Novel expression system for large-scale production and purification of recombinant class IIa bacteriocins and its application to piscicolin 126

Piscicolin 126 is a class IIa bacteriocin isolated from Carnobacterium piscicola JG126 that exhibits strong activity against Listeria monocytogenes. The gene encoding mature piscicolin 126 (m-pisA) was cloned into an Escherichia coli expression system and expressed as a thioredoxin-piscicolin 126 fusion protein that was purified by affinity chromatography. Purified recombinant piscicolin 126 was obtained after CNBr cleavage of the fusion protein followed by reversed-phase chromatography. Recombinant piscicolin 126 contained a single disulfide bond and had a mass identical to that of native piscicolin 126. This novel bacteriocin expression system generated approximately 26 mg of purified bacteriocin from 1 liter of E. coli culture. The purified recombinant piscicolin 126 acted by disruption of the bacterial cell membrane.     

Divergicin 750, a novel bacteriocin produced by Carnobacterium divergens 750

Abstract Divergicin 750, a bacteriocin produced by Carnobacterium divergens 750, preferentially inhibited the growth of strains of Carnobacterium and Enterococcus . Selected strains of Listeria monocytogenes and Clostridium perfringens were also inhibited. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation and sequential S-Sepharose, hydrophobic interaction and reversed phase chromatography. The complete amino acid sequence was determined by Edman degradation. The peptide consisted of 34 amino acid residues. The calculated ifM_r from the peptide sequence, 3447.7, agreed well with that obtained by mass spectrometry. Divergicin 750 did not show any sequence similarities to other known bacteriocins. The plasmid-located structural gene encoding divergicin 750 ( dvn750 ) was cloned and sequenced. The gene encoded a primary translation product of 63 amino acids with a deduced M _rmr = 6789.4 which is cleaved between amino acid residues 29 and 30 to yield the mature bacteriocin.     

Production of piscicolin 126 by Carnobacterium maltaromaticum UAL26 is controlled by temperature and induction peptide concentration

Carnobacterium maltaromaticum UAL26 produces the antimicrobial peptides (bacteriocins) piscicolin 126, first isolated from C. maltaromaticum JG126, and carnobacteriocin BM1, first isolated from C. maltaromaticum LV17. C. maltaromaticum UAL26 is especially inhibitory to strains of Listeria monocytogenes. Bacteriocin activity is not observable in the supernatant of cultures of UAL26 grown in liquid media at 25°C, but at temperatures less than 19°C bacteriocin activity can be detected. In contrast to JG126, the piscicolin 126 operon is downregulated in UAL26 at higher temperature, and piscicolin 126 mRNA is not detected when UAL26 is grown at 25°C. Bacteriocin production in UAL26 grown at 15°C can be induced by addition of 10⁻¹⁰ M of chemically synthesized piscicolin 126 induction peptide (PisN). However, induction of bacteriocin production in UAL26 grown at 25°C requires 10⁻⁷ M of PisN. The sequence of the piscicolin 126 operon in UAL26 contains 34 single nucleotide differences compared with the piscicolin 126 operon in JG126, including single nucleotide differences in the immunity, histidine kinase, dedicated ABC-transporter and accessory genes, as well as a single nucleotide deletion in the transport accessory gene. This deletion causes a frameshift, resulting in truncation of the PisE transport accessory protein in UAL26.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Isolation and Characterization of Carnocyclin A, a Novel Circular Bacteriocin Produced by Carnobacterium maltaromaticum UAL307

