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
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ABSTRACT |
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Keywords: lacticin 3147, lantibiotic production, immunity, modification enzymes
Abbreviations: ABC, ATP-binding cassette; AU, arbitrary units; CFS, cell-free supernatant
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INTRODUCTION |
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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.
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METHODS |
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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 manufacturers 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 (
ltnA2) and pOM39 (
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.
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RESULTS AND DISCUSSION |
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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 (34 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 (ltnA1) and pOM31 (
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
ltnA1 mutant by the simple addition of purified LtnA1, or by supplying purified LtnA2 to the
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 (ltnM1) and pOM32 (
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 (
ltnM1) or pOM32 (
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.
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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 (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 (
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
A and SacßA, with LtnA1 and Sac
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.
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ACKNOWLEDGEMENTS |
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Received 11 February 2000;
revised 20 May 2000;
accepted 30 May 2000.