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Osmoregulation and its importance to food-borne microorganisms
International Journal of Food Microbiology 74 (2002) 203 – 216 www.elsevier.com/locate/ijfoodmicro
Osmoregulation and its importance to food-borne microorganisms Conor P. O’Byrne, Ian R. Booth * Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, Scotland AB25 2ZD, UK Accepted 1 July 2001
Abstract The control of water activity has been used as a means of preserving foods for thousands of years. This preservation strategy presents food-borne microorganisms with serious problems, many of which relate to the management of water flow. Although the specific details of how each organism deals with these problems are different, several common themes have emerged. Bacteria induce specific responses, both physiological and genetic, to respond to either the loss or the gain of water, triggered by changes in the osmolarity of the environment. Many of the key systems have now been identified and the mechanisms of their regulation are beginning to be understood. Here we review recent developments in the field of bacterial osmoregulation with emphasis on key food-borne genera. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Osmoregulation; Compatible solutes; Betaine; Proline; Transport; Mechanosensitive channels; Regulation
1. Osmoregulation—fundamental principles The growth of bacterial cells requires the controlled flow of water. The major polymers of the cell, DNA, protein, RNA and lipids, are not osmotically active. However, growth is normally defined as the balanced increase in the mass of these polymers until such time as the cell achieves a critical mass and divides. However, the volume that is occupied by the newly synthesised polymers is created by expansion of the cell. This requires that water enter the cell to generate the force for expansion (Fig. 1a). Bacterial
*
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membranes are generally considered to exhibit a high permeability to water and bacteria lack mechanisms for pumping water between the cytoplasm the environment. As a consequence, the flow of water across the membrane is essentially passive and responds to gradients of osmotically active solutes. Thus, bacterial cells accumulate solutes in the cytoplasm to concentrations far higher than are required for the metabolism of the cell to ensure that the direction of water flow, during growth, is into the cell. Consequently, all growing bacterial cells exhibit a high, outwardly directed, turgor pressure that places the membrane in very close proximity to the expanding peptidoglycan wall. When bacteria are transferred to an environment with higher solute concentrations than are found in the cytoplasm (hyperosmotic shock), water exits from the cell (Fig. 1b) until the osmotic activity of the cytoplasm
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Fig. 1. Water flux during balanced growth (a), hyperosmotic stress (b), and hypoosmotic stress (c). The density of shading indicates the osmotically active solute concentration (greater shading = higher solute concentrations). See text for details. Open arrows indicate the turgor pressure forcing the contact between the membrane and the wall. Black arrowheads indicate the force leading to cell expansion or shrinkage in response to changes in turgor pressure. The broken lines indicate the new cell volume after initial water movement. In panel (c), the small arrows indicate solute efflux, via mechanosensitive channels to prevent excessive turgor (Booth and Louis, 1999). Note that the changes in cell volume are exaggerated in panels (b) and (c).
and the environment are balanced. There is loss of turgor and shrinkage of the cytoplasm may occur (Delamarche et al., 1999). The controlled accumulation of solutes allows the cell to restore turgor (Epstein, 1986; Booth et al., 1988). Even when cells have restored turgor the composition of the cytoplasm may not be optimal for enzyme activity and cells may exhibit limited growth potential and indeed may incur further stress through alterations in the pattern of metabolism (Dodd et al., 1997). Transfer to a dilute medium causes rapid water entry into the cell and must
be compensated by the release of solutes to the environment via mechanosensitive channels (Fig. 1c) (Berrier et al., 1992; Glaasker et al., 1996; reviewed in Booth and Louis, 1999). In essence, osmoregulation is about the control over the influx and efflux of solutes from the cell, with water movement being essentially passive. In recent years, it has become evident that some (and possibly all) bacteria possess water channels that accelerate water passage through the membrane (Calamita, 2000). The possession of such channels, and the regulation of their expression by the osmolarity of the growth medium (Calamita et al., 1998), suggests that the rate of water transfer across the membrane is also critical. Indeed, this consideration immediately draws attention to the fact that osmoregulation requires not just modulation of the activity of transport and enzyme systems, but also complex patterns of regulation of gene expression (Booth, 1993; Kempf and Bremer, 1998). It is increasingly clear that osmotic stress is used as a signal by many bacteria to prepare for more stringent conditions in the near future by the induction of more general systems of stress protection (Hengge-Aronis, 2000; see below). The food industry uses the water activity of many processed foods to control the potential of spoilage bacteria and to limit the survival of both spoilage and pathogenic organisms. It is rarely the case that water activity is used alone and other preservative regimes often go hand-in-hand with lowered water activity. While one can give good accounts of the mechanisms of osmoregulation, these arise from fundamental studies using a single variable, though often complemented by changes in the composition of the growth medium. A more accurate description of the performance of osmoregulation in the food context can only be gained by the analysis of combinations of variables. Although the survival of organisms under conditions mimicking foods has been analysed, such studies are rarely placed in the context of the success or failure of the organism’s elaborate adaptive mechanisms. There are two other areas in which osmoregulatory strategies impinge on the food industry, namely survival of drying and recovery of damaged organisms. It has been demonstrated that the incorporation of betaine (an osmoprotectant and compatible solute;
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see below) into the collection fluid and enumeration media when enumerating airborne bacteria increases significantly the recovery of viable organisms (Marthi and Lighthart, 1990). This suggests that betaine can aid the recovery of bacteria from dehydration. Such observations have clear implications for screening ‘‘clean’’ environments for the presence of organisms that might contaminate food processing plant. Related observations have shown that betaine can have beneficial effects when cells are required to survive drying. Lactic acid bacteria are often stored as freeze-dried cell suspensions. Kets et al. (1996) observed that a good correlation could be drawn between survival of drying and the prior accumulation of betaine.
2. Osmoregulation—the basic strategy Growth and/or survival of bacteria in high osmolarity environments usually requires them to accumulate compatible solutes (reviewed in Galinski and Truper, 1994; Kempf and Bremer, 1998; Poolman and Glaasker, 1998). These solutes are relatively inert organic molecules that can be accumulated by bacterial cells to molar levels, without impacting on the activity of the enzymes of the cell. The level of compatible solute accumulation is set by the environmental osmolarity, irrespective of whether the solutes are derived by their controlled accumulation from the environment or via their synthesis in the cytoplasm. Despite the fact that organisms differ widely in the range of osmolarity over which they will grow, the compatible solutes that they accumulate, and the molecular mechanisms used to control the expression of the genes for compatible solute accumulation, they all show essentially the same responses to osmotic shock. At low osmolarity, they accumulate salts, usually potassium glutamate, but other anions are also potentially important, particularly in foods. In the presence of benzoate and acetate, which are weak acid food preservatives, glutamate may be replaced by these anions (Roe et al., 1998). As the osmolarity of the environment is increased, the initial response is elevation of the cytoplasmic potassium glutamate pool. However, high concentrations of such salts are inimical to enzyme function and the cells initiate the accumulation of compatible solutes. It is well document-
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ed from in vitro studies that compatible solutes will restore the activity of enzymes that are incubated and assayed in the presence of high concentrations of salts (Pollard and Wyn Jones, 1979). However, in vivo, the accumulation of compatible solutes is usually accompanied by a decrease in the pools of salts (Sutherland et al., 1986). The major compatible solutes in the food-borne organisms consist of quaternary amines (e.g., betaine and carnitine), amino acids (e.g., proline), amino acid derivatives (e.g., proline betaine), sugars (e.g., trehalose and mannitol), and a range of peptides (e.g., prolyl-hydroxyproline). In addition, choline can be converted to betaine by some organisms (e.g., Landfald and Strøm, 1986) and most possess the ability to generate proline from peptides (though proteolysis may be a more limited activity). Some of these solutes are only available from the environment (e.g., choline, betaine and ectoine), while others can be either synthesized or transported (e.g., proline), whilst others are only available via synthesis (e.g., trehalose). The complex organic nature of food means that this represents a major potential source of compatible solutes (Fig. 2) and can be a limiting factor on the effectiveness of lowering water activity for food preservation.
Fig. 2. Sources of compatible solutes for food-borne bacteria. All of the compounds indicated outside the cell (indicated by dotted line) are available in foods and can either serve directly as compatible solutes or as precursors of solutes.
