Purification and Characterization of Bacteriocin Produced by a Strain of <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> TG2

The aims of this study were to purify and characterize a bacteriocin produced by a strain of Enterococcus faecalis TG2 and to test the safety of the strain. In this work, the active peptide was purified through precipitation with 70% saturated ammonium sulfate, cation-exchange chromatography, and gel filtration. The specific activity of purified bacteriocin was 30,073.42 AU/mg of protein, which corresponded to a 33.34-fold increase. The molecular mass of the purified bacteriocin was 6.3362 kDa determined by LC-MS/MS. The ten amino acid of N-terminal was MTRSKKLNLR and the ten amino acid of C-terminal was ATGGAAGWKS. The activity of the bacteriocin was unaffected by pH 2–10 and thermostable but was sensitive to proteolytic enzymes. The antimicrobial activity of the bacteriocin was not affected by metal ions. Tween-20, Tween-80, Triton X-100, and EDTA did not affect the bacteriocin activity and SDS was able to increase the activity of bacteriocin. Bacteriocin activity was not lost after treatment by < 8% NaCl. Inhibitory spectrum of the bacteriocin showed a wide range of activities against other lactic acid bacteria, food-spoilage, and food-borne pathogens. Ent. faecalis TG2 was sensitive to tetracycline and erythromycin but resistant to ampicillin, gentamicin, kanamycin, and chloramphenicol. Results from PCR indicated that Ent. faecalis TG2 did not harbor any virulence genes. The study suggests that Ent. faecalis TG2 and its bacteriocin might be used as bio-preservatives in food products.     

Bacteriocin PJ4 Active Against Enteric Pathogen Produced by Lactobacillus helveticus PJ4 Isolated from Gut Microflora of Wistar Rat (Rattus norvegicus): Partial Purification and Characterization of Bacteriocin

The increase of multidrug-resistant pathogens and the restriction on the use antibiotics due to its side effects have drawn attention to the search for possible alternatives. Bacteriocins are small antimicrobial peptides produced by numerous bacteria. Much interest has been focused on bacteriocins because they exhibit inhibitory activity against pathogens. Lactic acid bacteria possess the ability to synthesize antimicrobial compounds (like bacteriocin) during their growth. In this study, an antibacterial substance (bacteriocin PJ4) produced by Lactobacillus helveticus PJ4, isolated from rat gut microflora, was identified as bacteriocin. It was effective against wide assay of both Gram-positive and Gram-negative bacteria involved in various diseases, including Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Enterococcus faecalis, and Staphylococcus aureus. The antimicrobial peptide was relatively heat-resistant and also active over a wide pH range of 2–10. It has been partially purified to homogeneity using ammonium sulfate precipitation and size exclusion chromatography and checked on reverse-phase high-performance liquid chromatography. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of bacteriocin PJ4 purified through size exclusion chromatography resolved ~6.5 kDa protein with bacteriocin activity. The peptide is inactivated by proteolytic enzymes, trypsin, and lipase but not when treated with catalase, α-amylase, and pepsin. It showed a bactericidal mode of action against the indicator strains E. coli MTCC443, Lactobacillus casei MTCC1423, and E. faecalis DT48. Such characteristics indicate that this bacteriocin may be a potential candidate for alternative agents to control important pathogens.     

Antimicrobial leucocin analogues with a disulfide bridge replaced by a carbocycle or by noncovalent interactions of allyl glycine residues

The type IIa bacteriocins are antimicrobial peptides isolated from lactic acid bacteria that act as food preservation agents and have nanomolar activity against pathogens such as Listeria monocytogenes. Previous reports with mutant bacteriocins indicate that the conserved disulfide bridge between cysteine residues 9 and 14 in bacteriocins such as leucocin A (1) is critical for antibiotic properties, which are mediated by target membrane receptor proteins belonging to the mannose phosphotransferase (mpt) system. To examine whether the disulfide can be replaced by an olefin moiety, [9,14]-dicarba leucocin A (4) was made by on-resin ring closing metathesis of allyl glycine residues using a new protocol suitable for larger hydrophobic peptides. Carbocyclic analogue 4 still displays nanomolar activity but is about 10-fold less potent than 1. Surprisingly, the acyclic [9,14]-diallyl leucocin A (5) displays even higher antibiotic activity than 4 and is as effective as the parent, leucocin A (1). We attribute this activity to hydrophobic intermolecular interactions of the diallyl side chains of the acyclic bacteriocin 5 that assist realization of the correct conformation at the receptor active site. Such substitutions in other systems may allow linear acyclic peptides to mimic the biological activity of natural disulfide ring-containing parents.     

