An analysis of bacteriocins produced by lactic acid bacteria isolated from malted barley

AIMS: The aim of this study was to perform a detailed characterization of bacteriocins produced by lactic acid bacteria (LAB) isolated from malted barley. METHODS AND RESULTS: Bacteriocin activities produced by eight LAB, isolated from various types of malted barley, were purified to homogeneity by ammonium sulphate precipitation, cation exchange, hydrophobic interaction and reverse-phase liquid chromatography. Molecular mass analysis and N-terminal amino acid sequencing of the purified bacteriocins showed that four non-identical Lactobacillus sakei strains produced sakacin P, while four Leuconostoc mesenteroides strains were shown to produce bacteriocins highly similar or identical to leucocin A, leucocin C or mesenterocin Y105. Two of these bacteriocin-producing strains, Lb. sakei 5 and Leuc. mesenteroides 6, were shown to produce more than one bacteriocin. Lactobacillus sakei 5 produced sakacin P as well as two novel bacteriocins, which were termed sakacin 5X and sakacin 5T. The inhibitory spectrum of each purified bacteriocin was analysed and demonstrated that sakacin 5X was capable of inhibiting the widest range of beer spoilage organisms. CONCLUSION: All bacteriocins purified in this study were class II bacteriocins. Two of the bacteriocins have not been described previously in the literature while the remaining purified bacteriocins have been isolated from environments other than malted barley. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents a thorough analysis of bacteriocin-producing LAB from malt and demonstrates, for the first time, the variety of previously identified and novel inhibitory peptides produced by isolates from this environment. It also highlights the potential of these LAB cultures to be used as biological controlling agents in the brewing industry.     

Production of multiple bacteriocins from a single locus by gastrointestinal strains of Lactobacillus salivarius

Bacteriocins produced by Lactobacillus salivarius isolates derived from a gastrointestinal origin have previously demonstrated efficacy for in vivo protection against Listeria monocytogenes infection. In this study, comparative genomic analysis was employed to investigate the intraspecies diversity of seven L. salivarius isolates of human and porcine intestinal origin, based on the genome of the well-characterized bacteriocin-producing strain L. salivarius UCC118. This revealed a highly conserved megaplasmid-borne gene cluster in these strains involved in the regulation and secretion of two-component class IIb bacteriocins. However, considerable intraspecific variation was observed in the structural genes encoding the bacteriocin peptides. They ranged from close relatives of abp118, such as salivaricin P, which differs by 2 amino acids, to completely novel bacteriocins, such as salivaricin T, which is characterized in this study. Salivaricin T inhibits closely related lactobacilli and bears little homology to previously characterized salivaricins. Interestingly, the two peptides responsible for salivaricin T activity, SalTα and SalTβ, share considerable identity with the component peptides of thermophilin 13, a bacteriocin produced by Streptococcus thermophilus. Furthermore, the salivaricin locus of strain DPC6488 also encodes an additional novel one-component class IId anti-listerial bacteriocin, salivaricin L. These findings suggest a high level of redundancy in the bacteriocins that can be produced by intestinal L. salivarius isolates using the same enzymatic production and export machinery. Such diversity may contribute to their ability to dominate and compete within the complex microbiota of the mammalian gut.     

Wide-Inhibitory Spectra Bacteriocins Produced by Lactobacillus gasseri K7

The aim of our study was to determine the genetic characterization and classification of Lb. gasseri K7 bacteriocins, comparison with bacteriocins of the Lb. gasseri LF221 strain and other related strains. Bacteriocin-encoding genes were amplified by PCR, subjected to DNA sequencing, and BLAST sequence analysis was performed to search the database for homologous peptides. Lb. gasseri K7 produces two two-peptide bacteriocins, named gassericin K7 A and gassericin K7 B. Their nucleotide sequences were deposited at GenBank, under accession numbers EF392861 for the gassericin K7 A and AY307382 for the gassericin K7 B. Analysis of gene clusters of bacteriocins in Lb. gasseri K7 strain revealed a 100 percent sequence identity with bacteriocins in LF221 strain. An active peptide of gassericin K7 B is homologous to the complementary peptide of gassericin T, and a complementary peptide of gassericin K7 B is homologous to the active peptide of gassericin T. Another surprising finding was that the sakacin T-beta peptide is partly homologous to the active peptide of gassericin K7 A, while the other sakacin T peptide (alfa) is partly homologous to the complementary peptide of gassericin K7 B. Gassericins of Lb. gasseri K7 strain were both classified as two-peptide bacteriocins. Human probiotic strains Lb. gasseri K7 and LF221 are different isolates but with identical bacteriocin genes. They produce wide-inhibitory spectra bacteriocins that are new members of two-peptide bacteriocins with some homologies to other bacteriocins in this group. Described bacteriocins offer a great potential in applications in food industry, pharmacy and biomedicine.     

