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The aim of this work was to study the antifungal properties of durancins isolated from Enterococcus durans A5-11 and of their chemically synthesized fragments. Enterococcus durans A5-11 is a lactic acid bacteria strain isolated from traditional Mongolian airag cheese. This strain inhibits the growth of several fungi including Fusarium culmorum, Penicillium roqueforti and Debaryomyces hansenii. It produces two bacteriocins: durancin A5-11a and durancin A5-11b, which have similar antimicrobial properties. The whole durancins A5-11a and A5-11b, as well as their N- and C-terminal fragments were synthesized, and their antifungal properties were studied. C-terminal fragments of both durancins showed stronger antifungal activities than other tested peptides. Treatment of D. hansenii LMSA2.11.003 strain with 2 mmol l(-1) of the synthetic peptides led to the loss of the membrane integrity and to several changes in the ultra-structure of the yeast cells. Chemically synthesized durancins and their synthetic fragments showed different antimicrobial properties from each other. N-terminal peptides show activities against both bacterial and fungal strains tested. C-terminal peptides have specific activities against tested fungal strain and do not show antibacterial activity. However, the C-terminal fragment enhances the activity of the N-terminal fragment in the whole bacteriocins against bacteria., (© 2012 The Society for Applied Microbiology.)

Aims: To elucidate the molecular mechanism of action of the antimicrobial peptide subtilosin against the foodborne pathogen Listeria monocytogenes Scott A., Methods and Results: Subtilosin was purified from a culture of Bacillus amyloliquefaciens. The minimal inhibitory concentration of subtilosin against L. monocytogenes Scott A was determined by broth microdilution method. The effect of subtilosin on the transmembrane electrical potential (ΔΨ) and pH gradient (ΔpH), and its ability to induce efflux of intracellular ATP, was investigated. Subtilosin fully inhibited L. monocytogenes growth at a concentration of 19 μg ml(-1) . Subtilosin caused a partial depletion of the ΔΨ and had a similar minor effect on the ΔpH. There was no significant efflux of intracellular ATP., Conclusion: Subtilosin likely acts upon L. monocytogenes Scott A by perturbing the lipid bilayer of the cellular membrane and causing intracellular damage, leading to eventual cell death. Subtilosin's mode of action against L. monocytogenes Scott A differs from the one previously described for another human pathogen, Gardnerella vaginalis., Significance and Impact of the Study: This is the first report on the specific mode of action of subtilosin against L. monocytogenes and the first report of a bacteriocin with a species-specific mode of action., (No claim to US Government works. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.)

Aims: We have developed a PCR-based assay using custom designed panel of primers which allows rapid detection of class IIa bacteriocin-coding genes. To demonstrate the applicability of the developed assay, the method was applied on 40 metagenomic DNA preparations isolated from native microbiota of Polish artisanal cheeses produced in the Tatra Mountains., Methods and Results: The developed assay was designed on the basis of a large scale alignment of class IIa bacteriocin-coding genes. A panel of seven primer pairs with confirmed ability to detect class IIa bacteriocin-coding sequences was obtained. The following study has revealed a superb bacteriocinogenic potential of all forty analysed cheese samples., Conclusions: The majority of obtained sequences were lactic acid bacteria (LAB) related, although some sequences showed significant similarity to bacteriocin-coding sequences present in non-LAB bacteriocin producers. The results suggest that several potentially new bacteriocin-coding sequences were found., Significance and Impact of the Study: The developed assay can be extremely helpful in establishing whether isolates from the environment of interest have a potential of synthesizing antilisterial class IIa bacteriocins. Application of the approach may represent a useful tool contributing to ecological studies looking for valuable probiotic, bacteriocinogenic microbiota developing in foods., (© 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.)

