A system for the random mutagenesis of the two-peptide lantibiotic lacticin 3147: analysis of mutants producing reduced antibacterial activities

Lantibiotics are antimicrobial peptides that contain several unusual amino acids resulting from a series of enzyme-mediated posttranslational modifications. As a consequence of being gene-encoded, the implementation of peptide bioengineering systems has the potential to yield lantibiotic variants with enhanced chemical and physical properties. Here we describe a functional two-plasmid expression system which has been developed to allow random mutagenesis of the two-component lantibiotic, lacticin 3147. One of these plasmids contains a randomly mutated version of the two structural genes, ltnA1 and ltnA2, and the associated promoter, Pbac, while the other encodes the remainder of the proteins required for the biosynthesis of, and immunity to, lacticin 3147. To test this system, a bank of approximately 1,500 mutant strains was generated and screened to identify mutations that have a detrimental impact on the bioactivity of lacticin 3147. This strategy established/confirmed the importance of specific residues in the structural peptides and their associated leaders and revealed that a number of alterations which mapped to the -10 or -35 regions of Pbac abolished promoter activity.     

Overproduction of wild-type and bioengineered derivatives of the lantibiotic lacticin 3147

Lacticin 3147 is a broad-spectrum two-peptide lantibiotic whose genetic determinants are located on two divergent operons on the lactococcal plasmid pMRC01. Here we introduce each of 14 subclones, containing different combinations of lacticin 3147 genes, into MG1363 (pMRC01) and determine that a number of them can facilitate overproduction of the lantibiotic. Based on these studies it is apparent that while the provision of additional copies of genes encoding the biosynthetic/production machinery and the regulator LtnR is a requirement for high-level overproduction, the presence of additional copies of the structural genes (i.e., ltnA1A2) is not.     

Fate of the two-component lantibiotic lacticin 3147 in the gastrointestinal tract

The component peptides of lacticin 3147 were degraded by alpha-chymotrypsin in vitro with a resultant loss of antimicrobial activity. Activity was also lost in ileum digesta. Following oral ingestion, neither of the lacticin 3147 peptides was detected in the gastric, jejunum, or ileum digesta of pigs, and no lacticin 3147 activity was found in the feces. These observations suggest that lacticin 3147 ingestion is unlikely to have adverse effects, since it is probably inactivated during intestinal transit.     

Spontaneous resistance in Lactococcus lactis IL1403 to the lantibiotic lacticin 3147

The ability and frequency at which target organisms can develop resistance to bacteriocins is a crucial consideration in designing and implementing bacteriocin-based biocontrol strategies. Lactococcus lactis ssp. lactis IL1403 was used as a target strain in an attempt to determine the frequency at which spontaneously resistant mutants are likely to emerge to the lantibiotic lacticin 3147. Following a single exposure to lacticin 3147, resistant mutants only emerged at a low frequency (10(-8)-10(-9)) and were only able to withstand low levels of the bacteriocin (100 AU mL(-1)). However, exposure to increasing concentrations, in a stepwise manner, resulted in the isolation of eight mutants that were resistant to moderately higher levels of lacticin 3147 (up to 600 AU mL(-1)). Interestingly, in a number of cases cross-resistance to other lantibiotics such as nisin and lacticin 481 was observed, as was cross-resistance to environmental stresses such as salt. Finally, reduced adsorption of the bacteriocin in to the cell was documented for all resistant mutants.     

Cross-immunity and immune mimicry as mechanisms of resistance to the lantibiotic lacticin 3147

Lantibiotics are antimicrobial peptides that possess great potential as clinical therapeutic agents. These peptides exhibit many beneficial traits and in many cases the emergence of resistance is extremely rare. In contrast, producers of lantibiotics synthesize dedicated immunity proteins to provide self-protection. These proteins have very specific activities and cross-immunity is rare. However, producers of two peptide lantibiotics, such as lacticin 3147, face the unusual challenge of exposure to two active peptides (α and β). Here, in addition to establishing the contribution of LtnI and LtnFE to lacticin 3147 immunity, investigations were carried out to determine if production of a closely related lantibiotic (i.e. staphylococcin C55) or possession of LtnI/LtnFE homologues could provide protection. Here we establish that not only are staphylococcin C55 producers cross-immune to lacticin 3147, and therefore represent a natural repository of Staphylococcus aureus strains that are protected against lacticin 3147, but that functional immunity homologues are also produced by strains of Bacillus licheniformis and Enterococcus faecium . This result raises the spectre of resistance through immune mimicry, i.e. the emergence of lantibiotic-resistant strains from the environment resulting from the possession/acquisition of immunity gene homologues. These phenomena will have to be considered carefully when developing lantibiotics for clinical application.     

