Each peptide of the two-component lantibiotic lacticin 3147 requires a separate modification enzyme for activity

Olivia McAuliffe1, Colin Hill1 and R. Paul Ross2

Department of Microbiology and National Food Biotechnology Centre, University College Cork, Ireland1
National Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland2

Author for correspondence: Colin Hill. Tel: +353 21 902397. Fax: +353 21 903101. e-mail: c.hill{at}ucc.ie


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The genetic determinants for production and immunity to the two-component lantibiotic lacticin 3147 are encoded by a 12·6 kb region of the plasmid pMRC01. This region contains ten genes arranged in two divergent clusters; these include the structural genes and a number of genes whose products show significant similarity to proteins involved in the biosynthesis of other lantibiotics. Using a strategy of deletion and mutational analysis, the effect of disruption of a number of these genes was investigated. Inactivation of either of the structural genes, ltnA1 or ltnA2, resulted in mutants that were incapable of producing active lacticin 3147; however, the combination of the cell-free supernatant from both mutants resulted in a restoration of bacteriocin activity, confirming that processing and export of the structural peptides can occur independently. An unusual feature of the lacticin 3147 gene cluster is the presence of two lanM homologues, whose gene products are proposed to be involved in the dehydration and thioether-forming reactions which result in lanthionine bridge formation. Mutants created in the ltnM1 and ltnM2 genes were also incapable of lantibiotic production, confirming an essential role for these enzymes in the lacticin 3147 biosynthetic pathway and supporting the assertion that these proteins are modification enzymes. Interestingly, addition of purified LtnA1, but not purified LtnA2, to the cell-free supernatant of the ltnM1 mutant restored bacteriocin activity; in contrast, only purified LtnA2 could complement the cell-free supernatant of the ltnM2 mutant. Creation of a number of double mutants supported these findings, and confirmed that LtnM1 is required to produce mature LtnA1, while LtnM2 is required to produce mature LtnA2.

Keywords: lacticin 3147, lantibiotic production, immunity, modification enzymes

Abbreviations: ABC, ATP-binding cassette; AU, arbitrary units; CFS, cell-free supernatant


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Lacticin 3147 is a two-component bacteriocin produced by Lactococcus lactis subsp. lactis DPC3147 (Ryan et al., 1996 ; McAuliffe et al., 1998 ). Both components are required for the activity of the bacteriocin, which has been shown to result in the formation of ion-specific pores in the membrane of Gram-positive cells (McAuliffe et al., 1998 ). Lacticin 3147 belongs to the class of antimicrobials known as the lantibiotics (Ryan et al., 1999 ), a group of post-translationally modified peptides characterized by the presence of a high proportion of unusual amino acids, including the thioether amino acids lanthionine and ß-methyllanthionine (Schnell et al., 1988 ; Sahl et al., 1995 ). The presence of these residues was first identified in nisin (Berridge et al., 1952 ) and further analysis has revealed that lanthionine and ß-methyllanthionine originate from the sequence-specific dehydration of the hydroxyl amino acids serine (to didehydroalanine) and threonine (to didehydrobutyrine) (Ingram, 1969 ). These dehydrated amino acids possess electrophilic centres which enable them to react with neighbouring cysteine residues. The thioether lanthionine is formed when the double bond of didehydroalanine is attacked by the thiol group of cysteine; ß-methyllanthionine results when the reaction partner is didehydrobutyrine (Ingram, 1969 ). Some lantibiotics contain further modified residues, such as S-aminovinylcysteine in mature epidermin (Kupke et al., 1994 ) and D-alanine, first observed in mature lactocin S (Skaugen et al., 1994 ), and more recently identified in both lacticin 3147 peptides (Ryan et al., 1999 ).

