Nonheritable Antibiotic Resistance

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Chromosomal multiple antibiotic resistance in bacteria is a serious clinical problem. Our studies have shown that Escherichia coli becomes resistant to a variety of antibiotics, organic solvents and superoxides when the activities of any of three paralogous, but differently regulated, transcriptional activators, MarA, SoxS and Rob, are increased. These activators bind a sequence called the marbox which lies upstream of the promoters of a set of about 40 chromosomal genes called the mar/sox/rob regulon. The major goals of this project are to understand the regulation of these activators, the mechanisms whereby they activate the regulon promoters, and the mechanisms whereby the multiple antibiotic resistance is generated. A. The primary mechanism for the antibiotic resistance and organic solvent tolerance is the transcriptional activation of acrAB and tolC. These genes encode the major antibiotic efflux pump of Escherichia coli. Using transcriptional fusions and primer extension assays, we investigated the regulation of these operons by the repressor AcrR and by MarA, SoxS and Rob. We showed that tolC has two previously unidentified strong overlapping promoters which are activated by MarA. They are configured in a unique way so that the binding of an activator to a single marbox can activate transcription from either promoter. The marbox is 20 bp upstream of the -10 signal for RNA polymerase (Class I* configuration) at the tolC p4 promoter, but 30 bp upstream of the -10 signal (Class II configuration) at the tolC p3 promoter. Furthermore, we showed that three cis-acting elements are important in the regulation of acrAB and acrR: the marbox which allows activation of acrAB; a 24 bp inverted repeat which contains the acrAB promoter and part of the divergently transcribed acrR promoter, and which is the likely site of AcrR binding for repression of both acrAB and acrR; and a 22 bp inverted repeat that contains part of the acrR promoter. We found also that 2,2'-dipyridyl (which we previously found post-translationally activates Rob) down-regulates AcrR function. Constitutive expression of acrAB resulting from an acrR deletion minimally affects antibiotic resistance in the absence of tolC activation but leads to a hyper-resistant phenotype when tolC is activated. The tolC and acrAB marboxes coordinate the activation of the tripartite efflux pump. B. The different regulon genes are activated to different extents by the activators. We previously had determined that while some correlation exists between activity and the binding constant of the activator for the different marboxes, this correlation is insufficient to explain the differential activation. We have now placed the expression of MarA under the control of a lac promoter which is activated by IPTG, determined the relationship between IPTG concentration and the intracellular concentration of MarA, and examined the expression of a number of the regulon genes (using lacZ as a transcriptional reporter gene) as a function of growth in different concentrations of IPTG. The resulting data were used to develop mathematical models that yield insight into the mechanisms of activation at the different promoters. We found that the concentration of MarA required for activation varies by at least 30-fold for different promoters, thus identifying a previously unappreciated form of regulon control in which some genes are activated at a particular activator concentration whereas others are not significantly activated. The wild-type mar promoter itself is activated at the lowest concentration of MarA, reaching half-maximal stimulation at a concentration of about 900 MarA molecules/cell. The comparable number for the micF promoter is about 10,000 MarA/cell. None of the 15 other promoters tested exhibits a plateau in activity, even at the artificially highest levels of MarA (about 26,000 molecules/cell). To gain insight innto the diversity in activation of the regulon, we developed a mathematical model of MarA-dependent promoter activity. In the model, MarA either increases (attraction) or decreases (repulsion) the occupancy of RNA polymerase (RNAP) at the promoter, and either increases (acceleration) or decreases (retardation) the forward rate of transcription by RNAP once bound at the promoter. The best models of mar promoter activation combine attraction with acceleration. For other regulon promoters, models that combine repulsion with acceleration fit the data best. The results suggest that transcriptional activation can involve repulsion (a decrease in the occupancy of RNAP due to activator), an effect commonly associated with repression rather than activation. The results also suggest that acceleration (an increase in the forward rate of transcription due to activator) is an important part of activation. Acceleration combined with repulsion has not been previously appreciated as playing a role in activation.