Bacteriocin activity against various pathogens produced by Pediococcus pentosaceus VJ13 isolated from Idly batter

Bacteriocins, an antimicrobial peptide, is known to have wide spectrum antimicrobial activity against various pathogens. Because they are easily digested in the intestine, they are considered as safe and are widely used as food preservatives. Hence their purification and characterization have attracted considerable attraction, especially for those having activity against human pathogens. In this study, the bacteriocin produced by Pediococcus pentosaceus VJ13 was precipitated with cold acetone and purified by gel permeation chromatography and hydrophobic interaction chromatography. The bacteriocin exhibited antimicrobial activity against various pathogens, like Mycobacterium smegmatis, Klebsiella pneumonia, Clostridium perfringens and Staphylococcus epidermidis. The activity of bacteriocin was lost completely after treatment with protease, which revealed its proteinaceous nature. The bacteriocin was stable up to 100°C and exhibited antilisterial property which is a characteristic feature of class IIa bacteriocins. It was active within the pH range of 2–8 and stable against various chemicals and denaturants. Tricine SDS–PAGE revealed its molecular weight to be 4.0 kDa, where the corresponding activity against Listeria monocytogenes was also noted. Treatment of L. monocytogenes with bacteriocin decreased the viable cell count, and scanning electron microscope analysis revealed membrane pore formation that resulted in the release of intracellular content, suggesting its bactericidal effect. Copyright © 2013 John Wiley & Sons, Ltd.     

Elimination of Listeria monocytogenes in sausage meat by combination treatment: Radiation and radiation-resistant bacteriocins

Two new bacteria were isolated from human feces and were designated MT 104 and MT 162. They were able to produce bacteriocins that are active against five strains of Listeria monocytogenes. Bacteriocins produced by these isolated strains had 100% and 82.35% residual activity when they were treated by gamma radiation at doses of 4 and 40 kGy, respectively. A reduction of 1.0, 1.5 and 3 log CFU/g of L. monocytogenes was observed in sausage meat when treated with bacteriocins from MT 104, MT 162, and nisin, respectively. For synergic effect, the D₁₀ value in presence of the bacteriocins produced by MT 104 showed a 1.08 fold increased relative sensitivity of L. monocytogenes as compared to control after 5 days. The highest synergic effect was observed in presence of nisin which led to 1.61 fold increased relative sensitivity. Combined treatments with nisin and γ-irradiation showed a synergic antimicrobial effect in meat after 24 h and 5 days of storage. A synergic effect was observed only after 5 days at 4 °C for the bacteriocin from MT 104, as compared to the bacteriocin produced by MT 162 that had only an additive antimicrobial effect in all conditions.     

Transporter Protein-Guided Genome Mining for Head-to-Tail Cyclized Bacteriocins

Head-to-tail cyclized bacteriocins are ribosomally synthesized antimicrobial peptides that are defined by peptide backbone cyclization involving the N- and C- terminal amino acids. Their cyclic nature and overall three-dimensional fold confer superior stability against extreme pH and temperature conditions, and protease degradation. Most of the characterized head-to-tail cyclized bacteriocins were discovered through a traditional approach that involved the screening of bacterial isolates for antimicrobial activity and subsequent isolation and characterization of the active molecule. In this study, we performed genome mining using transporter protein sequences associated with experimentally validated head-to-tail cyclized bacteriocins as driver sequences to search for novel bacteriocins. Biosynthetic gene cluster analysis was then performed to select the high probability functional gene clusters. A total of 387 producer strains that encode putative head-to-tail cyclized bacteriocins were identified. Sequence and phylogenetic analyses revealed that this class of bacteriocins is more diverse than previously thought. Furthermore, our genome mining strategy captured hits that were not identified in precursor-based bioprospecting, showcasing the utility of this approach to expanding the repertoire of head-to-tail cyclized bacteriocins. This work sets the stage for future isolation of novel head-to-tail cyclized bacteriocins to serve as possible alternatives to traditional antibiotics and potentially help address the increasing threat posed by resistant pathogens.     

