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Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin, New Zealand
Correspondence
Ralph W. Jack
ralph.jack{at}stonebow.otago.ac.nz
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Riley & Wertz, 2002). Broadly speaking, bacteriocins fall into one of four main categories, depending on whether they are produced by (and therefore act primarily on) Gram-positive or Gram-negative bacteria, and whether they are of high (>10 kDa) or relatively low molecular mass (<10 kDa). There is a wealth of scientific literature concerning bacteriocins from three of these categories: (i) the >20 kDa bacteriocins of Gram-negative bacteria, epitomized by the plasmid-encoded colicins from Escherichia coli (Gillor et al., 2004; Kirkup & Riley, 2004); the <10 kDa antimicrobial peptides or microcins produced by Gram-negative species (Gillor et al., 2004); and (iii) the low-molecular-mass bacteriocins of Gram-positive bacteria (reviewed by Jack et al., 1998; Eijsink et al., 2002; Fimland et al., 2005; Cotter et al., 2005). However, comparatively little is known about the colicin equivalents of Gram-positive bacteria, since reports of high-molecular-mass bacteriocins from such bacteria are scarce, the most extensively studied being the peptidoglycan-hydrolysing enzyme lysostaphin from Staphylococcus simulans (reviewed by Navarre & Schneewind, 1999).
Amongst the Gram-positive bacteria, the lactic acid bacteria are especially prodigious bacteriocin producers. Indeed, in recent years there has been a seemingly exponential increase in reports of novel inhibitory substances from these organisms (Papagianni, 2003; Fimland et al., 2005). Typically, these bacteriocins are ribosomally synthesized peptides that are characterized by their small size (generally <6 kDa) and heat stability (Jack et al., 1995). On further investigation, some of these have proven to be extensively post-translationally modified, e.g. the lanthionine-containing bacteriocins, or ‘lantibiotics' (Schnell et al., 1988; Sahl & Bierbaum, 1998), while others remain unmodified (Chatterjee et al., 2005; Fimland et al., 2005).
Various species of the genus Streptococcus commonly produce bacteriocins, and those best characterized to date are from Streptococcus pyogenes (group A streptococcus), Streptococcus salivarius and Streptococcus mutans (Jack et al., 1995, 1998; Chatterjee et al., 2005), e.g. the lantibiotics streptococcin A-FF22 and salivaricin A (Ross et al., 1993; Jack et al., 1994), and the unmodified peptides mutacin N and mutacin IV (Balakrishnan et al., 2000; Qi et al., 2001). However, several relatively large, heat-labile bacteriocins have also been described, including zoocin A from Streptococcus equi subsp. zooepidemicus, millericin B from Streptococcus milleri, and streptococcin A-M57 from S. pyogenes (Simmonds et al., 1996; Beukes et al., 2000; Heng et al., 2004).
In an effort to categorize streptococcal bacteriocin producer strains, a bacteriocin typing (or bacteriocin fingerprinting) scheme has been devised to discriminate inhibitory strains on the basis of differences in their inhibitory activity against a set of standard indicator bacteria (Tagg & Bannister, 1979). Using this scheme, a set of nine so-called standard producer strains with distinctive inhibitory spectra were defined (Tagg & Bannister, 1979). One of these standard producer strains, Lancefield Group G streptococcus W2580 (standard producer P4), exhibits a narrow spectrum of inhibitory activity directed almost exclusively against the large-colony 
-haemolytic streptococci (Lancefield Groups A, C and G), the principal streptococcal pathogens of humans (Tagg & Bannister, 1979; Wong, 1981; Wong et al., 1981; Tagg & Wong, 1983), but not against a range of oral streptococci, neisseriae, lactobacilli, Corynebacterium diphtheriae, Bacillus spp., Listeria spp., staphylococci, and various Gram-negative bacteria (Wong, 1981; H. J. Baird, unpublished results). Preliminary studies of the inhibitory agent from strain W2580 (originally designated streptococcin G-2580) revealed that (i) it was inactivated both by treatment with trypsin and by heating, indicating that it may be essentially proteinaceous in nature; and (ii) its elution profile during gel-permeation chromatography was consistent with that of a protein of molecular mass 
18 kDa (Wong, 1981; Wong et al., 1981). Moreover, S. pyogenes treated with partially purified preparations of streptococcin G-2580 was shown to be killed without lysis (Wong, 1981; Wong et al., 1981). This mode of action is in direct contrast to that of other large streptococcal bacteriocins such as zoocin A, which lyse target cells via proteolytic cleavage of peptidoglycan (Simmonds et al., 1996). Strain W2580 has since been identified by 16S rDNA sequencing as Streptococcus dysgalactiae subsp. equisimilis (N. L. Ragland, unpublished results) and, consequently, its bacteriocin product (streptococcin G-2580) has been renamed dysgalacticin.