The events leading to the production of a mature lantibiotic begin with the synthesis of a gene-encoded precursor peptide composed of a leader peptide and a propeptide region. This latter region is extensively modified prior to processing of the leader peptide and translocation across the membrane, as first proposed for epidermin by Schnell et al. (1988) . The genes encoding the biosynthetic machinery of the lantibiotics characterized to date are arranged in either plasmid- or chromosomally-encoded gene clusters which encode a range of enzymes, including specific proteases (involved in removal of the leader sequence), regulatory proteins, immunity proteins and transport proteins [members of the ATP-binding cassette (ABC)-superfamily] (Jack et al., 1995 ; Sahl et al., 1995 ; Siezen et al., 1996 ). In addition, a number of genes have been identified whose products do not share sequence similarity with any known proteins. These include lanB and lanC in the nisin-like lantibiotics (Engelke et al., 1992 ; Gutowski-Eckel et al., 1994 ; Meyer et al., 1995 ), and lanM in the lantibiotic subclass containing lacticin 481 (Rince et al., 1994 ), lactocin S (Skaugen et al., 1997 ) and the two-component lantibiotics, cytolysin (Gilmore et al., 1994 ) and staphylococcin C55 (Navaratna et al., 1999 ). Disruption of these genes in various lantibiotic systems has revealed their essential role in biosynthesis, as production of active bacteriocin is abolished in their absence (Augustin et al., 1992 ; Klein et al., 1992 ; Gilmore et al., 1994 ; Siegers et al., 1996 ). It has been suggested, therefore, that these proteins are strong candidates as catalysts for the novel reactions responsible for the dehydration of hydroxyamino acids and subsequent thioether ring formation, although the molecular mechanisms involved remain to be elucidated. For example, it has been demonstrated that inactivation of PepC in a Pep 5-producing strain resulted in the secretion of completely dehydrated Pep 5 peptides; however, the majority of these peptides did not contain thioether amino acids (Meyer et al., 1995 ). More recently, Karakas Sen et al. (1999) reported the obvious importance of NisB in the dehydration of serine 33 to didehydroalanine 33 in mature nisin. Therefore, it is proposed that the dehydration of serine and threonine is catalysed by LanB, while LanC catalyses lanthionine ring formation. It is likely that the lanM gene products combine the functions of dehydration and ring formation, although this has yet to be proven.

The genes for lacticin 3147 production and immunity are encoded on a 60·2 kb conjugative plasmid, pMRC01; the complete sequence of pMRC01 has been reported previously (GenBank accession no. AE001272; Dougherty et al., 1998 ). It was proposed that up to ten ORFs identified in pMRC01 were involved in lacticin 3147 biosynthesis and immunity on the basis of sequence similarity to proteins from other lantibiotic biosynthetic pathways, including transport and modification proteins (Dougherty et al., 1998 ). An unusual feature is the presence of two lanM homologues within the gene cluster. Only one such gene is found in the cytolysin gene cluster (Gilmore et al., 1994 ). In the staphylococcin C55 system, one lanM homologue, sacM1, has been completely sequenced (Navaratna et al., 1999 ), while an incomplete ORF shows significant sequence similarity to ltnM2 (GenBank accession no. AF147744). The role of the lacticin 3147 gene clusters was confirmed on cloning a 12·6 kb region of pMRC01, containing the ten ORFs implicated in production and immunity, in L. lactis MG1363 (McAuliffe et al., 2000 ). The resultant strain, MG1363 (pOM02), exhibited wild-type immunity and produced similar levels of lacticin 3147 as did MG1363 harbouring the parental plasmid, pMRC01. A role in immunity has also been confirmed for the gene product of ltnI (McAuliffe et al., 2000 ). Here, the functional analysis of ltnM1 and ltnM2, genes whose products are proposed to be involved in the dehydration and thioether-forming reactions in mature lacticin 3147, is presented. We report the essential role of these putative modification enzymes in the production of active lacticin 3147, and demonstrate that each of the prepeptides of lacticin 3147 requires a separate modification enzyme.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and media.
The bacterial strains and plasmids used in this study are listed in Table 1. Lactococci were grown at 30 °C without aeration in M17 broth (Oxoid) supplemented with 0·5% glucose (GM17). Escherichia coli strains were grown in LB broth (Sambrook et al., 1989 ) at 37 °C with vigorous shaking. Agar media were prepared by adding 1·5% granulated agar (Difco) to broth, whereas overlay agar was prepared with 0·7% agar. Antibiotics used in the selection media were added at the following concentrations: ampicillin, 100 µg ml-1; and chloramphenicol, 20 µg ml-1 (E. coli) and 5 µg ml-1 (L. lactis). LB agar supplemented with 40 µg X-gal ml-1 and 40 µg IPTG ml-1 were used to screen for insertional inactivation of the {alpha}-lacZ gene in pUC19. Chemical reagents listed were obtained from Sigma.


