Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins.

A new bacteriocin has been isolated from an Enterococcus faecium strain. The bacteriocin, termed enterocin A, was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and mass spectrometry analysis. By combining the data obtained from amino acid and DNA sequencing, the primary structure of enterocin A was determined. It consists of 47 amino acid residues, and the molecular weight was calculated to be 4,829, assuming that the four cysteine residues form intramolecular disulfide bridges. This molecular weight was confirmed by mass spectrometry analysis. The amino acid sequence of enterocin A shared significant homology with a group of bacteriocins (now termed pediocin-like bacteriocins) isolated from a variety of lactic acid-producing bacteria, which include members of the genera Lactobacillus, Pediococcus, Leuconostoc, and Carnobacterium. Sequencing of the structural gene of enterocin A, which is located on the bacterial chromosome, revealed an N-terminal leader sequence of 18 amino acid residues, which was removed during the maturation process. The enterocin A leader belongs to the double-glycine leaders which are found among most other small nonlantibiotic bacteriocins, some lantibiotics, and colicin V. Downstream of the enterocin A gene was located a second open reading frame, encoding a putative protein of 103 amino acid residues. This gene may encode the immunity factor of enterocin A, and it shares 40% identity with a similar open reading frame in the operon of leucocin AUL 187, another pediocin-like bacteriocin.     

Purification, amino acid sequence and characterization of Bacthuricin F4, a new bacteriocin produced by Bacillus thuringiensis

AIMS: Purification and characterization of a new bacteriocin, Bacthuricin F4 of Bacillus thuringiensis. METHODS AND RESULTS: A newly isolated B. thuringiensis subsp. kurstaki strain BUPM4, was shown to produce a novel bacteriocin named Bacthuricin F4. The highest bacteriocin activity was found in the growth medium and evidenced in the late exponential growth phase. Bacthuricin F4 could be purified by a two-step procedure: ammonium sulphate precipitation of protein from culture supernatant followed by a reverse phase chromatography. Upon purification, the specific activity was increased 100-fold. This bacteriocin was heat-stable up to 70 degrees C and resisted up to pH 3.0. Bacthuricin F4 was sensitive to proteases demonstrating its proteinaceous nature. Its molecular mass, determined by mass spectrometry was 3160.05 Da. Direct N-terminal sequencing of Bacthuricin F4 revealed the following sequence: DWTXWSXL. The latter was unique in the databases. Bacthuricin F4 was active against Bacillus species while it had little or no effect on Gram-negative bacteria. CONCLUSIONS: A strain BUPM4 of B. thuringiensis subsp. kurstaki, was shown to produce a new bacteriocin named Bacthuricin F4 of both new molecular mass (3160.05 Da) and new amino acid terminal sequence. This is, to our knowledge, the first bacteriocin exhibiting such characteristics reported to be produced by B. thuringiensis. SIGNIFICANCE AND IMPACT OF THE STUDY: The bacteriocin produced by the B. thuringiensis strain BUPM4 respond to both criteria of thermostability and stability to low pHs. Thus, it could be used for the control of the related species of Bacillus harmful for agricultural products.     

Biochemical and genetic characterization of mundticin KS,an antilisterial peptide produced by Enterococcus mundtii NFRI 7393

Mundticin KS, a bacteriocin produced by Enterococcus mundtii NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen Listeria monocytogenes. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (mun locus) for mundticin KS production was cloned, and DNA sequencing revealed that the mun locus consists of three genes, designated munA, munB, and munC. The munA gene encodes a 58-amino-acid mundticin KS precursor, munB encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and munC encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by E. mundtii ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in Escherichia coli as recombinant peptides and found to be different in activity against certain Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus curvatus. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the mun locus in heterogeneous hosts such as E. faecium, L. curvatus, and Lactococcus lactis. Based on our results, the mun locus is located on a 50-kb plasmid, pML1, of E. mundtii NFRI 7393.     