Aim: The study aimed for the complete purification and recharacterization of the highly hydrophobic circular bacteriocins, gassericin A and reutericin 6., Methods and Results: Gassericin A and reutericin 6 were purified to homogeneity using previously described method and reverse-phase HPLC with an octyl column and eluents of aqueous acetonitrile and 2-propanol. Mass analysis, N-terminal sequencing and bacteriocin assay of the HPLC-purified bacteriocins showed the two bacteriocins had identical seamless circular structures with the same m/z value (5651) of [M + H](+) and both had the same specific activity. D/L-amino acid composition analysis using two distinct methods with the chiral fluorescent derivatization reagents (+)-1-(9-fluorenyl)ethyl chloroformate and O-phthalaldehyde/N-acetyl-L-cystein revealed neither gassericin A nor reutericin 6 contained D-alanine residues contrary to our previous results., Conclusion: Purified gassericin A and reutericin 6 are chemically identical circular molecules containing no D-alanine residues., Significance and Impact of the Study: The HPLC conditions developed in this study will facilitate advanced purification and correct characterization of other highly hydrophobic bacteriocins.

Aim: To identify the gene that encodes nigrescin, a bacteriocin produced by Prevotella nigrescens ATCC 25261., Methods and Results: Each open reading frame (ORF) of the nig gene cluster (nigA, nigB, nigC and nigD) was transferred into an expression vector. The recombinant proteins encoded by nigA, nigB, nigC and nigD were purified and assayed for bacteriocin activity against Porphyromonas gingivalis. The ORFs of the nig gene cluster in Pr. nigrescens ATCC 25261 were re-analysed. It revealed that the position of nig ORFs was similar to previously designated locations, except that the start codon of nigC was reassigned. The new nigC gene started at the nucleotide base position 2454 and stopped at position 3608 (the position designated is relative to the first nucleotide base of the nig locus) and putatively encoded a protein with a predicted molecular mass of 41.9 kDa. The N-terminal 6xHistidine-tag recombinant proteins of NigA, NigB, NigC and NigD were overexpressed in Escherichia coli BL21 star (DE3) and were purified using Ni-NTA resins. Only recombinant NigC showed inhibitory activity against P. gingivalis A244 with minimal inhibition concentration (MIC) of 40 microg ml(-1)., Conclusion: These results indicate that nigC is the gene that encodes nigrescin., Significance and Impact of the Study: This is the first report that indicates that the gene nigC codes for nigrescin, a bacteriocin produced by Pr. nigrescens ATCC 25261.

Aims: The identification of a new compound active against Agrobacterium tumefaciens., Methods and Results: The culture conditions of a newly isolated Bacillus subtilis strain, designed 14B, were optimized, as a first step, to produce its bacteriocin (termed Bac 14B) for the biocontrol of Agrobacterium spp., the causal agents of the crown gall disease. Bac 14B was then partially purified and biochemically characterized. Bacillus subtilis 14B was observed to produce an antibacterial compound having a protinaceous nature. As estimated by sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE), the semi-purified bacteriocin substance was found to be a monomeric protein with a molecular weight of 21 kDa. While the latter's antimicrobial activity was completely stable during exposure to a temperature range of up to 100 degrees C for 2 h, its initial activity was totally lost at 121 degrees C for 20 min. The maximum bacteriocin production (4096 AU ml(-1)) was recorded after 96 h-incubation in an optimized Luria Bertani medium supplemented with 10 g l(-1) glucose, 15 g l(-1) K(2)HPO(4) and 5 g l(-1) MgSO(4) 7H(2)O at 30 degrees C in a shaking flask culture. Interestingly, the B. subtilis 14B culture supernatant that contained the bacteriocin under study was proved efficient in reducing both the percentage of galled plants and the number of galls in tomato., Conclusion: The findings revealed that B. subtilis 14B and its bacteriocin are efficient in reducing the percentage of infections in plants caused by Ag. tumefaciens., Significance and Impact of the Study: The results could be useful for the nurserymen who are particularly interested in the biocontrol of the crown gall disease.

Aims: To isolate, characterize and identify bacteriocins from lactic acid bacteria in soil., Methods and Results: Thirty-four acid-producing bacteria were isolated from 87 soil samples. Antibacterial activities were detected, and one strain, L28-1 produced a bacteriocin that was active against some Gram-positive bacteria. L28-1 was identified as Enterococcus durans by 16S rDNA sequence analysis and API50CHL. This bacteriocin did not lose its activity after autoclaving (121 degrees C for 15 min), but was inactivated by protease K. The bacteriocin was purified by hydrophobic column chromatography, and Sep-Pak C(18). Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the partially purified bacteriocin contained numerous protein bands. Two bands that displayed antibacterial activities were c. 3.4 and 2.5 kDa in size. In this work, the 3.4-kDa bacteriocin was analysed with N-terminal amino acid and DNA sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. The results indicated that the 3.4-kDa bacteriocin of Ent. durans L28-1 is a new natural enterocin variant., Conclusions: Enterococcus durans L28-1 produced a new bacteriocin., Significance and Impact of the Study: This study reports a novel bacteriocin that is produced by Ent. durans that has potential for use as a food preservative.

