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.     

Partial Purification and Characterization of a Bacteriocin Produced by Propionibacterium thoenii

A partially purified bacteriocin produced by Propionibacterium thoenii designated propionicin PLG-1 was found to be active against closely related species and exhibited a broad spectrum of activity against other microorganisms. Propionicin PLG-1 was found to be heat labile, sensitive to several proteolytic enzymes, and stable at pH 3 to 9. Propionicin PLG-1 was isolated from solid medium, partially purified by ammonium sulfate precipitation, and purified further by gel filtration. Gel filtration experiments revealed that bacteriocin PLG-1 was present as two different protein aggregates with apparent molecular weights of more than 150,000 and approximately 10,000. Resolution of these protein aggregates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a protein common to both with an apparent molecular weight of 10,000.     

Molecular and Genetic Characterization of Propionicin F, a Bacteriocin from Propionibacterium freudenreichii

This work describes the purification and characterization of propionicin F, the first bacteriocin isolated from Propionibacterium freudenreichii. The bacteriocin has a bactericidal activity and is only active against strains of P. freudenreichii. Propionicin F appears to be formed through a processing pathway new to bacteriocins. The mass of the purified bacteriocin was determined by mass spectrometry, and the N-terminal amino acid sequence was determined by Edman degradation. Sequencing of pcfA, the bacteriocin structural gene, revealed that propionicin F corresponds to a 43-amino-acid peptide in the central part of a 255-amino-acid open reading frame, suggesting that mature propionicin F is excised from the probacteriocin by N- and C-terminal proteolytic modifications. DNA sequencing and Northern blot hybridizations revealed that pcfA is cotranscribed with genes encoding a putative proline peptidase and a protein from the radical S-adenosylmethionine family. A gene encoding an ABC transporter was also identified in close proximity to the bacteriocin structural gene. The potential role of these genes in propionicin F maturation and secretion is discussed.     

The unconventional antimicrobial peptides of the classical propionibacteria

The classical propionibacteria produce genetically unique antimicrobial peptides, whose biological activities are without equivalents, and to which there are no homologous sequences in public databases. In this review, we summarize the genetics, biochemistry, biosynthesis, and biological activities of three extensively studied antimicrobial peptides from propionibacteria. The propionicin T1 peptide constitutes a bona fide example of an unmodified general secretory pathway (sec)-dependent bacteriocin, which is bactericidal towards all tested species of propionibacteria except Propionibacterium freudenreichii. The PAMP antimicrobial peptide represents a novel concept within bacterial antagonism, where an inactive precursor protein is secreted in large amounts, and which activation appears to rely on subsequent processing by proteases in its resident milieu. Propionicin F is a negatively charged bacteriocin that displays an intraspecies bactericidal inhibition spectrum. The biosynthesis of propionicin F appears to proceed through a series of unusual events requiring both N- and C-terminal processing of a precursor protein, which probably requires the radical SAM superfamily enzyme PcfB.     

Heterologous Production of Antimicrobial Peptides in Propionibacterium freudenreichii

Heterologous bacteriocin production in Propionibacterium freudenreichii is described. We developed an efficient system for DNA shuttling between Escherichia coli and P. freudenreichii using vector pAMT1. It is based on the P. freudenreichii rolling-circle replicating plasmid pLME108 and carries the cml(A)/cmx(A) chloramphenicol resistance marker. Introduction of the propionicin T1 structural gene (pctA) into pAMT1 under the control of the constitutive promoter (P₄) yielded bacteriocin in amounts equal to those of the wild-type producer Propionibacterium thoenii 419. The P. freudenreichii clone showed propionicin T1 activity in coculture, killing 90% of sensitive bacteria within 48 h. The pamA gene from P. thoenii 419 encoding the protease-activated antimicrobial peptide (PAMP) was cloned and expressed in P. freudenreichii, resulting in secretion of the pro-PAMP protein. Like in the wild type, PAMP activation was dependent on externally added protease. Secretion of the antimicrobial peptide was obtained from a clone in which the pamA signal peptide and PAMP were fused in frame. The promoter region of pamA was identified by fusion of putative promoter fragments to the coding sequence of the pctA gene. The P₄ and P_pamp promoters directed constitutive gene expression, and activity of both promoters was enhanced by elements upstream of the promoter core region.     