Purification and cloning of sakacin 674, a bacteriocin from Lactobacillus sake Lb674

Abstract Sakacin 674, a bacteriocin produced by Lactobacillus sake Lb764 and which inhibits the growth of Listeria monocytogenes , was purified to homogeneity by ammonium sulphate precipitation and sequential ion exchange, hydrophobic interaction and reversed phase chromatography. The complete amino acid sequence of sakacin 674 was determined by Edman degradation. The bacteriocin consisted of 43 amino acid residues and had a calculated molecular mass of 4436.6 Da, which is in good agreement with the molecular mass of 4437.2 as determined by mass spectrometry. The structural gene encoding sakacin 674 ( sakR ) was located on the chromosome. This gene was cloned and sequenced. It encoded a primary translation product of 61 amino acid residues which was cleaved between amino acids 18 and 19 to yield the active sakacin 674. Sakacin 674 resembled other known bacteriocins and was very similar to sakacin P.     

Induction of bacteriocin production in Lactobacillus sake by a secreted peptide.

Lactobacillus sake LTH673 is known to produce a bacteriocin called sakacin P. Production of and immunity to sakacin P were found to depend on the presence of a protease-sensitive component that is produced by L. sake LTH673 itself. This component (called inducing factor [IF]) was purified from culture supernatants and shown to be a basic, nonbacteriocin peptide consisting of 19 amino acids, which in principle is capable of forming a highly amphiphilic helical structure. Circular dichroism studies showed that IF indeed could adopt a helical structure, but only in membrane-mimicking environments. Both purified IF and chemically synthesized IF induced expression of the structural gene for sakacin P and concomitant secretion of the gene product. In addition, IF induced its own production and immunity to sakacin P and related bacteriocins. These results indicate that bacteriocin production by L. sake LTH673 is controlled by an autoinduction pathway in which IF may function as a cell density signal.     

BlpC-regulated bacteriocin production in Streptococcus thermophilus

Streptococcus thermophilus B59671 produces a bacteriocin with anti-pediococcal activity, but genes required for its production are not characterized. Genome sequencing of S. thermophilus has identified a genetic locus encoding a quorum sensing (QS) system that regulates production of class II bacteriocins. However, in strains possessing this gene cluster, production of bacteriocin like peptides (Blp) was only observed when excess pheromone was provided. PCR analysis revealed this strain possessed blpC, which encodes the 30-mer QS pheromone. To investigate if BlpC regulates bacteriocin production in S. thermophilus B59671, an integrative vector was used to replace blpC with a gene encoding for kanamycin resistance and the resulting mutant did not inhibit the growth of Pediococcus acidilactici. Constitutive expression of blpC from a shuttle vector restored the bacteriocin production, confirming the blp gene cluster is essential for bacteriocin activity in S. thermophilus B59671.     

Cloning and nucleotide sequence of a gene from Lactobacillus sake Lb706 necessary for sakacin A production and immunity.

