Biophysical characterization of changes in amounts and activity of Escherichia coli cell and compartment water and turgor pressure in response to osmotic stress.

To obtain turgor pressure, intracellular osmolalities, and cytoplasmic water activity of Escherichia coli as a function of osmolality of growth, we have quantified and analyzed amounts of cell, cytoplasmic, and periplasmic water as functions of osmolality of growth and osmolality of plasmolysis of nongrowing cells with NaCl. The effects are large; NaCl (plasmolysis) titrations of cells grown in minimal medium at 0.03 Osm reduce cytoplasmic and cell water to approximately 20% and approximately 50% of their original values, and increase periplasmic water by approximately 300%. Independent analysis of amounts of cytoplasmic and cell water demonstrate that turgor pressure decreases with increasing osmolality of growth, from approximately 3.1 atm at 0.03 Osm to approximately 1.5 at 0.1 Osm and to less than 0.5 atm above 0.5 Osm. Analysis of periplasmic membrane-derived oligosaccharide (MDO) concentrations as a function of osmolality, calculated from literature analytical data and measured periplasmic volumes, provides independent evidence that turgor pressure decreases with increasing osmolality, and verifies that cytoplasmic and periplasmic osmolalities are equal. We propose that MDO play a key role in periplasmic volume regulation at low-to-moderate osmolality. At high growth osmolalities, where only a small amount of cytoplasmic water is observed, the small turgor pressure of E. coli demonstrates that cytoplasmic water activity is only slightly less than extracellular water activity. From these findings, we deduce that the activity of cytoplasmic water exceeds its mole fraction at high osmolality, and, therefore, conclude that the activity coefficient of cytoplasmic water increases with increasing growth osmolality and exceeds unity at high osmolality, presumably as a consequence of macromolecular crowding. These novel findings are significant for thermodynamic analyses of effects of changes in growth osmolality on biopolymer processes in general and osmoregulatory processes in particular in the E. coli cytoplasm.

Biophysical compensation mechanisms buffering E. coli protein-nucleic acid interactions against changing environments.

Escherichia coli adapts to changes in growth osmolarity of at least 100-fold by making large changes in the amounts of intracellular water and solutes, including cytoplasmic K+. A wide range of in vitro salt, solute and biopolymer concentrations should therefore be considered 'physiological'. Paradoxically, these large, osmotically induced changes in cytoplasmic K+ concentration do not greatly affect the equilibria and kinetics of cytoplasmic protein-nucleic acid interactions. Biophysical effects resulting from changes in the amount of cytoplasmic water (such as macromolecular crowding) and in the concentrations of other cytoplasmic solutes appear to compensate for the effects of changes in cytoplasmic K+ concentration and thereby maintain protein-nucleic acid equilibria and kinetics in the range required for in vivo function.

Responses of E. coli to osmotic stress: large changes in amounts of cytoplasmic solutes and water.

Escherichia coli is capable of growing in environments ranging from very dilute aqueous solutions of essential nutrients to media containing molar concentrations of salts or nonelectrolyte solutes. Growth in environments with such a wide range (at least 100-fold) of osmolarities poses significant physiological challenges for cells. To meet these challenges, E. coli adjusts a wide range of cytoplasmic solution variables, including the cytoplasmic amounts both of water and of charged and uncharged solutes.

Compensating effects of opposing changes in putrescine (2+) and K+ concentrations on lac repressor-lac operator binding: in vitro thermodynamic analysis and in vivo relevance.

Ion concentrations (K+, Glu-) in the cytoplasm of growing Escherichia coli cells increase strongly with increases in the osmolarity of a defined growth medium. While in vitro experiments demonstrate that the extent of protein-nucleic acid interactions (PNAI) depends critically on salt concentration, in vivo measurements indicate that cells maintain a relatively constant extent of PNAI independent of the osmolarity of growth. How do cells buffer PNAI against changes in the cytoplasmic environment? At high osmolarity, the increase in macromolecular crowding which accompanies the reduction in amount of cytoplasmic water in growing cells appears quantitatively sufficient to compensate for the increase in [K+]. At low osmolarity, however, changes in crowding appear to be insufficient to compensate for changes in [K+], and additional mechanisms must be involved. Here we report quantitative determinations of in vivo total concentrations of polyamines (putrescine(2+), spermidine(3+)) as a function of osmolarity (OsM) of growth, and in vitro binding data on the effects of putrescine concentration on a specific PNAI (lac repressor-lac operator) as a function of [K+]. The total concentration of putrescine in cytoplasmic water decreases at least eightfold from low osmolarity (approximately 64 mmol (l H2O)-1 at 0.03 OsM) to high osmolarity (approximately 8 mmol (l H2O)-1 at 1.02 OsM). Over this osmotic range the total [K+] increases from approximately 0.2 mol (l H2O)-1 to approximately 0.8 mol (lH2O)-1. We find that the effect of putrescine concentration on the repressor-operator interaction in vitro is purely competitive and is quantitatively described by a simple competition formalism in which lac repressor behaves a a specific-binding oligocation (ZR = 8+/-3). We demonstrate that this thermodynamic result is consistent with a structural analysis of the number of positively charged side-chains on two DNA binding domains of repressor which interact with the phosphodiester backbone of the operator site. Since this oligocation character of the binding surface of DNA-binding proteins appears to be general, we propose the competitive effects of putrescine and K+ concentrations on the strength of specific binding are general. At low osmolarity, compensating changes in putrescine and K+ concentration in response to changes in external osmolarity provide a general mechanism for E. coli to vary cytoplasmic osmolarity while maintaining a constant extent of PNAI.

