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PROKARYOTIC OSMOREGULATION:
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Genetics and Physiology Laszlo N. Csonka Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907
Andrew D. Hanson MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing Michigan 48824-1312; Institut de Recherche en Biologie Vegetale, 4101 Rue Sher
brooke Est, Montreal, Quebec HIX 2B2, Canada KEY WORDS:
osmotic control of gene expression, compatible solutes, turgor regulation, volume regulation, g rowth in high osmolality
CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
570
MECHANISMS UNDERLYING OSMOREGULATORY RESPONSES.... . . .. . . . . . . . .... Homeostasis vs Adaptation to Change. . . . . . . . . . . .... . . . . . . . . . . . . . . . . . ........................ Examination of Possible Signals . . . . . .. .... . . . . . . . . . :..........................................
571
PHYSIOLOGY OF OSMOTIC REGULATION . . . . .... . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . .. . Compatible Solutes Synthesized and/or Transported . . . . . . . . . . . . . . . . . ...................... Osmoregulation of the Periplasm . . . . . . . . ... . . . . . . . .... . . . . . . . ..... . . . . . . . .. . . . . ...............
581 581 588
GENES AND PROTEINS . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . The EnvZ/OmpR-Dependent Expression of the ompF and ompC Genes of E. coli K-12 .. . . . . . . . . .... . . . . . . ...... . . . . . . ...... . . . . . . . .... . . . ...................... The k dpABC operon of E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The proU Operon of E. coli and S. typhimurium . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . Other Osmoregulated Genes. . ................................... ...................... .........
588
571
573
589 592 593 597
Sed quis custodiet ipsos custodes [But who is to guard the guards themselves?] Decimus Junius Juvenal, Satires VI, 347
569
0066-4227/9 1 / 1001 -0569$02. 00
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Annu. Rev. Microbiol. 1991.45:569-606. Downloaded from www.annualreviews.org by Ball State University on 04/24/13. For personal use only.
INTRODUCTION The membranes that encompass cells are readily permeable to water but present a more effective barrier to most other solutes . Therefore, when the external concentration (strictly speaking, activity) of water changes because of increases or decreases in the concentrations of extracellular solutes that are excluded by the membrane, water moves out of or into the cells . This movement of water results in changes in the cellular volume (and hence, intracellular solute concentration) and/or pressure. Cells can carry out adap tive processes to restore these parameters to acceptable values. Because changes in the extracellular osmolality have the same physicochemical effects on cells from all biological kingdoms, the responses to osmotic shifts have considerable similarities in all organisms. In eukaryotes, the main ex perimental approaches with osmoregulation have, necessarily, been physi ological ones. Prokaryotic osmoregulation, however , is amenable to bio chemical-genetic analysis, making bacteria excellent model organisms for studying osmoregulation as a basic biological process. Current interest in bacterial osmoregulation is very strong. Various aspects of this topic have been covered in several recent reviews (19 , 44, 95 , 1 33) and in a book by Brown (28) , and new findings continually emerge. The study of osmoregulation has important applications in food microbiology (161, 1 68), plant-microbe interactions (32, 54, 55, 204), and medical microbiology ( 1 6, 1 7 , 35 , 36, 49, 50, 7 1 ) . Although recent interest has generated a great deal of information about the physiological and genetic responses to the osmolality of the environment in both bacteria and higher organisms, the signals that regulate these responses are understood poorly. This review discusses con cepts underlying the perception of osmoregulatory signals. The reader can consult the book by Brown (28) for a different treatment of this topic . We also summarize recent results in osmoregulation in mesophilic bacteria not dealt with in an earlier review (44) , but we do not cover in detail osmoregulation in the extreme halophilic bacteria, which has been reviewed by Brown (28) and Vreeland (195a). In this review, osmoregulation refers to active processes carried out during adaptation to the osmotic strength of the environment. There are two types of osmoregulatory phenomena: long-term or steady-state responses that are manifested during the growth of organisms at a constant osmolality, and short-term or transient responses that occur soon after changes in the external osmolality . By hyperosmotic shock, or osmotic shiftup, we mean an increase in the external osmolality, and by hypoosmotic shock or osmotic shiftdown, the opposite. Interestingly , all of the studies of osmoregulation have involved sudden shifts in osmolality; much information might be derived from gradual shift experiments .
BACTERIAL OSMOREGULATION
571
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MECHANISMS UNDERLYING OSMOREGULATORY RESPONSES The regulation of most biological responses depends on the recognition of signal molecules by specific receptors (for example, the induction of the lac operon by lactose). However, osmoregulation differs in that the information from the environment is not a specific molecule but a physicochemical parameter: the water activity of the exterior ( 1 1 1) . Regulation by physical rather than chemical signals occurs in only a few other systems in bacteria, such as the regulation of expression of genes by pressure (14), viscosity ( 1 26) , and temperature (138). In all these cases, very little concrete informa tion on the signal transduction is available. This section concerns the physical and chemical parameters that change in an osmotically stressed cell and how these may be used as signals to trigger osmoregulatory responses. The question of the signals for osmoregulation and allied responses is common to the physiology of prokaryotes and eukaryotes and has prompted work on animals, higher plants, algae, and fungi (28 , 40, 77, 200) . Accordingly, we first note some useful generalizations about osmoregula tion and cellular homeostatic mechanisms that have emerged from work on these organisms and point out a conceptual difficulty that bedevils studies of osmoregulation. We then give a brief classical treatment of the physicochemi cal changes in osmotically stressed cells, drawing on comparative physiology. In the final section, we consider the evidence for the response of specific bacterial systems to some of these changes.