View this table:
[in this window]
[in a new window]
 
Table 1. Bacterial strains and plasmids

 
Bacteriocin preparation.
Concentrated lacticin 3147 was prepared as described previously (McAuliffe et al., 1999 ) from supernatants of L. lactis subsp. lactis DPC3147 cultured in MRS broth (Oxoid). The activity of the concentrate was determined by critical dilution (Parente & Hill, 1992 ) using a well-diffusion assay. Basically, molten agar was cooled to 48 °C and seeded with the strain of interest (approx. 2x107 fresh overnight-grown cells). The inoculated medium was dispensed into sterile Petri plates, allowed to solidify and dried. Wells (approx. 4·6 mm in diameter) were made in the seeded agar plates. Aliquots (50 µl) of a twofold serial dilution of the bacteriocin preparation were dispensed into the wells and the plates incubated overnight at 30 °C. The arbitrary units per millilitre (AU ml-1) were calculated as the reciprocal of the last dilution that gave a distinct zone of inhibition (1/dilutionx20).

DNA preparation and transformation.
Plasmid DNA was isolated from E. coli strains using the Qiagen column purification kit, and from L. lactis using the method of Anderson & McKay (1983) . pMRC01 DNA (Ryan et al., 1996 ), which was used as template DNA in PCR, was purified by equilibrium centrifugation in CsCl/ethidium bromide gradients (Sambrook et al., 1989 ). L. lactis and E. coli were transformed by electroporation with a Gene-Pulser apparatus (Bio-Rad) as described by Wells et al. (1993) and Sambrook et al. (1989) , respectively.

General molecular techniques.
PCR was performed by following standard procedures using BIOTAQ DNA polymerase (Bioline) in a GeneAmp PCR Systems 2400 DNA thermal cycler (Perkin-Elmer). The Expand High Fidelity PCR System (Roche Biochemicals) was used to generate PCR products for cloning purposes. Thirty cycles were performed with the following conditions: 30 s at 90 °C, 1 min at 50 °C and 1 min at 72 °C. Where longer PCR products were sought, the Expand Long Template PCR System from Roche Biochemicals was used according to the manufacturer’s instructions. Oligonucleotide primers for PCR were synthesized using an Applied Biosystems PCR-MATE DNA synthesizer. Restriction digestion, Klenow treatment and DNA ligations were performed according to standard procedures (Sambrook et al., 1989 ). Restriction enzymes, the Klenow fragment of E. coli DNA polymerase I and T4 DNA ligase were purchased from New England Biolabs. Restriction-endonuclease-digested DNA was eluted from agarose gels using a GeneClean II kit from bio101.

Plasmid construction and mutagenesis.
The following strategies were used to analyse the genes in the lacticin 3147 gene cluster. Mutagenesis of ltnM1 was achieved by sub-cloning a 6·6 kb fragment from pOM02, representing approximately the first half of the bacteriocin-coding region (ltnEFIRA1A2M1), into pUC19. The resultant plasmid, pOM09, was digested with ClaI, the recessed 3' ends were filled in with the Klenow fragment from DNA polymerase I and the plasmid was religated to create pOM13. The 6·6 kb insert from pOM13, now with a frameshift in ltnM1, was subcloned into pCI372 containing a 6·0 kb fragment, representing the remainder of the bacteriocin-coding region, creating plasmid pOM18. A similar strategy was used to create pOM32; a 6·0 kb fragment from pOM02, containing the genes ltnTM2D, was cloned into pUC19, creating pOM10. pOM10 was subsequently digested with ClaI, filled-in and religated, creating the plasmid pOM30 with a frameshift in ltnM2. The mutated 6·0 kb fragment was used to replace the original fragment in pOM02, resulting in the plasmid pOM32. Plasmids pOM31 and pOM39 were generated as follows: in each case, primers with built-in restriction sites were designed to amplify the first half of the bacteriocin-coding region in two separate fragments, which resulted in deletion of part of the genes of interest, i.e. ltnA1 and ltnA2. These primers are listed in Table 2. The relevant fragments were amplified by High Fidelity PCR, ligated and used to replace the original 6·0 kb fragment in pOM02, creating pOM31 ({Delta}ltnA2) and pOM39 ({Delta}ltnA1). The double mutants, pOM33 and pOM47, were created by mutagenesis of ltnM2 in pOM31 and ltnM1 in pOM18, respectively, as described above for pOM32. Relevant plasmids are graphically represented in Fig. 1. Whenever PCR was used in plasmid construction, a number of independent clones were tested to confirm the associated phenotype.


