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Antimicrobial Agents and Chemotherapy, May 2009, p. 1964-1973, Vol. 53, No. 5
0066-4804/09/$08.00+0     doi:10.1128/AAC.01382-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Zhizeng Sun,^1,2 Jin Zhong,^1* Xiaobo Liang,^1, Jiale Liu,¹ Xiuzhu Chen,¹ and Liandong Huan^1*

State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China,¹ Graduate University, Chinese Academy of Sciences, Beijing 100039, China²

Received 15 October 2008/ Returned for modification 8 January 2009/ Accepted 27 February 2009

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Nisin is a 34-residue antibacterial peptide produced by Lactococcus lactis that is active against a wide range of gram-positive bacteria. In non-nisin-producing L. lactis, nisin resistance could be conferred by a specific nisin resistance gene (nsr), which encodes a 35-kDa nisin resistance protein (NSR). However, the mechanism underlying NSR-mediated nisin resistance is poorly understood. Here we demonstrated that the protein without the predicted N-terminal signal peptide sequence, i.e., NSRSD, could proteolytically inactivate nisin in vitro by removing six amino acids from the carboxyl "tail" of nisin. The truncated nisin (nisin^1-28) displayed a markedly reduced affinity for the cell membrane and showed significantly diminished pore-forming potency in the membrane. A 100-fold reduction of bactericidal activity was detected for nisin^1-28 in comparison to that for the intact nisin. In vivo analysis indicated that NSR localized on the cell membrane and endowed host strains with nisin resistance by degrading nisin as NSRSD did in vitro, whereas NSRSD failed to confer resistance upon the host strain. In conclusion, we showed that NSR is a nisin-degrading protease. This NSR-mediated proteolytic cleavage represents a novel mechanism for nisin resistance in non-nisin-producing L. lactis.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Nisin is a 34-residue antibacterial peptide and a widely used food preservative because of its potent bactericidal activity against a variety of food spoilage bacteria. It is produced by some strains of Lactococcus lactis and contains three dehydrated amino acids and five lanthionine (Lan) or β-methyllanthionine (MeLan) rings, for which reason the term lantibiotics (lanthionine-containing antibiotics) was coined (30). Nisin A and nisin Z are two well-studied natural variants of nisin, differing with a residue at position 27 (His for nisin A and Asn for nisin Z) but showing no difference in antibacterial activity. Studies have shown that nisin exerts its bactericidal activity by pore formation and disruption of cell wall synthesis by specific binding to lipid II, an essential intermediate in peptidoglycan synthesis (4, 14, 16, 46). In addition, nisin can induce cell autolysis (2) and inhibit the outgrowth of bacterial spores (13, 32).

However, the bactericidal efficacy of nisin has been compromised by the occurrence of nisin resistance in various bacteria. It could be acquired by repeated exposure of sensitive strains to increasing concentrations of nisin through alterations in the expression of genes involved in cell wall and cytoplasmic membrane biosynthesis, which is referred to as a physiological adaptation (12, 31, 36). In L. lactis with acquired nisin resistance, the ABC transporter might also be involved in removing nisin from the cytoplasmic membrane (23). Furthermore, a nisin-inactivating enzyme was found in several nisin-resistant strains of Bacillus subsp. (17). It was later identified as a dehydroalanine (Dha) reductase, reducing the carboxyl Dha to Ala to inactivate nisin as well as subtilin, a close relative of nisin in Bacillus subtilis (18). Nisin resistance in the nisin nonproducer L. lactis subsp. diacetylactis DRC3 was reported to be conferred by a specific nisin resistance gene (nsr), which is located on a 60-kb plasmid and encodes a 35-kDa nisin resistance protein (NSR) (10). Thereafter, several groups have isolated nisin-resistant lactococcal strains with nsr on the plasmid (28, 48). From fresh milk samples, our lab isolated several nisin-insensitive lactococcal strains, one of which was L. lactis subsp. lactis TS1640 (43). In the strain, a 47-kb plasmid, designated pTS50, was identified which encodes nisin resistance following transformation into L. lactis MG1363, a highly nisin-sensitive strain. A nisin resistance determinant was localized on a 1.9-kb EcoRI fragment by Southern blotting hybridization probed with a reported nsr sequence. The fragment was subcloned and sequenced. Its open reading frame encoding sequence is 99% identical to that reported previously (10), suggesting a common origin.