Homeostasis vs Adaptation to Change For active metabolism to occur, the intracellular milieu of any organism must remain relatively constant with respect to ionic composition, pH, and metabo lite levels; comparative work shows the limits to be strikingly similar among most species ( 175) . These requirements, particularly that for an intracellular K+ concentration of 100-1 50 ruM (40, 1 75) , set a minimum cytoplasmic osmolality of approximately 250 mosmol/kg (osmotic pressure 0.6 MPa). Thus, in media of low osmolality, those homeostatic mechanisms that keep the ion concentration, pH, and metabolite levels of the cytoplasm within the required limits dominate in the maintenance of intracellular osmolality (28, 1 99) . Therefore, osmoregulation is inseparable from general ionic and metabolic regulation, i.e. cytoplasmic osmolality is controlled by a diverse set of homeostatic feedback mechanisms that respond to various intracellular signals. Upon transfer to hyperosmotic medium, osmotic adaptation to change is required. The metabolism-imposed limits referred to above constrain the
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CSONKA & HANSON
extent to which most metabolites and inorganic ions can be used to fill the role of osmoregulatory solute (199, 202). Thus, following a shiftup in medium osmolality, accumulation of specialized osmolytes that are nontoxic ("com patible") at high concentrations is required (see below) (29, 1 99 , 202). Two general points about these adaptation responses are important. First, although the triggering of such responses at the level of gene expression or protein activity can be dramatic, it may be quite simple mechanistically-say the transient lifting of a negative control--compared to the homeostatic mech anisms that maintain the steady state (28). Second, the short-term responses to a shiftup in external osmolality must eventually be re-engaged with these homeostatic mechanisms to achieve the maintenance of a new osmotic steady state (40) . Summarizing, comparative physiology leads to the ideas that osmoregulatory mechanisms are inextricably linked to other cellular homeostatic mechanisms and that they are therefore likely to respond to more than one cellular signal. These generalizations are borne out by recent work in bacteria, as we shall see. Only a few studies have examined the transient responses to osmotic shifts in bacteria (8,9, 48, 97, 147, 158), perhaps largely for technical reasons. In these organisms, adaptation to osmotic shifts occurs in a much shorter time frame than subsequent exponential growth in the media of the new osmolality, so that it is simpler to study the long-term responses after the cells have completed osmotic adaptation than the transient responses that occur during the seconds or minutes required for adaptation to a new osmolality. However, our understanding of even the long-term osmoregulatory signals is un satisfactory. There is a conceptual difficulty in the long-term regulation of an osmoadap tive process in cells that are in complete osmotic equilibrium with their environment. We illustrate this with the ProP transport system of Escherichia coli. but it should be emphasized that all the other long-term osmotically regulated phenomena entail the same conceptual problem. The ProP system, which is a permease for proline and glycine betaine, is synthesized at a nearly constitutive level, but its activity is stimulated in cells growing exponentially in media of high osmolality (3 1 , 5 3 , 131), Interestingly, stimulation of the ProP system by high osmolality can be reproduced in vitro with membrane vesicles ( 1 0 1 , 13 1 ) . Because such vesicles can withstand only very small pressures, less than 1 % of the turgor pressure in whole cells ( 1 43), this observation suggests that turgor pressure is probably not the regulator of the activity of the ProP system. The signal is more likely to be membrane tension or some related parameter, which we address in more detail in the next section. In whole cells, the accumulation of compatible solutes (carried out in part by the ProP system itself) restores pressure and membrane tension to values very similar to those before the osmotic upshift, but nevertheless the
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BACTERIAL OSMOREGULATION
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ProP system remains in its activated state after the cells have completed osmotic adjustment. While one can envision that some change in membrane structure upon the loss of tension, such as an alteration in the contact of the ProP transport protein with lipids, might result in transient stimulation of the activity of this permease both in whole cells and in cell-free vesicles, it is more difficult to imagine how the ProP system could remain activated after the membrane has returned to its original condition as a result of osmotic adaptation. An additional regulator that responds to some persistent osmoreg ulatory signal is needed to explain the maintenance of the ProP system in its active form; Figure 1 schematically shows this control as an effector molecule that locks the proline porter in its active conformation. But herein lies the conceptual difficulty: whatever the signal is for the long-term regulation of the ProP system, it must itself be subject to regulation by the osmolality of the medium. A unifying hypothesis proposed that the primary osmoregulatory mech anism is a homeostatic control circuit that maintains turgor within a range that can support cell growth (19, 59). Self-regulation of turgor was postulated to result from the regulation of the intracellular K+ concentration, such that a drop in turgor pressure below a critical value would trigger increased uptake of K + and its excessive buildup would stimulate the excretion of this cation. A second part of the model proposed that the concentration of K + is the regulatory signal for all the other osmoregulatory responses, including the stimulation of the steady state activity of the ProP system in cells growing in media of high osmolality. However, the increase in the intracellular concen tration of K+ with increasing external osmolality is also one of the long-term osmoregulatory responses that persists after the turgor has been restored near or equal to its value prior to the osmotic shift. There is one additional level of complexity. Turgor has been proposed to regulate the rate of cell elongation (108). Because the growth rate of cells depends on other factors besides osmolality, such as the carbon or nitrogen source, temperature, etc, K + accumulation must also be regulated by growth rate (18Ia). Consequently, additional tiers of control must be invoked, deferring the question of what is the osmoregulatory signal. This deferral prompted the choice the quote from Juvenal as the theme for this review. ,
Examination of Possible Signals Figures 2 and 3 illustrate some physicochemical changes that might be used by gram-negative bacteria for osmoregulatory responses; they are examined below. In this discussion, we make the simplifying assumption that the physicochemical changes schematized in these two figures occur very rapidly after hyperosmotic shock, before any active processes of osmotic adaptation are initiated by the cell.
