Aminoglycosides are currently considered to be among the few antibiotics useful in the treatment of infections caused by Pseudomonas aeruginosa. The discrepancy between in vitro and in vivo susceptibility is a major problem in aminoglycoside therapy (6,8). The well-documented antagonism of aminoglycosides by divalent cations (18, 25) is one probable cause, and development of adaptive (nonmutational) resistance in vivo may also play a part. We recently showed that, in P. aeruginosa, induction of a major outer membrane protein, Hi, results in resistance to polymyxins and ethylenediaminetetraacetate (EDTA) (21). The increase in outer membrane protein Hi was accompanied by a decrease in cell envelope Mg2". Mutants which overproduce outer membrane protein Hi also show enhanced resistance to aminoglycosides (21), suggesting the involvement of an outer membrane site, which could be protected by outer membrane protein Hi, in the activity of these antibiotics. We report here that P. aeruginosa induced for outer membrane protein Hi, either as a result of adaptation to growth in low Mg2" or in mutants, has altered kinetics of aminoglycoside killing and uptake. Furthermore, we demonstrate that aminoglycosides can permeabilize outer membranes to lysozyme and a 8-lactam antibiotic by a mechanism which is extremely rapid and independent of aminoglycoside killing and energized uptake but is inhibited by Mg2". Our results suggest a mechanism of uptake across the outer membrane different from the previously characterized hydrophilic (porin-mediated) and hydrophobic pathways (22). MATERIALS AND METHODSBacterial strains. P. aeruginosa PA01 strain H103 and its derivatives H181 and H185 were previously described (21