Recent studies of nsr have been focused on the construction of food-grade vectors utilizing its nisin resistance phenotype (29), whereas virtually nothing is known about the underlying mechanism. It has been proposed that NSR-mediated nisin resistance might occur via proteolytic degradation of nisin for the presence of a C-terminal conserved tail-specific protease (TSPc) domain at the C terminus of NSR (7, 33). Tail-specific proteases are a group of endoproteases involved in processing their polypeptide substrates with nonpolar C termini. So far, the well-characterized tail-specific proteases include Tsp in Escherichia coli (19, 21, 39), CT441 in Chlamydia trachomatis (25, 26), and D1P in green alga (27) and in spinach (35). All of these proteases bear a conserved PDZ domain adjacent to the TSPc domain (Fig. 1A). Studies have shown that the PDZ domain is indispensable for binding of Tsp with nonpolar C termini of its peptide substrates and thus for the catalytic activity (1, 40). However, the domain is absent in NSR, the unique protein with a TSPc domain identified in L. lactis. In addition, nisin harbors charged and polar residues (Lys, His, and Ser) at the C terminus, which is also different from the sequence determinants of C-terminal substrates recognized by Tsp (20). Therefore, it is necessary to elucidate whether NSR could actually cause proteolysis of nisin and, if not, how NSR-mediated nisin resistance occurs in L. lactis.

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FIG. 1. Functional analysis of purified NSRSD. (A) Schematic structures of NSR and NSRSD in comparison with well-characterized tail-specific proteases. Striped blocks represent the C-terminal conserved tail-specific protease (TSPc) domain. Dotted blocks stand for the PDZ domain. Gray blocks in NSR and NSRSD represent hydrophobic regions. The N-terminal 27 amino acids of NSR including one hydrophobic region that constitute a predicted SP sequence have been deleted in NSRSD. (B) SDS-PAGE analysis of purified NSRSD. Lane 1, molecular size marker with masses in kDa indicated on the left; lane 2, noninduced bacterial lysate; lane 3, supernatant of induced bacterial lysate after sonication and centrifugation; lane 4, precipitate of induced bacterial lysate after sonication and centrifugation; lane 5, purified NSRSD. The positions of GST-NSRSD and NSRSD are indicated by arrows. (C) Determination of antibacterial activities of control (left) or NSRSD-digested nisin (right) against L. lactis MG1363 by the agar diffusion assay.

In this study, we demonstrate that the purified NSRSD (NSR without the predicted N-terminal signal peptide sequence) could proteolytically inactivate nisin in vitro by cleaving the peptide bond between MeLan²⁸ and Ser²⁹. The truncated nisin (nisin^1-28), compared with intact nisin, showed a markedly reduced affinity for the lactococcal membrane, a significantly diminished pore-forming potency in the target membrane, and a 100-fold-lower bactericidal activity against L. lactis MG1363. Western blotting hybridization revealed that both NSR and NSRSD were localized on the cell membrane of L. lactis. However, unlike the former, the anchored NSRSD had no ability to degrade nisin and therefore failed to endow the host strain with nisin resistance. Thus, we present a novel mechanism for nisin resistance in non-nisin-producing L. lactis.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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).

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TABLE 1. Bacterial strains and plasmids used in this study

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.

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TABLE 2. Primers used in this study

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 x 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 x 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 x g for 20 min. Soluble and insoluble proteins were separated by ultracentrifugation at 120,000 x 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 x 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 x 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 x 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 x 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 x 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 x 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.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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) x 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).