Molecular and genetic characterization of propionicin F, a bacteriocin from Propionibacterium freudenreichii

This work describes the purification and characterization of propionicin F, the first bacteriocin isolated from Propionibacterium freudenreichii. The bacteriocin has a bactericidal activity and is only active against strains of P. freudenreichii. Propionicin F appears to be formed through a processing pathway new to bacteriocins. The mass of the purified bacteriocin was determined by mass spectrometry, and the N-terminal amino acid sequence was determined by Edman degradation. Sequencing of pcfA, the bacteriocin structural gene, revealed that propionicin F corresponds to a 43-amino-acid peptide in the central part of a 255-amino-acid open reading frame, suggesting that mature propionicin F is excised from the probacteriocin by N- and C-terminal proteolytic modifications. DNA sequencing and Northern blot hybridizations revealed that pcfA is cotranscribed with genes encoding a putative proline peptidase and a protein from the radical S-adenosylmethionine family. A gene encoding an ABC transporter was also identified in close proximity to the bacteriocin structural gene. The potential role of these genes in propionicin F maturation and secretion is discussed.     

Prevalence of the genes encoding propionicin T1 and protease-activated antimicrobial peptide and their expression in classical propionibacteria

The purpose of this study was to investigate the frequency of production of the bacteriocin propionicin T1 and the protease-activated antimicrobial peptide (PAMP) and their corresponding genes in 64 isolates of classical propionibacteria. This study revealed that these genes are widespread in Propionibacterium jensenii and Propionibacterium thoenii but absent from the remaining species of classical propionibacteria that were studied. The pro-PAMP-encoding gene (pamA) was found in 63% of the P. jensenii strains and 61% of the P. thoenii strains, and all of these strains displayed PAMP activity. The propionicin T1-encoding gene (pctA) was present in 89% of the P. thoenii strains and 54% of the P. jensenii strains. All P. thoenii strains containing the pctA gene exhibited antimicrobial activity corresponding to propionicin T1 activity, whereas only 38% of the pctA-containing P. jensenii strains displayed this activity. Sequencing of the pctA genes revealed the existence of two allelic variants that differed in a single nucleotide in six strains of P. jensenii; in these strains the glycine at position 55 of propionicin T1 was replaced by an aspartate residue (A variant). No strains harboring the A variant showed any antimicrobial activity against propionicin T1-sensitive bacteria. An open reading frame (orf2) located immediately downstream from the pctA gene was absent in three strains containing the G variant of propionicin T1. Two of these strains showed low antimicrobial activity, while the third strain showed no antimicrobial activity at all. The protein encoded by orf2 showed strong homology to ABC transporters, and it has been proposed previously that this protein is involved in the producer immunity against propionicin T1. The limited antimicrobial activity exhibited by the strains lacking orf2 further suggests that this putative ABC transporter plays an important role in propionicin T1 activity.     

Isolation and purification of propionicin PLG-1, a bacteriocin produced by a strain of Propionibacterium thoenii.

Production of propionicin PLG-1 by Propionibacterium thoenii P127 was pH dependent, with maximal activity detected in supernatants of cultures grown at pH 7.0 Propionicin PLG-1 was purified by ion-exchange chromatography and isoelectric focusing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of propionicin PLG-1 purified through isoelectric focusing resolved a protein band with a molecular weight of 10,000. Propionicin PLG-1 was bactericidal to sensitive cells, demonstrating single-hit kinetics. The producing strain harbored a single plasmid (pLG1) with an approximate size of 250 kb. Preliminary data indicate that both propionicin PLG-1 and immunity to the bacteriocin are encoded on the chromosome. Exposure of strain P127 to acriflavine or to N-methyl-N'-nitro-N-nitrosoguanidine yielded isolates that no longer produced bacteriocin activity and isolates that were cured of the plasmid. However, loss of bacteriocin production was not correlated with loss of the plasmid. Isolates cured of the plasmid were phenotypically identical to plasmid-bearing cells in fermentation patterns, pigment production, and growth characteristics.     