Carnobacterium maltaromaticum UAL307, isolated from fresh pork, exhibits potent activity against a number of gram-positive organisms, including numerous Listeria species. Three bacteriocins were isolated from culture supernatant, and using matrix-assisted laser desorption ionization-time of flight mass spectrometry and Edman sequencing, two of these bacteriocins were identified as piscicolin 126 and carnobacteriocin BM1, both of which have previously been described. The remaining bacteriocin, with a molecular mass of 5,862 Da, could not be sequenced by traditional methods, suggesting that the peptide was either cyclic or N-terminally blocked. This bacteriocin showed remarkable stability over a wide temperature and pH range and was unaffected by a variety of proteases. After digestion with trypsin and α-chymotrypsin, the peptide was de novo sequenced by tandem mass spectrometry and a linear sequence deduced, consisting of 60 amino acids. Based on this sequence, the molecular mass was predicted to be 5,880 Da, 18 units higher than the observed molecular mass, which suggested that the peptide has a cyclic structure. Identification of the genetic sequence revealed that this peptide is circular, formed by a covalent linkage between the N and C termini following cleavage of a 4-residue peptide leader sequence. The results of structural studies suggest that the peptide is highly structured in aqueous conditions. This bacteriocin, named carnocyclin A, is the first reported example of a circular bacteriocin produced by Carnobacterium spp.     

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.     

Isolation and partial characterization of a bacteriocin produced by Lactobacillus plantarum BM‐1 isolated from a traditionally fermented Chinese meat product

Lactobacillus plantarum BM‐1 isolated from a traditionally fermented Chinese meat product was found to produce a novel bacteriocin that is active against a wide range of gram‐positive and gram‐negative bacteria. Production of the bacteriocin BM‐1 started early in the exponential phase and its maximum activity (5120 AU/mL) was recorded early during the stationary phase (16 hr). Bacteriocin BM‐1 is sensitive to proteolytic enzymes but stable in the pH range of 2.0–10.0 and heat‐resistant (15 min at 121°C). This bacteriocin was purified through pH‐mediated cell adsorption–desorption and cation‐exchange chromatography on an SP Sepharose Fast Flow column. The molecular weight of the purified bacteriocin BM‐1 was determined to be 4638.142 Da by electrospray ionization Fourier transform mass spectrometry. Furthermore, the N‐terminal amino acid sequence was obtained through automated Edman degradation and found to comprise the following 15 amino acid residues: H₂N‐Lys‐Tyr‐Tyr‐Gly‐Asn‐Gly‐Val‐Tyr‐Val‐Gly‐Lys‐His‐Ser‐Cys‐Ser. Comparison of this sequence with that of other bacteriocins revealed that bacteriocin BM‐1 contains the consensus YGNGV amino acid motif near the N‐terminus. Based on its physicochemical characteristics, molecular weight, and N‐terminal amino acid sequence, plantaricin BM‐1 is a novel class IIa bacteriocin.     

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.     

Characterization, Production, and Purification of Leucocin H, a Two-Peptide Bacteriocin from Leuconostoc MF215B

Leuconostoc MF215B was found to produce a two-peptide bacteriocin referred to as leucocin H. The two peptides were termed leucocin Hα and leucocin Hβ. When acting together, they inhibit, among others, Listeria monocytogenes, Bacillus cereus, and Clostridium perfringens. Production of leucocin H in growth medium takes place at temperatures down to 6°C and at pH below 7. The highest activity of leucocin H in growth medium was demonstrated in the late exponential growth phase. The bacteriocin was purified by precipitation with ammonium sulfate, ion-exchange (SP Sepharose) and reverse phase chromatography. Upon purification, specific activity increased 10⁵-fold, and the final specific activity was 2 × 10⁷ BU/OD₂₈₀. Amino acid composition analyses of leucocin Hα and leucocin Hβ indicated that both peptides consisted of around 40 amino acid residues. Their N-termini were blocked for Edman degradation, and the methionin residues of leucocin Hβ did not respond to Cyanogen Bromide (CNBr) cleavage. Absorbance at 280 nm indicated the presence of tryptophan residues and tryptophan-fracturing opened for partial sequencing by Edman degradation. From leucocin Hα, the sequence of 20 amino acids was obtained; from leucocin Hβ the sequence of 28 amino acid residues was obtained. No sequence homology to other known bacteriocins could be demonstrated. It also appeared that the two peptides themselves shared little or no sequence homology. The presence of soy oil did not affect the activity of leucocin H in agar. Received: 10 February 1999 / Accepted: 15 March 1999     

A food-grade process for isolation and partial purification of bacteriocins of lactic acid bacteria that uses diatomite calcium silicate.

