Cloning of the gene encoding Streptococcin A-FF22, a novel lantibiotic produced by Streptococcus pyogenes, and determination of its nucleotide sequence.

Streptococcin A-FF22 (SA-FF22) is a lantibiotic produced by Streptococcus pyogenes FF22. The nucleotide sequence of the SA-FF22 structural gene (scnA) was determined and shown to encode a 51-amino-acid prepeptide. The proteolytic processing site of the SA-FF22 prepeptide differs from that which characterizes other type A lantibiotics.     

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.     

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.     

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.     

In vitro reconstitution and substrate specificity of a lantibiotic protease

Lacticin 481 is a lanthionine-containing bacteriocin (lantibiotic) produced by Lactococcus lactis subsp. lactis. The final steps of lacticin 481 biosynthesis are proteolytic removal of an N-terminal leader sequence from the prepeptide LctA and export of the mature lantibiotic. Both proteolysis and secretion are performed by the dedicated ATP-binding cassette (ABC) transporter LctT. LctT belongs to the family of AMS (ABC transporter maturation and secretion) proteins whose prepeptide substrates share a conserved double-glycine type cleavage site. The in vitro activity of a lantibiotic protease has not yet been characterized. This study reports the purification and in vitro activity of the N-terminal protease domain of LctT (LctT150), and its use for the in vitro production of lacticin 481. The G(-2)A(-1) cleavage site and several other conserved amino acid residues in the leader peptide were targeted by site-directed mutagenesis to probe the substrate specificity of LctT as well as shed light upon the role of these conserved residues in lantibiotic biosynthesis. His 10-LctT150 did not process most variants of the double glycine motif and processed mutants of Glu-8 only very slowly. Furthermore, incorporation of helix-breaking residues in the leader peptide resulted in greatly decreased proteolytic activity by His 10-LctT150. On the other hand, His 10-LctT150 accepted all peptides containing mutations in the propeptide or at nonconserved positions of LctA. In addition, the protease domain of LctT was investigated by site-directed mutagenesis of the conserved residues Cys12, His90, and Asp106. The proteolytic activities of the resulting mutant proteins are consistent with a cysteine protease.     

Purification and Partial Characterization of Lacticin 481, a Lanthionine-Containing Bacteriocin Produced by Lactococcus lactis subsp. lactis CNRZ 481

Lacticin 481, a bacteriocin produced during the growth of Lactococcus lactis subsp. lactis CNRZ 481, was purified sequentially by ammonium sulfate precipitation, gel filtration, and preparative and analytical reversed-phase high-pressure liquid chromatography. Ammonium sulfate precipitations resulted in a 455-fold increase in total lacticin 481 activity. The entire purification protocol led to a 107, 506-fold increase in the specific activity of lacticin 481. On the basis of its electrophoretic pattern in sodium dodecyl sulfate-polyacrylamide gels, lacticin 481 appeared as a single peptide band of 1.7 kDa. However, dimers of 3.4 kDa also exhibiting lacticin activity were detected. Derivatives of the lacticin-producing strain which did not produce lacticin 481 (Bac^-) were sensitive to this bacteriocin (Bac^s) and failed to produce the 1.7-kDa band. Amino acid composition analysis of purified lacticin 481 revealed the presence of lanthionine residues, suggesting that lacticin 481 is a member of the lantibiotic family of antimicrobial peptides. Seven residues (K G G S G V I) were sequenced from the N-terminal portion of lacticin 481, and these did not shown any homology with nisin or other known bacteriocin sequences.     

The importance of the leader sequence for directing lanthionine formation in lacticin 481

Lantibiotics are post-translationally modified peptide antimicrobial agents that are synthesized with an N-terminal leader sequence and a C-terminal propeptide. Their maturation involves enzymatic dehydration of Ser and Thr residues in the precursor peptide to generate unsaturated amino acids, which react intramolecularly with nearby cysteines to form cyclic thioethers termed lanthionines and methyllanthionines. The role of the leader peptide in lantibiotic biosynthesis has been subject to much speculation. In this study, mutations of conserved residues in the leader sequence of the precursor peptide for lacticin 481 (LctA) did not inhibit dehydration and cyclization by lacticin 481 synthetase (LctM) showing that not one specific residue is essential for these transformations. These amino acids may therefore be conserved in the leader sequence of class II lantibiotics to direct other biosynthetic events, such as proteolysis of the leader peptide or transport of the active compound outside the cell. However, introduction of Pro residues into the leader peptide strongly affected the efficiency of dehydration, consistent with recognition of the secondary structure of the leader peptide by the synthetase. Furthermore, the presence of a hydrophobic residue at the position of Leu-7 appears important for enzymatic processing. Based on the data in this work and previous studies, a model for the interaction of LctM with LctA is proposed. The current study also showcases the ability to prepare other lantibiotics in the class II lacticin 481 family, including nukacin ISK-1, mutacin II, and ruminococcin A using the lacticin 481 synthetase. Surprisingly, a conserved Glu located in a ring that appears conserved in many class II lantibiotics, including those not belonging to the lacticin 481 subgroup, is not essential for antimicrobial activity of lacticin 481.     

