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1 Department of Microbiology and Immunology, Otago School of Medical Sciences, The University of Otago, PO Box 56, Dunedin, New Zealand
2 Department of Biochemistry, Otago School of Medical Sciences, The University of Otago, PO Box 56, Dunedin, New Zealand
3 Institut für Genomforschung und Systembiologie, Centrum für Biotechnologie, Universität Bielefeld, Universitätsstraße 25, 33615 Bielefeld, Germany
Correspondence
Ralph W. Jack
ralph.jack{at}otago.ac.nz
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Present address: AgResearch Ltd, Department of Biochemistry, Otago School of Medical Sciences, The University of Otago, PO Box 56, Dunedin, New Zealand. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), whilst others produce relatively lower molecular mass antimicrobial peptides (Mr<10 000) termed microcins (Heng & Jack, 2006). Similarly, a veritable plethora of modified and unmodified peptide bacteriocins (Mr<10 000) from Gram-positive bacteria have also been described, particularly those elaborated by the lactic acid bacteria (Bonelli et al., 2006; Nes et al., 2006; Heng et al., 2007). In recent years considerable research interest has focussed on the low molecular mass polypeptides from both Gram-positive and Gram-negative bacteria, in part because of their potential in pharmaceutical and nutraceutical applications (Cleveland et al., 2001; Ross et al., 2002; Gillor et al., 2004; Kirkup, 2006).
Despite the amount of research directed at the peptide bacteriocins of Gram-positive bacteria, microcins and colicins, surprisingly little is known about the higher molecular mass (Mr>10 000) bacteriocins from Gram-positive bacteria. Several bacteriocins that have proven to be cell-wall-hydrolysing (murolytic) enzymes, such as lysostaphin, zoocin A and stellalysin, have been reported from Gram-positive bacteria (Schindler & Schuhardt, 1964; Simmonds et al., 1996; Heng et al., 2006b). Historically, staphylococcin 1580 from Staphylococcus epidermidis (Jetten & Vogels, 1972a, b; Jetten et al., 1972) was reported to be a bacteriocin with a subunit molecular mass of 
20 kDa that formed multimeric structures of 300–400 kDa in conjunction with carbohydrate and lipid. However, more recent reisolation of the inhibitory agent revealed the antimicrobial activity to be epidermin, a peptide bacteriocin (2164.6 Da) belonging to the lantibiotic subgroup, which co-purified with the higher molecular mass species (Sahl, 1994). Two additional high molecular mass bacteriocins from Gram-positive bacteria have been reported: helveticin J from Lactobacillus helveticus 481 (Joerger & Klaenhammer, 1986, 1990) and a potentially related, unnamed compound from Lb. helveticus CNRZ450 (Thompson et al., 1996). The former was suggested to have a monomeric molecular mass of 37 kDa and to form aggregates of >300 kDa, whereas the latter was reported to be a bacteriocin with multimeric molecular mass of 30–50 kDa and potentially composed of 17 kDa monomers. Helveticin J is active against other lactic acid bacteria such as lactobacilli and lactococci, is chromosomally encoded and was shown to be inactivated by proteolytic digestion and heat (Joerger & Klaenhammer, 1986, 1990), features consistent with its size and proteinaceous nature.