View this table:
[in this window]
[in a new window]
 
Table 2. Primers used in plasmid construction

 


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1. Schematic representation of the organization of various constructs and the phenotype associated with these mutated derivatives. Arrows indicate ORFs and the proposed direction of transcription. Black arrows represent the ORFs remaining in the various subclones; grey arrows indicate those ORFs which have been deleted/mutated. 'X' indicates a frameshift mutation at this point; square brackets indicate where an ORF has been deleted. Important restriction sites are marked; SacI and SalI are engineered sites. Bac, bacteriocin production; +++, wild-type levels; –, no activity detected. *, Bacteriocin activity was restored by the addition of purified LtnA1 or LtnA2 as indicated.

 

   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The type-A group of lantibiotics have previously been classified into two subgroups based on the sequence of their leader peptides and the composition of their biosynthetic gene clusters (Jung, 1991 ; de Vos et al., 1995 ). Analysis of the products of the lacticin 3147 gene cluster (Dougherty et al., 1998 ) revealed prepeptides with ‘double-glycine’-type leader sequences, in addition to two LtnM proteins and LtnT, an ABC transporter with an N-terminal proteolytic domain. Consequently, the presence of such gene products clearly identifies lacticin 3147 as a member of the type-A(II) group, which also includes lacticin 481 (Rince et al., 1994 ) and another two-component lantibiotic, the haemolysin/bacteriocin cytolysin (Gilmore et al., 1994 ).

Cloning and expression of a 12·6 kb region which contains the lacticin 3147 ten-gene cluster to create the plasmid pOM02 has previously been described (McAuliffe et al., 2000 ). To gain an insight into the biosynthetic pathway of lacticin 3147, a strategy of deletion and/or mutational analysis was adopted to functionally analyse some of the genes in the lacticin 3147 gene cluster. A number of derivatives of pOM02 were constructed as described in the Methods section and are shown in Fig. 1. Each construct was isolated from E. coli XL1-Blue and electroporated into the non-producing, lacticin-sensitive strain L. lactis MG1363. Resultant transformants were confirmed by plasmid isolation and restriction analysis. Phenotypic analysis of the mutants involved determination of the levels of lacticin 3147 production and immunity, in addition to complementation between the products of strains harbouring different constructs.

Inactivation of either ltnA1 and ltnA2 results in loss of lacticin 3147 production
Previously, it has been shown that lacticin 3147 is a two-component bacteriocin composed of two small (3–4 kDa) peptides (McAuliffe et al., 1998 ). The genes ltnA1 and ltnA2 (originally referred to as ltnA and ltnB) were originally proposed as candidates for the lacticin 3147 structural genes (Dougherty et al., 1998 ) on the basis that they encode prepropeptides that are rich in serine, threonine and cysteine residues that could be acted on to give rise to dehydrated amino acids, and subsequently, lanthionine rings (Dougherty et al., 1998 ). In addition, ltnA1 and ltnA2 are located directly upstream of genes whose products are homologous to proteins involved in lantibiotic maturation, a context similar to the structural genes of some other lantibiotic gene clusters. Recent chemical analysis, including a combination of MS, amino acid analyses and N-terminal sequencing has confirmed ltnA1 and ltnA2 as the lacticin 3147 structural genes, and also confirmed the presence of lanthionine residues in each peptide (Ryan et al., 1999 ).

Plasmids pOM39 ({Delta}ltnA1) and pOM31 ({Delta}ltnA2) (Fig. 1) were created as described in the Methods section, using E. coli XL1-Blue as the intermediate host. Subsequent transformation of these plasmids into L. lactis MG1363 resulted in transformants that exhibited wild-type levels of immunity to exogenously applied lacticin 3147 (using a lacticin 3147 stock solution of 160000 AU ml-1; data not shown). It has been previously reported by Ryan et al. (1999) that LtnA1 has independent antimicrobial activity. However, this activity was only observed following a purification protocol which gave rise to high concentrations of LtnA1, concentrations which would not be reached in the culture supernatant. Therefore given that, at low concentrations, both peptide products are required for activity, deletion of either of the lacticin 3147 structural genes would be expected to result in a loss of the bacteriocin-producing phenotype. Predictably, neither mutant was able to generate active lacticin 3147, in that the transformants failed to inhibit a number of test strains including the hypersensitive indicator L. lactis subsp. cremoris AM2 in either direct or deferred antagonism assays (data not shown). However, it proved possible to restore lacticin 3147 activity to the cell-free supernatant (CFS) of the {Delta}ltnA1 mutant by the simple addition of purified LtnA1, or by supplying purified LtnA2 to the {Delta}ltnA2 CFS (data not shown). Furthermore, combining the CFS of both mutants also restored activity (data not shown). These results confirm that each mutant is capable of producing one modified and active peptide, and that processing and export of LtnA1 and LtnA2 can be carried out independently.