In this study, we have purified dysgalacticin to homogeneity, biochemically characterized the bacteriocin and determined the genetic basis for its production in strain W2580. We show that the bacteriocin is plasmid-encoded and, following heterologous expression in E. coli, confirm that purified recombinant dysgalacticin is solely responsible for the antimicrobial phenotype exhibited by strain W2580, and that it has a non-lytic mode of action. Furthermore, we demonstrate that dysgalacticin possesses a novel predicted secondary structure containing a single disulphide bond that is essential for its biological activity.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Streptococcal strains were routinely cultivated at 37 °C in a microaerophilic (5 % CO2 in air) atmosphere on BACa medium [Columbia blood agar base (Becton Dickinson) containing 4 % (v/v) human blood and 0.1 % (w/v) CaCO3] or in Todd–Hewitt broth (THB; Becton Dickinson). Escherichia coli DH5
(Hanahan, 1983), the host for all cloning and expression experiments, was propagated aerobically at 37 °C in LB broth or on LB agar (Sambrook & Russell, 2001). When required, antibiotics were added to the media at the following final concentrations: ampicillin, 100 µg ml–1; chloramphenicol, 15 µg ml–1.
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with S. pyogenes FF22 as the indicator strain. Suitable fractions were pooled and concentrated by membrane filtration (Microcon YM-10; Millipore), and then further fractionated by gel-permeation chromatography in 25 mM HEPES containing 150 mM NaCl (pH 7.2) through a Superdex 75 100/300 GL column (GE Healthcare Bio-Sciences) at a constant flow rate of 0.5 ml min–1. Appropriate fractions (0.5 ml) containing inhibitory activity were pooled, buffer-exchanged with 5 mM HEPES (pH 7.2) and concentrated by membrane filtration (Microcon YM-10).
Bioanalytical characterization of dysgalacticin and recombinant proteins.
The approximate mass and degree of homogeneity of various proteins was assessed by SDS-PAGE on pre-cast Novex 16 % Tris/Tricine gels (Invitrogen) in an XCell SureLock Mini-Cell apparatus (Invitrogen), using the manufacturer's protocols and reagents. Following electrophoresis, protein bands were visualized with Coomassie Brilliant Blue G250 (Sigma). MALDI-TOF MS and N-terminal amino acid sequencing (by automated Edman degradation) of purified dysgalacticin were carried out at the Protein Microchemistry Facility (Department of Biochemistry, University of Otago). MS was conducted with a Finnigan LaserMAT 2000 (Thermo BioAnalysis) mass analyser, according to the method described by Hubbard & McHugh (1996). The N-terminal amino acid sequence of dysgalacticin was determined by automated, repetitive Edman degradation on a Procise model 492 pulsed liquid/gas-phase micro-sequencer (Applied Biosystems), using the manufacturer's reagents, protocols and analysis software.
Plasmid curing experiments.