More recently, we have described the 17 kDa bacteriocin streptococcin A-M57 from Streptococcus pyogenes, identified during the sequencing of a novel plasmid (pDN571) isolated from a prototype M-57 strain (Heng et al., 2004) and the detailed characterization of dysgalacticin, a 21.5 kDa anionic, plasmid-encoded bacteriocin from Streptococcus dysgalactiae subsp. equisimilis (Heng et al., 2006). In the former study, we also identified two further putative proteins that were recognized during interrogation of public databases and were predicted to have both sequence and structural similarity to streptococcin A-M57: EF1097 from Enterococcus faecalis (accession no. AAO80897; Paulsen et al., 2003) and YpkK from Corynebacterium jeikeium (accession no. AAL85945; Tauch et al., 2004). Interestingly, these proteins share no similarity with those described above, indicating that they may represent a novel family of antibiotic proteins from Gram-positive bacteria. In this study, we report the expression of these proteins in Escherichia coli and present evidence that EF1097 and YpkK are antimicrobial proteins with structural similarity to, but different antimicrobial spectra from, streptococcin A-M57. Moreover, we show that the C-terminal portion of YpkK is the essential killing moiety.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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(Hanahan, 1983) was the host for all expression studies and was propagated aerobically at 37 °C, either in LB broth or on LB agar (Sambrook & Russell, 2001); when required ampicillin was added at 100 µg ml–1. The bacterial strains used in spectrum of activity studies (Table 3) were routinely propagated aerobically at 37 °C on Columbia Agar Base (Becton Dickinson) containing 5 % (v/v) whole human blood or in Todd–Hewitt broth (Difco) at the same temperature. Bacterial strains were maintained as frozen stock cultures stored at –80 °C in Todd–Hewitt broth containing 20 % (v/v) glycerol (Sigma-Aldrich).
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18 h. A zone of inhibition of the indicator lawn with a diameter of at least 5 mm was arbitrarily defined as positive inhibition. The quantitative MIC determination was carried out essentially as previously described (Staubitz et al., 2001) except that Tryptic Soy Broth (Difco) was used to support bacterial growth, the microtitre plates were not shaken and either Micrococcus luteus T-18 or Lactococcus lactis T-21 (Tagg & Bannister, 1979) was used to quantify the biological activity. The ability of the proteins to lyse either live or dead target bacteria using agar-immobilized or liquid culture-based assays was assessed essentially as previously described (Wirawan et al., 2007).
Recombinant DNA techniques, construction of recombinant expression systems and bioinformatic analyses.
PCR amplicons and plasmids from E. coli were purified using appropriate kits (Qiagen) according to the manufacturer's recommendations. Protocols for molecular biological techniques (cloning, plasmid transformation, etc.) were conducted as described by Sambrook & Russell (2001). The high-fidelity enzyme Platinum pfx DNA polymerase (Invitrogen) was used in accordance with the manufacturer's protocols in all PCR experiments; all primers (Table 1) were purchased from Invitrogen. DNA sequence analyses were carried out by the Allan Wilson Centre Genome Service (Palmerston North, New Zealand). Similarity searches used appropriate BLAST algorithms (Altschul et al., 1997; Schäffer et al., 2001), alignments were accomplished using CLUSTAL W (http://www.ebi.ac.uk/clustalw/; Chenna et al., 2003), and assembly and basic analyses of sequence data were carried out using GeneJockey II (Biosoft). In silico predictions of secretion signal peptides were accomplished using SignalP 3.0 (Vullo & Frasconi, 2004), whilst structural predictions were made using the suite of programs available on the Expert Protein Analysis System (ExPASy) proteomics server (http://expasy.org; Gasteiger et al., 2003) or the PredictProtein server (http://predictprotein.org; Rost et al., 2004).
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, 2006). In order to express full-length constructs, appropriate forward and reverse primers were used to amplify the correct product from the respective genomic DNA template, and the products were purified, digested, cloned into pQE80L and transformed into E. coli DH5. Plasmids were recovered from appropriate transformants, nucleotide sequenced to confirm their identity, and those with the correct inserts were used further as template in PCR for the generation of N- and C-terminal segment constructs and the expression of protein chimaeras. In order to construct the former, N-terminal segments were amplified using QIAF and appropriate reverse primers, whereas C-terminal segments were amplified using appropriate forward primers and QIAR; in each case the products were purified, digested with BamHI and HindIII, cloned into pQE80L and transformed into E. coli DH5 and appropriate transformants were selected and characterized prior to expression of the protein. In order to construct expression systems for chimaeras, the appropriate N-terminal segment was amplified by PCR using forward primers for the full-length constructs and an appropriate reverse primer incorporating a SalI restriction site, whereas the C-terminal segments were amplified using the appropriate forward primer incorporating a SalI restriction site and QIAR. Purified PCR products were restricted with SalI and ligated. The ligation mix was then used as template in a PCR amplification carried out using a forward primer appropriate for full-length amplification of the N-terminal segment (incorporating a BamHI restriction site) and a reverse primer appropriate for amplification of the C-terminal segment (incorporating a HindIII restriction site). The product was purified, restricted with BamHI and HindIII, ligated into pQE80L and then transformed into E. coli DH5. The identity of the inserts obtained was verified by sequencing of selected transformants.