Enterococcal cytolysin: a novel two component peptide system that serves as a bacterial defense against eukaryotic and prokaryotic cells

The cytolysin is a novel, two-peptide lytic toxin produced by some strains of Enterococcus faecalis. It is toxic in animal models of enterococcal infection, and associated with acutely terminal outcome in human infection. The cytolysin exerts activity against a broad spectrum of cell types including a wide range of gram positive bacteria, eukaryotic cells such as human, bovine and horse erythrocytes, retinal cells, polymorphonuclear leukocytes, and human intestinal epithelial cells. The cytolysin likely originated as a bacteriocin involved with niche control in the complex microbial ecologies associated with eukaryotic hosts. However, additional anti-eukaryotic activities may have been selected for as enterococci adapted to eukaryotic cell predation in water or soil ecologies. Cytolytic activity requires two unique peptides that possess modifications characteristic of the lantibiotic bacteriocins, and these peptides are broadly similar in size to most cationic eukaryotic defensins. Expression of the cytolysin is tightly controlled by a novel mode of gene regulation in which the smaller peptide signals high-level expression of the cytolysin gene cluster. This complex regulation of cytolysin expression may have evolved to balance defense against eukaryotic predators with stealth.     

Structure of subtilosin A,an antimicrobial peptide from Bacillus subtilis with unusual posttranslational modifications linking cysteine sulfurs to alpha-carbons of phenylalanine and threonine

The complete primary and three-dimensional solution structures of subtilosin A (1), a bacteriocin from Bacillus subtilis, were determined by multidimensional NMR studies on peptide produced using isotopically labeled [(13)C,(15)N]medium derived from Anabaena sp. grown on sodium [(13)C]bicarbonate and [(15)N]nitrate. Additional samples of 1 were also generated by separate incorporations of [U-(13)C,(15)N]phenylalanine and [U-(13)C,(15)N]threonine using otherwise unlabeled media. The results demonstrate that in addition to having a cyclized peptide backbone (N and C termini), three cross-links are formed between the sulfurs of Cys13, Cys7, and Cys4 and the alpha-positions of Phe22, Thr28, and Phe31, respectively. Such posttranslational linkage of a thiol to the alpha-carbon of an amino acid residue is very unusual in natural peptides or proteins. Subtilosin A (1) belongs to a new class of bacteriocins.     

Characterization of a New Bacteriocin from Lactobacillus plantarum LE5 and LE27 Isolated from Ensiled Corn

Bacteriocins are low molecular peptides with antimicrobial activity, which are of great interest as food bio-preservatives and for treating diseases caused by pathogenic bacteria. In this study, we present the characterization of bacteriocins produced by Lactobacillus plantarum LE5 and LE27 isolated from ensiled corn. Bacteriocins were purified through ammonium sulfate precipitation and double dialysis by using 12- and 1-kDa membranes. Bacteriocins showed activity against Listeria innocua, Listeria monocytogenes, and Enteroccocus faecalis. Molecular weight was estimated through Tricine-SDS-PAGE and overloading the gel onto Mueller-Hinton agar seeded with L. monocytogenes, showing an inhibition zone between 5 and 10 kDa. NanoLC-MS/MS analysis allowed the identification of UPF0291 protein (UniProtKB/Swiss-Prot Q88VI7), which is also presented in other lactic acid bacteria without assigned function. Ab initio modeling showed it has an α-helix-rich structure and a large positive-charged region. Bacteriocins were stable between 4 and 121 °C and pH 2 and 12, and the activity was inhibited by SDS and proteases. Mode of action assay suggests that the bacteriocin causes of target microorganism. Taken together, these results describe a possible new class IIa bacteriocin produced by L. plantarum, which has a wide stability to physicochemical conditions, and that could be used as an alternative for the control of foodborne diseases.     

