APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2003, p. 1–9 0099-2240/03/$08.00⫹0 DOI: 10.1128/AEM.69.1.1–9.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 69, No. 1
MINIREVIEWS A Postgenomic Appraisal of Osmotolerance in Listeria monocytogenes Roy D. Sleator, Cormac G. M. Gahan, and Colin Hill* Department of Microbiology and National Food Biotechnology Centre, University College Cork, Cork, Ireland plasm type, restricted mainly to members of the family Halobacteriaceae (for a review, see reference 26), and (ii) the organic-osmolyte type, an adaptation strategy which has as its hallmarks a minimal requirement for genetic change and a high degree of flexibility in allowing organisms to cope with fluctuations in external osmolarity (77). The organic-osmolyte solution is a biphasic response in which increased levels of K⫹ (and its counterion, glutamate) represent the primary response, followed by a dramatic increase in the cytoplasmic concentrations (⬎1 mol/kg of water [27]) of a number of socalled compatible solutes. Preferential exclusion is the basis for the compatibility of nature’s osmolytes (44). A model proposed by Bull and Breese (16) suggests that compatible solutes may raise the surface tension of water, increasing the cohesive forces between water molecules and making it energetically more difficult to break water-water interactions in favor of water-protein complexes. According to this model, the bulk water hydrates the protein, expelling the higher-surface-tension solute water from the protein surface, thus helping to maintain protein structure and function. In addition to the solute protection theory, Cayley et al. (17) proposed that it is the free cytoplasmic volume or unbound water which is the fundamental determinant of life at elevated osmolarity and that it is the secondary effect of volume increase (itself a consequence of preferential exclusion) which is most important in terms of the osmoprotective effects of compatible solutes. Thus, compatible solutes may play a dual role in osmoregulating cells—helping to restore cell volume while also stabilizing protein structure and function under adverse environmental conditions.
A characteristic feature of the intracellular food-borne pathogen Listeria monocytogenes is its ability to survive and even proliferate under a variety of hostile environmental conditions, particularly elevated osmolarity (10% NaCl) (47) and reduced temperature (⫺0.1°C) (75). Early physiological analysis revealed that this adaptation results, at least in part, from the accumulation of a restricted range of low-molecular-weight molecules termed osmolytes, or compatible solutes, owing to their compatibility with vital cellular processes at high internal concentrations (reviewed in reference 59). Initially restricted to betaine, carnitine, and proline (or proline-containing peptides), the list of compatible solutes promoting both salt and chill tolerances in Listeria has since been extended to include proline betaine, acetylcarnitine, gamma-butyrobetaine, and 3-dimethylsulfoniopropionate (6). Following the elegant biochemical and physiological analyses of the early 1990s, the listerial salt and chill stress response has recently become the focus of intense genetic analysis. A variety of innovative and imaginative molecular techniques led to the identification and subsequent characterization of some of the major genetic elements governing compatible-solute accumulation in Listeria. The recent publication of the listerial genome allows us for the first time to place the earlier experimentally generated data against the background of the in silico-based complete gene set. This exercise permits an informed overview of the complex interplay of systems controlling and enacting the listerial osmo- and cryostress responses. Beginning with a brief overview of the general principles underlying the biophysics of compatible-solute function, we review the development of our understanding of the mechanisms of compatible-solute accumulation in Listeria and use this information to attempt to construct a simplified model incorporating the known salt and chill stress response mechanisms of this ubiquitous and tenacious food-borne pathogen (Fig. 1.) In addition, we examine potential regulatory mechanisms governing osmolyte acquisition and suggest possible future directions in the field of listerial osmo- and cryotolerance.
PHYSIOLOGICAL ANALYSIS OF LISTERIAL OSMOTOLERANCE Some of the earliest physiological investigations into the mechanisms of listerial osmoadaptation involved analysis of the cytoplasmic contents of cultures grown in complex media following hyperosmotic shock, using various combinations of 13 C nuclear magnetic resonance spectroscopy, high-performance liquid chromatography, and amino acid analysis. In 1992, Patchett et al. (50) were the first to observe significant increases in the cytoplasmic concentrations of a number of potentially osmoprotective compounds following a sudden increase in the osmolarity of the bathing solution. At an external [NaCl] of 7.5% (wt/vol), the internal potassium (K⫹) concentration doubled (0.163 to 0.319 mM) while the concentration of glutamate was found to increase fivefold (52.01 to 280.85 mM). These data suggest that, as is the situation for the majority of bacteria, the accumulation of K⫹ (and its counterion, gluta-
BACTERIAL OSMOADAPTATION Osmoadaptation includes both the genetic and physiological manifestations of adaptation to low-water environments (26). In principle, two strategies of osmoadaptation have evolved to cope with life at elevated osmolarity (27): (i) the salt-in-cyto* Corresponding author. Mailing address: Department of Microbiology, University College Cork, Cork, Ireland. Phone: 353-21-4902397. Fax: 353-21-4903101. E-mail:
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FIG. 1. Overview of the listerial salt stress response. Components identified at the molecular level prior to the publication of the genome are separated from those mechanisms identified based on sequence analysis of the Listeria chromosome. The solid arrows indicate the fate of accumulated osmolytes within the cytoplasm, while the dashed arrows indicate probable targets for the transcriptional regulators B and KdpE (RR37).
