Laboratory of Microbial Gene Technology, Department of Chemistry and Biotechnology, Agricultural University of Norway, PO Box 5051, N-1432 s, Norway1
Norwegian Dairies Association, Centre for Research and Development, Oslo, Norway2
Department of Food Science and Technology, 240 Wiegand Hall, Oregon State University, Corvallis, OR 97331, USA3
Department of Immunology, Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, D-72076 Tübingen, Germany4
Author for correspondence: Helge Holo. Tel: +47 64949468. Fax: +47 64941465. e-mail: helge.holo{at}ikb.nlh.no
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ABSTRACT |
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Keywords: two-peptide bacteriocin, lactic acid bacteria
Abbreviations: BU, bacteriocin unit; Dha, 2,3-didehydroalanine; Dhb, 2,3-didehydrobutyrine; Lan, lanthionine; MeLan, 3-methyllanthionine; Plw, plantaricin W; TFA, trifluoroacetic acid
The GenBank accession number for the sequence reported in this paper is AY007251.
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INTRODUCTION |
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The bacteriocins from lactic acid bacteria are mostly small, heat-stable, hydrophobic and cationic peptides (Jack et al., 1995 ). The peptide bacteriocins are either normal, unmodified peptides or lantibiotics, the latter being defined as ribosomally synthesized peptides containing the thioether amino acids lanthionine (Lan) and 3-methyllanthionine (MeLan). Jung (1991)
divided the lantibiotics into two types: type A lantibiotics are elongated peptides with molecular masses of 21643488 Da, whereas type B lantibiotics are globular molecules with masses of 19592041. In addition to Lan and MeLan, the
,ß-unsaturated amino acids 2,3-didehydroalanine (Dha) and 2,3-didehydrobutyrine (Dhb) are frequently found in lantibiotics. The unusual amino acids in lantibiotics are formed as a result of post-translational modifications of precursor peptides. Serine and threonine residues are specifically dehydrated to give Dha and Dhb, respectively. The addition of cysteine thiol groups to the dehydrated residues leads to the formation of the intramolecular thioether bridges.
In general, the lantibiotics have wider inhibitory spectra than the non-lantibiotics. Nisin, produced by strains of Lactococcus lactis, is the best-studied lantibiotic and is also the only lantibiotic in practical use as an antimicrobial agent (Gross & Morell, 1971 ). The search for other bacteriocins of food-grade micro-organisms (lactic acid bacteria) is ongoing. Recently, a new lantibiotic lacticin 3147 produced by other strains of Lc. lactis was reported to have potential uses in food preservation as well as in veterinary medicine (Ryan et al., 1996
, 1998
, 1999
).
Insights into the mode of action and the structurefunction relationships is of great value in exploiting the antimicrobial properties of these peptides. The intramolecular rings formed by the thioether bonds give the lantibiotics unique structural properties. These bridges are probably also of great importance for their antimicrobial activities, and genetic engineering experiments have shown that mutations affecting the bridging pattern are detrimental to activity (Kuipers et al., 1996 ). In this report, we describe the two-peptide lantibiotic plantaricin W (Plw), which possesses several properties previously unseen among bacteriocins. Our data indicate that Plw belongs to a new family of two-peptide lantibiotics with conserved bridging patterns.
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METHODS |
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Determination of bacteriocin activity.
Sterile, cell-free culture supernatants were obtained by centrifugation (15000 g, 10 min) followed by incubation at 100 °C for 10 min. Bacteriocin activity was quantified by the microtitre plate assay (Holo et al., 1991 ). Each well of the microtitre plate contained 200 µl MRS broth, bacteriocin fractions at twofold dilutions, and the indicator organism, Lb. sakei NCDO 2714 (about 106 c.f.u. ml-1). The microtitre plate cultures were incubated overnight at 30 °C, after which growth inhibition of the indicator organism was measured spectrophotometrically at 600 nm. By definition, the activity of one bacteriocin unit (BU) causes 50% inhibition (50% of the growth of the control culture lacking bacteriocin addition) in this assay. An agar diffusion assay (Cintas et al., 1995
) was used for the examination of the inhibitory spectrum of the bacteriocin.
