Emiko Y. Rosenberg

Bile salts and fatty acids induce the expression of Escherichia coli AcrAB multidrug efflux pump through their interaction with Rob regulatory protein

AcrAB 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‐molecular‐weight 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.

AcrD of Escherichia coli Is an Aminoglycoside Efflux Pump

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 DeltaacrD cells accumulated higher levels of [(3)H]dihydrostreptomycin and [(3)H]gentamicin than did the parent strain.

Physical organization of lipids in the cell wall of Mycobacterium chelonae

Mycobacterial cell wall functions as an effective permeability barrier, making these bacteria resistant to most antibacterial agents. It has been assumed that this low permeability was due to the presence of a large amount of unusual lipids in the cell wall, but it was not known how these lipids are able to produce such an exceptional barrier. We report here the first experimental evidence on the physical arrangement of these lipids based on X‐ray diffraction studies of purified Mycobacterium chelonae cell wall, a result suggesting that the hydrocarbon chains of the cell‐wall lipids are arranged predominantly in a direction perpendicular to the cell wall surface, probably producing an asymmetric bilayer structure.

Outer membrane permeability and beta-lactamase stability of dipolar ionic cephalosporins containing methoxyimino substituents.

Some enteric bacteria, such as Enterobacter cloacae, can develop high-level resistance to broad-spectrum cephalosporins by overproducing their chromosomally encoded type I beta-lactamases. This is because these agents are hydrolyzed rapidly at pharmacologically relevant, low (0.1 to 1 microM), concentrations, owing to their high affinity for type I enzymes. In contrast, the more recently developed cephalosporins, with quaternary-nitrogen-containing substituents at the 3 position, show increased efficacy against beta-lactamase-overproducing strains and, indeed, have a much lower affinity for type I enzymes. However, the possible contribution of an improved outer membrane permeability in their increased efficacy has not been studied. We found by proteoliposome swelling assays that cefepime, cefpirome, and E-1040 all penetrated the porin channels of Escherichia coli and E. cloacae much more rapidly than did ceftazidime and at least as rapidly as did cefotaxime. Considering that the influx of anionic compounds such as cefotaxime and ceftazidime will be further retarded in intact cells, owing to the Donnan potential, we expect that the newer compounds will penetrate intact cells 2 to 10 times more rapidly than will cefotaxime and ceftazidime. The kinetic parameters of hydrolysis of these agents by E. cloacae beta-lactamase showed that at 0.1 microM, they were hydrolyzed much more slowly than was cefotaxime and at about the same rate as or a lower rate than was ceftazidime. The combination of these two effects explains nearly quantitatively why these newer agents are more effective against some of the beta-lactamase-overproducing gram-negative bacteria.

Functional reconstitution of lens gap junction proteins into proteoliposomees

SummaryMembranes rich in junction complexes were prepared from bovine lens, and the fragments of the membranes were reconstituted into proteoliposomes with a large excess of phosphatidylcholine and dicetylphosphate. The osmotic swelling behavior of these liposomes showed that the lens junction membranes contributed protein components that produced channels with a nominal diameter of 1.4 nm. Most preparations of lens junctions produced rates of osmotic swelling much slower than those found in proteoliposomes containing equivalent amounts ofEscherichia coli porin, and we discuss several possible explanations for this observation.

Specificity of the glucose channel formed by protein D1 of Pseudomonas aeruginosa

Protein D1 from the outer membrane of Pseudomonas aeruginosa was purified and reconstituted into proteoliposomes. Many small molecules were shown to diffuse through the D1 channel in the proteoliposomes, and the permeation rates of D-glucose, L-glucose, D-xylose, and L-xylose were much higher than expected for their size. This finding and the permeation rates of various glucose analogs suggest that, although the channel has a specific recognition site for glucose, it functions in a manner very different from that of the glucose carrier of erythrocytes.