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3. Compatible solutes in foods As discussed above, the accumulation of compatible solutes is commonly used as means of osmoregulation in bacteria. Therefore, the availability of these compounds in the environment plays a significant role in determining the growth rate of the bacterium under conditions of hyperosmotic stress. This is also the case for food-borne microorganisms; the compatible solute availability in a particular food is one of the factors that influences the growth of contaminating microbes, particularly in foods with a low water activity. What types of compatible solutes are commonly found in foods, and what is their effective concentration? Both plant material and various types of meat contain sufficiently high levels of the quaternary amine, glycine betaine, for it to act as a potential osmoprotectant (Verheul et al., 1997; Smith, 1996). Typically concentrations of betaine range from 0.3 to 0.5 nmol mg 1 fresh weight in meats. Fresh meat contains significant concentrations of free amino acids, which implies the availability of proline as an osmoprotectant (Beumer et al., 1994). The levels of carnitine in meat range from 0.2 to 1.0 nmol mg 1 fresh weight, though in vegetable matter carnitine is present at a concentration approximately 10-fold lower than this (Verheul et al., 1997; Smith, 1996). Other compatible solutes are also likely to be abundant in different foods, e.g. taurine in fish and crabs, which can serve as an osmoprotectant for Escherichia coli (McLaggan and Epstein, 1991). These concentrations of solutes are sufficient to allow bacterial pathogens to exploit these compounds as osmoprotectants, since the high affinity of the transport systems renders low (nM) amounts of betaine sufficient for protection (Koo and Booth, 1994). There are likely to be substantial pools of free amino acids in many foods and these will include proline, which can then be accumulated to alleviate the osmotic stress experienced by the cell. Similarly degradative enzymes that liberate choline and peptides from macromolecules make them available as precursors of compatible solute (Fig. 2). The direct accumulation of compatible solutes from meat has been studied for Listeria monocytogenes (Smith, 1996). When this organism grows on bologna at 7 BC it preferentially accumulates betaine
during the first 10 days of growth, with approximately twice as much betaine accumulated as carnitine during this period. Subsequently the pools of carnitine increase and after 20 days at 7 BC similar levels of both solutes can be detected in the cytoplasm (approx. 750 nmol mg cell protein 1). Similar data are obtained when betaine and carnitine accumulation is studied from a variety of other meats, though there is some variation in the relative pool sizes of these two solutes (Smith, 1996). The intracellular concentrations estimated in these studies represents approximately a 100-fold concentration of the solutes from the meat. Food, thus, constitutes a rich source of compatible solutes and their precursors. Consequently, higher osmolarities may be required to achieve inhibition of the resident pathogens and spoilage organisms than would be the case if either compatible solutes were absent or their accumulation could be prevented.
4. Lessons from different bacterial genera: case histories The essence of osmoregulation is controlled accumulation or release of solutes. The implication is that the membrane must be selectively permeable to this special group of solutes not just in the normal sense of selective permeability exhibited with organic molecules (e.g., stereospecificity and preferential accumulation or exclusion), but also in response to specific environmental cues. There is a mixed strategy employed across the bacterial genera in response to this problem—existing transport systems and enzymes may be activated/inhibited by cues, and/or the genes encoding their structural components may be induced/ repressed. Such mechanisms allow cells to modulate the rate of acquisition of compatible solutes. However, a universal finding is that, in addition to the rate of accumulation being set by the osmolarity of the environment, the pool of accumulated solute is also determined by the degree of osmotic stress. For example, the pool of betaine increases with the imposed osmotic stress (Perroud and Le Rudulier, 1985; Verheul et al., 1997). In the context of food, the accumulation of peptides by L. monocytogenes can also be seen to reflect this trend (see below) (Amezega et al., 1995). The regulatory mechanisms underpinning the
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setting of the pool size by the environment are poorly understood, but the indications are that they differ between organisms. Some case histories may serve to exemplify these differences. 4.1. E. coli and Salmonella typhimurium The growth of E. coli at high osmolarity is attended by the accumulation of either trehalose, when other compatible solutes are absent from the growth medium, or by any one of a range of solutes available from the growth medium (Larsen et al., 1987; Strøm and Kaasen, 1993). Trehalose synthetic enzymes are under the control of the rpoS sigma factor (Hengge-Aronis et al., 1991), which accumulates when cells are grown at high osmolarity (Jishage et al., 1996; Muffler et al., 1996). Cells that cannot accumulate trehalose, due either to mutations in rpoS or in the synthetic pathway (otsAB), exhibit a restricted growth range with respect to osmolarity (Strøm and Kaasen, 1993). Cells that accumulate trehalose are unable to grow as rapidly as those that have accumulated betaine, proline or ectoine. Proline can be available from protein digests, but E. coli is unable to derive proline from peptide mixtures, such as might be present in foods, despite the ability to use proline-containing peptides when incubated with single peptides (Amezega and Booth, 1999). Trehalose is only accumulated via its synthesis (Strøm and Kaasen, 1993). Although there are transport mechanisms for this sugar, the accumulated product is the sugar phosphate, which is not a compatible solute. It has been demonstrated that cells lacking periplasmic trehalase accumulate the sugar in the medium, which suggests that trehalose leaks from the cells possibly via mechanosensitive channels (Styrvold and Strøm, 1991). However, it not clear what mechanisms operate to control the pool size of this compatible solute. Proline, betaine and ectoine are accumulated via the ProP and ProU transport systems (Fig. 3) and may share a common efflux system (Koo et al., 1991). ProP and ProU are activated by osmotic stress and the structural genes are induced during growth at high osmolarity, especially in the absence of the compatible solute itself, which can act to repress expression of the structural genes (Cairney et al., 1985a,b). Again control over the pool size is poorly understood (Rancourt et al., 1984; Csonka, 1988; Koo et al., 1991; Lamark et al., 1992).
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4.2. Staphylococcus aureus In many respects osmoregulation in S. aureus resembles that found in the enteric bacteria. Proline and betaine are accumulated to high levels in response to osmotic stress, free peptides can serve as a source of proline, but this organism does not accumulate sugars as compatible solutes. On the whole, however, there can be seen to be two significant differences. Firstly, there is very much less evidence for regulation of gene expression by osmotic stress (see below). Secondly, the transport systems appear to be constitutively synthesised and to be activated by osmotic stress in a very specific way. There are multiple transport systems for betaine and proline; probably a single specific system each for betaine (BPI) and proline (PutP) and a less specific system that is strongly activated by osmotic stress and results in the accumulation of both proline and betaine (BPII) (Pourkomailian and Booth, 1992) (Fig. 3). Mutants lacking the BPII system are severely compromised in their growth at high osmolarity (Pourkomailian and Booth, 1994). In common with other organisms a complete understanding of the numbers and substrate specificity of each of the compatible solute transport systems may have to await the publication of the genome sequence. Simple kinetic analyses and limited genetic studies will not always reveal the true complexity of the transport options available to the cell (Kempf and Bremer, 1998). The most interesting feature of the BPII betaine– proline transport system is that, in addition to activation of the system by osmotic stress, there is also feedback inhibition by accumulated solute (Pourkomailian and Booth, 1994). Similar control over activity is found in lactic acid bacteria and in L. monocytogenes (see below). In contrast, the equivalent ProP and ProU transport systems of E. coli and S. typhimurium are activated by osmotic stress and remain active at the steady state with the putative efflux system becoming the focus of regulation of the pool (Koo et al., 1991). In contrast, in S. aureus, and in lactic acid bacteria, growth in broth leads to sufficient accumulation of solute that the principal betaine transport systems appear to be only poorly active (Graham and Wilkinson, 1992; Bae et al., 1993; Pourkomailian and Booth, 1994). Demonstration of the system’s
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Fig. 3. Schematic illustration of the compatible solute uptake systems of key food-borne bacteria. Compatible solute abbreviations are as follows: B, betaine; C, carnitine; P, proline; Ch, choline; E, ectoine; PB, proline betaine. SBP indicates the periplasmic substrate binding protein of ProU. PW denotes the peptidoglycan wall; OM, outer membrane; CM, cytoplasmic membrane and IM, inner membrane. * The BetT transporter is not present in S. typhimurium.
activity requires the imposition of osmotic stress that creates a demand for solute pools greater than already achieved, thus overcoming the feedback. Thus, con-
trol over the pool is achieved by the balance of the degree of activation vs. the strength of the feedback mechanism.
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4.3. L. monocytogenes One of the most fascinating features of this organism is the ability to use peptides as a source of compatible solutes. In the laboratory this is readily mimicked by the use of meat peptones as a growth substrate, which might be seen as a model for growth in pate and soft cheeses. Meat peptones contain hydroxyproline-containing peptides as well as glycine-rich peptides. Both of these are accumulated by L. monocytogenes, such that high concentrations both of the free amino acids and complete peptides are found in the cytoplasm of cells grown in high osmolarity medium (Amezega et al., 1995). The pools are sufficiently substantial to contribute significantly to turgor generation. In addition, the pool size is influenced positively by increased osmotic pressure. Provenance for the meat origin of the peptides, rather than synthesis, arises from the presence of hydroxyproline and from the absence of the peptides when cells are grown either with free amino acids or in defined medium. Betaine and carnitine are still the dominant compatible solutes for this organism and the accumulation of betaine leads to lowered peptide pools (Amezega et al., 1995). The accumulation of either betaine or carnitine strongly stimulates the growth of L. monocytogenes at high osmolarity. Recently significant progress has been made in identifying the systems used by L. monocytogenes to accumulate these compounds intracellularly (Ko and Smith, 1999; Sleator et al., 1999; Fraser et al., 2000) (Fig. 4). Betaine is accumulated both by a secondary transporter (BetL) and a substrate binding protein-dependent transporter that is driven by ATP (Gbu). Carnitine is transported by another substrate binding protein-dependent transporter, designated OpuC. Both Gbu and OpuC belong to the ABC transporter superfamily. The regulation of these systems at the genetic and biochemical levels has not yet been studied in detail but a number of interesting features have emerged. The accumulation of carnitine and betaine is subject to trans-inhibition. That is, pre-accumulation of carnitine regulates negatively the activity of the transport systems for either betaine or carnitine. Likewise, the pre-accumulation of betaine inhibits the activity of both the carnitine and betaine uptake system. This finding is in contrast to the betaine uptake systems in
Fig. 4. Osmotic activation of carnitine transport in L. monocytogenes. Schematic representation of feedback regulation of carnitine transport in L. monocytogenes. Rates of transport were measured in cells growing exponentially either in defined medium (DM) or in defined medium with added peptone (DMP) in a phosphate buffer in the presence of chloramphenicol (50 Ag ml 1) at 30 BC, as previously described (Fraser et al., in press). In DM, the rate of transport is high and is independent of the presence or absence of NaCl. With cells grown in DMP and measured in the absence of NaCl, the rate of transport is low due to feedback inhibition by solutes (S). Upon addition of NaCl feedback is relieved and the transport rate is high. Similar observations have been made for betaine transport in L. monocytogenes (Verheul et al., 1997).
