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Molecular mechanisms of bacteriocin evolution.

Microorganisms are engaged in a never-ending arms race. One consequence of this intense competition is the diversity of antimicrobial compounds that most species of bacteria produce. Surprisingly, little attention has been paid to the evolution of such extraordinary diversity. One class of antimicrobials, the bacteriocins, has received increasing attention because of the high levels of bacteriocin diversity observed and the use of bacteriocins as preservatives in the food industry and as antibiotics in the human health industry. However, little effort has been focused on evolutionary questions, such as what are the phylogenetic relationships among these toxins, what mechanisms are involved in their evolution, and how do microorganisms respond to such an arsenal of weapons? The focus of this review is to provide a detailed picture of our current understanding of the molecular mechanisms involved in the process of bacteriocin diversification.

Bacteriocins↗

The growth-inhibitory effect of the Enterococcus faecalis bacteriocin encoded by pAD1 extends to the oral streptococci.

Many strains of Enterococcus faecalis, a normal inhabitant of the oral cavity, elaborate a plasmid-encoded bacteriocin. The growth-inhibitory effect of this bacteriocin was observed to extend to a variety of pathogenic oral streptococci, including those that play a major role in tooth surface colonization and caries formation. These results suggest that transient colonization by bacteriocin-producing E. faecalis may effect shifts in oral colonization by susceptible organisms and that bacteriocin-producing E. faecalis may be a candidate for application in strain replacement therapy.

Bacteriocins↗

Gassericin A; an uncommon cyclic bacteriocin produced by Lactobacillus gasseri LA39 linked at N- and C-terminal ends.

Gassericin A, a bacteriocin that was produced by Lactobacillus gasseri LA39, was treated with lysylendopeptidase and 3-bromo-3-methyl-2-(2-nitrophenyl-mercapto)-3H-indole. The fragments were recovered by SDS-PAGE and sequenced. All amino acids of gassericin A were distributed by sequence analysis and the bacteriocin did not contain any modified amino acids. The amino acid sequence of gassericin A completely coincided with that found through the cloning of the structural gene. Gassericin A was shown to be a cyclic bacteriocin (class II) which is bound at the N- and C-terminal ends. This is the first example of a cyclic bacteriocin from lactobacilli lactic acid bacteria.

Amino Acid Sequence↗

Detection of genes encoding virulence factors and bacteriocins in fecal enterococci of poultry in Portugal.

Seventy-six Enterococcus isolates (43 E. faecalis, 30 E. faecium, two E. durans, and one E. hirae) recovered from fecal samples of poultry in a slaughterhouse (one isolate per fecal sample and one fecal sample per lot of animals) were studied for bacteriocin production and for the presence of genes encoding bacteriocins and virulence factors. The presence of genes encoding virulence factors (cpd, geE, fsr, ace, agg, and esp) and bacteriocins (entA, entB, entP, entQ, entAS-48, entL50A/B, cyl, and bac31) were studied by polymerase chain reaction in all enterococci. At least two virulence genes were detected in all 43 E. faecalis isolates, cpd and gelE being the most frequently detected genes (97.7%) followed by ace (62.8%), agg (39.5%), fsr (27.9%), and esp (2.3%). No virulence genes were detected in the other enterococcal species with the exception of one E. faecium and one E. durans isolates that harbored the gelE gene. Antimicrobial activity against eight indicator bacteria (including Listeria monocytogenes) was assayed in the enterococci, and 23 (30.3%) showed inhibitory activity against L. monocytogenes, the other 22 enterococci showing activity against indicator bacteria other than L. monocytogenes. Only the entA, entB, and cyl genes were detected in our study (entA + entB in nine E. faecium isolates and the cyl gene in seven E. faecalis isolates). A wide variety of virulence genes have been detected in fecal E. faecalis isolates from poultry, but not in the other enterococcal species. However, the presence of known bacteriocin structural genes is associated more with the E. faecium species.

Animals↗

Genetically engineered bacteriocins and their potential as the next generation of antimicrobials.

