Lacticin, a bacteriocin produced by Lactobacillus delbrueckii subsp. lactis

Twenty-one strains of Lactobacillus delbrueckii and L. helveticus were tested for bacteriocin production against each other. Lactobacillus delbrueckii subsp. lactis JCM 1106 and 1107 produced an inhibitory agent active against L. delbrueckii subsp. bulgaricus JCM 1002 and NIAI yB-62, L. delbrueckii subsp. lactis JCM 1248 and L. delbrueckii subsp. delbrueckii JCM 1012. Lactobacillus delbrueckii subsp. lactis JCM 1248 inhibited only the growth of L. delbrueckii subsp. bulgaricus NIAI yB-62. These agents were sensitive to proteolytic enzymes and heating (at 60°C for 10min). These agents were considered to be bacteriocins and designated lacticin A and B.     

Conjugal Transfer of Plasmid-Encoded Determinants for Bacteriocin Production and Immunity in Lactobacillus acidophilus 88

Lactobacillus acidophilus 88 produced a bacteriocin, designated lactacin F, that demonstrated inhibitory activity toward L. acidophilus 6032, L. lactis 970, L. helveticus 87, L. bulgaricus 1489, L. leichmanii 4797, L. fermentum 1750, and Streptococcus faecalis 19433. Production of lactacin F was pH dependent and could be maximized in MRS broth cultures maintained at pH 7.0. Lactacin F was heat stable and sensitive to ficin, proteinase K, trypsin, and Bacillus subtilis protease. L. acidophilus 88 harbored plasmids of 4 and 27 megadaltons. Variants of L. acidophilus 88 which were deficient in lactacin F production (Laf) and lactacin F immunity (Laf) retained the two resident plasmids. A Laf Laf derivative, L. acidophilus 89, was used as a recipient in agar surface mating experiments with L. acidophilus 88 (Laf Laf). Two types of Laf Laf transconjugants were recovered. One type (T-E) had acquired two plasmids of 68 (pPM68) and 52 (pPM52) megadaltons that were not detected in either the conjugal donor or the other type of Laf Laf transconjugants (T-89). Laf and Laf were unstable in the plasmid-bearing transconjugant. Plasmid analysis of Laf Laf variants revealed that pPM52 and pPM68 were cured with loss of Laf and Laf. Bacteriocin production and immunity phenotypes were genetically stable in Laf Laf transconjugants not harboring pPM52 and pPM68, suggesting chromosomal integration of the transferred determinants. The data demonstrated intragenic conjugation in L. acidophilus and provided direct evidence for involvement of transient plasmid determinants in Laf and Laf.     

Acidophilucin A, a new heat-labile bacteriocin produced by Lactobacillus acidophilus LAPT 1060

Lactobacillus acidophilus LAPT 1060, isolated from infant faeces, produced an antimicrobial agent active against six strains of Lactobacillus delbrueckii subsp. bulgaricus and six strains of Lactobacillus helveticus . The agent was sensitive to proteolytic enzymes and heat (10 min at 60°C) and is a bacteriocin and designated acidophilucin A.     

Reutericin 6, a new bacteriocin produced by Lactobacillus reuteri LA 6

Lactobacillus reuteri LA 6, isolated from infant faeces, produced an antimicrobial agent active against Lactobacillus acidophilus JCM 2125, Lactobacillus delbrueckii subsp. bulgaricus JCM 1002 and Lactobacillus delbrueckii subsp. lactis JCM 1148 and JCM 1248. The agent was sensitive to proteolytic enzymes and retained activity after heating at 100°C for 20 min. This agent was a bacteriocin and has been designated as reutericin 6. Reutericin 6 exceeds 200 kDa as determined by ultrafiltration studies. Activity against sensitive cells was both bacteriocidal and bacteriolytic.     

