistics

Downloads

Downloads per month over past year

Kettles, Rachel Andrea (2019). Regulatory mechanisms of MarA, the activator of multiple antibiotic resistance. University of Birmingham. Ph.D.

Preview

Kettles2019PhD.pdf
Text - Accepted Version
Available under License All rights reserved.
Download (10MB) | Preview

Abstract

The multiple antibiotic resistance (mar) operon in Escherichia coli is responsible for resistance to a broad range of antibacterial drugs. The mar operon is normally transcriptionally silent, but a faulty mar repressor (MarR) leads to constitutive expression of MarA (the mar activator). MarA transcriptionally regulates downstream targets leading to a mar phenotype of widespread resistance to antibiotics and other environmental stresses (e.g. oxidative stress, organic solvents and disinfectants). As the mar operon is conserved across a number of human pathogens, understanding the mechanisms through which it mediates antibiotic tolerance is essential.

A recent ChIP-seq analysis unveiled 33 targets of MarA, many of which were previously unknown. This work has characterised the regulation of one of these targets, ycgZ-ymgABC. The promoter upstream of ycgZ-ymgABC was found to be both σ70 and σ38-dependent. However, MarA activates transcription from this promoter in a σ70-dependent manner only, and was shown to act as a Class I activator. Furthermore, activation of ycgZ-ymgABC expression by MarA was shown to result in a reduction in biofilm formation, which may offer the cell alternative short-term survival strategies during antibiotic attack.

The requirements for activation of transcription at the regulatory region upstream of mlaFEDCB were investigated. Strict spacing and orientation requirements for MarA binding were observed; the MarA binding site (the marbox) only functions in the forward orientation, and cannot be moved more than 1 bp without loss of activation. Additionally, MarA was shown to require an UP element and contact with the C-terminal domain of RNAP for activation at this target.

Finally, the ChIP-seq targets, and a set of SoxS ChIP-exo targets from a separate study, were examined for binding by MarA and two related regulators, Rob and SoxS. These three proteins have an identical consensus site for DNA binding but bind non-consensus sites with hugely different affinities. It was noticed that SoxS requires a much closer match to the consensus site than MarA for optimal binding. We hypothesise here that this is due to a loss of amino acid side chains in SoxS that are key for hydrogen bonding interactions with the DNA backbone. To confirm this, we have shown here that MarA binds to the ycgZ promoter at a higher affinity than SoxS in high salt conditions only. At low salt conditions, hydrogen bonding is inhibited, significantly reducing MarA binding but not SoxS. This effect is dependent on residues E31 and Q58 of MarA, which make hydrogen bonding contacts with the DNA backbone; these contacts are lost in SoxS. Thus, this work predicts that intracellular salt conditions may influence the target preferences of these regulators.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):