Isolation of plantaricin W.
The bacteriocin was isolated from 1 litre cultures of strain LMG 2379 grown to early stationary phase. The cells were removed by centrifugation (10000 g for 10 min). Ten millilitres of SP Sepharose (Amersham Pharmacia Biotech) was added to the supernatant and the mixture was left overnight at 4 °C with constant stirring with a magnetic stirring bar. The mixture was then transferred to a chromatographic column, the matrix was washed with 200 ml 10 mM sodium phosphate (pH 7) and the bacteriocin was eluted in 20 ml 1 M NaCl. The eluate was applied to a column containing 2 g Amberlite XAD-16 (Supelco) equilibrated with water. After washing with 10 ml water and 10 ml 40% ethanol in water, the bacteriocin was eluted in 10 ml 70% 2-propanol containing 0·1% trifluoroacetic acid (TFA). This eluate was then subjected to reverse-phase chromatography on a Resource-RPC column, using an Äkta Purifier System (Amersham Pharmacia Biotech). The peptides were eluted from the column in a water/2-propanol gradient containing 0·1% TFA. Fractions showing synergistic activities were collected and purified separately by repeating the reverse-phase chromatography step.
Amino acid sequencing and composition analysis.
The purified peptides were hydrolysed and analysed on an amino acid analyser as described previously (Fykse et al., 1988 ). The amino acid sequence was determined by Edman degradation using an Applied Biosystems 477A automatic sequence analyser with an on-line 120A phenylthiohydantoin amino acid analyser (Cornwell et al., 1988
). To enable Edman degradation of Plwß, the peptide was chemically modified according to Meyer et al. (1994)
.
Mass spectral analysis.
MS analysis was performed on a Sciex API I electrospray mass spectrometer.
PCR and DNA sequencing.
DNA was prepared by the method of Anderson & McKay (1983) . Restriction enzymes and other DNA-modifying enzymes were used as recommended by the manufacturer (Promega). PCRs were performed with Dynazyme (Finnzymes) in a DNA thermal cycler (Perkin-Elmer). The DNA primers used for PCR and DNA sequencing are shown in Table 1
. The PCR products were purified by agarose gel electrophoresis and extracted from the gel with the GeneClean II kit (Bio 101). The PCR products were sequenced with the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer) and an ABI PRISM 377 DNA sequencer (Perkin-Elmer). The degenerate oligonucleotide primers W1 and W2 were constructed on the basis of the amino acid sequence of Plw
and used in a PCR to generate a 44 bp DNA fragment. This fragment was sequenced and new primers based on its sequence were synthesized. Further sequences were obtained largely as described previously (Casaus et al., 1997
); DNA of strain LMG 2379 was cut with EcoRV, DraI and RsaI, then ligated to plasmid vector Bluescript SK II (Stratagene) cut with HincII. The ligation products served as templates in PCRs using plw-specific primers in combination with vector-specific primers; the products obtained were sequenced.
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RESULTS |
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Sequences of the bacteriocin genes and elucidation of primary structure
The sequences of the genes encoding Plw and Plwß were obtained after the use of PCR and degenerate oligonucleotide primers synthesized on the basis of the amino acid sequence of Plw
. The sequence of the region (Fig. 2
) revealed that the two genes encoding each of the peptides are located next to each other. A putative promoter was identified upstream of plwßA, which is immediately preceded by plw
A, indicating that the two genes are organized in a transcriptional unit, like all other two-peptide bacteriocins studied.
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The role of the cystine bridge in Plw was studied by performing the microtitre assay in the presence of 10 mM mercaptoethanol to reduce the disulfide bond (data not shown). The activity of isolated Plw
was increased about fourfold by introducing mercaptoethanol into the assay. However, the thiol reagent caused no activity increase with Plwß or with mixtures of Plw
and Plwß.