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TABLE 3. Inhibition of bacterial growth by nisin Z

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 nisin1-28 (B). The presented values represent the means ± standard deviations of three independent determinations.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
There are overall two mechanisms by which bacteria can become resistant to nisin: a shielding mechanism to prevent it from reaching the target membrane and an enzymatic inactivation mechanism by modification or destruction of the antibiotics (3). All data published so far indicate that the former is the main mechanism, with the involvement of a number of genes, whereas the latter involves a Dha reductase, found in several strains of Bacillus subsp., that reduces Dha to Ala (18). However, the present study unveiled a lactococcal tail-specific protease, NSR, responsible for nisin resistance through proteolytic degradation of nisin.

It was demonstrated that the purified NSRSD inactivated nisin by hydrolytically removing its C-terminal "tail," which is consistent with the presence of a conserved TSPc domain at the C terminus of NSRSD. Prolonged incubation with NSRSD did not lead to further degradation of nisin beyond its cleavage into two fragments. Kinetic analysis of nisin cleavage by NSRSD yielded a K_m value of 290 µM, which is higher than that for substrate cleavage by Tsp (35 to 50 µM) (21). It is probably attributed to the fact that NSRSD, unlike Tsp, lacks a PDZ domain, which facilitates binding of proteases with their cognate substrates (40). However, the cleavage rate of NSRSD (k_cat/K_m = 20.85 x 10³ M^–1 s^–1) was much higher than those of Tsp (3.8 x 10³ M^–1 s^–1) or chymotrypsin (4.4 x 10³ M^–1 s^–1) for their substrate, a variant of Arc repressor (21). Additionally, in contrast to the preference of Tsp for substrates with small, uncharged or nonpolar C-terminal residues (20), nisin harbors large, positively charged and polar residues (His, Lys, and Ser) at the C terminus, reflecting the difference in substrate specificity between NSRSD and Tsp.

Western blot analysis showed that NSR localized specifically on the cell membrane of native and nsr recombinant lactococcal strains. It is conceivable that the TSPc domain of NSR resides on the extracellular side of the membrane, where it could contact and degrade nisin. The fact that NSRSD was also detected on the membrane fraction of the recombinant strain would suggest that the predicted N-terminal signal peptide region, if any, has no effect on protein-targeted localization. However, the evidence of NSRSD almost completely losing nisin cleavage activity in vivo (Fig. 6G) would mean that the N-terminal 27 amino acid residues of NSR are functionally nondisposable. Given that purified NSRSD could degrade nisin in vitro, it is speculated that the loss of the N-terminal region would change the structure/topology of NSR, probably with the TSPc domain of NSRSD redirecting to the cytoplasmic side of the membrane. And when the membrane barrier was removed, NSRSD in the lactococcal sonicates recovered its nisin cleavage activity (Fig. 7).

It has been reported that nisin acts on a model membrane at a micromolar concentration level whereas it exerts bactericidal activity at a nanomolar level in vivo, where lipid II is available as a specific docking molecule for binding (4). Studies so far have indicated that the N terminus of nisin is involved in binding with lipid II (5, 16, 46). The removal of the C-terminal five residues or a further nine residues from nisin to produce nisin^1-29 or nisin^1-20, respectively, leads to a ca. 16-fold or 110-fold decrease in bactericidal potency compared with that of intact nisin (6). In this study, we found that nisin^1-28, produced from the cleavage of nisin by NSR, also showed a 100-fold-reduced inhibitory activity against L. lactis MG1363. All these data indicate that the C-terminal "tail" of nisin plays a significant role in its antibacterial activity. Furthermore, Ser²⁹, as the sole hydroxyl residue that escapes dehydration during maturation of nisin, might be vital for bactericidal activity of nisin since its absence resulted in a marked decrease in bactericidal potency. However, there are instances indicating that unmodified Ser/Thr is not critical for biological activities of some other lantibiotics (8, 9). In this sense, the presence of a negatively charged carboxylate on the C terminus of MeLan²⁸ might be detrimental for interaction between nisin^1-28 and the negatively charged membrane.

