Bacteriocin inhibition of two glucose transport systems in Listeria monocytogenes

Listeria monocytogenes transports glucose by proton motive force-mediated and phosphoenolpyruvate-dependent phosphotransferase systems (PEP-dependent PTS). Inhibition of both systems by nisin, pediocin JD and leuconosin S is reported here for four strains of L. monocytogenes . Intracellular and extracellular adenosine triphosphate (ATP) and extracellular inorganic phosphate were measured in energized L. monocytogenes Scott A cells to determine whether inhibition of the PEP-dependent PTS might occur as a result of bacteriocin-induced leakage of intracellular components. Addition of nisin resulted in a decrease in intracellular ATP with an increase in extracellular ATP. Leuconosin S and pediocin JD induced a depletion of intracellular ATP. ATP efflux was low for the leuconosin S-treated cells and barely detectable for pediocin JD-treated cells. Addition of nisin, leuconosin S and pediocin JD induced efflux of inorganic phosphate. It appears that bacteriocin-mediated inhibition of the glucose PEP-dependent PTS occurs as a result of hydrolysis or efflux of ATP, PEP and other essential molecules from L. monocytogenes cells.     

Bacteriocins inhibit glucose PEP:PTS activity in Listeria monocytogenes by induced efflux of intracellular metabolites

Glucose transport by the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) of Listeria monocytogenes is inhibited by the bacteriocins nisin, pediocin JD and leuconocin S. To investigate the mechanism of inhibition, PTS activity assays were performed with permeabilized, bacteriocin-treated L. monocytogenes Scott A cells. In the presence of exogenous PEP, nisin stimulated the PTS while both pediocin JD and leuconocin S partially inhibited its activity. These results suggested that PTS enzymes were still active in bacteriocin-treated cells and that bacteriocin-induced PEP efflux may be a mechanism for inhibition of the PTS. To verify that PEP did efflux from bacteriocin-treated L. monocytogenes Scott A cells, intracellular and extracellular PEP were measured by HPLC. All three bacteriocins induced efflux of PEP. Nisin, pediocin JD and leuconocin S also induced efflux of AMP, ADP and ATP. These studies indicate that bacteriocin inhibition of the glucose PEP:PTS in L. monocytogenes is due to efflux of intracellular metabolites, particularly PEP.     

Collapse of the proton motive force in Listeria monocytogenes caused by a bacteriocin produced by Pediococcus acidilactici.

The effect of pediocin JD, a bacteriocin produced by Pediococcus acidilactici JD1-23, on the proton motive force and proton permeability of resting whole cells of Listeria monocytogenes Scott A was determined. Control cells, treated with trypsin-inactivated bacteriocin at a pH of 5.3 to 6.1, maintained a pH gradient and a membrane potential of approximately 0.65 pH unit and 75 mV, respectively. However, these gradients were rapidly dissipated in cells after exposure to pediocin JD, even though no cell lysis had occurred. The pH gradient and membrane potential of the producer cells were also unaffected by the bacteriocin. Whole cells treated with bacteriocin were twice as permeable to protons as control cells were. The results suggest that the inhibitory action of pediocin JD against L. monocytogenes is directed at the cytoplasmic membrane and that inhibition of L. monocytogenes may be caused by the collapse of one or both of the individual components of the proton motive force.     

Collapse of the proton motive force in Listeria monocytogenes caused by a bacteriocin produced by Pediococcus acidilactici.

The effect of pediocin JD, a bacteriocin produced by Pediococcus acidilactici JD1-23, on the proton motive force and proton permeability of resting whole cells of Listeria monocytogenes Scott A was determined. Control cells, treated with trypsin-inactivated bacteriocin at a pH of 5.3 to 6.1, maintained a pH gradient and a membrane potential of approximately 0.65 pH unit and 75 mV, respectively. However, these gradients were rapidly dissipated in cells after exposure to pediocin JD, even though no cell lysis had occurred. The pH gradient and membrane potential of the producer cells were also unaffected by the bacteriocin. Whole cells treated with bacteriocin were twice as permeable to protons as control cells were. The results suggest that the inhibitory action of pediocin JD against L. monocytogenes is directed at the cytoplasmic membrane and that inhibition of L. monocytogenes may be caused by the collapse of one or both of the individual components of the proton motive force.     