574
CSONKA & HANSON
h::){;" l--_�,_UT_ ... S � coo-
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Annu. Rev. Microbiol. 1991.45:569-606. Downloaded from www.annualreviews.org by Ball State University on 04/24/13. For personal use only.
Intracellular effector molecule
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Figure 1
A scheme based on the acti vation of the ProP proline transport system by a
h yp erosmotic shift. It illustrates how a change in membran e stretch mi ght switch on a process,
and h ow this process mi ght be m aintained by an oth er, p ersist ent signal. The other signal in this scheme i s an effector molecul e whose abi lity to bind t o the proline porter dep ends on hi gh
osmolality. (Frame 1) The membrane is in an expanded (stretched) state and causes the proline porter to adop t a c onformation in which it is inactive. (Frame 2) The membrane relaxes following
BACTERIAL OSMOREGULATION ISOTROPIC PRESSURE
575
Within the bulk liquid of the cell interior, changes in
hydrostatic pressure are isotropic (i.e. they are of uniform magnitude in all directions). As shown in Figure 3 , these changes are $ 0.5 MPa, which is so low compared with pressures having marked direct effects on biological processes that researchers view intracellular baroreceptors (Figure 2A) as unlikely in relation to osmoregulation (40, 9 1) . Nevertheless, small changes in hydrostatic pressure «
1 MPa) can bring about measurable changes in
protein-protein and protein-ligand interactions (88), and these might be ampli
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fied by cooperativity (l09). It is therefore interesting that in the barophilic gram-negative bacterium SS9, the ompH gene is substantially induced by a hydrostatic pressure of only 7 MPa (14), as this implies that the threshold pressure for response may be much lower. Overall, isotropic pressure changes may deserve some investigation as potential signals, for which pressure chambers should prove useful tools. PRESSURE D IFFERENTIAL ACROSS THE INNER M E M BRANE-PEPTIDOGLY CAN COMPLEX
In this case, the pressure is anisotropic, acting normal to the
wall. We refer to it as turgor pressure. It could be detected by a pressure (turgor) sensor located in the inner membrane, which responds to compres sion against the peptidoglycan sacculus (Figure 2B). The large tangential forces that develop within the wall to contain or oppose turgor pressure could also be used for its detection (70) (Figure 2C), as has been proposed for a peptidoglycan cleavage enzyme sensitive to bond angles in the substrate
(l08). Hydrostatic pressure acting outwards against the wall has been pro posed to be fundamental to cell growth and rigidity in bacteria, plants, and algae (40, 91, 108), and so appears at first sight an attractive candidate as a signal in osmoregulation. Nevertheless, wall-less algae (40), many naked animal cells (77 , 110), and vacuoles within plant cells (86) can also osmoreg ulate to control their volume in anisotonic media. Furthermore, as we discuss below, in gram-negative bacteria, the cytoplasmic membrane may not be in extensive contact with the peptidoglycan sacculus (106, 178). Therefore, one should keep in mind other possible signals when interpreting responses that apparently correlate with turgor changes. We now raise some relevant con siderations.
a
h yperosomotic shift; the proline porter assumes a new confi guration that imparts increased
transport activity and allows the intracellular effe ctor m olecule to bind to it. (Frame 3) The membrane retums to its original le ve l of stretch as osmoregulation restore s normal cell volume, but the porter resists the stretch because it is locked int o the active configuration by the bound
effector. The effector remains boun d a s long a s the cells are ke pt in the medium of h igh osmolality. (Frame 4) On return t o medium of low osmolality, the effector di ssociates from the porter, and the stretched membrane forces the porter back into the inactive conformation.