Plasmid-cured mutants of S. dysgalactiae strain W2580 were derived according to the method of Tagg & Wannamaker (1976) by growing the strain in THB containing doubling dilutions of novobiocin (1250 µg ml–1 to 5 µg ml–1) and 0.5 µg trypsin ml–1 (to destroy any residual bacteriocin activity). A total of approximately 200 colonies were picked from cultures grown in the presence of 5 and 10 µg novobiocin ml–1 and subjected to a deferred antagonism assay (Tagg & Wannamaker, 1976) using S. pyogenes FF22 as the indicator strain. Colonies displaying loss of inhibitory activity were then retested using a standard bacteriocin typing scheme (Tagg & Bannister, 1979). One plasmid-cured mutant, designated W2580C, which was indistinguishable from the wild-type strain by biochemical (API 20 Strep, bioMérieux) and serological (Oxoid Streptococcal Grouping Kit) testing, and by 16S rDNA sequencing (Wilson et al., 1990), was selected for further study.
Recombinant DNA techniques, sequencing of pW2580 and bioinformatic tools.
Genomic DNA from streptococcal strains, plasmid DNA (from E. coli) and PCR amplicons were purified using the appropriate Qiagen spin-column-based kits, according to the manufacturer's instructions. Plasmid DNA from S. dysgalacticae strain W2580 was obtained using the TempliPhi DNA amplification kit (Amersham Biosciences). Established protocols for molecular cloning in E. coli and genetic analysis (e.g. Southern hybridization) were conducted as described by Sambrook & Russell (2001). For PCR experiments, the high-fidelity Platinum Pfx DNA polymerase (Invitrogen) was used according to the supplier's recommendations. All nucleotide sequencing requirements were met either by the Waikato DNA Sequencing Facility (Hamilton, New Zealand) or by the Allan Wilson Centre Genome Service (Palmerston North, New Zealand).
pW2580 was completely sequenced using the strategy described by Heng et al. (2004). After initial enrichment of pW2580 DNA using TempliPhi DNA polymerase, the 2.2 kb PstI–XbaI and 1.8 kb EcoRI–XbaI plasmid-derived fragments were individually cloned into pFX3 (Xu et al., 1991) and partially sequenced. The remainder of the plasmid sequence was obtained by PCR-based primer walking, using a total of six oligonucleotide primers (Table 2). Assembly and basic analyses of nucleotide sequence data were accomplished using the GeneJockey II software package (Biosoft). Homology searches were conducted with the appropriate BLAST algorithms (Altschul et al., 1997). In silico predictions of (i) the secretion signal peptide and (ii) potential secondary structures of dysgalacticin were achieved using SignalP version 3.0 (Bendtsen et al., 2004) and various algorithms (Rost, 1996; McGuffin et al., 2000; Pollastri et al., 2002; Vullo & Frasconi, 2004) available on the PredictProtein server (http://predictprotein.org; Rost et al., 2004), respectively.
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). Purified PCR products were then digested with BamHI and HindIII, and inserted into the corresponding sites of the expression vector pQE-80L (Qiagen), yielding pQE-dysAM (Table 1). The presence of the desired coding sequences was verified by sequencing of the cloned insert with primers QIAF and QIAR (Table 2). E. coli QE-dysA (DH5 carrying pQE-dysAM) was grown in 2x yeast tryptone (YT) broth (Sambrook & Russell, 2001) containing 0.5 % (w/v) glucose to OD590 0.6, after which IPTG was added to a final concentration of 1 mM to induce expression. Following incubation for a further 4 h, cells were harvested by centrifugation (4000 g, 5 min), resuspended in one-thirtieth of their original volume of lysis buffer [50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 1 mg lysozyme ml–1 (pH 8.0)] and disrupted by repetitive sonication (15 bursts of 30 s; Ultrasonics model W-220F). Cell debris was then removed by centrifugation (31 000 g, 30 min, 4 °C) and the supernatant (crude protein preparation) was subjected to further purification by chromatographic separation. Recombinant dysgalacticin was purified by: (i) initial enrichment of hexahistidine-tagged recombinant dysgalacticin using nickel-nitriloacetate (Ni-NTA) agarose (Qiagen) under native (non-denaturing) conditions, according to the manufacturer's protocol; (ii) anion-exchange chromatography; (iii) removal of the hexahistidine tag by treatment with factor Xa protease (New England Biolabs) at a 50 : 1 substrate : enzyme ratio for 4 h at 22 °C; and (iv) gel-permeation chromatography. The anion-exchange and gel-permeation chromatographic steps were the same as those used for the purification of native dysgalacticin, except that the anion-exchange buffers contained 2 mM CaCl2 to facilitate factor Xa digestion without the need for buffer exchange. The purity of recombinant protein was assessed by SDS-PAGE.