mate) may constitute the primary response to hyperosmotic shock in Listeria. The other major cytoplasmic components of salt-stressed listerial cells are the amino acid proline, the trimethyl ammonium compound glycine betaine (trimethylglycine), and the structurally related trimethyl amino acid ␥-Ntrimethyl aminobutyrate (L-carnitine) (10, 39, 50). This increase is most likely a result of the so-called secondary response. Beumer et al. (10) demonstrated that, in addition to stimulating growth at elevated osmolarity, exogenously added betaine (1 mM), L-carnitine (1 mM), and proline (10 mM) also significantly enhanced growth rates at reduced temperatures (10°C). Present at relatively high concentrations in sugar beets and other foods of plant origin (40 to 400 mol/g [dry weight] under natural or experimental saline or dry conditions) (56), 130 M betaine stimulates growth of L. monocytogenes, resulting in an 11-fold increase in the growth rate at 8% NaCl and a 1.8-fold increase at 4°C (39). L-Carnitine is most commonly found in foods of animal origin (10), where it has been shown to function in long-chain fatty acid transport across the inner mitochondrial membrane (11). In addition, certain peptides, specifically the proline-containing di- and tripeptides prolylhydroxy-proline (PHP) and prolyl-glycyl-glycine (PGG), also function as effective osmoprotectants, causing an ⬃3-fold increase in the growth rate at 4% NaCl when supplied at a final concentration of 1 mM (2, 3). Increasing NaCl concentrations from 0 to 7.5% in defined medium containing peptone (an enzymatic hydrolysate of meat protein which provides mixtures of free amino acids and peptides) resulted in an increase in the intracellular concentrations of amino acids from 166 to 716 mM, primarily due to increases in the pools of glutamate, glutamine, aspartate, alanine, pro-
line, glycine, and hydroxyproline (50). Since the pools of glutamate, aspartate, and to some extent alanine are inversely related to the specific growth rate, Amezaga (3) claimed that the osmoprotective effect of peptone can be attributed principally to the accumulation of glycine, proline, and hydroxyproline. Analysis of the amino acid pools of cell lysates before and after acid hydrolysis revealed that the total amino acid pool was larger than the pool of free amino acids; thus, not all the peptides are hydrolyzed following uptake. The accumulated peptides are the dipeptide PHP and the tripeptide PGG (intracellular hydrolysis of some of the PGG to prolyl-glycine also resulted in the accumulation of prolyl-glycine). While the extents of growth stimulation were similar for the tripeptide PGG and the dipeptide PHP, competition studies revealed that the tripeptide might be the preferred substrate (2). While Amezaga et al. (2) provided evidence consistent with a protonmotive force (PMF) driven di- and tripeptide transporter, Verheul et al. (74) additionally demonstrated the existence of an ATP-dependent oligopeptide permease capable of transporting peptides of up to eight amino acid residues. Ko et al. (39) demonstrated that maximum betaine uptake occurs at ⬃4% NaCl, resulting in an uptake rate ⬃200 times higher than that observed in the absence of salt and 15-fold higher at 7 than at 30°C. The kinetics of betaine transport for salt- and chill-activated uptake initially suggested that the two transport activities might represent a single system. The lack of betaine uptake in cells subjected to osmotic stress with sucrose in the absence of added Na⫹ suggested a coupling of betaine transport to the Na⫹ gradient. In addition, the results of betaine uptake studies in the presence of the protein synthesis inhibitor chloramphenicol were indistinguishable from those in the absence of the antibiotic, suggesting that the transport
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system or systems are constitutive and that stimulation of transport results primarily from the activation of existing transporters (39). However, since Vmax values were two- to threefold greater if cells were grown under stress, it appears that there may be some degree of transcriptional control above the constitutive level (39). Verheul et al. (72) demonstrated that L-carnitine uptake occurs even under conditions of low osmolarity, reaching a peak at about 2% NaCl and remaining relatively constant at higher salt concentrations (1 M L-carnitine results in an approximate doubling of the growth rate at 4% NaCl [66]). In contrast to that of betaine, the uptake of L-carnitine decreases with decreasing temperature (i.e., 30 nmol/min/mg of protein at 30°C versus 3 nmol/min/mg of protein at 5°C [72]). Nonetheless, significant accumulation of L-carnitine was observed at 7°C (72). Smith (66) postulated that this might be due to a decrease in the rate of carnitine efflux. This accumulation of L-carnitine at low temperatures results in an increase in the specific growth rate; for example, in the absence of NaCl at 7°C, a generation time of 38 h was recorded in the presence of L-carnitine as opposed to 51 h in its absence (66). The high rate of L-carnitine uptake observed for cells grown in defined as well as complex media indicated that the transport system is constitutively expressed rather than induced by high concentrations of L-carnitine (72). Analysis of the effects of the H⫹ATPase inhibitor N,N⬘-dicyclohexylcarbodiimide on L-carnitine uptake showed a complete dissipation of the PMF, a slight increase in the intracellular [ATP] (127% of the control), and a resulting stimulation of L-carnitine transport (146% of the control). This observation, coupled with an inhibition of Lcarnitine transport in the presence of arsenate and vanadate, which decreases intracellular ATP levels, indicates that while transport is independent of PMF, ATP is required as the energy source for the uptake of L-carnitine. In addition to the temperature and osmolarity of the growth medium, Smith (66) demonstrated that the absolute amount of each osmolyte accumulated was dependent on the growth phase, i.e., while the initial osmolyte of choice was betaine, carnitine appears to play an increasingly important role in osmoregulation as the culture ages. Once the principal compatible solutes in Listeria had been identified and their uptake in vivo in whole cells had been studied, the physiological analysis of the mechanisms of Listeria osmotolerance turned to in vitro investigations, i.e., an examination of various transport systems in membrane vesicles, analogous to the cell-free systems used by enzymologists (65). Reduced-minus-oxidized difference spectra revealed peaks at ⬃425 nm, indicating the presence of heme proteins, and ⬃558 nm, suggesting a b-type cytochrome (65). Since electron transport chains are often terminated by cytochrome b (i.e., an O-type oxidase [4]), this observation suggested that energy could be generated in L. monocytogenes membrane vesicles by supplying electrons to an electron transport chain. To test this hypothesis, a transmembrane electrical potential (⌬) was generated using catalytic amounts of phenazine methosulfate as an electron carrier and ascorbate as the electron donor. This energization system generated transient membrane potentials ranging from 111 to 122 mV within the pH range of 5.5 to 7.5. Gerhardt et al. (28) demonstrated that not only could the uptake of [14C]glycine betaine be driven by