576
CSONKA & HANSON
Annu. Rev. Microbiol. 1991.45:569-606. Downloaded from www.annualreviews.org by Ball State University on 04/24/13. For personal use only.
.
t [Solute)
o
n
Figure 2
Inner membrane
E· '
•
Ow
Some physicochemical parameters that change in a gram-negative bacterial cell
subjected to an osmotic upshock, and possible sensors of such changes. (A) Hydrostatic pressure in the bulk liquid phase, detected by a baroreceptor. (B) Pressure differential across the inner membrane-peptidoglycan complex, detected by a turgor pressure sensor. (C) Tangential stress within the peptidoglycan layer, sensed by a wall stretch receptor. (D) Membrane surface area, sensed by
a
stretch receptor. (E, and Eo) Internal and external solute concentration or water
activity, sensed by chemoreeeptors.
The first of these concerns the relationship between turgor pressure and cellular volume, which according to the analogy of Brown (28), are as inseparable as space and time in relativity theory. We consider two situations: a hypothetical one in which the peptidoglycan sacculus is completely rigid (Figure 3, lines labeled R), and a second, more realistic one, in which the sacculus is elastic (Figure 3 , lines labeled E) so that it can shrink and swell along with the cytoplasm upon osmotic shifts . In the first case, the decline in pressure upon a hyperosmotic shock would be proportional to external osmo lality; the cytoplasmic volume, internal osmolality and membrane area would change very little until turgor fell to zero (Figure 3A, inset). In this scenario, turgor is the only one of these parameters that could be used to sense a modest shiftup « 0.5 MPa) in medium osmolality. Although previous treatments of osmoregulation in bacteria generally assumed that the sacculus is rigid (44, 196), several lines of evidence (see Figure 3 legend) indicate that this is not the case, and the sacculus may perhaps reach a state of zero tension only when more than half the cell water has been withdrawn. In this situation, turgor pressure would decline less steeply as the peptidoglycan shrank in step with the cytoplasm, with the relationship between the cellular volume and the external osmolality set by the volumetric elastic modulus of the sacculus. For simplicity in Figure 3 (lines labeled E), the volumetric elastic modulus of the sacculus is assumed to be constant, although it seems likely to increase as maximum cell volume is approached, as occurs in algal and plant cell walls (40). If the peptidoglycan sacculus is elastic, turgor will decline gradually
Annu. Rev. Microbiol. 1991.45:569-606. Downloaded from www.annualreviews.org by Ball State University on 04/24/13. For personal use only.
BACTERIAL OSMOREGULAnON
577
with increasing external osmolality (Figure 3A), and cytoplasmic volume, the concentration of internal solutes (Figure 3B), and membrane area (Figure 3C) will all change as soon as medium osmolality is raised. Contrast this with the buffering effect that a rigid sacculus has on these parameters. The point here is that if-as is most probably the case-the sacculus is elastic, then appreci able changes in internal solute concentration and membrane area will occur as well and at the same time as a decline in turgor, so that the cell might monitor three or more types of signals simultaneously . Complexities also arise from the osmotic regulation of the periplasmic space in gram-negative bacteria. The periplasm contains anionic polysaccha rides that generate a Donnan potential across the outer membrane ( 1 07, 1 78; see section on osmoregulation of the periplasm) . Hydrostatic pressure is generated in the periplasm by the accumulation of counterions (e.g. K + , Na+) for anionic polysaccharides, and it is presumably resisted externally by the wall on one side and the cytoplasm on the other. Perhaps one way to visualize this difficult concept is that the isotonic Donnan space in the periplasm behaves as an incompressible gel surrounding the cytoplasm ( 1 06) . The presence of such a space would not change the hydrostatic pressure exerted on the wall, but it would mean that the main pressure differential would not be at the inner membrane-peptidoglycan interface, but rather across the whole inner membrane-periplasm-outer membrane complex . As a corollary, the pressure in the periplasm must be nearly equal to the pressure in the cytoplasm ( 1 78), except for an insignificant pressure differential of -0 .003 MPa that can be supported by the lipid bilayer itself ( 143). If osmotic signal-sensing proteins are localized in the portions of the cytoplasmic membrane that contact the periplasm (as has been proposed for the transcriptional regulatory proteins EnvZ and KdpE; see below), then whether such proteins could indeed sense turgor pressure is questionable. However, our understanding of the structure of the periplasm is very tenuous, especially concerning whether the cytoplas mic membrane is in contact with the peptidoglycan sacculus over much (25 , 64) or over very little of its surface area ( 106) . INTERNAL SOLUTE LEVEL OR WATER ACTIVITY
As noted above, in cells with elastic walls, cytoplasmic volume would change with external osmolality as a continuous function, but in cells with rigid walls, it would decrease only after turgor is lost completely. In either case, once turgor is lost the cytoplasmic compartment follows the Boyle-Van't Hoff law, behaving as an osmometer ( 1 32) . Thus, in principle, either the cytoplasmic volume itself, the accompanying changes in the concentration of one or more solutes, or the activity (concentration) of water could serve as signals. These are the parame ters available as osmoregulatory signals to wall-less algal and animal cells, and evidence indicates sensing of the first two quantities in animals.
