APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1992, p. 2255-2259 0099-2240/92/072255-05$02.00/0
Vol. 58, No. 7
Copyright ©D 1992, American Society for Microbiology
Depletion of Proton Motive Force by Nisin in Listeria monocytogenes Cellst MARIA E. C. BRUNO,' ALAN KAISER,2 AND THOMAS J. MONTVILLE' 2* Department of Food Science' and Graduate Program in Microbiology and Molecular Genetics, 2 New Jersey Agricultural Experiment Station, Cook College, Rutgers, the State University of New Jersey, New Brunswick, New Jersey 08903-0231 Received 2 March 1992/Accepted 2 May 1992
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 (ZApH) and the membrane potential (A4) 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 .5 tag/ml completely dissipated PMF in cells at external pH values of 5.5 and 7.0. With 1 ig of nisin per ml, ApH was completely dissipated but A* decreased only slightly. The action of nisin on PMF in L. monocytogenes Scott A was both time and concentration dependent. Valinomycin depleted only A*, whereas nigericin and carbonyl cyanide m-chlorophenylhydrazone depleted only ApH, 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 ,ug/ml) also completely dissipated PMF in these strains. to ions and low-molecular-weight cellular compounds. Furthermore, the addition of nisin to intact cells and membrane vesicles leads to the immediate collapse of the membrane potential (A+i) (32). Recent studies by Gao et al. (14) demonstrated that nisin dissipates not only the membrane potential but also the pH gradient (ApH) in artificial liposomes and that nisin requires an energized membrane to exert its effect. In this paper, we elucidate for the first time the basal bioenergetic parameters of L. monocytogenes and report the influence of nisin and common ionophores on the pH gradient, membrane potential, and total proton motive force (PMF) in energized cells. We report that L. monocytogenes cells maintained basal bioenergetic parameters that were typical of facultative anaerobes and that nisin totally dissipated the membrane potential and pH gradient in energized cells, leading to the complete collapse of the PMF.
Listeria monocytogenes is a gram-positive, facultatively anaerobic rod that has emerged as an important food-borne pathogen (11, 33). Listeriosis can be life threatening to pregnant women, newborns, infants, and immunocompromised adults (27). L. monocytogenes has become a major concern to the food industry because of reports of listeriosis outbreaks associated with foods (7). The inherent biological characteristics of this pathogen make it difficult to control in foods (18). L. monocytogenes is widely distributed in the environment. It has been isolated from soils, plants, sewage, and water (7); it is able to grow under refrigeration conditions (15, 34, 38); it is resistant to relatively high levels of NaCl (9, 18); it can initiate growth at low pH values (9, 15); it can survive for long periods under dry conditions; and it is among the most heat resistant of vegetative bacterial cells (26). The use of bacteriocins produced by lactic acid bacteria that are generally recognized as safe may provide a means to control L. monocytogenes in foods. Bacteriocins are biologically active proteins with antimicrobial properties that vary in spectrum of activity, mode of action, genetic determinants, and biochemical characteristics (24, 37). Bacteriocin production has been associated with lactococci (36), lactobacilli, and pediococci from various foods. The effectiveness of nisin and other bacteriocins against L. monocytogenes has been extensively demonstrated (3-5, 8, 17, 25, 35, 39). Nisin, which is produced by certain Lactococcus lactis strains, is the most extensively studied bacteriocin of a gram-positive organism (19). Nisin has decades of safe international use (10) and is the only bacteriocin that is generally recognized as safe in the United States for use in processed cheese spreads (12). Early experiments regarding the mode of action of nisin pointed to the cytoplasmic membrane as the biological target (30). Nisin renders the cytoplasmic membranes of some bacterial species permeable
MATERIALS AND METHODS Culture conditions. L. monocytogenes Scott A, V7, and FRI-LM 103M were obtained from the Food Research Institute, Madison, Wis. Strains F5065' and ATCC 19115', which carry the plasmid pGK12, coding for chloramphenicol and erythromycin resistance, were kindly provided by P. M. Foegeding (North Carolina State University). All strains were maintained on Trypticase soy broth slants at 4°C and transferred monthly. Chloramphenicol and erythromycin were added to the media of strains carrying pGK12. For the determination of the pH gradient, membrane potential, and intracellular cell volume, L. monocytogenes was grown aerobically to the midlog phase at 30°C with shaking (150 rpm) in Trypticase soy broth supplemented with 0.5% glucose and 0.6% yeast extract. The midlog-phase cells were harvested by centrifugation (20,845 x g for 20 min), washed once in 0.9% NaCl, and resuspended in 0.1 M morpholinoethanesulfonic acid (MES) buffer containing 10 mM MgSO4 7H20 and 10 mM KCl at the appropriate pH to an A660 of 1.0 to 1.1. Determination of Vi. The intracellular volume (Vi) of L. monocytogenes Scott A was determined from the distribu-
* Corresponding author. t This is manuscript D-10971-1-92 from the New Jersey State Agricultural Experiment Station.