Preliminary mode-of-action studies.
In order to ascertain whether purified recombinant dysgalacticin kills target cells by lysis, we tested the effect of dysgalacticin on actively growing S. pyogenes strain FF22 by measuring changes in OD590 and cell viability, and compared this with cells treated with the known bacteriolytic agent zoocin A (Simmonds et al., 1996). Briefly, 50 ml pre-warmed THB broth was inoculated with 50 µl overnight culture of S. pyogenes strain FF22 and incubated at 37 °C without shaking. Samples were removed every hour for 4 h, after which, the culture was divided into three aliquots: the first was treated with the addition of 80 nM recombinant dysgalacticin, the second was treated with 10 nM purified recombinant zoocin A (kindly provided by R. Simmonds, University of Otago), and the third was untreated (negative control). Samples were then removed from each of the three aliquots after 10, 20, 40, 60 and 120 min. The OD590 was measured using a Novaspec-II spectrophotometer (Biochrom). Bacterial viability, expressed as c.f.u. ml–1, was assessed by spotting (in triplicate) 5 µl aliquots of serial dilutions (100 µl samples in 900 µl fresh THB) on blood agar plates. Following overnight incubation at 37 °C, colonies were enumerated.
Reductive alkylation of dysgalacticin.
In order to determine the oxidation state of the predicted cysteine residues of dysgalacticin, alkylation in the absence and presence of a reducing agent was carried out essentially as previously described (Jack et al., 1996). Briefly, recombinant dysgalacticin (3 µmol) was dissolved in 120 µl TA buffer [100 mM Tris/HCl (pH 8.0) containing 30 % (v/v) acetonitrile] and the sample was divided into three equal aliquots. The reducing agent 2-mercaptoethanol (Sigma) was then added in fivefold molar excess to one aliquot, and an equivalent volume of TA buffer was added to the remaining two portions of protein. After the samples had been mixed by gentle vortexing, they were incubated at room temperature for 1 h. Subsequently, 4-vinylpyridine (Sigma) was added in 20-fold molar excess to the reduced and one additional aliquot; an equivalent volume of TA buffer was added to the third portion and served as an untreated control. Following mixing, all samples were incubated for 2 h at room temperature. All three samples were desalted by gel-permeation chromatography in 5 mM HEPES (pH 7.2) on a 5 ml HiTrap desalting column (GE Healthcare Bio-Sciences), according to the manufacturer's instructions, and then assayed for antimicrobial activity (see above). Appropriate fractions were also analysed by MALDI-TOF MS to confirm incorporation of the 4-vinylpyridine moiety.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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22 kDa as judged by SDS-PAGE (data not shown). MALDI-TOF MS analysis revealed a single product with a mass of 21 492.5±19.3 Da, and N-terminal amino acid sequence analysis by automated Edman degradation generated the single sequence NETNNFAETQKEITTN(N)EA. A BLASTP search of publicly available sequence databases revealed no significant similarity between the N-terminal sequence of dysgalacticin and those of proteins of either known or unknown function, indicating that dysgalacticin is a novel antimicrobial agent.