Bacteriocins, including nisin, pediocin PO2, brevicin 286, and piscicolin 126, were extracted from fermentation media by adsorption onto Micro-Cel (a food-grade diatomite calcium silicate anticaking agent) and subsequent desorption. The optimal conditions for desorption of piscicolin 126 were determined and applied to other bacteriocins, and the relative purities of the desorbed preparations were compared. Piscicolin was not successfully desorbed from Micro-Cel at pH 1.0 to 12.0, with organic solvents, or by increase of ionic strength up to 1 M NaCl. However, 25 and 75% of the bacteriocin activity was desorbed by using 1% sodium deoxycholate and 1% sodium dodecyl sulfate (SDS), respectively. Higher levels (up to 100%) of desorption were achieved by repeated elution or by an increase in surfactant concentration. Desorption of piscicolin with 1/10 volume of SDS solution resulted in a preparation with 10 times concentration in activity, equivalent to that of ammonium sulfate preparations (409,600 to 819,200 activity units/ml). Determination of organic nitrogen (N) content revealed that the desorbed piscicolin preparations were substantially free of proteinaceous substances (approximately 92 to 99%) compared with original culture supernatants and ammonium sulfate preparations. Nisin, pediocin, and brevicin were also desorbed with 1% SDS with a similar level of purification.     

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.     

Isolation and characterization of a new bacteriocin,termed enterocin M,produced by environmental isolate <Emphasis Type="Italic">Enterococcus faecium</Emphasis> AL41

The new bacteriocin, termed enterocin M, produced by Enterococcus faecium AL 41 showed a wide spectrum of inhibitory activity against the indicator organisms from different sources. It was purified by (NH₄)₂SO₄ precipitation, cation-exchange chromatography and reverse phase chromatography (FPLC). The purified peptide was sequenced by N-terminal amino acid Edman degradation and a mass spectrometry analysis was performed. By combining the data obtained from amino acid sequence (39 N-terminal amino acid residues was determined) and the molecular weight (determined to be 4 628 Da) it was concluded that the purified enterocin M is a new bacteriocin, which is very similar to enterocin P. However, its molecular weight is different from enterocin P (4 701.25). Of the first 39 N-terminal residues of enterocin M, valine was found in position 20 and a lysine in position 35, while enterocin P has tryptophane residues in these positions.     

Adsorption of bacteriocins by ingestible silica compounds

Bacteriocins including nisin, pediocin PO2, brevicin 286 and piscicolin 126 were adsorbed from culture supernates by various food-grade porous silica anti-caking agents and the food colourant, titanium dioxide. All the porous silica (calcium silicate or silicon dioxide) materials showed substantial capacity in adsorbing bacteriocin activities from the culture supernate and biological activity was recovered in the adsorbents. In contrast, the food colourant titanium dioxide adsorbed most of the bacteriocin activity from the supernate, with minimal biological activity retained in the adsorbent. Experiments with piscicolin 126 showed that optimum adsorption could be achieved with Micro-Cel E within 30 min, independent of the supernate pH (2.0-10.0). Piscicolin activity of up to 5 × 10⁷ AU g^-1 of Micro-Cel E was obtained after adsorption from culture supernates and the adsorbed piscicolin demonstrated substantial biological activity against Listeria monocytogenes in both broth and a milk growth medium.     

Purification and characterization of a new bacteriocin isolated from a Carnobacterium sp.