The data obtained by amino acid sequencing of the modified Plwß were combined with amino acid composition analysis and DNA sequencing to elucidate the primary structure of the peptide. From Fig. 2, it can be seen that the 32 aa C-terminal part of the Plwß translation product corresponds to the isolated peptide. This peptide has a calculated molecular mass of 3207 Da and a pI of 9·96. With the exception of the three amino acids that are usually modified in lantibiotics, there is a good correlation between the amino acid composition of the unmodified peptide and that of Plwß (Table 3
). For Plw
, the data indicate that three Cys residues are involved in Lan/MeLan formation. Furthermore, all six hydroxy acid residues appeared to be modified, probably dehydrated. The molecular mass of Plwß was found, by MS, to be 3099 Da, which is in agreement with that for a modified peptide containing six dehydrated residues.
Homology with other bacteriocins
Computer-aided homology searches revealed that the Plwß prepeptides have sequence similarities with the prepeptides of two other two-peptide bacteriocins, namely lacticin 3147 (produced by Lc. lactis) and staphylococcin C55 (produced by Staphylococcus aureus). Both of these bacteriocins are two-peptide lantibiotics (Dougherty et al., 1998 ; Navaratna et al., 1998
, 1999
; Ryan et al., 1999
). In Fig. 3
, the corresponding prepeptides have been aligned. Staphylococcin C55 and lacticin 3147 are much more closely related to each other than to Plw, having 86·7 and 43·0% sequence identity for the
and ß propeptides, respectively. The Plw
propeptide showed 31·2% and 40·0% identity to the corresponding peptides of staphylococcin C55 and lacticin 3147, respectively. The values for the Plwß propeptide and its respective homologues were 31·6 and 26·5%. The alignments also show that most of the residues conserved in all three bacteriocins are located in the C-terminal parts of the peptides. Furthermore, they are amino acids that contribute to structural rigidity: Cys, Ser and Thr residues that can be involved in bridge formation and, in the case of the ß peptides, also Pro residues.
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DISCUSSION |
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Two-peptide bacteriocins have been isolated from many Gram-positive bacteria, in lactic acid bacteria in particular, and they are found amongst lantibiotics as well as non-lantibiotics. In most cases, it has been found that one of the two peptides exerts bacteriocin activity on its own, and that this activity is stimulated by the presence of the other peptide (Allison et al., 1994 ; Anderssen et al., 1998
; Jiménez-Díaz et al., 1995
; Marciset et al., 1997
; Navaratna et al., 1998
; Ryan et al., 1999
). Neither of the two peptides of lactococcin G has any bacteriocin activity (Nissen-Meyer et al., 1992
). In the Plw system, however, both peptides had inherent antimicrobial activities. In two-peptide bacteriocin systems, this has previously been described only for enterocin L-50, the two peptides of which are almost identical (Cintas et al., 1998
). In this regard, Plw is different from the related bacteriocins staphylococcin C55 and lacticin 3147 in that it is only the Plw
homologue that shows inhibitory activity on its own (Navaratna et al., 1998
; Ryan et al., 1999
).
The relationship established between the concentrations of each of the two Plw peptides in a mixture and the mixtures antmicrobial activity indicates that the two peptides cooperate in a 1:1 ratio. A 1:1 ratio between the two peptides has been reported to be optimal for several two-peptide bacteriocins lantibiotics as well as non-lantibiotics (Navaratna et al., 1998 ; Moll et al., 1996
; Marciset et al., 1997
; Anderssen et al., 1998
). This stoichiometry has usually been inferred from the mixing ratio giving the highest specific bacteriocin activity. At suboptimal mixing ratios, the doseresponse relationships have been found to differ significantly from that of the Plw system. In the case of staphylococcin C55, stimulation of the activity of the
peptide was observed only in mixtures with mixing ratios (ß/
) between 0·1 and 1 (Navaratna et al., 1998
). The enhancing factor, ThmB, of thermophilin 13 increased the activity of ThmA about 40-fold when the mixing ratio was 1, but an excess of ThmB was inhibitory to the bacteriocin activity (Marciset et al., 1997
). In the Plw system, on the other hand, an increasing and continuous response to the concentration of either peptide was seen, and the mixtures activities were directly related to their concentrations. The relationship between peptide concentrations and bacteriocin activity suggests that the activity was limited by each peptides affinity for its target, and that the concentation of the other peptide had no or little influence on this affinity.
