Novel Mechanism for Nisin Resistance via Proteolytic Degradation of Nisin by the Nisin Resistance Protein NSR

Bacterial strains, plasmids, and growth conditions.Bacterial strains and plasmids used in this study are listed in Table 1. Molecular cloning experiments were conducted using E. coli strain JM109. The bacteria were cultured in LB broth plus 100 μg/ml ampicillin (Amp) or erythromycin (Em) when necessary with aeration at 37°C. E. coli BL21(DE3) was used as the host strain for the expression of glutathione-S-transferase (GST)-fused proteins under the same growth conditions as for E. coli JM109. L. lactis strains were cultivated at 30°C in M17 medium (44) supplemented with 0.5% (wt/vol) glucose (GM17 medium), and 5 μg/ml Em was added when needed. In addition, unless otherwise indicated, 5 μg/ml and 1.25 μg/ml nisin A (Sigma) were used for selection of L. lactis TS1640 and L. lactis MG1363/pTS50, respectively. To produce solid media, agar was added into different media at a final concentration of 1.2% (wt/vol).

Manipulation of DNA.E. coli plasmid DNA was isolated by the alkali lysis method (38). Plasmids of L. lactis were isolated according to the method described previously by O'Sullivan and Klaenhammer (34). Restriction endonucleases, ligases, and polymerases were used according to the recommendations of the manufacturers. Linear DNA fragments were electrophoretically purified using a DEAE-cellulose membrane. Competent E. coli cells were prepared and transformed by standard techniques (38). L. lactis was transformed by electroporation as described previously (15). All other procedures were conducted according to the standard protocols.

Overexpression and purification of NSRSD in E. coli.To obtain a DNA fragment without the signal peptide (SP) coding sequence, i.e., nsrSD, pMG36e-PnsrM was used as the template for PCR with the primer pairs Nsr F1 and Nsr F2 (Table 2). The purified fragment was digested with EcoRI and XhoI and ligated into the same sites of pGEX-6P-1 (GE Healthcare) encoding for PreScission protease cleavable N-terminal GST-tagged fusion proteins. The resulting plasmid, designated pSZ311 (Table 1), was transformed into E. coli BL21(DE3) in which the expression of NSRSD was fused with GST and under the control of an isopropyl 1-thio-β-d-galactopyranoside (IPTG)-inducible promoter.

For the expression of GST-NSRSD, pSZ311-transformed E. coli BL21(DE3) was grown in LB broth containing 100 μg/ml Amp at 37°C until the optical density at 600 nm (OD₆₀₀) reached 0.6. Next, IPTG was added to a final concentration of 0.6 mM and the cultures were incubated at 16°C for 6 h under gentle shaking. The bacterial cells were collected by centrifugation, washed with ice-cold phosphate-buffered saline (PBS), and resuspended in 50 ml of PBS per liter of culture. The cells were disrupted by sonication, and the unbroken cells were removed by centrifugation at 10,000 × g for 10 min at 4°C. Next, GST-NSRSD in the supernatant was purified by column chromatography with glutathione-agarose beads (GE Healthcare), and the GST tag was cleaved on a column with PreScission Protease (GE Healthcare), following the manufacturer's instructions. Additional gel filtration chromatography was carried out on a Superdex 75 10/300 GL column (GE Healthcare) to remove traces of unspecific proteins in NSRSD. Finally, the concentration of NSRSD was determined using the bicinchoninic acid protein assay reagent (Pierce) with bovine serum albumin as a standard.

Assays for NSRSD activity in vitro.Fifty micrograms of pure nisin Z (a generous gift from Zhejiang Silver-Elephant Bio-engineering Co., Ltd., China) were incubated with 2 μg purified NSRSD in a 50-μl reaction buffer (50 mM Tris-HCl, pH 6.0) at 30°C for 6 h. The incubation without NSRSD was used as the control. After the reaction, 1 μg of the digested nisin Z or control sample in a volume of 20 μl was transferred into the wells lawned with the indicator strain L. lactis MG1363. The plate was kept at 4°C for 2 h for diffusion and incubated at 30°C overnight. Additionally, the two samples were subjected to the analyses of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and electrospray ionization mass spectrometry (ESI MS).

