As inhabitants of natural and artificial aqueous environments, bacteria survive dramatic changes in extracellular osmolality (for definitions of terms used in this review, see the glossary in Table 1). For example, soil bacteria survive periods of low and high rainfall, uropathogens survive urine concentration and dilution, and industrial organisms tolerate concentrated nutrient solutions as well as the extracellular accumulation of metabolic products. Bacteria respond both passively and actively to changes in the osmolality of their environment (Fig. 1 provides a summary of this phenomenon as it occurs in Escherichia coli).

Since the water permeability of the cytoplasmic membrane is high, imposed imbalances between turgor pressure and the osmolality gradient across the bacterial cell wall are short in duration. Changes in cell structure, organization, and composition that result from transmembrane water flux (Fig. 1, left column) trigger and are modulated by physiological responses (Fig. 1, right column). Bacteria respond to osmotic upshifts in three overlapping phases: dehydration (loss of some cell water) (phase I), adjustment of cytoplasmic solvent composition and rehydration (phase II), and cellular remodeling (phase III). Responses to osmotic downshifts are not yet well characterized, but they are also likely to proceed in three phases: water uptake (phase I), extrusion of water and cosolvents (phase II), and cytoplasmic cosolvent reaccumulation and cellular remodeling (phase III). Many of the cellular responses triggered by osmotic stimuli occur in parallel. Limited experimental evidence for sequential osmoregulatory processes (discussed below) offers insights into osmosensory mechanisms.

View larger version (45K): [in this window] [in a new window]   FIG. 1.   Phases of the osmotic stress response for E. coli K-12. Structural and physiological responses triggered by osmotic shifts (up or down) imposed at time zero proceed in parallel along the indicated, approximate timescales. The evidence supporting this scheme is discussed in the text.

Modulation of cytoplasmic solvent composition is the only response that is known to reverse the growth-inhibitory effects of osmotic shifts on bacteria. The following principles govern this process as it occurs in diverse bacteria, both gram positive and gram negative. (i) The concentrations of certain cytoplasmic solutes increase and decrease in response to osmotic up- and downshifts, respectively. (ii) There is a hierarchy of preference among the solutes that accumulate in response to an osmotic upshift. Particular zwitterionic organic cosolvents such as ectoine and glycine betaine are selected for this role over inorganic solutes such as K⁺. (iii) Osmoregulation of uptake, efflux, biosynthesis, and/or catabolism is required to modulate the cytoplasmic levels of these osmoregulatory solutes.

The genes and enzymes responsible for modulation of osmoregulatory solute levels have been identified in diverse bacteria. The mechanisms by which bacteria sense osmotic shifts (osmosensory mechanisms) and the basis for osmoregulatory solute selection are still poorly defined, however.

Chemosensors exploit the specificity of ligand-receptor interactions to detect the biochemistry of cellular environments, including both changes in nutrient supplies and signals with biological origins (Fig. 2). An osmosensor is a device that detects changes in extracellular water activity (direct osmosensing) or the resulting changes in cell structure (indirect osmosensing). In contrast to chemosensory mechanisms, osmosensory mechanisms cannot be based on stereospecific ligand-receptor interactions that occur at specific sites in receptor molecules. Instead, osmosensors are likely to be macromolecules that undergo changes in conformation and/or oligomerization in response to solvent changes or ensuing mechanical stimuli (Fig. 2). Our current knowledge suggests that osmosensors are located in the cytoplasmic membranes and nucleoids of bacteria. This review focuses on membrane-based osmosensors since they elicit primary osmoregulatory responses. In addition, current knowledge of the osmoregulation of transcription in bacteria has been reviewed whereas the basis for membrane-based osmosensing has not. The goals of this review are to define the properties of solvent-macromolecule and solvent-membrane interactions pertinent to osmosensing, to identify putative osmosensors and the changes that they detect, and to review our current understanding of cytoplasmic membrane-based osmosensory mechanisms in bacteria. This review is intended to stimulate broadly based research on osmosensing. Efforts have therefore been made to express microbiological, biochemical, and biophysical concepts in terms accessible to both specialists and nonspecialists. Readers who prefer textual explanations are therefore asked to accommodate the mathematical expressions preferred by those interested in the biophysical basis for osmosensing. This review also builds upon concepts developed by many other authors (2, 7, 25, 47, 74, 220, 228, 229, 256). Physiological responses to osmolality changes, to cooling-freezing, and to desiccation are related (44, 55, 124, 156). Only responses to osmolality changes are considered here, however. In addition, this review does not encompass the modifications to macromolecular structure that render organisms of the salt-in-cytoplasm type obligately halophilic (74).