Purification, partial characterization and plasmid-linkage of pediocin SJ-1, a bacteriocin produced by Pediococcus acidilactici

Pediococcus acidilactici SJ-1, isolated from a naturally-fermented meat product, produced an antibacterial agent active against selected strains of Lactobacillus spp., Clostridium perfringens and Listeria monocytogenes. The agent was bactericidal against sensitive indicators, and sensitive to proteolytic enzymes; it was identified as a bacteriocin, and was designated as pediocin SJ-1. It was stable over a wide pH range (3–9), and apparently most stable in the lower part of that range. At pH 3.6, pediocin SJ-1 was stable at heat-processing temperatures within the range 65–121°C; its activity decreased significantly, however, when it was heated at pH 7.0. The activity of pediocin SJ-1 on sensitive indicator cells was lost in the presence of α-amylase, suggesting that it contains a glyco moiety, necessary for its antibacterial action.
Native pediocin SJ-1 exists in the form of monomers and aggregates (with molecular weights in the range 80–150 kDa). Pediocin SJ-1 was purified 262-fold by direct application of cell-free supernatant fluids to a cation-exchange chromatography column, and was resolved by SDS-PAGE as a single peptide band with a MW of ca 4 kDa. The original pediocin SJ-1-producing strain (bac⁺) harbours three plasmids of 4.6, 23.5, and 45.7 MDa. Production of pediocin SJ-1, but not immunity to SJ-1, is associated with the 4.6 MDa plasmid.     

Salivaricin P, one of a family of two-component antilisterial bacteriocins produced by intestinal isolates of Lactobacillus salivarius

Lactobacillus salivarius DPC6005, a porcine intestinal isolate, produces a two-component bacteriocin, salivaricin P, with homology to ABP-118 produced by a human probiotic L. salivarius strain. Indeed, molecular characterization revealed that while the peptides Sln1 and ABP-118alpha are identical, their companion peptides (Sln2 and ABP-118beta, respectively) differ by two amino acids. This observation suggests that two-component bacteriocins may be a common feature of intestinal L. salivarius strains.     

Biochemical and genetic characterization of the two-peptide bacteriocin enterocin 1071 produced by Enterococcus faecalis FAIR-E 309

The structural genes for the two-peptide bacteriocin enterocin 1071 (Ent1071) in Enterococcus faecalis FAIR-E 309 were cloned. DNA sequence analysis showed that the enterocin 1071A (Ent1071A) peptide of strain FAIR-E 309 differed by two amino acids from the Ent1071A reported for E. faecalis BFE 1071 (E. Balla, L. M. T. Dicks, M. Du Toit, M. J. van der Merwe, and W. H. Holzapfel, Appl. Environ. Microbiol. 66:1298-1304, 2000), while the Ent1071B gene encoded identical peptides in these strains. However, resequencing of ent1071A from E. faecalis BFE 1071 showed that the Ent1071A peptide sequence reported previously was incorrect in two amino acids. Also, ent1071B in E. faecalis FAIR-E 309 encoded a prepeptide that was three amino acids shorter than that previously reported for E. faecalis BFE 1071 Ent1071B. A presumptive immunity gene (eni1071) was located downstream of the bacteriocin structural genes. This gene was cloned into the heterologous host E. faecalis ATCC 19433 and was shown to confer immunity. A truncated ABC transporter gene was located upstream of the Ent1071 structural genes.     

Biochemical and genetic characterization of propionicin T1, a new bacteriocin from Propionibacterium thoenii

