טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentLior Ben-Shalom
SubjectMAR (Multiple Antibiotic Resistance) Phenotype Regulation
in Escherichia coli and Salmonella
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professor Yaron Sima


Abstract

Bacteria often respond to environmental stresses by adaptive changes in the expression of proteins. The homologous bacterial regulators MarA, SoxS and Rob activate and repress a large common set of genes, resulting in multiple antibiotic resistance and superoxide resistance (mar phenotype). Each regulator is activated by a different stress signal. MarA is activated by phenolic compounds such as salicylate, SoxS by an oxidative stress imposed by compounds like paraquat and Rob is an abundant DNA-associated protein. MarA, SoxS and Rob positively regulate efflux of antimicrobial compounds by up-regulating of the AcrAB efflux pump, and negatively control influx by up-regulating micF, which involved in the regulation of the porin OmpF.

Our main goal was to study the regulation of marA, soxS and rob, and their regulated genes acrAB and micF in Escherichia coli and Salmonella enterica and to evaluate their role in antibiotic resistant clinical strains. We have developed a system for real-time monitoring of the transcriptional activation of the operons by reporter plasmids, in which gfp is under the control of one of the promoters. The resulting plasmids were transformed into the E. coli and Salmonella strains and into marR deficient mutants.

The promoters were active throughout the entire growth curve. However, the highest rates of normalized activity observed at the beginning of the stationary phase of growth. The strengths of basal transcription were ordered as micF>rob>acrAB>soxS, marRAB. Transcription profiles in Salmonella with and without inducers were very similar to those of E. coli, but induction levels of marA, acrAB and micF by salicylate, and micF by paraquat were significantly higher in Salmonella. The most important difference was the observation that transcription of marRAB, acrAB and rob in Salmonella enhanced when temperature increased from 30°C to 37°C, while their transcription declined in E. coli, demonstrating that in oppose to E. coli, the Salmonella mar system is more adapted to the host environment. Transcription profiles of the MarR mutants provided information about the function of MarA and Rob in the regulatory network. Kinetic studies showed that while genes responded immediately to salicylate, a delay of about cell cycle was observed following exposure to paraquat. In clinical multiple resistant strain which overexpressed AcrAB efflux pumps we also observed high basal transcription of micF, robA but not marRAB or soxS.

Comparing the network of regulation of these regulators provided additional information about how these regulators are controlled, and what are their roles in the stress response.