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this system in membrane vesicles, the resulting transport could generate an ⬃1,000-fold concentration gradient of betaine (inside-outside). Furthermore, the Km value for betaine (4.4 M) was comparable to that observed in whole cells. While Patchett et al. (51) failed to observe stimulation of betaine uptake in whole cells osmotically shocked with the nonionic solute sucrose, Gerhardt et al. (28) demonstrated that both sucrose and KCl were capable of eliciting active betaine uptake in membrane vesicles with a fixed concentration of NaCl or, more specifically, Na⫹. Thus, activation of this betaine permease, designated betaine porter I, appears to be dependent on osmotic shock rather than ionic strength and as such is relatively effective only when the osmotic stress is provided, or accompanied, by Na⫹. Given that transport generated a 1,000-fold concentration gradient of betaine, which is thermodynamically unfavorable, it became increasingly evident that uptake must be coupled to a favorable process. Using various combinations of the ion-selective ionophores valinomycin, nonactin, and monesin, which dissipate various components of the artificially generated membrane potential (⌬), it was found that ⌬ (positive on the outside) was absolutely required for transport. Therefore, coupling betaine transport to the ⌬-driven influx of Na⫹ allows the electric gradient to power transport of the neutral compatible solute. Since high intracellular concentrations of Na⫹ would surely be detrimental to the cell, the incoming Na⫹ ions have to be removed. To explain this phenomenon, Smith et al. (65) proposed a model based on the existence of an Na⫹-H⫹ antiporter that exchanges the accumulated Na⫹ ions for protons, which are presumably channeled into the electron transport system, generating energy for the cell. In the absence of a hypertonic buffering system, uptake rates were temperature dependent, exhibiting Arrhenius-type behavior within the range of 4 to 15°C. Maximum uptake was observed at 30°C, twice that observed at 7°C. Hence, the phenomenon of chill-activated (7°C optimum) betaine uptake observed in whole cells (39) could not be the result of porter I (28). This transporter alone thus fails to fully explain the general osmotic tolerance (osmotolerance in the absence of NaCl or Na⫹) or chill-activated betaine transport observed in whole cells (39). The existence of an additional betaine uptake system was proved using membrane vesicles prepared from cells grown under conditions of osmotic stress mediated by sucrose and assayed in the absence of Na⫹ (29). This system, designated betaine porter II, was found to be energized by ascorbatephenazine methosulfate and activated by hyperosmotic shock imposed by either sucrose or KCl, independent of the presence of Na⫹ (29). Given that uptake in the absence of Na⫹ was completely energy dependent and that, unlike porter I, no influx of betaine against its concentration gradient was observed, porter II is most likely a member of the ABC-type porters (33). In addition, in contrast to porter I, betaine porter II was found to be highly responsive to chill stress in that in the absence of osmotic activation, the permease could be activated by decreasing temperature within the range of 15 to 4°C, being maximal at 4°C and barely measurable at 15°C. An Arrhenius plot shows a negative activation energy over this temperature range, indicating that the system could be cryoactivated as well as osmotically activated. While the mechanism of activation remains obscure, it is possible that it results from either tem-
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perature-dependent changes in the lipid composition of the fluid portion of the membrane or changes in the topology of the membrane at lower temperatures. In any case, porter II most likely represents the chill-activated betaine transport system previously observed in whole cells by Ko et al. (39). MOLECULAR CHARACTERIZATION OF OSMOLYTE UPTAKE IN LISTERIA Some of the most recent dramatic advances in our understanding of the listerial salt stress response were achieved by using molecular approaches to isolate the genes governing compatible-solute accumulation and subsequently determining the role(s) of these gene-encoded systems in listerial osmotolerance (Fig. 1). Betaine uptake. (i) BetL (betaine porter I). The first genetic element linked to listerial osmotolerance was betL, identified by heterologous complementation of the betaine uptake mutant Escherichia coli MKH13 (60). Hydrophobicity analyses predict that the 507-residue BetL is an integral membrane protein possessing 12 transmembrane domains—a characteristic feature of secondary transporters (57). Indeed, a search for related proteins in the databases revealed significant sequence similarity to the secondary betaine uptake systems OpuD of Bacillus subtilis (57% identity) and BetP of Corynebacterium glutamicum (41% identity). An interesting feature of the betL gene is the presence of a consensus B-dependent promoter-binding site downstream of the vegetative A-dependent promoter, suggesting that in addition to being regulated at the protein level, at least a component of betaine uptake by BetL is regulated at the level of transcription (60). RNA slot blot analysis and reverse transcription-PCR transcription analysis proved that this was indeed the case, in that 15-min exposure to 4% NaCl resulted in a 1.6-fold increase in the level of betL transcription (61). While mutating betL resulted in a significant reduction in the growth rate of the mutant relative to the wild type at elevated osmolarities, no significant differences in the growth rate were observed at reduced temperatures, indicating that BetL is unlikely to play a role in betaine-dependent cryotolerance (61). This observed lack of chill tolerance, coupled with the fact that betL encodes a secondary transporter driven by ion-motive force, confirms that BetL activity corresponds to betaine porter I described by Mendum and Smith (48). The low level of betaine uptake observed against the ⌬betL background (⬃19% that of the wild type) provided additional evidence that betaine uptake in Listeria is mediated by more than one system, most likely the ATP-dependent betaine porter II system described by Gerhardt et al. (29). (ii) GbuABC (betaine porter II). Using a transposon-mediated mutagenesis strategy, Ko and Smith (40) isolated mutants of L. monocytogenes 10403S displaying significantly reduced growth rates under both osmotic and chill stress conditions and a concomitant reduction in betaine uptake (fourfold reduction relative to the wild type at elevated osmolarity and eightfold reduction under chill stress conditions). Analysis of the DNA flanking the transposon insertion site revealed three open reading frames (ORFs) exhibiting significant homology to the betaine uptake systems OpuA of B. subtilis (38) and ProU of E. coli (45). Interestingly, based on homology searches and the existence of a strong ribosomal binding site at the correct
