Mutations in the central cavity and periplasmic domain affect efflux activity of the resistance-nodulation-division pump EmhB from Pseudomonas fluorescens cLP6a

Abstract

The EmhABC efflux system in Pseudomonas fluorescens cLP6a is homologous to the multidrug and solvent efflux systems belonging to the resistance-nodulation-division (RND) family and is responsible for polycyclic aromatic hydrocarbon transport, antibiotic resistance, and toluene efflux. To gain a better understanding of substrate transport in RND efflux pumps, the EmhB pump was subjected to mutational analysis. Mutagenesis of amino acids within the central cavity of the predicted three-dimensional structure of EmhB showed selective activity towards antibiotic substrates. An A384P/A385Y double mutant showed increased susceptibility toward rhodamine 6G compared to the wild type, and F386A and N99A single mutants showed increased susceptibility to dequalinium compared to the wild type. As well, the carboxylic acid side chain of D101, located in the central cavity region, was found to be essential for polycyclic aromatic hydrocarbon transport and resistance to all antibiotic substrates of EmhB. Phenylalanine residues located within the periplasmic pore domain were also targeted for mutagenesis, and the F325A and F281A mutations significantly impaired efflux activity for all EmhB substrates. One mutation (A206S) in the outer membrane protein docking domain increased antibiotic resistance and toluene tolerance, demonstrating the important role of this domain in transport activity. These data demonstrate the roles of the central cavity and periplasmic domains in the function of the RND efflux pump EmhB.

FIG. 1.

Predicted structure of the EmhB efflux pump, modeled using the known crystal structure of the E. coli pump AcrB (PDB entry 1IWG [19]). (A) The view of the predicted EmhB trimer from above the plane of the inner membrane shows the presence of a pore extending through the pump. (B) The transmembrane domain (TMD), pore domain, and outer membrane protein docking domain (OMPDD) are indicated on the side view of the EmhB trimer, with the rear monomer removed to facilitate viewing. A central cavity is present at the center of the pump above the transmembrane domain. This figure and Fig. 3, 4, and 5 were drawn using the DS ViewerPro version 6.0 software (Accelrys).

FIG. 2.

Expression of mutant EmhB_His proteins in P. fluorescens cLP6a-1 relative to EmhB_His expression. Cell extracts (10 μg total protein) from P. fluorescens cLP6a-1 carrying the mutant EmhB_His proteins were separated by SDS-PAGE and subjected to immunoblotting with an antibody against the histidine tag. The proteins are indicated above the lanes.

FIG. 3.

Predicted model of the central cavity in EmhB. (A) The central cavity is viewed from the side parallel to the membrane, and the front monomer of EmhB is removed for ease of viewing. The N99, D101, F386, F458, and F459 residues lining the cavity are indicated. The A384 and A385 residues are not shown for ease of viewing. (B) A top view of the central cavity perpendicular to the plane of the membrane shows a ring formed by the residues F386, F458, and F459.

FIG. 4.

Predicted structure of the periplasmic pore domain of EmhB. The pore domain is viewed from the top of the protein perpendicular to the plane of the membrane, showing the positions of the central pore, vestibules, and clefts. The locations of the residues targeted for mutagenesis are indicated.

FIG. 5.

Predicted model of the outer membrane protein docking domain of EmhB. The domain is viewed from the top of the protein perpendicular to the plane of the membrane (A) and from the side of the protein with the rear monomer removed (B). The location of the A206 residue is indicated.