The dysgalacticin structural gene is plasmid-encoded
In order to localize the dysgalacticin structural gene, oligonucleotide OP4-A (Table 2), synthesized based on the first seven amino acid residues of dysgalacticin, was used as a probe in Southern hybridization analyses. When EcoRI-digested genomic DNA of strain W2580 was probed, OP4-A hybridized not only to a fragment of 3.0 kb but also to a band migrating below that of chromosomal DNA in an undigested sample (data not shown), indicating an extrachromosomal location for the dysgalacticin structural gene. Furthermore, when W2580 genomic DNA treated with TempliPhi DNA polymerase (which preferentially amplifies plasmid DNA as concatemers) was subjected to restriction endonuclease analysis (yielding unit-length plasmid fragments), a single 3.1 kb band was observed by agarose gel electrophoresis (data not shown). Recognition sites for the restriction enzymes EcoRI, PstI and HindIII, but not BamHI, were present in the plasmid (Fig. 1).
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) was isolated following novobiocin treatment. Together, these results demonstrate that the genetic determinant responsible for dysgalacticin production is located on the indigenous plasmid of S. dysgalactiae W2580, designated pW2580.
Nucleotide sequence analysis of pW2580 and identification of dysA, the dysgalacticin structural gene
The complete nucleotide sequence of pW2580 was determined and totalled 3043 bp with a mol% G+C content of 35 mol%, slightly lower than the 39–41 mol% determined for S. dysgalactiae (Hardie, 1986). To the best of our knowledge, pW2580 is the first plasmid from this organism to be completely sequenced. We arbitrarily assigned the first nucleotide of the unique EcoRI site (Fig. 1) as the starting point for plasmid annotation. BLAST searches identified four genetic elements (Fig. 1), all associated with plasmid replication, and which classify pW2580 as a new member of the pMV158 rolling-circle plasmid family (Moscoso et al., 1995): (i) copG (nt 86–220) encodes a putative 5 kDa CopG repressor protein, probably responsible for maintaining plasmid copy number; (ii) repB (nt 283–906) which specifies the 24 kDa RepB replication initiation protein; (iii) dso (nt 2893–2991) or double-strand origin of replication, which is nicked by RepB during initiation of plasmid replication; and (iv) sso (nt 2394–2567) or single-strand (lagging-strand) origin, essential for conversion of the ssDNA intermediate to its double-stranded form (del Solar et al., 1998; Khan, 2005). The putative sso of pW2580 contains a highly conserved 14 bp sequence, termed RSB (5'-TTTATGCCGTGAAA-3'; nt 2399–2412) that is believed to be the site of interaction with RNA polymerase during initiation of lagging-strand synthesis (Kramer et al., 1998; Khan, 2005). The RSB is also a distinctive feature of ssoA, the most commonly encountered sso in rolling-circle plasmids isolated from Gram-positive species, irrespective of plasmid family (Khan, 2005). Each component of the replication machinery of pW2580 displays significant homology (between 86 and 97 % identity) with its respective counterpart from the prototype of the plasmid family, pMV158 (Moscoso et al., 1995), as well as other streptococcal members of the pMV158 family, including pSSU1 from Streptococcus suis (Takamatsu et al., 2000), and pER13 and pSMQ172 from Streptococcus thermophilus (Solow & Somkuti, 2000; Turgeon & Moineau, 2000).