Characterization of proteolytic activity of NSRSD against nisin Z.For quantitative analyses of nisin Z and its digested products, 20 μl of the aliquots were injected onto a SinoChrom ODS-BP 5-μm, 4.6-mm by 250-mm reverse-phase column (Elitehplc, Dalian, China) in a Waters 600 high-performance liquid chromatography (HPLC) system. Peptides were eluted at a flow rate of 1 ml/min using a linear gradient of 10 to 50% acetonitrile containing 0.1% trifluoroacetic acid (TFA) (vol/vol/vol) for 15 min and then 90% acetonitrile in 0.1% TFA for 5 min. The eluates were monitored with a fluorescence detector at 220 nm, and the amount of nisin Z was calculated using a calibration curve made by injecting 20 μl of pure nisin Z at concentrations ranging from 18 to 600 μM.

Kinetic constants for cleavage of nisin Z were determined by mixing 43 nM NSRSD with 15 to 600 μM nisin Z in reaction buffer (50 mM Tris-HCl, pH 6.0) at 30°C for 2 min. The reaction mixture was quenched with 0.1% TFA and analyzed by reverse-phase HPLC (RP-HPLC) as described above. The unit of cleavage was determined in μM/min by recording the rate of appearance of one digested product. By fitting the data to a Lineweaver-Burk double-reciprocal plot, the apparent kinetic parameters K_m and k_cat were obtained.

Overexpression of NSR and NSRSD in L. lactis.The plasmid pMG36e-PnsrM was used as the template to amplify the P59 promoter with the primer pair P59 F and P59 R (Table 2). The purified product was digested with EcoRI and SacI and cloned into pUC18 for the plasmid pUC18-P59 (Table 1). Then, nsr was amplified from pMG36e-PnsrM using the primer pair Nsr F2 and Nsr R2 (Table 2) and cloned downstream of P59 in pUC18-P59 to generate the plasmid pUC18-P59-nsr (Table 1). An EcoRI- and PstI-digested fragment containing P59-nsr was subcloned into pMG36e restricted by the same enzymes, yielding the lactococcal expression plasmid pSZ531 (Table 1). Similarly, nsrSD was amplified from pMG36e-PnsrM by PCR with the oligonucleotides Nsr F3 and Nsr R2 (Table 2) and cloned downstream of P59 in pUC18-P59 to produce the plasmid pUC18-P59-nsrSD (Table 1). Then, an EcoRI- and PstI-digested fragment containing P59-nsrSD was ligated into the corresponding sites in pMG36e, resulting in an nsrSD expression vector designated pSZ537 (Table 1).

The constitutive expression vectors pSZ531 and pSZ537 were separately transformed into L. lactis MG1363 through electroporation. For nsr or nsrSD expression, overnight cultures of L. lactis MG1363 carrying the respective plasmids were diluted 100-fold in GM17 broth containing 5 μg/ml Em and incubated at 30°C. Cells were harvested at the late exponential phase of growth.

Localization analysis of NSR and NSRSD in L. lactis.Total protein extracts from cell cultures (containing supernatants) were fractionated following a slightly modified procedure of Piard et al. (37). Briefly, 100 ml of late-exponential-phase culture was centrifuged at 8,000 × g for 5 min at 4°C. The supernatant and the cells were processed separately. The supernatant was filtered through 0.2-μm-pore-size filters to remove bacteria, and proteins from 4 ml of the filtrate were precipitated with trichloroacetic acid at a final concentration of 16% (wt/vol). The resulting pellet was washed twice with 2 ml of acetone and dissolved in 100 μl of 50 mM NaOH. The cell pellets were washed once with ice-cold PBS and resuspended in 5 ml PBS. The cells were disrupted by sonication, and intact cells were removed by centrifugation at 6,000 × g for 20 min. Soluble and insoluble proteins were separated by ultracentrifugation at 120,000 × g for 2 h. The supernatant (cytoplasmic fraction) and the viscous pellet (cell membrane fraction) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to Western blot analysis using mouse antiserum against NSRSD. The binding of the antiserum was probed with a secondary goat anti-mouse immunoglobulin G conjugated with horseradish peroxidase (Sino-American Biotechnology, Henan, China) and visualized using enhanced chemiluminescence (Applygen, Beijing, China).