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tion of 3H-labeled water and '4C-labeled inulin as described by Rottenberg (31). The calculated Vi was 1.80 RI/mg (dry weight) of cells. This value was used for all strains in the calculation of the membrane potential and the pH gradient. Determination of pH gradient and membrane potential. The membrane potential (interior negative) and pH gradient (interior alkaline) were determined by the transmembrane distribution of 3H-labeled tetraphenylphosphonium bromide (TPP) and [14C]salicylic acid, respectively, as described by Rottenberg (31). Corrections for nonspecific label binding and radioactivity attributable to extracellular fluid trapped in the pellets were determined from the extent of label accumulation in control cells treated with 7% butanol for 60 min. The counts from control cells were subtracted from the experimental values (23). Cells suspended in 0.1 M MES buffer containing 10 mM MgSO4 7H20 and 10 mM KCI were first energized for 15 min by adding glucose to a final concentration of 10 mM. Energized cells (1 ml) were immediately dispensed into microcentrifuge tubes containing 7.5 puM [14C]salicylic acid (0.375 pCi) and [3H]water (3.75 puCi) or 6.8 puM [3H]TPP (1.5 puCi) and incubated for 15 min at 20°C to equilibrate the probes. Separate experiments (data not shown) demonstrated that the probes equilibrated within 5 min. In the experiments with nisin and ionophores, whose working solutions were prepared in appropriate diluents, energized cells were dispensed into tubes containing the test compound at the appropriate concentration and incubated for 30 min at 20°C before the probes were added. For the experiment on the time dependence of nisin action, energized cells were incubated with nisin for specified intervals before the radioisotopes were added and equilibrated. After the cells were incubated with the probes, 0.4 ml of silicone oil (96% mixture with octane, final density, 1.02 g/ml) was added to the suspension. The cells and the supernatant were separated by centrifugation through the silicone oil layer at 13,000 x g for 6 min. For the pH gradient determination only, samples (100 pul) of the supernatant top layer were removed for measuring radioactivity. For the pH gradient and membrane potential determinations, the aqueous layer and most of the silicone oil were removed with a pipette. Residual silicone oil was removed by swabbing the tube with a cotton swab. Then the bottom portions of the tubes containing the cell pellets were cut off directly into scintillation vials containing 5 ml of scintillation cocktail (Ecosint H; National Diagnostics) and 0.5 ml of 0.6 N perchloric acid. Radioactivity was measured with a Beckman LS1801 scintillation counter equipped with a dual-label counting program. Chemicals. Trypticase soy broth without dextrose was purchased from Becton Dickinson (Cockeysville, Md.). Yeast extract was obtained from Difco Laboratories (Detroit, Mich.). Radioactively labeled [3H]TPP (45.0 Ci/mmol), [14C]salicylic acid (56.0 mCi/mmol), [3H]water (1.0 mCi/ml), and [14C]inulin (2.0 mCi/g) were obtained from Du Pont Co. (Wilmington, Del.). Silicone oil (density, 1.05 g/ml) and octane anhydrous were purchased from Aldrich Chemical Co., Inc. (Milwaukee, Wis.). Glucose, perchloric acid, potassium chloride, magnesium sulfate, and butanol were obtained from Fisher Scientific Co. (Pittsburgh, Pa.). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.). Purified nisin (approximate activity, 39 x 106 IU/g) was kindly provided by Aplin and Barrett Ltd. (Trowbridge, England).
200
AS
150
E
100 50
-
7
5.5.0
5.5
6.0
6.5
7.0
7.5
External pH
FIG. 1. Basal bioenergetic parameters (ZApH, A*Ji, and PMF [Ap]) in energized cells of L. monocytogenes Scott A. Error bars represent the standard deviations of four replicates.
RESULTS AND DISCUSSION
Determination of Vi. There was no significant difference in the Vi values of L. monocytogenes Scott A determined in triplicate at external pH values (pHe) ranging from 5.5 to 7.0 (data not shown). Therefore, the overall average of 12 determinations was used to calculate the Vi of 1.80 ± 0.33 pul/mg of cell dry weight. This is lower than the Vi of 2.97 ± 0.11 pul/mg reported by Ita and Hutkins (20). However, cell volumes change considerably under various metabolic conditions (31); Ita and Hutkins used overnight cultures, whereas in this study we used midlog-phase glycolyzing cells, which might be smaller. Nevertheless, the PMF calculations are relatively insensitive to variations in Vi. Determination of the basal bioenergetic parameters. The PMF is composed of the membrane potential and the pH gradient, as given by the equation PMF = A - ZApH, where Z equals 2.3 (RT/F) and R, T, and F are as described previously (16). The basal bioenergetic parameters for L. monocytogenes Scott A were determined over the pHe range of 5.5 to 7.0. At pHe 5.5, the ZApH (alkaline inside) was as high as 104.0 mV and decreased to 25.7 mV at pHe 7.0. The Al4 (negative inside) increased from -65.9 mV at pHe 5.5 to -116.2 mV at pHe 7.0 (Fig. 1). The interconversion of AtJ and ZApH over this pHe range led to the maintenance of a reasonably constant PMF around -160 mV, which is typical of facultative anaerobes (21). L. monocytogenes Scott A cells maintained their internal pH (pHi) at approximately 7.5 over a pHe range of 5.5 to 7.0. This is within the pHi range reported for neutrophiles (6). Because the pHi was constant, the ApH decreased from 1.7 pH units at pHe 5.5 to 0.4 pH unit at pHe 7.0. In contrast, Ita and Hutkins (20) report that the ApH was constant at 0.2 pH unit for L. monocytogenes Scott A cells between pHe 5.5 and 6.5 during growth when pHe was maintained by adding weak organic acids. These differences may be explained by the nature of the experimental systems used. In our experiments, cells were resuspended in a biological buffer (MES) designed to minimize interference with the metabolic processes of the cells. This buffer did not cause acidification of the cytoplasm. Ita and Hutkins (20) grew cells in media to which weak organic acids were continually added. The neutral species of these organic acids diffuse across the cell membrane, whereas the ionic species are impermeable (31).
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NISIN-MEDIATED DEPLETION OF PMF
TABLE 1. Influence of nisin concentration on the PMF of L. monocytogenes Scott A at pH 5.5 and 7.oa
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200
pH and nisin concn (>g/ml)
ZApH
-AW
-PMF
~~~(mV)
(mV)
(mV)
150
pH 5.5 Control (no nisin) 10.0 5.0 1.0
104.4 0.0 0.0 0.0
77.2 0.0 0.0 65.4
181.6 0.0 0.0 65.4
E 100
pH 7.0 Control (no nisin) 10.0 5.0 1.0
47.2 0.0 0.0 0.0
99.2 0.0 0.0 64.8
146.4 0.0 0.0 64.8
Ap
50
0.0
a The results represent the averages of two experiments.