Expression, purification, modification and bioanalytical characterization of recombinant proteins.
The expression and purification of all recombinant proteins, as well as the reductive alkylation of selected proteins, was carried out as previously described (Jack et al., 1996; Heng et al., 2004, 2006). SDS-PAGE analyses were carried out by electrophoretic separation on precast 16 % Tris-Tricine gels (Invitrogen) using the manufacturer's apparatus, chemicals and protocols, and protein bands were visualized with Coomassie brilliant blue G250 (Bio-Rad). Bioanalytical characterization of recombinant proteins involved mass spectrometry and, where necessary, N-terminal amino acid sequence analysis; this work was undertaken by the Centre for Protein Research (University of Otago).
Assessment of the distribution of ef1097 and ypkK.
Various clinical isolates of Enterococcus faecalis and Enterococcus faecium from our laboratory collection were used in colony PCR incorporating the primer pair EF1097-F3 and EF1097-R3 to detect ef1097. In order to detect ypkK, PCR were performed on clinical isolates of the nosocomial pathogen C. jeikeium obtained from the University Hospitals, Leuven (Belgium), and at the Université Louis Pasteur, Strasbourg (France), with the primer pair YpkK-F3/YpkK-R3 (Table 1
), obtained from Operon Biotechnologies (Cologne, Germany). In each case, PCR amplification was carried out using Taq DNA polymerase according to standard procedures, and products generated were separated by electrophoresis on 1 % agarose gels and visualized by transillumination following staining with ethidium bromide. E. coli DH5 with and without pQE80L-enterococcin V583 served as controls for ef1097 amplifications, whilst purified plasmid DNA of pB85766 and pK64 (Tauch et al., 2004) served as controls for the identification of ypkK.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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, b). In the present study the predicted mature forms of ScnM57 (i.e. ScnM57[Lys28–Phe179] or SA-M57), EF1097 (i.e. EF1097[Ser35–Ser170]) and YpkK (i.e. YpkK[Ala28–Trp164]; Table 2) were individually expressed as recombinant proteins in E. coli, purified and characterized to confirm their identity. All three recombinant proteins had demonstrable antimicrobial activity against a variety of bacterial strains (Table 3) with MICs against Lactococcus lactis of 64 nM, 4 nM and 2 nM for SA-M57, EF1097 and YpkK, respectively. Interestingly, some bacterial strains such as Micrococcus luteus, Lc. lactis and several species of listeriae appeared susceptible to all three antibiotics, whilst other indicators were targeted in a highly specific manner. Still others, for example all staphylococcal species tested, were insensitive to the effects of the three antibiotics. Similarly, no antimicrobial activity could be detected when SA-M57, EF1097[Ser35–Ser170] or YpkK[Ala28–Trp164] were tested against a variety of Gram-negative bacteria (not shown). Since their current names reflect their unknown status, as well as the observation that the mature, biologically active proteins are not the primary translation product of their respective gene but are revealed only after cleavage of a leader peptide, we suggest that EF1097[Ser35–Ser170]) be renamed enterococcin V583 and YpkK[Ala28–Trp164] be renamed corynicin JK to adequately reflect their bacteriocinogenic nature.