A bacteriocin-producing Carnobacterium sp. was isolated from fish. The bacteriocin, termed carnocin UI49, was purified to homogeneity by a four-step purification procedure, including hydrophobic interaction chromatography and reverse-phase chromatography. Carnocin UI49 has a bactericidal mode of action. It was shown to be heat tolerant and stable between pH 2 and 8. At pH above 8, carnocin UI49 was rapidly inactivated. Amino acid analysis revealed a composition of about 35 to 37 amino acids in addition to an unidentified peak which migrates at the position of lanthionine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggests a molecular weight of about 4,500 to 5,000. Mass spectrometry gave a molecular weight of 4,635, which is about 1,000 larger than that calculated from the amino acid analysis data. Performic acid oxidation of carnocin UI49, followed by amino acid hydrolysis, revealed the presence of cysteic acid. The sequence of the first seven amino acid residues was determined to be N-Gly-Ser-Glu-Ile-Gln-Pro-Arg. After the seventh amino acid, carnocin UI49 was not available for further Edman degradation. The results suggest that carnocin UI49 belongs to the class of bacteriocins termed lantibiotics.     

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.     

A versatile system for the expression of nonmodified bacteriocins in Escherichia coli

AIMS: To develop a method and plasmid vectors suitable for expression of class II bacteriocins from Escherichia coli. METHODS AND RESULTS: The expression vector pSuV1 was constructed by inserting the PelB secretion signal coding sequence and a number of restriction endonuclease sites for cloning, into pTYB1. Codon optimized genes encoding the active mature region of each bacteriocin were constructed and inserted into pSuV1. Transfer of these constructs to a host expressing T7 RNA polymerase allowed for expression of secreted mature or fusion forms of the bacteriocins. Generation of the fusion, to the adjacent intein-chitin-binding domain gene, was achieved by removal of a small intervening BseRI fragment. The bacteriocins BacR1, divercin V41, enterocin P, pediocin PA-1 and piscicolin 126 were expressed from this system. For piscicolin 126, expression levels of 200 microg l(-1) in the mature form and 1100 microg l(-1) when cleaved from the fusion partner were achieved. All expressed bacteriocins displayed antimicrobial activity. CONCLUSIONS: Several class II bacteriocins have been expressed in E. coli using purpose designed plasmid vectors described here. SIGNIFICANCE AND IMPACT OF THE STUDY: This method provides a common expression system capable of producing a range of different class II bacteriocins. It allows researchers to study class II bacteriocins without access to the original producer strain, the native bacteriocin gene, or highly specific heterologous producing strains. Resulting expression levels are as high or higher than those previously reported for related bacteriocins.     

Double-glycine-type leader peptides direct secretion of bacteriocins by ABC transporters: colicin V secretion in Lactococcus lactis

Many non-lantibiotic bacteriocins of lactic acid bacteria are produced as precursors which have N-terminal leader peptides that share similarities in amino acid sequence and contain a conserved processing site of two glycine residues in positions -1 and -2. A dedicated ATP-binding cassette (ABC) transporter is responsible for the proteolytic cleavage of the leader peptides and subsequent translocation of the bacteriocins across the cytoplasmic membrane. To investigate the role that these leader peptides play in the recognition of the precursor by the ABC transporters, the leader peptides of leucocin A, lactococcin A or colicin V were fused to divergicin A, a bacteriocin from Carnobacterlum divergens that is secreted via the cell's general secretion pathway. Production of divergicin was monitored when these fusion constructs were introduced into Leuconostoc gelidum, Lactococcus lactis and Escherichia coli, which carry the secretion apparatus for leucocin A, lactococcins A and B, and colicin V, respectively. The different leader peptides directed the production of divergicin in the homologous hosts. In some cases production of divergicin was also observed when the leader peptides were used in heterologous hosts. For ABC-transporter-dependent secretion in E. coli the outer membrane protein TolC was required. Using this strategy, colicin V was produced in L. lactis by fusing this bacteriocin behind the leader peptide of leucocin A.     

