Characterization of YvcC (BmrA), a multidrug ABC transporter constitutively expressed in Bacillus subtilis.
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.
Steinfels
Emmanuelle
E
Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS-UCBL et IFR 128, 7 passage du Vercors, 69367 Lyon Cedex 07, France.
Orelle
Cédric
C
Fantino
Jean-Raphaël
JR
Dalmas
Olivier
O
Rigaud
Jean-Louis
JL
Denizot
François
F
Di Pietro
Attilio
A
Jault
Jean-Michel
JM
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
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