Compared with intact nisin, nisin^1-28 displays a markedly decreased affinity for the cell membrane (Fig. 6D and F) and a significantly reduced capacity for potassium efflux induction (Fig. 8A and B). Presently it is unknown whether the former is attributed to the decreased affinity of nisin^1-28 for lipid II. Nevertheless, the latter is an indicator of its abolished pore-forming potency, which might rationalize its significantly reduced antibacterial activity.

In short, for the first time, we provide direct evidence for a novel nisin resistance mechanism conferred by NSR: proteolytically cleaving the antibacterial peptide by removal of the C-terminal "tail." Furthermore, it is worthwhile to elucidate whether this proteolytic mechanism could be applied to the occurrence of other structure-related lantibiotic resistance mechanisms, such as that for subtilin, though either of their cognate immunity systems provides no cross-tolerance against the other lantibiotic (41, 42). Further studies are still required to decipher the active pocket, i.e., the TSPc of NSR, and how to engineer nisin to counteract bacterial resistance.

 
We thank Chengshu Wang (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) for critical reading of the manuscript. We greatly appreciate the gift of pure nisin Z from Zhejiang Silver-Elephant Bio-engineering Co., Ltd., China.

This work was supported by grants from the National Program for High Technology Research and Development of China (2006AA10Z319) and the Key Project of the Chinese Academy of Sciences (KSCXZ-YW-G-016).

 
* Corresponding author. Mailing address: Institute of Microbiology, Chinese Academy of Sciences, A3 Datun Road, Chaoyang District, Beijing 100101, China. Phone: (86) 10 6480 7439. Fax: (86) 10 6480 7401. E-mail for Jin Zhong: zhongj{at}im.ac.cn. E-mail for Liandong Huan: huanld{at}im.ac.cn

Published ahead of print on 9 March 2009.