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location, two of the three ORFs identified (gbuA and gbuC) start with the alternative initiation codon TTG (also found for betL). Given the observed dependence of the system on ATP (independent of Na⫹) as the driving force for betaine uptake and the apparent link to betaine-dependent cryotolerance (29, 40), it seems likely that GbuABC activity corresponds to betaine porter II described by Gerhardt et al. (29). The fact that betaine uptake was not completely abolished against the ⌬gbu background is consistent with the previous physiological data suggesting that betaine uptake results from two independent systems, the Na⫹-dependent porter I (BetL) and the ATPdependent betaine porter II (GbuABC). Convincing evidence that this is indeed the case was obtained by assaying the ⌬gbu mutant for betaine uptake in the absence of Na⫹. While the absence of Na⫹ had only a minor effect on betaine uptake in the wild-type strain, it reduced the rate of uptake in the ⌬gbu mutant to about 1% that of the parent, indicating that nearly all the residual betaine uptake observed for ⌬gbu is due to BetL. Further evidence that BetL and GbuABC constitute the principal betaine uptake systems was provided by the work of Wemekamp-Kamphuis et al. (76), in which a mutant of L. monocytogenes LO28 carrying deletions in both betL and gbu exhibits almost no detectable betaine uptake. Exhibiting completely different biochemical properties, each transporter appears to play a unique role in the osmotic adaptation of the organism. Mendum and Smith (48) demonstrated that betaine porter I (BetL) appears to be responsible for the majority of betaine uptake immediately following osmotic upshock, thus providing immediate protection, most likely by activation of preexisting enzyme. However, while BetL does provide some long-term protection against low levels of salt stress (ⱕ3% NaCl; a result of transcriptional up-regulation of the betL gene) (61), it is the ATP-dependent porter II (GbuABC) that plays the dominant role in long-term osmoadaptation, particularly at higher salt concentrations (ⱖ4% NaCl). Carnitine uptake. (i) OpuC. The four genes encoding OpuC, the principal carnitine uptake system in Listeria, were independently identified by two groups (24, 64). In addition to possessing a single B-dependent promoter, similar to the situation for betL, gbuA, and gbuC, the recurring feature of alternative start codons is also observed for the opuCA gene (in this case, GTG rather than TTG is the alternative signal). Mutating opuC results in a significant reduction in both betaine and carnitine uptake, particularly at elevated osmolarity (64). This observed role for OpuC in betaine transport might account for the low-level betaine uptake (⬃1% that of the wild type) observed in the absence of BetL and GbuABC activities and is significant given that the B. subtilis OpuC homologue was originally identified as a betaine uptake system (43). While mutating opuC results in a dramatic reduction in carnitine uptake and a resulting inability to utilize carnitine as an effective osmoprotectant, carnitine uptake against the ⌬opuC background is not completely abolished, indicating the existence of at least one additional transporter for this osmolyte. This apparent degeneracy illustrates the importance of carnitine uptake in Listeria (B. subtilis, by comparison, despite possessing multiple systems for betaine uptake, depends solely on OpuC for carnitine accumulation) and is particularly sig-
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nificant given that carnitine uptake (via OpuC) has recently been shown to play a role in listerial pathogenesis (64). (ii) OpuB. The residual osmolyte uptake observed against a triple-mutant (⌬betL ⌬gbu ⌬opuC) background suggests at least one additional osmolyte uptake system dedicated primarily to the accumulation of carnitine (76). Given the highly conserved nature of the Bacillus carnitine and choline uptake systems, opuC and opuB, respectively, which are separated by only 4.3 kb on the B. subtilis chromosome, Kappes and coworkers (37) suggested that the two systems most likely arose from a common ancestor. Interestingly, analysis of the sequence flanking the listerial opuC homologue reveals significant synteny with that of Bacillus, in that a listerial opuB homologue occurs approximately 2.4 kb downstream of the opuC operon. Sequence analysis of opuB (accession number AF432069) revealed the presence of two ORFs oriented in the same direction and overlapping by 4 nucleotides. Flanked by stem-loop structures and preceded by a consensus B-dependent promoter-binding site, this tight genetic organization suggests that both ORFs (designated opuBA and opuBB, respectively) constitute an operon, which like betL and opuC, may form part of the B regulon. The encoded OpuB proteins exhibit a functional organization similar to that of BusA (OpuA) of Lactococcus lactis. Representing a new functional organization within ABC transporters, the second gene of the operon, opuBB, encodes a 505-residue protein (OpuBB) exhibiting a fusion of the transmembrane and substrate binding domains and a swapping of the N- and C-terminal domains of the substrate binding domain. While the B. subtilis OpuB homologue transports choline, which is subsequently converted to betaine by two intracellular enzymes (GbsA and GbsB), the results of accumulation studies using the triple mutant (⌬betL ⌬gbu ⌬opuC) point to a possible role for OpuB in carnitine uptake in L. monocytogenes LO28 (76). The generation of a quadruple mutant (under way in our laboratory) will make it possible to investigate in more detail the roles of the major osmolytes, particularly betaine and carnitine, under several stress conditions, as this strain would be predicted to be completely impaired in its ability to accumulate either osmolyte. Peptide transport: OppA. Uptake studies revealed the existence of two separate systems dedicated to the accumulation