Role of the glycine betaine and carnitine transporters in adaptation of Listeria monocytogenes to chill stress in defined medium

The food-borne pathogen Listeria monocytogenes proliferates at refrigeration temperatures, rendering refrigeration ineffective in the preservation of Listeria-contaminated foods. The uptake and intracellular accumulation of the potent compatible solutes glycine betaine and carnitine has been shown to be a key mediator of the pathogen's cold-tolerant phenotype. To date, three compatible solute systems are known to operate in L. monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and the carnitine transporter OpuC. We investigated the specificity of each transporter towards each compatible solute at 4 degrees C by examining mutant derivatives of L. monocytogenes 10403S that possess each of the transporters in isolation. Kinetic and steady-state compatible solute accumulation data together with growth rate experiments demonstrated that under cold stress glycine betaine transport is primarily mediated by Gbu and that Gbu-mediated betaine uptake results in significant growth stimulation of chill-stressed cells. BetL and OpuC can serve as minor porters for the uptake of betaine, and their action is capable of providing a small degree of cryotolerance. Under cold stress, carnitine transport occurs primarily through OpuC and results in a high level of cryoprotection. Weak carnitine transport occurs via Gbu and BetL, conferring correspondingly weak cryoprotection. No other transporter in L. monocytogenes 10403S appears to be involved in transport of either compatible solute at 4 degrees C, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown at that temperature.     

Gbu glycine betaine porter and carnitine uptake in osmotically stressed Listeria monocytogenes cells

The food-borne pathogen Listeria monocytogenes grows actively under high-salt conditions by accumulating compatible solutes such as glycine betaine and carnitine from the medium. We report here that the dominant transport system for glycine betaine uptake, the Gbu porter, may act as a secondary uptake system for carnitine, with a K(m) of 4 mM for carnitine uptake and measurable uptake at carnitine concentrations as low as 10 microM. This porter has a K(m) for glycine betaine uptake of about 6 micro M. The dedicated carnitine porter, OpuC, has a K(m) for carnitine uptake of 1 to 3 microM and a V(max) of approximately 15 nmol/min/mg of protein. Mutants lacking either opuC or gbu were used to study the effects of four carnitine analogs on growth and uptake of osmolytes. In strain DP-L1044, which had OpuC and the two glycine betaine porters Gbu and BetL, triethylglycine was most effective in inhibiting growth in the presence of glycine betaine, but trigonelline was best at inhibiting growth in the presence of carnitine. Carnitine uptake through OpuC was inhibited by gamma-butyrobetaine. Dimethylglycine inhibited both glycine betaine and carnitine uptake through the Gbu porter. Carnitine uptake through the Gbu porter was inhibited by triethylglycine. Glycine betaine uptake through the BetL porter was strongly inhibited by trigonelline and triethylglycine. These results suggest that it is possible to reduce the growth of L. monocytogenes under osmotically stressful conditions by inhibiting glycine betaine and carnitine uptake but that to do so, multiple uptake systems must be affected.     

Osmotic and chill activation of glycine betaine porter II in Listeria monocytogenes membrane vesicles

Listeria monocytogenes is a foodborne pathogen known for its tolerance to conditions of osmotic and chill stress. Accumulation of glycine betaine has been found to be important in the organism's tolerance to both of these stresses. A procedure was developed for the purification of membranes from L. monocytogenes cells in which the putative ATP-driven glycine betaine permease glycine betaine porter II (Gbu) is functional. As is the case for the L. monocytogenes sodium-driven glycine betaine uptake system (glycine betaine porter I), uptake in this vesicle system was dependent on energization by ascorbate-phenazine methosulfate. Vesicles lacking the gbu gene product had no uptake activity. Transport by this porter did not require sodium ion and could be driven only weakly by artificial gradients. Uptake rates could be manipulated under conditions not affecting secondary transport but known to affect ATPase activity. The system was shown to be both osmotically activated and cryoactivated. Under conditions of osmotic activation, the system exhibited Arrhenius-type behavior although the uptake rates were profoundly affected by the physical state of the membrane, with breaks in Arrhenius curves at approximately 10 and 18 degrees C. In the absence of osmotic activation, the permease could be activated by decreasing temperature within the range of 15 to 4 degrees C. Kinetic analyses of the permease at 30 degrees C revealed K(m) values for glycine betaine of 1.2 and 2.9 microM with V(max) values of 2,200 and 3,700 pmol/min. mg of protein under conditions of optimal osmotic activation as mediated by KCl and sucrose, respectively.     