Aims: To purify and to characterize the antimicrobial compound cerein 8A., Methods and Results: Cerein 8A was isolated by ammonium sulfate precipitation, 1-butanol extraction and ion-exchange chromatography. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. The purified substance corresponded to a 26 kDa peptide band. The native protein eluted at the void volume of Sephadex G-100, but within the included volume when a 1.5 mol l(-1) NaCl buffer was used, indicating that cerein 8A aggregates extracellularly. The antimicrobial activity was lost by treatment with proteases and heat. The ultraviolet spectrum was typical of a polypeptide and the infrared spectrum indicates that the peptide contains acyl group(s) in its structure. Intact Bacillus cereus spores were sensitive to cerein 8A at 1600 AU ml(-1)., Conclusions: Cerein 8A show distinct properties from other antimicrobial peptides of B. cereus, and has a significant inhibitory effect on spores., Significance and Impact of the Study: The characterization of a substance active against important pathogens addresses an important aspect of food safety.

Aims: To characterize the antimicrobial activity produced by Prevotella nigrescens ATCC 25261, and to evaluate its safety on cultured gingival fibroblasts., Methods and Results: An antimicrobial activity was obtained from purifying the culture supernatant of Pr. nigrescens ATCC 25261. Purification of the active compound was achieved with ammonium sulphate precipitation followed by anion-exchange and gel filtration chromatography. As revealed by SDS-PAGE, the active fraction was relatively homogeneous, showing a protein with an approximate molecular weight of 41 kDa. The antimicrobial compound, named nigrescin, exhibited a bactericidal mode of action against Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, and Actinomyces spp. Nigrescin was stable in a pH range between 6.5 and 9.5, at 100 degrees C for 10 min, and resistant to lyophilization. But its activity was lost after proteinase K treatment. Despite at very high concentrations beyond the minimum inhibitory concentration (MIC), nigrescin was not toxic to the gingival fibroblasts., Conclusion: Nigrescin is a novel bacteriocin produced by Pr. nigrescens ATCC 25261. It exhibits antimicrobial activity against species that are implicated in periodontal diseases. The absence of toxicity on the gingival fibroblasts suggests the possibility in using of nigrescin for an application in periodontal treatment., Significance and Impact of the Study: Novel evidence on nigrescin would make Pr. nigrescens ATCC 25261 attractive in biotechnological applications as an antimicrobial agent in clinical dentistry.

Aims: Physiological and molecular analysis such as PCR species-specific and randomly amplified polymorphic PCR (RAPD-PCR) have been used for typing of Lactobacillus plantarum strains from typical wine must., Methods and Results: Phenotypic tests such as API 50CH and evaluation of D-L-lactate production from glucose were used to perform a preliminary characterization of lactobacilli. Furthermore, 18 strains of lactobacilli were analyzed by PCR species-specific oligonucleotides based on short sequences of the recA gene., Conclusions: Four strains were identified as belonging to the L. plantarum species and were further analysed by RAPD-PCR. The RAPD-PCR profiles were similar in all strains that had positive results for species-specific PCR, suggesting that the four L. plantarum strains were closely related., Significance and Impact of the Study: Using PCR species-specific as a preliminary screening test and then RAPD-PCR can be as considered the most reliable method of performing a rapid and correct typing of L. plantarum from wine must.