A novel type of immunity protein, NukH, for the lantibiotic nukacin ISK-1 produced by Staphylococcus warneri ISK-1

Staphylococcus warneri ISK-1 produces a lantibiotic, nukacin ISK-1. The nukacin ISK-1 gene cluster consists of at least six genes, nukA, -M, -T, -F, -E, and -G, and two open reading frames, ORF1 and ORF7 (designated nukH). Sequence comparisons suggested that NukF, -E, -G, and -H contribute to immunity to nukacin ISK-1. We investigated the immunity levels of recombinant Lactococcus lactis expressing nukFEG and nukH against nukacin ISK-1. The co-expression of nukFEG and nukH resulted in a high degree of immunity. The expression of either nukFEG or nukH conferred partial immunity against nukacin ISK-1. These results suggest that NukH contributes cooperatively to self-protection with NukFEG. The nukacin ISK-1 immunity system might function against another lantibiotic, lacticin 481. Western blot analysis showed that NukH expressed in Staphylococcus carnosus was localized in the membrane. Peptide release/bind assays indicated that the recombinant L. lactis expressing nukH interacted with nukacin ISK-1 and lacticin 481 but not with nisin A. These findings suggest that NukH contributes cooperatively to host immunity as a novel type of lantibiotic-binding immunity protein with NukFEG.     

Use of lacticin 481 to facilitate delivery of the bacteriophage resistance plasmid, pCBG104 to cheese starters

AIMS: Use of lacticin 481 to facilitate the conjugal transfer of the bacteriophage resistance plasmid pCBG104 to various starter cultures. METHODS AND RESULTS: A raw milk isolate of Lactococcus was found to harbour determinants for lacticin 481 production and immunity and phage resistance on a plasmid designated pCBG104. The lacticin 481 was successfully used to mobilize the phage resistance determinant to a variety of cheese starters enabling the formation of highly phage resistant starters. In addition, it facilitated the stacking of a number of phage resistance genes, namely a type I restriction modification system, a phage abortive infection system and a phage adsorption blocking system in a single Lactococcus strain without the use of recombinant techniques. The transconjugants were all shown to produce lacticin 481 and to contain the entire 481 operon. Subsequently one transconjugant was selected and successfully used for large-scale cheddar cheese manufacture. CONCLUSIONS: Lacticin 481 could be used as a food-grade selectable marker to facilitate the introduction of advantageous traits to starter cultures for industrial food fermentations. SIGNIFICANCE AND IMAPCT OF THE STUDY: Food-grade selectable markers greatly facilitate the introduction of various advantageous traits to starter cultures for industrial food fermentation. Indeed self-cloning which is becoming increasingly important for strain improvement has a requirement for the identification and demonstration of the utility of tools such as lacticin 481.     

Characterization of the lacticin 481 operon: the Lactococcus lactis genes lctF, lctE, and lctG encode a putative ABC transporter involved in bacteriocin immunity.

The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis strains. The genetic determinants of lacticin 481 production are organized as an operon encoded by a 70-kb plasmid. We previously reported the first three genes of this operon, lctA, lctM, and lctT, which are involved in the bacteriocin biosynthesis and export (A. Rincé, A. Dufour, S. Le Pogam, D. Thuault, C. M. Bourgeois, and J.-P. Le Pennec, Appl. Environ. Microbiol. 60:1652-1657, 1994). The operon contains three additional open reading frames: lctF, lctE, and lctG. The hydrophobicity profiles and sequence similarities strongly suggest that the three gene products associate to form an ABC transporter. When the three genes were coexpressed into a lacticin 481-sensitive L. lactis strain, the strain became resistant to the bacteriocin. This protection could not be obtained when any of the three genes was deleted, confirming that lctF, lctE, and lctG are all necessary to provide immunity to lacticin 481. The quantification of the levels of immunity showed that lctF, lctE, and lctG could account for at least 6% and up to 100% of the immunity of the wild-type lacticin 481 producer strain, depending on the gene expression regulation. The lacticin 481 biosynthesis and immunity systems are discussed and compared to other lantibiotic systems.     