CSONKA & HANSON
578
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Further
Quick links to online content Annu. Rev. Microbiol. 1991.45:569-606 Copyright © 1991 by Annual Reviews Inc. All rights reserved
PROKARYOTIC OSMOREGULATION:
Annu. Rev. Microbiol. 1991.45:569-606. Downloaded from www.annualreviews.org by Ball State University on 04/24/13. For personal use only.
Genetics and Physiology Laszlo N. Csonka Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907
Andrew D. Hanson MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing Michigan 48824-1312; Institut de Recherche en Biologie Vegetale, 4101 Rue Sher
brooke Est, Montreal, Quebec HIX 2B2, Canada KEY WORDS:
osmotic control of gene expression, compatible solutes, turgor regulation, volume regulation, g rowth in high osmolality
CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
570
MECHANISMS UNDERLYING OSMOREGULATORY RESPONSES.... . . .. . . . . . . . .... Homeostasis vs Adaptation to Change. . . . . . . . . . . .... . . . . . . . . . . . . . . . . . ........................ Examination of Possible Signals . . . . . .. .... . . . . . . . . . :..........................................
571
PHYSIOLOGY OF OSMOTIC REGULATION . . . . .... . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . .. . Compatible Solutes Synthesized and/or Transported . . . . . . . . . . . . . . . . . ...................... Osmoregulation of the Periplasm . . . . . . . . ... . . . . . . . .... . . . . . . . ..... . . . . . . . .. . . . . ...............
581 581 588
GENES AND PROTEINS . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . The EnvZ/OmpR-Dependent Expression of the ompF and ompC Genes of E. coli K-12 .. . . . . . . . . .... . . . . . . ...... . . . . . . ...... . . . . . . . .... . . . ...................... The k dpABC operon of E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The proU Operon of E. coli and S. typhimurium . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . Other Osmoregulated Genes. . ................................... ...................... .........
588
571
573
589 592 593 597
Sed quis custodiet ipsos custodes [But who is to guard the guards themselves?] Decimus Junius Juvenal, Satires VI, 347
569
0066-4227/9 1 / 1001 -0569$02. 00
570
CSONKA & HANSON
Annu. Rev. Microbiol. 1991.45:569-606. Downloaded from www.annualreviews.org by Ball State University on 04/24/13. For personal use only.
INTRODUCTION The membranes that encompass cells are readily permeable to water but present a more effective barrier to most other solutes . Therefore, when the external concentration (strictly speaking, activity) of water changes because of increases or decreases in the concentrations of extracellular solutes that are excluded by the membrane, water moves out of or into the cells . This movement of water results in changes in the cellular volume (and hence, intracellular solute concentration) and/or pressure. Cells can carry out adap tive processes to restore these parameters to acceptable values. Because changes in the extracellular osmolality have the same physicochemical effects on cells from all biological kingdoms, the responses to osmotic shifts have considerable similarities in all organisms. In eukaryotes, the main ex perimental approaches with osmoregulation have, necessarily, been physi ological ones. Prokaryotic osmoregulation, however , is amenable to bio chemical-genetic analysis, making bacteria excellent model organisms for studying osmoregulation as a basic biological process. Current interest in bacterial osmoregulation is very strong. Various aspects of this topic have been covered in several recent reviews (19 , 44, 95 , 1 33) and in a book by Brown (28) , and new findings continually emerge. The study of osmoregulation has important applications in food microbiology (161, 1 68), plant-microbe interactions (32, 54, 55, 204), and medical microbiology ( 1 6, 1 7 , 35 , 36, 49, 50, 7 1 ) . Although recent interest has generated a great deal of information about the physiological and genetic responses to the osmolality of the environment in both bacteria and higher organisms, the signals that regulate these responses are understood poorly. This review discusses con cepts underlying the perception of osmoregulatory signals. The reader can consult the book by Brown (28) for a different treatment of this topic . We also summarize recent results in osmoregulation in mesophilic bacteria not dealt with in an earlier review (44) , but we do not cover in detail osmoregulation in the extreme halophilic bacteria, which has been reviewed by Brown (28) and Vreeland (195a). In this review, osmoregulation refers to active processes carried out during adaptation to the osmotic strength of the environment. There are two types of osmoregulatory phenomena: long-term or steady-state responses that are manifested during the growth of organisms at a constant osmolality, and short-term or transient responses that occur soon after changes in the external osmolality . By hyperosmotic shock, or osmotic shiftup, we mean an increase in the external osmolality, and by hypoosmotic shock or osmotic shiftdown, the opposite. Interestingly , all of the studies of osmoregulation have involved sudden shifts in osmolality; much information might be derived from gradual shift experiments .