Aims: To investigate the antimicrobial activity of a strain of Lactobacillus helveticus., Methods and Results: The culture supernatant fluid Lact. helveticus G51 showed antimicrobial activity against thermophilic strains of Lactobacillus. Purification of the active compound was achieved after gel filtration and ion exchange chromatography. As revealed by SDS-PAGE, active fractions were relatively homogeneous, showing a protein with a molecular mass of 12.5 kDa. The antimicrobial compound was heat labile, inactivated by proteolytic enzymes and had a bactericidal mode of action., Conclusion: The antimicrobial activity expressed by Lact. helveticus G51 was correlated with the production of a bacteriocin with properties that were different to other helveticins., Significance and Impact of the Study: The study has provided further data on Lact. helveticus bacteriocins. The strong activity of the bacteriocin towards various thermophilic lactobacilli warrants further investigation for its potential to obtain attenuated cultures for the enhancement of the cheese-ripening process.

Aims: Detection and identification of new antagonistic activities towards Bacillus cereus and relatives., Methods and Results: Twenty Bacillus thuringiensis strains were screened for their capacity to express bacteriocin-like agents. Strain BMG1.7, isolated from soil, showed an antagonistic activity called thuricin 7. Thuricin 7 was active against several species of the genus Bacillus, including three of the four known B. thuringiensis/B. cereus bacteriocin producers, as well as against Streptococcus pyogenes and Listeria monocytogenes strains. Antimicrobial activity was lost after treatment with proteinase K. The active protein had an apparent molecular weight of 11.6 kDa, and was secreted at the end of the exponential growth phase. Thuricin 7 retained 55% of the activity after incubation at 98 degrees C for 30 min. The mode of action of thuricin 7 was shown to be bactericidal and bacteriolytic., Conclusion: Thuricin 7 is a novel bacteriocin produced by a newly isolated Bacillus thuringiensis strain BMG1.7., Significance and Impact of the Study: The characteristics of thuricin 7 indicate that it is a new bacteriocin which may have interesting biotechnological applications due to its relatively large activity spectrum.

Aims: To characterize polyfermenticin SCD, a newly identified bacteriocin of Bacillus polyfermenticus SCD., Methods and Results: Bacillus polyfermenticus SCD was identified as a bacteriocin producer with a bactericidal activity against Bacillus subtilis IFO 12113. Polyfermenticin SCD, named tentatively as the bacteriocin produced by B. polyfermenticus SCD, showed a narrow spectrum of activity against Gram-positive and Gram-negative bacteria, a yeast and moulds. Production of polyfermenticin SCD in a 5 l jar fermenter followed typical kinetics of primary metabolite synthesis. The antibacterial activity of polyfermenticin SCD on sensitive indicator cells disappeared completely by treatment with proteinase K, which indicates its proteinaceous nature. Polyfermenticin SCD seemed to be very stable throughout the pH range of 2.0 to 9.0, and it was relatively heat labile compared with other bacteriocins. Direct detection of polyfermenticin SCD activity on SDS-PAGE suggested that it had an apparent molecular mass of about 14.3 kDa., Conclusions: Bacillus polyfermenticus SCD produced relatively heat-labile polyfermenticin SCD with a narrow spectrum of activity., Significance and Impact of the Study: Bacillus polyfermenticus SCD is a commercial probiotic which has been used for the treatment of long-term intestinal disorders. New findings on polyfermenticin SCD will be valuable in the evaluation of commercial probiotics. Polyfermenticin SCD can be used to control Bacillus spoilage organisms as a biological control agent.

A solvent extraction method was developed to concentrate lacidin from the culture of Lactobacillus acidophilus OSU133. The new method concentrates the bacteriocin at the interface between chloroform and the aqueous culture of the producing bacterium. Compared with other extraction procedures, the new method effectively recovers higher bacteriocin yield and results in relatively clean preparations. Recovery of lacidin by the chloroform extraction procedure, compared with ammonium sulphate precipitation and cell acidification methods, was >10-fold and about 100-fold greater, respectively. The new extraction procedure saves time and is easy to perform. This method is also effective in recovering subtilin, bacillicin, pediocin and nisin from cultures of Bacillus subtilis ATCC 6633, B. subtilis OSY1115/C, Pediococcus acidilactici PO2 and Lactococcus lactis ATCC 11454, respectively.