The gene products of ltnM1 and ltnM2 are also essential for the production of active lacticin 3147
Two large genes, ltnM1 and ltnM2, are present downstream of the structural genes, separated by an ORF encoding the putative bacteriocin translocator, ltnT (Fig. 1). These genes encode the proteins LtnM1 (980 amino acids) and LtnM2 (814 amino acids), respectively (Dougherty et al., 1998 ). Both LtnM1 and LtnM2 share sequence similarity with CylM, an enzyme proposed to be involved in the post-translational modification of the haemolysin/bacteriocin, cytolysin (Gilmore et al., 1994 ), and a protein implicated in lacticin 481 modification, LctM (Rince et al., 1994 ).

The role of the products of the ltnM1 and ltnM2 genes in the biosynthesis of lacticin 3147 was investigated by creation of frameshift mutations within each of these genes in pOM02, creating the plasmids pOM18 ({Delta}ltnM1) and pOM32 ({Delta}ltnM2). This was achieved by filling-in ClaI sites in each of the genes, as described in the Methods section. The resultant plasmids were then introduced into L. lactis MG1363 by electroporation, and the phenotypic properties of a number of transformants examined. In all cases, MG1363 harbouring either pOM18 ({Delta}ltnM1) or pOM32 ({Delta}ltnM2) was immune to exogenously applied lacticin 3147 (160000 AU ml-1; Fig. 2a). In addition, neither mutant was capable of producing active lacticin 3147. This confirmed that while both LtnM1 and LtnM2 play an important role in lacticin 3147 production, they do not contribute to immunity. This supports the initial presumption that these proteins are indeed modification enzymes.



View larger version (78K):
[in this window]
[in a new window]
 
Fig. 2. (a) Inhibitory activity of a twofold serial dilution of concentrated lacticin 3147 (160000 AU ml-1) against L. lactis MG1363(pOM18) ({Delta}ltnM1; upper panel), and the plasmid-free L. lactis MG1363 (lower panel). (b) Activity of the cell-free supernatant of L. lactis MG1363(pOM18) (well {Delta}M1) and L. lactis MG1363(pOM32) (well {Delta}M2) against the sensitive indicator L. lactis subsp. cremoris HP. Heat-treated supernatant (25 µl) was added to each well. {Delta}M1+{Delta}M2, 25 µl supernatant from each culture was added to this well. (c) Activity of (1) 25 µl FPLC-derived fraction corresponding to purified LtnA2; (2) 25 µl cell-free supernatant of L. lactis MG1363(pOM33) ({Delta}ltnA2M2); (3) combination of 25 µl each of both (1) and (2), assayed against L. lactis subsp. cremoris HP.

 
LtnA1 and LtnA2 require separate LtnM enzymes for activity
Although there was no activity associated with the CFS of the ltnM1 or ltnM2 mutants (Fig. 2b), it was possible to restore bacteriocin activity by combining the CFS of both strains in a single well (Fig. 2b). If the CFS from each mutant was placed in closely adjacent wells, a zone of inhibition was observed between the wells (Fig. 2b). This suggested that each mutant was producing a single active peptide. Confirmation of this, and an indication of which peptide is being produced, was provided when CFS of both the ltnM1 and ltnM2 mutants were combined with purified FPLC fractions corresponding to the lacticin 3147 components LtnA1 and LtnA2. On combining CFS from the ltnM1 mutant with fractions corresponding to the A1 peptide, bacteriocin activity was restored against L. lactis subsp. cremoris HP (Fig. 2C), but not on combination with the A2 peptide (data not shown). In contrast, inhibition was observed only when the A2 peptide was added to the CFS from the ltnM2 mutant (data not shown).