Extensive post-translational modification, including serine to D-alanine conversion, in the two-component lantibiotic, lacticin 3147

Lacticin 3147 is a two-component bacteriocin produced by Lactococcus lactis subspecies lactis DPC3147. In order to further characterize the biochemical nature of the bacteriocin, both peptides were isolated which together are responsible for the antimicrobial activity. The first, LtnA1, is a 3,322 Da 30-amino acid peptide and the second component, LtnA2, is a 29-amino acid peptide with a mass of 2,847 Da. Conventional amino acid analysis revealed that both peptides contain the thioether amino acid, lanthionine, as well as an excess of alanine to that predicted from the genetic sequence of the peptides. Chiral phase gas chromatography coupled with mass spectrometry of amino acid composition indicated that both LtnA1 and LtnA2 contain D-alanine residues and amino acid sequence analysis of LtnA1 confirmed that the D-alanine results from post-translational modification of a serine residue in the primary translation product. Taken together, these results demonstrate that lacticin 3147 is a novel, two-component, D-alanine containing lantibiotic that undergoes extensive post-translational modification which may account for its potent antimicrobial activity against a wide range of Gram-positive bacteria.     

Complete alanine scanning of the two-component lantibiotic lacticin 3147: generating a blueprint for rational drug design

Lantibiotics are post-translationally modified antimicrobial peptides which are active at nanomolar concentrations. Some lantibiotics have been shown to function by targeting lipid II, the essential precursor of cell wall biosynthesis. Given that lantibiotics are ribosomally synthesized and amenable to site-directed mutagenesis, they have the potential to serve as biological templates for the production of novel peptides with improved functionalities. However, if a rational approach to novel lantibiotic design is to be adopted, an appreciation of the roles of each individual amino acid (and each domain) is required. To date no lantibiotic has been subjected to such rigorous analysis. To address this issue we have carried out complete scanning mutagenesis of each of the 59 amino acids in lacticin 3147, a two-component lantibiotic which acts through the synergistic activity of the peptides LtnA1 (30 amino acids) and LtnA2 (29 amino acids). All mutations were performed in situ in the native 60 kb plasmid, pMRC01. A number of mutations resulted in the elimination of detectable bioactivity and seem to represent an invariable core within these and related peptides. Significantly however, of the 59 amino acids, at least 36 can be changed without resulting in a complete loss of activity. Many of these are clustered to form variable domains within the peptides. The information generated in this study represents a blue-print that will be critical for the rational design of lantibiotic-based antimicrobial compounds.     

Homologues and bioengineered derivatives of LtnJ vary in ability to form D-alanine in the lantibiotic lacticin 3147

Ltnα and Ltnβ are individual components of the two-peptide lantibiotic lacticin 3147 and are unusual in that, although ribosomally synthesized, they contain d-amino acids. These result from the dehydration of l-serine to dehydroalanine by LtnM and subsequent stereospecific hydrogenation to d-alanine by LtnJ. Homologues of LtnJ are rare but have been identified in silico in Staphylococcus aureus C55 (SacJ), Pediococcus pentosaceus FBB61 (PenN), and Nostoc punctiforme PCC73102 (NpnJ, previously called NpunJ [P. D. Cotter et al., Proc. Natl. Acad. Sci. U. S. A. 102:18584-18589, 2005]). Here, the ability of these enzymes to catalyze d-alanine formation in the lacticin 3147 system was assessed through heterologous enzyme production in a ΔltnJ mutant. PenN successfully incorporated d-alanines in both peptides, and SacJ modified Ltnα only, while NpnJ was unable to modify either peptide. Site-directed mutagenesis was also employed to identify residues of key importance in LtnJ. The most surprising outcome from these investigations was the generation of peptides by specific LtnJ mutants which exhibited less bioactivity than those generated by the ΔltnJ strain. We have established that the reduced activity of these peptides is due to the inability of the associated LtnJ enzymes to generate d-alanine residues in a stereospecific manner, resulting in the presence of both d- and l-alanines at the relevant locations in the lacticin 3147 peptides.     

