Osmosensing by Bacteria

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Science's STKE  17 Oct 2006:
Vol. 2006, Issue 357, pp. pe43
DOI: 10.1126/stke.3572006pe43


Osmosensors are proteins that sense environmental osmotic pressure. They mediate or direct osmoregulatory responses that allow cells to survive osmotic changes and extremes. Bacterial osmosensing transporters sense high external osmotic pressure and respond by mediating organic osmolyte uptake, hence cellular rehydration. Detailed studies of osmosensing transporters OpuA, BetP, and ProP suggest that they sense and respond to different osmotic pressure–dependent cellular properties. These studies also suggest that each protein has a cytoplasmic osmosensory or osmoregulatory domain, but that these domains differ in structure and function. It is not yet clear whether each transporter represents a distinct osmosensory mechanism or whether different research groups are approaching the same mechanism by way of different paths. Principles emerging from this research will apply to other osmosensors, including those that initiate signal transduction cascades in prokaryotes and eukaryotes.

Recent articles on transporter OpuA of Lactococcus lactis exemplify insights into osmosensing that are emerging from research on bacterial systems (1, 2). Cellular osmoregulation is a key stress response. Osmosensors, which are proteins that detect osmotic pressure (Π), mediate or direct responses that allow cells to survive transient or sustained changes in environmental osmotic pressure. Like plants, soil bacteria use these mechanisms to survive floods and drought. Like renal cells, bacteria infecting mammalian urinary tracts survive urine concentration and dilution.

As extracellular osmotic pressure rises or falls, transmembrane water fluxes concentrate or dilute the cytoplasm, disrupting cell structure and function. Cells respond by actively adjusting the distributions of selected solutes across the cytoplasmic membrane (3). In bacteria, these solutes include K+ and organic osmolytes that are also protein stabilizers (for instance, polyols such as trehalose, amino acids such as proline, and amino acid derivatives such as glycine betaine) (3, 4). Although K+ is ubiquitous, the organic osmolytes are preferred, perhaps because they more effectively restore cellular hydration (5). Thus, osmoregulatory responses counter imposed water fluxes and mitigate their consequences. Organisms have arrays of osmoregulatory systems that appear to be functionally redundant (Fig. 1).

Fig. 1.

Osmoregulatory systems of E. coli. Aquaporin AqpZ mediates transmembrane water flux. K+ transporters TrkA(G/H)/SapD and KdpFABC mediate K+ accumulation in response to high osmotic pressure. KdpD (a membrane-integral sensor kinase) and KdpE (a cytoplasmic response regulator) constitute a two-component regulatory system that controls kdpFABC transcription in response to K+ supply and osmotic stress. Suppression of glutamate catabolism leads to its accumulation as K+ counterion. Transporters ProP, ProU, BetT, and BetU mediate organic osmolyte accumulation at high osmotic pressure. ProU is an ortholog of OpuA from L. lactis. BetT and BetU are orthologs of BetP from C. glutamicum. Enzymes BetA and BetB mediate glycine betaine synthesis from choline, and enzymes OtsA and OtsB mediate trehalose synthesis from glucose at high osmotic pressure. Mechanosensitive channels MscL and MscS mediate solute efflux in response to decreasing osmotic pressure.

The osmotic pressure of an aqueous solution is proportional to its water activity and is determined by the activities (but not the identities) of all its solutes. Thus, the solutes are also cosolvents, contributing to the osmotic pressure like anonymous members of a collective. To operate like the ligand-specific receptors (chemosensors) that initiate other signal transduction cascades, a direct osmosensor would detect water activity. However, osmotic shifts alter many cellular properties, any of which could be detected by an indirect osmosensor. These include cell volume, turgor pressure, and membrane strain as well as the concentrations of individual solutes, the ionic strength, and the crowding of macromolecules in the cytoplasm (3). Electrolytes contribute collectively to ionic strength and macromolecules contribute collectively to crowding. Mechanosensitive channels are indirect osmosensors that are opened by the increasing membrane strain that occurs when water flows into cells. Solutes then exit, with water in pursuit (6).

Cells faced with dehydration accumulate solutes through synthesis or transport (Fig. 1). Water follows, restoring cellular hydration and volume. The proteins that respond to dehydration by synthesizing or transporting solutes are controlled genetically and biochemically (3). Here, I address the biochemistry of osmosensing and osmoregulation by transporters of organic osmolytes. Principles emerging from the study of these transporters will apply to other osmosensors, including those such as KdpD (Fig. 1) that initiate signal transduction cascades osmoregulating gene expression.