Once inside the cell, the membrane-permeable neutral species dissociate and acidify the cytoplasm. Influence of nisin on PMF. Ruhr and Sahl (32) demonstrated that nisin dissipates the Aq, of several gram-positive species and suggested that dissipation of the pH gradient was likely to occur. We examined the influence of nisin on PMF in energized L. monocytogenes Scott A cells at pHe 5.5 and 7.0 so that maximal ZApH and A* values would be generated in untreated cells. Nisin at final concentrations of 10.0 and 5.0 ,ug/ml totally dissipated ZApH and A* at pHe 5.5 and 7.0 (Table 1). Under the same conditions, the addition of 1.0 jig of nisin per ml also led to the total dissipation of ZApH but only partially depleted A,. At pHe 5.5, 1.0 jig of nisin per ml slightly decreased A4i from -77.2 to -65.4 mV, whereas at PHe 7.0 the result was more pronounced, with A4i decreasing from -99.2 to -64.8 mV. Gao et al. (14) recently demonstrated that both ApH and A*, are dissipated by nisin in artificial liposomes. Time and concentration dependence of nisin-mediated PMF dissipation. The influence of nisin at 1.0 ,ug/ml on PMF in energized L. monocytogenes Scott A cells was determined at PHe 7.0 for time periods ranging from 30 s to 30 min (Fig. 2). ZApH was practically dissipated and A* was markedly decreased from -107.4 mV to -77.3 mV within 5 min. 200 -
1 50 -
Ap
0.5
1.5
1.0
2.0
2.5
Nisin Concentration (pg/mi) FIG. 3. Concentration dependence of nisin on ZApH, A/, and total PMF (Ap) in energized cells of L. monocytogenes Scott A at pH 6.5.
Further incubation led to the complete dissipation of ZApH and to an additional reduction of Au, to -64.2 mV after 30 min of exposure to nisin. This time interval was used for concentration dependence studies, which were conducted at PHe 6.5 because ApH and A* contribute equally to the PMF at this pH. The addition of 0.5 ,ug of nisin per ml caused a modest reduction of ZApH and A*, (Fig. 3). Nevertheless, with 1.0 ,ug of nisin per ml, ZApH was almost dissipated and Aup was drastically reduced from -85.4 mV to -25.4 mV. There were no detectable differences between the results with nisin concentrations of 1.0 and 1.5 ,ug/ml, whereas 2.5 jig of nisin per ml completely dissipated the two components of PMF. These results are consistent with the time and concentration dependencies found in other experimental systems (14, 32). Influence of various ionophores on PMF in L. monocytogenes Scott A cells. The influences of the ionophores nigericin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and valinomycin on PMF of L. monocytogenes Scott A were determined at pHe 6.5 and compared with the action of nisin under the same assay conditions (Table 2). lonophores have been extensively used to control either one or both components of PMF by increasing the ionic permeability of the membranes (1, 13, 14, 29). Nigericin carries out the antiport of K+ for H+ and can therefore be used to dissipate pH gradients in an electroneutral manner (16). The addition of 2.0 ,uM nigericin to L. monocytogenes Scott A cells caused the total dissipation of ZApH and a 21% increase in Ali. TABLE 2. Influence of nisin, nigericin, CCCP, and valinomycin on the PMF in energized cells of L. monocytogenes Scott A at
>100
pH 6.5w Additive
ZApH (mV) ± ± ± ± ±
-Ail (mV)
-PMF (mV)
30
169.8 ± 16.3 + ± 0.0 ± 0.0 ± 101.8 ± 6.4 30.7 ± 14.6 + ± 53.5 ± 2.7 a The results are the averages of at least three experiments ± the standard
FIG. 2. Influence of nisin (1.0 ,ug/ml) on the PMF in energized cells of L. monocytogenes Scott A as a func tion of time at pH 7.0.
deviations. b These values were obtained in the presence of 200 mM KCI; the control values for ZApH and Aqi were 20 and 15 mV, respectively, lower than the control values given above.
Control 2.5 ,ug of nisin ml-1 2.0 p.M nigericin 5.0 ,uM valinomycinb 240 ,uM CCCP
50
0
5
10
15
20
25
Time (min.)
85.7 0.0 0.0 30.7 0.0
18.1 0.0 0.0 14.6 0.0
84.1 0.0 101.8 0.0 53.5
6.2 0.0 6.4 0.0 2.7
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TABLE 3. Influence of nisin on the PMF in energized cells of five different strains of L. monocytogenes at pH 6.5a Treatment and strain
No nisin Scott A V7 FRI-LM 103M
F5069r ATCC 19115r 2.5 p.g of nisin ml-' Scott A V7 FRI-LM 103M F5069r ATCC 19115r
ZApH (mV)
-Aqi (mV)
-PMF (mV)
85.5 92.5 86.0 85.6 75.0
87.8 76.8 63.9 90.5 91.8
173.6 169.3 149.9 176.1 166.7
0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0
a The values are the averages of two experiments.