E. coli, ProP and ProU, whose activity is apparently not regulated by intracellular substrate concentrations (Koo et al., 1991). Recently, the presence of peptone in the medium has also been found to lead to trans inhibition in L. monocytogenes. Some component of peptone (peptides or free amino acids) is accumulated and the activity of the carnitine uptake system is inhibited (Fraser et al., 2000). Trans inhibition can be overcome by osmotic stimulation; subjecting cells to hyperosmotic shock (in the presence of chloramphenicol) leads to an activation of the uptake systems. This activation is only seen when the compatible solute transport systems are trans inhibited; without solute pre-accumulation the activity of the transporters
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in independent of the medium osmolarity (Verheul et al., 1997; Fraser et al., 2000) (Fig. 4). The molecular basis for these observations remains unclear. One possibility is that the accumulated solutes interact with the cytoplasmic face of the transport systems and inhibit them directly. A second possibility is that pre-accumulated solute affects cell turgor and the transporters respond directly to changes in turgor (perhaps by sensing lateral forces in the cell membrane). Increased turgor arising from the intracellular accumulation of any solute would then lead to inhibition of transporter activity. In this regard it is interesting that the activity of the OpuA betaine transporter of Lactococcus lactis, which is related to Gbu and OpuC from L. monocytogenes (Fraser et al., 2000), is activated by membrane perturbation (Van der Heide and Poolman, 2000). Further studies will be required to determine the molecular basis for trans inhibition of compatible solute uptake in L. monocytogenes. 4.4. Lactic acid bacteria: There are strong similarities between osmoregulation in L. monocytogenes and the lactic acid bacteria. Betaine and carnitine are the two major compatible solutes and the uptake systems show strong activation by osmotic stress. The molecular mechanism of activation of the major betaine transporter, the ATP-de pendent OpuA system, has been investigated in detail in Lactococcus lactis. The purified OpuA system has been reconstituted in liposomes and has been shown to both sense, and respond to, changes in osmotic pressure (Van der Heide and Poolman, 2000). Similar data have been obtained for the simpler ProP system of E. coli (Milner et al., 1994). This means that all of the elements required for osmotic activation of transport are integral to the proteins of the OpuA and ProP. Preliminary data suggest that the OpuA system senses and responds to changes in the physical state of the membrane. Amphiphiles, which insert into the membrane and perturb the curvature of the membrane in a manner predicted to occur in response to osmotic stress, activate OpuA in the absence of osmotic stress (Van der Heide and Poolman, 2000). Similar observations have been made for mechano-transducing channels (see below), which are activated by high turgor.
5. Compatible solutes and cryoprotection In addition to their role as osmoprotectants compatible solutes have also been implicated in cryotolerance. The quaternary amines betaine and carnitine are both found to accumulate at higher levels in L. monocytogenes when this organism is grown at refrigeration temperatures, even in the absence of osmotic stress (Ko et al., 1994; Smith, 1996). A null mutation in gbu, which encodes an ATP-dependent betaine transporter in L. monocytogenes, abolishes the accumulation of betaine at refrigeration temperatures (Ko and Smith, 1999). In addition, the growth of this gbu mutant is markedly impaired at 7 BC (Ko and Smith, 1999), demonstrating the cryoprotective role of betaine in L. monocytogenes. In general, compounds that act as good osmoprotectants in L. monocytogenes are also found to have a cryoprotective effect (Bayles and Wilkinson, 2000). The molecular basis of this cryoprotective effect is not yet known but it is not confined to this food-borne pathogen; the cyanobacterium Synechococcus displays an increased growth rate at low temperatures when engineered to synthesise betaine in the cytoplasm (Deshnium et al., 1997). Betaine accumulation in the plant Arabidopsis thaliana also enhances its ability to tolerate low temperatures. Specifically, the sensitivity of the wild-type plant to light when incubated at 5 BC is not seen in a plant expressing the choline oxidase gene (the product of which catalyses the conversion of choline to betaine) from the bacterium Arthrobacter globiformis (Hayashi et al., 1997). Similar results have recently been obtained with a transgenic plant overproducing glycine betaine (Holmstrøm et al., 2000). A number of possible explanations for the ability of betaine and carnitine to act as cryoprotectants have been suggested. The stabilising effects of compatible solutes on protein structure are well known (Santoro et al., 1992). Ko et al. (1994) have proposed that chill stress may lead to a destabilisation of the tertiary structure of some proteins (either by affecting the mature fully folded structure or by affecting the structure during folding) and this effect may be ameliorated by the presence of compatible solutes. However, no biochemical data are yet available to support this idea. Another possibility is that presence of high concentrations of compatible solute may alter
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the membrane fluidity. Evidence from in vitro studies indicates that at high concentrations (1 M) betaine, proline and trehalose have the ability to reduce the temperature of the liquid crystalline to gel phase transition that occurs in unilamellar lipid vesicles (Rudolph et al., 1986). Deshnium et al. (1997) have also shown that in Synechococcus the lipid phase transition temperature is reduced when the intracellular levels of betaine are elevated. In L. monocytogenes betaine also has an effect on membrane fluidity; cells grown at 5 BC in the presence of betaine show a small increase in anteiso-C15:0 lipids, and these lipids are known to play a role in membrane fluidity in this pathogen (Annous et al., 1997). In E. coli, the synthesis of cyclopropane fatty acids is under the control of the RpoS sigma factor (Wang and Cronan, 1994; Eichel et al., 1999), which accumulates during growth at high osmolarity and low temperature (HenggeAronis, 2000). RpoS levels are reduced by growth with betaine. Thus, betaine could also have effects on the lipid phase transition of enteric bacteria. Further studies will be necessary to determine which of these effects is sufficient to account the cryoprotective effect of betaine.
6. Osmotic stress: induced cross protection Bacteria often coordinately regulate their responses to environmental stress through the activation/ induced expression of specific transcription factors, which are then responsible for triggering the appropriate transcriptional responses. Specific stress-inducible sigma factors are often central to these global responses. In E. coli and S. typhimurium as well as other Gram-negative species the RpoS sigma factor (jS) plays a key role in coordinately regulating gene expression in response to environmental stresses (Loewen et al., 1998; Hengge-Aronis, 2000). Osmotic stress is known to lead to elevated levels of RpoS in the cell (Hengge-Aronis, 1993). This occurs by increasing the rate of translation of the rpoS mRNA and increasing the stability of the jS protein (Jishage et al., 1996; Muffler et al., 1996). In E. coli the jS regulon includes over 50 different genes and the products of these genes confer resistance to wide range of stress conditions including osmotic stress, oxidative stress, starvation and low pH stress (Hengge-Aronis,
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2000). It is clear, therefore, that specific environmental triggers, which lead to elevated levels of jS in the cell, have the potential to confer cross-protection against multiple stresses. It is possible, for example, that an osmotic stress imposed on a bacterium as part of a food preservation regime could inadvertently increase its ability to tolerate other stresses that may also be relevant in the context of food preservation. An increased virulence potential is another possible consequence of jS activation; several genes that contribute to virulence are under jS control (Hengge-Aronis, 2000). Since genes associated with pathogenesis can form part of the regulons under the control of the stress sigma factors, it seems possible that growth under mild stress may potentiate the pathogenic potential of the surviving organisms. Paradoxically, the effect of betaine in reversing RpoS accumulation (HenggeAronis, 1993) may prevent cells from reaching both their potential protected state and developing their pathogenic potential in foods containing adequate supplies of the compatible solute. The reverse scenario also applies; stresses that lead to increased jS levels will also impact the ability of the cell to survive osmotic stress and on the expression of pathogenicity determinants in the surviving organisms. The diversity of systems affected by RpoS has so far precluded animal model experiments that relate to the expression of the specific pathogenicity determinants. In Gram-positive pathogens such as S. aureus and L. monocytogenes, as well as in Bacillus subtilis, many genes with stress-related functions are under the transcriptional control of the sigma factor SigB (jB) (Price, 2000). Like jS, the products of the genes transcribed by jB act to protect cells from a diverse range of environmental stresses, including low pH, oxidative stress, freeze –thaw damage, starvation and high temperature (Vo¨lker et al., 1999). The levels of free jB in the cell are carefully regulated by a complex cascade of regulatory factors, whose ultimate role is to regulate the activity of the anti-sigma factor RsbW (Price, 2000). Various environmental stresses are found to liberate jB from RsbW in B. subtilis, effectively raising its intracellular concentration and allowing it to interact with core RNA polymerase in order to direct transcription of the appropriate genes. The levels of sigB transcript are also elevated in response to environmental stress stimuli (Becker et al., 1998). Hyperosmotic shock is one of the principal
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stresses that leads to increased intracellular levels of jB, and sigB mutants of both B. subtilis (Vo¨lker et al., 1999) and L. monocytogenes (Becker et al., 1998) are found to be impaired in their ability to grow and survive under conditions of high medium osmolarity. In the case of L. monocytogenes it appears that the osmotic sensitivity is due to a defect in compatible solute transport; the transport of the quaternary amines betaine and carnitine is impaired in the absence of jB (Becker et al., 1998; Fraser and O’Byrne, unpublished observations). The overlap between the osmotic stress response and the heat shock response is emphasized by the finding that the j32 and the jH regulons of E. coli are also stimulated by hyperosmotic stress (Bianchi and Baneyx, 1999). The induction of these regulons appears to play a role in allowing cells repair the protein mis-folding that occurs during the loss of turgor following an osmotic shock. It has been proposed that this system operates as an emergency response, allowing the cells the opportunity to induce a jS-directed osmoadaptation response (Bianchi and Baneyx, 1999).