The osmosensing transporters ProP (7), BetP (8), and OpuA (9) have been purified and reconstituted in proteoliposomes (artificial phospholipid vesicles). Each protein represents a different transporter family with a different energy supply (Table 1). Chemically diverse, membrane-impermeant solutes contribute in parallel to the osmotic pressure of the external medium and to the activation of each transporter. Because this occurs in both cells and proteoliposomes, each transporter acts as an osmosensor and osmoregulator in vivo and in vitro. Activation of these osmosensing transporters coincides with the osmotic inactivation of other membrane enzymes such as LacY, the lactose permease of Escherichia coli, that are not osmosensors or osmoregulators (Fig. 2). This makes sense: Osmoregulation is necessary because increasing osmotic pressure affects all cell constituents and impairs the functions of many constituents. Systems such as ProP, BetP, and OpuA can both sense this problem and respond, correcting cellular hydration.

Table 1.

Representative osmosensing transporters.

Fig. 2.

ProP activates as LacY inactivates under osmotic stress. Measurements of the uptake of radiolabeled lactose and radiolabeled proline by intact bacteria (12, 38) and by cytoplasmic membrane vesicles (24) reveal that the osmotic activation of ProP (red line) coincides with the osmotic inactivation of LacY (black line).

What signal(s) do these transporters detect? What are their structural responses to extracellular osmotic pressure? Recent analyses of OpuA, BetP, and ProP by different laboratories suggest that each detects a different osmotic pressure–dependent cellular property (Table 1 and Fig. 3). Each lab ascribes a key role to a cytoplasmic regulatory domain, but the domain structures and the ascribed roles differ. Does each transporter represent a distinct osmosensory mechanism, or is each lab approaching the same mechanism by way of a different path?

Fig. 3.

Models for osmosensing by bacterial osmosensing transporters. Osmosensing by OpuA (A), BetP (B), and ProP (C) is illustrated in the context of a generic model. Details of transporter structure and oligomeric state are provided in Table 1 but not shown here. Each transporter can exist in at least two conformations: (i) inactive, represented by the rectangular proteins in the top row, and (ii) active, represented by the ovoid proteins in the bottom row. The equilibria between these states are influenced by properties of the internal solvent that are determined by the osmotic pressure of the external solvent (Low Π and High Π). Relevant properties of the internal solvent are specified below. Stippling denotes increased external solute concentration and elevated osmotic pressure. Each transporter has a cytoplasmic C-terminal domain, implicated in osmoregulation, that is denoted as a small rectangular or ovoid appendage. (A) Poolman and his colleagues propose that OpuA is an ionic strength sensor (1). OpuA activates when increasing internal ionic strength (shift from light to dark blue) releases C-terminal CBS domains from the membrane surface. (B) Krämer and his colleagues propose that BetP is a K+-specific chemosensor (10, 13). BetP activates when internal K+ is concentrated (increasing number of green dots), altering the conformation and interactions of the C terminus. (C) I propose that ProP senses its own hydration. ProP activates when it is partially dehydrated, retaining water molecules that contribute to the pathway for H+-transport (focusing of red dots within the transporter). Dehydration occurs because the water activity decreases (lightening of red background). Large, internal molecules also contribute, perhaps through crowding or steric exclusion from protein-associated water pools (increasingly crowded gray shapes).

The solvents internal and external to proteoliposomes can be manipulated at will, with or without imposing osmotic gradients that would alter the topology of the bounding membrane (10). Because all three transporters can activate at constant membrane area, we know that they do not respond to changes in membrane strain (1012) and they are probably not designed to regulate turgor pressure. Surprisingly, all three respond with specificity to the internal solvent (1012). At osmotic pressures that would activate when applied externally, internal inorganic ions activate whereas small organic solutes do not. The required ion concentrations are high (up to half molar) and depend on the specific lipid composition of the proteoliposome.

Glycine betaine transport by OpuA accelerates as the salt concentration of the medium bathing its cytoplasmic surface increases (11). In recent articles, Poolman and his colleagues designated OpuA an ionic strength sensor because diverse salts elicited this response and divalent cations were more effective than monovalent cations (1) (Fig. 3A). Among the latter, K+ was most effective, followed by Na+ and Li+, but Rb+ and Cs+ strongly inhibited transport (1). K+ was denoted the physiologically relevant cation.