View larger version (36K): [in this window] [in a new window]   FIG. 2.   Chemosensing versus osmosensing. Many biosensors are molecules whose conformations change between an "off" and an "on" conformation in response to a change in the biosensor environment. Chemosensors detect the biochemistry of cellular environments, including changes in nutrient supplies and signals with biological origins. The proportion of chemosensor molecules in the "on" conformation increases when the appropriate ligand binds to a specific site on the chemosensor surface. Osmosensors detect changes in extracellular water activity (direct osmosensing) or resulting changes in cell composition or structure (indirect osmosensing). At least some osmosensors are expected to change their conformations in response to solvent changes. The proportion of osmosensor molecules in the "on" conformation would then increase when the sensor surface was exposed to a suitably altered solvent (depicted here by a change from a white to a shaded background). Alternatively, sensing could involve a change in oligomeric state (for example, ligand- or solvent-induced dimerization).

SOLVENTS AND THEIR INTERACTIONS WITH BIOMOLECULES

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Solvent Properties Pertinent to Osmosensing

For the purpose of this review, the chemical potential of water can be expressed as

(1)

where µ_w* is the chemical potential of water under standard conditions, R is the gas constant, T is the temperature (Kelvin), a_w is the activity of water, _w is the partial molar volume of water, and P is the pressure.

Water and its solutes can be characterized by their activities (a_i), where

(2)

_i and x_i are the activity coefficient and the mole fraction of the ith constituent, respectively, and O < a_i < 1. In a physicochemically ideal system, the solvent activity approaches 1 and the solute concentrations (expressed as mole fractions or as the more familiar molar units), which approach zero, can be used as effective proxies for their activities. Experimental systems are often designed to approximate ideality, so that simplifying assumptions can be made about the resulting chemical and physical phenomena. Although some laboratory media and some natural microbial environments can be described as ideal solutions, many are nonideal. The aqueous compartments within microorganisms are certainly nonideal. Nonideality has important consequences for osmosensing (see "Solvent-macromolecule interactions").

The ionic strength and osmotic pressure of the solvent arise through collective contributions of ionic solutes and of all solutes, respectively. Solutes that are present at high concentrations have the greatest effects on osmolality and (if they are ionic) ionic strength. The osmotic pressure of an aqueous solution () is defined as

(3)

Note that definitions of osmotic pressure vary. This definition differs from those used in some previous reviews on bacterial osmoregulation (47, 178) and desiccation tolerance (221). It is used here for reasons discussed by Nobel (200). Osmotic pressure can also be expressed in terms of the osmolality (units of osmoles/kg of solvent or osmolal:

(4)

Osmolarity, the sum of the concentrations of osmotically active solutes in solution, provides a convenient approximation for osmolality:

(5)

where the c_i are the molar concentrations of the osmotically active solutes. Osmolarity and osmolality are not equal, and osmolality can be measured but not calculated (272).

Diverse cosolvents are present outside and inside bacteria. NaCl and sugars (or other polyols) are the most prevalent and abundant extracellular cosolvents in natural environments. K⁺, organic anions, compatible solutes, proteins, and nucleic acids are the predominant intracellular cosolvents (28, 29, 74, 257, 304). The intracellular solvent of most bacteria is thus differentiated from the extracellular solvent by including a restricted array of low-molecular-weight cosolvents and a high concentration of macromolecular cosolvents. To understand osmosensing, it is necessary to deduce both the solvent changes to which osmosensors are actually exposed in vivo and the mechanisms by which those solvent changes can trigger changes in osmosensor conformation. Osmosensors located in the cytoplasmic membrane or nucleoid are exposed to the constrained solvent environments of the cytoplasm, membrane, and/or periplasm, not to the extracellular aqueous medium.