of peptides (i.e., a PMF-driven di- and tripeptide transporter [71] and an ATP-dependent oligopeptide permease [74]). Prior to the publication of the genome sequence, only the oligopeptide permease, OppA, had been fully characterized at the molecular level (14). Originally identified as a partial ORF adjacent to the yjbD and mecA genes on a 3-kb XbaI DNA fragment from L. monocytogenes LO28 (14), the oppA operon encompasses five genes, the highest similarities to which, in terms of peptide identity and operon organization, were found to be the oligopeptide permease opp operon of B. subtilis. As is the situation with betL, opuC, gbuA, and gbuC, the first codon in the predicted ORF encoded by oppA starts with an alternative initiation codon, in this case GTG. The encoded protein (OppA), presumably a lipoprotein attached to the external part of the cytoplasmic membrane, exhibits significant homologies to the substrate binding domains of bacterial oligopeptide transport systems (⬃32% identity to OppA in B. subtilis), as well as to the pheromone-binding proteins (TraC)
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of several conjugative plasmids of Enterococcus faecalis (37% identity). Between oppA and the next ORF, oppB, is a 277-bp noncoding sequence, which contains a potential stem-loop structure located 30 bp downstream from the oppA stop codon. This is particularly significant, since although all the opp genes are transcribed as part of an operon, oppA is predominantly expressed alone. For the remaining genes in the operon, the intercistronic regions are short or absent, with two overlapping regions between oppB and oppC and between oppD and oppF. While OppB and OppC are predicted to form integral membrane proteins with five and six transmembrane-spanning domains, respectively, the remaining ORFs, encoding OppD and OppF, exhibit high similarity to several ATP-binding proteins. Functional inactivation of the oppA operon resulted in a mutant exhibiting resistance to bialaphos, a toxic peptide derivative, thus demonstrating that OppA is functional and capable of transporting peptide analogues. Further evidence that OppA mediates the transport of oligopeptides was obtained by testing bacterial growth in a minimal defined medium where valine, an essential amino acid in L. monocytogenes, was replaced by valine-containing peptides. As expected, the oppA mutant was unable to use peptides longer than three residues, while both the mutant and wild type were capable of growth with a valine-containing tripeptide as the sole source of the amino acid. This is consistent with the findings of Verheul et al. (71) that L. monocytogenes possesses a distinct system for the transport of di- and tripeptides. Another interesting feature of the oppA mutant is its apparent sensitivity to chill stress. While the wild-type strain reached maximum cell density after ⬃15 days, the mutant failed to grow at this temperature during a period of 20 days. In addition, transcriptional analysis revealed that oppA is expressed at 5°C to a higher level than is seen at 37°C. Furthermore, the transcript at 5°C is shorter than that at physiological temperatures (⬃1.8 versus 2 kb), suggesting the presence of an alternative oppA promoter that is specifically activated at lower temperatures (14). While the reason for oppA-dependent growth under chill stress conditions is unclear, a number of possible explanations have been proposed. It has been speculated, for example, that the OppA system might transport specific oligopeptides which act as “cold” pheromones, activating a signal transduction pathway specific for bacterial replication at low temperatures (14). The opp systems of B. subtilis and E. faecalis, for example, are required for sensing the competence pheromone CSF (67) and the pheromones necessary for induction of conjugation, respectively (42). Alternatively, the accumulated peptides may provide a source of proline, previously shown to confer chill stress resistance on Listeria (10). Osmolyte synthesis. Despite confusion in the literature arising from the indiscriminate use of the word “synthesis” to describe situations in which precursor molecules (such as choline in the case of betaine synthesis) are enzymatically converted to the final compatible solute, de novo osmolyte synthesis is rare, being confined largely to oxygenic and anoxygenic phototrophic eubacteria (35). Perhaps the beststudied osmolyte synthesis systems, involving the enzymatic conversion of precursor molecules to the final functional compatible solutes, are those of glycine betaine and proline. While previous investigations suggested that Listeria is incapable of
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synthesizing betaine from choline (3), homology searches of the listerial genome reveal a number of genes exhibiting significant sequence similarities to gbsA (lmo0913, lmo0383, and lmo1179) and gbsB (pduQ, lmo1634, lmo1166, and lmo0554), encoding a glycine betaine aldehyde dehydrogenase and a type III alcohol dehydrogenase, respectively, both of which are involved in the enzymatic conversion of choline to betaine in B. subtilis (12, 13). The lack of a discernible phenotype for betaine synthesis in Listeria suggests that the genes may not be expressed, at least under the physiological conditions tested. In E. coli, for example, betaine synthesis is inhibited under anaerobic conditions by ArcA, the regulatory component of the two-component system ArcA-ArcB, which controls the activities of genes repressed under anaerobic conditions (41). Prior to the publication of the genome, only one listerial osmolyte synthesis system, that of proline, was partially characterized at the molecular level (62). For the majority of bacteria, proline is synthesized from glutamate via three enzymatic reactions catalyzed by ␥-glutamyl kinase (proB), ␥-glutamyl phosphate reductase (proA), and ⌬1-pyrroline-5-carboxylate reductase (P5CR; proC). In general, the proB and proA genes constitute an operon, which is distant from proC on the chromosome (59). The listerial proBA operon was isolated by heterologous complementation of the E. coli proline auxotroph, CSH26 (⌬proBA). Analysis of the genome reveals two proC candidate genes, lmo0396 and lmo1387, encoding proteins with similarities to P5CR2 (34% identity) and P5CR3 (35% identity) of B. subtilis. This degeneracy of potential systems is particularly significant given that Belitsky et al. (9) also recently reported the existence of multiple genes for the last step of proline biosynthesis in B. subtilis. Mutating the proBA operon resulted in proline auxotrophy, with upwards of 10 mM proline required to restore growth to wild-type levels, thus suggesting that unlike B. subtilis (OpuE) or E. coli (PutP), Listeria probably lacks a high-affinity proline