The dysgalacticin structural gene, designated dysA, was located between nt 1102 and 1764 but in the opposite transcriptional orientation with respect to the other ORFs identified in pW2580 (Fig. 1). The dysA gene, which encodes the 220 aa (24.5 kDa) dysgalacticin preprotein DysA, was flanked (i) upstream by a potential promoter consisting of hexanucleotide motifs resembling –35 and –10 regions (separated by a 17 bp spacer), and (ii) downstream by an inverted repeat sequence that could function as a rho-independent transcriptional terminator (Fig. 2). As anticipated, DysA contained the sequence obtained by N-terminal sequencing of dysgalacticin (Fig. 2). However, the first amino acid of mature dysgalacticin corresponds to the twenty-ninth amino acid of DysA, indicating that the first 28 aa may constitute a secretion signal peptide. This finding was not unexpected, as all streptococcal bacteriocins characterized to date are exported either by dedicated ATP-binding cassette transporters, e.g. streptin (Wescombe & Tagg, 2003) and the non-lantibiotic mutacins (Hale et al., 2005), or by the general secretory (Sec-dependent) pathway, e.g. zoocin A (Simmonds et al., 1997). Indeed, the putative signal peptide of DysA displayed the typical hallmarks of proteins secreted by the Sec-dependent translocation pathway (Tjalsma et al., 2004; van Roosmalen et al., 2004) including: (i) several positively charged amino acids at the N-terminal (N-domain) end; (ii) a central core (H-domain) of primarily hydrophobic amino acids; and (iii) a C-terminal (C-domain) portion that contains a conserved proline residue at position –6 (Fig. 2). These observations were in perfect agreement with the results independently obtained using the SignalP signal peptide prediction algorithm (Bendtsen et al., 2004).
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In addition to the bacteriocin structural gene, most (if not all) bacteriocin-associated genetic loci usually contain genes that encode immunity factors conferring producer self-protection. For example, zif, which encodes the zoocin A immunity factor, is located adjacent to zooA, the zoocin A structural gene (Simmonds et al., 1997). Interestingly, the pW2580-free derivative, S. dysgalactiae strain W2580C, was not only defective in its ability to produce dysgalacticin, but also sensitive to the effects of the bacteriocin (either in a deferred-antagonism assay or with exogenously added inhibitor), implying that the gene(s) encoding the dysgalacticin immunity factor is (are) also plasmid-borne. However, as pW2580 appeared to contain only dysA and those genetic elements necessary for its replication, the mechanism of immunity to dysgalacticin may be atypical and thus remains enigmatic.
Detection of dysA variants in S. dysgalactiae subsp. equisimilis
Forty-two presumptive S. dysgalactiae strains available in our culture collection, which were isolated from clinical sources (Dunedin Public Hospital and the University of Minnesota, Minneapolis, MN, USA) and from Dunedin schoolchildren (Ragland & Tagg, 1990) were screened for the production of inhibitory activity using a standard deferred-antagonism protocol (Tagg & Bannister, 1979). Of these, nine exhibited inhibitory activity similar to that of strain W2580, whilst the remainder were non-inhibitory. All 42 strains were subjected to PCR analyses to detect the presence of dysA or pW2580-like plasmids using the following primer pairs (Table 2): (i) DysA-intF/DysA-intR, which amplifies an internal fragment of dysA; (ii) DysAUpF/DysA-ExpR, yielding the entire dysA locus; and (iii) P4-PR1/RepB-R, specific for repB of pW2580. Whereas PCR amplicons were obtained with all primer combinations from the nine inhibitory strains (data not shown), none was detected from non-inhibitory strains. In order to ascertain if these nine dysgalacticin-producing strains harboured dysA elements identical to that of strain W2580, nucleotide sequence analysis of these nine dysA homologues was conducted. Three nucleotide changes were observed in the dysgalacticin-coding sequence, but only resulting in two conservative amino acid changes (Leu–25
Ile, Thr+22Ser), in each case apparently not affecting the inhibitory activity displayed by the producer strains. Taken collectively, our results demonstrate that: (i) the production of dysgalacticin is correlated with the presence of pW2580-like plasmids, and (ii) dysgalacticin appears to be highly conserved.
