SummaryAcrAB of Escherichia coli , an archetype among bacterial multidrug efflux pumps, exports an extremely wide range of substrates including solvents, dyes, detergents and antimicrobial agents. Its expression is regulated by three XylS/AraC family regulators, MarA, SoxS and Rob. Although MarA and SoxS regulation works by the alteration of their own expression levels, it was not known how Rob, which is constitutively expressed, exerts its regulatory action. We show here that the induction of the AcrAB efflux pump by decanoate and the more lipophilic unconjugated bile salts is mediated by Rob, and that the low-molecularweight inducers specifically bind to the C-terminal, non-DNA-binding domain of Rob. Induction of Rob is not needed for induction of AcrAB, and we suggest that the inducers act by producing conformational alterations in pre-existing Rob, as was suggested recently (Rosner, Dangi, Gronenborn and Martin, J Bacteriol 184: 1407-1416, 2002). Decanoate and unconjugated bile salts, which are present in the normal habitat of E. coli , were further shown to make the bacteria more resistant to lipophilic antibiotics, at least in part because of the induction of the AcrAB efflux pump. Thus, it is likely that E. coli is protecting itself by the Rob-mediated upregulation of AcrAB against the harmful effects of bile salts and fatty acids in the intestinal tract.

Rates of diffusion of uncharged and charged solute molecules through porin channels were determined by using liposomes reconstituted from egg phosphatidylcholine and purified Escherichia coli porins OmpF (protein 1a), OmpC (protein 1b), and PhoE (protein E). All three porin proteins appeared to produce channels of similar size, although the OmpF channel appeared to be 7 to 9% larger than the OmpC and PhoE channels in an equivalent radius. Hydrophobicity of the solute retarded the penetration through all three channels in a similar manner. The presence of one negative charge on the solute resulted in about a threefold reduction in penetration rates through OmpF and OmpC channels, whereas it produced two- to tenfold acceleration of diffusion through the PhoE channel. The addition of the second negatively charged group to the solutes decreased the diffusion rates through OmpF and OmpC channels further, whereas diffusion through the PhoE channel was not affected much. These results suggest that PhoE specializes in the uptake of negatively charged solutes. At the present level of resolution, no sign of true solute specificity was found in OmpF and OmpC channels; peptides, for example, diffused through both of these channels at rates expected from their molecular size, hydrophobicity, and charge. However, the OmpF porin channel allowed influx of more solute molecules per unit time than did the equivalent weight of the OmpC porin when the flux was driven by a concentration gradient of the same size. This apparent difference in "efficiency" became more pronounced with larger solutes, and it is likely to be the consequence of the difference in the sizes of OmpF and OmpC channels.

AcrD, a transporter belonging to the resistance-nodulation-division family, was shown to participate in the efflux of aminoglycosides. Deletion of the acrD gene decreased the MICs of amikacin, gentamicin, neomycin, kanamycin, and tobramycin by a factor of two to eight, and ⌬acrD cells accumulated higher levels of [ 3 H]dihydrostreptomycin and [ 3 H]gentamicin than did the parent strain.Multidrug efflux pumps play an important role in establishing the intrinsic level of resistance of gram-negative bacteria to a number of agents (16,17). Many of these pumps belong to the resistance-nodulation-division (RND) family, whose members have been known to pump out mostly lipophilic or amphiphilic molecules or toxic divalent cations (16,19). The Escherichia coli genome contains several genes coding for RND transporters. Among these, acrB is constitutively expressed and is largely responsible for the intrinsic resistance of E. coli to a very wide range of compounds, including lipophilic drugs, detergents (including bile salts), and dyes (8,10,18,20). The gene acrF has a high degree of similarity to acrB (77% identity at the amino acid level, with no gaps) and is also expected to pump out a wide variety of lipophilic and amphiphilic agents. Indeed, AcrF overexpression strains can be isolated as suppressors of acrAB mutants (J. Xu, M. L. Nilles, and K. P. Bertrand, Abstr. 93rd Gen. Meet. Am. Soc. Microbiol. 1993, abstr. K-169, p. 290, 1993 and seem to have a resistance phenotype similar to that of the acrAB ϩ wild-type strain. However, disruption of the acrF gene in the wild-type strain does not produce a drug hypersusceptibility phenotype (8), suggesting that acrF is weakly expressed in wild-type E. coli.Disruption of the acrD gene also did not result in hypersusceptibility to lipophilic and amphiphilic drugs (8). However, recently RND-type transporters that pump out very hydrophilic compounds, aminoglycosides, have been observed in Burkholderia pseudomallei (13) and in Pseudomonas aeruginosa (1). Furthermore, comparison of the P. aeruginosa aminoglycoside efflux pump MexY sequence with open reading frames in the E. coli genome sequence using the gapped BLAST program (2) shows that AcrD has the highest similarity score at the amino acid level. In light of these findings, we have reexamined the drug susceptibility of an acrD disruption mutant and found it to be hypersusceptible to a variety of aminoglycosides.Deletion-insertion of the acrD gene was done as follows. Plasmid pBW7, which corresponds to vector pEMBL8(ϩ) containing a 5.5-kb fragment of E. coli K-12 chromosome with the dapE (msgB) gene and the upstream acrD gene (21), was obtained from D. Ang. This plasmid was digested with ClaI (which cuts at position 710 of the acrD gene) and EcoRV (which cuts at position 2370), creating a deletion within acrD of 1,660 bp, and the AvaI-EcoRI fragment of pBR322 containing the tet gene was inserted in this interval. The plasmid was linearized and used to transform JC7623 [K-12 argE3 hisG4 leuB6 ⌬(gpt-proA) thr-1 thi-1 ara-14 galK2...

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A B s T R A c T Nutrients usually cross the outer membrane of Escherichia coli by diffusion through water-filled channels surrounded by a specific class of protein, porins. In this study, the rates of diffusion of hydrophilic nonelectrolytes, mostly sugars and sugar alcohols, through the porin channels were determined in two systems, (a) vesicles reconstituted from phospholipids and purified porin and (b) intact cells of mutant strains that produce many fewer porin molecules than wild-type strains. The diffusion rates were strongly affected by the size of the solute, even when the size was well within the "exclusion limit" of the channel. In both systems, hexoses and hexose disaccharides diffused through the channel at rates 50-80% and 2-4%, respectively, of that of a pentose, arabinose. Application of the Renkin equation to these data led to the estimate that the pore radius is -0.6 nm, if the pore is assumed to be a hollow cylinder. The results of the study also show that the permeability of the outer membrane of the wildtype E. coli cell to glucose and lactose can be explained by the presence of porin channels, that a significant fraction of these channels must be functional or "open" under our conditions of growth, and that even 105 channels per cell could become limiting when E. coli tries to grow at a maximal rate on low concentrations of slowly penetrating solutes, such as disaccharides.