Effects of cosolvents on the behavior of water at interfaces (and their effects on proteins) have been described, systematically but empirically, in terms of the Hofmeister effect (41). Both ionic and nonionic cosolvents can be characterized in this way as kosmotropic (water structure making, e.g. phosphate, ammonium, sucrose, and glycerol), close to neutral (e.g., Na⁺ and Cl), or chaotropic (water structure breaking, e.g., SCN and urea). For example, kosmotropes and chaotropes tend to stabilize and destabilize native protein conformations, respectively (for a further discussion, see below). Effects of kosmotropes and chaotropes on protein function may be additive or mutually compensatory (see, e.g., references 292 and 308).

Efforts by biochemists and biophysicists to systematically describe and explain water-cosolvent-macromolecule-solid-matrix interactions now offer additional perspectives and tools to students of osmoregulation (Fig. 3). Included are studies of (i) very low affinity ligand-receptor interactions and the impacts of diverse cosolvents on macromolecular stability, solubility, and assembly explained in terms of preferential exclusion of certain solvent constituents from macromolecular surfaces (275); (ii) the significance of hydration changes, probed by varying the osmotic pressure, for macromolecular interactions and functions (209); and (iii) the impacts of crowding and confinement on the structures and interactions of macromolecules (182, 297). Each rests on the concept that "the local environment will influence reactions taking place in a biological medium when reactants, transition state complexes, and products interact unequally with background species" (311). In the context of osmosensing, the reactants and products would be the forms ("off" and "on") of an osmosensor and the background species constituting the local environment would include water, cosolvents, and structural elements.

Preferential interaction. Some interactions among water, cosolvents, and osmosensors may be more appropriately considered in terms of preferential binding or exchange than in terms of classical binding theory. The latter describes the fractional occupancy of a specific receptor site(s) on a macromolecule by one or more ligands. This occupancy may vary from 0 (no binding) to the number of sites, which is usually small. In terms of preferential binding or exchange, the reference state is a macromolecule in pure water with all exposed surface sites hydrated (no sites occupied by cosolvent). A gradual exchange of water for cosolvent occurs as the cosolvent is added to the system. At any cosolvent concentration, the fractional occupancy of surface sites by cosolvent exceeds, matches, or falls short of the proportion (mole fraction) of cosolvent molecules in bulk solution if the surface sites prefer cosolvent over water, show no preference, or prefer water over cosolvent, respectively. This situation differs substantially from that treated by classical binding theory. Each solvent-macromolecule interaction is weak, but the number of sites involved is large and hence the potential impact of solvent-cosolvent exchange on macromolecular structure and function is also large. For example, it has been estimated that lysozyme offers a total of 266 surface sites for solvent and/or cosolvent occupancy (276). In addition, the global behavior of the macromolecule-solvent system is an average over weak interactions among macromolecule, cosolvent, and water at a large number of nonidentical surface sites.

View larger version (45K): [in this window] [in a new window]   FIG. 3.   Solvent effects on macromolecular conformation. If different forms (conformations) of a macromolecule (or osmosensor) interact differently with their solvent, solvent changes will alter the distribution of the macromolecule (or sensor) population among those forms. Such effects could constitute a basis for osmosensing, as illustrated in Fig. 2. Simultaneous exposure of membrane-based osmosensors to multiple solvent environments would further enhance the scope for modulation of their structure by solvent changes. Three aspects of solvent-macromolecule interactions that have been demonstrated to influence macromolecular conformation are illustrated in this figure. For preferential exclusion, if water and cosolvent interact differently with the macromolecular surface, a change in cosolvent composition can trigger a change in macromolecular conformation and/or oligomerization. For example, an increased concentration of one or more cosolvents (indicated by a darker grey background) that are preferentially excluded from the sensor surface (indicated by a halo) favors a conformational change that results in a decreased sensor surface area (indicated by a change from square to round). For hydration changes, if entry of cosolvent molecules to macromolecule-associated solvent pools is restricted (as indicated by the relative sizes of the grey cosolvent molecules and the water-filled cleft on the macromolecular surface), an increase in cosolvent concentration will cause macromolecule-associated solvent to be extracted and macromolecular conformation to be altered (closing the water-filled cleft and possibly also causing a more generalized conformational change, indicated by a change from square to round). For macromolecular crowding or confinement within matrices, in crowded solutions (those containing high concentrations or networks of macromolecules) compact or globular conformations of a test macromolecule (or osmosensor) are favored. Changes in crowding of the bacterial cytoplasm, where macromolecules occupy as much as 50% of the available volume (indicated as a change in the concentration of oblong, grey molecules comparable in size to the test macromolecule), may therefore favor changes in conformation of osmosensor molecules (again indicated as a change from square to round).