Similar results have been reported for EDTA-treated Escherichia coli cells, in which nigericin (2.0 ,uM) decreases ZApH to near zero with a slight increase in Al (1). The uncoupling agent CCCP is a protonophore that equilibrates pHi and pHe (13). The addition of 240 ,M CCCP to L. monocytogenes cells led to the complete dissipation of ZApH and a 36% decrease in Ai\. Valinomycin mediates the electrogenic uniport of K+. When added to cells that have established a Ad,, valinomycin permits the uptake of K+ with the concurrent dissipation of Ai* (16), although the K+ flux is also influenced by other K+ pumps and the permeability of the membrane to other ions. The addition of 5.0 ,uM valinomycin to L. monocytogenes Scott A cells resuspended in buffer containing 10 mM KCI did not change Ati but caused a slight reduction in ZApH (data not shown). Since valinomycin action depends on the K+ concentration, its influence on PMF could only be observed when the KCI concentration was increased to 200 mM. In the buffer system containing 200 mM KCI, the values for Al and ZApH in the absence of any ionophore were lower than those in the presence of 10 mM KCI. This is because, even in the absence of ionophores, the addition of K+ leads to a decrease in A* that is partially compensated for by an increase in ZApH (2, 22). However, at high K+ concentrations (>30 mM), both ZApH and A4j are decreased (1). In our study, the addition of 5.0 ,uM valinomycin to L. monocytogenes Scott A cells in the presence of 200 mM KCl caused the total dissipation of AV and a 53% reduction in ZApH. Finally, the addition of 2.5 jig of nisin per ml totally dissipated the two components of PMF in L. monocytogenes Scott A cells, regardless of the concentration of KCI in the buffer. Influence of nisin on the PMF of other L. monocytogenes strains. The influence of nisin (2.5 ,ug/ml) on the PMF of five L. monocytogenes strains was determined at pHe 6.5. The basal bioenergetic parameters were very similar for all strains, except for FRI-LM 103M, which maintained a comparatively lower PMF (Table 3). The addition of 2.5 ,ug of nisin per ml totally dissipated PMF in Scott A cells. Likewise, in strains V7, FRI-LM 103M, F5065r, and ATCC 19115r, the PMF was completely depleted by 2.5 ,ug of nisin per ml. These data suggest that L. monocytogenes strains are quite nisin sensitive, in contrast to the relative insensitivity and strain-variable response shown by Clostridium botulinum (28). Nisin has been approved for use in processed cheese
spreads at maximum additio-t levels of 10,000 IU/ml to prevent the outgrowth of C. botulinum spores (10). We report here that nisin at 2.5 jig/ml (100 IU/ml) depleted PMF in five L. monocytogenes strains. During this period the culture viability decreased by 5 log cycles (data not shown). These data demonstrate that nisin is very potent against L. monocytogenes and strongly support its use in foods. However, the appropriate nisin levels for use in a given food system must be established by challenge studies with L. monocytogenes in that specific product. ACKNOWLEDGMENTS This research was supported by State Appropriations, U.S. Hatch Act Funds, and by grant 91-37201-6796 from the U.S. Department of Agriculture CSRS NRI Food Safety Program. Maria E. C. Bruno is grateful for the support of CAPES (Brazilian Post-Graduate Education Federal Agency). We thank C. Lewus for technical assistance on some of these studies. REFERENCES 1. Ahmed, S., and I. R. Booth. 1983. The use of valinomycin, nigericin and trichlorocarbanilide in control of proton motive force in Escherichia coli cells. Biochem. J. 212:105-112. 2. Bakker, E. P., and W. E. Mangerich. 1981. Interconversion of components of the bacterial proton motive force by electrogenic potassium transport. J. Bacteriol. 147:820-826. 3. Benkerroum, N., and W. E. Sandine. 1988. Inhibitory action of nisin against Listenia monocytogenes. J. Dairy Sci. 71:32373245. 4. Berry, E. D., R. W. Hutkins, and R. W. Mandigo. 1991. The use of bacteriocin-producing Pediococcus acidilactici to control postprocessing Listeria monocytogenes contamination in frankfurters. J. Food Prot. 54:681-686. 5. Berry, E. D., M. B. Liewen, R. W. Mandigo, and R. W. Hutkins. 1990. Inhibition of Listenia monocytogenes by bacteriocinproducing Pediococcus during the manufacture of fermented semidry sausage. J. Food Prot. 53:194-197. 6. Booth, I. R. 1985. Regulation of cytoplasmic pH in bacteria. Microbiol. Rev. 49:359-378. 7. Brackett, R. E. 1988. Presence and persistence of Listenia monocytogenes in food and water. Food Technol. 42:162-164. 8. Carminati, D., G. Giraffa, and M. G. Bossi. 1989. Bacteriocinlike inhibitors of Streptococcus lactis against Listenia monocytogenes. J. Food Prot. 52:614-617. 9. Conner, D. E., R. E. Brackett, and L. R. Beuchat. 1986. Effect of temperature, sodium chloride, and pH on growth of Listenia monocytogenes in cabbage juice. Appl. Environ. Microbiol. 52:59-63. 10. Delves-Broughton, J. 1990. Nisin and its uses as a food preservative. Food Technol. 44:110-112. 11. Farber, J. M., and P. I. Peterkin. 1991. Listenia monocytogenes, a food-borne pathogen. Microbiol. Rev. 55:476-511. 12. Federal Register. 1988. Nisin preparation: affirmation of GRAS status as a direct human food ingredient. Code of Federal Regulations, chapter 21. Part 184. 53:11247-11251. 13. Foster, J. W., and H. K. Hall. 1991. Inducible pH homeostasis and the acid tolerance response of Salmonella typhimurium. J. Bacteriol. 173:5129-5135. 14. Gao, F. H., T. Abee, and W. N. Konings. 1991. Mechanism of action of the peptide antibiotic nisin in liposomes and cytochrome c oxidase-containing proteoliposomes. Appl. Environ. Microbiol. 57:2164-2170. 15. George, S. M., B. M. Lund, and T. T. Brocklehurst. 1988. The effect of pH and temperature on initiation of growth of Listeria monocytogenes. Lett. Appl. Microbiol. 6:153-156. 16. Harold, F. M. 1986. The vital force: a study of bioenergetics. W. H. Freeman and Co., New York. 17. Harris, L. J., M. A. Daeschel, M. E. Stiles, and T. R. Klaenhammer. 1989. Antimicrobial activity of lactic acid bacteria against Listeria monocytogenes. J. Food Prot. 52:614-617.