Detection of the bacteriocin propionicin PLG-1 with polyvalent anti-PLG-1 antiserum

Polyclonal antibodies against the bacteriocin propionicin PLG-1 were produced in rabbits at high titer (256,000 to 512,000, as determined by indirect enzyme-linked immunosorbent assay [ELISA]). Anti-PLG-1 antiserum neutralized the antimicrobial activity of PLG-1 preparations in a well diffusion assay. Cross-reacting protein was detected using an indirect ELISA of the culture supernatant from a fed-batch fermentation of the producer strain Propionibacterium thoenii P127 within the first 24 h of incubation, but bacteriocin activity was not detected in the same culture until 217 h of incubation. Culture supernatants from 156 strains of classical dairy propionibacteria were tested by indirect ELISA at 5 and 12 days of incubation for production of cross-reacting protein and by well diffusion assay for bacteriocin activity. Cross-reacting protein was detected in 52 strains: all of the tested strains of P. thoenii, most of the strains of Propionibacterium jensenii, and a minority of the Propionibacterium acidipropionici and Propionibacterium freudenreichii strains. Of these 52 strains, only 4 strains of P. thoenii showed bacteriocin activity in a well diffusion assay. Eight bacteriocin-negative mutants of strain P127 were negative in both ELISA and well diffusion assays. Western blot analysis showed that three protein bands bound anti-PLG-1 antibodies in culture supernatants: a 9.1-kDa band that is assumed to be the PLG-1 monomer and 16.2- and 27.5-kDa bands that may be precursors, multimers, or complexes of PLG-1.     

Identification of the Propionicin F Bacteriocin Immunity Gene (pcfI) and Development of a Food-Grade Cloning System for Propionibacterium freudenreichii

This report describes the first functional analysis of a bacteriocin immunity gene from Propionibacterium freudenreichii and its use as a selection marker for food-grade cloning. Cloning of the pcfI gene (previously orf5 [located as part of the pcfABC propionicin F operon]) rendered the sensitive host 1,000-fold more tolerant to the propionicin F bacteriocin. The physiochemical properties of the 127-residue large PcfI protein resemble those of membrane-bound immunity proteins from bacteriocin systems found in lactic acid bacteria. The high level of immunity conferred by pcfI allowed its use as a selection marker for plasmid transformation in P. freudenreichii. Electroporation of P. freudenreichii IFO12426 by use of the pcfI expression plasmid pSL102 and propionicin F selection (200 bacteriocin units/ml) yielded 10⁷ transformants/μg DNA. The 2.7-kb P. freudenreichii food-grade cloning vector pSL104 consists of the pLME108 replicon, a multiple cloning site, and pcfI expressed from the constitutive P_pampS promoter for selection. The pSL104 vector efficiently facilitated cloning of the propionicin T1 bacteriocin in P. freudenreichii. High-level propionicin T1 production (640 BU/ml) was obtained with the IFO12426 strain, and the food-grade propionicin T1 expression plasmid pSL106 was maintained by ~91% of the cells over 25 generations in the absence of selection. To the best of our knowledge this is the first report of an efficient cloning system that facilitates the generation of food-grade recombinant P. freudenreichii strains.     

Purification and Genetic Characterization of Enterocin I from Enterococcus faecium 6T1a, a Novel Antilisterial Plasmid-Encoded Bacteriocin Which Does Not Belong to the Pediocin Family of Bacteriocins

Enterocin I (ENTI) is a novel bacteriocin produced by Enterococcus faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation. The bacteriocin is active against many olive spoilage and food-borne gram-positive pathogenic bacteria, including clostridia, propionibacteria, and Listeria monocytogenes. ENTI was purified to homogeneity by ammonium sulfate precipitation, binding to an SP-Sepharose fast-flow column, and phenyl-Sepharose CL-4B and C₂/C₁₈ reverse-phase chromatography. The purification procedure resulted in a final yield of 954% and a 170,000-fold increase in specific activity. The primary structure of ENTI was determined by amino acid and nucleotide sequencing. ENTI consists of 44 amino acids and does not show significant sequence similarity with any other previously described bacteriocin. Sequencing of the entI structural gene, which is located on the 23-kb plasmid pEF1 of E. faecium 6T1a, revealed the absence of a leader peptide at the N-terminal region of the gene product. A second open reading frame, ORF2, located downstream of entI, encodes a putative protein that is 72.7% identical to ENTI. entI and ORF2 appear to be cotranscribed, yielding an mRNA of ca. 0.35 kb. A gene encoding immunity to ENTI was not identified. However, curing experiments demonstrated that both enterocin production and immunity are conferred by pEF1.     