Listeria monocytogenes Scott A transports glucose by high-affinity and low-affinity glucose transport systems.

Listeria monocytogenes transported glucose by a high-affinity phosphoenolpyruvate-dependent phosphotransferase system and a low-affinity proton motive force-mediated system. The low-affinity system (Km = 2.9 mM) was inhibited by 2-deoxyglucose and 6-deoxyglucose, whereas the high-affinity system (Km = 0.11 mM) was inhibited by 2-deoxyglucose and mannose but not 6-deoxyglucose. Cells and vesicles artificially energized with valinomycin transported glucose or 2-deoxyglucose at rates greater than those of de-energized cells, indicating that a membrane potential could drive uptake by the low-affinity system.     

Depletion of proton motive force by nisin in Listeria monocytogenes cells.

The basal proton motive force (PMF) levels and the influence of the bacteriocin nisin on the PMF were determined in Listeria monocytogenes Scott A. In the absence of nisin, the interconversion of the pH gradient (Z delta pH) and the membrane potential (delta psi) led to the maintenance of a fairly constant PMF at -160 mV over the external pH range 5.5 to 7.0. The addition of nisin at concentrations of greater than or equal to 5 micrograms/ml completely dissipated PMF in cells at external pH values of 5.5 and 7.0. With 1 microgram of nisin per ml, delta pH was completely dissipated but delta psi decreased only slightly. The action of nisin on PMF in L. monocytogenes Scott A was both time and concentration dependent. Valinomycin depleted only delta pH, whereas nigericin and carbonyl cyanide m-chlorophenylhydrazone depleted only delta psi, under conditions in which nisin depleted both. Four other L. monocytogenes strains had basal PMF parameters similar to those of strain Scott A. Nisin (2.5 micrograms/ml) also completely dissipated PMF in these strains.     

Uncoupler-Resistant Glucose Uptake by the Thermophilic Glycolytic Anaerobe Thermoanaerobacter thermosulfuricus (Clostridium thermohydrosulfuricum)

The transport of glucose across the bacterial cell membrane of Thermoanaerobacter thermosulfuricus (Clostridium thermohydrosulfuricum) Rt8.B1 was governed by a permease which did not catalyze concomitant substrate transport and phosphorylation and thus was not a phosphoenolpyruvate-dependent phosphotransferase. Glucose uptake was carrier mediated, could not be driven by an artificial membrane potential (Δψ) in the presence or absence of sodium, and was not sensitive to inhibitors which dissipate the proton motive force (Δp; tetrachlorosalicylanilide, N,N-dicyclohexylcarboiimide, and 2,4-dinitrophenol), and no uptake of the nonmetabolizable analog 2-deoxyglucose could be demonstrated. The glucokinase apparent K_m for glucose (0.21 mM) was similar to the K_t (affinity constant) for glucose uptake (0.15 mM), suggesting that glucokinase controls the rate of glucose uptake. Inhibitors of ATP synthesis (iodoacetate and sodium fluoride) also inhibited glucose uptake, and this effect was due to a reduction in the level of ATP available to glucokinase for glucose phosphorylation. These results indicated that T. thermosulfuricus Rt8.B1 lacks a concentrative uptake system for glucose and that uptake is via facilitated diffusion, followed by ATP-dependent phosphorylation by glucokinase. In T. thermosulfuricus Rt8.B1, glucose is metabolized by the Embden-Meyerhof-Parnas pathway, which yields 2 mol of ATP (G. M. Cook, unpublished data). Since only 1 mol of ATP is used to transport 1 mol of glucose, the energetics of this system are therefore similar to those found in bacteria which possess a phosphotransferase.     