Sodium-driven, osmotically activated glycine betaine transport in Listeria monocytogenes membrane vesicles.

Transport of the osmoprotectant and cryoprotectant glycine betaine was investigated in membrane vesicles of Listeria monocytogenes. Uptake-driving transmembrane potentials ranging from 111 to 122 mV within the pH range of 5.5 to 7.5 could be generated by the electron donor system ascorbate-phenazine methosulfate but not by the electron donor system ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine. Transport was dependent on both high concentrations of sodium ion and the presence of a hypertonic solute gradient. Arrhenius-type temperature activation was observed. Lineweaver-Burk plots indicated a Km of 4.4 microM for glycine betaine and a Vmax of 700 pmol/min x mg of protein. The Michaelis constant for NaCl depended on the solute used to maintain a constant hyperosmotic pressure, and the Km values were 200 and 75 mM when KCl and sucrose were employed, respectively. Transport was 65% lower in vesicles derived from cells grown under stress provided by KCI rather than NaCl and approximately 94% lower in vesicles derived from cells that were not grown under osmotic stress. This porter appears to be specific for glycine betaine, since neither proline, carnitine, nor choline inhibited uptake effectively. Kinetic studies using ionophores and artificial gradients indicate that glycine betaine is cotransported with sodium ion.     

Identification of an ATP-driven, osmoregulated glycine betaine transport system in Listeria monocytogenes.

The ability of the gram-positive, food-borne pathogen Listeria monocytogenes to tolerate environments of elevated osmolarity and reduced temperature is due in part to the transport and accumulation of the osmolyte glycine betaine. Previously we showed that glycine betaine transport was the result of Na(+)-glycine betaine symport. In this report, we identify a second glycine betaine transporter from L. monocytogenes which is osmotically activated but does not require a high concentration of Na(+) for activity. By using a pool of Tn917-LTV3 mutants, a salt- and chill-sensitive mutant which was also found to be impaired in its ability to transport glycine betaine was isolated. DNA sequence analysis of the region flanking the site of transposon insertion revealed three open reading frames homologous to opuA from Bacillus subtilis and proU from Escherichia coli, both of which encode glycine betaine transport systems that belong to the superfamily of ATP-dependent transporters. The three open reading frames are closely spaced, suggesting that they are arranged in an operon. Moreover, a region upstream from the first reading frame was found to be homologous to the promoter regions of both opuA and proU. One unusual feature not shared with these other two systems is that the start codons for two of the open reading frames in L. monocytogenes appear to be TTG. That glycine betaine uptake is nearly eliminated in the mutant strain when it is assayed in the absence of Na(+) is an indication that only the ATP-dependent transporter and the Na(+)-glycine betaine symporter occur in L. monocytogenes.     

Two evolutionarily closely related ABC transporters mediate the uptake of choline for synthesis of the osmoprotectant glycine betaine in Bacillus subtilis