To further substantiate the proposition that LtnM1 acts to create active LtnA1, while LtnM2 acts on LtnA2, we were able to show that addition of the CFS from the ltnA2 mutant restored activity to the CFS of the ltnM1 mutant, but not to the ltnM2 mutant (data not shown). As expected, combining the CFS from the ltnA1 mutant with that of the ltnM2 mutant, but not the ltnM1 mutant, also resulted in a restoration of lacticin 3147 activity.

If the modification of each prepeptide is truly independent then it should be expected that double ltnA1M1 and ltnA2M2 mutants should each produce one active peptide, modified A1 and A2, respectively. To confirm this theory, two such double mutants were created. Firstly, plasmid pOM33 ({Delta}ltnA2M2, Fig. 1) was created to eliminate any possibility that LtnM1 plays a role in the production of A2. As expected, the complete absence of the A2 peptide combined with a non-functional LtnM2 resulted in an MG1363 derivative which did not produce active lacticin 3147. However, providing the A2 peptide restored activity to this construct, as did the CFS from either the ltnM1 and ltnA1 mutants. The opposite was also true in that CFS from transformants of MG1363 harbouring pOM47 ({Delta}ltnA1M1) could be complemented by purified LtnA1 as well as CFS from the ltnM2 and ltnA2 mutants. These data confirm that LtnM1 acts solely on LtnA1, and LtnM2 solely on LtnA2.

The LanM proteins represent a novel group of enzymes which have not been identified outside a lantibiotic context, and therefore, it has been proposed that these enzymes catalyse the novel reactions involved in thioether ring formation. Disruption of the lanM gene in other systems resulted in a Bac- phenotype, establishing a crucial role for the LanM protein in lantibiotic biosynthesis (Gilmore et al., 1994 ; Rince et al., 1994 ; Skaugen et al., 1997 ); however, their function is as yet speculative. Similarly, inactivation of ltnM1 or ltnM2 by introducing a frameshift abolished lacticin 3147 production. A feature of the lacticin 3147 gene cluster is the presence of two ltnM genes. It could be envisaged that one LtnM protein is responsible for dehydration of the hydroxy amino acids, while the other catalyses thioether ring formation, much like the LanB and LanC enzymes in the type-A(I) group (for a review see Sahl et al., 1995 ). However, the absence of a second modification enzyme in the lacticin 481, lactocin S and cytolysin systems makes this unlikely. A more probable scenario is that each lacticin 3147 prepeptide requires a separate modification enzyme. There is no significant sequence homology between the lacticin 3147 and cytolysin peptides, whereas protein sequence alignments show that LtnA1 and LtnA2 are very closely related to the staphylococcin C55 components Sac{alpha}A and SacßA, with LtnA1 and Sac{alpha}A sharing 83% identity and LtnA2 and SacßA sharing 44% identity (Navaratna et al., 1999 ). Furthermore, in the cytolysin system, both peptide components are much more related to each other than is the case for lacticin 3147 or staphylococcin C55. This may explain why cytolysin can rely on only one LanM modification enzyme, while two modification enzymes may be necessary in both the lacticin 3147 and staphylococcin C55 systems.

The presence of two modification genes and two structural genes within the lacticin gene cluster presented a number of possible scenarios. First, both prepeptides could be modified sequentially by both M proteins; secondly, either one of the M proteins may have modified both prepeptides (with the second M protein redundant); or thirdly, each prepeptide may simply have been modified by the first M protein encountered within the cell. However, on the basis of the various knockout experiments presented here, we conclude that the only possibility which is supported by the evidence is that each prepeptide requires a dedicated modification enzyme. Given the low level of identity between LtnA1 and A2 peptides this also provides the most logical explanation of the requirement for two modification enzymes. Further analysis of the peptide products from the various knockout mutants will shed further light on the precise interactions between prepeptides and modification proteins.