Cloning, phenotypic expression, and DNA sequence of the gene for lactacin F, an antimicrobial peptide produced by Lactobacillus spp

Lactacin F is a heat-stable bacteriocin produced by Lactobacillus acidophilus 11088. A 63-mer oligonucleotide probe deduced from the N-terminal lactacin F amino acid sequence was used to clone the putative laf structural gene from plasmid DNA of a lactacin F-producing transconjugant, L. acidophilus T143. One clone, NCK360, harbored a recombinant plasmid, pTRK160, which contained a 2.2-kb EcoRI fragment of the size expected from hybridization experiments. An Escherichia coli-L. acidophilus shuttle vector was constructed, and a subclone (pTRK162) containing the 2.2-kb EcoRI fragment was introduced by electroporation into two lactacin F-negative strains, L. acidophilus 89 and 88-C. Lactobacillus transformants containing pTRK162 expressed lactacin F activity and immunity. Bacteriocin produced by the transformants exhibited an inhibitory spectrum and heat stability identical to those of the wild-type bacteriocin. An 873-bp region of the 2.2-kb fragment was sequenced by using a 20-mer degenerate lactacin F-specific primer to initiate sequencing from within the lactacin F structural gene. Analysis of the resulting sequence identified an open reading frame which could encode a protein of 75 amino acids. The 25 N-terminal amino acids for lactacin F were identified within the open reading frame along with an N-terminal extension, possibly a signal sequence. The lactacin F N-terminal sequence, through the remainder of the open reading frame (57 amino acids; 6.3 kDa), correlated extremely well with composition analyses of purified lactacin F which also predicted a size of 51 to 56 amino acid residues. Molecular characterization of lactacin F identified a small hydrophobic peptide that may be representative of a common bacteriocin class in lactic acid bacteria.     

Characterization and purification of helveticin J and evidence for a chromosomally determined bacteriocin produced by Lactobacillus helveticus 481.

Lactobacillus helveticus 481 produced an antimicrobial agent active against five closely related species. The sensitive indicators included L. helveticus 1846 and 1244, L. bulgaricus 1373 and 1489, and L. lactis 970. The antimicrobial compound was active at neutral pH under aerobic or anaerobic conditions, was sensitive to proteolytic enzymes and heat (30 min at 100 degrees C), and demonstrated a bactericidal mode of action against sensitive indicators. These data confirmed that antimicrobial activity of L. helveticus 481 was mediated by a bacteriocin, designated helveticin J. Production of helveticin J was maximized in an anaerobic fermentor held at a constant pH of 5.5. Ultrafiltration experiments on culture supernatants containing the bacteriocin revealed that helveticin J was present as an aggregate with a molecular weight in excess of 300,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of helveticin J purified through Sephadex chromatography resolved a 37,000-dalton protein band with bacteriocin activity. L. helveticus 481 was shown to harbor a single 8-megadalton plasmid (pMJ1008). Isolates cured of pMJ1008 were phenotypically identical to plasmid-bearing cells in fermentation patterns, helveticin J activity, and immunity spectra. The data provided evidence for a chromosomal location of helveticin J and host immunity determinants.     

Molecular characterization of antimicrobial compound produced by Lactobacillus acidophilus AA11

Approximately 63 strains of Lactobacillus acidophilus were isolated from Egyptian home-made cheese and examined for production of antagonism. Only eight strains demonstrated inhibitory activity against spoilage microorganisms (i.e. Staphylococcus aureus and Bacillus cereus) and pathogens (i.e. E. coli, Salmonella sp. and Shigella sp.). Lactobacillus acidophilus AA11 produced higher antimicrobial activity with a wide range of inhibition. The agent AA11 was sensitive to proteolytic enzymes and retained full activity after 30 min at 100 degrees C. Activity against sensitive cells was bactericidal but not bacteriolytic. The compound was produced during growth phase and could be extracted from the culture supernatant fluids with n-butanol. 12% SDS-PAGE analysis of 40% ammonium sulphate precipitated agent showed two peptides with molecular weights of approximately 36 kDa and approximately 29 kDa. No plasmid was identified in Lactobacillus acidophilus AA11 indicating that the genes encoding the inhibitory agent were located on the chromosome. These characteristics identify the inhibitory substance as a bacteriocin, designated acidocin AA11 and confer the agent an application potential as a biopreservative.     