BACTERIAL OSMOREGULATION
571
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MECHANISMS UNDERLYING OSMOREGULATORY RESPONSES The regulation of most biological responses depends on the recognition of signal molecules by specific receptors (for example, the induction of the lac operon by lactose). However, osmoregulation differs in that the information from the environment is not a specific molecule but a physicochemical parameter: the water activity of the exterior ( 1 1 1) . Regulation by physical rather than chemical signals occurs in only a few other systems in bacteria, such as the regulation of expression of genes by pressure (14), viscosity ( 1 26) , and temperature (138). In all these cases, very little concrete informa tion on the signal transduction is available. This section concerns the physical and chemical parameters that change in an osmotically stressed cell and how these may be used as signals to trigger osmoregulatory responses. The question of the signals for osmoregulation and allied responses is common to the physiology of prokaryotes and eukaryotes and has prompted work on animals, higher plants, algae, and fungi (28 , 40, 77, 200) . Accordingly, we first note some useful generalizations about osmoregula tion and cellular homeostatic mechanisms that have emerged from work on these organisms and point out a conceptual difficulty that bedevils studies of osmoregulation. We then give a brief classical treatment of the physicochemi cal changes in osmotically stressed cells, drawing on comparative physiology. In the final section, we consider the evidence for the response of specific bacterial systems to some of these changes.
Homeostasis vs Adaptation to Change For active metabolism to occur, the intracellular milieu of any organism must remain relatively constant with respect to ionic composition, pH, and metabo lite levels; comparative work shows the limits to be strikingly similar among most species ( 175) . These requirements, particularly that for an intracellular K+ concentration of 100-1 50 ruM (40, 1 75) , set a minimum cytoplasmic osmolality of approximately 250 mosmol/kg (osmotic pressure 0.6 MPa). Thus, in media of low osmolality, those homeostatic mechanisms that keep the ion concentration, pH, and metabolite levels of the cytoplasm within the required limits dominate in the maintenance of intracellular osmolality (28, 1 99) . Therefore, osmoregulation is inseparable from general ionic and metabolic regulation, i.e. cytoplasmic osmolality is controlled by a diverse set of homeostatic feedback mechanisms that respond to various intracellular signals. Upon transfer to hyperosmotic medium, osmotic adaptation to change is required. The metabolism-imposed limits referred to above constrain the
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extent to which most metabolites and inorganic ions can be used to fill the role of osmoregulatory solute (199, 202). Thus, following a shiftup in medium osmolality, accumulation of specialized osmolytes that are nontoxic ("com patible") at high concentrations is required (see below) (29, 1 99 , 202). Two general points about these adaptation responses are important. First, although the triggering of such responses at the level of gene expression or protein activity can be dramatic, it may be quite simple mechanistically-say the transient lifting of a negative control--compared to the homeostatic mech anisms that maintain the steady state (28). Second, the short-term responses to a shiftup in external osmolality must eventually be re-engaged with these homeostatic mechanisms to achieve the maintenance of a new osmotic steady state (40) . Summarizing, comparative physiology leads to the ideas that osmoregulatory mechanisms are inextricably linked to other cellular homeostatic mechanisms and that they are therefore likely to respond to more than one cellular signal. These generalizations are borne out by recent work in bacteria, as we shall see. Only a few studies have examined the transient responses to osmotic shifts in bacteria (8,9, 48, 97, 147, 158), perhaps largely for technical reasons. In these organisms, adaptation to osmotic shifts occurs in a much shorter time frame than subsequent exponential growth in the media of the new osmolality, so that it is simpler to study the long-term responses after the cells have completed osmotic adaptation than the transient responses that occur during the seconds or minutes required for adaptation to a new osmolality. However, our understanding of even the long-term osmoregulatory signals is un satisfactory. There is a conceptual difficulty in the long-term regulation of an osmoadap tive process in cells that are in complete osmotic equilibrium with their environment. We illustrate this with the ProP transport system of Escherichia coli. but it should be emphasized that all the other long-term osmotically regulated phenomena entail the same conceptual problem. The ProP system, which is a permease for proline and glycine betaine, is synthesized at a nearly constitutive level, but its activity is stimulated in cells growing exponentially in media of high osmolality (3 1 , 5 3 , 131), Interestingly, stimulation of the ProP system by high osmolality can be reproduced in vitro with membrane vesicles ( 1 0 1 , 13 1 ) . Because such vesicles can withstand only very small pressures, less than 1 % of the turgor pressure in whole cells ( 1 43), this observation suggests that turgor pressure is probably not the regulator of the activity of the ProP system. The signal is more likely to be membrane tension or some related parameter, which we address in more detail in the next section. In whole cells, the accumulation of compatible solutes (carried out in part by the ProP system itself) restores pressure and membrane tension to values very similar to those before the osmotic upshift, but nevertheless the
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BACTERIAL OSMOREGULATION