Proteoliposomes were used to explore the phospholipid dependence of OpuA (11). Among the properties examined, only headgroup charge influenced osmotic activation. Anionic lipid [e.g., phosphatidylglycerol (PG)] was required both for activity and to suppress OpuA activity at low osmotic pressure. In addition, the osmotic pressure at which OpuA became active (the activation threshold) increased with PG concentration. Subunit OpuAA includes dual cytoplasmic CBS (cystathionine-β-synthase) domains followed by an anionic, C-terminal tail (1). Interacting effects of phospholipid composition and OpuAA modifications led to the proposal that ionic strength controls interactions of the CBS domains with the membrane surface (1). They are released from the membrane and the transporter activates as internal ionic strength rises with external osmotic pressure (Fig. 3A). The anionic tail is a tuner that reduces the threshold ionic strength by repelling the anionic membrane surface.

Krämer and his colleagues designated BetP a K+-specific chemosensor because, among monovalent cations internal to proteoliposomes, K+, Rb+, and Cs+ activate BetP more effectively than do Na+, NH4+, or choline (10, 13) (Fig. 3B). The osmotic activation threshold rises with the anionic lipid (PG) content of the membrane, in vivo and in vitro (8, 14, 15). The cytoplasmic, anionic C terminus of BetP is implicated in K+ sensing (16, 17). After expression in E. coli or reconstitution in E. coli lipid (30% PG), BetP variants with altered C termini, particularly replacements for Glu572, were active but not responsive to osmotic pressure (16). The impacts of Glu572 substitutions were attenuated in PG-supplemented proteoliposomes and in C. glutamicum (87% PG) (15, 16). The Krämer lab has proposed that increasing K+ activates BetP by altering the conformation of the C terminus and thereby its interaction with the membrane and perhaps the transporter itself (16, 17) (Fig. 3B). An unknown mechanism suppresses BetP activity when cells have adapted to osmotic stress by accumulating glycine betaine (18).

ProP is a H+-solute symporter similar in tertiary structure to other MFS (major facilitator superfamily) members such as LacY (1921). K+ promotes ProP activity in vivo (22, 23). This effect can be attributed to the K+ requirement for maintenance of the proton motive force under osmotic stress (24, 25). K+, Na+, Li+, and Cs+ chlorides are equally effective as ProP activators inside proteoliposomes (12). Internal poly(ethylene)glycols (PEGs) also activate ProP at constant K phosphate concentration, and this activation is dependent on PEG size (12).

Paired replicas of the C terminus from E. coli ProP form antiparallel α-helical coiled coils in vitro (26), and an analogous structure may link adjacent ProP monomers in vivo (2730). ProP orthologs with and without coiled coils exist, and representatives of both groups osmoregulate (31). A lower osmotic pressure is required to activate orthologs and variants with the coiled coil than is required to activate those without it (27, 32). The osmotic activation threshold and the cardiolipin content of the membrane increase as the phosphatidyl ethanolamine content decreases when E. coli is cultivated in media of increasing osmotic pressure (32). Thus, the coiled-coil domain of ProP appears to tune the activation threshold, perhaps by altering interactions of the C terminus with the membrane (32). This represents osmotic adaptation, not osmosensing per se.

How does ProP sense osmotic pressure? I propose that ProP activity reflects ProP hydration. Increasing external osmotic pressure increases competition among cytoplasmic or proteoliposome contents for water of hydration (Fig. 3C). Experiments show that, despite their membrane integration, MFS members such as LacY and ProP are flexible and highly hydrated with more extensive cytoplasmic than periplasmic surface exposure (21, 33, 34). These proteins are likely to share an alternating access mechanism in which two helix bundles rock around an axis in the membrane plane, opening the substrate binding site sequentially to the periplasm and cytoplasm (35). In LacY, the coupled H+ transport involves key acidic and basic residues and likely associated water molecules (34). Waters are integral to other H+ transport reactions, as shown beautifully in crystal structures of bacteriorhodopsin (36), an osmotic pressure–sensitive (37), light-driven H+ pump. I postulate that, as osmotic pressure increases, key water molecules are lost from these proteins. Most are inhibited by dehydration, but dehydration creates a pathway for H+ transport through ProP or activates it by means of more global structural change.

This proposal raises challenging questions: Why do inorganic electrolytes and PEGs inside proteoliposomes activate ProP more than small organic osmolytes do? Do large PEGs and cytoplasmic proteins activate ProP only by competing for water of hydration? Do macromolecular crowding or steric exclusion of these large solutes from water pools within ProP also contribute (3, 12)?

Differences among OpuA, BetP, and ProP also raise important questions. Do cytoplasmic K+ concentration, ionic strength, and water activity vary independently under physiological conditions, requiring systems that can specifically detect and modulate each property? How well do the reported in vitro responses of these transporters correspond to their in vivo behavior? Ultimately, we aim to understand how each signal is transduced, altering transporter activity.


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