presence of NaCl (Anderson et al., 1991). The molecular basis for this phenomenon is not well understood but it is thought to result from the reduced intracellular concentration of water. It seems likely that water molecules, which become energized by supplying heat, have a potent ability to interact with proteins and accelerate their denaturation. A number of in vitro studies have also found that increased thermostability of proteins is frequently observed when the water activity of the solvent is reduced and that compatible solutes enhance thermostability (Santoro et al., 1992; Knubovets et al., 1999; Castro, 2000). In addition, at least part of this protection may be caused by increased RpoS activity in cells incubated at high osmolarity (Jenkins et al., 1990). In the context of food and food-borne pathogens, therefore, it is essential that osmotic effects on thermosensitivity be taken into account when designing heating regimes for the inactivation of contaminating microbes.
7. Consequences of osmotic stress: thermotolerance
To this point we have mainly dealt with the response to hyperosmotic stress. We have emphasised the need for the selective permeability of the membrane but also for the tight control over solute pools. It has been known for many years that bacteria subjected to rapid hypoosmotic shock, i.e. a sudden decrease in osmolarity, release their pools of amino acids and cations (Britten and McClure, 1962). It has been clear also that this release is often uncontrolled leading to a requirement, at least in the case of E. coli, for the cells to re-accumulate jettisoned nutrients and cations (Berrier et al., 1992; Schleyer et al., 1993). The putative mechanism for this release of solutes was mechanosensitive channels in the bacterial cytoplasmic membrane. The channels were discovered during electrophysiological analysis of giant protoplasts of E. coli (and subsequently several other Gram-positive bacteria). Berrier et al. (1992) demonstrated the potential importance of these channels with the observation that gadolinium ions, which inhibit the activity of mechanosensitive channels, prevent or slow the release of solutes. Subsequently, by a combination of high quality protein biochemistry and molecular biology it has been substantiated that most bacteria have several
Osmotic stress presents specific physical problems for all cells. In addition to the well-studied problems associated with water imbalance (discussed above), other interesting phenomena are also observed in osmotically stressed bacterial cells. Numerous studies have shown that the rate of thermal inactivation of bacteria is closely linked to the osmolarity of the medium. In most cases a reduced water activity leads to an increase in thermotolerance. In L. monocytogenes, for example the loss of viability associated with mild heat stress (56 – 60 BC) is markedly reduced if NaCl is added to the heating menstruum (Anderson et al., 1991; Juneja and Eblen, 1999; Knight et al., 1999). Similar protective effects are observed when S. enteritidis and E. coli O157:H7 (Blackburn et al., 1997) or S. aureus (Shebuski et al., 2000) are heattreated in the presence of increasing salt concentrations. When differential scanning calorimetry (DSC) is used to examine macromolecular changes that occur in heated L. monocytogenes cells, the major changes observed (presumably denaturation events) are shifted to higher temperatures when cells are heated in the
8. Coping with hypoosmotic stress — a new dimension to osmoregulation
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such channels and that they play a very important role in survival of severe hypoosmotic shock (Levina et al., 1999). When bacteria are subjected to hypoosmotic shock, the turgor pressure rises very rapidly due to the influx of water down the solute gradient (i.e., higher solute concentrations in the cytoplasm than outside leads to water influx) (Fig. 1). Although the peptidoglycan wall is a semi-elastic structure it is likely that it is already stretched during growth and possibly close to its elastic limit (Yao et al., 1999). Consequently, the sudden increase in pressure on the wall has the potential to cause wall disruption leading to cell lysis (Levina et al., 1999). The recent work on mechanosensitive channels has revealed for E. coli that there are a number of channels each with a different pressure threshold at which they become activated. Although not proven this is probably true for most free-living bacteria. The severity of the turgor increase determines which channel opens (in E. coli MscM, followed by MscS and then finally MscL; Blount et al., 1996). Since these channels have large electrical conductances in the open state it is considered that they create a large diameter pore in the membrane through which solutes flow indiscriminately. The rapid release of solutes eliminates the driving force for water entry and thus relieves the pressure on the cell wall. In this way the mechanosensitive channels operate to preserve the integrity of the cell. Similar observations have been made for a range of Grampositive bacteria and this accords with the known existence of mechanosensitive channels in these bacteria (Glaasker et al., 1996; Verheul et al., 1997; E. Bremer, personal communication). Clearly the lack of discrimination in the channels between different solutes offers both a hazard to the cell and a potential opportunity to the food microbiologist. The ability of small ions to move through the channels essentially means that the preservation of homeostasis (pH, solute balance, osmolarity, ion, etc.) requires that the channels remain tightly closed until required. When the channels open aberrantly the result can be either growth inhibition (Blount et al., 1997; Ou et al., 1998) or death (Levina et al., 1999). An example of this is that E. coli cells subjected to hypoosmotic shock when incubated at acid pH suffer a several log reduction in viability compare with cells transferred iso-osmotically to acid pH (Levina et al.,
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1999). The imposition of hypoosmotic shock is not in itself a readily realisable treatment for many processed foods. However, compounds that lead to loss of tight control over the channels could potentiate cell death in processed foods when combined with a low pH ( < 5.5) that is often used in such products. Even when the pH is not low enough to kill cells the loss of the permeability barrier may be sufficient to reduce the growth rate. Mechanosensitive channels can be activated by a variety of membrane active compounds, such as local anaesthetics (Martinac et al., 1990). However, while these are not likely to be accepted for addition to food it appears that at least one class of food preservative may act by this route. Butyl paraben is an effective inhibitor of the growth of E. coli cells. Mutants that lack the MscL mechanosensitive channel appear to be less sensitive to butyl paraben, which suggests that either this protein is the target or that other changes in the membrane structure consequent upon loss of MscL are the reason for the altered sensitivity (Stokes and Booth, unpublished data). A search for other membrane active compounds may identify other activators that have significant potential as food preservatives.
Acknowledgements In memorium: The ideas presented in this paper have been shaped by many people, most of whom have left their marks in the literature. Gordon was engaged with this field from the early days of the discovery of the osmoregulated proU promoter and continued to make contributions that are particularly important in the context of the food industry. In reviewing his published work I was struck by the fact that, compared to the work on quorum sensing, his published work on osmoregulation is small. However, in addition to the seminal study on ProU (Park et al., 1989), Gordon was the major contributor to debates on death due to inimical stresses (Dodd et al., 1997) (including low Aw; Armstrong-Buisseret et al., 1995) and the role of RpoS in survival (Rees et al., 1996). Much of this work lives on through work arising from his discussions with close colleagues. The authors wish to thank their close colleagues who have contributed to this review either directly through their published work or indirectly through their comments. I.R.B. is a Wellcome Trust Research
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Leave Fellow. The BBSRC Food Directorate, The Wellcome Trust and Aberdeen Royal Hospitals Trust have supported the work of the two groups. References Amezega, M.-R., Booth, I.R., 1999. Osmoprotection of Escherichia coli by peptone is mediated by the uptake and accumulation of free proline but not of proline-containing peptides. Appl. Environ. Microbiol. 65, 5272 – 5278. Amezega, M.-R., Davidson, I., McLaggan, D., Verheul, A., Abee, T., Booth, I.R., 1995. The role of peptide metabolism in the growth of Listeria monocytogenes ATCC 23074 at high osmolarity. Microbiology 141, 41 – 49. Anderson, W.A., Hedges, N.D., Jones, M.V., Cole, M.B., 1991. Thermal inactivation of Listeria monocytogenes studied by differential scanning calorimetry. J. Gen. Microbiol. 137, 1419 – 1424. Annous, B.A., Becker, L.A., Bayles, D.O., Labeda, D.P., Wilkinson, B.J., 1997. Critical role of anteiso-C15:0 fatty acid in the growth of Listeria monocytogenes at low temperatures. Appl. Environ. Microbiol. 63, 3887 – 3894. Armstrong-Buisseret, L., Cole, M.B., Stewart, G.S.A.B., 1995. A homologue to the Escherichia coli alkyl hydroperoxide reductase AhpC is induced by osmotic upshock in Staphylococcus aureus. Microbiology 141, 1655 – 1661. Bae, J.H., Anderson, S.H., Miller, K.J., 1993. Identification of a high-affinity glycine betaine transport system in Staphylococcus aureus. Appl. Environ. Microbiol. 59, 2734 – 2736. Bayles, D.O., Wilkinson, B.J., 2000. Osmoprotectants and cryoprotectants for Listeria monocytogenes. Lett. Appl. Microbiol. 30, 23 – 27. Becker, L.A., Sevket Cetin, M., Hutkins, R.W., Benson, A.K., 1998. Identification of the gene encoding the alternative sigma factor jB from Listeria monocytogenes and its role in osmotolerance. J. Bacteriol. 180, 4547 – 4554. Berrier, C., Coulombe, A., Szabo, I., Zoratti, M., Ghazi, A., 1992. Gadolinium ion inhibits loss of metabolites induced by osmotic shock and large stretch-activated channels in bacteria. Eur. J. Biochem. 206, 559 – 565. Beumer, R.R., Te Giffel, M.C., Cox, L.J., Rombouts, F.M., Abee, T., 1994. Effect of exogenous proline, betaine and carnitine on growth of Listeria monocytogenes in a minimal medium. Appl. Environ. Microbiol. 60, 1359 – 1363. Bianchi, A.A., Baneyx, F., 1999. Hyperosmotic shock induces the j32 and jE stress regulons of Escherichia coli. Mol. Microbiol. 34, 1029 – 1038. Blackburn, C.W., Curtis, L.M., Humpheson, L., Billon, C., McClure, P.J., 1997. Development of thermal inactivation models for Salmonella enteritidis and Escherichia coli O157:H7 with temperature, pH and NaCl as controlling factors. Int. J. Food Microbiol. 38, 31 – 44. Blount, P., Sukharev, S.I., Moe, P.C., Nagle, S.K., Kung, C., 1996. Towards an understanding of the structural and functional properties of MscL, a mechanosensitive channel in bacteria. Biol. Cell 87, 1 – 8.
