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Several mutants of Escherichia coli affecting aerobic energy generation and energization of the bacterial membrane have been examined for their effect on streptomycin and gentamicin accumulation and susceptibility. A heme-deficient mutant (K207) and two mutants [colicin K insensitive] and NR-70) associated with defective aerobic active transport were associated with decreased transport of streptomycin and gentamicin and increased resistance to those antibiotics. These mutants also exhibited increased resistance to several other aminoglycoside antibiotics, but not the aminocyclitol spectinomycin. The same observations were made with a ubiquinone-deficient mutant, but a strA derivative of this mutant was shown additionally to be saturable for streptomycin accumulation at a concentration four or more times lower than that required for saturation of the parent. A mutant uncoupled for adenosine 5'-triphosphate synthesis from electron transport and membrane Mg-adenosine 5'-triphosphatase deficient was hypersensitive to those aminoglycosides tested and spectinomycin, and showed enhanced transport of streptomycin and gentamicin. A variety of compounds structurally related to streptomycin were examined at high concentrations for inhibition of streptomycin uptake in a strA mutant ofE. coli K-12 SA 1306, but no evidence for competition was detected, suggesting the absence of a common transport carrier. Four different divalent cations were shown to inhibit streptomycin and gentamicin accumulation in E. coli K-12 SA 1306. Divalent cations were shown to inhibit uptake of these two drugs in two bacterial species with distinct cell wall structures, Pseudomonas aeruginosa and Staphylococcus aureus, and to inhibit streptomycin uptake in spheroplasts of streptomycin-susceptible and -resistant E. coli. However, calcium had almost no inhibitory effect on streptomycin uptake by the ubiquinone-deficient mutant E. coli AN66. These and previous findings have been used to formulate a model for aminoglycoside entry into bacteria using a low-affinity membranous complex involved in membrane energization that includes respiratory quinones, which probably act to bind and transport aminoglycosides across the cell membrane. This phase of transport is associated with the lowest accumulation rate (termed energy-dependent phase I) that is rate limiting for susceptibility. It is further proposed that subsequent association of the membrane-bound aminoglycoside with higher-affinity binding sites on membrane-associated ribosomes carrying out a normal ribosomal cycle and protein synthesis results in a more rapid transport rate (termed energy-dependent phase II). The increased rate could result from a state of membrane energization analogous to that causing enhanced aminoglycoside transport rates seen in the uncoupled mutant, AN120. How this model explains the mechanism by which enzymatically modified aminoglycosides render cells resistant to unmodified aminoglycosides is also discussed.The action of the aminoglycoside antibiotics cumulation by bacter...

The effects of a set of conditions on aminoglycoside uptake were determined. Membrane vesicles either with a membrane potential (A0,) of -125 mV (adequate to drive lysine uptake) or with succinate, lactate, or phenazine methosulfate did not accumulate gentamicin unless components of protein synthesis were included. Ribosomally resistant (rpsL) Escherichia coli cells demonstrated energy-dependent phase II uptake similar to that of a streptomycin-susceptible strain of E. coli when treated with 100 ,ug of puromycin per ml. Puromycin (100 ,ug/ml) also increased the uptake of the cationic compounds polyamine and arginine. These studies support a role of protein synthesis in aminoglycoside uptake and in the development of energy-dependent phase II. A* of cells did not increase either at the initiation of or during energy-denpendent phase II, showing that energydependent phase II is not due to an elevation of A+. In a Bacillus subtilis system, significant streptomycin uptake requires a threshold value of A* which varies depending upon the concentration of streptomycin used. At 25 ,ug/ml, the uptake of streptomycin reached maximal levels after exceeding the threshold value, whereas at 100 p.g/ml there was a gradual increase of the uptake to the maximal after the threshold value was exceeded. Several studies supported the view that electron transport has a specific role other than its requirement to produce the cellular &*. The uptake of gentamicin was stimulated to a greater extent by phenazine methosulfate-ascorbate than by the ionophore nigericin in strains of E. coli, although nigericin stimulated Ai, to a greater degree. Cells with 25% of the normal quinone concentration had values identical to cells with the normal quinone concentration, but the quinone-deficient cells had a significantly lower rate of gentamicin uptake. KCN prevented gentamicin uptake but did not prevent the development of A+. The effects of ubiquinone depletion in an E. coli strain were more evident on gentamicin uptake than on ATP-driven glutamine transport or proton motive force-driven proline transport, consistent with a specific requirement for quinones in aminoglycoside uptake. A detailed explanation of the mechanism of accumulation of streptomycin and gentamicin and a proposed mechanism for killing bacterial cells by these agents have been provided.The uptake of the aminoglycoside antibiotics streptomycin and gentamicin has been shown to be influenced by a complex set of conditions. It has been shown that the kinetics of uptake involve an initial energy-independent phase associated with ionic binding to the cell surface and cytoplasmic membrane. This is followed by two energy-dependent phases, a slow initial rate of uptake termed energy-dependent phase I (EDP-I) and a second accelerated rate termed energy-dependent phase II (EDP-II). Initiation of the latter phase requires binding to ribosomes (reviewed in references 2 and 11).The most effective energy source has been demonstrated to be electron transport involving quinone oxidation-reducti...