Hydration forces. Osmotic pressure has been used as a tool to probe the biological significance of macromolecular hydration, i.e., of "hydration forces." For example, the addition of osmotically active, polymeric cosolvent (e.g., polyethylene glycol [PEG]) to the surrounding medium can suppress ion channel opening and alter both K_D and K_M for the interaction of glucose with hexokinase (209). These data are interpreted as showing that the entry of cosolvent molecules to macromolecule-associated solvent pools is restricted and that solvent compartments which differ in osmolality are thus created. Thus, macromolecules may adjust to their solvent environments by assuming states that balance the (unfavorable) exclusion of cosolvent from inaccessible solvent pools against the (unfavorable) juxtaposition of macromolecular constituents. For example, withdrawal of solvent from intramolecular pools of low osmolality may force macromolecules to adopt different (and less energetically favorable) conformations than are assumed to be present in the absence of cosolvent (Fig. 3).

Pressure effects on the specificity of ligand-receptor interactions. Both osmotic and hydrostatic pressures have been used to perturb DNA-protein interactions (235). Stresses imposed with osmotic and hydrostatic pressures are fundamentally different; osmotic stress causes water molecules to be transferred from cosolvent-inaccessible to cosolvent-accessible regions around or within macromolecules, whereas hydrostatic pressure changes the weight density of the entire system. For some restriction endonucleases, DNA recognition sequence specificity changed as osmotic pressure was increased by the addition of diverse, low-molecular-weight cosolvents including sucrose, glycerol, 2-propanol, and N-methylformamide (0 to 3 osmolal, but with significant effect at 1 osmolal) (see, e.g., reference 234). These effects were reversed by elevated hydrostatic pressure (up to 500 atm). They were interpreted as indicating differential involvement of water at the enzyme-DNA interface for different DNA sequences. Hydration has also been recognized as playing a critical role in carbohydrate-protein interactions (152). Such behavior differs fundamentally from that described by preferential binding or exchange theory, however. It involves small numbers of water molecules that mediate high-affinity enzyme-substrate recognition at specific enzyme and substrate sites.

Crowding and confinement. Considerations of macromolecular crowding and confinement may also be used to explain "background" effects on macromolecular structures and interactions (182, 311). They define the collective impact of macromolecules, whether soluble or present as structural elements, on cellular processes. Garner and Burg (77) have reviewed these concepts from a physiological perspective. The term "crowding" refers to effects of high collective volume occupancy by macromolecules on the structures and functions of individual macromolecular species with which they interact only weakly and nonspecifically. Such effects may be biologically significant since macromolecules occupy as much as 50% of the cytoplasmic volume. The term "confinement" refers to effects of entrapment within a subcellular matrix on macromolecular function.

Solvent effects on membrane structure. Biophysicists and biochemists have long been fascinated and puzzled by the diversity and complex phase behavior of membrane phospholipids. The net charge, hydrogen-bonding capacity, and hydration of each head group, the length and unsaturation of each acyl chain, and the resulting overall shape of each phospholipid molecule all contribute to the phospholipid-solvent interactions which determine phase behavior in aqueous phospholipid dispersions (57, 60, 78, 85, 155). Each phospholipid (and each phospholipid mixture) has a particular propensity to form the lamellar (L) phase characteristic of biological membranes versus alternative arrangements, including the nonlamellar hexagonal (H_I or H_II) or cubic phases. Phospholipid phase transitions, for example, the transitions from gel (L) to liquid crystalline (L) lamellar phases and from the lamellar phase to the hexagonal phase, are characterized by phase transition temperatures (for these examples, T_M and T_H, respectively). Phospholipid phase behavior (often indicated by phase transition temperatures) is also influenced by amphiphiles which insert among phospholipid molecules (e.g., alcohols, fatty acids, detergents, anaesthetics, and peptides) and by such solvent properties as pH, ionic strength, and cosolvent composition.