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.     

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.     

Conjugal transfer of the determinants for bacteriocin (lacticin 481) production and immunity in Lactococcus lactis subsp. lactis CNRZ 481

Abstract The lacticin 481-producer (Lct⁺), L. lactis subsp. lactis (L. lactis ) CNRZ 481 harbours 5 plasmids of 6.5, 7.5, 20, 37 and 69 kb. Novobiocin treatment of L. lactis 481 led to the appearance of lacticin 481 deficient variants which had all lost the 69 kb plasmid. Conjugal transfer of the lacticin 481 structural gene ( lct ) into the plasmid free strain L. lactis IL1441 yielded Lct⁺ transconjugants at a 10⁻⁴ frequency, which carried a plasmid with an apparent size of 120–130 kb. Southern hybridization analyses showed that the lct gene was located on the 69 kb plasmid in L. lactis 481 and on the 120–130 kb plasmid in the transconjugants. The lct gene was in higher copy number in transconjugants than in the parental strain resulting in two-fold higher lacticin 481 production in the former strain.     

Change of cell wall chitin content in amphotericin B resistant Kluyveromyces strains

Lacticin 481 is produced by Lactococcus lactis subsp. lactis and belongs to subgroup AII of the lanthionine-containing bacteriocins. The putative homodimeric LctT involved in lacticin 481 production shares significant similarities with the 'LcnC' protein encoded by 'lcnC', located on the chromosome of the lactic acid bacterium, L. lactis IL1403. LctT and 'LcnC' belong to the recently defined family of AMS (ABC transporter maturation and secretion) proteins. Inactivation of the 'lcnC' gene demonstrates that it is not responsible for the weak lacticin 481 production observed in a strain expressing only the precursor peptide LctA, and the modification enzyme LctM. This result indicates that the two AMS proteins, 'LcnC' and LctT, are not interchangeable in the machinery of processing/export of lacticin 481.     

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.     

Macedocin,a food-grade lantibiotic produced by Streptococcus macedonicus ACA-DC 198

Streptococcus macedonicus ACA-DC 198, a strain isolated from Greek Kasseri cheese, produces a food-grade lantibiotic named macedocin. Macedocin has a molecular mass of 2,794.76 +/- 0.42 Da, as determined by electrospray mass spectrometry. Partial N-terminal sequence analysis revealed 22 amino acid residues that correspond with the amino acid sequence of the lantibiotics SA-FF22 and SA-M49, both of which were isolated from the pathogen Streptococcus pyogenes. Macedocin inhibits a broad spectrum of lactic acid bacteria, as well as several food spoilage and pathogenic bacteria, including Clostridium tyrobutyricum. It displays a bactericidal effect towards the most sensitive indicator strain, Lactobacillus sakei subsp. sakei LMG 13558(T), while the producer strain itself displays autoinhibition when it is grown under conditions that do not favor bacteriocin production. Macedocin is active at pHs between 4.0 and 9.0, and it retains activity even after incubation for 20 min at 121 degrees C with 1 atm of overpressure. Inhibition of macedocin by proteolytic enzymes is variable.     

A lacticin 481-producing adjunct culture increases starter lysis while inhibiting nonstarter lactic acid bacteria proliferation during Cheddar cheese ripening

AIMS: The main aim of this study was to exploit a lacticin 481 producing strain, Lactococcus lactis CNRZ481, as an adjunct for Cheddar cheese manufacture, to increase starter cell lysis and control nonstarter lactic acid bacteria (NSLAB) proliferation in cheese. METHODS AND RESULTS: Lactococcus lactis CNRZ481 was exploited as an adjunct to L. lactis HP for the manufacture of Cheddar cheese at pilot scale (450 l). In these trials, inclusion of the adjunct strain did not compromise acid production by L. lactis HP and cheese was successfully manufactured within 5 h. Experimental cheese exhibited levels of lactate dehydrogenase (LDH) up to five-fold higher than control cheese and a significant reduction in NSLAB growth was also observed throughout the ripening period. CONCLUSIONS: The aims of the study were accomplished as (i) greater enzyme release was achieved through lacticin 481-induced lysis which was associated with an improved flavoured cheese as assessed by a commercial grader and (ii) NSLAB growth was controlled, thus reducing the risk of off-flavour development. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of lacticin 481-producing adjuncts for cheese manufacture may prove beneficial for manufacturers who aim to achieve faster ripening through premature and elevated intracellular enzyme release while minimizing inconsistencies in cheese quality because of NSLAB activity.