Present address: Department of Pediatric Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1
1. Beebe, K. D., J. Shin, J. Peng, C. Chaudhury, J. Khera, and D. Pei. 2000. Substrate recognition through a PDZ domain in tail-specific protease. Biochemistry 39:3149-3155.[CrossRef][Medline]
2
2. Bierbaum, G., and H. G. Sahl. 1985. Induction of autolysis of staphylococci by the basic peptide antibiotics Pep 5 and nisin and their influence on the activity of autolytic enzymes. Arch. Microbiol. 141:249-254.[CrossRef][Medline]
3
3. Breukink, E., and B. de Kruijff. 2006. Lipid II as a target for antibiotics. Nat. Rev. Drug Discov. 5:321-332.[CrossRef][Medline]
4
4. Breukink, E., I. Wiedemann, C. van Kraaij, O. P. Kuipers, H. Sahl, and B. de Kruijff. 1999. Use of the cell wall precursor lipid II by a pore-forming peptide antibiotic. Science 286:2361-2364.[Abstract/Free Full Text]
5
5. Brotz, H., M. Josten, I. Wiedemann, U. Schneider, F. Gotz, G. Bierbaum, and H. G. Sahl. 1998. Role of lipid-bound peptidoglycan precursors in the formation of pores by nisin, epidermin and other lantibiotics. Mol. Microbiol. 30:317-327.[CrossRef][Medline]
6
6. Chan, W. C., M. Leyland, J. Clark, H. M. Dodd, L. Y. Lian, M. J. Gasson, B. W. Bycroft, and G. C. Roberts. 1996. Structure-activity relationships in the peptide antibiotic nisin: antibacterial activity of fragments of nisin. FEBS Lett. 390:129-132.[CrossRef][Medline]
7
7. Chatterjee, C., M. Paul, L. Xie, and W. A. van der Donk. 2005. Biosynthesis and mode of action of lantibiotics. Chem. Rev. 105:633-684.[CrossRef][Medline]
8
8. Cooper, L. E., A. L. McClerren, A. Chary, and W. A. van der Donk. 2008. Structure-activity relationship studies of the two-component lantibiotic haloduracin. Chem. Biol. 15:1035-1045.[CrossRef][Medline]
9
9. Cotter, P. D., L. H. Deegan, E. M. Lawton, L. A. Draper, P. M. O'Connor, C. Hill, and R. P. Ross. 2006. Complete alanine scanning of the two-component lantibiotic lacticin 3147: generating a blueprint for rational drug design. Mol. Microbiol. 62:735-747.[CrossRef][Medline]
10
10. Froseth, B. R., and L. L. McKay. 1991. Molecular characterization of the nisin resistance region of Lactococcus lactis subsp. lactis biovar diacetylactis DRC3. Appl. Environ. Microbiol. 57:804-811.[Abstract/Free Full Text]
11
11. Gasson, M. J. 1983. Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J. Bacteriol. 154:1-9.[Abstract/Free Full Text]
12
12. Gravesen, A., K. Sorensen, F. M. Aarestrup, and S. Knochel. 2001. Spontaneous nisin-resistant Listeria monocytogenes mutants with increased expression of a putative penicillin-binding protein and their sensitivity to various antibiotics. Microb. Drug Resist. 7:127-135.[CrossRef][Medline]
13
13. Gut, I. M., A. M. Prouty, J. D. Ballard, W. A. van der Donk, and S. R. Blanke. 2008. Inhibition of Bacillus anthracis spore outgrowth by nisin. Antimicrob. Agents Chemother. 52:4281-4288.[Abstract/Free Full Text]
14
14. Hasper, H. E., N. E. Kramer, J. L. Smith, J. D. Hillman, C. Zachariah, O. P. Kuipers, B. de Kruijff, and E. Breukink. 2006. An alternative bactericidal mechanism of action for lantibiotic peptides that target lipid II. Science 313:1636-1637.[Abstract/Free Full Text]
15
15. Holo, H., and I. F. Nes. 1989. High-frequency transformation, by electroporation, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl. Environ. Microbiol. 55:3119-3123.[Abstract/Free Full Text]
16
16. Hsu, S. T., E. Breukink, E. Tischenko, M. A. Lutters, B. de Kruijff, R. Kaptein, A. M. Bonvin, and N. A. van Nuland. 2004. The nisin-lipid II complex reveals a pyrophosphate cage that provides a blueprint for novel antibiotics. Nat. Struct. Mol. Biol. 11:963-967.[CrossRef][Medline]