Sakacin A is an antilisterial bacteriocin produced by Lactobacillus sake Lb706. In order to identify genes involved in sakacin A production and immunity, the plasmid fraction of L. sake Lb706 was shotgun cloned directly into a sakacin A-nonproducing and -sensitive variant, L. sake Lb706-B, by using the broad-host-range vector pVS2. Two clones that produced sakacin A and were immune to the bacteriocin were obtained. A DNA fragment of approximately 1.8 kb, derived from a 60-kb plasmid of strain Lb706 and present in the inserts of both clones, was necessary for restoration of sakacin A production and immunity in strain Lb706-B. The sequence of the 1.8-kb fragment from one of the clones was determined. It contained one large open reading frame, designated sakB, potentially encoding a protein of 430 amino acid residues. Hybridization and nucleotide sequence analyses revealed that the cloned sakB complemented a mutated copy of sakB present in strain Lb706-B. The sakB gene mapped 1.6 kb from the previously cloned structural gene for sakacin A (sakA) on the 60-kb plasmid. The putative SakB protein shared 22% amino acid sequence identity (51% similarity if conservative changes are considered) to AgrB, the deduced amino acid sequence of the Staphylococcus aureus gene agrB. The polycistronic agr (accessory gene regulator) locus is involved in the regulation of exoprotein synthesis in S. aureus. Similar to the AgrB protein, SakB had some features in common with a family of transmembrane histidine protein kinases, involved in various adaptive response systems of bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular analysis of the 21-kb bacteriocin-encoding plasmid pEF1 from Enterococcus faecium 6T1a

The complete 21,344-bp DNA sequence of the bacteriocin-encoding plasmid pEF1 from Enterococcus faecium 6T1a was determined. Thirty-four putative open reading frames which could code for proteins longer than 42 amino acids were found. Those included the structural genes encoding for the previously described bacteriocins enterocin I and J (also named as enterocins L50A and L50B). After comparison to sequences in public databases, analysis of the gene organization of pEF1 suggests a modular structure with three different functional domains: the replication region, the bacteriocin region and the mobilization plus UV-resistance region. This genetic mosaic structure most probably evolved through recombination events promoted by transposable elements. The hypothesis that the bacteriocin cluster on pEF1 could act as a functional plasmid stabilization module in E. faecium 6T1a is discussed.     

Purification and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici.

A bacteriocin produced by Pediococcus acidilactici has been purified to homogeneity by a rapid and simple four-step purification procedure which includes ammonium sulphate precipitation, chromatography with a cation-exchanger and Octyl Sepharose, and reverse-phase chromatography. The purification resulted in an approximately 80,000-fold increase in the specific activity and about a 6-fold increase in the total activity. The amino acid composition and sequencing data indicated that the bacteriocin contained 43-44 amino acid residues. The predicted M(r) and isolectric point of the bacteriocin are about 4600 and 8.6, respectively. Comparing the amino acid sequence of this bacteriocin with the sequences of leucocin A-UAL 187, sakacin P and curvacin A (bacteriocins produced by Leuconostoc gelidum, Lactobacillus sake and Lactobacillus curvatus, respectively) revealed that all four bacteriocins had in their N-terminal region the sequence Tyr-Gly-Asn-Gly-Val-Xaa-Cys, indicating that this concensus sequence is of fundamental importance for this group of bacteriocins. The bacteriocin from P. acidilactici and sakacin P were very similar, having at least 25 common amino acid residues. The sequence similarity was greatest in the N-terminal half of the molecules--17 of the first 19 residues were common--indicating the fundamental importance of this region. Leucocin A-UAL 187 and curvacin A had, respectively, at least 16 and 13 amino acid residues in common with the bacteriocin from P. acidilactici.     

Heterologous coproduction of enterocin A and pediocin PA-1 by Lactococcus lactis: detection by specific peptide-directed antibodies