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) indicate that the polypeptides share little similarity in the N-terminal half of their sequences and only minimal similarity in the C-terminal portion. However, it has previously been noted that they do contain conserved predicted secondary structural similarity in the latter portion (Heng et al., 2004). Moreover, whilst the full-length SA-M57, enterococcin V583 and corynicin JK have predicted acidic pI values of 4.8, 4.9 and 5.4, respectively, the N-terminal portions of each appear to be more acidic (pI 4.1, 3.8 and 4.0, respectively) whilst the individual C-terminal segments are basic (pI 9.6, 10.0 and 9.8, respectively). These observations lead us to hypothesize that such antibiotic proteins may be composed of two portions: N-terminal segments with diverse structure and sequences responsible for targeting the diverse ranges of susceptible bacteria, and C-terminal portions with highly conserved secondary structure (but not primary structure) responsible for the killing action of the proteins.
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) that are predicted in silico to form a disulphide. We have previously shown that the related antibiotic protein dysgalacticin contains a disulphide that is essential for biological activity (Heng et al., 2006). In the present study, differential S-alkylation in the presence or absence of a reducing agent demonstrated that (i) recombinant SA-M57, enterococcin V583 and corynicin JK each contain a single disulphide bond and (ii) this bond is essential for biological activity (Table 4). This result further demonstrates the structural similarities conserved amongst the C-terminal portions of all members of this novel family of antimicrobial proteins and also supports the hypothesis that the C-terminal portion is essential for killing activity. In silico structure predictions for all three polypeptides indicate that, whilst the N-terminal segment is relatively unstructured, the C-terminal region may be composed of two helical elements, separated by a flexible loop region (Heng et al., 2004, 2006). Since the conserved Cys residues are located immediately before the first and immediately after the second putative helix, it seems reasonable to assume that the covalent bridge formed by the disulphide holds the helices parallel to one another and stabilizes the overall conformation of this region.
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). None of the purified recombinant N-terminal segments showed antibacterial activity at any concentration tested. In addition, we observed neither agonistic nor antagonistic effects when susceptible cells were either pre- or concomitantly treated with the N-terminal segment and its cognate full-length polypeptide. This latter result may indicate that the N-terminal portion is not (solely) responsible for target specificity.
By contrast, attempts to express either ScnM57[Val106–Phe179] or EF1097[Val94–Ser170] were unsuccessful; the reasons for this were not further investigated, but appeared to involve toxicity to the heterologous host as it failed to thrive after induction. We did however successfully express, isolate and characterize the C-terminal portion of YpkK. Interestingly, YpkK[Val88–Trp164] was biologically active in a spot assay (Fig. 2A) and had a MIC against M. luteus of 72 nM, a value only sixfold less than that for corynicin JK (MIC 12 nM). In a further experiment, approximately equal amounts of purified, recombinant YpkK[Ala28–Ser87] and YpkK[Val88–Trp164] were mixed, but this had neither an agonistic nor an antagonistic effect on the biological activity of the mixture. Similarly, YpkK[Ala28–Ser87] was neither agonistic nor antagonistic to corynicin JK when the two were mixed in approximately equal proportions. Surprisingly, YpkK[Val88–Trp164] also showed the same spectrum of activity as corynicin JK (not shown), indicating that the determinants of target specificity lie, at least partially, within this portion of the polypeptide and not the N-terminal region as hypothesized. The reduced antimicrobial activity of YpkK[Val88–Trp164] compared with corynicin JK also indicates that the N-terminal portion is at least indirectly involved in biological activity. Since the N- and C-terminal segments have opposite (and therefore attractive) pI, it is possible that the N-terminal segment interacts with and stabilizes the C-terminal portion or that it interacts with a possible ‘receptor’, thereby better localizing the C-terminal region to its target.