Study of antimicrobial activity amongLactobacillus helveticus strains using three different assays

The antimicrobial activity of 18Lactobacillus helveticus strains as well as one control strain, the bacteriocin producingLactobacillus helveticus 481, was tested with three different inhibition assays. FourLactobacillus helveticus strains had antimicrobial activity against seven otherLactobacillus helveticus strains and twoLactobacillus delbrueckil strains while the remaining sevenLactobacillus helveticus strains were indifferent. Inhibition was also observed againstLactococcus andLeuconostoc species, due to production of organic acids or hydrogen peroxide. The strains with the highest antimicrobial activity produced a heat sensitive proteinacious bacteriocin with a narrow species activity spectrum against only thermophilicLactobacillus strains.     

蒙古戈壁地区自然发酵乳中乳酸菌的分离鉴定

从采集自蒙古国戈壁地区的6份自然发酵乳中分离到14株乳酸菌,经过形态特征,生理生化特性,糖发酵试验和乳酸旋光性的测定,鉴定结果:乳酸球菌5株,包括Lactococcus lactissubsp.cremoris 1株,Pedio-coccus.(后缩写为Ped.).urinaeequi3株,Pediococcus.pentosaceus1株;乳杆菌9株,包括Lactobacillus.(后缩写为L.)helveticus8株,Lactobacillus.delbrueckii.subsp.bulgaricus1株。蒙古国戈壁地区自然发酵乳中的优势菌为Lactobacillus.helveticus,其次为Pediococcus.urinaeequi。     

Purification and partial characterization of lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088.

Lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088 (NCK88), was purified and characterized. Lactacin F is heat stable, proteinaceous, and inhibitory to other lactobacilli as well as Enterococcus faecalis. The bacteriocin was isolated as a floating pellet from culture supernatants brought to 35 to 40% saturation with ammonium sulfate. Native lactacin F was sized at approximately 180 kDa by gel filtration. Column fractions having lactacin F activity were examined by electron microscopy and contained micelle-like globular particles. Purification by ammonium sulfate precipitation, gel filtration, and high-performance liquid chromatography resulted in a 474-fold increase in specific activity of lactacin F. The purified bacteriocin was identified as a 2.5-kDa peptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lactacin F peptide retained activity after extraction from SDS-PAGE gel slices, confirming the identity of the 2.5-kDa peptide. Variants of NCK88 that failed to exhibit lactacin F activity did not produce the 2.5-kDa band. Sequence analysis of purified lactacin F identified 25 N-terminal amino acids containing an arginine residue at the N terminus. Composition analysis indicates that lactacin F may contain as many as 56 amino acid residues.     

Bacteriocin-mediated inhibition of Clostridium botulinum spores by lactic acid bacteria at refrigeration and abuse temperatures.

The bacteriocinogenicity of Lactococcus lactis ATCC 11454, Pediococcus pentosaceus ATCC 43200, P. pentosaceus ATCC 43201, Lactobacillus plantarum BN, L. plantarum LB592, L. plantarum LB75, and Lactobacillus acidophilus N2 against Clostridium botulinum spores at 4, 10, 15, and 35 degrees C was investigated by modified deferred and simultaneous antagonism methods. All the strains, except L. acidophilus N2, produced inhibition zones on lawns of C. botulinum spores at 30 degrees C. L. plantarum BN, L. lactis ATCC 11454, and P. pentosaceus ATCC 43200 and 43201 were bacteriocinogenic at 4, 10, and 15 degrees C. Supplementation of brain heart infusion agar with 0 to 5% NaCl increased the radii of inhibition zones during simultaneous antagonism assays. Detectable bacteriocin activities were extracted from freeze-thawed agar cultures of L. plantarum BN and L. lactis ATCC 11454 which had been grown at 4 and 10 degrees C. These results suggest that low levels of L. plantarum BN or L. lactis ATCC 11454, in the presence of 3 or 4% NaCl, could be formulated into minimally processed refrigerated food products for protection against possible botulism hazards.     