Genome level analysis of bacteriocins of lactic acid bacteria

Bacteriocins are antimicrobial peptides which are ribosomally synthesized by mainly all bacterial species. LABs (lactic acid bacteria) are a diverse group of bacteria that include around 20 genera of various species. Though LABs have a tremendous potential for production of anti-microbial peptides, this group of bacteria is still underexplored for bacteriocins. To study the diversity among bacteriocin encoding clusters and the putative bacteriocin precursors, genome mining was performed on 20 different species of LAB not reported to be bacteriocin producers. The phylogenetic tree of gyrB, rpoB, and 16S rRNA were constructed using MEGA6 software to analyze the diversity among strains. Putative bacteriocins operons identified were found to be diverse and were further characterized on the basis of physiochemical properties and the secondary structure. The presence of at least two cysteine residues in most of the observed putative bacteriocins leads to disulphide bond formation and provide stability. Our data suggests that LABs are prolific source of low molecular weight non modified peptides.     

Isolation of a Bacteriocin-Producing Lactococcus lactis and Application of Its Bacteriocin to Manage Spoilage Bacteria in High-Value Marine Fish Under Different Storage Temperatures

The bacteriocins of lactic acid bacteria have considerable potential for biopreservation. The Lactococcus lactis strain PSY2 (GenBank account no. JF703669) isolated from the surface of marine perch Perca flavescens produced antibacterial activity against pathogenic and spoilage-causing Gram-positive and Gram-negative bacteria viz. Arthrobacter sp., Acinetobacter sp., Bacillus subtilis, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa and Staphylococcus aureus and possessed broad inhibitory spectrum. The biopreservative efficacy of the bacteriocin PSY2 was evaluated using fillets of reef cod, Epinephelus diacanthus. The fillets (10?g) were sprayed with 2.0?ml of 1,600?AU/ml bacteriocin, wrapped and kept under different storage temperatures viz., 4, 0 and ?18?°C. The biopreservative extended the shelf-life of fillets stored at 4?°C to >21?days as against <14?days observed in the untreated samples. The total count of spoilage bacteria was reduced by 2.5 logarithmic units in the treated sample during the 14th day of storage as against the control. Chemical analysis revealed a significant change (P?<?0.05) in the pH value, free fatty acid (as % oleic acid), total volatile base nitrogen and total methyl amine content in the treated samples. The overall acceptability in terms of sensory attributes was significantly higher in the bacteriocin-treated samples stored for 21?days at 4?°C while the untreated samples became unacceptable by the 14th day. The biopreservative gave no significant effect at ?18?°C. Thus, the bacteriocin derived from L. lactis PSY2 gave increased protection against spoilage bacteria and offers an alternative for the preservation of high-value sea foods.     

Genetic engineering in Streptomyces roseosporus to produce hybrid lipopeptide antibiotics

Daptomycin is a lipopeptide antibiotic produced by a nonribosomal peptide synthetase (NRPS) in Streptomyces roseosporus. The holoenzyme is composed of three subunits, encoded by the dptA, dptBC, and dptD genes, each responsible for incorporating particular amino acids into the peptide. We introduced expression plasmids carrying dptD or NRPS genes encoding subunits from two related lipopeptide biosynthetic pathways into a daptomycin nonproducing strain of S. roseosporus harboring a deletion of dptD. All constructs successfully complemented the deletion in trans, generating three peptide cores related to daptomycin. When these were coupled with incomplete methylation of 1 amino acid and natural variation in the lipid side chain, 18 lipopeptides were generated. Substantial amounts of nine of these compounds were readily obtained by fermentation, and all displayed antibacterial activity against gram-positive pathogens.     