Glucose uptake by the cellulolytic ruminal anaerobe Bacteroides succinogenes.

Glucose uptake by Bacteroides succinogenes S85 was measured under conditions that maintained anaerobiosis and osmotic stability. Uptake was inhibited by compounds which interfere with electron transport systems, maintenance of proton or metal ion gradients, or ATP synthesis. The most potent inhibitors were proton and metal ionophores. Oxygen strongly inhibited glucose uptake. Na+ and Li+, but not K+, stimulated glucose uptake. A variety of sugars, including alpha-methylglucoside, did not inhibit glucose uptake. Only cellobiose and 2-deoxy-D-glucose were inhibitory, but neither behaved as a competitive inhibitor. Metabolism of both sugars appeared to be responsible for the inhibition. Cells grown in cellobiose medium transported glucose at one-half the rate of glucose-grown cells. Spheroplasts transported glucose as well as whole cells, indicating glucose uptake is not dependent on a periplasmic glucose-binding protein. Differences in glucose uptake patterns were detected in cells harvested during the transition from the lag to the log phase of growth compared with cells obtained during the log phase. These differences were not due to different mechanisms for glucose uptake in the cell types. Based on the results of this study, B. succinogenes contains a highly specific, active transport system for glucose. Evidence of a phosphoenolpyruvate-glucose phosphotransferase system was not found.     

Absence of a putative mannose-specific phosphotransferase system enzyme IIAB component in a leucocin A-resistant strain of Listeria monocytogenes, as shown by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Leucocin A is a class IIa bacteriocin produced by Leuconostoc spp. that has previously been shown to inhibit the growth of Listeria monocytogenes. A spontaneous resistant mutant of L. monocytogenes was isolated and found to be resistant to leucocin A at levels in excess of 2 mg/ml. The mutant showed no significant cross-resistance to nontype IIa bacteriocins including nisaplin and ESF1-7GR. However, there were no inhibition zones found on a lawn of the mutant when challenged with an extract containing 51,200 AU of pediocin PA-2 per ml as determined by a simultaneous assay on the sensitive wild-type strain. DNA and protein analysis of the resistant and susceptible strains were carried out using silver-stained amplified fragment length polymorphism (ssAFLP) and one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Two-dimensional SDS-PAGE clearly showed a 35-kDa protein which was present in the sensitive but absent from the resistant strain. The N-terminal end of the 35-kDa protein was sequenced and found to have an 83% homology to the mannose-specific phosphotransferase system enzyme IIAB of Streptococcus salivarius.     

Behavior of Listeria monocytogenes in wiener exudates in the presence of Pediococcus acidilactici H or pediocin AcH during storage at 4 or 25 degrees C.

Exudative fluids were collected from packages of five brands of all-beef wieners and inoculated to contain 10(4) to 10(5) CFU of a three-strain (Scott A, V7, and 101M) mixture of Listeria monocytogenes per ml. Listeriae were inactivated (decrease of 0.61 to 3.8 log10 CFU/ml) in all five exudates held at 4 degrees C for 29 days. L. monocytogenes grew (increase of 1.7 to 3.6 log10 CFU/ml) in two of five exudates held at 25 degrees C for 6 days. Exudate was inoculated with a derivative of Pediococcus acidilactici H (designated JBL1095) or treated with pediocin AcH (a bacteriocin) as a novel approach to control the growth of L. monocytogenes in wiener exudates. Initially, pediocin AcH caused rapid death (decrease of 0.74 log10 CFU/ml in 2 h) of L. monocytogenes in exudate held at 4 degrees C, but thereafter the inactivation was similar to that in control exudate (L. monocytogenes only) or exudate containing L. monocytogenes plus JBL1095. At 25 degrees C, L. monocytogenes grew in the presence of JBL1095 during the first 64 h of incubation, but thereafter the numbers of the pathogen decreased appreciably (5.84 log10 CFU/ml in 3 days). In the presence of pediocin AcH, there was a gradual decrease in numbers of L. monocytogenes throughout the storage period at 25 degrees C. These data indicate that added biopreservatives can potentiate and amplify the intrinsic listeriostatic or listericidal activity of wiener exudate.     

