Molecular Sieving by the Bacillus megaterium Cell Wall and Protoplast

Abstract

Passive permeabilities of the cell wall and protoplast of Bacillus megaterium strain KM were characterized by use of 50 hydrophilic probing molecules (tritiated water, sugars, dextrans, glycols, and polyglycols) which varied widely in size. Weight per cent uptake values ( R w ) were measured at diffusional equilibrium under conditions that negated the influences of adsorption or active transport. Plots of R w for intact cells as a function of number-average molecular weight ( ¯M n ) or Einstein-Stokes hydrodynamic radius ( ¯r ES ) of the solutes showed three phases: a protoplast uptake phase with a polydisperse exclusion threshold of ¯M n = 0.6 × 10 3 to 1.1 × 10 3 , ¯r ES = 0.6 to 1.1 nm; a cell wall uptake phase with a polydisperse exclusion threshold of ¯M n = 0.7 × 10 5 to 1.2 × 10 5 , ¯r ES ≅ 8.3 nm; and a total exclusion phase. Isolated cell walls showed only the latter two phases. However, it became evident that the cell wall selectively passed only the smallest molecules in a heterodisperse polymer sample. When the molecular-weight distributions of polyglycol samples ( ¯M n = 1,000, 1,450, and 3,350) were determined by analytical gel chromatography before and after uptake by intact cells or isolated cell walls, a quasi-monodisperse exclusion threshold was obtained corresponding to M n = 1,200, r ES = 1.1 nm. The permeability of isolated protoplasts was assessed by the relative ability of solutes to effect osmotic stabilization. An indefinite exclusion threshold, evident even with monodisperse sugars, was attributed to lengthwise orientation of the penetrating rod-shaped molecules. Altogether, the best estimate of the limiting equivalent porosity of the protoplast was 0.4 to 0.6 nm in radius and of the cell wall, 1.1 nm.