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). These experiments were based on the premise that if target specificity is defined by the N-terminal portion, then swapping potential ‘domains’ might alter the spectrum of activity of a given C-terminal segment and further clarify the role of the individual N-terminal portions. Surprisingly, only two of these recombinant chimaeras, namely ScnM57[Lys28–Lys105]/EF1097[Val94–Ser170] and ScnM57[Lys28–Lys105]/YpkK[Val88–Trp164], were expressed in the heterologous host as judged by SDS-PAGE. Again, the reasons for the failure of the remaining four to express in E. coli were not further investigated, but in this case it did not appear to involve host toxicity as the host grew as well as controls expressing full-length constructs during the expression phase. Both expressed chimaeras were isolated and analytically characterized to confirm their identity, and their potential antimicrobial activity was assessed. Surprisingly, neither showed any detectable antibacterial activity against any of the indicator strains tested. This result could indicate that there is an intimate and customized association between the N- and C-terminal segments of these antibiotics that is critical for full antimicrobial activity.
SA-M57, enterococcin V583, corynicin JK and YpkK[Val88–Trp164] kill sensitive bacteria without lysis
A small number of high molecular mass bacteriocins have been reported that lyse susceptible bacteria. For example, the glycyl-glycine endopeptidase lysostaphin from Staphylococcus simulans hydrolyses the transpeptide bridges of certain staphylococcal cell walls (Schindler & Schuhardt, 1964; Zygmunt & Tavormina, 1972), whereas the bacteriocins zoocin A from Streptococcus zooepidemicus (Simmonds et al., 1996, 1997) and stellalysin from Streptococcus constellatus (Heng et al., 2006b) are thought to act in a similar manner against various susceptible streptococci. In addition, the cyclic antimicrobial peptide uberolysin from various Streptococcus uberis strains kills susceptible cells by inducing their lysis (Wirawan et al., 2007). By contrast, we have previously shown that the 21.5 kDa antibiotic protein dysgalacticin kills susceptible cells without lysis (Heng et al., 2006). In the present study we assessed the killing action of SA-M57, enterococcin V583, corynicin JK and YpkK[Val 88–Trp164]. When the proteins were applied to agar-immobilized cell suspensions (Wirawan et al., 2007) of a variety of either live or heat-killed sensitive and non-sensitive indicator strains, we observed no indications of target cell lysis, although further growth of live, sensitive indicators was inhibited as expected. Moreover, addition of either 20 nM corynicin JK or 60 nM YpkK[Val88–Trp164] to a mid-exponential-phase liquid culture of Lc. lactis T-21 inhibited further growth but did not result in a decrease in optical density (Fig. 2B). Similarly, the optical density of a heat-killed suspension of Lc. lactis T-21 did not change after treatment with either corynicin JK or YpkK[Val 88-Trp164] at the same concentrations (Fig. 2C). In both cases, controls consisting of lysozyme-treated Lc. lactis T-21 showed characteristic loss of optical density, consistent with the murolytic mode of action of this enzyme. Taken together these results indicate that the tested proteins are neither murolytic enzymes nor mediators of lysis induction in sensitive cells; however, the mechanism by which they kill susceptible bacteria, like the basis for their target specificity, remains enigmatic.
Distribution of the genes encoding SA-M57, enterococcin V583 and corynicin JK
SA-M57 has been previously reported to be widely distributed amongst M type 57 Streptococcus pyogenes, as both the gene (scnM57) and plasmid on which it is carried (pDN571), as well as biologically active SA-M57, were identified in all 37 clinical isolates tested (Heng et al., 2004). In contrast, PCR screening of 33 independent clinical isolates of C. jeikeium identified ypkK in just two strains, indicating a relatively restricted distribution of this gene in corynebacterial isolates. This finding is surprising given that the plasmid on which ypkK may be carried (pK43-like family) is common among clinical isolates (Tauch et al., 2004). Furthermore, antibacterial activity consistent with corynicin JK could not be detected from C. jeikeium K64 in a modified deferred antagonism test, raising the possibility that ypkK either is not expressed in this strain, or is only expressed under as yet undefined culture conditions.