Assays for nisin cleavage activity of NSR or NSRSD expressed in L. lactis.To investigate whether NSR or NSRSD could degrade nisin in vivo, a series of peptide release assays were performed according to the method described by Stein et al. (42). Flasks containing 50 ml of GM17 medium (with 5 μg/ml Em added when needed; no nisin A was added for L. lactis TS1640 and L. lactis MG1363/pTS50) were inoculated with a 1/100 volume of overnight precultures and incubated at 30°C until the OD₆₀₀ reached 0.6. Cells were harvested by centrifugation at 4,000 × g for 10 min, washed twice with 50 mM Tris-HCl (pH 6.0), centrifuged again, and resuspended in an incubation buffer (50 mM sodium phosphate buffer [pH 6.0], 1 M NaCl, and 1% [wt/vol] glucose) at 1-ml aliquots in microcentrifuge tubes. Nisin Z (100 μg) was added, and the aliquots were incubated for 30 min at 30°C with gentle shaking. After incubation, the aliquots were centrifuged for 10 min at full speed in a microcentrifuge, and the supernatants were collected. The harvested cell pellets were washed with the incubation buffer, gently mixed with 1 ml of 20% acetonitrile in water containing 0.1% TFA, and incubated with gentle shaking at 30°C for 5 min. The cells were removed by centrifugation at 12,000 × g for 10 min, and the supernatants were collected. The collected supernatants (200 μl each) were subjected to RP-HPLC analysis as described above.

Additionally, overnight precultures of recombinant L. lactis MG1363 strains were diluted to 1/100 in 10 ml of fresh GM17 medium containing 5 μg/ml Em and incubated at 30°C until the OD₆₀₀ reached 1.0. Cells were harvested by centrifugation at 8,000 × g for 10 min, washed once with 50 mM Tris-HCl (pH 6.0), and resuspended in 0.5 ml wash buffer. The cells were disrupted by sonication, and intact cells were removed by centrifugation at 6,000 × g for 20 min. Next, nisin Z was added to the suspension to a final concentration of 50 μg/ml. After incubation at 30°C for 30 min, 20 μl of the aliquots were tested for antibacterial activity against L. lactis MG1363 by agar diffusion assay as described above.

Purification of nisin Z-derivatized fragments.Nisin Z (10 mg) was incubated with 20 μg NSRSD in 10 ml buffer (50 mM Tris-HCl, pH 6.0) at 30°C for 24 h. After the reaction, the mixture was lyophilized and reconstituted with 2 ml deionized water. The desired fragments, nisin Z^1-28 and nisin Z^29-34, were purified using semipreparative RP-HPLC with a C₁₈ SinoChrom ODS-BP 10-μm, 10-mm by 250-mm column (Elitehplc, Dalian, China). The column was eluted with a linear gradient of 10 to 50% acetonitrile in 0.1% TFA for 15 min and then 90% acetonitrile containing 0.1% TFA for 10 min at the flow rate of 2 ml/min. Most of the solvent was removed by rotative evaporation under vacuum, followed by lyophilization. Then, nisin Z^1-28 was dissolved in 0.02 M HCl, while nisin Z^29-34 was dissolved in deionized water, which was subjected to Edman degradation sequencing using an Applied Biosystems 491 automatic sequencer.

Assays for antibacterial activities of nisin Z and nisin Z^1-28.Antibacterial activities of nisin Z and nisin Z^1-28 were tested against various lactococcal strains by a method slightly modified from that of Kuipers et al. (24). Briefly, overnight cultures of indicator organisms were diluted in GM17 broth (with 5 μg/ml Em added when needed; no nisin A was added for L. lactis TS1640 and L. lactis MG1363/pTS50) to an OD₆₀₀ of 0.025 and were divided into aliquots of 5 ml. These tubes were inoculated with different amounts of nisin Z or nisin Z^1-28 and were incubated at 30°C without shaking. Outgrowth was measured when the culture without nisin Z or nisin Z^1-28 had reached an OD₆₀₀ of 0.8. The lowest concentration of nisin Z or nisin Z^1-28 that resulted in no obvious growth of the tested strains (OD₆₀₀ < 0.04) was taken as the MIC.

Determination of potassium efflux.Potassium efflux was determined according to the method of Xiao et al. (47) with slight modification. L. lactis MG1363 was cultured in GM17 medium plus 2.5 mM KCl at 30°C to mid-exponential phase (OD₆₀₀ = 0.6 to 0.7) for cell dry weight (CDW) and potassium efflux determinations. Cells were harvested by centrifugation at 10,000 × g at 4°C for 10 min, washed with 0.1 M morpholine ethanesulfonic acid buffer at pH 6.3 containing 0.2% (wt/vol) glucose and 0.6 mM KCl, and resuspended in the same volume of the washing buffer. Then, purified nisin Z or nisin Z^1-28 was added to the cell suspension at different concentrations. After treatment for 1 h at room temperature, the suspension was centrifuged at 10,000 × g for 10 min. The collected supernatants were applied to an atomic absorption spectrometer (5100PC; Perkin-Elmer, Ueberlingen, Germany) for determination of the potassium content. Potassium efflux was expressed in μmol/mg CDW, and each treatment was performed in triplicate.