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Osmoregulation and its importance to food-borne microorganisms Conor P. O’Byrne, Ian R. Booth * Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, Scotland AB25 2ZD, UK Accepted 1 July 2001
Abstract The control of water activity has been used as a means of preserving foods for thousands of years. This preservation strategy presents food-borne microorganisms with serious problems, many of which relate to the management of water flow. Although the specific details of how each organism deals with these problems are different, several common themes have emerged. Bacteria induce specific responses, both physiological and genetic, to respond to either the loss or the gain of water, triggered by changes in the osmolarity of the environment. Many of the key systems have now been identified and the mechanisms of their regulation are beginning to be understood. Here we review recent developments in the field of bacterial osmoregulation with emphasis on key food-borne genera. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Osmoregulation; Compatible solutes; Betaine; Proline; Transport; Mechanosensitive channels; Regulation
1. Osmoregulation—fundamental principles The growth of bacterial cells requires the controlled flow of water. The major polymers of the cell, DNA, protein, RNA and lipids, are not osmotically active. However, growth is normally defined as the balanced increase in the mass of these polymers until such time as the cell achieves a critical mass and divides. However, the volume that is occupied by the newly synthesised polymers is created by expansion of the cell. This requires that water enter the cell to generate the force for expansion (Fig. 1a). Bacterial
*
Corresponding author. Tel.: +44-1224-273-152; fax: +441224-273-144. E-mail address: [email protected] (I.R. Booth).
membranes are generally considered to exhibit a high permeability to water and bacteria lack mechanisms for pumping water between the cytoplasm the environment. As a consequence, the flow of water across the membrane is essentially passive and responds to gradients of osmotically active solutes. Thus, bacterial cells accumulate solutes in the cytoplasm to concentrations far higher than are required for the metabolism of the cell to ensure that the direction of water flow, during growth, is into the cell. Consequently, all growing bacterial cells exhibit a high, outwardly directed, turgor pressure that places the membrane in very close proximity to the expanding peptidoglycan wall. When bacteria are transferred to an environment with higher solute concentrations than are found in the cytoplasm (hyperosmotic shock), water exits from the cell (Fig. 1b) until the osmotic activity of the cytoplasm
0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 ( 0 1 ) 0 0 6 8 1 - X
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Fig. 1. Water flux during balanced growth (a), hyperosmotic stress (b), and hypoosmotic stress (c). The density of shading indicates the osmotically active solute concentration (greater shading = higher solute concentrations). See text for details. Open arrows indicate the turgor pressure forcing the contact between the membrane and the wall. Black arrowheads indicate the force leading to cell expansion or shrinkage in response to changes in turgor pressure. The broken lines indicate the new cell volume after initial water movement. In panel (c), the small arrows indicate solute efflux, via mechanosensitive channels to prevent excessive turgor (Booth and Louis, 1999). Note that the changes in cell volume are exaggerated in panels (b) and (c).
and the environment are balanced. There is loss of turgor and shrinkage of the cytoplasm may occur (Delamarche et al., 1999). The controlled accumulation of solutes allows the cell to restore turgor (Epstein, 1986; Booth et al., 1988). Even when cells have restored turgor the composition of the cytoplasm may not be optimal for enzyme activity and cells may exhibit limited growth potential and indeed may incur further stress through alterations in the pattern of metabolism (Dodd et al., 1997). Transfer to a dilute medium causes rapid water entry into the cell and must
be compensated by the release of solutes to the environment via mechanosensitive channels (Fig. 1c) (Berrier et al., 1992; Glaasker et al., 1996; reviewed in Booth and Louis, 1999). In essence, osmoregulation is about the control over the influx and efflux of solutes from the cell, with water movement being essentially passive. In recent years, it has become evident that some (and possibly all) bacteria possess water channels that accelerate water passage through the membrane (Calamita, 2000). The possession of such channels, and the regulation of their expression by the osmolarity of the growth medium (Calamita et al., 1998), suggests that the rate of water transfer across the membrane is also critical. Indeed, this consideration immediately draws attention to the fact that osmoregulation requires not just modulation of the activity of transport and enzyme systems, but also complex patterns of regulation of gene expression (Booth, 1993; Kempf and Bremer, 1998). It is increasingly clear that osmotic stress is used as a signal by many bacteria to prepare for more stringent conditions in the near future by the induction of more general systems of stress protection (Hengge-Aronis, 2000; see below). The food industry uses the water activity of many processed foods to control the potential of spoilage bacteria and to limit the survival of both spoilage and pathogenic organisms. It is rarely the case that water activity is used alone and other preservative regimes often go hand-in-hand with lowered water activity. While one can give good accounts of the mechanisms of osmoregulation, these arise from fundamental studies using a single variable, though often complemented by changes in the composition of the growth medium. A more accurate description of the performance of osmoregulation in the food context can only be gained by the analysis of combinations of variables. Although the survival of organisms under conditions mimicking foods has been analysed, such studies are rarely placed in the context of the success or failure of the organism’s elaborate adaptive mechanisms. There are two other areas in which osmoregulatory strategies impinge on the food industry, namely survival of drying and recovery of damaged organisms. It has been demonstrated that the incorporation of betaine (an osmoprotectant and compatible solute;
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see below) into the collection fluid and enumeration media when enumerating airborne bacteria increases significantly the recovery of viable organisms (Marthi and Lighthart, 1990). This suggests that betaine can aid the recovery of bacteria from dehydration. Such observations have clear implications for screening ‘‘clean’’ environments for the presence of organisms that might contaminate food processing plant. Related observations have shown that betaine can have beneficial effects when cells are required to survive drying. Lactic acid bacteria are often stored as freeze-dried cell suspensions. Kets et al. (1996) observed that a good correlation could be drawn between survival of drying and the prior accumulation of betaine.
2. Osmoregulation—the basic strategy Growth and/or survival of bacteria in high osmolarity environments usually requires them to accumulate compatible solutes (reviewed in Galinski and Truper, 1994; Kempf and Bremer, 1998; Poolman and Glaasker, 1998). These solutes are relatively inert organic molecules that can be accumulated by bacterial cells to molar levels, without impacting on the activity of the enzymes of the cell. The level of compatible solute accumulation is set by the environmental osmolarity, irrespective of whether the solutes are derived by their controlled accumulation from the environment or via their synthesis in the cytoplasm. Despite the fact that organisms differ widely in the range of osmolarity over which they will grow, the compatible solutes that they accumulate, and the molecular mechanisms used to control the expression of the genes for compatible solute accumulation, they all show essentially the same responses to osmotic shock. At low osmolarity, they accumulate salts, usually potassium glutamate, but other anions are also potentially important, particularly in foods. In the presence of benzoate and acetate, which are weak acid food preservatives, glutamate may be replaced by these anions (Roe et al., 1998). As the osmolarity of the environment is increased, the initial response is elevation of the cytoplasmic potassium glutamate pool. However, high concentrations of such salts are inimical to enzyme function and the cells initiate the accumulation of compatible solutes. It is well document-
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ed from in vitro studies that compatible solutes will restore the activity of enzymes that are incubated and assayed in the presence of high concentrations of salts (Pollard and Wyn Jones, 1979). However, in vivo, the accumulation of compatible solutes is usually accompanied by a decrease in the pools of salts (Sutherland et al., 1986). The major compatible solutes in the food-borne organisms consist of quaternary amines (e.g., betaine and carnitine), amino acids (e.g., proline), amino acid derivatives (e.g., proline betaine), sugars (e.g., trehalose and mannitol), and a range of peptides (e.g., prolyl-hydroxyproline). In addition, choline can be converted to betaine by some organisms (e.g., Landfald and Strøm, 1986) and most possess the ability to generate proline from peptides (though proteolysis may be a more limited activity). Some of these solutes are only available from the environment (e.g., choline, betaine and ectoine), while others can be either synthesized or transported (e.g., proline), whilst others are only available via synthesis (e.g., trehalose). The complex organic nature of food means that this represents a major potential source of compatible solutes (Fig. 2) and can be a limiting factor on the effectiveness of lowering water activity for food preservation.
