CHARACTERIZATION OF THE CYTOPLASM OF ESCHERICHIA-COLI-K-12 AS A FUNCTION OF EXTERNAL OSMOLARITY - IMPLICATIONS FOR PROTEIN DNA INTERACTIONS INVIVO

The water-accessible volumes, the amounts of all significant osmolytes, and the protein concentration in the cytoplasm of aerobically grown Escherichia coli K-12 have been determined as a function of the osmolarity of the minimal growth medium. The volume of cytoplasmic water (V ̄cyto) decreases linearly with increasing osmolarity from 2.23(±0.12) μl/mg dry weight in cells grown at 0.10 osm to 1.18(±0.06) μl/mg dry weight at 1.02 osm. Above 0.28 osm, growth rate decreases linearly with increasing osmolarity. The growth rate extrapolates to zero at an osmolarity of approximately 1.8, corresponding to an estimated V ̄cyto of 0.5(±0.2) μl/mg dry weight. Measurements of V ̄cyto in titrations of non-growing cells with the plasmolyzing agent NaCl were used to obtain volumes of "bound" water (presumably water of macromolecular hydration) and cytoplasmic osmotic coefficients for cells grown in medium of low (0.10 osm) and moderate (0.28 osm) osmolarity. The volume of bound water V ̄b is similar in the two osmotic conditions (V ̄b = 0.40(±0.04) μl/mg dry wt), and corresponds to approximately 0.5 g H2O/g cytoplasmic macromolecule. Since V ̄cyto decreases with increasing osmolarity, whereas V ̄b appears to be independent of osmolarity, water of hydration becomes a larger fraction of V ̄cyto as the osmolarity of the growth medium increases. Growth appears to cease at the osmolarity where V ̄cyto is approximately equal to V ̄b. K+ and glutamate (Glu-) are the only significant cytoplasmic osmolytes in cells grown in medium of low osmolarity. The amount of K+ greatly exceeds that of Glu-. Analysis of cytoplasmic electroneutrality indicates that the cytoplasm behaves like a concentrated solution of the K+ salt of cytoplasmic polyanions, in which the amount of additional electrolyte (K+ Glu-) increases with increasing osmolarity. As the osmolarity of the growth medium becomes very low, the cytoplasm approaches an electrolyte-free K+-polyanion solution. In vivo osmotic coefficients were determined from the variation of V ̄cyto with external osmolarity in plasmolysis titrations of non-growing cells. The values obtained (φ = 0.54(±0.06) for cells grown at 0.10 osm and φ = 0.71(±0.11) at 0.28 osm) indicate a high degree of non-ideality of intracellular ions arising from coulombic interactions between K+ and cytoplasmic polyanions. Analysis of these osmotic coefficients using polyelectrolyte theory indicates that the thermodynamic activity of cytoplasmic K+ increases from approximately 0.14 m in cells grown at an external osmolarity of 0.10 osm to approximately 0.76 m at 1.02 osm. The amounts of protein and nucleic acid per unit cell dry weight appear to be independent of external osmolarity. Since V ̄cyto decreases with external osmolarity, the concentration of cytoplasmic protein increases from approximately 200 mg/ml in cells grown at 0.10 osm to approximately 320 mg/ml at 1.02 osm. The RNA concentration increases from approximately 75 mg/ml to approximately 120 mg/ml over this range. Scaled particle theory predicts that this increase in macromolecular concentration will cause the activity coefficients of typical cytoplasmic proteins to increase by several orders of magnitude. Such an increase in activity coefficients of DNA-binding proteins with increasing osmolarity appears to be sufficient to counter the expected effect of the concomitant increase in the thermodynamic activity of K+ on protein-DNA interactions. This compensation may explain the anomalous lack of dependence of protein-DNA interactions in vivo on [K+] and external osmolarity. Differential crowding effects as a function of external osmolarity should affect cell phenotype. For example, the ability of high osmolarity to restore the wild-type phenotype of certain conditional lethal mutants of E. coli (classified as "osmotic remedial" mutants) may be a manifestation of the effect of increased macromolecular crowding on the stability and assembly of proteins in the cytoplasm of cells grown at high osmolarity. © 1991.