Nucleotide sequence accession number.The subcloned and sequenced EcoRI fragment was assigned GenBank accesion no. AY219902.

Expression of NSRSD in E. coli.To obtain a sufficient amount of NSR for biochemical characterization, it was fused with GST and expressed in E. coli. However, the expression level and solubility of GST-NSR were too low for it to be purified (data not shown), which was probably due to the adverse effect of a putative SP sequence composed of 27 amino acid residues at the N terminus (predicted by the software program SignalP 3.0). To test the expression without the SP region (the product designated NSRSD), E. coli BL21(DE3) was transformed by the expression vector pSZ311, in which expression of NSRSD was fused with GST. After induction with IPTG, GST-NSRSD was detected mainly in the soluble fraction of cell lysate by SDS-PAGE (Fig. 1B, lane 3). NSRSD was purified to homogeneity by GST affinity purification, PreScission protease cleavage, and gel filtration chromatography (Fig. 1B, lane 5).

In vitro degradation of nisin by NSRSD and identification of the cleavage site.To test whether purified NSRSD has proteolytic activity against nisin, it was incubated with purified nisin and then tested against L. lactis MG1363. As shown in Fig. 1C, antibacterial activity of treated nisin decreased by a large margin compared with that of the control. Subsequent MALDI-TOF MS analyses revealed the absence of nisin (3,330.6 Da) but the presence of one molecule with a smaller molecular mass of 2,697.0 Da in the reaction buffer (Fig. 2A and B). With the help of ESI MS, another molecule was detected, with a molecular mass of 652.6 Da (Fig. 2C). Meanwhile, doubly protonated molecular ions of the above molecules were further analyzed with ESI MS, resulting in two molecular masses of 1,349.8 Da and 326.9 Da (Fig. 2C), respectively. These two fragments were then purified using semipreparative RP-HPLC, and the small one was sequenced by Edman degradation analysis. Five amino acids of its N terminus were identified as Ser-Ile-His-Val-X (X represents a modified amino acid), an exact match to the residues from Ser²⁹ to Dha³³ of nisin. The data indicated that NSRSD can degrade nisin by hydrolyzing the peptide bond between MeLan²⁸ and Ser²⁹, thus resulting in the loss of antibacterial activity of nisin. Hence, NSRSD-catalyzed proteolysis of nisin is shown in Fig. 3.

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FIG. 2.

Mass spectrometric analyses of nisin and its degraded products after cleavage by NSRSD. (A) MALDI-TOF MS analysis of nisin. (B) MALDI-TOF MS analysis of NSRSD-cleaved nisin. (C) ESI-MS analysis of NSRSD-cleaved nisin. The peak with a molecular mass of 326.9 Da represents the doubly protonated ion of the peak with a molecular mass of 652.6 Da (M₁). In addition, the peak with a molecular mass of 1,349.8 Da is equivalent to the doubly protonated ion of the peak with a molecular mass of 2,697.0 Da (M₂) detected by MALDI-TOF MS in panel B.

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FIG. 3.

Proteolysis of nisin catalyzed by NSRSD. The shadowed residues indicate amino acids which have undergone posttranslational modifications. A-S-A, lanthionine; Abu-S-A, β-methyllanthionine; Dha, 2,3-didehydroalanine; Dhb, 2,3-didehydrobutyrine. Molecular masses of intact and NSRSD-cleaved nisin are indicated in parentheses to the right of the corresponding peptides.

Characterization of proteolytic activity of NSRSD against nisin.RP-HPLC was used to analyze nisin and its digested products. As shown in Fig. 4, nisin and its two digested fragments were completely separated, with a retention time of 16.5 min for nisin and 9.8 min and 17.3 min for the two cleaved products, respectively. This facilitated the integration of each peak and the quantification of corresponding peptides.

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FIG. 4.

RP-HPLC profiling for intact and NSRSD-cleaved nisin. (A) RP-HPLC profile of intact nisin. (B) RP-HPLC profile of nisin cleaved by NSRSD. Nisin and its cleaved peptides were separated with a reverse-phase column. The star represents intact nisin, and arrows indicate the two fragments of nisin degraded by NSRSD.