Fig. 2. Sources of compatible solutes for food-borne bacteria. All of the compounds indicated outside the cell (indicated by dotted line) are available in foods and can either serve directly as compatible solutes or as precursors of solutes.
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3. Compatible solutes in foods As discussed above, the accumulation of compatible solutes is commonly used as means of osmoregulation in bacteria. Therefore, the availability of these compounds in the environment plays a significant role in determining the growth rate of the bacterium under conditions of hyperosmotic stress. This is also the case for food-borne microorganisms; the compatible solute availability in a particular food is one of the factors that influences the growth of contaminating microbes, particularly in foods with a low water activity. What types of compatible solutes are commonly found in foods, and what is their effective concentration? Both plant material and various types of meat contain sufficiently high levels of the quaternary amine, glycine betaine, for it to act as a potential osmoprotectant (Verheul et al., 1997; Smith, 1996). Typically concentrations of betaine range from 0.3 to 0.5 nmol mg 1 fresh weight in meats. Fresh meat contains significant concentrations of free amino acids, which implies the availability of proline as an osmoprotectant (Beumer et al., 1994). The levels of carnitine in meat range from 0.2 to 1.0 nmol mg 1 fresh weight, though in vegetable matter carnitine is present at a concentration approximately 10-fold lower than this (Verheul et al., 1997; Smith, 1996). Other compatible solutes are also likely to be abundant in different foods, e.g. taurine in fish and crabs, which can serve as an osmoprotectant for Escherichia coli (McLaggan and Epstein, 1991). These concentrations of solutes are sufficient to allow bacterial pathogens to exploit these compounds as osmoprotectants, since the high affinity of the transport systems renders low (nM) amounts of betaine sufficient for protection (Koo and Booth, 1994). There are likely to be substantial pools of free amino acids in many foods and these will include proline, which can then be accumulated to alleviate the osmotic stress experienced by the cell. Similarly degradative enzymes that liberate choline and peptides from macromolecules make them available as precursors of compatible solute (Fig. 2). The direct accumulation of compatible solutes from meat has been studied for Listeria monocytogenes (Smith, 1996). When this organism grows on bologna at 7 BC it preferentially accumulates betaine
during the first 10 days of growth, with approximately twice as much betaine accumulated as carnitine during this period. Subsequently the pools of carnitine increase and after 20 days at 7 BC similar levels of both solutes can be detected in the cytoplasm (approx. 750 nmol mg cell protein 1). Similar data are obtained when betaine and carnitine accumulation is studied from a variety of other meats, though there is some variation in the relative pool sizes of these two solutes (Smith, 1996). The intracellular concentrations estimated in these studies represents approximately a 100-fold concentration of the solutes from the meat. Food, thus, constitutes a rich source of compatible solutes and their precursors. Consequently, higher osmolarities may be required to achieve inhibition of the resident pathogens and spoilage organisms than would be the case if either compatible solutes were absent or their accumulation could be prevented.
4. Lessons from different bacterial genera: case histories The essence of osmoregulation is controlled accumulation or release of solutes. The implication is that the membrane must be selectively permeable to this special group of solutes not just in the normal sense of selective permeability exhibited with organic molecules (e.g., stereospecificity and preferential accumulation or exclusion), but also in response to specific environmental cues. There is a mixed strategy employed across the bacterial genera in response to this problem—existing transport systems and enzymes may be activated/inhibited by cues, and/or the genes encoding their structural components may be induced/ repressed. Such mechanisms allow cells to modulate the rate of acquisition of compatible solutes. However, a universal finding is that, in addition to the rate of accumulation being set by the osmolarity of the environment, the pool of accumulated solute is also determined by the degree of osmotic stress. For example, the pool of betaine increases with the imposed osmotic stress (Perroud and Le Rudulier, 1985; Verheul et al., 1997). In the context of food, the accumulation of peptides by L. monocytogenes can also be seen to reflect this trend (see below) (Amezega et al., 1995). The regulatory mechanisms underpinning the
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setting of the pool size by the environment are poorly understood, but the indications are that they differ between organisms. Some case histories may serve to exemplify these differences. 4.1. E. coli and Salmonella typhimurium The growth of E. coli at high osmolarity is attended by the accumulation of either trehalose, when other compatible solutes are absent from the growth medium, or by any one of a range of solutes available from the growth medium (Larsen et al., 1987; Strøm and Kaasen, 1993). Trehalose synthetic enzymes are under the control of the rpoS sigma factor (Hengge-Aronis et al., 1991), which accumulates when cells are grown at high osmolarity (Jishage et al., 1996; Muffler et al., 1996). Cells that cannot accumulate trehalose, due either to mutations in rpoS or in the synthetic pathway (otsAB), exhibit a restricted growth range with respect to osmolarity (Strøm and Kaasen, 1993). Cells that accumulate trehalose are unable to grow as rapidly as those that have accumulated betaine, proline or ectoine. Proline can be available from protein digests, but E. coli is unable to derive proline from peptide mixtures, such as might be present in foods, despite the ability to use proline-containing peptides when incubated with single peptides (Amezega and Booth, 1999). Trehalose is only accumulated via its synthesis (Strøm and Kaasen, 1993). Although there are transport mechanisms for this sugar, the accumulated product is the sugar phosphate, which is not a compatible solute. It has been demonstrated that cells lacking periplasmic trehalase accumulate the sugar in the medium, which suggests that trehalose leaks from the cells possibly via mechanosensitive channels (Styrvold and Strøm, 1991). However, it not clear what mechanisms operate to control the pool size of this compatible solute. Proline, betaine and ectoine are accumulated via the ProP and ProU transport systems (Fig. 3) and may share a common efflux system (Koo et al., 1991). ProP and ProU are activated by osmotic stress and the structural genes are induced during growth at high osmolarity, especially in the absence of the compatible solute itself, which can act to repress expression of the structural genes (Cairney et al., 1985a,b). Again control over the pool size is poorly understood (Rancourt et al., 1984; Csonka, 1988; Koo et al., 1991; Lamark et al., 1992).
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4.2. Staphylococcus aureus In many respects osmoregulation in S. aureus resembles that found in the enteric bacteria. Proline and betaine are accumulated to high levels in response to osmotic stress, free peptides can serve as a source of proline, but this organism does not accumulate sugars as compatible solutes. On the whole, however, there can be seen to be two significant differences. Firstly, there is very much less evidence for regulation of gene expression by osmotic stress (see below). Secondly, the transport systems appear to be constitutively synthesised and to be activated by osmotic stress in a very specific way. There are multiple transport systems for betaine and proline; probably a single specific system each for betaine (BPI) and proline (PutP) and a less specific system that is strongly activated by osmotic stress and results in the accumulation of both proline and betaine (BPII) (Pourkomailian and Booth, 1992) (Fig. 3). Mutants lacking the BPII system are severely compromised in their growth at high osmolarity (Pourkomailian and Booth, 1994). In common with other organisms a complete understanding of the numbers and substrate specificity of each of the compatible solute transport systems may have to await the publication of the genome sequence. Simple kinetic analyses and limited genetic studies will not always reveal the true complexity of the transport options available to the cell (Kempf and Bremer, 1998). The most interesting feature of the BPII betaine– proline transport system is that, in addition to activation of the system by osmotic stress, there is also feedback inhibition by accumulated solute (Pourkomailian and Booth, 1994). Similar control over activity is found in lactic acid bacteria and in L. monocytogenes (see below). In contrast, the equivalent ProP and ProU transport systems of E. coli and S. typhimurium are activated by osmotic stress and remain active at the steady state with the putative efflux system becoming the focus of regulation of the pool (Koo et al., 1991). In contrast, in S. aureus, and in lactic acid bacteria, growth in broth leads to sufficient accumulation of solute that the principal betaine transport systems appear to be only poorly active (Graham and Wilkinson, 1992; Bae et al., 1993; Pourkomailian and Booth, 1994). Demonstration of the system’s
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Fig. 3. Schematic illustration of the compatible solute uptake systems of key food-borne bacteria. Compatible solute abbreviations are as follows: B, betaine; C, carnitine; P, proline; Ch, choline; E, ectoine; PB, proline betaine. SBP indicates the periplasmic substrate binding protein of ProU. PW denotes the peptidoglycan wall; OM, outer membrane; CM, cytoplasmic membrane and IM, inner membrane. * The BetT transporter is not present in S. typhimurium.
activity requires the imposition of osmotic stress that creates a demand for solute pools greater than already achieved, thus overcoming the feedback. Thus, con-
trol over the pool is achieved by the balance of the degree of activation vs. the strength of the feedback mechanism.