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ProP system remains in its activated state after the cells have completed osmotic adjustment. While one can envision that some change in membrane structure upon the loss of tension, such as an alteration in the contact of the ProP transport protein with lipids, might result in transient stimulation of the activity of this permease both in whole cells and in cell-free vesicles, it is more difficult to imagine how the ProP system could remain activated after the membrane has returned to its original condition as a result of osmotic adaptation. An additional regulator that responds to some persistent osmoreg ulatory signal is needed to explain the maintenance of the ProP system in its active form; Figure 1 schematically shows this control as an effector molecule that locks the proline porter in its active conformation. But herein lies the conceptual difficulty: whatever the signal is for the long-term regulation of the ProP system, it must itself be subject to regulation by the osmolality of the medium. A unifying hypothesis proposed that the primary osmoregulatory mech anism is a homeostatic control circuit that maintains turgor within a range that can support cell growth (19, 59). Self-regulation of turgor was postulated to result from the regulation of the intracellular K+ concentration, such that a drop in turgor pressure below a critical value would trigger increased uptake of K + and its excessive buildup would stimulate the excretion of this cation. A second part of the model proposed that the concentration of K + is the regulatory signal for all the other osmoregulatory responses, including the stimulation of the steady state activity of the ProP system in cells growing in media of high osmolality. However, the increase in the intracellular concen tration of K+ with increasing external osmolality is also one of the long-term osmoregulatory responses that persists after the turgor has been restored near or equal to its value prior to the osmotic shift. There is one additional level of complexity. Turgor has been proposed to regulate the rate of cell elongation (108). Because the growth rate of cells depends on other factors besides osmolality, such as the carbon or nitrogen source, temperature, etc, K + accumulation must also be regulated by growth rate (18Ia). Consequently, additional tiers of control must be invoked, deferring the question of what is the osmoregulatory signal. This deferral prompted the choice the quote from Juvenal as the theme for this review. ,
Examination of Possible Signals Figures 2 and 3 illustrate some physicochemical changes that might be used by gram-negative bacteria for osmoregulatory responses; they are examined below. In this discussion, we make the simplifying assumption that the physicochemical changes schematized in these two figures occur very rapidly after hyperosmotic shock, before any active processes of osmotic adaptation are initiated by the cell.
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Figure 1
A scheme based on the acti vation of the ProP proline transport system by a
h yp erosmotic shift. It illustrates how a change in membran e stretch mi ght switch on a process,
and h ow this process mi ght be m aintained by an oth er, p ersist ent signal. The other signal in this scheme i s an effector molecul e whose abi lity to bind t o the proline porter dep ends on hi gh
osmolality. (Frame 1) The membrane is in an expanded (stretched) state and causes the proline porter to adop t a c onformation in which it is inactive. (Frame 2) The membrane relaxes following
BACTERIAL OSMOREGULATION ISOTROPIC PRESSURE
575
Within the bulk liquid of the cell interior, changes in
hydrostatic pressure are isotropic (i.e. they are of uniform magnitude in all directions). As shown in Figure 3 , these changes are $ 0.5 MPa, which is so low compared with pressures having marked direct effects on biological processes that researchers view intracellular baroreceptors (Figure 2A) as unlikely in relation to osmoregulation (40, 9 1) . Nevertheless, small changes in hydrostatic pressure «
1 MPa) can bring about measurable changes in
protein-protein and protein-ligand interactions (88), and these might be ampli
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fied by cooperativity (l09). It is therefore interesting that in the barophilic gram-negative bacterium SS9, the ompH gene is substantially induced by a hydrostatic pressure of only 7 MPa (14), as this implies that the threshold pressure for response may be much lower. Overall, isotropic pressure changes may deserve some investigation as potential signals, for which pressure chambers should prove useful tools. PRESSURE D IFFERENTIAL ACROSS THE INNER M E M BRANE-PEPTIDOGLY CAN COMPLEX
In this case, the pressure is anisotropic, acting normal to the
wall. We refer to it as turgor pressure. It could be detected by a pressure (turgor) sensor located in the inner membrane, which responds to compres sion against the peptidoglycan sacculus (Figure 2B). The large tangential forces that develop within the wall to contain or oppose turgor pressure could also be used for its detection (70) (Figure 2C), as has been proposed for a peptidoglycan cleavage enzyme sensitive to bond angles in the substrate
(l08). Hydrostatic pressure acting outwards against the wall has been pro posed to be fundamental to cell growth and rigidity in bacteria, plants, and algae (40, 91, 108), and so appears at first sight an attractive candidate as a signal in osmoregulation. Nevertheless, wall-less algae (40), many naked animal cells (77 , 110), and vacuoles within plant cells (86) can also osmoreg ulate to control their volume in anisotonic media. Furthermore, as we discuss below, in gram-negative bacteria, the cytoplasmic membrane may not be in extensive contact with the peptidoglycan sacculus (106, 178). Therefore, one should keep in mind other possible signals when interpreting responses that apparently correlate with turgor changes. We now raise some relevant con siderations.
a
h yperosomotic shift; the proline porter assumes a new confi guration that imparts increased
transport activity and allows the intracellular effe ctor m olecule to bind to it. (Frame 3) The membrane retums to its original le ve l of stretch as osmoregulation restore s normal cell volume, but the porter resists the stretch because it is locked int o the active configuration by the bound
effector. The effector remains boun d a s long a s the cells are ke pt in the medium of h igh osmolality. (Frame 4) On return t o medium of low osmolality, the effector di ssociates from the porter, and the stretched membrane forces the porter back into the inactive conformation.