17
17. Jarvis, B. 1967. Resistance to nisin and production of nisin-inactivating enzymes by several Bacillus species. J. Gen. Microbiol. 47:33-48.[Abstract/Free Full Text]
18
18. Jarvis, B., and J. Farr. 1971. Partial purification, specificity and mechanism of action of the nisin-inactivating enzyme from Bacillus cereus. Biochim. Biophys. Acta 227:232-240.[Medline]
19
19. Keiler, K. C., and R. T. Sauer. 1995. Identification of active site residues of the Tsp protease. J. Biol. Chem. 270:28864-28868.[Abstract/Free Full Text]
20
20. Keiler, K. C., and R. T. Sauer. 1996. Sequence determinants of C-terminal substrate recognition by the Tsp protease. J. Biol. Chem. 271:2589-2593.[Abstract/Free Full Text]
21
21. Keiler, K. C., K. R. Silber, K. M. Downard, I. A. Papayannopoulos, K. Biemann, and R. T. Sauer. 1995. C-terminal specific protein degradation: activity and substrate specificity of the Tsp protease. Protein Sci. 4:1507-1515.[Medline]
22
22. Kramer, N. E., E. J. Smid, J. Kok, B. de Kruijff, O. P. Kuipers, and E. Breukink. 2004. Resistance of Gram-positive bacteria to nisin is not determined by lipid II levels. FEMS Microbiol. Lett. 239:157-161.[CrossRef][Medline]
23
23. Kramer, N. E., S. A. van Hijum, J. Knol, J. Kok, and O. P. Kuipers. 2006. Transcriptome analysis reveals mechanisms by which Lactococcus lactis acquires nisin resistance. Antimicrob. Agents Chemother. 50:1753-1761.[Abstract/Free Full Text]
24
24. Kuipers, O. P., H. S. Rollema, W. M. Yap, H. J. Boot, R. J. Siezen, and W. M. de Vos. 1992. Engineering dehydrated amino acid residues in the antimicrobial peptide nisin. J. Biol. Chem. 267:24340-24346.[Abstract/Free Full Text]
25
25. Lad, S. P., J. Li, J. da Silva Correia, Q. Pan, S. Gadwal, R. J. Ulevitch, and E. Li. 2007. Cleavage of p65/RelA of the NF-kappaB pathway by Chlamydia. Proc. Natl. Acad. Sci. USA 104:2933-2938.[Abstract/Free Full Text]
26
26. Lad, S. P., G. Yang, D. A. Scott, G. Wang, P. Nair, J. Mathison, V. S. Reddy, and E. Li. 2007. Chlamydial CT441 is a PDZ domain-containing tail-specific protease that interferes with the NF-B pathway of immune response. J. Bacteriol. 189:6619-6625.[Abstract/Free Full Text]
27
27. Liao, D. I., J. Qian, D. A. Chisholm, D. B. Jordan, and B. A. Diner. 2000. Crystal structures of the photosystem II D1 C-terminal processing protease. Nat. Struct. Biol. 7:749-753.[CrossRef][Medline]
28
28. Liu, C. Q., M. L. Harvey, and N. W. Dunn. 1997. Cloning of a gene encoding nisin resistance from Lactococcus lactis subsp. lactis M189 which is transcribed from an extended 210 promoter. J. Gen. Appl. Microbiol. 43:67-73.[CrossRef][Medline]
29
29. Liu, C. Q., P. Su, N. Khunajakr, Y. M. Deng, S. Sumual, W. S. Kim, J. E. Tandianus, and N. W. Dunn. 2005. Development of food-grade cloning and expression vectors for Lactococcus lactis. J. Appl. Microbiol. 98:127-135.[Medline]
30
30. Lubelski, J., R. Rink, R. Khusainov, G. N. Moll, and O. P. Kuipers. 2008. Biosynthesis, immunity, regulation, mode of action and engineering of the model lantibiotic nisin. Cell Mol. Life Sci. 65:455-476.[CrossRef][Medline]
31
31. Mantovani, H. C., and J. B. Russell. 2001. Nisin resistance of Streptococcus bovis. Appl. Environ. Microbiol. 67:808-813.[Abstract/Free Full Text]
32
32. Montville, T. J., T. De Siano, A. Nock, S. Padhi, and D. Wade. 2006. Inhibition of Bacillus anthracis and potential surrogate bacilli growth from spore inocula by nisin and other antimicrobial peptides. J. Food Prot. 69:2529-2533.[Medline]
33