The discovery of penicillin by Fleming in 1928 was an historical milestone in the fight against infectious disease. Over the following fifty years, pharmaceutical companies discovered and developed over 100 antibiotics effective against a wide range of human pathogens. More recently, the dramatic rise in antibiotic-resistant pathogens has stimulated renewed efforts to identify, develop or redesign antibiotics active against these multi-resistant bacteria. This review focuses on such efforts directed at one large and highly diverse family of toxins, the bacteriocins, which hold great promise as the next generation of antimicrobials. The majority of bacteriocins differ from traditional antibiotics in one critical way: they have a relatively narrow killing spectrum and are, therefore, toxic only to bacteria closely related to the producing strain. Accordingly, they can be considered "designers drugs" that target specific bacterial pathogens. In this review we focus on recent attempts to generate custom designed bacteriocins using genetic engineering techniques. These efforts illustrate the potential of genetically-modified bacteriocins to solve some of the most challenging problems in disease control.

Animals↗

Peptide AS-48: prototype of a new class of cyclic bacteriocins.

After the discovery of bacteriocin AS-48, a 70-residue cyclic peptide produced by Enterococcus faecalis subsp. liquefaciens, some naturally-occurring cyclic proteins from bacteria have been reported. AS-48 is encoded by the 68-kb pheromone-responsive plasmid pMB2, and the gene cluster involved in production and immunity has been identified and sequenced. This peptide exerts a bactericidal action on sensitive cells (most of the Gram-positive and some Gram-negative bacteria). Its target is the cytoplasmic membrane, in which it opens pores, leading to the dissipation of the proton motive force and cell death, a mechanism similar to that proposed for the action of defensins or, most generally, cationic antibacterial peptides. This fact, together with its remarkable stability and solubility over a wide pH range, suggest that this bacteriocin could be a good candidate as a natural food preservative. The amino acid composition of purified AS-48 shows the absence of modified or dehydrated residues, making it clearly different from lantibiotics. Bacteriocin AS-48 also differs from defensins in that it does not contain cysteines and consequently no disulfide bridges, which makes is high stability even more remarkable. Composition analysis of AS-48 shows a high proportion of basic to acidic amino acids, conferring to this peptide a strong basic character, with an isoelectric point close to 10.5. Determination of the AS-48 structural gene DNA sequence, together with the sequences of AS-48 protease digestion fragments and mass spectrometry determinations, allowed us to determine unambiguously the cyclic structure of the molecule, being the first example of a posttranslational modification in which a cyclic structure arises from a "head-to-tail" linkage. We have solved the three-dimensional structure of AS-48 in solution, and it consists of a globular arrangement of five alpha-helices enclosing a compact hydrophobic core. Interestingly, the head-to-tail peptide link between Trp-70 and Met-1 lies in the middle of alpha-helix 5, which is shown to have a pronounced effect on the stability of the three-dimensional structure. Analysis of structure-function relationship allowed us to propose models to understand the aspects of the molecular function of AS-48. The purpose of this work is to review recent developments in our understanding about the biochemical and biological characteristics and structure of this unusual type of bacteriocin.

Animals↗

Inhibition of psychrotrophic organisms by propionicin PLG-1, a bacteriocin produced by Propionibacterium thoenii.

Propionibacterium thoenii strain P127, which produces the bacteriocin propionicin PLG-1, was grown in a skim milk medium and produced bacteriocin in that medium. No bacteriocin activity was detected in skim milk medium in which strain P127-1, a bacteriocin-negative variant of strain P127, had been grown. Five psychrotrophic spoilage or pathogenic organisms (one strain each of Listeria monocytogenes, Pseudomonas fluorescens, Vibrio parahaemolyticus, Yersinia enterocolitica, and one strain of Corynebacterium sp.) were incubated for 24 h in laboratory medium, nonfermented skim milk, and skim milk that had been fermented by strain P127 or P127-1. Strains were inhibited only in the skim milk fermented by strain P127, as evidenced by loss in numbers of viable cells after 24 h at 10 degrees C and less growth than in other media after 24 h at optimal growth temperatures. Growth of selected strains was delayed or slowed during prolonged incubation (21 d) at 10 degrees C. Propionicin PLG-1 shows promise as a preservative for food products.

Animals↗

Inhibition of Listeria monocytogenes by in situ produced and semipurified bacteriocins of Carnobacterium spp. on vacuum-packed, refrigerated cold-smoked salmon.

