Multidrug Efflux Pump MdtBC of Escherichia coli Is Active Only as a B2C Heterotrimer

RND (resistance-nodulation-division) family transporters in Gram-negative bacteria frequently pump out a wide range of inhibitors and often contribute to multidrug resistance to antibiotics and biocides. An archetypal RND pump of Escherichia coli, AcrB, is known to exist as a homotrimer, and this construction is essential for drug pumping through the functionally rotating mechanism. MdtBC, however, appears different because two pump genes coexist within a single operon, and genetic deletion data suggest that both pumps must be expressed in order for the drug efflux to occur. We have expressed the corresponding genes, with one of them in a His-tagged form. Copurification of MdtB and MdtC under these conditions showed that they form a complex, with an average stoichiometry of 2:1. Unequivocal evidence that only the trimer containing two B protomers and one C protomer is active was obtained by expressing all possible combinations of B and C in covalently linked forms. Finally, conversion into alanine of the residues, known to form a proton translocation pathway in AcrB, inactivated transport only when made in MdtB, not when made in MdtC, a result suggesting that MdtC plays a different role not directly involved in drug binding and extrusion.Bacterial multidrug resistance is a major public health problem (10, 17). One widespread resistance mechanism involves the multidrug resistance (MDR) transporters. Among these, the resistance-nodulation-cell division (RND) family transporters, such as the AcrAB-TolC system in Escherichia coli, play a major role in drug resistance in Gram-negative bacteria because they allow the direct extrusion of drug molecules into extracellular space, and because they sometimes confer an increased level of tolerance to an astonishingly wide range of toxic compounds (18). In general, an RND-type exporter protein (such as AcrB), located in the inner membrane, forms a tripartite complex with a periplasmic adaptor protein, such as AcrA, and a homotrimeric outer membrane channel, such as TolC (18). The drug efflux process requires the presence of all three components. The crystallographic structures of AcrB (13, 14, 22, 24), AcrA (11, 27), and TolC (2, 8) are known, and models of the tripartite complex have been proposed (6, 27).AcrB is a homotrimeric transporter (14) located in the inner membrane and uses the proton gradient as the energy source (31). The homotrimeric structure is thought to be functionally important, or even essential, as each protomer appears to undergo a series of mandatory conformational alterations during the process of drug export, often called “functionally rotating mechanism,” as deduced from the structure of the asymmetric trimers of AcrB (13, 22, 24). This mechanism was also supported by the observation that, in a trimer in which protomers were covalently linked to each other, inactivation of one protomer alone abolishes the activity of the entire trimeric complex (29).Not all RND-type transporters, however, follow this homotrimeric organization. The mdtABC genes of E. coli encode an RND system that is unusual in that it contains two different RND pump genes, mdtB and mdtC, in addition to its own adaptor gene, mdtA. Previous genetic studies have demonstrated that the deletion of either of the two RND pump genes abolishes (1) the resistance to β-lactams, novobiocin, and bile salt derivatives, like deoxycholate, or narrows the range of pump substrates (15), a result suggesting that the functional unit is likely a heteromultimeric pump formed by MdtB/MdtC proteins. However, no direct data have so far been presented supporting the interaction between these proteins or the stoichiometry of the complex. Because the heterooligomeric composition of this pump was unexpected based on the accepted notion of how the homotrimeric pump functions by the functionally rotating mechanism, we examined here the nature of the MdtBC complex in detail.In this study, we first purified the oligomeric transporter by labeling either MdtB or MdtC with a His tag. We obtained a trimeric complex(es) containing both MdtB and MdtC in an approximately 2:1 ratio. However, we could not rule out the possibility that there were mixtures of trimers containing different ratios of the B and C proteins. We therefore utilized the recently introduced technology of creating covalently linked trimers (29), and we show here that the only active trimers are those containing two units of MdtB and one unit of MdtC.