33. O'Driscoll, J., F. Glynn, G. F. Fitzgerald, and D. van Sinderen. 2006. Sequence analysis of the lactococcal plasmid pNP40: a mobile replicon for coping with environmental hazards. J. Bacteriol. 188:6629-6639.[Abstract/Free Full Text]
34
34. O'Sullivan, D. J., and T. R. Klaenhammer. 1993. Rapid mini-prep isolation of high-quality plasmid DNA from Lactococcus and Lactobacillus spp. Appl. Environ. Microbiol. 59:2730-2733.[Abstract/Free Full Text]
35
35. Oelmuller, R., R. G. Herrmann, and H. B. Pakrasi. 1996. Molecular studies of CtpA, the carboxyl-terminal processing protease for the D1 protein of the photosystem II reaction center in higher plants. J. Biol. Chem. 271:21848-21852.[Abstract/Free Full Text]
36
36. Peschel, A., M. Otto, R. W. Jack, H. Kalbacher, G. Jung, and F. Gotz. 1999. Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides. J. Biol. Chem. 274:8405-8410.[Abstract/Free Full Text]
37
37. Piard, J. C., I. Hautefort, V. A. Fischetti, S. D. Ehrlich, M. Fons, and A. Gruss. 1997. Cell wall anchoring of the Streptococcus pyogenes M6 protein in various lactic acid bacteria. J. Bacteriol. 179:3068-3072.[Abstract/Free Full Text]
38
38. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
39
39. Silber, K. R., K. C. Keiler, and R. T. Sauer. 1992. Tsp: a tail-specific protease that selectively degrades proteins with nonpolar C termini. Proc. Natl. Acad. Sci. USA 89:295-299.[Abstract/Free Full Text]
40
40. Spiers, A., H. K. Lamb, S. Cocklin, K. A. Wheeler, J. Budworth, A. L. Dodds, M. J. Pallen, D. J. Maskell, I. G. Charles, and A. R. Hawkins. 2002. PDZ domains facilitate binding of high temperature requirement protease A (HtrA) and tail-specific protease (Tsp) to heterologous substrates through recognition of the small stable RNA A (ssrA)-encoded peptide. J. Biol. Chem. 277:39443-39449.[Abstract/Free Full Text]
41
41. Stein, T., S. Heinzmann, S. Dusterhus, S. Borchert, and K. D. Entian. 2005. Expression and functional analysis of the subtilin immunity genes spaIFEG in the subtilin-sensitive host Bacillus subtilis MO1099. J. Bacteriol. 187:822-828.[Abstract/Free Full Text]
42
42. Stein, T., S. Heinzmann, I. Solovieva, and K. D. Entian. 2003. Function of Lactococcus lactis nisin immunity genes nisI and nisFEG after coordinated expression in the surrogate host Bacillus subtilis. J. Biol. Chem. 278:89-94.[Abstract/Free Full Text]
43
43. Tang, S., X. Chen, W. Yang, M. Chen, and L. Huan. 2001. Isolation and characterization of a plasmid pTS50, which encodes nisin resistance determinant in Lactococcus lactis TS1640. Wei Sheng Wu Xue Bao 41:536-541.[Medline]
44
44. Terzaghi, B. E., and W. E. Sandine. 1975. Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol. 29:807-813.[Medline]
45
45. van de Guchte, M., J. M. van der Vossen, J. Kok, and G. Venema. 1989. Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis. Appl. Environ. Microbiol. 55:224-228.[Abstract/Free Full Text]
46
46. Wiedemann, I., E. Breukink, C. van Kraaij, O. P. Kuipers, G. Bierbaum, B. de Kruijff, and H. G. Sahl. 2001. Specific binding of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity. J. Biol. Chem. 276:1772-1779.[Abstract/Free Full Text]
47
47. Xiao, H., X. Chen, M. Chen, S. Tang, X. Zhao, and L. Huan. 2004. Bovicin HJ50, a novel lantibiotic produced by Streptococcus bovis HJ50. Microbiology 150:103-108.[Abstract/Free Full Text]
48
48. Yun, X. X., J. Hu, and Q. Chen. 2006. Screening, isolation and identification of nisin resistance determinant gene in strains of Lactococcus lactis. Nan Fang Yi Ke Da Xue Xue Bao 26:839-842.[Medline]

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Antimicrobial Agents and Chemotherapy, May 2009, p. 1964-1973, Vol. 53, No. 5
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