Biosynthesis of the compatible solute glycine betaine in Bacillus subtilis confers a considerable degree of osmotic tolerance and proceeds via a two-step oxidation process of choline, with glycine betaine aldehyde as the intermediate. We have exploited the sensitivity of B. subtilis strains defective in glycine betaine production against glycine betaine aldehyde to select for mutants resistant to this toxic intermediate. These strains were also defective in choline uptake, and genetic analysis proved that two mutations affecting different genetic loci (opuB and opuC) were required for these phenotypes. Molecular analysis allowed us to demonstrate that the opuB and opuC operons each encode a binding protein-dependent ABC transport system that consists of four components. The presumed binding proteins of both ABC transporters were shown to be lipoproteins. Kinetic analysis of [14C]-choline uptake via OpuB (K(m) = 1 microM; Vmax = 21 nmol min-1 mg-1 protein) and OpuC (K(m) = 38 microM; Vmax = 75 nmol min-1 mg-1 protein) revealed that each of these ABC transporters exhibits high affinity and substantial transport capacity. Western blotting experiments with a polyclonal antiserum cross-reacting with the presumed substrate-binding proteins from both the OpuB and OpuC transporter suggested that the expression of the opuB and opuC operons is regulated in response to increasing osmolality of the growth medium. Primer extension analysis confirmed the osmotic control of opuB and allowed the identification of the promoter of this operon. The opuB and opuC operons are located close to each other on the B. subtilis chromosome, and their high sequence identity strongly suggests that these systems have evolved from a duplication event of a primordial gene cluster. Despite the close relatedness of OpuB and OpuC, these systems exhibit a striking difference in substrate specificity for osmoprotectants that would not have been predicted readily for such closely related ABC transporters.     

Characterization of glycine betaine porter I from Listeria monocytogenes and its roles in salt and chill tolerance

Listeria monocytogenes is a pathogenic bacterium that can grow at low temperatures and elevated osmolarity. The organism survives these stresses by the intracellular accumulation of osmolytes: low-molecular-weight organic compounds which exert a counterbalancing force. The primary osmolyte in L. monocytogenes is glycine betaine, which is accumulated from the environment via two transport systems: glycine betaine porter I, an Na(+)-glycine betaine symporter; and glycine betaine porter II, an ATP-dependent transporter. The biochemical characteristics of glycine betaine porter I were investigated in a mutant strain (LTG59) lacking the ATP-dependent transporter. At 4% NaCl, glycine betaine uptake in LTG59 was about fivefold lower than in strain DP-L1044, which has both transporters, indicating that the ATP-dependent transporter is the primary means by which glycine betaine enters the cell. In the absence of osmotic stress, cold-activated uptake by both transporters was most rapid between 7 and 12 degrees C, but a larger fraction of the total uptake was via the ATP-dependent transporter than was observed under salt-stressed conditions. Twelve glycine betaine analogs were tested for their ability to inhibit glycine betaine uptake and growth of stressed cultures. Carnitine, dimethylglycine, and gamma-butyrobetaine appear to inhibit the ATP-dependent transporter, while trigonelline and triethylglycine primarily inhibit glycine betaine porter I. Triethylglycine was also able to retard the growth of osmotically stressed L. monocytogenes grown in the presence of glycine betaine.     

Changes in Listeria monocytogenes membrane fluidity in response to temperature stress

Listeria monocytogenes is a food-borne pathogen that has been implicated in many outbreaks associated with ready-to-eat products. Listeria adjusts to various stresses by adjusting its membrane fluidity, increasing the uptake of osmoprotectants and cryoprotectants, and activating the sigma(B) stress factor. The present work examines the regulation of membrane fluidity through direct measurement based on fluorescent anisotropy. The membrane fluidities of L. monocytogenes Scott A, NR30, wt10403S, and cld1 cells cultured at 15 and 30 degrees C were measured at 15 and 30 degrees C. The membrane of the cold-sensitive mutant (cld1) was more rigid than the membranes of the other strains when grown at 30 degrees C, but when grown at 15 degrees C, it was able to adjust its membrane to approach the rigidity of the other strains. The difference in rigidities, as determined at 15 and 30 degrees C, was greater in liposomes than in whole cells. The rates of fluidity adjustment and times required for whole cells to adjust to a different temperature were similar among strains but different from those of liposomes. This suggests that the cells had a mechanism for homeoviscous adaptation that was absent in liposomes.     