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Vol. 58, No. 7
Copyright ©D 1992, American Society for Microbiology
Depletion of Proton Motive Force by Nisin in Listeria monocytogenes Cellst MARIA E. C. BRUNO,' ALAN KAISER,2 AND THOMAS J. MONTVILLE' 2* Department of Food Science' and Graduate Program in Microbiology and Molecular Genetics, 2 New Jersey Agricultural Experiment Station, Cook College, Rutgers, the State University of New Jersey, New Brunswick, New Jersey 08903-0231 Received 2 March 1992/Accepted 2 May 1992
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 (ZApH) and the membrane potential (A4) 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 .5 tag/ml completely dissipated PMF in cells at external pH values of 5.5 and 7.0. With 1 ig of nisin per ml, ApH was completely dissipated but A* decreased only slightly. The action of nisin on PMF in L. monocytogenes Scott A was both time and concentration dependent. Valinomycin depleted only A*, whereas nigericin and carbonyl cyanide m-chlorophenylhydrazone depleted only ApH, 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 ,ug/ml) also completely dissipated PMF in these strains. to ions and low-molecular-weight cellular compounds. Furthermore, the addition of nisin to intact cells and membrane vesicles leads to the immediate collapse of the membrane potential (A+i) (32). Recent studies by Gao et al. (14) demonstrated that nisin dissipates not only the membrane potential but also the pH gradient (ApH) in artificial liposomes and that nisin requires an energized membrane to exert its effect. In this paper, we elucidate for the first time the basal bioenergetic parameters of L. monocytogenes and report the influence of nisin and common ionophores on the pH gradient, membrane potential, and total proton motive force (PMF) in energized cells. We report that L. monocytogenes cells maintained basal bioenergetic parameters that were typical of facultative anaerobes and that nisin totally dissipated the membrane potential and pH gradient in energized cells, leading to the complete collapse of the PMF.
Listeria monocytogenes is a gram-positive, facultatively anaerobic rod that has emerged as an important food-borne pathogen (11, 33). Listeriosis can be life threatening to pregnant women, newborns, infants, and immunocompromised adults (27). L. monocytogenes has become a major concern to the food industry because of reports of listeriosis outbreaks associated with foods (7). The inherent biological characteristics of this pathogen make it difficult to control in foods (18). L. monocytogenes is widely distributed in the environment. It has been isolated from soils, plants, sewage, and water (7); it is able to grow under refrigeration conditions (15, 34, 38); it is resistant to relatively high levels of NaCl (9, 18); it can initiate growth at low pH values (9, 15); it can survive for long periods under dry conditions; and it is among the most heat resistant of vegetative bacterial cells (26). The use of bacteriocins produced by lactic acid bacteria that are generally recognized as safe may provide a means to control L. monocytogenes in foods. Bacteriocins are biologically active proteins with antimicrobial properties that vary in spectrum of activity, mode of action, genetic determinants, and biochemical characteristics (24, 37). Bacteriocin production has been associated with lactococci (36), lactobacilli, and pediococci from various foods. The effectiveness of nisin and other bacteriocins against L. monocytogenes has been extensively demonstrated (3-5, 8, 17, 25, 35, 39). Nisin, which is produced by certain Lactococcus lactis strains, is the most extensively studied bacteriocin of a gram-positive organism (19). Nisin has decades of safe international use (10) and is the only bacteriocin that is generally recognized as safe in the United States for use in processed cheese spreads (12). Early experiments regarding the mode of action of nisin pointed to the cytoplasmic membrane as the biological target (30). Nisin renders the cytoplasmic membranes of some bacterial species permeable
MATERIALS AND METHODS Culture conditions. L. monocytogenes Scott A, V7, and FRI-LM 103M were obtained from the Food Research Institute, Madison, Wis. Strains F5065' and ATCC 19115', which carry the plasmid pGK12, coding for chloramphenicol and erythromycin resistance, were kindly provided by P. M. Foegeding (North Carolina State University). All strains were maintained on Trypticase soy broth slants at 4°C and transferred monthly. Chloramphenicol and erythromycin were added to the media of strains carrying pGK12. For the determination of the pH gradient, membrane potential, and intracellular cell volume, L. monocytogenes was grown aerobically to the midlog phase at 30°C with shaking (150 rpm) in Trypticase soy broth supplemented with 0.5% glucose and 0.6% yeast extract. The midlog-phase cells were harvested by centrifugation (20,845 x g for 20 min), washed once in 0.9% NaCl, and resuspended in 0.1 M morpholinoethanesulfonic acid (MES) buffer containing 10 mM MgSO4 7H20 and 10 mM KCl at the appropriate pH to an A660 of 1.0 to 1.1. Determination of Vi. The intracellular volume (Vi) of L. monocytogenes Scott A was determined from the distribu-
* Corresponding author. t This is manuscript D-10971-1-92 from the New Jersey State Agricultural Experiment Station.