View larger version (83K): [in this window] [in a new window]   FIG. 4.   Solvent effects on membranes. Membrane strain is the relative displacement of membrane constituents in response to an imposed stress. Such strain may be communicated to osmosensors by the phospholipid bilayer. (A) Intrinsic membrane strain arises because phospholipid monolayers that have an intrinsic tendency to be curved must flatten in order to associate and form phospholipid bilayers in aqueous solution (satisfying the requirements of the hydrophobic effect). This phenomenon has a number of potential consequences including the possible existence of a lateral pressure, exerted in the bilayer plain, that is higher in the membrane core than at the membrane surface (see the text for a discussion of this phenomenon). (B) Changes in cosolvent composition (indicated in grey) may modulate intrinsic membrane strain by acting in the ways illustrated in Fig. 3. Such changes could be transmitted to (and modify the conformations of) osmosensors that are integral membrane proteins. (C) Since biomembranes are fluid, topologically closed, and differentially permeable to water and cosolvents, changes of intra- or extracellular water activity cause changes in membrane shape. Such changes may alter mechanically imposed membrane strain in ways that change the conformations of osmosensors that are integral membrane proteins.

Biomembrane permeability. For small deviations from equilibrium, the net rate of volume flux across a membrane in response to a hydrostatic or osmotic driving force can be described in terms of an osmotic permeability coefficient (P_f [centimeters per second]) characteristic of the membrane and a reflection coefficient ( [dimensionless]) characteristic of the membrane and the cosolvent used to impose the osmotic gradient (71, 200). The flow of volume across a membrane separating compartment 1 from compartment 2 can be described as

(6)

where J_v is the water flux (cubic centimeters per second), A is the membrane surface area (square centimeters), _w is the partial molar volume of water, R is the gas constant, T is the temperature (Kelvin), P₁ and P₂ are the hydrostatic pressures (atmospheres) in compartments 1 and 2, respectively, _i (0 < _i < 1) describes the relative rates at which the ith cosolvent and water cross the membrane (via all available pathways) and Osm_i1 and Osm_i2 are the osmolalities due to the ith cosolvent in compartments 1 and 2, respectively. The reflection coefficient (_i) varies from close to 0 for a highly permeant cosolvent to 1 for a cosolvent which does not cross the cell surface (e.g., glycerol has a low  and sucrose has a high  with respect to the surface of E. coli [see below]). The significance of  can be grasped by recognizing that a cosolvent flux sufficiently rapid (with respect to solvent flux) to collapse the osmolality gradient will attenuate the solvent flux (J_v). In terms which are important for osmosensing, P_f and _i contribute to the rate and extent of solvent flux and hence to the duration of imposed osmotic gradients (Osm), the rate of change of hydrostatic pressure (P), and the rate at and degree to which a plastic, membrane-bounded osmotic compartment will be deformed in response to an osmotic shift.

3

2

2

Mechanical properties of membranes. A membrane can be defined, mechanically, as "a material with a very small thickness in comparison with its radii of curvature which separates two adjacent, liquid-like domains and supports the stresses created by the embedding medium" (19). To understand membrane-associated osmosensors, it may become necessary to link our understanding of membrane molecular structure and dynamics (with a length scale up to a few nanometers) with our understanding of the membrane as a continuum of condensed matter (with a length scale greater than 500 nm). Barriers to the attainment of that goal include both difficulties of communication among investigators and technical limits to the study of phenomena which occur on the relevant length scale and timescale (19).

(7)

Osmoregulators are devices that implement the response of an organism to changing environmental osmolality. An individual device may both detect and respond to solvent changes (i.e., osmosensors may also be osmoregulators). Alternatively, osmosensors and osmoregulators may be separate and communication between or among them may require additional signal transduction machinery. The high water permeability and solute selectivity of biomembranes ensure that changes in extracellular osmolality imposed with membrane-impermeant cosolvents trigger extensive changes in cell structure and chemistry. Thus, in principle osmosensors may detect changes in extracellular water activity (direct osmosensing) or they may detect and elicit responses tailored to address secondary consequences of osmotic shifts (indirect osmosensing). It is therefore expected that an array of osmosensors may detect and control a temporal cascade of cellular changes and osmoregulatory responses. As a result, the "osmotic history" of each cell will determine its response to new osmotic stimuli (76, 261).