Osmoprotection by carnitine in a Listeria monocytogenes mutant lacking the OpuC transporter: evidence for a low affinity carnitine uptake system

A deletion mutant of Listeria monocytogenes lacking OpuC, an ABC transporter responsible for the uptake of the compatible solute carnitine, was constructed and carnitine transport assays confirmed that carnitine transport was defective in this mutant. However, the mutant retained the ability to derive osmoprotection from carnitine, suggesting the presence of a second uptake system for this compatible solute. Measurement of intracellular carnitine pools during balanced growth confirmed that the opuC mutant accumulated high levels of carnitine. These pools were only achieved in the mutant when high levels (1 mM) of carnitine were present extracellularly. When a lower level (100 microM) was supplied in the medium the mutant failed to accumulate a substantial intracellular pool and failed to derive osmoprotection from carnitine. These data suggest the presence of a second low affinity carnitine uptake system in this osmotolerant pathogen.     

Betaine and carnitine uptake systems in Listeria monocytogenes affect growth and survival in foods and during infection

AIMS: To establish the relative importance of the osmo- and cryoprotective compounds glycine betaine and carnitine, and their transporters, for listerial growth and survival, in foods and during infection. METHODS AND RESULTS: A set of Listeria monocytogenes mutants with single, double and triple mutations in the genes encoding the principal betaine and carnitine uptake systems (gbu, betL and opuC, respectively) was used to determine the specific contribution of each transporter to listerial growth and survival. Food models were chosen to represent high-risk foods of plant and animal origin i.e. coleslaw and frankfurters, which have previously been linked to major human outbreaks of listeriosis. BALB/c mice were used as an in vivo model of infection. Interestingly, while betaine appeared to confer most protection in foods, the hierarchy of transporter importance differs depending on the food type: Gbu>BetL>OpuC for coleslaw, as opposed to Gbu>OpuC>BetL in frankfurters. By contrast in the animal model, OpuC and thus carnitine, appears to play the dominant role, with the remaining systems contributing little to the infection process. CONCLUSIONS: This study demonstrates that the individual contribution of each system appears dependent on the immediate environment. In foods Gbu appears to play the dominant role, while during infection OpuC is most important. SIGNIFICANCE AND IMPACT OF THE STUDY: It is envisaged that this information may ultimately facilitate the design of effective control measures specifically targeting this pathogen in foods and during infection.     

Listeria monocytogenes sigma B regulates stress response and virulence functions

While the stress-responsive alternative sigma factor sigma(B) has been identified in different species of Bacillus, Listeria, and Staphylococcus, the sigma(B) regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify sigma(B)-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidate sigma(B)-dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted sigma(B)-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and Delta sigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significant sigma(B)-dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-fold-higher expression in the wild type). Of the 55 genes exhibiting sigma(B)-dependent expression, 54 were preceded by a sequence resembling the sigma(B) promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the sigma(B)-dependent nature of a subset of eight selected promoter regions. Notably, the sigma(B)-dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, sigma(B) also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest that sigma(B) contributes to L. monocytogenes gene expression during infection.     

Glycine betaine improves Listeria monocytogenes tolerance to desiccation on parsley leaves independent of the osmolyte transporters BetL, Gbu and OpuC

Aims: To investigate the effect of glycine betaine (GB) on the survival of Listeria monocytogenes on leaf surfaces under low relative humidity (RH). Methods and Results: The addition of GB (≥25 mmol l^?1) improved the survival of L. monocytogenes under low RH on parsley leaves, thus suggesting that GB can improve the tolerance of L. monocytogenes to desiccation. Ten times less GB was needed to improve L. monocytogenes survival under low RH on nonbiological surfaces compared with parsley leaves, suggesting that, on the leaf surface, L. monocytogenes may have to compete for the available GB with autochthonous bacteria and/or the plant itself. Wild type and mutants carrying deletions in the three GB uptake systems, BetL, Gbu and OpuC, behaved similarly with and without added GB on parsley leaves (P > 0·05). In addition, preaccumulation of GB, triggered by osmotic stress prior to inoculation, failed to improve survival under low RH compared with osmotic stress without GB accumulation. Conclusions: Exogenous GB had a protective effect on L. monocytogenes cells from desiccation during survival on parsley leaves. This effect was independent of intracellular GB accumulation by the known uptake systems. Significance and Impact of the Study: Presence of GB could improve the survival of L. monocytogenes to desiccation on leaf surfaces and nonbiological surfaces.     