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.
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Ow
Some physicochemical parameters that change in a gram-negative bacterial cell
subjected to an osmotic upshock, and possible sensors of such changes. (A) Hydrostatic pressure in the bulk liquid phase, detected by a baroreceptor. (B) Pressure differential across the inner membrane-peptidoglycan complex, detected by a turgor pressure sensor. (C) Tangential stress within the peptidoglycan layer, sensed by a wall stretch receptor. (D) Membrane surface area, sensed by
a
stretch receptor. (E, and Eo) Internal and external solute concentration or water
activity, sensed by chemoreeeptors.
The first of these concerns the relationship between turgor pressure and cellular volume, which according to the analogy of Brown (28), are as inseparable as space and time in relativity theory. We consider two situations: a hypothetical one in which the peptidoglycan sacculus is completely rigid (Figure 3, lines labeled R), and a second, more realistic one, in which the sacculus is elastic (Figure 3 , lines labeled E) so that it can shrink and swell along with the cytoplasm upon osmotic shifts . In the first case, the decline in pressure upon a hyperosmotic shock would be proportional to external osmo lality; the cytoplasmic volume, internal osmolality and membrane area would change very little until turgor fell to zero (Figure 3A, inset). In this scenario, turgor is the only one of these parameters that could be used to sense a modest shiftup « 0.5 MPa) in medium osmolality. Although previous treatments of osmoregulation in bacteria generally assumed that the sacculus is rigid (44, 196), several lines of evidence (see Figure 3 legend) indicate that this is not the case, and the sacculus may perhaps reach a state of zero tension only when more than half the cell water has been withdrawn. In this situation, turgor pressure would decline less steeply as the peptidoglycan shrank in step with the cytoplasm, with the relationship between the cellular volume and the external osmolality set by the volumetric elastic modulus of the sacculus. For simplicity in Figure 3 (lines labeled E), the volumetric elastic modulus of the sacculus is assumed to be constant, although it seems likely to increase as maximum cell volume is approached, as occurs in algal and plant cell walls (40). If the peptidoglycan sacculus is elastic, turgor will decline gradually
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BACTERIAL OSMOREGULAnON
577
with increasing external osmolality (Figure 3A), and cytoplasmic volume, the concentration of internal solutes (Figure 3B), and membrane area (Figure 3C) will all change as soon as medium osmolality is raised. Contrast this with the buffering effect that a rigid sacculus has on these parameters. The point here is that if-as is most probably the case-the sacculus is elastic, then appreci able changes in internal solute concentration and membrane area will occur as well and at the same time as a decline in turgor, so that the cell might monitor three or more types of signals simultaneously . Complexities also arise from the osmotic regulation of the periplasmic space in gram-negative bacteria. The periplasm contains anionic polysaccha rides that generate a Donnan potential across the outer membrane ( 1 07, 1 78; see section on osmoregulation of the periplasm) . Hydrostatic pressure is generated in the periplasm by the accumulation of counterions (e.g. K + , Na+) for anionic polysaccharides, and it is presumably resisted externally by the wall on one side and the cytoplasm on the other. Perhaps one way to visualize this difficult concept is that the isotonic Donnan space in the periplasm behaves as an incompressible gel surrounding the cytoplasm ( 1 06) . The presence of such a space would not change the hydrostatic pressure exerted on the wall, but it would mean that the main pressure differential would not be at the inner membrane-peptidoglycan interface, but rather across the whole inner membrane-periplasm-outer membrane complex . As a corollary, the pressure in the periplasm must be nearly equal to the pressure in the cytoplasm ( 1 78), except for an insignificant pressure differential of -0 .003 MPa that can be supported by the lipid bilayer itself ( 143). If osmotic signal-sensing proteins are localized in the portions of the cytoplasmic membrane that contact the periplasm (as has been proposed for the transcriptional regulatory proteins EnvZ and KdpE; see below), then whether such proteins could indeed sense turgor pressure is questionable. However, our understanding of the structure of the periplasm is very tenuous, especially concerning whether the cytoplas mic membrane is in contact with the peptidoglycan sacculus over much (25 , 64) or over very little of its surface area ( 106) . INTERNAL SOLUTE LEVEL OR WATER ACTIVITY
As noted above, in cells with elastic walls, cytoplasmic volume would change with external osmolality as a continuous function, but in cells with rigid walls, it would decrease only after turgor is lost completely. In either case, once turgor is lost the cytoplasmic compartment follows the Boyle-Van't Hoff law, behaving as an osmometer ( 1 32) . Thus, in principle, either the cytoplasmic volume itself, the accompanying changes in the concentration of one or more solutes, or the activity (concentration) of water could serve as signals. These are the parame ters available as osmoregulatory signals to wall-less algal and animal cells, and evidence indicates sensing of the first two quantities in animals.
CSONKA & HANSON
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