A collection of propionibacteria was screened for bacteriocin production. A new bacteriocin named propionicin T1 was isolated from two strains of Propionibacterium thoenii. This bacteriocin shows no sequence similarity to other bacteriocins. Propionicin T1 was active against all strains of Propionibacterium acidipropionici, Propionibacterium thoenii, and Propionibacterium jensenii tested and also against Lactobacillus sake NCDO 2714 but showed no activity against Propionibacterium freudenreichii. The bacteriocin was purified, and the N-terminal part of the peptide was determined with amino acid sequencing. The corresponding gene pctA was sequenced, and this revealed that propionicin T1 is produced as a prebacteriocin of 96 amino acids with a typical sec leader, which is processed to give a mature bacteriocin of 65 amino acids. An open reading frame encoding a protein of 424 amino acids was found 68 nucleotides downstream the stop codon of pctA. The N-terminal part of this putative protein shows strong similarity with the ATP-binding cassette of prokaryotic and eukaryotic ABC transporters, and this protein may be involved in self-protection against propionicin T1. Propionicin T1 is the first bacteriocin from propionibacteria that has been isolated and further characterized at the molecular level.     

Identification and partial characterization of tochicin, a bacteriocin produced by Bacillus thuringiensis subsp tochigiensis

Bacillus thuringiensis subsp tochigiensis HD868 was identified as a bacteriocin producer which exhibited a bactericidal effect against closely related species. This bacteriocin designated as tochicin, was partially purified by 75% ammonium sulfate precipitation followed by subsequent dialysis. This partially purified tochicin showed a narrow antibacterial spectrum of activity against most of 20 typical B. thuringiensis strains and a strain of B. cereus, but not against other bacteria and yeasts tested. The antibacterial activity of tochicin on sensitive indicator cells disappeared completely by proteinase K treatment (1 mg ml⁻¹), which indicates its proteinaceous nature. Tochicin was very stable throughout the range of pH 3.0–9.0 and was relatively heat-stable at 90°C, but bacteriocin activity was not detected after boiling for 30 min. The relationship between cell growth and bacteriocin production was studied in a semi-defined medium. Tochicin activity was detected at the mid-log growth phase, reached the maximum at the early stationary phase, but decreased after the stationary phase. Direct detection of tochicin activity on sodium dodecyl sulfate-polyacrylamide gel suggested it has an apparent molecular mass of about 10.5 kDa. Tochicin exhibited a bactericidal activity against B. thuringiensis subsp thompsoni HD522 in phosphate buffer (pH 7.0). Received 02 December 1996/ Accepted in revised form 25 August 1997     

Detection and characterization of cerein 7, a new bacteriocin produced by Bacillus cereus with a broad spectrum of activity

A bacteriocin-producing strain of Bacillus cereus was identified and isolated from a soil sample. The bacteriocin could be purified by a two-step procedure: ammonium sulfate precipitation of culture supernatants followed by a butanol extraction step, the antibiotic was recovered from the organic phase. The peptidic nature of the bacteriocin was proven by its sensitivity to proteolytic enzymes; its molecular mass, determined by mass spectrometry, was 3940 Da; and its amino-terminal sequence (GWGDVL) is unique in the databases. The compound was active against most Gram-positive but not Gram-negative bacteria. This is to our knowledge the first bacteriocin with these characteristics reported to be produced by B. cereus.     

Structural analysis and characterization of lacticin Q, a novel bacteriocin belonging to a new family of unmodified bacteriocins of gram-positive bacteria

Lactococcus lactis QU 5 isolated from corn produces a novel bacteriocin, termed lacticin Q. By acetone precipitation, cation-exchange chromatography, and reverse-phase high-performance liquid chromatography, lacticin Q was purified from the culture supernatant of this organism, and its molecular mass was determined to be 5,926.50 Da by mass spectrometry. Subsequent analyses of amino acid and DNA sequences revealed that lacticin Q comprised 53 amino acid residues and that its N-terminal methionine residue was formylated. In contrast to most bacteriocins produced by gram-positive bacteria, lacticin Q had no N-terminal extensions such as leader or signal sequences. It showed 66% and 48% identity to AucA, a hypothetical protein from Corynebacterium jeikeium plasmid pA501, and aureocin A53, a bacteriocin from Staphylococcus aureus A53, respectively. The characteristics of lacticin Q were determined and compared to those of nisin A. Similar to nisin A, lacticin Q exhibited antibacterial activity against various gram-positive bacteria. Lacticin Q was very stable against heat treatment and changes in pH; in particular, it was stable at alkaline pH values, while nisin A was inactivated. Moreover, lacticin Q induced ATP efflux from a Listeria sp. strain in a shorter time and at a lower concentration than nisin A, indicating that the former affected indicator cells in a different manner from that of the latter. The results described here clarified the fact that lacticin Q belongs to a new family of class II bacteriocins and that it can be employed as an alternative to or in combination with nisin A.     