Purification and partial characterization of lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088

Lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088 (NCK88), was purified and characterized. Lactacin F is heat stable, proteinaceous, and inhibitory to other lactobacilli as well as Enterococcus faecalis. The bacteriocin was isolated as a floating pellet from culture supernatants brought to 35 to 40% saturation with ammonium sulfate. Native lactacin F was sized at approximately 180 kDa by gel filtration. Column fractions having lactacin F activity were examined by electron microscopy and contained micelle-like globular particles. Purification by ammonium sulfate precipitation, gel filtration, and high-performance liquid chromatography resulted in a 474-fold increase in specific activity of lactacin F. The purified bacteriocin was identified as a 2.5-kDa peptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lactacin F peptide retained activity after extraction from SDS-PAGE gel slices, confirming the identity of the 2.5-kDa peptide. Variants of NCK88 that failed to exhibit lactacin F activity did not produce the 2.5-kDa band. Sequence analysis of purified lactacin F identified 25 N-terminal amino acids containing an arginine residue at the N terminus. Composition analysis indicates that lactacin F may contain as many as 56 amino acid residues.     

Bacteriocin-mediated inhibition of Clostridium botulinum spores by lactic acid bacteria at refrigeration and abuse temperatures.

The bacteriocinogenicity of Lactococcus lactis ATCC 11454, Pediococcus pentosaceus ATCC 43200, P. pentosaceus ATCC 43201, Lactobacillus plantarum BN, L. plantarum LB592, L. plantarum LB75, and Lactobacillus acidophilus N2 against Clostridium botulinum spores at 4, 10, 15, and 35 degrees C was investigated by modified deferred and simultaneous antagonism methods. All the strains, except L. acidophilus N2, produced inhibition zones on lawns of C. botulinum spores at 30 degrees C. L. plantarum BN, L. lactis ATCC 11454, and P. pentosaceus ATCC 43200 and 43201 were bacteriocinogenic at 4, 10, and 15 degrees C. Supplementation of brain heart infusion agar with 0 to 5% NaCl increased the radii of inhibition zones during simultaneous antagonism assays. Detectable bacteriocin activities were extracted from freeze-thawed agar cultures of L. plantarum BN and L. lactis ATCC 11454 which had been grown at 4 and 10 degrees C. These results suggest that low levels of L. plantarum BN or L. lactis ATCC 11454, in the presence of 3 or 4% NaCl, could be formulated into minimally processed refrigerated food products for protection against possible botulism hazards.     

Conjugal transfer of plasmid pIP501 from Lactococcus lactis to Lactobacillus delbrückii subsp. bulgaricus and Lactobacillus helveticus

Plasmid pIP501 was transferred by conjugation from Lactococcus lactis to Lactobacillus delbrückii subsp. bulgaricus and Lactobacillus helveticus. Only Lb. delbrückii subsp. bulgaricus transconjugants could act as a donor in crosses with Lc. lactis. No Lactobacillus transconjugants were detected after inter- or intra-species Lactobacillus crosses. Plasmid pIP501 has undergone no detectable deletion or rearrangement during transfer from Lc. lactis to Lactobacillus strains.     

Conjugal transfer of plasmid pIP501 from Lactococcus lactis to Lactobacillus delbruckii subsp. bulgaricus and Lactobacillus helveticus

Abstract Plasmid pIP501 was transferred by conjugation from Lactococcus lactis to Lactobacillus delbrückii subsp. bulgaricus and Lactobacillus helveticus . Only Lb. delbrückii subsp. bulgaricus transconjugants could act as a donor in crosses with Lc. lactis . No Lactobacillus transconjugants were detected after inter- or intra-species Lactobacillus crosses. Plasmid pIP501 has undergone no detectable deletion or rearrangement during transfer from Lc. lactis to Lactobacillus strains.     

Metabolism of pyruvate and citrate in lactobacilli

Lactobacillus acidophilus, L. bulgaricus, L. casei, L. delbrueckii , L. lactis and L. plantarum contained a pyruvate oxidase for the oxidation of pyruvate to acetyl phosphate and acetate. The presence of an acetate kinase converted the acetyl phosphate to acetate. L. casei and L. plantarum produced lactate and acetoin, in addition to acetate, under the conditions used while L. casei also produced diacetyl. L. casei and L. plantarum were the only species to utilize citrate. L. helveticus and L. helveticus subsp. jugurti did not utilize pyruvate under the conditions used.     

Kinetic studies of the action of lactacin F, a bacteriocin produced by Lactobacillus johnsonii that forms poration complexes in the cytoplasmic membrane.

