SOXS

Abstract: Triclosan (Irgasan) is a broad spectrum antimicrobial agent used in handsoaps, toothpastes, fabrics, and plastics. It inhibits lipid biosynthesis in Escherichia coli, probably by action upon enoyl reductase (FabI) (McMurry L.M., Oethinger M. and Levy S.B. (1988) Nature 394, 531–532). We report here that overexpression of the multidrug efflux pump locus acrAB, or of marA or soxS, both encoding positive regulators of acrAB, decreased susceptibility to triclosan 2-fold. Deletion of the acrAB locus increased the susceptibility to triclosan approximately 10-fold. Four of five clinical E. coli strains which overexpressed marA or soxS also showed enhanced triclosan resistance. The acrAB locus was involved in the effects of triclosan upon both cell growth rate and cell lysis.

Abstract: The soxRS oxidative stress regulon of Escherichia coli is triggered by superoxide (O2.-) generating agents or by nitric oxide through two consecutive steps of gene activation. SoxR protein has been proposed as the redox sensing gene activator that triggers this cascade of gene expression. We have now characterized two forms of SoxR: Fe-SoxR contained non-heme iron (up to 1.6 atoms per monomer); apo-SoxR was devoid of Fe or other metals. The spectroscopic properties of Fe-SoxR indicated that it contains a redox active iron-sulfur (FeS) cluster that is oxidized upon extraction from E. coli. Fe-SoxR and apo-SoxR bound the in vivo target, the soxS promoter, with equal affinities and protected the same region from DNase I in vitro. However, only Fe-SoxR stimulated transcription initiation at soxS in vitro > 100-fold, similar to the activation of soxS expression in vivo. This stimulation occurred at a step after the binding of RNAP and indicates a conformational effect of oxidized Fe-SoxR on the soxS promoter. The variable redox state of the SoxR FeS cluster may thus be employed in vivo to modulate the transcriptional activity of this protein in response to specific types of oxidative stress.

Abstract: Fluoroquinolone resistance is mediated by target changes (DNA gyrase and/or topoisomerase IV) and/or decreased intracellular accumulation. The genes (gyrA/gyrB/parC/parE) and proteins of DNA topoisomerase IV show great similarity, both at the nucleotide and amino acid sequence level to those of DNA gyrase. It has been shown that there are hotspots, called the quinolone resistance determining region (QRDR), for mutations within gyrA and parC. Based on the Escherichia coli co-ordinates, the hotspots most favoured for giving rise to decreased susceptibility and/or full resistance to quinolones are at serine 83 and aspartate 87 of gyrA, and at serine 79 and aspartate 83 for parC. Few mutations in gyrB or parE/grlB of any bacteria have been described. Efflux of fluoroquinolones is the major cause of decreased accumulation of these agents; for Staphylococcus aureus, the efflux pump involved in norfloxacin resistance is NorA, and for Streptococcus pneumoniae, PmrA. By analysis of minimum inhibitory concentration (MIC) data derived in the presence and absence of the efflux inhibitor reserpine, it has been shown that up to 50% of ciprofloxacin-resistant clinical isolates of S. pneumoniae may possess enhanced efflux. This suggests that efflux may be an important mechanism of clinical resistance in this species. In Pseudomonas aeruginosa, several efflux operons have been demonstrated genetically and biochemically. These operons are encoded by mex (Multiple EffluX) genes: mexAmexB-oprM, mexCD-OprJ system and mexEF-oprN system. The E. coli efflux pump is the acrAB-tolC system. Both the mar operon and the sox operon can give rise to multiple antibiotic resistance. It has been shown that mutations giving rise to increased expression of the transcriptional activators marA and soxS affect the expression of a variety of different genes, including ompF and acrAB. The net result is that expression of OmpF is reduced and much less drug is able to enter the cell; expression of acrAB is increased, enhancing efflux from the cell.

Abstract: The soxR locus of Escherichia coli K12 mediates transcriptional activation of a complex oxidative stress regulon in response to superoxide-generating (redox-cycling) agents. We have cloned the soxR locus, which is positioned near the uvrA gene at 92.2 min on the genetic map, by monitoring complementation of a delta soxR mutation. Subclones from the soxR region in the delta soxR strain simultaneously restored cellular resistance to the redox-cycling agent phenazine methosulfate and inducibility of at least two of the regulon proteins, glucose-6-phosphate dehydrogenase and endonuclease IV, by paraquat, another redox-cycling agent. DNA sequence analysis revealed the presence of two genes involved in activating the soxR regulon. These genes, named soxR and soxS, are arranged divergently with their 5' ends separated by only 85 bp. The predicted 12.9-kDa SoxS protein is related to the AraC family of one-component gene regulators, but corresponds only to the putative DNA-binding regions of these proteins. The 17.1-kDa SoxR protein bears significant homology only to the MerR family of proteins including a predicted DNA-binding helix-turn-helix and a cluster of cysteine residues positioned similarly to those that regulate the activity of MerR in response to Hg2+. This suggests that SoxR could be a metal-binding gene regulator that acts as the intracellular sensor for superoxide. SoxS is evidently the proximal activator of the regulon genes: antibiotic resistance and high-level expression of at least three of the regulon proteins was effected in vivo by the individual expression of SoxS, but not of SoxR, whether or not the cells were exposed to paraquat. These data, together with the recently reported paraquat-inducibility of the soxS gene (Wu, I., and Weiss, B. (1990) J. Bacteriol. 173, 2864-2871), indicate that SoxR and SoxS may constitute a novel type of two-component regulatory system in which the two proteins act sequentially to activate transcription of the various regulon genes in response to superoxide stress.