Using a diagnostic PCR with appropriate primers (Table 1) we have screened 18 independent isolates and found ef1097 in 10 of 11 strains of Ent. faecalis (including the genome sequence reference strain Ent. faecalis V583: Paulsen et al., 2003) and four of seven strains of Ent. faecium. This result would indicate that, like scnM57, ef1097 is widely distributed amongst natural isolates of enterococci. Moreover, antibacterial activity consistent with enterococcin V583 could also be detected from Ent. faecalis V583 in a modified deferred antagonism test, confirming that the gene is expressed, at least in this strain. In a recent study (Bourgogne et al., 2006), the FsrABC system of enterococci, which is a quorum-sensing regulator of enterococcal virulence (Qin et al., 2000, 2001), was shown to upregulate ef1097 expression (at the level of mRNA) upon entry into stationary phase. The broad distribution of ef1097 in enterococci, coupled with the observation that its expression is tightly regulated together with known virulence factors, may indicate that it is of importance to the producing bacterium and could also indicate that its production is related to enterococcal virulence.
SA-M57, enterococcin V583 and corynicin JK may be part of a broader family of antibiotic proteins from Gram-positive bacteria
Interestingly, recent interrogation of currently available databases would suggest that at least four further potential family members may also be currently documented as proteins of unknown function: the hypothetical proteins BCZK5154 from Bacillus cereus E33L (accession no. YP_086722; Han et al., 2006), BcerKBAB4DRAFT_0813 from Bacillus weihenstephanensis KBAB4 (accession no. ZP_01187444; A. Lapidus and others, 2006, DOE Joint Genome Institute, unpublished) and BL05338 from Bacillus licheniformis ATCC 14580 (accession no. YP_080680; Rey et al., 2004), as well as the putative ‘integral membrane protein’ from Lactobacillus plantarum WCFS1 (accession no. NP_786248; Kleerebezem et al., 2004). Although they share little sequence similarity, in silico analysis of these four sequences (not shown) indicates that their translated products are all likely to yield mature proteins of approximately 150 amino acids following cleavage of 30 amino acid signal peptides. Moreover, the predicted proteins are composed of two distinct portions, the C-terminal segments of which share similar predicted secondary structure and contain two conserved cysteine residues. Thus, it would seem that the hitherto unrecognized ability of Gram-positive bacteria to produce large proteinaceous antibiotics (bacteriocins) is relatively widely distributed. Indeed, it is tempting to speculate that this family of protein antibiotics may represent the Gram-positive equivalent of the colicin family in Gram-negative bacteria (Cascales et al., 2007).
Conclusion
We have demonstrated that the hypothetical proteins EF1097 and YpkK previously identified in the genome sequences of Ent. faecalis and C. jeikeium (Paulsen et al., 2003; Tauch et al., 2004) are precursors of antimicrobial proteins with similarities to the bacteriocins streptococcin A-M57 (Heng et al., 2004) and dysgalacticin (Heng et al., 2006). We have also shown that dysgalacticin, SA-M57, enterococcin V583 and corynicin JK may form (part of) a family of antibiotic proteins from Gram-positive bacteria that share little sequence similarity whilst maintaining conserved secondary structural elements. Information gained from the variety of recombinant protein segments and chimaeras used in this study indicates that the conserved secondary structure of the C-terminal portion of this family of protein antibiotics may be critical in mediating their killing activity and that target cell death does not involve lysis. Thus, our future studies will be directed at dissecting the basis for their target specificity and defining the mechanism by which the proteins kill susceptible cells.
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ACKNOWLEDGEMENTS |
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Edited by: D. M. Gordon
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REFERENCES |
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Received 18 June 2007;
accepted 24 July 2007.
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); 10 nM corynicin JK (
); 60 nM YpkK[Val88–Trp164] (
); or 1 mg lysozyme ml–1 (lysis control, 
). (C) Corynicin JK and YpkK[Val88–Trp164] do not lyse heat-killed Lc. lactis T-21 in suspension. Heat-killed Lc. lactis T-21 was treated at time 0 with: water (untreated control,