Bacteriocin diversity in Streptococcus and Enterococcus.
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Bacteriocins↗
Biomedical subjects
Helge Holo
Publications and source records attributed to Helge Holo.
Purification and sequencing of cerein 7B, a novel bacteriocin produced by Bacillus cereus Bc7.
Cerein 7B is a new bacteriocin produced simultaneously with cerein 7A by Bacillus cereus Bc7 in liquid brain heart infusion cultures. Both bacteriocins are not synergistic. The two peptides have been purified to homogeneity by hydrophobic interaction, cation exchange and reverse-phase liquid chromatography. They can be distinguished by their N-terminal amino acid sequences N-Gly-Trp-Gly-Asp-Val-Leu (7A) and N-Gly-Trp-Trp-Asn-Ser-Trp-Gly-Lys (7B). Pre-cerein 7B is 74 amino acids long and contains an 18 aminoacid double-glycine type leader sequence that is removed to produce the mature bacteriocin. The leader peptide sequence is related to that of sec-independent secretion signals suggesting that cerein 7B belongs to class II sec-independent bacteriocins.
Amino Acid Sequence↗
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.
Antimicrobial Cationic Peptides↗
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.
Amino Acid Sequence↗
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.
ATP-Binding Cassette Transporters↗
Enterolysin A, a cell wall-degrading bacteriocin from Enterococcus faecalis LMG 2333.
A novel antimicrobial protein, designated enterolysin A, was purified from an Enterococcus faecalis LMG 2333 culture. Enterolysin A inhibits growth of selected enterococci, pediococci, lactococci, and lactobacilli. Antimicrobial activity was initially detected only on solid media, but by growing the bacteria in a fermentor under optimized production conditions (MRS broth with 4% [wt/vol] glucose, pH 6.5, and a temperature between 25 and 35 degrees C), the bacteriocin activity was increased to 5,120 bacteriocin units ml(-1). Enterolysin A production was regulated by pH, and activity was first detected in the transition between the logarithmic and stationary growth phases. Killing of sensitive bacteria by enterolysin A showed a dose-response behavior, and the bacteriocin has a bacteriolytic mode of action. Enterolysin A was purified, and the primary structure was determined by combined amino acid and DNA sequencing. This bacteriocin is translated as a 343-amino-acid preprotein with an sec-dependent signal peptide of 27 amino acids, which is followed by a sequence corresponding to the N-terminal part of the purified protein. Mature enterolysin A consists of 316 amino acids and has a calculated molecular weight of 34,501, and the theoretical pI is 9.24. The N terminus of enterolysin A is homologous to the catalytic domains of different cell wall-degrading proteins with modular structures. These include lysostaphin, ALE-1, zoocin A, and LytM, which are all endopeptidases belonging to the M37 protease family. The N-terminal part of enterolysin A is linked by a threonine-proline-rich region to a putative C-terminal recognition domain, which shows significant sequence identity to two bacteriophage lysins.
Amino Acid Sequence↗
Production of class II bacteriocins by lactic acid bacteria; an example of biological warfare and communication.
Lactic acid bacteria (LAB) fight competing Gram-positive microorganisms by secreting anti-microbial peptides called bacteriocins. Peptide bacteriocins are usually divided into lantibiotics (class I) and non-lantibiotics (class II), the latter being the main topic of this review. During the past decade many of these bacteriocins have been isolated and characterized, and elements of the genetic mechanisms behind bacteriocin production have been unravelled. Bacteriocins often have a narrow inhibitory spectrum, and are normally most active towards closely related bacteria likely to occur in the same ecological niche. Lactic acid bacteria seem to compensate for these narrow inhibitory spectra by producing several bacteriocins belonging to different classes and having different inhibitory spectra. The latter may also help in counteracting the possible development of resistance mechanisms in target organisms. In many strains, bacteriocin production is controlled in a cell-density dependent manner, using a secreted peptide-pheromone for quorum-sensing. The sensing of its own growth, which is likely to be comparable to that of related species, enables the producing organism to switch on bacteriocin production at times when competition for nutrients is likely to become more severe. Although today a lot is known about LAB bacteriocins and the regulation of their production, several fundamental questions remain to be solved. These include questions regarding mechanisms of immunity and resistance, as well as the molecular basis of target-cell specificity.
Amino Acid Sequence↗
Characterization of the genetic locus responsible for the production of ABP-118, a novel bacteriocin produced by the probiotic bacterium Lactobacillus salivarius subsp. salivarius UCC118.
