Multidrug efflux transporters, especially those that belong to the resistance-nodulation-division (RND) family, often show very broad substrate specificity and play a major role both in the intrinsic antibiotic resistance and, with increased levels of expression, in the elevated resistance of Gram-negative bacteria. However, it has not been possible to determine the kinetic behavior of these important pumps so far. This is partly because these pumps form a tripartite complex traversing both the cytoplasmic and outer membranes, with an outer membrane channel and a periplasmic adaptor protein, and it is uncertain if the behavior of an isolated component protein reflects that of the protein in this multiprotein complex. Here we use intact cells of Escherichia coli containing the intact multiprotein complex AcrB-AcrA-TolC, and measure the kinetic constants for various cephalosporins, by assessing the periplasmic concentration of the drug from their rate of hydrolysis by periplasmic ␤-lactamase and the rate of efflux as the difference between the influx rate and the hydrolysis rate. Nitrocefin efflux showed a Km of about 5 M with little sign of cooperativity. For other compounds (cephalothin, cefamandole, and cephaloridine) that showed lower affinity to the pump, however, kinetics showed strong positive cooperativity, which is consistent with the rotating catalysis model of this trimeric pump. For the very hydrophilic cefazolin there was little sign of efflux.cephalosporin ͉ drug efflux pump ͉ multidrug resistance M ultidrug eff lux pumps of the resistance-nodulationdivision (RND) family in Gram-negative bacteria can pump out a surprisingly wide range of antimicrobial compounds (1, 2). For example, AcrB of Escherichia coli, which has been studied most extensively as a prototype of similar pumps, exports a number of dyes, detergents, chloramphenicol, tetracyclines, macrolides, novobiocin, fluoroquinolones, and organic solvents. Importantly, it also pumps out ␤-lactams, some of which cannot easily cross the cytoplasmic membrane and thus stay in the periplasm (3). However, in the 13 years since their discovery (4, 5), no success was achieved in the determination of kinetic behavior of these pumps, although the affinity of some substrates could be assessed on a relative scale by their activity as competitive inhibitors in a proteoliposome reconstitution assay (6). Recently an attempt to measure the binding of dyes to purified AcrB was made by using fluorescence polarization (7); however, it is unclear if the experiments measured binding to active sites or to generally hydrophobic pockets in the protein.The knowledge of kinetic constants is essential in our attempts to understand the contribution of the pumps to drug resistance in a quantitative manner. Furthermore, recent crystallographic studies of AcrB (8-10) suggest that each protomer in this trimeric transporter undergoes a succession of conformational changes that are dependent on the conformations of the neighbors, that is, a functionally rotating mechanism. This m...

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The major outer-membrane proteins RagA and RagB of Porphyromonas gingivalis are considered to form a receptor complex functionally linked to TonB. In this study, P. gingivalis mutants with ragA, ragB or both deleted were constructed from strain W83 as the parent to examine the physiological and pathological functions of RagA and RagB. The double-deletion mutant completely lacked both RagA and RagB, whereas the DragA mutant reduced RagB expression considerably and the DragB mutant produced degraded RagA. Growth of the three mutants in a nutrient-rich medium and synthetic media containing digested protein as a unique nutrient source was similar to that of the parental strain; however, both the DragA and DragAB mutants exhibited very slow growth in a synthetic medium containing undigested, native protein, and the two mutants tended to lose their viability during experiments, although gingipain (protease) activities were unchanged in the mutants. A mouse model showed that the DragB mutant had reduced virulence. Cell-surface labelling with biotin and dextran revealed that both RagA and RagB localized on the outermost cell surface. A cross-linking experiment using wild-type P. gingivalis showed that RagA and RagB were closely associated with each other. Furthermore, co-immunoprecipitation confirmed that RagA and RagB formed a protein-protein complex. These results suggest that physically associated RagA and RagB may stabilize themselves on the cell surface and function as active transporters of large degradation products of protein and in part as a virulence factor.

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