Biological and molecular characterization of a two-peptide lantibiotic produced by Lactococcus lactis IFPL105

The lactic acid bacterium Lactococcus lactis IFPL105 secretes a broad spectrum bacteriocin produced from the 46 kb plasmid pBAC105. The bacteriocin was purified to homogeneity by ionic and hydrophobic exchange and reverse-phase chromatography. Bacteriocin activity required the complementary action of two distinct peptides (alpha and beta) with average molecular masses of 3322 and 2848 Da, respectively. The genes encoding the two peptides were cloned and sequenced and were found to be identical to the ltnAB genes from plasmid pMRC01 of L. lactis DPC3147. LtnA and LtnB contain putative leader peptide sequences similar to the known 'double glycine' type. The predicted amino acid sequence of mature LtnA and LtnB differed from the amino acid content determined for the purified alpha and beta peptides in the residues serine, threonine, cysteine and alanine. Post-translational modification, and the formation of lanthionine or methyllanthionine rings, could partly explain the difference. Hybridization experiments showed that the organization of the gene cluster in pBAC105 responsible for the production of the bacteriocin is similar to that in pMRC01, which involves genes encoding modifying enzymes for lantibiotic biosynthesis and dual-function transporters. In both cases, the gene clusters are flanked by IS946 elements, suggesting an en bloc transposition. The findings from the isolation and molecular characterization of the bacteriocin provide evidence for the lantibiotic nature of the two peptides.     

Saturation mutagenesis of selected residues of the α‐peptide of the lantibiotic lacticin 3147 yields a derivative with enhanced antimicrobial activity

The lantibiotic lacticin 3147 consists of two ribosomally synthesized and post‐translationally modified antimicrobial peptides, Ltnα and Ltnβ, which act synergistically against a wide range of Gram‐positive microorganisms. We performed saturation mutagenesis of specific residues of Ltnα to determine their functional importance. The results establish that Ltnα is more tolerant to change than previously suggested by alanine scanning mutagenesis. One substitution, LtnαH23S, was identified which improved the specific activity of lacticin 3147 against one pathogenic strain, Staphylococcus aureus NCDO1499. This represents the first occasion upon which the activity of a two peptide lantibiotic has been enhanced through bioengineering.     

Heterologous expression of lacticin 3147 in Enterococcus faecalis: comparison of biological activity with cytolysin

Lacticin 3147 is a broad-spectrum, two-component, lanthionine-containing bacteriocin produced by Lactococcus lactis DPC3147 which has widespread food and biomedical applications as a natural antimicrobial. Other two-component lantibiotics described to date include cytolysin and staphylococcin C55. Interestingly, cytolysin, produced by Enterococcus faecalis, has an associated haemolytic activity. The objective of this study was to compare the biological activity of lacticin 3147 with cytolysin. The lacticin 3147-encoding determinants were heterologously expressed in Ent. faecalis FA2-2, a plasmid-free strain, to generate Ent. faecalis pOM02, thereby facilitating a direct comparison with Ent. faecalis FA2-2.pAD1, a cytolysin producer. Both heterologously expressed lacticin 3147 and cytolysin exhibited a broad spectrum of activity against bacterial targets. Furthermore, enterococci expressing active lacticin 3147 did not exhibit a haemolytic activity against equine blood cells. The results thus indicate that the lacticin 3147 biosynthetic machinery can be heterologously expressed in an enterococcal background resulting in the production of the bacteriocin with no detectable haemolytic activity.     

Isolation and characterization of enterocin W, a novel two-peptide lantibiotic produced by Enterococcus faecalis NKR-4-1

Enterococcus faecalis NKR-4-1 isolated from pla-ra produces a novel two-peptide lantibiotic, termed enterocin W, comprising Wα and Wβ. The structure of enterocin W exhibited similarity with that of plantaricin W. The two peptides acted synergistically, and their order of binding to the cell membrane was important for their inhibitory activity.     

Generation of food-grade lactococcal starters which produce the lantibiotics lacticin 3147 and lacticin 481

Transconjugant lactococcal starters which produce both lantibiotics lacticin 3147 and lacticin 481 were generated via conjugation of large bacteriocin-encoding plasmids. A representative of one of the resultant strains proved more effective at killing Lactobacillus fermentum and inhibiting the growth of Listeria monocytogenes LO28H than either of the single bacteriocin-producing parental strains, demonstrating the potential of these transconjugants as protection cultures for food safety applications.     

Inhibition of Listeria monocytogenes in cottage cheese manufactured with a lacticin 3147-producing starter culture

The efficacy of using a lacticin 3147-producing starter as a protective culture to improve the safety of cottage cheese was investigated. This involved the manufacture of cottage cheese using Lactococcus lactis DPC4268 (control) and L. lactis DPC4275, a bacteriocin-producing transconjugant strain derived from DPC4268. A number of Listeria monocytogenes strains, including a number of industrial isolates, were assayed for their sensitivity to lacticin 3147. These strains varied considerably with respect to their sensitivity to the bacteriocin. One of the more tolerant strains, Scott A, was used in the cottage cheese study; the cheese was subsequently inoculated with approximately 10(4) L. monocytogenes Scott A g-1. The bacteriocin concentration in the curd was measured at 2560 AU ml-1, and bacteriocin activity could be detected throughout the 1 week storage period. In cottage cheese samples held at 4 degrees C, there was at least a 99.9% reduction in the numbers of L. monocytogenes Scott A in the bacteriocin-containing cheese within 5 d, whereas in the control cheeses, numbers remained essentially unchanged. At higher storage temperatures, the kill rate was more rapid. These results demonstrate the effectiveness of lacticin 3147 as an inhibitor of L. monocytogenes in a food system where post-manufacture contamination by this organism could be problematic.     