Biochemical and Genetic Characterization of Coagulin, a New Antilisterial Bacteriocin in the Pediocin Family of Bacteriocins, Produced by Bacillus coagulans I4

A plasmid-linked antimicrobial peptide, named coagulin, produced by Bacillus coagulans I₄ has recently been reported (B. Hyronimus, C. Le Marrec and M. C. Urdaci, J. Appl. Microbiol. 85:42–50, 1998). In the present study, the complete, unambiguous primary amino acid sequence of the peptide was obtained by a combination of both N-terminal sequencing of purified peptide and the complete sequence deduced from the structural gene harbored by plasmid I₄. Data revealed that this peptide of 44 residues has an amino acid sequence similar to that described for pediocins AcH and PA-1, produced by different Pediococcus acidilactici strains and 100% identical. Coagulin and pediocin differed only by a single amino acid at their C terminus. Analysis of the genetic determinants revealed the presence, on the pI₄ DNA, of the entire 3.5-kb operon of four genes described for pediocin AcH and PA-1 production. No extended homology was observed between pSMB74 from P. acidilactici and pI₄ when analyzing the regions upstream and downstream of the operon. An oppositely oriented gene immediately dowstream of the bacteriocin operon specifies a 474-amino-acid protein which shows homology to Mob-Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from gram-positive bacteria. This is the first report of a pediocin-like peptide appearing naturally in a non-lactic acid bacterium genus.     

Isolation, partial purification and characterization of a bacteriocin produced by a newly isolated Bacillus subtilis strain

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.     

Isolation and characterization of a bacteriocin produced by Bacillus stearothermophilus strain NU-10.

Broth-grown cultures of Bacillus stearothermophilus strain NU-10 produce a bacteriocin which exerts lethal activity on other strains of the bacterium. Optimal production occurs during late maximum stationary phase of growth, at neutral pH, and 55-65 degrees C. The bacteriocin can be substantially purified by a combination of precipitations, centrifugations, and gel filtrations. The thermocin is composed of protein and carbohydrate. It is partially destroyed by proteolytic enzymes but is resistant to DNase, RNase, and various chemical treatments. The bacteriocin has a small molecular weight and exhibits considerable thermostability.     

Purification and characterization of enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA 4.

A simple two-step procedure was developed to obtain pure enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA 4. Chemical and genetic characterization revealed that the primary structure of enterocin 4 is identical to that of peptide antibiotic AS-48 from Enterococcus faecalis S-48. In contrast to the reported inhibitory spectrum of AS-48, enterocin 4 displayed no activity against gram-negative bacteria.     

Purification and partial characterization of bacillocin 490, a novel bacteriocin produced by a thermophilic strain of Bacillus licheniformis

Background  

Applications of bacteriocins as food preservatives have been so far limited, principally because of their low antimicrobial activity in foods. Nisin is the only bacteriocin of significant use, but applications are restricted principally because of its very low activity at neutral or alkaline pH. Thus the isolation of new bacteriocins active in foods is desirable.     

Isolation and characterization of a bacteriocin produced by Cicer-Rhizobium

A constitutively expressed bacteriocin from Cicer-Rhizobium was purified to homogeneity. The purified preparation yielded a homogenous protein with a molecular weight of about 29 kDa. This protein was heat stable, unaffected by nucleases and was found to have an iso-electric point (pI) of 4.6. The N-terminal sequence of the protein was found to be M-N-N-N-Y-R-E-L-L-P-I-I-G-P-P-W-A-E-I-E, sharing 78% homology with linocin M18. Bacteriocin bioactivity was correlated with the presence of a 29 kDa protein in the growth diffusates of the culture. A mutant strain unable to produce this bacteriocin was found to have a statistically significant reduction in nodule occupancy and competitiveness against the wild type and indigeneous populations under unsterile conditions. Bacteriocin production by the mutant carrying the complement clone pJNP365 was found to be stable even in an unsterile environment.     