The following discussion is designed to place putative bacterial osmosensors within the cascade of osmotically induced changes to cell structure and physiology, thereby identifying the stimuli to which each osmosensor may (or may not) respond. This process of correlation and elimination is important, since the list of stimuli which could, in principle, be detected by each osmosensor is long (Table 2).

When subjected to an increase or decrease in the osmolality of the suspending medium (an osmotic upshift or downshift, respectively), E. coli cells change as illustrated in Fig. 1. This review focuses primarily on membrane-based sensory processes that occur during phase I of this response. Phases II and III are considered only to the extent that they illustrate the physiological objectives met by osmoregulatory processes and the relevant changes in cell structure associated with steady-state exposure to media of various osmolalities. The timescales of these events have not yet been fully defined, and the interpretation of existing evidence concerning the timescales of phases I and II is subject to ambiguities (see Appendix). Nevertheless, the events listed as phase I of the response to an osmotic upshift can be considered to occur within milliseconds to minutes of an osmotic shift. Phase II (which also begins on imposition of an osmotic shift) extends until approximately 40 min or 10 to 20 min after the shift for bacteria cultivated in the absence and presence of osmoprotectants, respectively. Phase III is highly condition dependent and can extend for 1 h or more after the shift. The timescales for water uptake (phase I) and release of cytoplasmic cosolvents (phase II) after an osmotic downshift are at or below the limits of the experimental techniques that have been applied to date. These phases are complete within 1 to 2 min, however, and the ensuing cosolvent reaccumulation (phase III) is complete within 10 to 20 min after the shift. The evidence on which these estimates are based is discussed further below.

Most studies have focused on effects of osmotic upshifts or of osmotic downshifts or of steady-state adaptation to either of these conditions. Osmotic upshifts and downshifts have usually been applied to bacteria cultivated in low- and high-osmolality media, respectively. Additional insight regarding osmosensing will be gained by considering, as a continuum, cellular responses to both increases and decreases in medium osmolality (see, for example, Parker's assessment of changes in cell volume versus macromolecular crowding as triggers of regulatory volume changes in canine erythrocytes [207]). Such studies will be facilitated by examining organisms with sufficient osmotolerance to permit the application of significant osmotic shifts, both up and down, after cosolvent loading (e.g., Lactobacillus plantarum [80, 81] and Halomonas elongata).

Bacterial responses have been examined at different growth phases (usually either mid-exponential or late exponential phase) and with different growth status during the experiment (growing or partially or fully nutrient deprived). Significant cellular remodeling accompanies both long-term (minutes to hours) osmoadaptation and the transition to stationary-phase growth. Thus, the patterns outlined in Fig. 1 will be refined as the short- and long-term effects of osmolality, growth phase, and nutrient status are disentangled.

Turgor pressure (P) is defined as the hydrostatic pressure difference which balances the osmotic pressure (or osmolality) difference between cell interior (i) and exterior (o), rendering the chemical potentials (µ_w) of intracellular and extracellular water (see equation 1) equal at equilibrium. For cells which can be treated as two-compartment systems:

(8)

where P is hydrostatic pressure. Imposed changes in P, Osm_i or Osm_o cause solvent flux across the cell surface to reestablish the equilibrium described by equation 8. On the basis of measured osmotic water permeability coefficients for phospholipid bilayers and membrane vesicles, equilibration is expected to occur within seconds of an osmotic shift (305). Since large cells (e.g., those of plants and some eukaryotic microorganisms) can be impaled, P and Osm_i can be manipulated directly (89, 102, 210). Such techniques may also be applicable to giant bacterial cells created by mutation and/or antibiotic treatment. The rate of solvent flux and the participation of particular cosolvents in passive cellular adjustment to osmotic shifts are illustrated (for fluxes of small magnitude) by equation 6.

Turgor pressure imposes stress on the cell surface. For a spherical cell with a very thin surface layer against which turgor pressure is exerted, the relationship between turgor pressure and the imposed stress (S) would be

(9)

where r is the cell radius and h is the thickness of the surface layer. The surface layers of many bacteria are quite thick in relation to their radii of curvature, however, and for nonspherical cells, surface stress varies as a function of both location and direction along the cell surface in a manner which cannot be simply modeled (274).