Translocation and metabolism of glycine betaine by barley plants in relation to water stress

The glycine betaine which accumulated in shoots of young barley plants (Hordeum vulgare L.) during an episode of water stress did not undergo net destruction upon relief of stress, but its distribution among plant organs changed. During stress, betaine accumulated primarily in mature leaves, whereas it was found mainly in young leaves after rewatering. Well-watered, stressed, and stressed-rewatered plants were supplied with [methyl-¹⁴C]betaine (8.5 nmol) via an abraded spot on the second leaf blade, and incubated for 3 d. In all three treatments the added ¹⁴C migrated more or less extensively from the second leaf blade, but was recovered quantitatively from various plant organs in the form of betaine; no labeled degradation products were found in any organ. When 0.5 mol of [methyl-¹⁴C]betaine was applied via an abraded spot to the second leaf blades of well-watered, mildly-stressed, and stressed-rewatered plants, ¹⁴C was translocated out of the blades at velocities of about 0.2–0.3 cm/min which were similar to velocities found for applied [¹⁴C]sucrose. Heat-girdling of the sheath prevented export of [¹⁴C]betaine from the blade. When 0.5 mol [³H]sucrose and 0.5 mol [¹⁴C]betaine were suppled simultaneously to second leaf blades, the ³H/¹⁴C ratio in the sheath tissue was the same as that of the supplied mixture. After supplying tracer [¹⁴C]betaine aldehyde (the immediate precursor of betaine) to the second leaf blade, the ¹⁴C which was translocated into the sheath was in the form of betaine. Thus, betaine synthesized by mature leaves during stress behaves as an inert end product and upon rewatering is translocated to the expanding leaves, most probably via the phloem. Accordingly, it is suggested that the level of betaine in a barley plant might serve as a useful cumulative index of the water stress experienced during growth.     

Identification and disruption of BetL, a secondary glycine betaine transport system linked to the salt tolerance of Listeria monocytogenes LO28

The trimethylammonium compound glycine betaine (N,N, N-trimethylglycine) can be accumulated to high intracellular concentrations, conferring enhanced osmo- and cryotolerance upon Listeria monocytogenes. We report the identification of betL, a gene encoding a glycine betaine uptake system in L. monocytogenes, isolated by functional complementation of the betaine uptake mutant Escherichia coli MKH13. The betL gene is preceded by a consensus sigmaB-dependent promoter and is predicted to encode a 55-kDa protein (507 amino acid residues) with 12 transmembrane regions. BetL exhibits significant sequence homologies to other glycine betaine transporters, including OpuD from Bacillus subtilis (57% identity) and BetP from Corynebacterium glutamicum (41% identity). These high-affinity secondary transporters form a subset of the trimethylammonium transporter family specific for glycine betaine, whose substrates possess a fully methylated quaternary ammonium group. The observed Km value of 7.9 microM for glycine betaine uptake after heterologous expression of betL in E. coli MKH13 is consistent with values obtained for L. monocytogenes in other studies. In addition, a betL knockout mutant which is significantly affected in its ability to accumulate glycine betaine in the presence or absence of NaCl has been constructed in L. monocytogenes. This mutant is also unable to withstand concentrations of salt as high as can the BetL+ parent, signifying the role of the transporter in Listeria osmotolerance.     