Isolation and characterization of pediocin L50, a new bacteriocin from Pediococcus acidilactici with a broad inhibitory spectrum.

Lactic acid bacteria were isolated from Spanish dry-fermented sausages and screened for bacteriocin production. About 10% of the isolates produced antimicrobial substances when grown on solid media, but only 2% produced detectable activity in liquid media. Strain L50, identified as Pediococcus acidilactici, showed the strongest inhibitory activity and was active against members of all of the gram-positive genera tested. The strain produced a heat-stable bacteriocin when grown at 8 to 32 degrees C but not at 45 degrees C. The bacteriocin was purified to homogeneity. Its mass was determined to be 5,250.11 +/- 0.30 by electrospray mass spectrometry. The N terminus of the bacteriocin was blocked for sequencing by Edman degradation, but a partial sequence of 42 amino acids was obtained after cleavage of the peptide by cyanogen bromide. The sequence showed no similarity to those of other bacteriocins. Pediocin L50 appears to contain modified amino acids but not lanthionine or methyl-lanthionine.     

Coagulin, a bacteriocin-like inhibitory substance produced by Bacillus coagulans I4

A protease-sensitive antibacterial substance produced by Bacillus coagulans I₄ strain, isolated from cattle faeces, was classified as a bacteriocin-like inhibitory substance and named coagulin. The inhibitory spectrum included B. coagulans and unrelated bacteria such as Enterococcus , Leuconostoc , Oenococcus , Listeria and Pediococcus . Coagulin was stable at 60 °C for 90 min, at a pH ranging from 4 to 8 and appeared to be unaffected by α-amylase, lipase or organic solvents (10% v/v). Coagulin exhibited a bactericidal and a bacteriolytic mode of action against indicator cells. The apparent molecular mass was estimated to be about 3–4 kDa by SDS-PAGE. The B. coagulans I₄ strain harbours a plasmid, pI₄, approximately 14 kb in size. Novobiocin curing experiments yielded two derivatives that no longer produced the bacteriocin-like inhibitory substance. Plasmid content of these two derivatives showed that one had lost pI₄,whereas the second harboured a deleted form of this plasmid, thus suggesting a plasmid location for the genes for coagulin production.     

Characterization of lactosporin, a novel antimicrobial protein produced by Bacillus coagulans ATCC 7050

Aims:  To characterize the antimicrobial protein produced by Bacillus coagulans used in the probiotic dietary supplement (Lactospore^® Probiotic, Sabinsa Corp., Piscataway, NJ, USA).
Methods and Results:  Bacillus coagulans ATCC 7050 was grown at 37°C for 18 h. The cell free supernatant was concentrated 10-fold (lactosporin preparation, LP). The antimicrobial activity of LP was confirmed against Micrococcus luteus ATCC 10420 in a well diffusion assay. The proteinaceous nature of LP was determined following exposure to different enzymes. The activity of LP was pH-dependent but stable to heat. The isoelectric point of LP was determined to be 3·5–4·0. PCR analyses showed no similarity between lactosporin and known antimicrobial proteins produced by the Bacillus spp.
Conclusions:  Lactosporin is a novel antimicrobial protein. Initial characterization indicates that it may fall outside of the conventional classification of class I and II bacteriocins. Loss of activity after exposure to a number of proteolytic enzymes and lipase suggest that lactosporin may posses a lipid moiety which contributes to its inhibitory activity.
Significance and Impact of the Study:  The unique characteristics of lactosporin, including its antimicrobial activity against pathogenic micro-organisms, indicate that it may have potential for application in foods and personal care products.