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tion of 3H-labeled water and '4C-labeled inulin as described by Rottenberg (31). The calculated Vi was 1.80 RI/mg (dry weight) of cells. This value was used for all strains in the calculation of the membrane potential and the pH gradient. Determination of pH gradient and membrane potential. The membrane potential (interior negative) and pH gradient (interior alkaline) were determined by the transmembrane distribution of 3H-labeled tetraphenylphosphonium bromide (TPP) and [14C]salicylic acid, respectively, as described by Rottenberg (31). Corrections for nonspecific label binding and radioactivity attributable to extracellular fluid trapped in the pellets were determined from the extent of label accumulation in control cells treated with 7% butanol for 60 min. The counts from control cells were subtracted from the experimental values (23). Cells suspended in 0.1 M MES buffer containing 10 mM MgSO4 7H20 and 10 mM KCI were first energized for 15 min by adding glucose to a final concentration of 10 mM. Energized cells (1 ml) were immediately dispensed into microcentrifuge tubes containing 7.5 puM [14C]salicylic acid (0.375 pCi) and [3H]water (3.75 puCi) or 6.8 puM [3H]TPP (1.5 puCi) and incubated for 15 min at 20°C to equilibrate the probes. Separate experiments (data not shown) demonstrated that the probes equilibrated within 5 min. In the experiments with nisin and ionophores, whose working solutions were prepared in appropriate diluents, energized cells were dispensed into tubes containing the test compound at the appropriate concentration and incubated for 30 min at 20°C before the probes were added. For the experiment on the time dependence of nisin action, energized cells were incubated with nisin for specified intervals before the radioisotopes were added and equilibrated. After the cells were incubated with the probes, 0.4 ml of silicone oil (96% mixture with octane, final density, 1.02 g/ml) was added to the suspension. The cells and the supernatant were separated by centrifugation through the silicone oil layer at 13,000 x g for 6 min. For the pH gradient determination only, samples (100 pul) of the supernatant top layer were removed for measuring radioactivity. For the pH gradient and membrane potential determinations, the aqueous layer and most of the silicone oil were removed with a pipette. Residual silicone oil was removed by swabbing the tube with a cotton swab. Then the bottom portions of the tubes containing the cell pellets were cut off directly into scintillation vials containing 5 ml of scintillation cocktail (Ecosint H; National Diagnostics) and 0.5 ml of 0.6 N perchloric acid. Radioactivity was measured with a Beckman LS1801 scintillation counter equipped with a dual-label counting program. Chemicals. Trypticase soy broth without dextrose was purchased from Becton Dickinson (Cockeysville, Md.). Yeast extract was obtained from Difco Laboratories (Detroit, Mich.). Radioactively labeled [3H]TPP (45.0 Ci/mmol), [14C]salicylic acid (56.0 mCi/mmol), [3H]water (1.0 mCi/ml), and [14C]inulin (2.0 mCi/g) were obtained from Du Pont Co. (Wilmington, Del.). Silicone oil (density, 1.05 g/ml) and octane anhydrous were purchased from Aldrich Chemical Co., Inc. (Milwaukee, Wis.). Glucose, perchloric acid, potassium chloride, magnesium sulfate, and butanol were obtained from Fisher Scientific Co. (Pittsburgh, Pa.). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.). Purified nisin (approximate activity, 39 x 106 IU/g) was kindly provided by Aplin and Barrett Ltd. (Trowbridge, England).
200
AS
150
E
100 50
-
7
5.5.0
5.5
6.0
6.5
7.0
7.5
External pH
FIG. 1. Basal bioenergetic parameters (ZApH, A*Ji, and PMF [Ap]) in energized cells of L. monocytogenes Scott A. Error bars represent the standard deviations of four replicates.
RESULTS AND DISCUSSION
Determination of Vi. There was no significant difference in the Vi values of L. monocytogenes Scott A determined in triplicate at external pH values (pHe) ranging from 5.5 to 7.0 (data not shown). Therefore, the overall average of 12 determinations was used to calculate the Vi of 1.80 ± 0.33 pul/mg of cell dry weight. This is lower than the Vi of 2.97 ± 0.11 pul/mg reported by Ita and Hutkins (20). However, cell volumes change considerably under various metabolic conditions (31); Ita and Hutkins used overnight cultures, whereas in this study we used midlog-phase glycolyzing cells, which might be smaller. Nevertheless, the PMF calculations are relatively insensitive to variations in Vi. Determination of the basal bioenergetic parameters. The PMF is composed of the membrane potential and the pH gradient, as given by the equation PMF = A - ZApH, where Z equals 2.3 (RT/F) and R, T, and F are as described previously (16). The basal bioenergetic parameters for L. monocytogenes Scott A were determined over the pHe range of 5.5 to 7.0. At pHe 5.5, the ZApH (alkaline inside) was as high as 104.0 mV and decreased to 25.7 mV at pHe 7.0. The Al4 (negative inside) increased from -65.9 mV at pHe 5.5 to -116.2 mV at pHe 7.0 (Fig. 1). The interconversion of AtJ and ZApH over this pHe range led to the maintenance of a reasonably constant PMF around -160 mV, which is typical of facultative anaerobes (21). L. monocytogenes Scott A cells maintained their internal pH (pHi) at approximately 7.5 over a pHe range of 5.5 to 7.0. This is within the pHi range reported for neutrophiles (6). Because the pHi was constant, the ApH decreased from 1.7 pH units at pHe 5.5 to 0.4 pH unit at pHe 7.0. In contrast, Ita and Hutkins (20) report that the ApH was constant at 0.2 pH unit for L. monocytogenes Scott A cells between pHe 5.5 and 6.5 during growth when pHe was maintained by adding weak organic acids. These differences may be explained by the nature of the experimental systems used. In our experiments, cells were resuspended in a biological buffer (MES) designed to minimize interference with the metabolic processes of the cells. This buffer did not cause acidification of the cytoplasm. Ita and Hutkins (20) grew cells in media to which weak organic acids were continually added. The neutral species of these organic acids diffuse across the cell membrane, whereas the ionic species are impermeable (31).
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TABLE 1. Influence of nisin concentration on the PMF of L. monocytogenes Scott A at pH 5.5 and 7.oa
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200
pH and nisin concn (>g/ml)
ZApH
-AW
-PMF
~~~(mV)
(mV)
(mV)
150
pH 5.5 Control (no nisin) 10.0 5.0 1.0
104.4 0.0 0.0 0.0
77.2 0.0 0.0 65.4
181.6 0.0 0.0 65.4
E 100
pH 7.0 Control (no nisin) 10.0 5.0 1.0
47.2 0.0 0.0 0.0
99.2 0.0 0.0 64.8
146.4 0.0 0.0 64.8
Ap
50
0.0
a The results represent the averages of two experiments.
