(34c) Coordinated Regulation of Intrinsic Multiple Antibiotic Resistance in Escherichia Coli

Bacterial resistance to antibiotics has been a persistent problem in clinical and public health situations for decades. While most bacteria acquire antibiotic resistance via genes encoded in mobile genetic elements such as plasmids and transposons, many bacterial species possess intrinsic mechanisms for resistance to antibiotics, organic solvents, oxidative stressors, and household disinfectants. In the enteric bacteria Escherichia coli and other closely related bacterial species, such as pathogenic forms of E. coli and Salmonella enterica, this resistance is mediated by the activation of chromosomally encoded efflux systems, cytoplasmic reducing enzymes, and large-scale changes in cellular metabolism. Governing these cellular responses is three transcriptional regulators of the multiple antibiotic resistance (marA), superoxide (soxS), and rob regulons ? collectively known as the marA/soxS/rob regulon. Although these networks respond to different environmental queues, marA, soxS, and rob are known to regulate the expression of many downstream genetic targets, as well as regulating the expression of each other. This complex interlocked genetic circuitry is believed to provide the ability to sense and respond to broad classes of toxic chemicals.

In this work, we have employed mixed strategy of experimental genetics coupled with computational modeling to develop a quantitative description of the interlocked marA/soxS/rob regulon, a primary determinant of intrinsic antibiotic resistance in E. coli. Performing comprehensive deletion and complementation analysis we systematically determined all cross-regulatory and auto-regulatory interactions in the marA, soxS, and rob genetic circuits. Using this refined set of regulatory interactions, we have formulated a more accurate model for activation of the marA/soxS/rob regulon on exposure to known antimicrobial inducers. Finally, we have examined the role of the interlocked marA/soxS/rob genetic circuitry in providing additive and synergistic response to multiple environmental queues.

Although our current understanding of the marA/soxS/rob genetic circuit has largely relied on the use of bacteriostatic antibiotics such as tetracycline and chloramphenicol, recent genetic and biochemical evidence has suggested that on exposure to bacteriocidal antibiotics superoxide is generated in the cytoplasm of E. coli cells. In the context of the marA/soxS/rob regulon, superoxides are known activators of the soxS regulon providing a critical link between the intrinsic response to bacteriostatic and bacteriocidal antibiotics. Taken together, the results of this work will allow for a more thorough understanding of the mechanism of intrinsic resistance to clinically relevant antibiotics and further our understanding of these types of resistance mechanisms in related human and animal pathogens.