Antibodies against enterocin A were obtained by immunization of rabbits with synthetic peptides PH4 and PH5 designed, respectively, on the N- and C-terminal amino acid sequences of enterocin A and conjugated to the carrier protein KLH. Anti-PH4-KLH antibodies not only recognized enterocin A but also pediocin PA-1, enterocin P, and sakacin A, three bacteriocins which share the N-terminal class IIa consensus motif (YGNGVXC) that is contained in the sequence of the peptide PH4. In contrast, anti-PH5-KLH antibodies only reacted with enterocin A because the amino acid sequences of the C-terminal parts of class IIa bacteriocins are highly variable. Enterocin A and/or pediocin PA-1 structural and immunity genes were introduced in Lactococcus lactis IL1403 to achieve (co)production of the bacteriocins. The level of production of the two bacteriocins was significantly lower than that obtained by the wild-type producers, a fact that suggests a low efficiency of transport and/or maturation of these bacteriocins by the chromosomally encoded bacteriocin translocation machinery of IL1403. Despite the low production levels, both bacteriocins could be specifically detected and quantified with the anti-PH5-KLH (anti-enterocin A) antibodies isolated in this study and the anti-PH2-KLH (anti-pediocin PA-1) antibodies previously generated (J. M. Martínez, M. I. Martínez, A. M. Suárez, C. Herranz, P. Casaus, L. M. Cintas, J. M. Rodríguez, and P. E. Hernández, Appl. Environ. Microbiol. 64:4536-4545, 1998). In this work, the availability of antibodies for the specific detection and quantification of enterocin A and pediocin PA-1 was crucial to demonstrate coproduction of both bacteriocins by L. lactis IL1403(pJM04), because indicator strains that are selectively inhibited by each bacteriocin are not available.     

Differences in susceptibility of Listeria monocytogenes strains to sakacin P,sakacin A,pediocin PA-1, and nisin

Two hundred strains of Listeria monocytogenes collected from food and the food industry were analyzed for susceptibility to the class IIa bacteriocins sakacin P, sakacin A, and pediocin PA-1 and the class I bacteriocin nisin. The individual 50% inhibitory concentrations (IC(50)) were determined in a microtiter assay and expressed in nanograms per milliliter. The IC(50) of sakacin P ranged from 0.01 to 0.61 ng ml(-1). The corresponding values for pediocin PA-1, sakacin A, and nisin were 0.10 to 7.34, 0.16 to 44.2, and 2.2 to 781 ng ml(-1), respectively. The use of a large number of strains and the accuracy of the IC(50) determination revealed patterns not previously described, and for the first time it was shown that the IC(50) of sakacin P divided the L. monocytogenes strains into two distinct groups. Ten strains from each group were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and amplified fragment length polymorphism. The results from these studies essentially confirmed the grouping based on the IC(50) of sakacin P. A high correlation was found between the IC(50) of sakacin P and that of pediocin PA-1 for the 200 strains. Surprisingly, the correlation between the IC(50) of the two class IIa bacteriocins sakacin A and sakacin P was lower than the correlation between the IC(50) of sakacin A and the class I bacteriocin nisin.     

Diversity of bacteriocins and activity spectrum in Streptococcus pneumoniae

The production of bacteriocins can be favorable for colonization of the host by eliminating other bacterial species that share the same environment. In Streptococcus pneumoniae, the pnc (blp) locus encoding putative bacteriocins, immunity, and export proteins is controlled by a two-component system similar to the comCDE system required for the induction of genetic competence. A detailed comparison of the pnc clusters of four genetically distinct isolates confirmed the great plasticity of this locus and documented several repeat sequences. Members of the multiple-antibiotic-resistant Spain23F-1 clone, one member of the Spain9V-3 clone, sensitive 23F strain 2306, and the TIGR4 strain produced bactericidal substances active against other gram-positive bacteria and in some cases against S. pneumoniae as well. However, other strains did not show activity against the indicator strains despite the presence of a bacteriocin cluster, indicating that other factors are required for bacteriocin activity. Analysis of strain 2306 and mutant derivatives of this strain confirmed that bacteriocin production was dependent on the two-component regulatory system and genes involved in bacteriocin transport and processing. At least one other bacteriocin gene, pncE, is located elsewhere on the chromosome and might contribute to the bacteriocin activity of this strain.     

Purification and amino acid sequence of sakacin A, a bacteriocin from Lactobacillus sake Lb706.

Sakacin A, a bacteriocin produced by Lactobacillus sake Lb706 and which inhibits the growth of Listeria monocytogenes, was purified to homogeneity by ammonium sulphate precipitation and ion-exchange, hydrophobic-interaction and reversed-phase chromatography. The complete amino acid sequence of sakacin A was determined by Edman degradation. The bacteriocin consisted of 41 amino acid residues and had a calculated M(r) of 4308.7, which is in good agreement with the value determined by mass spectrometry. The structural gene encoding sakacin A (sakA) was cloned and sequenced. The gene encoded a primary translation product of 59 amino acid residues which was cleaved between amino acids 18 and 19 to yield the active sakacin A. Sakacin A shared some sequence similarities with other bacteriocins.     