Listeria monocytogenes inhibition by Carnobacterium strains and crude bacteriocins on sterile and commercial vacuum-packed cold-smoked salmon stored at 4 degrees C and 8 degrees C was investigated. Carnobacterium piscicola V1 was bactericidal against L. monocytogenes at the two temperatures, whereas Carnobacterium divergens V41 presented a bacteriostatic effect. C. piscicola SF668 delayed L. monocytogenes growth at 8 degrees C and had a bacteriostatic effect at 4 degrees C. Listeria growth was not affected by a non-bacteriocin-producing C. piscicola. Crude extracts of piscicocins were bactericidal at 4 degrees C and 8 degrees C. Listeria growth was delayed by divercin V41 at 8 degrees C and was inhibited at 4 degrees C. Nisin delayed Listeria growth at 8 degrees C and was bacteriostatic at 4 degrees C. The present study demonstrates that L. monocytogenes growth could be prevented on vacuum-packed cold-smoked salmon by Carnobacterium and associated bacteriocins at chilled temperatures. Moreover, no product spoilage could be observed with the use of such bacteriocin-producing strains as demonstrated by good sensorial analyses and low biogenic amine production.

Animals↗

Optimization of bacteriocin ST311LD production by Enterococcus faecium ST311LD, isolated from spoiled black olives.

Bacteriocin ST311LD is approximately 2.3 kDa in size. Low levels of bacteriocin activity were recorded in BHI and M17 broth (800 AU/ml) and in 10% (w/v) soy milk (3,200 AU/ml). No bacteriocin production was recorded in 10% (w/v) molasses, despite good growth. Optimal levels (12,800 AU/ml) were detected in MRS broth which had been supplemented with tryptone (20.0 g/l), saccharose (5.0 or 10.0 g/l) or vitamin C (1 ppm). Increased potassium levels did not result in higher levels of activity, and glycerol (1.0 g/l) inhibited the production of bacteriocin ST311LD.

Bacteriocins↗

Shigella flexneri strains produce bacteriocins active against members of the human microbial intestinal flora.

The principal aim of this work was to detect the bacteriocinogenic capacity of S. flexneri strains on members of the human intestinal flora. A total of 49 bacteriocinogenic S. flexneri strains were isolated from individuals of both sexes and different ages. The bacteriocins were detected by means of the drop method using E. coli and B. fragilis as target strains. The serotypes of the S. flexneri were determined. The producer capacity of bacteriocins was analysed in 10 different colonies of the same cellular clone and also the arbitrary units were determined. The highest number of bacteriocinogenic S. flexneri strains were obtained from diarrhoeal individuals from 0-10 years old and the S. flexneri serotype 2a was the most abundant. It was demonstrated that E. coli and B. fragilis isolated from the normal intestinal flora of healthy individuals were susceptible to the bacteriocinogenic S. flexneri strains. By means of the determination of arbitrary units per ml of the bacteriocin, it was demonstrated that colonies from a single colony isolate of a same clone of bacteriocinogenic S. flexneri produce different quantities of bacteriocin.

Adolescent↗

[Staphylococcal protein A, bacteriocin, DNase and lactamases of methicillin-resistant Staphylococcus].

231 Methicillin-Resistant Staphylococcus (MRS) and 76 Methicillin-Susceptible Staphylococcus (MSS) strains were investigated for Staphylococcal Protein A, bacteriocin, DNase and beta lactamase production properties. It was found that 73.6% of the strains of MRS were positive for protein A, 3.8% for bacteriocin, 76.2% for DNase and 84.4% for beta lactamase production. And it was found that 61.8% of MSS strains were positive for protein A, 11.8% for bacteriocin, 55.2% for DNase and 57.9% for beta lactamase production. Staphylococcal protein A, DNase and lactamase production. Staphylococcal protein A, DNase and lactamase production were found to be significantly higher in MRS strains than MSS strains and bacteriocin production was found to be higher in MSS strains than MRS strains according to Chi Square Test.

Bacteriocins↗

[The biological activity and physicochemical properties of a new bacteriocin from a strain of Pseudomonas cepacia 5779].

Pseudomonas cepacia 5779 bacteriocin (cepaciacin) whose producer was revealed due to application of the special screening system has been studied for its certain biological and physicochemical properties. Possessing a narrow range of action, it inhibits only the P. cepacia strains. Its biosynthesis occurs more intensely on the rich nutrient media, the highest quantities of cepaciacine being revealed at the terminal stage of the produced log growth. UV irradiation or mitomycin C introduction into the medium stimulated biosynthesis of this bacteriocin. Cepaciacin P. cepacia 5779 is a complex consisting of several protein subunits and carbon part. The protein-carbohydrate ratio is 3:1. The molecular weight of the complex is 1.8 x 10(6) Da. Lipopolysaccharides isolated from the indicator strain being added, cepaciacine loses its activity. This bacteriocin is stable in the narrow range of pH, thermolabile, decomposes under the effect of proteases and is, evidently, a representative of a new type of the bacteriocin-like substances.