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4.3. L. monocytogenes One of the most fascinating features of this organism is the ability to use peptides as a source of compatible solutes. In the laboratory this is readily mimicked by the use of meat peptones as a growth substrate, which might be seen as a model for growth in pate and soft cheeses. Meat peptones contain hydroxyproline-containing peptides as well as glycine-rich peptides. Both of these are accumulated by L. monocytogenes, such that high concentrations both of the free amino acids and complete peptides are found in the cytoplasm of cells grown in high osmolarity medium (Amezega et al., 1995). The pools are sufficiently substantial to contribute significantly to turgor generation. In addition, the pool size is influenced positively by increased osmotic pressure. Provenance for the meat origin of the peptides, rather than synthesis, arises from the presence of hydroxyproline and from the absence of the peptides when cells are grown either with free amino acids or in defined medium. Betaine and carnitine are still the dominant compatible solutes for this organism and the accumulation of betaine leads to lowered peptide pools (Amezega et al., 1995). The accumulation of either betaine or carnitine strongly stimulates the growth of L. monocytogenes at high osmolarity. Recently significant progress has been made in identifying the systems used by L. monocytogenes to accumulate these compounds intracellularly (Ko and Smith, 1999; Sleator et al., 1999; Fraser et al., 2000) (Fig. 4). Betaine is accumulated both by a secondary transporter (BetL) and a substrate binding protein-dependent transporter that is driven by ATP (Gbu). Carnitine is transported by another substrate binding protein-dependent transporter, designated OpuC. Both Gbu and OpuC belong to the ABC transporter superfamily. The regulation of these systems at the genetic and biochemical levels has not yet been studied in detail but a number of interesting features have emerged. The accumulation of carnitine and betaine is subject to trans-inhibition. That is, pre-accumulation of carnitine regulates negatively the activity of the transport systems for either betaine or carnitine. Likewise, the pre-accumulation of betaine inhibits the activity of both the carnitine and betaine uptake system. This finding is in contrast to the betaine uptake systems in
Fig. 4. Osmotic activation of carnitine transport in L. monocytogenes. Schematic representation of feedback regulation of carnitine transport in L. monocytogenes. Rates of transport were measured in cells growing exponentially either in defined medium (DM) or in defined medium with added peptone (DMP) in a phosphate buffer in the presence of chloramphenicol (50 Ag ml 1) at 30 BC, as previously described (Fraser et al., in press). In DM, the rate of transport is high and is independent of the presence or absence of NaCl. With cells grown in DMP and measured in the absence of NaCl, the rate of transport is low due to feedback inhibition by solutes (S). Upon addition of NaCl feedback is relieved and the transport rate is high. Similar observations have been made for betaine transport in L. monocytogenes (Verheul et al., 1997).
E. coli, ProP and ProU, whose activity is apparently not regulated by intracellular substrate concentrations (Koo et al., 1991). Recently, the presence of peptone in the medium has also been found to lead to trans inhibition in L. monocytogenes. Some component of peptone (peptides or free amino acids) is accumulated and the activity of the carnitine uptake system is inhibited (Fraser et al., 2000). Trans inhibition can be overcome by osmotic stimulation; subjecting cells to hyperosmotic shock (in the presence of chloramphenicol) leads to an activation of the uptake systems. This activation is only seen when the compatible solute transport systems are trans inhibited; without solute pre-accumulation the activity of the transporters
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in independent of the medium osmolarity (Verheul et al., 1997; Fraser et al., 2000) (Fig. 4). The molecular basis for these observations remains unclear. One possibility is that the accumulated solutes interact with the cytoplasmic face of the transport systems and inhibit them directly. A second possibility is that pre-accumulated solute affects cell turgor and the transporters respond directly to changes in turgor (perhaps by sensing lateral forces in the cell membrane). Increased turgor arising from the intracellular accumulation of any solute would then lead to inhibition of transporter activity. In this regard it is interesting that the activity of the OpuA betaine transporter of Lactococcus lactis, which is related to Gbu and OpuC from L. monocytogenes (Fraser et al., 2000), is activated by membrane perturbation (Van der Heide and Poolman, 2000). Further studies will be required to determine the molecular basis for trans inhibition of compatible solute uptake in L. monocytogenes. 4.4. Lactic acid bacteria: There are strong similarities between osmoregulation in L. monocytogenes and the lactic acid bacteria. Betaine and carnitine are the two major compatible solutes and the uptake systems show strong activation by osmotic stress. The molecular mechanism of activation of the major betaine transporter, the ATP-de pendent OpuA system, has been investigated in detail in Lactococcus lactis. The purified OpuA system has been reconstituted in liposomes and has been shown to both sense, and respond to, changes in osmotic pressure (Van der Heide and Poolman, 2000). Similar data have been obtained for the simpler ProP system of E. coli (Milner et al., 1994). This means that all of the elements required for osmotic activation of transport are integral to the proteins of the OpuA and ProP. Preliminary data suggest that the OpuA system senses and responds to changes in the physical state of the membrane. Amphiphiles, which insert into the membrane and perturb the curvature of the membrane in a manner predicted to occur in response to osmotic stress, activate OpuA in the absence of osmotic stress (Van der Heide and Poolman, 2000). Similar observations have been made for mechano-transducing channels (see below), which are activated by high turgor.
5. Compatible solutes and cryoprotection In addition to their role as osmoprotectants compatible solutes have also been implicated in cryotolerance. The quaternary amines betaine and carnitine are both found to accumulate at higher levels in L. monocytogenes when this organism is grown at refrigeration temperatures, even in the absence of osmotic stress (Ko et al., 1994; Smith, 1996). A null mutation in gbu, which encodes an ATP-dependent betaine transporter in L. monocytogenes, abolishes the accumulation of betaine at refrigeration temperatures (Ko and Smith, 1999). In addition, the growth of this gbu mutant is markedly impaired at 7 BC (Ko and Smith, 1999), demonstrating the cryoprotective role of betaine in L. monocytogenes. In general, compounds that act as good osmoprotectants in L. monocytogenes are also found to have a cryoprotective effect (Bayles and Wilkinson, 2000). The molecular basis of this cryoprotective effect is not yet known but it is not confined to this food-borne pathogen; the cyanobacterium Synechococcus displays an increased growth rate at low temperatures when engineered to synthesise betaine in the cytoplasm (Deshnium et al., 1997). Betaine accumulation in the plant Arabidopsis thaliana also enhances its ability to tolerate low temperatures. Specifically, the sensitivity of the wild-type plant to light when incubated at 5 BC is not seen in a plant expressing the choline oxidase gene (the product of which catalyses the conversion of choline to betaine) from the bacterium Arthrobacter globiformis (Hayashi et al., 1997). Similar results have recently been obtained with a transgenic plant overproducing glycine betaine (Holmstrøm et al., 2000). A number of possible explanations for the ability of betaine and carnitine to act as cryoprotectants have been suggested. The stabilising effects of compatible solutes on protein structure are well known (Santoro et al., 1992). Ko et al. (1994) have proposed that chill stress may lead to a destabilisation of the tertiary structure of some proteins (either by affecting the mature fully folded structure or by affecting the structure during folding) and this effect may be ameliorated by the presence of compatible solutes. However, no biochemical data are yet available to support this idea. Another possibility is that presence of high concentrations of compatible solute may alter
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the membrane fluidity. Evidence from in vitro studies indicates that at high concentrations (1 M) betaine, proline and trehalose have the ability to reduce the temperature of the liquid crystalline to gel phase transition that occurs in unilamellar lipid vesicles (Rudolph et al., 1986). Deshnium et al. (1997) have also shown that in Synechococcus the lipid phase transition temperature is reduced when the intracellular levels of betaine are elevated. In L. monocytogenes betaine also has an effect on membrane fluidity; cells grown at 5 BC in the presence of betaine show a small increase in anteiso-C15:0 lipids, and these lipids are known to play a role in membrane fluidity in this pathogen (Annous et al., 1997). In E. coli, the synthesis of cyclopropane fatty acids is under the control of the RpoS sigma factor (Wang and Cronan, 1994; Eichel et al., 1999), which accumulates during growth at high osmolarity and low temperature (HenggeAronis, 2000). RpoS levels are reduced by growth with betaine. Thus, betaine could also have effects on the lipid phase transition of enteric bacteria. Further studies will be necessary to determine which of these effects is sufficient to account the cryoprotective effect of betaine.
