Regulatory mechanisms of MarA, the activator of multiple antibiotic resistance

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

The Escherichia coli MarA protein regulates the ycgZ-ymgABC operon to inhibit biofilm formation

The Escherichia coli marRAB operon is a paradigm for chromosomally encoded antibiotic resistance. The operon exerts its effect via an encoded transcription factor called MarA that modulates efflux pump and porin expression. In this work, we show that MarA is also a regulator of biofilm formation. Control is mediated by binding of MarA to the intergenic region upstream of the ycgZ-ymgABC operon. The operon, known to influence the formation of curli fibres and colanic acid, is usually expressed during periods of starvation. Hence, the ycgZ-ymgABC promoter is recognised by σ38 (RpoS)-associated RNA polymerase (RNAP). Surprisingly, MarA does not influence σ38 -dependent transcription. Instead, MarA drives transcription by the housekeeping σ70 -associated RNAP. The effects of MarA on ycgZ-ymgABC expression are coupled with biofilm formation by the rcsCDB phosphorelay system, with YcgZ, YmgA and YmgB forming a complex that directly interacts with the histidine kinase domain of RcsC

The multiple antibiotic resistance operon of enteric bacteria controls DNA repair and outer membrane integrity

Transcription factors MarR and MarA confer multidrug resistance in enteric bacteria by modulating efflux pump and porin expression. Here, Sharma et al. show that MarA also upregulates genes required for lipid trafficking and DNA repair, thus reducing antibiotic entry and quinolone-induced DNA damage

Improved Production and Biophysical Analysis of Recombinant Silicatein-α

Silicatein-α is a hydrolase found in siliceous sea sponges with a unique ability to condense and hydrolyse silicon–oxygen bonds. The enzyme is thus of interest from the perspective of its unusual enzymology, and for potential applications in the sustainable synthesis of siloxane-containing compounds. However, research into this enzyme has previously been hindered by the tendency of silicatein-α towards aggregation and insolubility. Herein, we report the development of an improved method for the production of a trigger factor-silicatein fusion protein by switching the previous hexahistidine tag for a Strep-II tag, resulting in 244-fold improvement in protein yield compared to previous methods. Light scattering and thermal denaturation analyses show that under the best storage conditions, although oligomerisation is never entirely abolished, these nanoscale aggregates of the Strep-tagged protein exhibit improved colloidal stability and solubility. Enzymatic assays show that the Strep-tagged protein retains catalytic competency, but exhibits lower activity compared to the His6-tagged protein. These results suggest that the hexahistidine tag is capable of non-specific catalysis through their imidazole side chains, highlighting the importance of careful consideration when selecting a purification tag. Overall, the Strep-tagged fusion protein reported here can be produced to a higher yield, exhibits greater stability, and allows the native catalytic properties of this protein to be assessed

Analysis of Outcomes in Ischemic vs Nonischemic Cardiomyopathy in Patients With Atrial Fibrillation A Report From the GARFIELD-AF Registry

IMPORTANCE Congestive heart failure (CHF) is commonly associated with nonvalvular atrial fibrillation (AF), and their combination may affect treatment strategies and outcomes