ABP-118, a small heat-stable bacteriocin produced by Lactobacillus salivarius subsp. salivarius UCC118, a strain isolated from the ileal-caecal region of the human gastrointestinal tract, was purified to homogeneity. Using reverse genetics, a DNA fragment specifying part of ABP-118 was identified on a 10769 bp chromosomal region. Analysis of this region revealed that ABP-118 was a Class IIb two-peptide bacteriocin composed of Abp118alpha, which exhibited the antimicrobial activity, and Abp118beta, which enhanced the antimicrobial activity. The gene conferring strain UCC118 immunity to the action of ABP-118, abpIM, was identified downstream of the abp118beta gene. Located further downstream of abp118beta, several ORFs were identified whose deduced proteins resembled those of proteins involved in bacteriocin regulation and secretion. Heterologous expression of ABP-118 was achieved in Lactobacillus plantarum, Lactococcus lactis and Bacillus cereus. In addition, the abp118 locus encoded an inducing peptide, AbpIP, which was shown to play a role in the regulation of ABP-118 production. This novel bacteriocin is, to the authors' knowledge, the first to be isolated from a known human probiotic bacterium and to be characterized at the genetic level.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
Plantaricin W from Lactobacillus plantarum belongs to a new family of two-peptide lantibiotics.
Plantaricin W (Plw) is a new two-peptide bacteriocin, from Lactobacillus plantarum, which inhibits a large number of Gram-positive bacteria. The two peptides, Plwalpha (comprising 29 residues) and Plwbeta (comprising 32 residues), were isolated from the culture supernatants and characterized. The individual peptides had low antimicrobial activity but acted synergistically, and synergism was seen at all mixing ratios tested. The data indicate that the two peptides work in a 1:1 ratio. Chemical analyses showed that both peptides are lantibiotics, but two unmodified cysteines and one serine residue were present in Plwalpha, and Plwbeta contained one cysteine residue. The Plw structural genes were sequenced and shown to encode prepeptides with sequence similarities to two other two-peptide lantibiotics, namely staphylococcin C55 and lacticin 3147. The conserved residues are mainly serines, threonines and cysteines that can be involved in intramolecular thioether bond formation in the C-terminal parts of the molecules. This indicates that these bacteriocins are members of a new family of lantibiotics with common bridging patterns, and that the ring structures play an important functional role. Based on the data a structural model is presented in which each peptide has a central lanthionine and two overlapping thioether bridges close to their C-termini.
Amino Acid Sequence↗
Characterization and production of amylovorin L471, a bacteriocin purified from Lactobacillus amylovorus DCE 471 by a novel three-step method.
The strongly hydrophobic bacteriocin amylovorin L471 from Lactobacillus amylovorus DCE 471 was isolated and purified to homogeneity from complex culture broth by a novel, rapid and simple three-step protocol including (i) ammonium sulphate precipitation, (ii) chloroform/methanol extraction/precipitation and (iii) reversed-phase HPLC, the only chromatographic step involved. The molecular mass of the peptide was determined to be 4876.9 Da by electrospray mass spectrometric analysis. N-terminal amino acid sequencing identified 35 amino acid residues as being identical to the N-terminal sequence of lactobin A, a bacteriocin from another L. amylovorus strain. These non-identical strains produce bacteriocins that display small differences in molecular mass and inhibitory spectrum. The amino acid sequence of amylovorin L471 shared significant homology with lactacin X, one of the two bactericidal peptides produced by Lactobacillus johnsonii VPI11088. A purified amylovorin L471 preparation permitted confirmation of the inhibitory spectrum previously established with a crude extract. It displayed a bactericidal mode of action on lactobacilli after an extremely rapid adsorption to the target cells. Two Listeria spp. were only weakly sensitive. Amylovorin L471 appears to be produced constitutively. Ethanol not only stimulated specific bacteriocin production but also prevented adsorption of the bacteriocin molecules to the producer cells upon prolonged fermentation. The latter result supports the hypothesis that the apparent inactivation of bacteriocin observed during the stationary phase of batch fermentations is due to adsorption.
Amino Acid Sequence↗
Enterocin B, a new bacteriocin from Enterococcus faecium T136 which can act synergistically with enterocin A.
The strain Enterococcus faecium T136 produces two bacteriocins, enterocin A, a member of the pediocin family of bacteriocins, and a new bacteriocin termed enterocin B. The N-terminal amino acid sequences of enterocins A and B were determined, and the gene encoding enterocin B was sequenced. The primary translation product was a 71 aa peptide containing a leader peptide of the double-glycine type which is cleaved off to give mature enterocin B of 53 aa. Enterocin B does not belong to the pediocin family of bacteriocins and shows strong homology to carnobacteriocin A. However, sequence similarities in their leader peptides and C-termini suggest that enterocin B and carnobacteriocin A are related to bacteriocins of the pediocin family. Enterocins A and B had only slightly different inhibitory spectra, and both were active against a wide range of Gram-positive bacteria, including listeriae, staphylococci and most lactic acid bacteria tested. Both had bactericidal activities, but survival at a frequency of 10(-4)-10(-2) was observed when sensitive cultures were exposed to either bacteriocin. The number of survivors was drastically reduced when a mixture of the two bacteriocins was added to the cells.
Amino Acid Sequence↗