The Molecular Basis Of Antibiotic Resistance

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The marRAB multiple antibiotic resistance operon of Escherichia coli controls the expression of a large number of genes resulting in low level antibiotic and superoxide resistance through a complex network of reactions. MarR auto-represses the mar operon but is inactivated upon interaction with salicylate, losing its DNA binding capacity. This, in turn, results in derepression of the operon and expression of MarA, which activates the transcription of some 40 to 60 promoters (the mar/sox/rob regulon) including the marRAB promoter itself (auto-activation). We have focused recently on the capacity of MarA to activate these disparate promoters in an attempt to develop a mathematical model to describe the network. We find that the concentration of MarA and/or SoxS necessary to result in half-maximum stimulation varies by at least 30-fold for the different promoters and is similar but not identical for the two activators. Thus, at intermediate concentrations of the activators, while some genes in the regulon exhibit significant activation, others remain dormant. We refer to this type of regulation as hierarchical activation. A mathematical model has been developed to characterize this phenomenon quantitatively. The remarkable conclusion of this modeling is that the activation of most genes of the regulon is best accounted for by a very large increase in the forward reaction rate (clearance) accompanied by a decrease (rather than an increase) in the affinity of RNA polymerase for the promoter in the presence of the activator. We and others have previously proposed models for the interaction of MarA with RNA polymerase requisite for transcriptional activation. The above analyses have thrown into doubt some aspects of those models. Consequently we are currently engaged in an extensive analysis of MarA mutants employing the above technique. In addition we have carried out an extensive analysis of the two promoters believed to regulate the three genes primarily responsible for antibiotic resistance, tolC and acrAB. This work was carried out in collaboration principally with Drs. J.L. Rosner and Michael Wall (Computer and Computational Sciences & Bioscience, Los Alamos National Laboratory, Mail Stop B256,Los Alamos NM 87545 USA).