title: Characterization of YvcC (BmrA), a multidrug ABC transporter constitutively expressed in Bacillus subtilis.
abstract: The involvement of transporters in multidrug resistance of bacteria is an increasingly challenging problem, and most of the pumps identified so far use the protonmotive gradient as the energy source. A new member of the ATP-binding cassette (ABC) family, known in Bacillus subtilis as YvcC and homologous to each half of mammalian P-glycoprotein and to LmrA of Lactococcus lactis, has been studied here. The yvcC gene was constitutively expressed in B. subtilis throughout its growth, and a knockout mutant showed a lower rate of ethidium efflux than the wild-type strain. Overexpression of yvcC in Escherichia coli allowed the preparation of highly enriched inverted-membrane vesicles that exhibited high transport activities of three fluorescent drugs, namely, Hoechst 33342, doxorubicin, and 7-aminoactinomycin D. After solubilization with n-dodecyl beta-D-maltoside, the hexahistidine-tagged YvcC was purified by a one-step affinity chromatography, and its ability to bind many P-glycoprotein effectors was evidenced by fluorescence spectroscopy experiments. Collectively, these results showed that YvcC is a multidrug ABC transporter functionally active in wild-type B. subtilis, and YvcC was therefore renamed BmrA for Bacillus multidrug resistance ATP. Besides, reconstitution of YvcC into liposomes led to the highest, vanadate-sensitive, ATPase activity reported so far for an ABC transporter. Interestingly, such a high ATP hydrolysis proceeds with a positive cooperativity mechanism, a property only found so far with ABC importers.
authors.affiliation: Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS-UCBL et IFR 128, 7 passage du Vercors, 69367 Lyon Cedex 07, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Steinfels
authors.forename: Cédric
authors.initials: C
authors.lastname: Orelle
authors.forename: Jean-Raphaël
authors.initials: JR
authors.lastname: Fantino
authors.forename: Olivier
authors.initials: O
authors.lastname: Dalmas
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Rigaud
authors.forename: François
authors.initials: F
authors.lastname: Denizot
authors.forename: Attilio
authors.initials: A
authors.lastname: Di Pietro
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Jault
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001562:Benzimidazoles / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects; D003609:Dactinomycin / Q000031:analogs & derivatives* / Q000378:metabolism; D004317:Doxorubicin / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects; D004996:Ethidium / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012110:Reserpine / Q000494:pharmacology; D016415:Sequence Alignment; D014638:Vanadates / Q000494:pharmacology
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D001562:Benzimidazoles; D008081:Liposomes; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; C075041:Bmr protein, Bacillus subtilis; D003609:Dactinomycin; D014638:Vanadates; C025942:7-aminoactinomycin D; D004317:Doxorubicin; D012110:Reserpine; D000255:Adenosine Triphosphate; D004996:Ethidium; C017807:bisbenzimide ethoxide trihydrochloride