Characterization of YvcC (BmrA), a Multidrug ABC Transporter Constitutively Expressed in Bacillus subtilis

• Published in: Biochemistry (Easton) Vol. 43; no. 23; pp. 7491 - 7502
• Main Authors: Steinfels, Emmanuelle, Orelle, Cédric, Fantino, Jean-Raphaël, Dalmas, Olivier, Rigaud, Jean-Louis, Denizot, François, Di Pietro, Attilio, Jault, Jean-Michel
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 15.06.2004
• Subjects: Adenosine Triphosphate - metabolism; Amino Acid Sequence; ATP-Binding Cassette Transporters - chemistry; ATP-Binding Cassette Transporters - genetics; ATP-Binding Cassette Transporters - isolation & purification; ATP-Binding Cassette Transporters - metabolism; Bacillus subtilis; Bacillus subtilis - drug effects; Bacillus subtilis - genetics; Bacillus subtilis - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Benzimidazoles - metabolism; Biochemistry, Molecular Biology; Biological Transport - drug effects; Cell Membrane - drug effects; Dactinomycin - analogs & derivatives; Dactinomycin - metabolism; Doxorubicin - metabolism; Drug Resistance, Bacterial; Escherichia coli; Escherichia coli - drug effects; Ethidium - metabolism; Lactococcus lactis; Life Sciences; Liposomes - chemistry; Liposomes - metabolism; Membrane Transport Proteins - chemistry; Membrane Transport Proteins - genetics; Membrane Transport Proteins - isolation & purification; Membrane Transport Proteins - metabolism; Molecular Sequence Data; Reserpine - pharmacology; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sequence Alignment; Vanadates - pharmacology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0362018

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 β-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.