Behavior of Listeria monocytogenes in wiener exudates in the presence of Pediococcus acidilactici H or pediocin AcH during storage at 4 or 25 degrees C

Exudative fluids were collected from packages of five brands of all-beef wieners and inoculated to contain 10(4) to 10(5) CFU of a three-strain (Scott A, V7, and 101M) mixture of Listeria monocytogenes per ml. Listeriae were inactivated (decrease of 0.61 to 3.8 log10 CFU/ml) in all five exudates held at 4 degrees C for 29 days. L. monocytogenes grew (increase of 1.7 to 3.6 log10 CFU/ml) in two of five exudates held at 25 degrees C for 6 days. Exudate was inoculated with a derivative of Pediococcus acidilactici H (designated JBL1095) or treated with pediocin AcH (a bacteriocin) as a novel approach to control the growth of L. monocytogenes in wiener exudates. Initially, pediocin AcH caused rapid death (decrease of 0.74 log10 CFU/ml in 2 h) of L. monocytogenes in exudate held at 4 degrees C, but thereafter the inactivation was similar to that in control exudate (L. monocytogenes only) or exudate containing L. monocytogenes plus JBL1095. At 25 degrees C, L. monocytogenes grew in the presence of JBL1095 during the first 64 h of incubation, but thereafter the numbers of the pathogen decreased appreciably (5.84 log10 CFU/ml in 3 days). In the presence of pediocin AcH, there was a gradual decrease in numbers of L. monocytogenes throughout the storage period at 25 degrees C. These data indicate that added biopreservatives can potentiate and amplify the intrinsic listeriostatic or listericidal activity of wiener exudate.     

Development of an improved chemically defined minimal medium for Listeria monocytogenes.

A chemically defined minimal medium for Listeria monocytogenes has been developed by modification of Welshimer's medium. The growth factors required by L. monocytogenes Scott A are leucine, isoleucine, arginine, methionine, valine, cysteine (each at 100 mg/liter), riboflavin and biotin (each at 0.5 micrograms/ml), thiamine (1.0 micrograms/ml), and thioctic acid (0.005 micrograms/ml). Growth was stimulated by 20 micrograms of Fe3+ per ml as ferric citrate. Glucose (1%) and glutamine (600 mg/liter) are required as primary sources of carbon and nitrogen. Glucose could not be replaced by various organic acids or amino acids. Of several sugars tested, fructose, mannose, cellobiose, trehalose, maltose (weak), glycerol (weak), and the amino sugars glucosamine, N-acetylglucosamine, and N-acetylmuramic acid supported growth in the absence of glucose. Evidence was found that chitin and cell walls of starter bacteria (Lactococcus lactis) supported survival of L. monocytogenes, which suggests that the pathogen may obtain carbon and energy sources during colonization of some foods, such as cheeses, by assimilating bacteria or molds that are present.     

Transport of sugars by the fat body of the silkworm, Bombyx mori

The transport of glucose and α-methyl glucoside into the fat body of the silkworm, Bombyx mori L., has been studied. Glucose is transported into the tissue by a mechanism similar to facilitated diffusion and α-methyl glucoside by a diffusion process. The uptake of these sugars is neither energy dependent nor coupled to a phosphotransferase system.     

Enhanced control of Listeria monocytogenes by in situ-produced pediocin during dry fermented sausage production.