Lipopolysaccharide (LPS) from smooth strains of Pseudomonas aeruginosa 503, PAZ1, PA01715, PA01716, and Z61 was fractionated by gel filtration chromatography. LPS samples from the first four strains, all PAO1 derivatives, separated into three major size populations, whereas LPS from strain Z61, a Pac K799/WT mutant strain, separated into two size populations. When column fractions were applied to sodium dodecyl sulfate-polyacrylamide gels in their order of elution, molecules of decreasing size were resolved, and the ladder of molecules with different-length 0 antigens formed a diagonal across the gel. The LPS from the PAO1 derivatives contained two distinct sets of bands, distinguished on the gels as two sets of diagonals. The set of bands with the faster mobility, the B bands, was found in column fractions comprising the three major amino sugar-containing peaks. In the sample from strain 503, a fourth minor peak which contained B bands was resolved. The slower-moving set of bands, the A bands, were recovered in a minor peak. LPS from strain Z61 contained only one set of bands, with the higher-molecular-weight molecules eluting from the column in a volume similar to that of the B bands of the PAO1 strains. Analysis of the fractions of LPS from all strains indicated that less than 8% of the LPS molecules had a long, attached 0 antigen. Analysis of the peak that contained mainly A bands indicated a lack of reactive amino sugar and phosphate, although heptose and 2-keto-3-deoxyoctulosonic acid were detected. Reaction of isolated fractions with monoclonal antibody specific for the PA01 0-antigen side chain indicated that only the B bands from the PAO1 strains were antigenically reactive. The bands from strain Z61 showed no reactivity. The data suggest that the A and B bands from the PAO1 strains are antigenically distinct. We propose that PAO1 strains synthesize two types of molecules that are antigenically different.Lipopolysaccharide (LPS), a major component of the outer membranes of gram-negative bacteria, is important in the structure (33, 37) and function (33,36) of this membrane. Structural microheterogeneity in several regions of LPS molecules from members of the family Enterobacteriaceae (2,14,22,37,38,48,51) and Pseudomonas aeruginosa strains (33, 57) has been demonstrated. Of the several methods used to separate the subclasses of LPS from individual strains, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (20,24,45,48) and gel filtration (9, 26, 30, 32, 34, 48) are the best. Either of these two methods by themselves, however, may be insufficient to completely characterize the high-and low-molecular-weight fractions of LPS. Peterson and McGroarty (48) demonstrated that the SDS-PAGE of the column fractions of samples from Salmonella typhimurium, Salmonella minnesota, and Escherichia coli was instrumental in characterizing the various-sized fractions. Analysis of the isolated fractions allowed for the estimation of the average number of 0-antigen repeat units per LPS from each of the si...

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