Structural and functional study of the phenicol-specific efflux pump FloR belonging to the major facilitator superfamily.
The florfenicol-chloramphenicol resistance gene floR from Salmonella enterica was previously identified and postulated to belong to the major facilitator (MF) superfamily of drug exporters. Here, we confirmed a computer-predicted transmembrane topological model of FloR, using the phoA gene fusion method, and classified this protein in the DHA12 family (containing 12 transmembrane domains) of MF efflux transporters. We also showed that FloR is a transporter specific for structurally associated phenicol drugs (chloramphenicol, florfenicol, thiamphenicol) which utilizes the proton motive force to energize an active efflux mechanism. By site-directed mutagenesis of specific charged residues belonging to putative transmembrane segments (TMS), two residues essential for active efflux function, D23 in TMS1 and R109 in TMS4, were identified. Of these, the acidic residue D23 seems to participate directly in the affinity pocket involved in phenicol derivative recognition. A third residue, E283 in TMS9, seems to be necessary for correct membrane folding of the transporter.
UR86 Bio-Agresseurs, Santé, Environnement, Institut National de la Recherche Agronomique, Centre de Recherche de Tours, 37380 Nouzilly, France.
Martine
M
Braibant
Jacqueline
J
Chevalier
Elisabeth
E
Chaslus-Dancla
Jean-Marie
JM
Pagès
Axel
A
Cloeckaert
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002701:Chloramphenicol / Q000494:pharmacology*; D002702:Chloramphenicol Resistance; D003198:Computer Simulation; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D012486:Salmonella typhimurium / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013839:Thiamphenicol / Q000031:analogs & derivatives* / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C501927:FloR protein, E coli; D002701:Chloramphenicol; C035534:florfenicol; D013839:Thiamphenicol