Like those of phospholipid membranes, the mechanical properties of cell surfaces may in principle be described in terms of both their stiffness (or viscosity) and their elastic moduli (their ability to undergo various reversible deformations). For example, bacterial murein layers are both stiffer and more elastic than cytoplasmic membranes (as discussed by Thwaites and Mendelson [274]). For a spherical cell with a single, thin, elastic surface layer, the relationship between surface stress and strain would be

(10)

where k is a modulus describing the elasticity of the cell surface material and A/A is the strain, i.e., the proportional change in the cell surface area in response to the imposed stress. In the simplest case, k would be constant regardless of the position on the cell surface, orientation on the cell surface, or magnitude of the applied stress.

Equations 9 and 10 can be combined to relate cell surface strain (A/A) and the stress imposed by turgor pressure (P) for a spherical cell:

(11)

The following points emerge from this relationship. (i) The surfaces of larger cells experience greater strain than do those of smaller cells for a given stress (turgor pressure [P]), elastic modulus (k), and surface layer thickness (h). (ii) Thicker cell surfaces expand and contract less (have lower strain) under a given stress (turgor pressure [P]) than do thinner surfaces for a given elastic modulus (k) and cell size (r). (iii) In cells with elastic surfaces (small k), changes in extracellular osmolality may be accommodated by both water fluxes and adjustments in cell surface area. The time courses of solvent flux and of structural change after an osmotic perturbation are then determined by P_f, _i, k, and viscosity coefficients which determine the rates at which structural changes can occur.

By using a microscopic pressure probe to measure and manipulate turgor pressure, osmotic responses and stress-strain relationships have been examined for large cells of plants and eukaryotic microbes. Such analyses suggest that for those systems at least, _i, P_f, and k can vary with turgor pressure (89, 200, 210). Cellular properties such as surface stress and elastic modulus cannot readily be deduced from measurements of turgor pressure, osmotic gradients, and cell dimensions for nonspherical cells or for cells in which the elastic modulus varies with position on the cell surface, orientation on the cell surface, or magnitude of the applied stress. Since bacterial turgor pressure cannot readily be manipulated or measured (see Appendix), current prospects for quantifying surface stress and strain in whole bacteria are poor. However, pertinent information can be obtained by examining the mechanical properties of cellular constituents (see, e.g., reference 274).

The relationships outlined above suggest that the kinetics of osmotically induced solvent flux, of associated structural and biochemical changes, and hence of the time constants required of osmosensory and osmoregulatory processes can be adjusted by remodeling the cell surface to alter its reflection coefficient (_i) and osmotic permeability coefficient (P_f) or its mechanical properties (its stiffness and elastic moduli). For example, reducing the reflection coefficient for a cosolvent would facilitate its rapid equilibration across the cytoplasmic membrane, reducing or eliminating osmotically induced water flux. For a benign cosolvent (e.g., glycerol), such equilibration may be more favorable to cellular survival and growth than cosolvent exclusion. Reduction of the osmotic permeability coefficient (P_f) would slow cellular dehydration in response to osmotic upshifts. Adjustment of surface stiffness and elasticity, either in general or within local cell surface regions, would modulate the degree to which swelling, shrinkage, and shape changes occur in response to extracellular osmolality changes.

Passive structural responses of bacteria to osmolality changes. How are the structures of bacterial cells influenced by extracellular osmolality changes? Measurements of many cellular properties are pertinent to this topic. They include turgor pressure; cellular, cytoplasmic, and periplasmic volume (cell partitioning); cell density (not population density); cell size, shape and ultrastructure; and the mechanical properties of cell surface layers. Relevant techniques are discussed in the Appendix. Some bacteria have been shown to maintain turgor pressure (references 45 and 294 and references cited therein). For example, the turgor pressure for E. coli cells cultivated in standard, defined media is believed to be 3 to 5 atm. Although the mechanical properties of bacteria have not been fully analyzed (for reasons discussed above), studies of osmotic effects on cell size and hydration offer some insight into their osmotic relations.

Cell surface modifications and osmosensing. Does structural remodeling of the cell surface influence the osmotic stress response? Genetic screening has revealed genes encoding E. coli cell surface molecules whose expression is modulated by extracellular osmolality (Table 3). In most cases, steady-state levels of these elements have been reported but their influence on the kinetics of the osmotic stress response has not.