Production of recombinant dysgalacticin by E. coli
As a means of obtaining larger amounts of dysgalacticin to facilitate subsequent biochemical studies, and also to establish whether the 21.5 kDa protein isolated from S. dysgalactiae W2580 is the sole agent responsible for the antimicrobial activity of this strain, recombinant dysgalacticin was produced by heterologous expression in E. coli. In order to accomplish this, an expression cassette consisting of the dysA gene (excluding the nucleotide tract encoding the signal peptide), preceded by a 5' nucleotide extension encoding a short linker sequence followed by a recognition site for factor Xa protease, was cloned into the hexahistidine-tag-encoding vector pQE-80L (Qiagen). The resulting construct, pQE-dysAM, facilitated the production of a recombinant protein that could be purified using successive chromatographic separations. Hexahistidine-tagged recombinant dysgalacticin was enriched by nickel ion affinity and subsequent anion-exchange chromatography and, following removal of the hexahistidine tag with factor Xa protease, recombinant dysgalacticin was obtained after fractionation by gel-permeation chromatography. Purified recombinant dysgalacticin was biologically active and its activity spectrum was indistinguishable from that of S. dygalactiae strain W2580 in a deferred antagonism test (data not shown). Taken together, these results conclusively demonstrate that: (i) dysgalacticin is solely responsible for the antibacterial activity exhibited by S. dysgalactiae strain W2580, and (ii) purified recombinant dysgalacticin is suitable for subsequent biochemical characterization experiments.
Dysgalacticin kills sensitive S. pyogenes without inducing lysis
The large bacteriocins of Gram-positive bacteria can generally be divided into the bacteriolytic enzymes, such as lysostaphin and zoocin A (Simmonds et al., 1997; Navarre & Schneewind, 1999), and the bacteriocins that kill by non-lytic means. However, to date, only one large bacteriocin, namely helveticin J from Lactobacillus helveticus (Joerger & Klaenhammer, 1986), appears to fulfil the criteria of the latter category, although nothing is known of its structure and physical properties. It has previously been suggested that partially purified dysgalacticin acts to kill S. pyogenes strain FF22 in a non-lytic manner, based on examination of treated cells by Gram staining and by electron microscopy (Wong et al., 1981; Wong, 1981). In the present study, we treated S. pyogenes strain FF22 with either purified recombinant dysgalacticin or the bacteriolytic protein zoocin A (Simmonds et al., 1996) and monitored both the OD590 and the viable cell count of the culture. As shown in Fig. 3, the OD590 of dysgalacticin-treated cells remained static, whilst the cell viability decreased dramatically immediately after addition of the bacteriocin. In contrast, zoocin A treatment of S. pyogenes resulted in a rapid decrease in both OD590 and viable cell count, consistent with the bacteriolytic mode of action of this bacteriocin (Simmonds et al., 1996). Taken collectively, these observations confirm that dysgalacticin kills sensitive target cells by a non-lytic mechanism.
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) and these, according to the DISULFIND algorithm (Vullo & Frasconi, 2004), are predicted to form a disulphide bond. It is interesting to note that the putative disulphide (cystine) in dysgalacticin flanked the predicted strongly helical region (Fig. 2) and could, therefore, play a role in maintaining the structural stability of the C-terminal portion of the bacteriocin.
Disulphides are thought to be important to the biological activity of a number of antimicrobial proteins from Gram-positive bacteria (Ennahar et al., 2000). Alkylation with 4-vinylpyridine in the absence and presence of a reducing agent has been shown to be a useful technique in determining both the oxidation state of cysteine residues and the role they play in the biological activity of bacteriocins such as piscicolin 126 (Jack et al., 1996). Under the conditions employed, alkylation only occurs when the polypeptide being studied contains cysteine (free thiol); thus, if cystine is naturally present, S-pyridethyl adducts are observed (e.g. by MS) only on alkylation following reduction. Conversely, if the encoded Cys residues are naturally present as cysteine, alkylation will occur irrespective of prior reduction.
