Selection of a Highly Monensin-Resistant Prevotella bryantii Subpopulation with Altered Outer Membrane Characteristics

Abstract

ABSTRACT Prevotella bryantii cultures treated with monensin grew more slowly than untreated cultures, but only if the monensin concentration was greater than 1 μM. Cultures that were repeatedly transferred (eight transfers or 25 doublings) with monensin always grew rapidly, even at a 10 μM concentration. The amount of monensin needed to facilitate half-maximal potassium depletion ( K d ) from monensin-selected cells was 16-fold greater than “unadapted” wild-type cultures (3,200 versus 200 nM). Cells taken from continuous culture had a K d of 100 nM, and these inocula could not grow in batch culture when 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 10 μM monensin, the K d did not reach its maximum value (3,200 nM) until after eight transfers (25 doublings). K d declined when monensin was removed, and it took eight transfers to reach the control value (200 nM). The most probable number of wild-type cells was 1,000-fold lower than of the monensin-selected cells, but calculations based on relative growth advantage and K d indicated that the wild-type culture had 1 to 10% highly monensin-resistant cells. Cell pellets of wild-type cultures were more difficult to disperse than were monensin-selected cells, and water-soluble phenol extracts of monensin-selected cells had 1.8-fold more anthrone-reactive material than did the wild type. Wild-type 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 were not agglutinated by lysozyme. Wild-type colonies taken from high-dilution agar roll tubes retained the lysozyme agglutination phenotype even if transferred with monensin, and monensin-selected colonies were never agglutinated. These observations indicated that wild-type P. bryantii cultures had a subpopulation with different outer membrane characteristics and increased monensin resistance.