Kinetic parameters for NSRSD cleavage of nisin were determined by using a reaction (conducted at 30°C and pH 6.0) with nisin at concentrations ranged from 15 μM to 600 μM. Fitting the data to a Lineweaver-Burk double-reciprocal plot yields kinetic parameters, i.e., K_m = 290 (±19) μM; k_cat = 6.05 (±0.02) s⁻¹; and k_cat/K_m = 20.85 (±1.34) × 10³ M⁻¹ s⁻¹.

Cellular localization of NSR and NSRSD in L. lactis.Hydropathy profile analysis suggested that NSR was an integral membrane protein, with a strongly hydrophobic domain at the amino terminus, i.e., the signal sequence, and a weakly hydrophobic region in the middle (Fig. 1A). To test whether NSR is localized on the cell membrane, total protein extracts of L. lactis were fractionated into supernatant, cell membrane, and cytoplasmic fractions, respectively. Western blot analysis demonstrated that NSR, with a calculated molecular mass of 35.8 kDa, was detected solely in the cell membrane fraction of either native strain L. lactis TS1640 or recombinant L. lactis MG1363, which had been transformed with the plasmid designated pSZ531, containing the nsr gene (Fig. 5, lanes 3 and 6). For the strain transformed with pTS50, the nsr-containing plasmid that was originally isolated from L. lactis TS1640, NSR was also detected in the cell membrane fraction (data not shown).

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FIG. 5.

Subcellular localization of NSR and NSRSD in L. lactis. Western blot analysis of NSR and NSRSD in ultracentrifugation-fractionated samples of different L. lactis strains. Lanes 1 to 3, supernatant and cytoplasmic and cell membrane fractions of L. lactis TS1640 (a native NSR expression strain); lanes 4 to 6, supernatant and cytoplasmic and cell membrane fractions of L. lactis MG1363/pSZ531 (a recombinant NSR expression strain); lanes 7 to 9, supernatant and cytoplasmic and cell membrane fractions of L. lactis MG1363/pSZ537 (a recombinant NSRSD expression strain). The positions of NSR and NSRSD are indicated by arrows.

A plasmid designated pSZ537 was constructed to assess the contribution of the N-terminal hydrophobic domain to the localization of NSR. In this lactococcal expression plasmid, nsrSD, encoding the protein without the N-terminal hydrophobic sequence, was placed under the control of a P59 promoter. After transformation into L. lactis MG1363, NSRSD was detected only in the membrane fraction of the cell extract, with a slightly faster mobility shift than NSR (Fig. 5, lane 9). This excludes the possibility that the N-terminal hydrophobic domain of NSR should be posttranslationally cleaved before cell membrane localization.

Degradation of nisin by NSR or NSRSD expressed in L. lactis.The in vivo nisin cleavage activity of NSR or NSRSD was investigated with a series of peptide release assays. After incubation of L. lactis cells with nisin, the peptide, its derivatives in the culture supernatant, and those associated with the cell pellet were analyzed by RP-HPLC. For the nisin-sensitive L. lactis strain MG1363, about two-thirds of the applied nisin was found intact in the supernatant and one-third was attached to the cell membrane (Fig. 6A and B). In contrast, when applied to a naturally nisin-resistant strain, L. lactis TS1640, only a small amount of nisin was found attached to the cell while a large amount of degraded products of nisin was detected in the culture supernatant (Fig. 6C and D). Similarly, incubation of nisin with NSR-expressing L. lactis MG1363 led to the degradation of nisin, with its degraded products being present only in the culture supernatant (Fig. 6E and F). On the contrary, for the strain expressing NSRSD, in addition to the detection of intact nisin in both fractions, a small amount of degraded products was also found in the supernatant (Fig. 6G and H). The RP-HPLC pattern for recombinant L. lactis MG1363 transformed with the empty vector pMG36e was identical to that for the host strain. pTS50-transformed L. lactis MG1363 displayed the same degradation profile as L. lactis TS1640 after incubation with nisin (data not shown). In conclusion, our data showed that membrane-localized NSR could degrade nisin in vivo and the substrate cleavage site is identical to that in vitro judging from the retention time of degraded products on RP-HPLC. The degraded nisin derivatives lost affinity for the cell membrane since they were detected exclusively in the culture supernatant.

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FIG. 6.