transporter (no such homologue can be found in the EGDe genome). While mutating proBA resulted in a salt-sensitive phenotype, particularly at higher salt concentrations (⬎6% NaCl), directed mutagenesis of the listerial proB gene leading to proline overproduction conferred only increased osmotolerance in E. coli but not in Listeria (63). REGULATION No discussion of osmolyte accumulation would be complete without at least a brief overview of the possible mechanisms regulating the systems involved. It is likely that control of osmolyte accumulation in Listeria is achieved at the transcriptional, translational, and posttranslational levels. While sequence similarity suggests that B is regulated similarly in L. monocytogenes and B. subtilis, activation of B in L. monocytogenes was found to be uniquely responsive to temperature and osmotic stress, whereas its activity in B. subtilis is only moderately induced in the case of osmolarity and almost completely undetectable in the case of rapid cold shock (7, 15). This link with the listerial B homologue and the salt and chill stress responses, coupled with the existence of putative Bdependent promoter sites upstream of betL, opuC, and opuB, suggests that at least a component of osmolyte uptake in L. monocytogenes forms part of the B regulon. In support of this
proposal, Becker et al. (7, 8) demonstrated that a B mutant of L. monocytogenes is significantly impaired in its ability to use betaine and carnitine as osmo- and cryoprotectants. Given that neither betaine nor carnitine uptake is completely abolished against the B mutant background, it seems unlikely that B represents the sole transcriptional regulator of the listerial salt and chill stress responses. In betL, for example, the presence of a substrate-inducible B-independent promoter (A) upstream of the B-dependent promoter might compensate for the loss of B, as is the case for opuE in B. subtilis (68). This adoption of “redundant” promoters, and indeed whole gene systems, may have led to a dissemination of the role of B in coordinating transcriptional control among other regulatory systems. One such group of alternative regulators is the GntR transcription regulator family. Possessing 19 members, this group represents the largest family of transcriptional regulatory proteins in L. monocytogenes (30). In L. lactis NCDO763, a gene located upstream of busA (opuA) codes for a GntR transcriptional regulator, which has been shown to act as a repressor of busA (opuA) expression (D. Obis, A. Guillot, and M.-Y. Mistou, abstr. Comp. Biochem. Physiol. A 126:S105, 2000). This is particularly significant given that sequence analysis downstream of the listerial proBA operon revealed a gene exhibiting significant homology (31% identity over 77 residues) to ydfD, a member of the GntR transcription regulator family of B. subtilis (62). In addition to transcriptional regulators, Listeria, like other organisms, may possess proteins that merely modulate transcriptional activity. A functional homologue of H-NS (involved in transcriptional control of proU in E. coli) has also been identified in Listeria; this homologue, named FlaR, appears to play a role in modulating the superhelicity of DNA and is itself induced under conditions of hyperosmotic stress (58). In addition, with 15 histidine kinases and 16 response regulators (30), it is likely that at least one of the resulting two-component systems in L. monocytogenes may also play a role in coordinating the listerial salt and chill stress responses, analogous to the EnvZ-OmpR or PhoP-PhoQ two-component systems of Salmonella. While transcriptional control through alternative sigma factors, and various other regulatory proteins, plays a major role in gene regulation, the final yield of protein product is also determined by translation efficiency. The strength of the ribosomal binding site, the choice of initiation codon, and spacing differences all determine the efficiency with which the ribosomal binding site is recognized by the ribosome (70). While the initiation codon of 91% of E. coli genes is ATG (31), other codons (TTG, GTG, and, more recently, CTG) have also been shown to function in this role (1, 32). The relative translational yields from the three initiation codons are found to be on the order of ATG ⬎ GTG ⬎ TTG in E. coli and ATG ⬎ TTG ⬎ GTG in B. subtilis (1, 55). In E. coli and, to a lesser extent, B. subtilis (due possibly to the existence of stronger Shine-Dalgarno sequences in that organism), it is apparent that the use of non-ATG initiation codons serves to limit expression at the translational level (55). The converse is of course also true; changing the unusual start codon, GTG, of the Pseudomonas putida putidaredoxin reductase gene to ATG resulted in an 18-fold increase in the level of expression of the encoded protein when expressed against the E. coli background (52). These results are particularly significant given that five of the
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known listerial salt stress genes (betL, opuCA, gbuA, gbuC, and oppA) are preceded by alternative initiation codons and as such are presumably regulated, at least to some extent, at the level of translation. Verheul et al. (73) demonstrated that, in addition to being regulated at the transcriptional and translational levels, osmolyte uptake in L. monocytogenes is also controlled at the protein level by a novel osmolyte-sensing mechanism in which uptake of both betaine and carnitine is subject to inhibition by preaccumulated solute. Cytoplasmic betaine inhibits not only the transport of external betaine but also that of carnitine and vice versa. The observed trans inhibition is alleviated upon osmotic upshock, which suggests that alterations in membrane structure are transmitted to the allosteric binding sites for betaine and carnitine of both transporters at the inner surface of the membrane. The linkage of the trans-inhibitory effect to the osmotic strength of the environment is also observed in Lactobacillus plantarum (53) and Staphylococcus aureus (54, 69) and thus may form part of a general strategy to tune the intracellular osmolarity and maintain the cell turgor within certain limits. FUTURE PROSPECTS While it is clear from the data presented in this communication that the secondary response to hyperosmotic shock (i.e., osmoprotectant accumulation) in Listeria has been thoroughly investigated (34, 59) at both the gene and protein levels, the primary response (i.e., the initial phase of osmoadaptation, involving K⫹ uptake) has received considerably less attention. Other than a study by Kallipolitis and Ingmer (36) which identified RR37, a response regulator component of a two-component system exhibiting significant homology to kdpDE (involved in the transcriptional control of the high-affinity