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

Abstract

Prevotella bryantii cultures treated with monensin grew more slowly than untreated cultures, but only if the monensin concentration was greater than 1 microM. Cultures that were repeatedly transferred (eight transfers or 25 doublings) with monensin always grew rapidly, even at a 10 microM 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 microM 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.

FIG. 1

(a) Effect of monensin on the initial growth rate of wild-type (○) or monensin-selected (●) P. bryantii B₁4 batch cultures. (b) Effect of monensin on the initial growth rate of inocula that were taken from continuous culture (D = 0.1 h⁻¹). Open symbols (□) show inocula from a continuous culture that had not been treated with monensin, and closed symbols (■) indicate inocula that were obtained from a continuous culture that had been treated with monensin (10 μM). In each case, the initial cell density was 0.2 optical density units.

FIG. 2

(a) Relationship between intracellular potassium and the growth rate of wild-type cultures without monensin (○), wild-type cultures treated with 10 μM monensin (●), and monensin-selected cultures that were treated with 10 μM monensin (▴). (b) Relationship between intracellular ATP and the growth rate.

FIG. 3

(a) Double reciprocal plot of 1/monensin concentration versus 1/potassium depletion from wild-type (○) or monensin-selected (●) P. bryantii B₁4 cultures. The amount of ionophore needed to catalyze half-maximal potassium depletion is determined from −1/K_d, the intercept of the abscissa. (b) K_d of wild-type cultures (▴) transferred with 10 μM monensin and monensin-selected cultures (▵) transferred without monensin. Dotted lines indicate wild-type (bottom) and monesin-selected (top) cultures.

FIG. 4

Optical density (○) and intracellular potassium (▴) of wild-type P. bryantii continuous cultures (D = 0.1 h⁻¹, pH 6.7). Monensin (10 μM) was added directly to the culture vessel at time zero and at 25 h.

FIG. 5

Effect of lysozyme on wild-type (left test tube and upper micrograph) and monensin-selected (right test tube and lower micrograph) P. bryantii cultures. Cultures (test tubes) were centrifuged (120 × g, 2 min, room temperature). The microscopic magnification (micrographs) is ×1,800.

FIG. 6

Inhibition of lysozyme (0.1 μg/ml) activity by lipopolysaccharide extracts from wild-type (○) and monensin-selected (●) P. bryantii. The initial velocity (V_o) is expressed as the decrease in optical density of M. luteus cells per minute, and the initial S_o is the concentration of M. luteus cells in milligrams per milliliter. The closed triangles (▴) show the effect of lysozyme on M. luteus when no lipopolysaccharide extract was added.

FIG. 7

(a) Optical density of wild-type (○) or monensin-selected (●) P. bryantii cultures that were grown with 10 μM monensin. The arrow shows the fourfold relative growth advantage. (b) Calculated changes in K_d based on initial populations of 0.1, 1, or 10% monensin-resistant cells. Measured K_d values are indicated (▴).