   ACKNOWLEDGEMENTS
 
The authors thank Máire Ryan for supplying the purified lacticin 3147 peptides. This work was funded by the Non-Commissioned Food Research Programme, operated by the Irish Department of Agriculture, Food and Forestry, and BioResearch Ireland.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Anderson, D. G. & McKay, L. L. (1983). Simple and rapid methods for isolating large plasmid DNA from lactic streptococci. Appl Environ Microbiol 46, 549-552.[Medline]

Augustin, J., Rosenstein, R., Wieland, B., Schneider, U., Schnell, N., Engelke, G., Entian, K.-D. & Götz, F. (1992). Genetic analysis of biosynthetic genes and epidermin-negative mutants of Staphylococcus epidermidis. Eur J Biochem 204, 1149-1154.[Abstract]

Berridge, N. J., Newton, G. C. F. & Abraham, E. P. (1952). Purification and nature of the antibiotic nisin. Biochem J 52, 529-535.[Medline]

Dougherty, B. A., Hill, C., Weidman, J. F., Richardson, D. R., Venter, J. C. & Ross, R. P. (1998). Sequence and analysis of the 60 kb conjugative, bacteriocin-producing plasmid pMRC01 from Lactococcus lactis DPC3147. Mol Microbiol 29, 1029-1038.[Medline]

Engelke, G., Gutowski-Eckel, Z., Hammelmann, M. & Entian, K.-D. (1992). Biosynthesis of the lantibiotic nisin: genomic organization and membrane localization of the NisB protein. Appl Environ Microbiol 58, 3730-3743.[Abstract]

Gasson, M. J. (1983). Plasmid complements of Streptococcus lactis NCDO712 and other lactic streptococci after protoplast curing. J Bacteriol 154, 1-9.[Medline]

Gilmore, M. S., Segarra, R. A., Booth, M. C., Bogie, C. P., Hall, L. R. & Clewell, D. B. (1994). Genetic structure of the Enterococcus faecalis plasmid pAD1-encoded cytolytic toxin system and its relationship to lantibiotic determinants. J Bacteriol 176, 7335-7344.[Abstract]

Gutowski-Eckel, Z., Klein, C., Siegers, K., Bohm, K., Hammelmann, M. & Entian, K.-D. (1994). Growth phase-dependent regulation and membrane localisation of SpaB, a protein involved in biosynthesis of the lantibiotic subtilin. Appl Environ Microbiol 60, 1-11.[Abstract]

Hayes, F., Daly, C. & Fitzgerald, G. F. (1990). Identification of the minimal replicon of Lactococcus lactis subsp. lactis UC317 plasmid pCI305. Appl Environ Microbiol 56, 202-209.

Ingram, L. C. (1969). Synthesis of the antibiotic nisin: formation of lanthionine and ß-methyllanthionine. Biochim Biophys Acta 184, 216-219.[Medline]

Jack, R. W., Tagg, J. R. & Ray, B. (1995). Bacteriocins of gram-positive bacteria. Microbiol Rev 59, 171-200.[Abstract]

Jung, G. (1991). Lantibiotics: a survey. In Nisin and Novel Lantibiotics , pp. 320-332. Edited by G. Jung & H.-G. Sahl. Leiden, The Netherlands: ESCOM.

Karakas Sen, A., Narbad, A., Horn, N., Dodd, H. M., Parr, A. J., Colquhoun, I. & Gasson, M. J. (1999). Post-translational modification of nisin: the involvement of NisB in the dehydration process. Eur J Biochem 261, 524-532.[Abstract/Free Full Text]

Klein, C., Kaletta, C., Schnell, N. & Entian, K.-D. (1992). Analysis of genes involved in biosynthesis of the lantibiotic subtilin. Appl Environ Microbiol 58, 132-142.[Abstract]

Kupke, T., Kempter, C., Gnau, V., Jung, G. & Götz, F. (1994). Mass spectroscopic analysis of a novel enzymatic reaction: oxidative decarboxylation of the lantibiotic precursor peptide EpiA catalyzed by the flavoprotein EpiD. J Biol Chem 269, 5653-5659.[Abstract/Free Full Text]

Leenhouts, K. J., Buist, G., Bolhuis, A., ten Berge, A., Kiel, J., Mierau, I., Dabrowska, M., Venema, G. & Kok, J. (1996). A gene system for generating unlabeled gene replacements in bacterial chromosomes. Mol Gen Genet 253, 217-224.[Medline]

McAuliffe, O., Ryan, M. P., Ross, R. P., Hill, C., Breeuwer, P. & Abee, T. (1998). Lacticin 3147, a broad-spectrum bacteriocin which selectively dissipates the membrane potential. Appl Environ Microbiol 64, 439-445.[Abstract/Free Full Text]