In order to assess the oxidation state of Cys132 and Cys186, recombinant dysgalacticin was exposed to the alkylating agent 4-vinylpyridine, in either the absence or presence of the reducing agent 2-mercaptoethanol (Jack et al., 1996). Samples that were treated with 2-mercaptoethanol prior to alkylation exhibited a marked loss of biological activity against the indicator organism S. pyogenes strain FF22 (Fig. 4). In contrast, untreated dysgalacticin, or that treated with the 4-vinylpyridine alone, showed essentially the same level of biological activity (Fig. 4). Furthermore, MS of the reaction products generated showed an increase in mass of 210 Da (indicating alkylation at two sites) only in protein that had been reduced prior to exposure to the alkylating agent. These results provide experimental evidence that dysgalacticin contains a disulphide essential for its biological activity.
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). These two bacteriocins do not share any similarity with respect to their primary amino acid sequences but, surprisingly, the predicted secondary structure of SA-M57 is similar to that of dysgalacticin, i.e. a flexible N-terminal portion and a predominantly helical C-terminal region containing a putative disulphide (between Cys87 and Cys148). Reductive alkylation experiments with purified recombinant SA-M57 confirmed the presence of an essential disulphide (data not shown), strongly supporting the proposed function of the disulphide in facilitating a biologically active protein conformation. Unlike dysgalacticin, however, SA-M57 is not active against S. pyogenes, but rather has a spectrum of activity that includes Micrococcus luteus, Lactococcus lactis (and its different subspecies), certain Listeria spp., Bacillus megaterium and Staphylococcus simulans (Heng et al., 2004). Interestingly, SA-M57 (but not dysgalacticin) shares limited sequence similarity with two hypothetical proteins of unknown function (Heng et al., 2004): EF1097 from the genome sequence of Enterococcus faecalis V583 (Paulsen et al., 2003), and YpkK from the sequence of a large virulence-associated plasmid harboured by Corynebacterium jeikeium (Tauch et al., 2004). It remains to be determined whether EF1097 and YpkK possess antimicrobial activity.
Based on the similarity of their predicted secondary structures, dysgalacticin and SA-M57 (and possibly EF1097 and YpkK) may represent the first members of a novel family of antimicrobial agents, the target specificity of which may be dictated by their primary amino acid sequences (i.e. the variable N-terminal segment), whilst their killing function could be determined by their shared secondary structure, especially of the C-terminal portion. Such a model is compatible with that proposed for the well-studied lytic bacteriocin zoocin A, which is composed of an N-terminal peptidase (catalytic) domain and a C-terminal substrate-binding (targeting) domain, separated by a short linker sequence (Simmonds et al., 1997). Recent studies with recombinant fragments of zoocin A have also shown that each individual domain can function independently (Lai et al., 2002), indicating that a similar approach may be of value in determining whether dysgalacticin and its related proteins are indeed composed of distinct domains.
Conclusion
In conclusion, we have utilized a combination of protein purification, reverse genetics and heterologous expression (in E. coli) to characterize dysgalacticin, a novel plasmid-encoded, large non-lytic bacteriocin that is presumably exported via a Sec-dependent protein secretion system. In light of the observation that strain W2580C (the plasmid-cured mutant) is sensitive to the effects of the bacteriocin, it is tempting to speculate that the ecological role of dysgalacticin is analogous to that of the colicins, i.e. as a selective agent to maintain pW2580 in the bacterial population (Kirkup & Riley, 2004). In addition, dysgalacticin may represent the prototype of a new family of antimicrobial proteins, differing in their antimicrobial spectra, but possessing similar predicted secondary structures. Our ongoing studies are now focused on obtaining a greater understanding of structure–function relationships within this unusual new class of antimicrobial proteins.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 6 January 2006;
accepted 23 March 2006.
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, untreated S. pyogenes culture (negative control); 
, S. pyogenes treated with dysgalacticin; 
, S. pyogenes treated with zoocin A.