Molecular Mechanisms of Anticancer Activity of N-Glycosides of Indolocarbazoles LCS-1208 and LCS-1269

Novel indolocarbazole derivatives named LCS were synthesized by our research group. Two of them were selected as the most active anticancer agents in vivo. We studied the mechanisms of anticancer activity in accordance with the previously described effects of indolocarbazoles. Cytotoxicity was estimated by MTT assay. We analyzed LCS-DNA interactions by circular dichroism in cholesteric liquid crystals and fluorescent indicator displacement assay. The effect on the activity of topoisomerases I and II was studied by DNA relaxation assay. Expression of interferon signaling target genes was estimated by RT-PCR. Chromatin remodeling was analyzed–the effect on histone H1 localization and reactivation of epigenetically silenced genes. LCS-induced change in the expression of a wide gene set was counted by means of PCR array. Our study revealed the cytotoxic activity of the compounds against 11 cancer cell lines and it was higher than in immortalized cells. Both compounds bind DNA; binding constants were estimated—LCS-1208 demonstrated higher affinity than LCS-1269; it was shown that LCS-1208 intercalates into DNA that is typical for rebeccamycin derivatives. LCS-1208 also inhibits topoisomerases I and IIα. Being a strong intercalator and topoisomerase inhibitor, LCS-1208 upregulates the expression of interferon-induced genes. In view of LCSs binding to DNA we analyzed their influence on chromatin stability and revealed that LCS-1269 displaces histone H1. Our analysis of chromatin remodeling also included a wide set of epigenetic experiments in which LCS-1269 demonstrated complex epigenetic activity. Finally, we revealed that the antitumor effect of the compounds is based not only on binding to DNA and chromatin remodeling but also on alternative mechanisms. Both compounds induce expression changes in genes involved in neoplastic transformation and target genes of the signaling pathways in cancer cells. Despite of being structurally similar, each compound has unique biological activities. The effects of LCS-1208 are associated with intercalation. The mechanisms of LCS-1269 include influence on higher levels such as chromatin remodeling and epigenetic effects.     

Two enzymes catalyze the maturation of a lasso peptide in Escherichia coli

Microcin J25 (MccJ25) is a gene-encoded lasso peptide secreted by Escherichia coli which exerts a potent antibacterial activity by blocking RNA polymerase. Here we demonstrate that McjB and McjC, encoded by genes in the MccJ25 gene cluster, catalyze the maturation of MccJ25. Requirement for both McjB and McjC was shown by gene inactivation and complementation assays. Furthermore, the conversion of the linear precursor McjA into mature MccJ25 was obtained in vitro in the presence of McjB and McjC, all proteins being produced by recombinant expression in E. coli. Analysis of the amino acid sequences revealed that McjB could possess proteolytic activity, whereas McjC would be the ATP/Mg(2+)-dependent enzyme responsible for the formation of the Gly1-Glu8 amide bond. Finally, we show that putative lasso peptides are widespread among Proteobacteria and Actinobacteria.     

Antiinfluenza virus activity of a bacteriocin produced by Lactobacillus delbrueckii

A novel antibacterial substance produced by Lactobacillus delbrueckii has been isolated and characterized (1). The inhibitory agent corresponded to the criteria for bacteriocins. It was active against lactic acid bacteria (LAB) species and several food-borne pathogens. The cell-free supernatant was purified by HPLC gel-filtration. Three preparations at different purification steps were tested for activity on the reproduction of influenza virus A/chicken/Germany, strain Weybridge (H7N7) and strain Rostock (H7N1) in cell cultures of chicken embryo fibroblasts (CEF). The inhibitory effect was shown to be highly selective and specific. Expression of viral glycoproteins hemagglutinin, neuraminidase, and nucleoprotein on the surface of infected cells, virus-induced cytopathic effect, infectious virus yield, and hemagglutinin production were all reduced at nontoxic concentrations of the crude preparation (B1). B1 did not protect cells from infection, did not affect adsorption, and slightly inhibited viral penetration into infected cells. The purification did not enhance the cellular toxicity and increased about 870-fold the virus-inhibitory activity. No inactivating effect on extracellular virus was found.     

