title: Directed evolution of a bacterial efflux pump: adaptation of the E. coli TolC exit duct to the Pseudomonas MexAB translocase.
abstract: Bacterial multidrug efflux pumps operate by periplasmic recruitment and opening of TolC family outer membrane exit ducts by cognate inner membrane translocases. Directed evolution of active hybrid pumps was achieved by challenging a library of mutated, shuffled TolC variants to adapt to the non-cognate Pseudomonas MexAB translocase, and confer resistance to the efflux substrate novobiocin. Amino acid substitutions in MexAB-adapted TolC variants that endowed high resistance were recreated independently, and revealed that MexAB-adaptation was conferred only by substitutions located in the lower alpha-helical barrel of TolC, specifically the periplasmic equatorial domain and entrance coiled coils. These changes converge to the native MexAB partner OprM, and indicate an interface key to the function and diversity of efflux pumps.
authors.affiliation: Cambridge University Department of Pathology, Tennis Court Road, Cambridge CB2 1QP, UK.
authors.forename: Evert
authors.initials: E
authors.lastname: Bokma
authors.forename: Eva
authors.initials: E
authors.lastname: Koronakis
authors.forename: Sune
authors.initials: S
authors.lastname: Lobedanz
authors.forename: Colin
authors.initials: C
authors.lastname: Hughes
authors.forename: Vassilis
authors.initials: V
authors.lastname: Koronakis
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods; D024881:Drug Resistance, Bacterial / Q000187:drug effects / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D050506:Mutant Chimeric Proteins / Q000235:genetics* / Q000378:metabolism; D009675:Novobiocin / Q000378:metabolism / Q000494:pharmacology; D019897:Periplasm / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C118598:MexA protein, Pseudomonas aeruginosa; C118597:MexB protein, Pseudomonas aeruginosa; D050506:Mutant Chimeric Proteins; C073126:tolC protein, E coli; D009675:Novobiocin