Identification of a new plasmid-encoded sec-dependent bacteriocin produced by Listeria innocua 743

Listeria innocua 743 produces an inhibitory activity demonstrating broad-spectrum inhibition of Listeria monocytogenes isolates. Gel-electrophoretic analysis of culture supernatants indicated that two inhibitors with different molecular weights were produced by this strain. Insertion of Tn917 into a 2.9 Kb plasmid (pHC743) generated mutants with either an impaired ability or a loss in ability to produce one of the inhibitors. Sequence analysis of the transposon insertion regions revealed the presence of two continuous open reading frames, the first encoding a new pediocin-like bacteriocin (lisA) and the second encoding a protein homologous with genes involved in immunity toward other bacteriocins (lisB). Translation of the bacteriocin gene (lisA) initiates from a noncanonical start codon and encodes a 71-amino-acid prebacteriocin which lacked the double glycine leader peptidase processing site common in other type II bacteriocins. Alignment of the sequence with the processed N termini of related bacteriocins suggests that the mature bacteriocin consists of 43 amino acids, with a predicted molecular mass of 4,484 Da. Mutants containing insertions into lisA were sensitive to the inhibitor, indicating that lisAB forms a single operon and that lisB represents the immunity protein. Cloning of an amplicon containing the lisAB operon into Escherichia coli resulted in expression and export of the bacteriocin. This finding confirms that the phenotype is dependent on the structural and immunity gene only and that export of this bacteriocin is sec dependent. This is the first confirmation of bacteriocin production in a Listeria spp., and it is of interest that this bacteriocin is closely related to the pediocin family of bacteriocins produced by lactic acid bacteria.     

Identification and nucleotide sequence of genes involved in the synthesis of lactocin 705, a two-peptide bacteriocin from Lactobacillus casei CRL 705

The structural gene determinants of lactocin 705, a bacteriocin produced by Lactobacillus casei CRL 705, have been amplified from a plasmid of approximately 35 kb and sequenced. Lactocin 705 is a class IIb bacteriocin, whose activity depends upon the complementation of two peptides (705alpha and 705beta) of 33 amino acid residues each. These peptides are synthesized as precursors with signal sequences of the double-glycine type, which exhibited high identities with the leader peptides of plantaricin S and J from Lactobacillus plantarum, brochocin C from Brochotrix campestris, sakacin P from Lactobacillus sake, and the competence stimulating peptides from Streptococcus gordonii and Streptococcus mitis. However, the two mature bacteriocins 705alpha and 705beta do not show significant similarity to other sequences in the databases.     

Characterization of a New Bacteriocin Operon in Sakacin P-Producing Lactobacillus sakei, Showing Strong Translational Coupling between the Bacteriocin and Immunity Genes

Previous studies of genes involved in the production of sakacin P by Lactobacillus sakei Lb674 revealed the presence of an inducible promoter downstream of the known spp gene clusters. We show here that this promoter drives the expression of an operon consisting of a bacteriocin gene (sppQ), a cognate immunity gene (spiQ), another gene with an unknown function (orf4), and a pseudoimmunity gene containing a frameshift mutation (orf5). The leader peptide of the new one-peptide bacteriocin sakacin Q contains consensus elements that are typical for so-called “double-glycine” leader peptides. The mature bacteriocin shows weak similarity to the BrcA peptide of the two-peptide bacteriocin brochocin C. Sakacin Q has an antimicrobial spectrum that differs from that of sakacin P, thus expanding the antimicrobial properties of the producer strain. The genes encoding sakacin Q and its cognate immunity protein showed strong translational coupling, which was investigated in detail by analyzing the properties of a series of β-glucuronidase fusions. Our results provide experimental evidence that production of the bacteriocin and production of the cognate immunity protein are tightly coregulated at the translational level.