Detection and partial sequence of phytochrome genes in the ferns Anemia phyllitidis (L.)Sw (Schizaeaceae) and Dryopteris filix-mas L. (Polypodiaceae) by using polymerase-chain reaction technology.

Phytochrome controls several developmental steps during formation and differentiation of the fern gametophyte, including spore germination, morphogenesis of the gametophyte or differentiation of the sexual cells. To obtain information about the amino acid sequence and the regulation of phytochrome expression at the gene level, two degenerated oligonucleotides against well conserved amino acid regions were designed after an optimal alignment of the known phytochrome sequences. These primers were tested against DNA isolated from Arabidopsis thaliana, and the polymerase-chain reaction (PCR) products were cloned and sequenced. The DNA fragment produced with this method proved to be identical with a phytochrome-A-gene fragment from A. thaliana, and hence this fragment was used in further experiments to prove whether amplified DNA from fern species contains phytochrome-like DNA. With this procedure we successfully detected and cloned gene fragments both from gametophytes of Anemia phyllitidis and Dryopteris filix-mas, cultured for 7 days under vegetative conditions. In addition, poly(A)+ RNA was prepared from 7-day-old gametophytes of A. phyllitidis, induced to differentiate antheridia under generative conditions. This poly(A)+ RNA was transcribed into complementary DNA and used together with both phytochrome specific primers in a PCR experiment. We thereby obtained another DNA fragment. These data strongly suggest that A. phyllitidis has at least two phytochrome genes, and that at least one of them is expressed in light-grown gametophytes.     

Novel avilamycin derivatives with improved polarity generated by targeted gene disruption

The oligosaccharide antibiotics avilamycin A and C are produced by Streptomyces viridochromogenes Tu57. Both consist of a heptasaccharide chain, which is attached to a polyketide-derived dichloroisoeverninic acid moiety. They show excellent antibiotic activity against Gram-positive bacteria. Both molecules are modified by O-methylation at different positions, which contributes to poor water solubility and difficulties in galenical drug development. In order to generate novel avilamycin derivatives with improved polarity and improved pharmacokinetic properties, we generated a series of mutants with one, two, or three mutated methyltransferase genes. Based on the structure of the novel avilamycin derivatives, the exact function of three methyltransferases, AviG2, AviG5, and AviG6, involved in avilamycin biosynthesis could be assigned.     

Lantibiotics—Ribosomally Synthesized Biologically Active Polypeptides containing Sulfide Bridges and α,β-Didehydroamino Acids

Lantibiotics are polycyclic peptide antibiotics containing intrachain sulfide bridges, formed from the thioether groups of the amino acids lanthionine and β-methyllanthionine. They also contain α,β-unsaturated amino acids such as didehydroalanine and didehydroaminobutyric acid. A knowledge of the lantibiotic biosynthetic steps and the enzymes involved makes possible a gene technological construction of analogous highly modified polypeptides. To the family of lantibiotics belong nisin, an important food preservative, epidermin, a highly specific therapeutic agent against acne, a series of enzyme inhibitors, as well as immunologically interesting active peptides. Lantibiotics are produced by ribosomal synthesis, starting from inactive precursor proteins (prelantibiotics). The latter are post-translationally converted into the active peptide antibiotics through enzymic modifications. The modifying enzymes effect dehydrations at the serine and threonine residues and stereospecific additions of the cysteine thiol groups to the resulting α,β-unsaturated double bonds, which lead to the formation of several sulfide bridges. Upon subsequent proteolytic cleavage of the leader peptide, the biologically active lantibiotic is formed. Conformational analyses of the lantibiotics, as well as of their prepeptides, enables one to obtain information about the mechanism and steps of the biosynthesis. Antibodies against synthetic prepeptide sequences, and modern instrumental methods for the analysis of peptides, allow structural elucidation of the biosynthetic intermediates.