Once inside the cell, the membrane-permeable neutral species dissociate and acidify the cytoplasm. Influence of nisin on PMF. Ruhr and Sahl (32) demonstrated that nisin dissipates the Aq, of several gram-positive species and suggested that dissipation of the pH gradient was likely to occur. We examined the influence of nisin on PMF in energized L. monocytogenes Scott A cells at pHe 5.5 and 7.0 so that maximal ZApH and A* values would be generated in untreated cells. Nisin at final concentrations of 10.0 and 5.0 ,ug/ml totally dissipated ZApH and A* at pHe 5.5 and 7.0 (Table 1). Under the same conditions, the addition of 1.0 jig of nisin per ml also led to the total dissipation of ZApH but only partially depleted A,. At pHe 5.5, 1.0 jig of nisin per ml slightly decreased A4i from -77.2 to -65.4 mV, whereas at PHe 7.0 the result was more pronounced, with A4i decreasing from -99.2 to -64.8 mV. Gao et al. (14) recently demonstrated that both ApH and A*, are dissipated by nisin in artificial liposomes. Time and concentration dependence of nisin-mediated PMF dissipation. The influence of nisin at 1.0 ,ug/ml on PMF in energized L. monocytogenes Scott A cells was determined at PHe 7.0 for time periods ranging from 30 s to 30 min (Fig. 2). ZApH was practically dissipated and A* was markedly decreased from -107.4 mV to -77.3 mV within 5 min. 200 -
1 50 -
Ap
0.5
1.5
1.0
2.0
2.5
Nisin Concentration (pg/mi) FIG. 3. Concentration dependence of nisin on ZApH, A/, and total PMF (Ap) in energized cells of L. monocytogenes Scott A at pH 6.5.
Further incubation led to the complete dissipation of ZApH and to an additional reduction of Au, to -64.2 mV after 30 min of exposure to nisin. This time interval was used for concentration dependence studies, which were conducted at PHe 6.5 because ApH and A* contribute equally to the PMF at this pH. The addition of 0.5 ,ug of nisin per ml caused a modest reduction of ZApH and A*, (Fig. 3). Nevertheless, with 1.0 ,ug of nisin per ml, ZApH was almost dissipated and Aup was drastically reduced from -85.4 mV to -25.4 mV. There were no detectable differences between the results with nisin concentrations of 1.0 and 1.5 ,ug/ml, whereas 2.5 jig of nisin per ml completely dissipated the two components of PMF. These results are consistent with the time and concentration dependencies found in other experimental systems (14, 32). Influence of various ionophores on PMF in L. monocytogenes Scott A cells. The influences of the ionophores nigericin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and valinomycin on PMF of L. monocytogenes Scott A were determined at pHe 6.5 and compared with the action of nisin under the same assay conditions (Table 2). lonophores have been extensively used to control either one or both components of PMF by increasing the ionic permeability of the membranes (1, 13, 14, 29). Nigericin carries out the antiport of K+ for H+ and can therefore be used to dissipate pH gradients in an electroneutral manner (16). The addition of 2.0 ,uM nigericin to L. monocytogenes Scott A cells caused the total dissipation of ZApH and a 21% increase in Ali. TABLE 2. Influence of nisin, nigericin, CCCP, and valinomycin on the PMF in energized cells of L. monocytogenes Scott A at
>100
pH 6.5w Additive
ZApH (mV) ± ± ± ± ±
-Ail (mV)
-PMF (mV)
30
169.8 ± 16.3 + ± 0.0 ± 0.0 ± 101.8 ± 6.4 30.7 ± 14.6 + ± 53.5 ± 2.7 a The results are the averages of at least three experiments ± the standard
FIG. 2. Influence of nisin (1.0 ,ug/ml) on the PMF in energized cells of L. monocytogenes Scott A as a func tion of time at pH 7.0.
deviations. b These values were obtained in the presence of 200 mM KCI; the control values for ZApH and Aqi were 20 and 15 mV, respectively, lower than the control values given above.
Control 2.5 ,ug of nisin ml-1 2.0 p.M nigericin 5.0 ,uM valinomycinb 240 ,uM CCCP
50
0
5
10
15
20
25
Time (min.)
85.7 0.0 0.0 30.7 0.0
18.1 0.0 0.0 14.6 0.0
84.1 0.0 101.8 0.0 53.5
6.2 0.0 6.4 0.0 2.7
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TABLE 3. Influence of nisin on the PMF in energized cells of five different strains of L. monocytogenes at pH 6.5a Treatment and strain
No nisin Scott A V7 FRI-LM 103M
F5069r ATCC 19115r 2.5 p.g of nisin ml-' Scott A V7 FRI-LM 103M F5069r ATCC 19115r
ZApH (mV)
-Aqi (mV)
-PMF (mV)
85.5 92.5 86.0 85.6 75.0
87.8 76.8 63.9 90.5 91.8
173.6 169.3 149.9 176.1 166.7
0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0
a The values are the averages of two experiments.
