Vol. 143, No. 2 JOURNAL OF BACTERIOLOGY, Aug. 1980, p. 872-878 0021-9193/80/08-0872/07$02.00/0 Outer Membrane Protein Hi of Pseudomonas aeruginosa: Involvement in Adaptive and Mutational Resistance to Ethylenediaminetetraacetate, Polymyxin B, and Gentamicin THALIA I. NICAS AND ROBERT E. W. HANCOCK* Department of Microbiology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1 W5 It is well established that Pseudomonas aeruginosa cells grown in Mg2+- deficient medium acquire nonmutational resistance to the chelator ethylenedia- minetetraacetate and to the cationic antibiotic polymyxin B; this type of resistance can be reversed by transferring the cells to Mg2+-sufficient medium for a few generations. Stable mutants resistant to polymyxin B were isolated and shown to have also gained ethylenediaminetetraacetate resistance. Both the mutants and strains grown on Mg2e-deficient medium had greatly enhanced levels of outer membrane protein Hi when compared with the wild-type strain or with revertants grown in Mg2+-sufficient medium. It was determined that in all strains and at all medium Mg2+ concentrations, the cell envelope Mg2+ concentration varied in- versely with the amount of protein Hi. In addition, the increase in protein Hi in the mutants was associated with an increase in resistance to another group of cationic antibiotics, the aminoglycosides, e.g., gentamicin. We propose that protein Hi acts by replacing Mg2+ at a site on the lipopolysaccharide which can otherwise be attacked by the cationic antibiotics or ethylenediaminetetraacetate. In recent years considerable difficulty has been experienced in the treatment of Pseudo- monas aeruginosa infections, due to the differ- ence between in vitro and in vivo antibiotic susceptibilities (4, 6). Two possible causes are the development of adaptive (nonmutational) resistance in vivo (as demonstrated for certain antibiotics in vitro [1, 9, 18]) and the antagonism of antipseudomonal antibiotics, such as poly- myxin B and aminoglycosides, by Mg2" and Ca2" (16, 23). Brown and Melling (1) first demon- strated that growth of sensitive P. aeruginosa in Mg2-limited medium resulted in the acqui- sition of resistance to polymyxin B and EDTA (see also reference 9). The latter observation was particularly interesting, since P. aeruginosa is unusually susceptible to lysis by EDTA (c.f. enteric bacteria [19]). The fact that the Ca2+- specific chelator ethylene glycol-bis(,8-amino- ethyl ether)-N,N-tetraacetate was relatively in- effective (13, 19), combined with the results for Mg2+-limited cells, suggested a key role for Mg2' in EDTA and polymyxin B lyses. Recently, the separation of outer and inner membranes of P. aeruginosa in the absence of EDTA was re- ported by Hancock and Nikaido (11). Six to eight major outer membrane proteins were shown to be present, depending on the growth conditions (10). We report here that Mg2+-lim- ited cells which have acquired resistance to poly- myxin B and EDTA, as well as polymyxin B- resistant mutants, have greatly increased levels of outer membrane protein Hi. We suggest that protein Hi replaces Mg2e at a site on the lipo- polysaccharide (LPS) and protects this site from EDTA and polymyxin B attacks. MATERIALS AND METHODS Media and growth conditions. The growth me- dium was BM2 minimal medium (9) containing 10 yIM FeSO4 and 20 mM succinate or 0.4% (wt/vol) glucose. Mg2+-deficient medium contained 0.02 mM MgSO4, whereas Mg2e-sufficient medium contained 0.5 or 5 mM MgSO4. Control experiments demonstrated that in these media protein HI increased throughout the growth phase to a maximum in stationary phase. Cells grew at the same rate in both Mg2`-sufficient and Mg2+-deficient media, although the final yields of cells differed. Strains and mutant isolation. Strains H181 and H185 were independently isolated from P. aeruginosa PAO1 strain H103 (10) after diethyl sulfate mutagen- esis by selection on BM2 minimal agar plates (9) containing 10MuM FeSO4, 20 mM potassium succinate, 0.5 mM MgSO4, and 50,ug of polymyxin B sulfate per ml. The revertant H207 and five similar revertants of H181 and H185 were isolated from old cultures of H181 and H185 by screening for loss of polymyxin B resistance. The levels of resistance to polymyxin B on BM2 succinate agar plates were 0.8, 75, 75, and 0.8 ,ug/ ml for H103, H181, H185, and H207, respectively. Bacteriophages and aeroginocins. All methods used in the handling of bacteriophages were described previously (12). A pilus-deficient strain of P. aerugi- 872