RP-HPLC profiles of nisin and its cleaved products by membrane-localized NSR or NSRSD in vivo. Different L. lactis strains were incubated with nisin. After centrifugation, nisin or its degraded fragments in the supernatant or attached to the cell pellet were analyzed by RP-HPLC. Asterisks represent intact nisin, and arrows indicate the two fragments of nisin degraded by membrane-localized NSR or NSRSD. Panels A, C, E, and G show peptides detected in the supernatants of L. lactis MG1363, L. lactis TS1640, L. lactis MG1363/pSZ531, and L. lactis MG1363/pSZ537, respectively. Panels B, D, F, and H show peptides attached to the cell pellets of L. lactis MG1363, L. lactis TS1640, L. lactis MG1363/pSZ531, and L. lactis MG1363/pSZ537, respectively.

Although NSRSD localized on the cell membrane (Fig. 5, lane 9), it displayed little nisin cleavage activity in vivo (Fig. 6G). Given that purified NSRSD could efficiently degrade nisin in vitro (Fig. 4B), it was postulated that its loss of activity in vivo was probably attributed to cytoplasmic localization of the active domain, i.e., the TSPc domain. To test this hypothesis, the cell membrane was disrupted by sonication, after which the cytoplasmic and membrane fractions were tested for their nisin cleavage activities. As shown in Fig. 7 (sample 4 and sample 6), NSRSD displayed nisin cleavage activity similar to that of NSR, which was reflected by the reduced antibacterial activity of nisin after incubation with lactococcal sonicates containing NSR or NSRSD.

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FIG. 7.

Antibacterial activity of nisin against L. lactis MG1363 after incubation with lactococcal sonicates. Con, nisin (1 μg) incubated in 50 mM Tris-HCl (pH 6.0); 1, 3, and 5, sonicates of L. lactis MG1363/pMG36e, L. lactis MG1363/pSZ531, and L. lactis MG1363/pSZ537, respectively; 2, 4, and 6, nisin (1 μg) incubated with sonicates of L. lactis MG1363/pMG36e, L. lactis MG1363/pSZ531, and L. lactis MG1363/pSZ537, respectively.

Growth inhibitory activities of nisin and nisin^1-28 against L. lactis.To further establish the connection between the ability of NSR to degrade nisin and its contribution to nisin resistance in L. lactis, the MIC of nisin was determined by growth inhibitory assays. In general, the increase of the MIC is positively correlated with nisin-degrading activity (Table 3). Therefore, it can be confirmed that NSR contributes to nisin resistance in L. lactis. The exception of L. lactis TS1640 would imply that there are additional mechanisms conferring nisin resistance (Table 3). However, when the strain was incubated overnight in GM17 medium without nisin, it became less resistant to the antibacterial peptide, with an MIC of 3 μg/ml, though expression of NSR was not influenced (data not shown). Therefore, after repeated exposure to nisin during screening, L. lactis TS1640 evolved unstable physiological adaptations, which were lost once the nisin pressure was removed (3, 22).

The inhibitory activity of nisin^1-28 was assayed and resulted in an MIC of 6 μg/ml against L. lactis MG1363, 100-fold higher than that of the intact nisin molecule (0.06 μg/ml). This implies that the C-terminal “tail” of nisin plays an essential role in its antibacterial activity.

Potassium efflux induced by nisin and nisin^1-28.To evaluate the pore-forming potency of nisin and nisin^1-28, potassium efflux assays were carried out. As shown in Fig. 8A, nisin caused potassium efflux from L. lactis MG1363 in a concentration-dependent manner. A significant efflux of potassium (0.54 μmol) per mg of CDW was induced by using nisin at a concentration (0.1 μg/ml) slightly in excess of the MIC (0.06 μg/ml). When a higher concentration of nisin (0.5 μg/ml) was applied, the efflux reached a maximum of 1.4 μmol/mg CDW, indicating a total intracellular potassium efflux. In contrast, nisin^1-28 induced less than 40% efflux (0.5 μmol/mg CDW) of the intracellular potassium even when the applied peptide reached 100 μg/ml (Fig. 8B), 16 times higher than the MIC (6 μg/ml). Increasing the concentration of nisin^1-28 up to 200 μg/ml did not result in further-activated potassium efflux (data not shown). Therefore, removal of the C-terminal “tail” of nisin resulted in a remarkably decreased potency in forming pores in the membrane.

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FIG. 8.

Potassium efflux assays of L. lactis MG1363 induced by nisin (A) or nisin^1-28 (B). The presented values represent the means ± standard deviations of three independent determinations.