Propionicin SM1, a bacteriocin from Propionibacterium jensenii DF1: isolation and characterization of the protein and its gene

We purified a bacteriocin from the cell-free supernatant of Propionibacterium jensenii DF1 isolated from Swiss raw milk, and named it propionicin SM1. The heat-stable protein was strongly bactericidal against P. jensenii DSM20274. On the basis of the N-terminal amino acid sequence of the purified protein, a degenerate oligonucleotide probe was designed to locate and clone the corresponding gene of P. jensenii DF1. It hybridized exclusively with the DF1l-resident plasmid pLME106, but not with chromosomal DNA. Sequencing of the 6.9-kb plasmid revealed the targeted amino acid sequence within an open reading frame (ORF4) of 207 amino acids (molecular mass, 22,865 Da). The corresponding gene was named ppnA. It encodes the prepeptide PpnA that is processed to the mature protein (19,942 Da) propionicin SM1. No sequence homology is detectable with known proteins. However, the proposed leader peptide sequence containing 27 amino acids has typical signal peptide features and shows good homology to the leader peptide of Usp45, a protein excreted from Lactococcus lactis (VAN ASSELDONK et al., 1993). Plasmid pLME106 contains at least 9 ORFs, some exhibiting significant homologies to plasmid-encoded functions from other bacteria. The highest identity values were found for ORF1 with the theta replicase (acc. no. U39878) of Brevibacterium linens (58.8%) and ORF6 with the recombinase/invertase (acc. no. AF060871) found in Rhodococcus rhodochrous (46.4%).     

Weissellicin 110, a Newly Discovered Bacteriocin from Weissella cibaria 110, Isolated from Plaa-Som, a Fermented Fish Product from Thailand

Weissella cibaria 110, isolated from the Thai fermented fish product plaa-som, was found to produce a bacteriocin active against some gram-positive bacteria. Bacteriocin activity was not eliminated by exposure to high temperatures or catalase but was destroyed by exposure to the proteolytic enzymes proteinase K and trypsin. The bacteriocin from W. cibaria 110 was purified, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified bacteriocin contained one protein band that was approximately 2.5 kDa in size. Mass spectrometry analysis showed the mass of the peptide to be approximately 3,487.8 Da. N-terminal amino acid sequence analysis was performed, and 27 amino acids were identified. Because it has no similarity to other known bacteriocins, this bacteriocin was defined as a new bacteriocin and termed weissellicin 110.     

Genus- and Species-Specific PCR-Based Detection of Dairy Propionibacteria in Environmental Samples by Using Primers Targeted to the Genes Encoding 16S rRNA

PCR assays with primers targeted to the genes encoding 16S rRNA were developed for detection of dairy propionibacteria. Propionibacterium thoenii specific oligonucleotide PT3 was selected after partial resequencing. Tests allowed the detection of less than 10 cells per reaction from milk and cheese and 10² cells per reaction from forage and soil.     

An antimicrobial peptide is produced by extracellular processing of a protein from Propionibacterium jensenii

A protease-activated antimicrobial peptide (PAMP) and its inactive precursor were purified from the culture supernatant of Propionibacterium jensenii LMG 3032 and characterized at the molecular level. PAMP is a 64-amino-acid cationic peptide of 6,383 Da with physicochemical features similar to those of bacteriocins from gram-positive bacteria. This peptide displayed bactericidal activity against several propionibacteria and lactobacilli. DNA sequencing indicated that the PAMP-encoding gene (pamA) is translated as a proprotein of 198 amino acids with an N-terminal signal peptide of 27 amino acids and that PAMP constitutes the C-terminal part of this precursor. The amino acid sequence of pro-PAMP showed no similarity to those of other known proteins. By using activity tests and mass spectrometry, we showed that PAMP was formed upon protease treatment of the precursor protein. The propionibacteria produced the PAMP precursor constitutively during growth up to a level of approximately 4 mg/liter, but the producing bacteria were unable to activate the precursor. The requirement for an external protease represents a novel strategy for generating antimicrobial peptides.     

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.     

Nitrite reduction to nitrous oxide by propionibacteria: Detoxication mechanism

Characteristics of dissimilatory nitrate reduction by Propionibacterium acidi-propionici, P. freudenreichii, P. jensenii, P. shermanii and P. thoenii were studied. All strains reduced nitrate to nitrite and further to N₂O. Recovery of added nitrite-N as N₂O-N approached 100%, so that no other end product existed in a significant quantity. Specific rates of N₂O production were 3 to 6 orders of magnitude lower than specific rates of N₂ production by common denitrifiers. Oxygen but not acetylene inhibited N₂O production in P. acidi-propionici and P. thoenii. Nitrite reduction rates were generally higher than nitrate reduction rates. The enzymes involved in nitrate and nitrite reduction were either constitutive or derepressed by anacrobiosis. Nitrate stimulated synthesis of nitrate reductase in P. acidi-propionici. Specific growth rates and growth yields were increased by nitrate. At 10 mM, nitrite was toxic to all strains, and at 1 mM its effect ranged from none to total inhibition. No distinction was obvious between incomplete forms of denitrification and dissimilatory nitrate reduction to ammonia. N₂O production from nitrite by propionibacteria may represent a detoxication mechanism rather than a part of an energy transformation system.     