Identification of a novel two-peptide lantibiotic, haloduracin, produced by the alkaliphile Bacillus halodurans C-125

Complete genome sequencing of the alkaliphilic bacterium Bacillus halodurans C-125 revealed the presence of several genes homologous to those involved in the production of lantibiotic peptides. Additional bioinformatic analysis identified a total of eleven genes, spanning a 15 kbp region, potentially involved in the production, modification, immunity and transport of a two-peptide lantibiotic. Having established that strain C-125 exhibited antimicrobial activity against a wide range of Gram-positive bacteria, it was demonstrated through peptide purification, MS and site-directed mutagenesis that this activity was indeed attributable to the production of a lantibiotic encoded by these genes. This antimicrobial has been designated haloduracin and represents the first occasion wherein production of two-peptide lantibiotic has been associated with a Bacillus sp. It is also the first example of a lantibiotic of any kind to be produced by an alkaliphilic species.     

The mode of action of the lantibiotic lacticin 3147--a complex mechanism involving specific interaction of two peptides and the cell wall precursor lipid II

Lacticin 3147 is a two-peptide lantibiotic produced by Lactococcus lactis in which both peptides, LtnA1 and LtnA2, interact synergistically to produce antibiotic activities in the nanomolar concentration range; the individual peptides possess marginal (LtnA1) or no activity (LtnA2). We analysed the molecular basis for the synergism and found the cell wall precursor lipid II to play a crucial role as a target molecule. Tryptophan fluorescence measurements identified LtnA1, which is structurally similar to the lantibiotic mersacidin, as the lipid II binding component. However, LtnA1 on its own was not able to substantially inhibit cell wall biosynthesis in vitro; for full inhibition, LtnA2 was necessary. Both peptides together caused rapid K(+) leakage from intact cells; in model membranes supplemented with lipid II, the formation of defined pores with a diameter of 0.6 nm was observed. We propose a mode of action model in which LtnA1 first interacts specifically with lipid II in the outer leaflet of the bacterial cytoplasmic membrane. The resulting lipid II:LtnA1 complex is then able to recruit LtnA2 which leads to a high-affinity, three-component complex and subsequently inhibition of cell wall biosynthesis combined with pore formation.     

IS1675, a novel lactococcal insertion element, forms a transposon-like structure including the lacticin 481 lantibiotic operon

Two copies of IS1675, a novel lactococcal insertion element from the IS4 family, are present on a 70-kb plasmid, where they frame the lantibiotic lacticin 481 operon. The whole structure could be a composite transposon designated Tn5721. This study shows that the lacticin 481 operon does not include any regulatory gene and provides a new example of a transposon-associated bacteriocin determinant. We identified five other IS1675 copies not associated with the lacticin 481 operon. The conservation of IS1675 flanking sequences suggested a 24-bp target site.     

Continuous production of lacticin 3147 and nisin using cells immobilized in calcium alginate

Bacteriocinogenic strains, Lactococcus lactis subsp. lactis DPC 3147 and L. lactis DPC 496, producing lacticin 3147 and nisin, respectively, were immobilized in double-layered calcium alginate beads. These beads were inoculated into MRS broth at a ratio of 1:4 and continuously fermented for 180 h. Free cells were used to compare the effect of immobilization on bacteriocin production. After equilibrium was reached, a flow rate of 580 ml h(-1) was used in the immobilized cell (IC), and 240 ml h(-1) in free-cell (FC) bioreactors. Outgrowth from beads was observed after 18 h. Bacteriocin production peaked at 5120 AU ml(-1) in both IC and FC bioreactors. However, FC production declined after 80 h to 160 AU ml(-1) at the end of the fermentation. Results of this study indicate that immobilization offers the possibility of a more stable and long-term means of producing lacticin 3147 in laboratory media than with free cells.     

Maturation by LctT is required for biosynthesis of full-length lantibiotic lacticin 481

In lantibiotic lacticin 481 biosynthesis, LctT cleaves the precursor peptide and exports mature lantibiotic. Matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed that a truncated form of lacticin 481 is produced in the absence of LctT or after cleavage site inactivation. Production of truncated lacticin 481 is 4-fold less efficient, and its specific activity is about 10-fold lower.