K⫹ uptake system KdpFABC of E. coli), no other gene systems involved in K⫹ uptake in Listeria had been identified prior to the publication of the genome. A comparative genomic approach involving homology searches of the listerial chromosomal DNA sequence reveals that L. monocytogenes possesses two K⫹ transporters, a high-affinity system, KdpABC, which exhibits significant similarity to Kdp of E. coli and a low-affinity system, Lmo0993, which resembles both the KtrII system of Enterococcus hirae (32% identity) and the TrkG and TrkH subunits of the E. coli Trk system (20). In addition to the so-called primary and secondary responses to salt stress, a number of osmotolerance-related genes have recently been described which have no apparent involvement in compatible-solute accumulation (21), thus representing a new and interesting facet of the listerial salt stress response. Okada et al. (49), for example, demonstrated that mutating relA {a gene encoding guanosine tetraphosphate and guanosine pentaphosphate [(p)ppGpp] synthase} significantly reduces growth of L. monocytogenes at elevated osmolarity. Since growth of the mutant was apparently unaffected in defined medium with 4% added NaCl, supplemented with either betaine or carnitine, the authors suggest that an appropriate intracellular concentration of (p)ppGpp is essential for full osmotolerance and that its mechanism differs from that for the accumulation of compatible solutes. Similarly, Bayles and Wonderling (D. O. Bayles and L. D. Wonderling, Abstr. 100th
7
Gen. Meet. Am. Soc. Microbiol., abstr. K-16, 2000) isolated an NaCl-sensitive Tn917-induced mutant, with the htrA gene (encoding a serine protease) disrupted, whose ability to utilize compatible solutes is also apparently unaffected. While the hyperosmotic-stress response has been the focus of much intense genetic and physiological analysis in recent years, the listerial hypoosmotic-stress response (i.e., adaptation to a decrease in osmolarity) has until now been largely ignored. For the majority of bacteria studied to date, rapid increases in the water activity of the external environment are countered by a combination of solute and water efflux (59). Stretch-activated, or mechanosensitive, channels mediate rapid solute efflux under hypoosmotic-stress conditions. While patch clamp analysis reveals that E. coli possesses between three and five mechanosensitive channels, only two, MscS and MscL, have been identified at the molecular level. Interestingly, a search for related systems on the listerial genome reveals two genes: lmo2064, which exhibits significant similarity to mscL of E. coli (43% identity at the protein level), and lmo1013, encoding a protein which is 35% identical to a putative mechanosensitive channel from Streptococcus pneumoniae. In addition to mechanosensitive channels, recent evidence suggests that bacteria, like higher plants and animals, possess aquaporins, specific water channels that facilitate rapid water efflux and influx, thus alleviating water stress without dissipating the transmembrane potential (23). Two Listeria genes, glpF and lmo1539, exhibit similarity to known genes for water transporters. While detailed analysis of the Listeria genome thus has the potential to provide important information concerning hitherto-unknown mechanisms of listerial osmotolerance, the story is by no means complete. The sequenced strain EGDe, for example, may lack one or more systems employed by other listerial strains. This is the case for the betaine uptake system BetU of the uropathogenic E. coli strain HU734, which is absent from the sequenced strain K-12 (19). As well as the potential absence of particular genes from the genome of the sequenced strain, significant strain variation in relation to the activity and/or regulation of specific osmolyte uptake systems has also been reported in Listeria (22, 64), thus limiting the information that can be gleaned from the genome sequence of a single strain, at least in the context of a global understanding of the listerial salt stress response. In addition, while homology based on DNA sequence alone provides some indication of protein structure and possible enzymatic function, further detailed physiological analysis will be required to fully characterize the isolated systems. To this end, it is hoped that structural analysis of the proteins encoded by these novel genetic elements (e.g., Lmo2064 [MscL]), using techniques such as X-ray diffraction studies coupled with functional reconstitution into artificial membranes, will provide important insights into the structures and functions of the isolated systems. Rigorous kinetic analysis of the activation mechanisms, in relation to structural studies of the system components, should indicate whether certain structural modifications have a role in osmoregulation or indeed osmosensing—the next major challenge facing the study of the listerial salt stress response. Given the diversity of the systems involved and the existence of multiple physiological signals (internal and external osmolarity, turgor pressure, or related parameters, such as membrane tension [59]), the mechanism of osmo-
8
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sensing remains the most challenging and as yet largely unexplored area of not only listerial osmoadaptation but also that of other bacteria, as well as higher plants and animals. The role of compatible solutes in listerial pathogenesis has been the focus of much attention in recent times and as such provides a new and interesting dimension to the study of the listerial salt stress response; in this respect, Listeria is an excellent model for the study of bacterial osmotolerance from the perspective of an important intracellular pathogen, as opposed to B. subtilis, where environmental survival is most likely the emphasis. In addition to their role in salt and chill tolerance, there is increasing evidence to suggest that osmoprotective compounds, together with their transport and synthesis systems, may function as important virulence factors for certain pathogenic bacteria, e.g., ProP in E. coli (18) and PutP in S. aureus (5). Recent work in our laboratory suggests that inactivating OpuC (and consequently reducing carnitine uptake) in L. monocytogenes LO28 results in a significant reduction in the colonization of the upper small intestine and subsequent systemic infection following peroral inoculation (64). Interestingly, this effect appears to be strain specific and seems to be less dramatic in a knockout mutant of L. monocytogenes ScottA, again highlighting strain differences. Indeed, recent analysis of L. monocytogenes strains with multiple mutations has revealed that OpuC, and not Gbu or BetL, is the principal osmolyte uptake system required during infection in murine models (76). The