McAuliffe, O., Hill, C. & Ross, R. P. (1999). Inhibition of Listeria monocytogenes in cottage cheese manufactured with a lacticin-3147 producing starter culture. J Appl Microbiol 86, 251-256.[Medline]

McAuliffe, O., Hill, C. & Ross, R. P. (2000). Identification and overexpression of ltnI a novel gene, which confers immunity to the two-component lantibiotic lacticin 3147. Microbiology 146, 129-138.[Abstract/Free Full Text]

Meyer, C., Bierbaum, G., Heidrich, C., Reis, M., Süling, J., Iglesias-Wind, M. I., Kempter, C., Molitor, E. & Sahl, H.-G. (1995). Nucleotide sequence of the lantibiotic Pep5 biosynthetic gene cluster and functional analysis of PepP and PepC: evidence for a role of PepC in thioether formation. Eur J Biochem 232, 478-489.[Abstract]

Navaratna, M. A., Sahl, H.-G. & Tagg, J. R. (1999). Identification of genes encoding two-component lantibiotic production in Staphylococcus aureus strain C55 and other phage group II S. aureus strains and demonstration of an association with the exfoliative toxin B gene. Infect Immun 67, 4268-4271.[Abstract/Free Full Text]

Parente, E. & Hill, C. (1992). A comparison of factors affecting the production of two bacteriocins from lactic acid bacteria. J Appl Bacteriol 73, 290-298.

Rince, A., Dufour, A., LePogam, S., Thuault, D., Bourgeois, C. M. & LePennec, J. P. (1994). Cloning, expression and nucleotide sequence of genes involved in production of lactococcin DR, a bacteriocin from Lactococcus lactis subsp. lactis. Appl Environ Microbiol 60, 1652-1657.[Abstract]

Ryan, M. P., Rea, M. C., Hill, C. & Ross, R. P. (1996). An application in cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147. Appl Environ Microbiol 62, 612-619.[Abstract]

Ryan, M. P., Jack, R. W., Josten, M., Sahl, H.-G., Jung, G., Ross, R. P. & Hill, C. (1999). Extensive post-translational modification, including serine to D-alanine conversion, in the two-component lantibiotic, lacticin 3147. J Biol Chem 274, 37544-37550.[Abstract/Free Full Text]

Sahl, H.-G., Jack, R. W. & Bierbaum, G. (1995). Biosynthesis and biological activities of lantibiotics with unique post-translational modifications. Eur J Biochem 230, 827-853.[Abstract]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Schnell, N., Entian, K.-D., Schneider, U., Götz, F., Zähner, H., Kellner, R. & Jung, G. (1988). Prepeptide sequence of epidermin, a ribosomally synthesized antibiotic with four sulphide rings. Nature 333, 276-278.[Medline]

Siegers, K., Heinzmann, S. & Entian, K.-D. (1996). Biosynthesis of lantibiotic nisin: post-translational modification of its prepeptide occurs at a multimeric membrane-associated lanthionine synthetase complex. J Biol Chem 271, 12294-12301.[Abstract/Free Full Text]

Siezen, R. J., Kuipers, O. P. & de Vos, W. M. (1996). Comparison of lantibiotic gene clusters and encoded proteins. Antonie Leeuwenhoek 69, 171-184.

Skaugen, M., Nissen-Meyer, J., Jung, G., Stevanovic, S., Sletten, K., Abildgaard, C. I. M. & Nes, I. F. (1994). In vivo conversion of L-serine to D-alanine in a ribosomally synthesized polypeptide. J Biol Chem 269, 27183-27185.[Abstract/Free Full Text]

Skaugen, M., Abildgaard, C. I. M. & Nes, I. F. (1997). Organization and expression of a gene cluster involved in the biosynthesis of the lantibiotic lactocin S. Mol Gen Genet 253, 674-686.[Medline]

de Vos, W. M., Kuipers, O. P., van der Meer, J. R. & Siezen, R. J. (1995). Maturation pathway of nisin and other lantibiotics: post-translationally modified antimicrobial peptides exported by gram-positive bacteria. Mol Microbiol 17, 427-437.[Medline]

Wells, J. M., Wilson, P. W. & Page, R. W. F. (1993). Improved cloning vectors and transformation procedure for Lactococcus lactis. J Appl Bacteriol 74, 629-636.[Medline]

Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33, 103-119.[Medline]

Received 11 February 2000; revised 20 May 2000; accepted 30 May 2000.