Study of Multidrug Efflux System Protein Degradation in E. coli Using Transposons Library

Abstract

AcrB is a trimeric multidrug efflux pump in E. coli belonging to the Resistance‐Nodulation‐Division (RND) super family. We have previously found that the introduction of a small peptide tag, the ssrA tag, to the C‐terminus of AcrB lead to efficient degradation of the protein. Soluble proteases ClpP and ClpX have been found to be involved in the degradation. However, a significant reduction in the amount of AcrB‐ssrA could still be detected in knockout strains lacking functional ClpXP. We speculate that the reduction of AcrB‐ssrA level is due to another protease/proteases. This study focuses on the identification of other proteins capable of degrading AcrB‐ssrA. Transposons libraries containing random gene disruption were generated and screened for clones displaying increased expression of AcrB‐ssrA. Our hypothesis is that the increased activity of AcrB‐ssrA protein is due to a mutation or inactivation of a gene that is involved in the degradation of AcrB‐ssrA. The elevated level of AcrB‐ssrA expression was then confirmed using anti‐AcrB antibody and minimum inhibitory concentration assays. Several candidate genes have been identified, which we are currently characterizing for their potential role in AcrB‐ssrA degradation.Support or Funding InformationNSF grant CHE‐1709381This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.