To determine whether pediocin is produced and has effective antilisterial activity during food fermentation, six sausage fermentation trials were conducted with antibiotic-resistant, pediocin-producing (Bac+) Pediococcus acidilactici PAC 1.0 (Strr Rifr) and an isogenic pediocin-negative (Bac-) derivative used as a control. Meat was inoculated (ca. 10(5) CFU/g) with a composite of five Listeria monocytogenes strains, each electrotransformed with pGK12 (Cmr Emr). P. acidilactici and L. monocytogenes populations were selectively enumerated by plating on media with antibiotics. This study indicated that the dry sausage fermentation process can reduce L. monocytogenes populations. Effective inactivation of L. monocytogenes was observed when the pH at the end of the fermentation portion of the process was less than 4.9. Pediocin was responsible for part of the antilisterial activity during the fermentation in each of the six trials. Furthermore, inhibition of L. monocytogenes during drying was enhanced in the presence of pediocin in the three trials in which L. monocytogenes could be detected throughout the drying process. Thus, pediocin production contributed to an increase in safety during both the fermentation and drying portions of sausage manufacturing.     

Enhanced control of Listeria monocytogenes by in situ-produced pediocin during dry fermented sausage production.

To determine whether pediocin is produced and has effective antilisterial activity during food fermentation, six sausage fermentation trials were conducted with antibiotic-resistant, pediocin-producing (Bac+) Pediococcus acidilactici PAC 1.0 (Strr Rifr) and an isogenic pediocin-negative (Bac-) derivative used as a control. Meat was inoculated (ca. 10(5) CFU/g) with a composite of five Listeria monocytogenes strains, each electrotransformed with pGK12 (Cmr Emr). P. acidilactici and L. monocytogenes populations were selectively enumerated by plating on media with antibiotics. This study indicated that the dry sausage fermentation process can reduce L. monocytogenes populations. Effective inactivation of L. monocytogenes was observed when the pH at the end of the fermentation portion of the process was less than 4.9. Pediocin was responsible for part of the antilisterial activity during the fermentation in each of the six trials. Furthermore, inhibition of L. monocytogenes during drying was enhanced in the presence of pediocin in the three trials in which L. monocytogenes could be detected throughout the drying process. Thus, pediocin production contributed to an increase in safety during both the fermentation and drying portions of sausage manufacturing.     

Glucokinase contributes to glucose phosphorylation in d-lactic acid production by Sporolactobacillus inulinus Y2-8

Sporolactobacillus inulinus, a homofermentative lactic acid bacterium, is a species capable of efficient industrial d-lactic acid production from glucose. Glucose phosphorylation is the key step of glucose metabolism, and fine-tuned expression of which can improve d-lactic acid production. During growth on high-concentration glucose, a fast induction of high glucokinase (GLK) activity was observed, and paralleled the patterns of glucose consumption and d-lactic acid accumulation, while phosphoenolpyruvate phosphotransferase system (PTS) activity was completely repressed. The transmembrane proton gradient of 1.3–1.5 units was expected to generate a large proton motive force to the uptake of glucose. This suggests that the GLK pathway is the major route for glucose utilization, with the uptake of glucose through PTS-independent transport systems and phosphorylation of glucose by GLK in S. inulinus d-lactic acid production. The gene encoding GLK was cloned from S. inulinus and expressed in Escherichia coli. The amino acid sequence revealed significant similarity to GLK sequences from Bacillaceae. The recombinant GLK was purified and shown to be a homodimer with a subunit molecular mass of 34.5?kDa. Strikingly, it demonstrated an unusual broad substrate specificity, catalyzing phosphorylation of 2-deoxyglucose, mannitol, maltose, galactose and glucosamine, in addition to glucose. This report documented the key step concerning glucose phosphorylation of S. inulinus, which will help to understand the regulation of glucose metabolism and d-lactic acid production.     

Glucose uptake in Clostridium beijerinckii NCIMB 8052 and the solvent-hyperproducing mutant BA101.