The bacteriocin lactacin F is bactericidal against Lactobacillus delbrueckii, Lactobacillus helveticus, and Enterococcus faecalis. Activity against L. delbrueckii was recently shown to be dependent on two peptides, LafA and LafX, which are encoded within the lactacin F operon (T. R. Klaenhammer, FEMS Microbiol. Rev. 12:39-87, 1993). It has been proposed that two peptides form an active lactacin F complex. In this study, the action of lactacin F against E. faecalis ATCC 19443 and the effects of various environmental parameters were investigated in detail. Addition of lactacin F induced the loss of K+ from cells of L. delbrueckii, Lactobacillus johnsonii 88-4, and E. faecalis, while the lactacin F producer L. johnsonii VPI 11088 was not affected by the bacteriocin. Lactacin F caused an immediate loss of cellular K+, depolarization of the cytoplasmic membrane, and hydrolysis of internal ATP in E. faecalis. Lactacin F induced loss of K+ in 3,3',4',5-tetrachlorosalicylanilide-treated cells, indicating that pores are formed in the absence of a proton motive force. ATP hydrolysis was not due to dissipation of the proton motive force but was most likely caused by efflux of inorganic phosphate, resulting in a shift of the ATP hydrolysis equilibrium. Action of lactacin F was optimal at acidic pH values and was reduced in the presence of di- and trivalent cations. The lanthanide gadolinium (Gd3+) prevented action of lactacin F completely at a concentration of 0.2 mM. Lactacin F-induced loss of cell K+ was severely reduced at low temperatures, presumably as a result of increased ordering of the lipid hydrocarbon chains in the cytoplasmic membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the amylovorin locus of Lactobacillus amylovorus DCE 471, producer of a bacteriocin active against Pseudomonas aeruginosa, in combination with colistin and pyocins

Lactobacillus amylovorus DCE 471 produces amylovorin L, a bacteriocin with an antibacterial activity against some strains of the Lactobacillus lineage. Based on the sequence of one active peptide, a gene encoding active amylovorin L was cloned and sequenced. Genome walking allowed us to sequence a larger fragment of 7577 bp of genomic DNA, with 12 predicted ORFs. The previously characterized amylovorin L peptide-encoding gene is preceded by another gene encoding a small polypeptide with a typical bacteriocin-processing double-glycine site, suggesting that amylovorin L is a two-component class IIb bacteriocin (amylovorin Lalpha/beta). Lalpha and Lbeta show the highest similarity to gassericin T from Lactobacillus gasseri SBT2055 and BlpN from Streptococcus pneumoniae R6, respectively, and to LafA and LafX, which form the lactacin F bacteriocin of Lactobacillus johnsonii NCC 533. As for other lactic acid bacteria bacteriocins, amylovorin L showed no activity against the Gram-negative opportunistic pathogen Pseudomonas aeruginosa on its own, but showed synergistic inhibitory activity when used in combination with the peptide antibiotic colistin, and, remarkably, with the P. aeruginosa soluble bacteriocins, pyocins S1 and S2.     

Exopolysaccharides produced by lactic acid bacteria of kefir grains

A Lactobacillus delbrueckii subsp. bulgaricus HP1 strain with high exopolysaccharide activity was selected from among 40 strains of lactic acid bacteria, isolated from kefir grains. By associating the Lactobacillus delbrueckii subsp. bulgaricus HP1 strain with Streptococcus thermophilus T15, Lactococcus lactis subsp. lactis C15, Lactobacillus helveticus MP12, and Sacharomyces cerevisiae A13, a kefir starter was formed. The associated cultivation of the lactobacteria and yeast had a positive effect on the exopolysaccharide activity of Lactobacillus delbrueckii subsp. bulgaricus HP1. The maximum exopolysaccharide concentration of the starter culture exceeded the one by the Lactobacillus delbrueckii subsp. bulgaricus HP1 monoculture by approximately 1.7 times, and the time needed to reach the maximum concentration (824.3 mg exopolysacharides/l) was shortened by 6 h. The monomer composition of the exopolysaccharides from the kefir starter culture was represented by glucose and galactose in a 1.0:0.94 ratio, which proves that the polymer synthesized is kefiran.