In lantibiotics, most Cys, Ser and Thr residues have undergone post-translational modifications, forming specific Lan/MeLan bridges and, usually, ,ß-unsaturated amino acids. By comparing the amino acid contents of the isolated peptides with those expected from the DNA sequences, it was clear that most of the Ser, Thr and Cys residues in Plw were modified. However, unmodified Cys was found in both of the Plw peptides: one residue was in Plwß and two were in Plw
. In lantibiotics, unmodified Cys residues have previously been demonstrated only in sublancin 168 (Paik et al., 1998
). Furthermore, all of the Ser and Thr residues of Plwß were modified, and our data indicate the presence of three
,ß-unsaturated amino acid residues and three Lan/MeLan cross-links. The number of thioether bridges appears to be the same in Plw
, but this peptide contained no
,ß-unsaturated amino acid residues and, more remarkably, an unmodified Ser residue. This is the first example of the presence of both a Cys and a Ser in a lantibiotic.
A number of two-peptide bacteriocins have been found in lactic acid bacteria; they show a remarkable diversity of primary structure. The two peptides of Plw show significant sequence similarities with the corresponding peptides of two other two-component lantibiotics, namely lacticin 3147 (from Lc. lactis) and staphylococcin C55 (from S. aureus) (Navaratna et al., 1999 ; Ryan et al., 1999
). We suggest that these three bacteriocins constitute a new family of bacteriocins. They belong to the type A lantibiotics, which are linear peptides, as opposed to the circular type B lantibiotics (Jung, 1991
). The type A lantibiotics have been grouped into two classes, namely class AI and class AII (de Vos et al., 1995
). This classification is mainly based on the properties of the leader peptide and the mechanism by which the bacteriocin is exported. Class AII lantibiotics are all translated with leader peptides of the so-called double-glycine type, characterized by two conserved glycine residues at positions -1 and -2. This kind of leader is also prevalent in non-lantibiotic peptide bacteriocins from Gram-positive and Gram-negative bacteria (H
varstein et al., 1994
). The export of bacteriocins containing double-glycine-type leaders involves a dedicated ABC transporter containing an N-terminal proteolytic domain which cleaves off the leader peptide (H
varstein et al., 1995
). The leader peptides of the bacteriocins exported by these ABC transporters are characterized by consensus elements occurring at defined distances relative to the cleavage site (H
varstein et al., 1995
). The plw
A gene encodes a peptide of 59 aa, and the sequence of the N-terminal extension has most of the characteristics of a leader peptide of the double-glycine type. On the other hand, the amino acid sequence preceding the N-terminal residue of mature Plwß did not conform to such a leader. With one exception (staphylococcin C55ß; Navaratna et al., 1999
) (Fig. 3
), the Gly at position -2 relative to the processing site is present in all of the leader peptides of the Gly-Gly type studied to date, and cleavage after the sequence Ala-Arg would be a violation of this rule. However, genes encoding ABC transporters with the characteristics of transporters of bacteriocins with double-glycine leaders are found downstream of the structural genes for lacticin 3147 (Dougherty et al., 1998
) and staphylococcin C55 (Navaratna et al., 1999
), as well as Plw (GenBank accession number AY007251). This suggests that these bacteriocins are indeed class AII lantibiotics. In the Plwß prepeptide, an alternative processing site could be identified between positions -6 and -7 (Fig. 3
). Thus, the sequence encompassing the first 29 residues in the Plwß prepeptide could constitute a putative leader peptide of the double-glycine type. With such a leader peptide, the processing site would be after the sequence Gly-Ala, and eight of the nine consensus elements for double-glycine leader peptides would be present. The only consensus element missing is a hydrophobic residue at position -4, and this element is also absent from the leader peptides of lactococcin G
and the lantibiotic sublancin 168 (Paik et al., 1998
). We suggest, therefore, that Plwß is synthesized with a 29 aa leader, processed by an ABC transporter and then subjected to further proteolytic attack, resulting in the removal of the six N-terminal residues. A similar situation is seen with plantaricin A. The sequence of isolated plantaricin A indicates that a few amino acid residues are removed from the N-terminus after cleavage of the prepeptide at the Gly-Gly processing site (Diep et al., 1994
).
