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bioRxiv · 10.1101/570408

The Anaerobic Efflux Pump MdtEF-TolC Confers Resistance to Cationic Biocides

Novoa, D.·Conroy-Ben, O.
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Abstract

The E. coli RND transporter MdtEF-TolC is a tri-partite efflux pump that exports toxic substances. Little is known of the full range of substrate specificity of the anaerobic efflux pump, but MdtF shares similar homology and substrate specificity to the major RND efflux protein AcrB. To determine the substrate range of the anaerobic efflux pump MdtEF-TolC, E. coli mutants were exposed to 210 different biocides, and growth was monitored. This approach was used to validate AcrAB-TolC substrates and discover new chemicals transported by the major antibiotic efflux protein. Results showed that overexpression of MdtEF conferred resistance to the same substrates as AcrAB-TolC, but were limited to cationic amino-based biocides. Alignment of the amino acids lining the distal pocket of MdtF and AcrB revealed a more acidic isoelectric point (pI) of an order of magnitude in MdtF, whereas the proximal pocket and external cleft were homologous and displayed identical pIs. This analysis suggests that pH, which determines acid-base speciation, and the distal pocket surface proteins play a role in MdtF substrate specificity.\n\nImportanceHigh-throughput screening of E. coli mutants revealed that the substrates of the anaerobic efflux pump MdtEF-TolC are the same cationic biocides exported by AcrAB-TolC. Comparison of the protein sequences of the distal pocket, proximal pocket, and external cleft of the two RND proteins showed homology in amino acid surface charge and isoelectric point. Residue differences within the distal pocket are responsible for a more acidic pI and greater negative charge of the inner membrane protein MdtF surface, and support the findings of transport of cationic substances.

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Novoa, D., Conroy-Ben, O.. 2019-03-09. The Anaerobic Efflux Pump MdtEF-TolC Confers Resistance to Cationic Biocides. https://doi.org/10.1101/570408

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