6. Osmotic stress: induced cross protection Bacteria often coordinately regulate their responses to environmental stress through the activation/ induced expression of specific transcription factors, which are then responsible for triggering the appropriate transcriptional responses. Specific stress-inducible sigma factors are often central to these global responses. In E. coli and S. typhimurium as well as other Gram-negative species the RpoS sigma factor (jS) plays a key role in coordinately regulating gene expression in response to environmental stresses (Loewen et al., 1998; Hengge-Aronis, 2000). Osmotic stress is known to lead to elevated levels of RpoS in the cell (Hengge-Aronis, 1993). This occurs by increasing the rate of translation of the rpoS mRNA and increasing the stability of the jS protein (Jishage et al., 1996; Muffler et al., 1996). In E. coli the jS regulon includes over 50 different genes and the products of these genes confer resistance to wide range of stress conditions including osmotic stress, oxidative stress, starvation and low pH stress (Hengge-Aronis,
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2000). It is clear, therefore, that specific environmental triggers, which lead to elevated levels of jS in the cell, have the potential to confer cross-protection against multiple stresses. It is possible, for example, that an osmotic stress imposed on a bacterium as part of a food preservation regime could inadvertently increase its ability to tolerate other stresses that may also be relevant in the context of food preservation. An increased virulence potential is another possible consequence of jS activation; several genes that contribute to virulence are under jS control (Hengge-Aronis, 2000). Since genes associated with pathogenesis can form part of the regulons under the control of the stress sigma factors, it seems possible that growth under mild stress may potentiate the pathogenic potential of the surviving organisms. Paradoxically, the effect of betaine in reversing RpoS accumulation (HenggeAronis, 1993) may prevent cells from reaching both their potential protected state and developing their pathogenic potential in foods containing adequate supplies of the compatible solute. The reverse scenario also applies; stresses that lead to increased jS levels will also impact the ability of the cell to survive osmotic stress and on the expression of pathogenicity determinants in the surviving organisms. The diversity of systems affected by RpoS has so far precluded animal model experiments that relate to the expression of the specific pathogenicity determinants. In Gram-positive pathogens such as S. aureus and L. monocytogenes, as well as in Bacillus subtilis, many genes with stress-related functions are under the transcriptional control of the sigma factor SigB (jB) (Price, 2000). Like jS, the products of the genes transcribed by jB act to protect cells from a diverse range of environmental stresses, including low pH, oxidative stress, freeze –thaw damage, starvation and high temperature (Vo¨lker et al., 1999). The levels of free jB in the cell are carefully regulated by a complex cascade of regulatory factors, whose ultimate role is to regulate the activity of the anti-sigma factor RsbW (Price, 2000). Various environmental stresses are found to liberate jB from RsbW in B. subtilis, effectively raising its intracellular concentration and allowing it to interact with core RNA polymerase in order to direct transcription of the appropriate genes. The levels of sigB transcript are also elevated in response to environmental stress stimuli (Becker et al., 1998). Hyperosmotic shock is one of the principal
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stresses that leads to increased intracellular levels of jB, and sigB mutants of both B. subtilis (Vo¨lker et al., 1999) and L. monocytogenes (Becker et al., 1998) are found to be impaired in their ability to grow and survive under conditions of high medium osmolarity. In the case of L. monocytogenes it appears that the osmotic sensitivity is due to a defect in compatible solute transport; the transport of the quaternary amines betaine and carnitine is impaired in the absence of jB (Becker et al., 1998; Fraser and O’Byrne, unpublished observations). The overlap between the osmotic stress response and the heat shock response is emphasized by the finding that the j32 and the jH regulons of E. coli are also stimulated by hyperosmotic stress (Bianchi and Baneyx, 1999). The induction of these regulons appears to play a role in allowing cells repair the protein mis-folding that occurs during the loss of turgor following an osmotic shock. It has been proposed that this system operates as an emergency response, allowing the cells the opportunity to induce a jS-directed osmoadaptation response (Bianchi and Baneyx, 1999).
presence of NaCl (Anderson et al., 1991). The molecular basis for this phenomenon is not well understood but it is thought to result from the reduced intracellular concentration of water. It seems likely that water molecules, which become energized by supplying heat, have a potent ability to interact with proteins and accelerate their denaturation. A number of in vitro studies have also found that increased thermostability of proteins is frequently observed when the water activity of the solvent is reduced and that compatible solutes enhance thermostability (Santoro et al., 1992; Knubovets et al., 1999; Castro, 2000). In addition, at least part of this protection may be caused by increased RpoS activity in cells incubated at high osmolarity (Jenkins et al., 1990). In the context of food and food-borne pathogens, therefore, it is essential that osmotic effects on thermosensitivity be taken into account when designing heating regimes for the inactivation of contaminating microbes.
7. Consequences of osmotic stress: thermotolerance
To this point we have mainly dealt with the response to hyperosmotic stress. We have emphasised the need for the selective permeability of the membrane but also for the tight control over solute pools. It has been known for many years that bacteria subjected to rapid hypoosmotic shock, i.e. a sudden decrease in osmolarity, release their pools of amino acids and cations (Britten and McClure, 1962). It has been clear also that this release is often uncontrolled leading to a requirement, at least in the case of E. coli, for the cells to re-accumulate jettisoned nutrients and cations (Berrier et al., 1992; Schleyer et al., 1993). The putative mechanism for this release of solutes was mechanosensitive channels in the bacterial cytoplasmic membrane. The channels were discovered during electrophysiological analysis of giant protoplasts of E. coli (and subsequently several other Gram-positive bacteria). Berrier et al. (1992) demonstrated the potential importance of these channels with the observation that gadolinium ions, which inhibit the activity of mechanosensitive channels, prevent or slow the release of solutes. Subsequently, by a combination of high quality protein biochemistry and molecular biology it has been substantiated that most bacteria have several
Osmotic stress presents specific physical problems for all cells. In addition to the well-studied problems associated with water imbalance (discussed above), other interesting phenomena are also observed in osmotically stressed bacterial cells. Numerous studies have shown that the rate of thermal inactivation of bacteria is closely linked to the osmolarity of the medium. In most cases a reduced water activity leads to an increase in thermotolerance. In L. monocytogenes, for example the loss of viability associated with mild heat stress (56 – 60 BC) is markedly reduced if NaCl is added to the heating menstruum (Anderson et al., 1991; Juneja and Eblen, 1999; Knight et al., 1999). Similar protective effects are observed when S. enteritidis and E. coli O157:H7 (Blackburn et al., 1997) or S. aureus (Shebuski et al., 2000) are heattreated in the presence of increasing salt concentrations. When differential scanning calorimetry (DSC) is used to examine macromolecular changes that occur in heated L. monocytogenes cells, the major changes observed (presumably denaturation events) are shifted to higher temperatures when cells are heated in the
8. Coping with hypoosmotic stress — a new dimension to osmoregulation
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such channels and that they play a very important role in survival of severe hypoosmotic shock (Levina et al., 1999). When bacteria are subjected to hypoosmotic shock, the turgor pressure rises very rapidly due to the influx of water down the solute gradient (i.e., higher solute concentrations in the cytoplasm than outside leads to water influx) (Fig. 1). Although the peptidoglycan wall is a semi-elastic structure it is likely that it is already stretched during growth and possibly close to its elastic limit (Yao et al., 1999). Consequently, the sudden increase in pressure on the wall has the potential to cause wall disruption leading to cell lysis (Levina et al., 1999). The recent work on mechanosensitive channels has revealed for E. coli that there are a number of channels each with a different pressure threshold at which they become activated. Although not proven this is probably true for most free-living bacteria. The severity of the turgor increase determines which channel opens (in E. coli MscM, followed by MscS and then finally MscL; Blount et al., 1996). Since these channels have large electrical conductances in the open state it is considered that they create a large diameter pore in the membrane through which solutes flow indiscriminately. The rapid release of solutes eliminates the driving force for water entry and thus relieves the pressure on the cell wall. In this way the mechanosensitive channels operate to preserve the integrity of the cell. Similar observations have been made for a range of Grampositive bacteria and this accords with the known existence of mechanosensitive channels in these bacteria (Glaasker et al., 1996; Verheul et al., 1997; E. Bremer, personal communication). Clearly the lack of discrimination in the channels between different solutes offers both a hazard to the cell and a potential opportunity to the food microbiologist. The ability of small ions to move through the channels essentially means that the preservation of homeostasis (pH, solute balance, osmolarity, ion, etc.) requires that the channels remain tightly closed until required. When the channels open aberrantly the result can be either growth inhibition (Blount et al., 1997; Ou et al., 1998) or death (Levina et al., 1999). An example of this is that E. coli cells subjected to hypoosmotic shock when incubated at acid pH suffer a several log reduction in viability compare with cells transferred iso-osmotically to acid pH (Levina et al.,
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1999). The imposition of hypoosmotic shock is not in itself a readily realisable treatment for many processed foods. However, compounds that lead to loss of tight control over the channels could potentiate cell death in processed foods when combined with a low pH ( < 5.5) that is often used in such products. Even when the pH is not low enough to kill cells the loss of the permeability barrier may be sufficient to reduce the growth rate. Mechanosensitive channels can be activated by a variety of membrane active compounds, such as local anaesthetics (Martinac et al., 1990). However, while these are not likely to be accepted for addition to food it appears that at least one class of food preservative may act by this route. Butyl paraben is an effective inhibitor of the growth of E. coli cells. Mutants that lack the MscL mechanosensitive channel appear to be less sensitive to butyl paraben, which suggests that either this protein is the target or that other changes in the membrane structure consequent upon loss of MscL are the reason for the altered sensitivity (Stokes and Booth, unpublished data). A search for other membrane active compounds may identify other activators that have significant potential as food preservatives.
Acknowledgements In memorium: The ideas presented in this paper have been shaped by many people, most of whom have left their marks in the literature. Gordon was engaged with this field from the early days of the discovery of the osmoregulated proU promoter and continued to make contributions that are particularly important in the context of the food industry. In reviewing his published work I was struck by the fact that, compared to the work on quorum sensing, his published work on osmoregulation is small. However, in addition to the seminal study on ProU (Park et al., 1989), Gordon was the major contributor to debates on death due to inimical stresses (Dodd et al., 1997) (including low Aw; Armstrong-Buisseret et al., 1995) and the role of RpoS in survival (Rees et al., 1996). Much of this work lives on through work arising from his discussions with close colleagues. The authors wish to thank their close colleagues who have contributed to this review either directly through their published work or indirectly through their comments. I.R.B. is a Wellcome Trust Research
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