The role of L-carnitine and glycine betaine in the survival and sub-lethal injury of non-growing Listeria monocytogenes cells during chilled storage

AIMS: To determine the role played by previous growth in the presence of osmolytes on the subsequent survival and sub-lethal injury of L. monocytogenes during long-term chilled storage in a model buffer system. METHODS AND RESULTS: Four Listeria monocytogenes strains were grown separately to stationary phase in Listeria minimal medium (DM) alone or in DM with 4% NaCl alone, or both these media supplemented with 1 mM L-carnitine and/or 1 mM glycine betaine. Cells were resuspended in phosphate buffered saline (pH 5.5) and stored for four weeks at 4 degrees C. Initially, and at weekly intervals, samples were plated on both Tryptic Soy Agar and Tryptic Soy Agar with 4% NaCl to determine total numbers and degree of sub-lethal injury in the populations. The numbers of cells within all strains after growth to stationary phase, except one which increased ( approximately 2 log cfu ml-1, P < 0.05) in the presence of NaCl, were not influenced significantly by previous growth conditions (P > 0.05). During subsequent chilled storage, however, numbers of all strains grown in the presence of NaCl remained constant while those grown in its absence decreased. The rate and magnitude of the decrease in cell numbers was strain dependent. The initial percentage of sub-lethal injury increased significantly in all strains when grown previously in the presence of L-carnitine (P < 0.05). During subsequent chilled storage sub-lethal injury increased for all strains in a manner that was strain dependent, but not related to the previous growth conditions. CONCLUSION: Previous growth in the presence of osmolytes of NaCl, but not osmolytes alone, increases the subsequent survival, but not percentage sub-lethal injury, of L. monocytogenes during subsequent chilled storage in buffer. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that risks associated with L. monocytogenes in chilled food may be influenced by the individual life histories of the cells.     

Influence of interactions between temperature,ferric ammonium citrate and glycine betaine on the growth of Listeria monocytogenes in a defined medium

AIMS: To investigate interactions, if any, between temperature, ferric ammonium citrate and glycine betaine on the growth of Listeria monocytogenes in modified Pine's medium (Pine et al. 1989). METHODS AND RESULTS: Modified Pine's medium containing 0, 0.044, 0.088 or 0.176 g l(-1) ferric ammonium citrate, and 0 or 1 mM glycine betaine, was inoculated with each of two L. monocytogenes strains and incubated at 4, 25 or 37 degrees C. The optical density at 600 nm, and cell numbers, were determined at appropriate time intervals. At 4 degrees C, but not other temperatures, increasing ferric ammonium citrate resulted in improved growth in the absence, but not the presence, of glycine betaine. The presence of glycine betaine was inhibitory at 25 and 37 degrees C, but not at 4 degrees C. CONCLUSIONS: Interactions affecting the growth kinetics of L. monocytogenes were apparent between the parameters investigated. SIGNIFICANCE AND IMPACT OF THE STUDY: Limitations on the use of modified Pine's medium, and the significance of iron metabolism at lower temperatures, were revealed.     

Comparative proteomic analysis of Listeria monocytogenes tolerance to bile stress

The ability of the food-borne pathogen Listeria monocytogenes to tolerate bile is critical to its successful infection and colonization in the human gastrointestinal tract. Using comparative proteomics, a total of 48 proteins were identified in this study in the presence of moderate (0.3 %) or high (3 %) level of bile salts in the wild-type strain EGD. Identified proteins fell into 14 functional categories covering most of the biochemical functions of bacterial cells, indicating that there were complex physiological mechanisms involved in L. monocytogenes tolerance of bile stress. Among them, 16, 14, and 18 proteins were expressed differently in the isogenic deletion mutants of L. monocytogenes EGDΔsigB, EGDΔprfA, and EGDΔprfAΔsigB, respectively, compared with their parent strain EGD at corresponding concentrations of bile salts. All proteins identified in EGDΔsigB and EGDΔprfAΔsigB were all down-expressed in the presence of bile salts, whereas several proteins were up-expressed in EGDΔprfA, in particular at the high level of bile (3 %), indicating that SigB plays an essential positive role in L. monocytogenes tolerance of bile stress and that the negative effect of PrfA may facilitate its survival in bile in the gastrointestinal tract before its successful colonization and invasion.