Isolation and Characterization of Pediocin AcH Chimeric Protein Mutants with Altered Bactericidal Activity

A collection of pediocin AcH amino acid substitution mutants was generated by PCR random mutagenesis of DNA encoding the bacteriocin. Mutants were isolated by cloning mutagenized DNA into an Escherichia coli malE plasmid that directs the secretion of maltose binding protein-pediocin AcH chimeric proteins and by screening transformant colonies for bactericidal activity against Lactobacillus plantarum NCDO955 (K. W. Miller, R. Schamber, Y. Chen, and B. Ray, 1998. Appl. Environ. Microbiol. 64:14–20, 1998). In all, 17 substitution mutants were isolated at 14 of the 44 amino acids of pediocin AcH. Seven mutants (N5K, C9R, C14S, C14Y, G37E, G37R, and C44W) were completely inactive against the pediocin AcH-sensitive strains L. plantarum NCDO955, Listeria innocua Lin11, Enterococcus faecalis M1, Pediococcus acidilactici LB42, and Leuconostoc mesenteroides Ly. A C24S substitution mutant constructed by other means also was inactive against these bacteria. Nine other mutants (K1N, W18R, I26T, M31T, A34D, N41K, H42L, K43N, and K43E) retained from <1% to ~60% of wild-type activity when assayed against L. innocua Lin11. One mutant, K11E, displayed ~2.8-fold-higher activity against this indicator. About one half of the mutations mapped to amino acids that are conserved in the pediocin-like family of bacteriocins. All four cysteines were found to be required for activity, although only C9 and C14 are conserved among pediocin-like bacteriocins. Several basic amino acids as well as nonpolar amino acids located within the hydrophobic C-terminal region also were found to be important. The mutations are discussed in the context of structural models that have been proposed for the bacteriocin.     

Weissellicin 110, a newly discovered bacteriocin from Weissella cibaria 110, isolated from plaa-som, a fermented fish product from Thailand

Weissella cibaria 110, isolated from the Thai fermented fish product plaa-som, was found to produce a bacteriocin active against some gram-positive bacteria. Bacteriocin activity was not eliminated by exposure to high temperatures or catalase but was destroyed by exposure to the proteolytic enzymes proteinase K and trypsin. The bacteriocin from W. cibaria 110 was purified, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified bacteriocin contained one protein band that was approximately 2.5 kDa in size. Mass spectrometry analysis showed the mass of the peptide to be approximately 3,487.8 Da. N-terminal amino acid sequence analysis was performed, and 27 amino acids were identified. Because it has no similarity to other known bacteriocins, this bacteriocin was defined as a new bacteriocin and termed weissellicin 110.     

Heterologous production of antimicrobial peptides in Propionibacterium freudenreichii

Heterologous bacteriocin production in Propionibacterium freudenreichii is described. We developed an efficient system for DNA shuttling between Escherichia coli and P. freudenreichii using vector pAMT1. It is based on the P. freudenreichii rolling-circle replicating plasmid pLME108 and carries the cml(A)/cmx(A) chloramphenicol resistance marker. Introduction of the propionicin T1 structural gene (pctA) into pAMT1 under the control of the constitutive promoter (P4) yielded bacteriocin in amounts equal to those of the wild-type producer Propionibacterium thoenii 419. The P. freudenreichii clone showed propionicin T1 activity in coculture, killing 90% of sensitive bacteria within 48 h. The pamA gene from P. thoenii 419 encoding the protease-activated antimicrobial peptide (PAMP) was cloned and expressed in P. freudenreichii, resulting in secretion of the pro-PAMP protein. Like in the wild type, PAMP activation was dependent on externally added protease. Secretion of the antimicrobial peptide was obtained from a clone in which the pamA signal peptide and PAMP were fused in frame. The promoter region of pamA was identified by fusion of putative promoter fragments to the coding sequence of the pctA gene. The P4 and Ppamp promoters directed constitutive gene expression, and activity of both promoters was enhanced by elements upstream of the promoter core region.