Amino Acids↗

Staphylococcosis of turkeys. 4. Characterization of a bacteriocin produced by an interfering Staphylococcus.

A Staphylococcus epidermidis isolate designated strain 115, which is used as an interfering agent against staphylococcosis of turkeys, produces a bacteriocin that was partially purified and characterized in this study. This bacteriocin diffused through agar media, but it was not found in appreciable quantities in the supernatant fluid of broth cultures. Extraction of the bacterial cells with 7 M urea, 1% sodium dodecyl sulfate, or 1% Triton X-100 caused considerable amounts of the bacteriocin to go into solution. This substance was partially purified by selective chemical extraction and by gel filtration chromatography using a Sephacryl S-300 column. This bacteriocin had two active forms: an aggregate, and a small-molecular-weight form estimated by gel filtration chromatography to be less than 6500. Activity was not affected by heat, repeated freeze-thaw cycles, pH 2 and pH 10, or a variety of proteolytic enzymes, nucleases, a lipase, and lysozyme.

Animals↗

Bacteriocin typing of some Lactobacillus species of the subgenus betabacterium.

One hundred and forty cultures of L. fermenti, L. brevis, and L. buchneri were tested by the method of delayed antagonism for sensitivity to 39 bacteriocins produced by lactobacillus strains of different species. According to their bacteriocin sensitivity patterns, 84 L. fermenti, 43 L. brevis and 13 L. buchneri cultures were differentiated into 26, 18 and 10 bacteriocin types, respectively. Bacteriocin typing allows not only intraspecific differentiation of L. fermenti, L. brevis and L. buchneri cultures but also a subdivision of their biochemical-physiological variants.

Bacteriocins↗

Isolation and preliminary characterization of Streptococcus cremoris (strain 202) bacteriocin.

Ability to produce bacteriocin (lactostrepcin) is demonstrated in Streptococcus cremoris. However, lactostrepcin was found to be produced by only one strain (strain 202) out of the 17 S. cremoris strains tested. This bacteriocin is preliminarily characterized, it differs from other lactic streptococci bacteriocins. The strain 202 bacteriocin acts on many strains of lactic streptococci, strains of Bacillus cereus, and some strains of Leuconostoc and Lactobacillus. It was shown that strain 202 contains some plasmids, but we were not able by preliminary observations to prove the plasmid localization of the determinant(s) for lactostrepcin production by this strain. In persistent lactostrepcin-negative variants of strain 202, in comparison with the parental strain, the plasmid pattern may be changed or unchanged.

Bacteria↗

Bacteriocine typing of Providencia isolates.

A method of typing isolates of Providencia is described based on the sensitivities of the organisms to bacteriocines. Twelve standard bacteriocine producing strains were selected from a large number of isolates tested and from these liquid bacteriocine preparations were obtained. The activities of these preparations on over 300 isolates were determined and from this information a bacteriocine typing system has been developed.

Bacteriocins↗

Studies concerning the sensitivity of Klebsiella strains to a set of Klebsiella bacteriocins.

Optimal crude preparations of Klebsiella bacteriocins were obtained bu U.V. irradiation of bacteriocin producer strains. Of 308 clinical isolates of Klebsiella 88.9% could be typed on the basis of their sensitivity to different Klebsiella bacteriocins. The method affords further development of bacteriocin typing as a suitable procedure for hospital epidemiological investigation.

Bacteriocins↗

[Sensitivity of the lactobacilli of the subgenus Streptobacterium to bacteriocins of lactobacilli of different species].

Sensitivity of 167 cultures of Lactobacilli of Streptobacterium to 39 bacteriocins produced by L. acidophilus, L. jugurti, L. salivarius, L. lactis, L. helveticus, L. casei, L. plantarum, L. fermenti, L. brevis and L. buchneri was studied. It was found that the cultures of L. casei were sensitive to 30 types of bacteriocins, while the cultures of L. plantarum were sensitive to 9 bacteriocin types. 89.6 per cent of the cultures of L. casei and all cultures of L. plantarum were sensitive to one or several bacteriocins produced by Lactobacilli of different species.

Bacteriocins↗