Glucose uptake and accumulation by Clostridium beijerinckii BA101, a butanol hyperproducing mutant, were examined during various stages of growth. Glucose uptake in C. beijerinckii BA101 was repressed 20% by 2-deoxyglucose and 25% by mannose, while glucose uptake in C. beijerinckii 8052 was repressed 52 and 28% by these sugars, respectively. We confirmed the presence of a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) associated with cell extracts of C. beijerinckii BA101 by glucose phosphorylation by PEP. The PTS activity associated with C. beijerinckii BA101 was 50% of that observed for C. beijerinckii 8052. C. beijerinckii BA101 also demonstrated lower PTS activity for fructose and glucitol. Glucose phosphorylation by cell extracts derived from both C. beijerinckii BA101 and 8052 was also dependent on the presence of ATP, a finding consistent with the presence of glucokinase activity in C. beijerinckii extracts. ATP-dependent glucose phosphorylation was predominant during the solventogenic stage, when PEP-dependent glucose phosphorylation was dramatically repressed. A nearly twofold-greater ATP-dependent phosphorylation rate was observed for solventogenic stage C. beijerinckii BA101 than for solventogenic stage C. beijerinckii 8052. These results suggest that C. beijerinckii BA101 is defective in PTS activity and that C. beijerinckii BA101 compensates for this defect with enhanced glucokinase activity, resulting in an ability to transport and utilize glucose during the solventogenic stage.     

Characterization of physicochemical forces involved in adhesion of Listeria monocytogenes to surfaces

This study investigated the physicochemical forces involving the adhesion of Listeria monocytogenes to surfaces. A total of 22 strains of L. monocytogenes were compared for relative surface hydrophobicity with the salt aggregation test. Cell surface charges and hydrophobicity of L. monocytogenes Scott A were also determined by electrophoretic mobility, hydrophobic-interaction chromatography, and contact angle measurements. Electrokinetic measurements indicated that the strain Scott A has a negative electrophoretic mobility. Physicochemical characterization of L. monocytogenes by various methods indicates that this microorganism is hydrophilic. All L. monocytogenes strains tested with the salt aggregation test method aggregated a at very high ammonium sulfate molarities. The hydrophobicity-interaction chromatography results show that L. monocytogenes Scott A cells do not adhere to octyl-Sepharose unless the pH is low. Results from contact angle measurements showed that the surface free energy of strain Scott A was 65.9 mJ.m-2, classifying this microorganism as a hydrophilic bacterium. In addition, the interfacial free energy of adhesion of L. monocytogenes Scott A estimated for polypropylene and rubber was lower than that for glass and stainless steel. However, these theoretical implications could not be correlated with the attachment capabilities of L. monocytogenes.     

Ability of the Listeria monocytogenes strain Scott A to cause systemic infection in mice infected by the intragastric route

Listeriosis is an important food-borne disease that causes high rates of morbidity and mortality. For reasons that are not clear, most large outbreaks of human listeriosis involve Listeria monocytogenes serotype 4b. Relatively little is known about the pathogenesis of listeriosis following gastrointestinal exposure to food-borne disease isolates of L. monocytogenes. In the present study, we investigated the pathogenesis of systemic infection by the food-borne isolate Scott A in an intragastric (i.g.) mouse challenge model. We found that the severity of infection with L. monocytogenes Scott A was increased in mice made neutropenic by administration of monoclonal antibody RB6-8C5. This observation was similar to a previous report on a study with the laboratory strain L. monocytogenes EGD. Prior administration of sodium bicarbonate did not enhance the virulence of L. monocytogenes strain Scott A for i.g. inoculated mice. Following i.g. inoculation of mice, two serotype 4b strains of L. monocytogenes (Scott A and 101M) achieved a greater bacterial burden in the spleen and liver and elicited more severe histopathological damage to those organs than did a serotype 1/2a strain (EGD) and a serotype 1/2b stain (CM). Of the four strains tested, only strain CM exhibited poor survival in synthetic gastric fluid in vitro. The other three strains exhibited similar patterns of survival at pHs of greater than 5 and relatively rapid (<30 min) loss of viability at pHs of less than 5.0. Growth of L. monocytogenes Scott A at temperatures of 12.5 to 37 degrees C did not affect its ability to cause systemic infection in i.g. inoculated mice. These observations suggest that the serotype 4b L. monocytogenes strains Scott A and 101M possess one or more virulence determinants that make them better able to cause systemic infection following inoculation via the g.i. tract than do the serotype 1/2 strains EGD and CM.