Distribution and ecology of the generalist lactic acid bacterium Carnobacterium maltaromaticum in different freshwater habitats: Metabolic and antagonistic abilities

We explored the distribution, metabolic and antagonistic activities of Carnobacterium maltaromaticum, isolated from freshwater locations in Denmark during winter or early spring. This species was widely distributed in such habitats although it was relatively rare in low pH locations. Isolates possessed a diverse metabolism, potentially enabling functional capacities independent of habitat. The intraspecies competition showed a relatively high degree of mostly low-intensity interactions, which overall were not correlated with phylogeny or location. Only a few isolates exhibited broad-spectrum inhibition activity, targeting species from other genera and families, including one isolate that exhibited a broad inhibitory activity due to H₂O₂ production. Bioinformatic analyses revealed that the frequency of bacteriocinogenic systems was low, and only one unmodified bacteriocin, piscicolin 126, correlated with phenotypic antagonistic activity. Furthermore, most potential bacteriocin gene complexes were not complete. Overall, this study showed C. maltaromaticum to be a generalist (nomadic) species with a constant presence in freshwater habitats, especially those with pH values >5. General metabolic properties did not suggest a strong degree of adaptation to the freshwater environment, and bacteriocin-mediated antagonistic activities appeared to play a minimal ecological role.     

Enterocin T, a novel class IIa bacteriocin produced by Enterococcus sp. 812

Enterococcus sp. 812, isolated from fresh broccoli, was previously found to produce a bacteriocin active against a number of Gram-positive bacteria, including Listeria monocytogenes. Bacteriocin activity decreased slightly after autoclaving (121 °C for 15 min), but was inactivated by protease K. Mass spectrometry analysis revealed the bacteriocin mass to be approximately 4,521.34 Da. N-terminal amino acid sequencing yielded a partial sequence, NH₂-ATYYGNGVYXDKKKXWVEWGQA, by Edman degradation, which contained the consensus class IIa bacteriocin motif YGNGV in the N-terminal region. The obtained partial sequence showed high homology with some enterococcal bacteriocins; however, no identical peptide or protein was found. This peptide was therefore considered to be a novel bacteriocin produced by Enterococcus sp. 812 and was termed enterocin T.     

Characterization and Structure Analysis of a Novel Bacteriocin,Lacticin Z,Produced by Lactococcus lactis QU 14

A novel bacteriocin, lacticin Z, produced by Lactococcus lactis QU 14 isolated from a horse’s intestinal tract was identified. Lacticin Z was purified through a three step procedure comprised of hydrophobic-interaction, cation-exchange chromatography, and reverse-phase HPLC. ESI-TOF MS determined the molecular mass of lacticin Z to be 5,968.9 Da. The primary structure of lacticin Z was found to consist of 53 amino acid residues without any leader sequence or signal peptide. Lacticin Z showed homology to lacticin Q from L. lactis QU 5, aureocin A53 from Staphylococcus aureus A53, and mutacin BHT-B from Streptococcus rattus strain BHT. It exhibited a nanomolar range of MICs against various Gram-positive bacteria, and the activity was completely stable up to 100 °C. Unlike many of other LAB bacteriocins, the stability of lacticin Z was emphasized under alkaline conditions rather than acidic conditions. All the results indicated that lacticin Z belongs to a novel type of bacteriocin.     

Characterization, Production, and Purification of Carnocin H, a Bacteriocin Produced by Carnobacterium 377

Carnocin H, a bacteriocin produced by a Carnobacterium sp., inhibited lactic acid bacteria, clostridia, enterococci, and some Staphylococcus aureus strains. Some strains of Listeria and Pediococcus were also sensitive to carnocin H. The bacteriocin was produced during the late stationary growth phase. Carnocin H was purified by cation exchange chromatography and reverse phase chromatography. Amino acid sequence and composition indicate that carnocin H is a novel bacteriocin belonging to the class II bacteriocins. The bacteriocin consists of approximately 75 amino acid residues with a highly cationic N-terminal containing six succeeding lysines. Activity, as measured by agar diffusion zones, was reduced at increased pH values, levels of indicator bacteria, NaCl, agar, and soy oil.