A new procedure combining Triton X-114 phase partitioning and cation exchange chromatography was developed to purify a bacteriocin from a complex culture medium. This pediocin-like bacteriocin, secreted by Carnobacterium divergens and named divercin V41, was entirely recovered in the lower detergent-rich phase whereas all other substances (compounds from culture medium, bacterial metabolites) remained in the upper detergent-poor phase. Subsequent cation-exchange chromatography of the TX-114-rich phase allowed recovery of the pure active bacteriocin and also detergent removing. This new purification method is versatile, fast (only two steps) and can be carried out on whole broth.

A wild type micro-organism producing antibacterial substances has been isolated from a Chinese fermented soybean seasoning and identified as Bacillus subtilis. A crude antibacterial preparation (CABP) was obtained by ammonium sulphate precipitation. Isoelectric focusing assay revealed at least four antimicrobial components in the CABP. However, in SDS-PAGE analysis, only one peptide band displayed antimicrobial activity against pathogenic Bacillus cereus and Listeria monocytogenes. This inhibitory peptide had a molecular weight of approximately 3.4 kDa and a pI value of approximately 4.7. Results of this study suggest that at least one antimicrobial substance produced by this wild type strain of B. subtilis may be a new bacteriocin. Its sensitivity to gastric peptidases and activity against the food-borne pathogens make this bacteriocin potentially useful as an antimicrobial agent in foods.

Bacteria isolated from radish were identified as Lactococcus lactis subsp. cremoris R and their bacteriocin was designated lactococcin R. Lactococcin R was sensitive to some proteolytic enzymes (proteinase-K, pronase-E, proteases, pepsin, alpha-chymotrypsin) but was resistant to trypsin, papain, catalase, lysozyme and lipase, organic solvents, or heating at 90 degrees C for 15, 30 and 60 min, or 121 degrees C for 15 min. Lactococcin R remained active after storage at -20 and -70 degrees C for 3 months and after exposure to a pH of 2-9. The molecular weight of lactococcin R was about 2.5 kDa. Lactococcin R was active against many food-borne pathogenic and food spoilage bacteria such as Clostridium, Staphylococcus, Listeria, Bacillus, Micrococcus, Enterococcus, Lactobacillus, Leuconostoc, Streptococcus and Pediococcus spp., but was not active against any Gram-negative bacteria. Lactococcin R was produced during log phase and reached a maximum activity (1600 AU ml-1) at early stationary phase. The highest lactococcin R production was obtained in MRS broth with 0.5% glucose, at 6.5-7.0 initial pH values, 30 degrees C temperature and 18-24-h incubation times. Lactococcin R adsorbed maximally to its heat-killed producing cells at pH 6-7 (95%). Crude lactococcin R at 1280 AU ml-1 was bactericidal, reducing colony counts of Listeria monocytogenes by 99.98% in 3 h. Lactococcin R should be useful as a biopreservative to prevent growth of food-borne pathogenic and food spoilage bacteria in ready-to-eat, dairy, meat, poultry and other food products. Lactococcin R differs from nisin in having a lower molecular weight, 2.5 kDa vs 3.4 kDa, and in being sensitive to pepsin and alpha-chymotrypsin to which nisin is resistant.

Lactobacillus plantarum BFE 905 isolated from 'Waldorf' salad produced a bacteriocin termed plantaricin D which was active against Lact. sake and Listeria monocytogenes strains. Plantaricin D was heat stable, retaining activity after heating at 121 degrees C. The bacteriocin was inactivated by alpha-chymotrypsin, trypsin, pepsin and proteinase K, but not by papain and other non-proteolytic enzymes tested. Plantaricin D was stable at pH values ranging from 2.0 to 10.0. The bacteriocin inhibited growth of L. monocytogenes in automated turbidity assays. Although Lact. Plantarum BFE 905 harboured plasmids ranging in size from 3 to 55 kilobase pairs, loss of bacteriocin production could not be correlated with plasmid loss. A role for bacteriocin-producing Lact. plantarum of vegetable origin in assuring the safety of vegetable foods is suggested.

Enterocin CRL 35, a bacteriocin produced by Enterococcus faecium CRL 35 that inhibits food-borne pathogens, was purified by precipitation with (NH4)2SO4, gel filtration, ion exchange and reverse phase chromatography. The partial N-terminal amino acid sequence indicated a strong homology with other 'pediocin-like bacteriocins' previously described.