Selection of a highly monensin-resistant Prevotella bryantii subpopulationwith altered outer membrane characteristics

Prevotella bryantii cultures treated with monensin grew more Slowly than un treated cultures, but only if the monensin concentration was greater than 1 mu M. Cultures-that were;repeatedly transferred (eight transfers or 25 dou blings) with monensin always grew rapidly, even at a 10 mu M concentration, The amount of monensin needed to facilitate half-maximal potassium depleti on (K-d) from monensin-selected cells was 16-fold greater than "unadapted" wild-type cultures (3,200 versus 200 nM). Cells taken from continuous cultu re had a K-d of 100 nM, and these inocula could not grow in batch culture w hen the monensin concentration was greater than 300 nM, Continuous cultures treated with monensin nearly washed out, but the surviving cells had a K-d of 1,300 nM. When wild-type cells were transferred in batch culture with 1 0 mu M monensin, the K-d did not reach its maximum value (3,200 nM) until a fter eight transfers (25 doublings), K-d declined when monensin was removed , and it took eight transfers to reach the control value (200 nhl), The mos t probable number of wild-type cells was 1,000-fold lower than of the monen sin-selected cells, but calculations based on relative growth advantage and K-d indicated that the wild-type culture had 1 to 10% highly monensin-resi stant cells. Cell pellets of wild-type cultures were more difficult to disp erse than were monensin-selected cells, and water-soluble phenol extracts o f monensin-selected cells had 1.8-fold more anthrone-reactive material than did the wild type. Wildtype cultures that were washed in Tris buffer (pH 8 .0) released little alkaline phosphatase and were agglutinated by lysozyme. Monensin-selected cultures leaked ninefold more alkaline phosphatase and w ere not agglutinated by lysozyme, Wild-type colonies taken from high-diluti on agar roll tubes retained the lysozyme agglutination phenotype even if tr ansferred with monensin, and monensin-selected colonies were never agglutin ated, These observations indicated that wild-type P, bryantii cultures had a:subpopulation with different outer membrane characteristics and increased monensin resistance.