The thioether bridging patterns have been determined for several lantibiotics, and homologous lantibiotics have conserved bridging patterns (Jack et al., 1998 ). Also, the few engineered variants altered at the sites involved in ring formation showed little or no bacteriocin activity (Kuipers et al., 1996
). This indicates an important role for Lan/MeLan in lantibiotic function. However, the thioether bridging pattern has not been established for any two-peptide lantibiotic.
The enzymic formation of dehydrated amino acid residues and that of thioether bonds are specific processes. For unknown reasons, the thioether bonds in type A lantibiotics are formed between Cys residues and Ser- or Thr-derived residues located N-terminally to their partners (de Vos et al., 1995 ; Jack et al., 1998
). This restricts the number of potential thioether bridges in the Plw peptides. The model presented in Fig. 5
shows a Lan cross-link between residues 8 and 18 in Plw
. As expected, the unmodified Ser residue is located close to the C-terminus and is not conserved within the family. The model also shows two overlapping thioether bridges in the C-terminal part of the Plw
molecule. We consider this structure to be more likely than the alternative, which has one ring enclosed within the other a pattern never seen in a type A lantibiotic. Furthermore, overlapping thioether bridges are found in most type A lantibiotics with a known bridging pattern (Jack et al., 1998
). The exceptions are lactocin S (Skaugen et al., 1994
), which has two bridges, and, of course, sublancin, which has only one bridge (Paik et al., 1998
).
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The high numbers of hydroxy amino acid residues and Cys residues in the lacticin 3147 and staphylococcin C55 propeptides make it impossible to predict their bridging patterns directly from their sequences. Like Plw, they have three thioether bridges in each of their peptides (Navaratna et al., 1998 ; Ryan et al., 1999
). Furthermore, the residues conserved among all three bacteriocins are mainly restricted to those that can be involved in thioether bridge formation. These findings, coupled with the fact that thioether bridges appear to be of vital importance for function, strongly suggest that the bridging patterns have been conserved within the family. Thus, the thioether bridging patterns suggested in Fig. 5
also apply to lacticin 3147 and staphylococcin C55. However, the cystine is unique to the Plw
molecule. The overlapping cross-links give the Plw
molecule two rigid parts joined by the peptide bond between residues 18 and 19. The corresponding peptides of staphylococcin C55 and lacticin 3147 would lack the cystine bridge and would have more-flexible N-terminal parts. Although reduction of the cystine bridge increased the activity of isolated Plw
about fourfold, it had no effect on the activities of mixtures of Plw
and Plwß. Several type A lantibiotics have been found to form pores in bacterial cytoplasmic membranes. It has been suggested that the peptides remain surface bound during the pore-formation process and enter the membrane in a bent conformation (van den Hooven et al., 1996a
, b
). The peptides contain a central hinge region, and it has been shown for nisin, epidermin and Pep5 that this flexible region is essential for activity (Sahl et al., 1995
). Apparently, such a flexible region is not important for Plw activity.
As discussed above, we suggest that the bridging patterns shown are common to all three members of this new family of two-peptide lantibiotics. We have taken advantage of the low sequence similarities between Plw and its relatives to pinpoint residues contributing to structural rigidity. Our data have enabled us to present the first model for the localization of thioether cross-links in two-peptide lantibiotics; these data will also be useful in elucidating the structurefunction relationships of such antimicrobial compounds.
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ACKNOWLEDGEMENTS |
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Received 22 August 2000;
revised 13 November 2000;
accepted 15 November 2000.