Similar results have been reported for EDTA-treated Escherichia coli cells, in which nigericin (2.0 ,uM) decreases ZApH to near zero with a slight increase in Al (1). The uncoupling agent CCCP is a protonophore that equilibrates pHi and pHe (13). The addition of 240 ,M CCCP to L. monocytogenes cells led to the complete dissipation of ZApH and a 36% decrease in Ai\. Valinomycin mediates the electrogenic uniport of K+. When added to cells that have established a Ad,, valinomycin permits the uptake of K+ with the concurrent dissipation of Ai* (16), although the K+ flux is also influenced by other K+ pumps and the permeability of the membrane to other ions. The addition of 5.0 ,uM valinomycin to L. monocytogenes Scott A cells resuspended in buffer containing 10 mM KCI did not change Ati but caused a slight reduction in ZApH (data not shown). Since valinomycin action depends on the K+ concentration, its influence on PMF could only be observed when the KCI concentration was increased to 200 mM. In the buffer system containing 200 mM KCI, the values for Al and ZApH in the absence of any ionophore were lower than those in the presence of 10 mM KCI. This is because, even in the absence of ionophores, the addition of K+ leads to a decrease in A* that is partially compensated for by an increase in ZApH (2, 22). However, at high K+ concentrations (>30 mM), both ZApH and A4j are decreased (1). In our study, the addition of 5.0 ,uM valinomycin to L. monocytogenes Scott A cells in the presence of 200 mM KCl caused the total dissipation of AV and a 53% reduction in ZApH. Finally, the addition of 2.5 jig of nisin per ml totally dissipated the two components of PMF in L. monocytogenes Scott A cells, regardless of the concentration of KCI in the buffer. Influence of nisin on the PMF of other L. monocytogenes strains. The influence of nisin (2.5 ,ug/ml) on the PMF of five L. monocytogenes strains was determined at pHe 6.5. The basal bioenergetic parameters were very similar for all strains, except for FRI-LM 103M, which maintained a comparatively lower PMF (Table 3). The addition of 2.5 ,ug of nisin per ml totally dissipated PMF in Scott A cells. Likewise, in strains V7, FRI-LM 103M, F5065r, and ATCC 19115r, the PMF was completely depleted by 2.5 ,ug of nisin per ml. These data suggest that L. monocytogenes strains are quite nisin sensitive, in contrast to the relative insensitivity and strain-variable response shown by Clostridium botulinum (28). Nisin has been approved for use in processed cheese
spreads at maximum additio-t levels of 10,000 IU/ml to prevent the outgrowth of C. botulinum spores (10). We report here that nisin at 2.5 jig/ml (100 IU/ml) depleted PMF in five L. monocytogenes strains. During this period the culture viability decreased by 5 log cycles (data not shown). These data demonstrate that nisin is very potent against L. monocytogenes and strongly support its use in foods. However, the appropriate nisin levels for use in a given food system must be established by challenge studies with L. monocytogenes in that specific product. ACKNOWLEDGMENTS This research was supported by State Appropriations, U.S. Hatch Act Funds, and by grant 91-37201-6796 from the U.S. Department of Agriculture CSRS NRI Food Safety Program. Maria E. C. Bruno is grateful for the support of CAPES (Brazilian Post-Graduate Education Federal Agency). We thank C. Lewus for technical assistance on some of these studies. REFERENCES 1. Ahmed, S., and I. R. Booth. 1983. The use of valinomycin, nigericin and trichlorocarbanilide in control of proton motive force in Escherichia coli cells. Biochem. J. 212:105-112. 2. Bakker, E. P., and W. E. Mangerich. 1981. Interconversion of components of the bacterial proton motive force by electrogenic potassium transport. J. Bacteriol. 147:820-826. 3. Benkerroum, N., and W. E. Sandine. 1988. Inhibitory action of nisin against Listenia monocytogenes. J. Dairy Sci. 71:32373245. 4. Berry, E. D., R. W. Hutkins, and R. W. Mandigo. 1991. The use of bacteriocin-producing Pediococcus acidilactici to control postprocessing Listeria monocytogenes contamination in frankfurters. J. Food Prot. 54:681-686. 5. Berry, E. D., M. B. Liewen, R. W. Mandigo, and R. W. Hutkins. 1990. Inhibition of Listenia monocytogenes by bacteriocinproducing Pediococcus during the manufacture of fermented semidry sausage. J. Food Prot. 53:194-197. 6. Booth, I. R. 1985. Regulation of cytoplasmic pH in bacteria. Microbiol. Rev. 49:359-378. 7. Brackett, R. E. 1988. Presence and persistence of Listenia monocytogenes in food and water. Food Technol. 42:162-164. 8. Carminati, D., G. Giraffa, and M. G. Bossi. 1989. Bacteriocinlike inhibitors of Streptococcus lactis against Listenia monocytogenes. J. Food Prot. 52:614-617. 9. Conner, D. E., R. E. Brackett, and L. R. Beuchat. 1986. Effect of temperature, sodium chloride, and pH on growth of Listenia monocytogenes in cabbage juice. Appl. Environ. Microbiol. 52:59-63. 10. Delves-Broughton, J. 1990. Nisin and its uses as a food preservative. Food Technol. 44:110-112. 11. Farber, J. M., and P. I. Peterkin. 1991. Listenia monocytogenes, a food-borne pathogen. Microbiol. Rev. 55:476-511. 12. Federal Register. 1988. Nisin preparation: affirmation of GRAS status as a direct human food ingredient. Code of Federal Regulations, chapter 21. Part 184. 53:11247-11251. 13. Foster, J. W., and H. K. Hall. 1991. Inducible pH homeostasis and the acid tolerance response of Salmonella typhimurium. J. Bacteriol. 173:5129-5135. 14. Gao, F. H., T. Abee, and W. N. Konings. 1991. Mechanism of action of the peptide antibiotic nisin in liposomes and cytochrome c oxidase-containing proteoliposomes. Appl. Environ. Microbiol. 57:2164-2170. 15. George, S. M., B. M. Lund, and T. T. Brocklehurst. 1988. The effect of pH and temperature on initiation of growth of Listeria monocytogenes. Lett. Appl. Microbiol. 6:153-156. 16. Harold, F. M. 1986. The vital force: a study of bioenergetics. W. H. Freeman and Co., New York. 17. Harris, L. J., M. A. Daeschel, M. E. Stiles, and T. R. Klaenhammer. 1989. Antimicrobial activity of lactic acid bacteria against Listeria monocytogenes. J. Food Prot. 52:614-617.
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