Analyses of thermodynamic data for concentrated hemoglobin solutions using scaled particle theory: implications for a simple two-state model of water in thermodynamic analyses of crowding in vitro and in vivo.

Quantitative description of the thermodynamic consequences of macromolecular crowding (excluded volume nonideality) is an important component of analyses of the thermodynamics and kinetics of noncovalent interactions of biopolymers in vivo and in concentrated polymer solutions in vitro. By analyzing previously published thermodynamic data, we have investigated extensively the comparative applicability of two forms of scaled particle theory (SPT). In both forms, macromolecules are treated as hard spheres, but MSPT, introduced by Ross and Minton, treats the solvent as a structureless continuum, whereas bulk water molecules are included explicitly as hard spheres in BSPT, an approach developed by Berg. Here we use both MSPT and BSPT to calculate the excluded volume component of the macromolecular activity coefficient of hemoglobin (Hb) at concentrations up to 509 mg/ml by fitting osmotic pressure data for Hb and sedimentation equilibrium data for Hb and sickle-cell Hb (HbS). Both forms of SPT also are used here to analyze the effects of other globular proteins (BSA and Hb) on the solubility of HbS. In applying MSPT and BSPT to analyze macromolecular crowding, the extent of hydration delta Hb (in gH2O/gprotein) is introduced as an adjustable parameter to specify the effective (hard sphere) radius of hydrated Hb. In our nonlinear least-squares fittings based on BSPT, the hard sphere radius of bulk water molecules is either fixed at 1.375 A or floated. Although both forms of SPT yield good fittings (with different values of delta Hb) at Hb concentrations up to 350 mg/ml, only BSPT gives good fittings of all available Hb osmotic pressure data as well as of the sedimentation equilibrium and solubility data. Only BSPT predicts values for delta Hb (approximately 0.5-0.6 g/g) in the range obtained for Hb from hydrodynamic measurements (approximately 0.36-0.78 g/g). These findings indicate the applicability, at least in the context of BSPT, of a simple two-state classification of water (bulk water and water of macromolecular hydration) as a basis for interpreting excluded volume nonideality in concentrated solutions of globular proteins.

K(+)-ribosome interactions determine the large enhancements of 39K NMR transverse relaxation rates in the cytoplasm of Escherichia coli K-12.

As a probe of physical chemical properties of the intracellular environment, we measured 39K NMR transverse relaxation rates in concentrated cell slurries of Escherichia coli K-12 grown in minimal medium over a range of osmolarities (from 0.1 to 1.0 OsM) and after plasmolysis. The 39K transverse relaxation at a resonance frequency of approximately 18.67 MHz is biexponential under all conditions, and 100% of the expected signal intensity is detected. Both components of the 39K NMR transverse relaxation are very fast, and the difference between the fast and slow relaxation rates is very large compared to previous measurements on 23Na and 39K in protein and nucleic acid solutions in vitro. The 39K transverse relaxation rates decrease as the osmolarity of the growth media increases but increase dramatically when cells grown in 0.1 OsM media are plasmolyzed at 1.0 OsM. The homogeneous nature and the 100% visibility of the 39K signal indicate the existence of fast exchange among the multiple, magnetically distinguishable populations of 39K which probably exist in the cytoplasm. The absence of static quadrupolar splitting of the cytoplasmic 39K signal (as indicated by a single peak in the spectrum) indicates that the cytoplasm, as probed by 39K NMR, behaves like a concentrated but isotropic nucleic acid solution rather than an anisotropic nucleic acid liquid crystal. To understand the origins of the striking NMR relaxation behavior of 39K in viable cells, we have investigated NMR transverse relaxation rates of 39K (and also 23Na and 35Cl) in E. coli 50S and 70S ribosome solutions in vitro. At concentrations of ions and of ribosomes that to the extent possible mimic those of the cytoplasm of E. coli, we find that 39K, 23Na, and 35Cl transverse relaxation rates all exhibit biexponential behavior, and 39K and 23Na exhibit the large magnitudes and the large difference between the slow and the fast relaxation rates observed in viable cells. These polyanionic ribosome solutions are the only in vitro model system discovered to date that exhibits 39K transverse relaxation rates comparable to those in viable cells. We conclude that K(+)-ribosome interactions are the dominant source of the NMR properties of K+ in E. coli.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of the cytoplasm of Escherichia coli K-12 as a function of external osmolarity. Implications for protein-DNA interactions in vivo.

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 (Vcyto) decreases linearly with increasing osmolarity from 2.23(+/- 0.12) microliters/mg dry weight in cells grown at 0.10 OSM to 1.18(+/- 0.06) microliters/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 Vcyto of 0.5(+/- 0.2) microliters/mg dry weight. Measurements of Vcyto 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 Vb is similar in the two osmotic conditions (Vb = 0.40(+/- 0.04) microliters/mg dry wt), and corresponds to approximately 0.5 g H2O/g cytoplasmic macromolecule. Since Vcyto decreases with increasing osmolarity, whereas Vb appears to be independent of osmolarity, water of hydration becomes a larger fraction of Vcyto as the osmolarity of the growth medium increases. Growth appears to cease at the osmolarity where Vcyto is approximately equal to Vb. 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 Vcyto with external osmolarity in plasmolysis titrations of non-growing cells. The values obtained (phi = 0.54(+/- 0.06) for cells grown at 0.10 OSM and phi = 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.(ABSTRACT TRUNCATED AT 400 WORDS)