RUI: Unraveling the physiological roles of multidrug efflux pumps in bacteria

The University Corporation, Northridge, Northridge CA

Investigators

Abstract

This project will determine how a multidrug efflux (MDR) pump of the bacterium Escherichia coli, regulates gene expression and cell physiology. Efflux is an essential function in all life forms. Gram-negative bacteria such as E. coli possess MDR pumps that are highly-conserved and play a pivotal role in cell physiology and bacterial adaptation to internal and environmental changes. These MDR pumps have a unique ability to expel a wide range of molecules out of the cell. These molecules include toxic compounds entering the cells from the outside such as bile salts and antibiotics; as well as molecules internally produced by these bacteria involved in functions such as bacterial growth, signaling, and capturing iron or other limited nutrients. Among MDR pumps, the AcrAB-TolC pump of E. coli is the most well-known because it is the main MDR pump in this bacterium, which is an important model organism, gut commensal, pathogen, and is widely used in biotechnology. This pump was first studied because of its role in conferring antibiotic resistance by effluxing different antibiotics. Recent findings indicate that this pump also plays a central role in regulating gene expression and bacterial physiology; however, how this pump controls these functions remains unknown. By identifying the molecules internally produced by E. coli that are effluxed by the AcrAB-TolC pump, and how this pump and its regulators control gene expression and physiology, this project will make multiple significant contributions to science and the overall well-being of society. More specifically, this project will advance our understanding of how bacteria maintain their internal balance and adapt to new environments, and offer novel insights into how to bioengineer bacteria to improve the production of biofuels and other compounds of industrial interest. The broader outcomes of this project stem from its integration into a re-designed Microbial Physiology course at California State University, Northridge (CSUN). CSUN is a primarily undergraduate and minority-serving institution. Thus, this project will expand the number of students from diverse backgrounds that have access to hands-on research, which is essential to train and inspire the next generation of scientist and engineers. MDR pumps regulate gene expression, metabolism, stress responses, bacterial motility, and other physiological processes. However, the molecular mechanisms that allow these pumps to control all these processes remain unknown. Especially intriguing is the question of how MDR pumps, which are located in the cell envelope, can regulate gene expression. Our central hypothesis is that the main function of the AcrAB-TolC pump is to efflux cellular metabolites, whereas the transcriptional repressor AcrR senses the concentrations of different pump substrates and then acts as a global regulator of gene expression. This hypothesis is based on recent findings from the PI’s laboratory and other groups. To attain the overall objective, we will pursue two specific aims: 1) Identify the genes directly regulated by AcrR and the metabolites that control AcrR function; and 2) Identify the metabolites that bind to AcrAB-TolC and the effect of AcrB-AcrZ interactions in modulating the function of this pump. We will accomplish these aims by employing bioinformatics and electrophoretic mobility shift assays to identify genes directly regulated by AcrR; as well as fluorescence polarization binding assays to identify the cellular metabolites effluxed by AcrAB-TolC, determine the effect of the small protein AcrZ on substrate recognition by this pump, and identify which pump substrates bind to and regulate AcrR function. Ultimately, this project will uncover the functional link between pump function and gene regulation by identifying which cellular metabolites are both pump substrates and AcrR ligands. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.