Small RNA regulation of a multidrug efflux pump (750.1)

Multidrug efflux pumps are a major mechanism used by bacteria to export drugs and toxic compounds from the cell. One of the most important efflux pumps in E. coli is the AcrAB‐TolC system. In E. coli and among other enteric organisms, acrAB and tolC deletion mutants are known to be hypersensitive to antibiotics, and very little has been done to investigate regulation at the post‐transcriptional level of these efflux genes. Small ribonucleic acids (sRNAs) are known to be major post‐transcriptional regulators in bacteria, they usually enhance or repress translation by binding to the 5’ untranslated region (UTR) of mRNA targets with the help of a small chaperone protein, Hfq. In this study we hypothesized that Hfq‐dependent sRNAs might regulate acrA, acrB, and/or tolC. Translational fusions of the 5’ UTR and start of the open reading frame for each gene with lacZ were constructed and transformed with a sRNA plasmid library to test for sRNA regulation, measured by beta‐galactosidase assays. RyeB, a sRNA expressed during stationary phase, was found to repress the expression of tolC, which encodes the outer membrane protein of many multidrug resistant efflux pumps. Compensatory mutants were used to validate that the repression was by direct base pairing, upstream from the ribosomal binding site. We have confirmed by Western blot analysis that RyeB regulation of tolC at the mRNA level leads to a decrease in TolC protein levels. Our results suggest that the decreased amounts of TolC in stationary phase is due to the increase in expression of RyeB, and this may limit efflux of some compounds under these conditions. Grant Funding Source : National Institutes of Health, Intramural Research