oligopeptide system OppA also plays a role in the virulence potential of Listeria. In the absence of OppA, bacterial growth was delayed in macrophages in vitro, as well as in the organs of mice during the early phase of infection (14). Quantitative electron microscopy studies confirmed that OppA might be implicated in phagosomal escape, since only 21% of the mutants had reached the macrophage cytoplasm 3 h postinfection compared with 41% for the wild type (14). While osmolyte transport has thus clearly been linked to the virulence potential of L. monocytogenes, the role of osmolyte synthesis in listerial pathogenesis is less obvious. Inactivation of the proBA locus reduces salt tolerance in a complex broth of elevated osmolarity, but it does not appear to affect the virulence potential of the organism when administered to mice via either the intraperitoneal or peroral route (62). This finding reflects that of an earlier study in which Marquis et al. (46), using an uncharacterized proline auxotroph, showed that proline auxotrophy fails to result in reduced virulence, suggesting that the host tissue contains a relatively abundant source of free proline or proline-containing peptides. Furthermore, manipulation of the system to induce proline overproduction also failed to alter the virulence potential of L. monocytogenes (63). The advent of elegant and imaginative techniques for detecting genes expressed both in vivo (25) and at elevated osmolarities, allied to the use of gene chip technology (based on the genome sequence), offers the possibility that a more complete understanding of the relationship between osmostress and virulence is within reach. In conclusion, it is hoped that a detailed understanding of the listerial salt stress response may ultimately lead to the design of effective control measures for this pathogen, either deliberately subverting its essential osmoregulatory systems, i.e., smugglin technology (a term used to describe the use of
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antimicrobial compounds which structurally mimic compatible solutes, e.g., triethylglycine, which inhibits betaine uptake via both BetL and GbuABC [48]), or by limiting the availability of osmolytes, thereby preventing the growth and survival of this food-borne pathogen in products destined for human consumption. ACKNOWLEDGMENTS We acknowledge the financial assistance of the Irish Government under the National Development Plan 2000-2006. We also acknowledge J. M. Wood (University of Guelph, Guelph, Ontario, Canada) for interesting and stimulating discussions. REFERENCES 1. Ambulos, N. P., Jr., T. Smith, W. Mulbry, and P. S. Lovett. 1990. CUG as a mutant start codon for cat-86 and xylE in Bacillus subtilis. Gene 94:125–128. 2. Amezaga, M.-R., I. Davidson, D. McLaggan, A. Verheul, T. Abee, and I. R. Booth. 1995. The role of peptide metabolism in the growth of Listeria monocytogenes ATCC 23074 at high osmolarity. Microbiology 141:41–49. 3. Amezaga, M.-R. 1996. The adaptation of Listeria monocytogenes to osmotic stress. Ph.D. thesis. University of Aberdeen, Aberdeen, Scotland. 4. Anraku, Y. 1988. Bacterial electron transport chains. Annu. Rev. Biochem. 57:101–132. 5. Bayer, A. S., S. N. Coulter, C. K. Stover, and W. R. Schwan. 1999. Impact of the high-affinity proline permease gene (putP) on the virulence of Staphylococcus aureus in experimental endocarditis. Infect. Immun. 67:740–744. 6. Bayles, D. O., and B. J. Wilkinson. 2000. Osmoprotectants and cryoprotectants for Listeria monocytogenes. Lett. Appl. Microbiol. 30:23–27. 7. Becker, L. A., M. S. C ¸ etin, R. W. Hutkins, and A. K. Benson. 1998. Identification of the gene encoding the alternative sigma factor B from Listeria monocytogenes and its role in osmotolerance. J. Bacteriol. 180:4547–4554. 8. Becker, L. A., S. N. Evans, R. W. Hutkins, and A. K. Benson. 2000. Role of B in adaptation of Listeria monocytogenes to growth at low temperature. J. Bacteriol. 182:7083–7087. 9. Belitsky, B. R., J. Brill, E. Bremer, and A. L. Sonenshein. 2001. Multiple genes for the last step of proline biosynthesis in Bacillus subtilis. J. Bacteriol. 183:4389–4392. 10. Beumer, R. R., M. C. Te Giffel, L. J. Cox, F. M. Rombouts, and T. Abee. 1994. Effect of exogenous proline, betaine, and carnitine on growth of Listeria monocytogenes in a minimal medium. Appl. Environ. Microbiol. 60:1359– 1363. 11. Bieber, L. L. 1988. Carnitine. Annu. Rev. Biochem. 57:261–283. 12. Boch, J., B. Kempf, and E. Bremer. 1994. Osmoregulation in Bacillus subtilis: synthesis of the osmoprotectant glycine betaine from exogenously provided choline. J. Bacteriol. 176:5364–5371. 13. Boch, J., B. Kempf, R. Schmid, and E. Bremer. 1996. Synthesis of the osmoprotectant glycine betaine in Bacillus subtilis: characterization of the gbsAB genes. J. Bacteriol. 178:5121–5129. 14. Boreze´e, E., E. Pellegrini, and P. Berche. 2000. OppA of Listeria monocytogenes, an oligopeptide-binding protein required for bacterial growth at low temperature and involved in intracellular survival. Infect. Immun. 68:7069– 7077. 15. Boylan, S. A., A. R. Redfield, M. S. Brody, and C. W. Price. 1993. Stressinduced activation of the B transcription factor of Bacillus subtilis. J. Bacteriol. 175:7931–7937. 16. Bull, H. B., and K. Breese. 1974. Surface tension of amino acid solutions: a hydrophobicity scale of the amino acid residues. Arch. Biochem. Biophys. 161:665–670. 17. Cayley, S., B. A. Lewis, and M. T. Record, Jr. 1992. Origins of the osmoprotective properties of betaine and proline in Escherichia coli K-12. J. Bacteriol. 174:1586–1595. 18. Culham, D. E., C. Dalgado, C. L. Gyles, D. Mamelak, S. Maclellan, and J. M. Wood. 1998. Osmoregulatory transporter ProP influences colonization of the urinary tract by Escherichia coli. Microbiology 144:91–102. 19. Culham, D. E., A. Lu, M. Jishage, K. A. Krogfelt, A. Ishihama, and J. M. Wood. 2001. The osmotic stress response and virulence in pyelonephritis isolates of Escherichia coli: contributions of RpoS, ProP, ProU and other systems. Microbiology 147:1657–1670. 20. Dosch, D. C., G. L. Helmer, S. H. Sutton, F. F. Salvacion, and W. Epstein. 1991. Genetic analysis of potassium transport loci in Escherichia coli: evidence for three constitutive systems mediating uptake of potassium. J. Bacteriol. 173:687–696. 21. Duche´, O., F. Tre´moulet, P. Glaser, and J. Labadie. 2002. Salt stress proteins induced in Listeria monocytogenes. Appl. Environ. Microbiol. 68:1491–1498. 22. Dykes, G. A., and S. M. Moorhead. 2000. Survival of osmotic and acid stress by Listeria monocytogenes strains of clinical or meat origin. Int. J. Food Microbiol. 56:161–166. 23. Engel, A., Y. Fujiyoshi, and P. Agre. 2000. The importance of aquaporin water channel protein structures. EMBO J. 19:800–806.
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