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Djamel Drider - One of the best experts on this subject based on the ideXlab platform.
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Heterologous Biosynthesis of Five New Class II Bacteriocins From Lactobacillus paracasei CNCM I-5369 With Antagonistic Activity Against Pathogenic Escherichia coli Strains
Frontiers in Microbiology, 2020Co-Authors: Yanath Belguesmia, Kamel Bendjeddou, Isabelle Kempf, Rabah Boukherroub, Djamel DriderAbstract:Lactobacillus paracasei CNCM I-5369 isolated from a traditional Algerian dairy product produces extracellular inhibitory substances, namely, Bacteriocins, which are active against a panel of pathogenic Escherichia coli strains. This activity was observed only at a narrow pH 4.5-5, and resulted to be heat stable and sensitive to the action of proteolytic enzymes, which indicate a proteinaceous nature. This new strain has a genome of 2,752,975 bp, with a 46.6% G + C ratio and contains at least 2664 coding sequences. The Bagel software analysis identified five open reading frames (ORFs) that are translated to new Class II Bacteriocin. Each ORF was cloned in frame with a His-tag tail and expressed in E. coli BL21 (DE3) (pLysS) strain. Of note, each fusion protein carrying any of these ORFs at the C- or N-terminal position resulted to be active against E. coli 184 strain used as target organism. This manuscript reports the first multi-Bacteriocinogenic strain producing five new Class II Bacteriocins with activity against Gram-negative bacilli (GNB), namely, E. coli. Heterologous expression and activity of each new Class II Bacteriocin were demonstrated.
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Production of recombinant Bacteriocin divercin V41 by high cell density Escherichia coli batch and fed-batch cultures
Applied Microbiology and Biotechnology, 2007Co-Authors: Selcuk Yildirim, Djamel Drider, Daniel Konrad, Ségolène Calvez, Hervé Prévost, Christophe LacroixAbstract:To increase the yield of heterologous production of the Class II Bacteriocin DvnRV41 with Escherichia coli Origami (DE3) (pLysS/pCR03), induction of Bacteriocin gene expression was optimized by varying the inducer isopropyl β- d -thiogalactopyranoside (IPTG) concentration (0–2 mM), and controlled batch and fed-batch cultures were tested on a 2-L scale. A concentration of 0.5 mM IPTG was found to be optimal for cell growth and Bacteriocin production. Shake flask cultivation of E. coli Origami (DE3) (pLysS/pCR03) gave biomass and Bacteriocin yields of 1.54 ± 0.06 g cdw/l and 18 ± 1 mg DvnRV41/l, respectively. Biomass (2.70 ± 0.06 and 6.8 ± 0.6 g cdw/l, respectively) and Bacteriocin yields (30 and 74 mg DvnRV41 per liter, respectively) were both increased with batch and fed-batch compared to shake flask cultures. Bacteriocin yields reported in this study are among the highest published for other heterologous expression systems in shake flasks.
R.p. Ross - One of the best experts on this subject based on the ideXlab platform.
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a multiBacteriocin cheese starter system comprising nisin and lacticin 3147 in lactococcus lactis in combination with plantaricin from lactobacillus plantarum
Applied and Environmental Microbiology, 2017Co-Authors: Susan Mills, Colin Hill, Wilco C Meijer, Carmel Griffin, Paula M Oconnor, L M Serrano, R.p. RossAbstract:Functional starter cultures demonstrating superior technological and food safety properties are advantageous to the food fermentation industry. We evaluated the efficacies of single- and double-Bacteriocin-producing starters of Lactococcus lactis capable of producing the Class I Bacteriocins nisin A and/or lacticin 3147 in terms of starter performance. Single producers were generated by mobilizing the conjugative bacteriophage resistance plasmid pMRC01, carrying lacticin genetic determinants, or the conjugative transposon Tn5276, carrying nisin genetic determinants, to the commercial starter L. lactis CSK2775. The effect of Bacteriocin coproduction was examined by superimposing pMRC01 into the newly constructed nisin transconjugant. Transconjugants were improved with regard to antimicrobial activity and bacteriophage insensitivity compared to the recipient strain, and the double producer was immune to both Bacteriocins. Bacteriocin production in the starter was stable, although the recipient strain proved to be a more efficient acidifier than transconjugant derivatives. Overall, combinations of Class I Bacteriocins (the double producer or a combination of single producers) proved to be as effective as individual Bacteriocins for controlling Listeria innocua growth in laboratory-scale cheeses. However, using the double producer in combination with the Class II Bacteriocin producer Lactobacillus plantarum or using the lacticin producer with the Class II producer proved to be most effective for reducing bacterial load. As emergence of Bacteriocin tolerance was reduced 10-fold in the presence of nisin and lacticin, we suggest that the double producer in conjunction with the Class II producer could serve as a protective culture providing a food-grade, multihurdle approach to control pathogenic growth in a variety of industrial applications.IMPORTANCE We generated a suite of single- and double-Bacteriocin-producing starter cultures capable of generating the Class I Bacteriocin lacticin 3147 or nisin or both Bacteriocins simultaneously via conjugation. The transconjugants exhibited improved bacteriophage resistance and antimicrobial activity. The single producers proved to be as effective as the double-Bacteriocin producer at reducing Listeria numbers in laboratory-scale cheese. However, combining the double producer or the lacticin-producing starter with a Class II Bacteriocin producer, Lactobacillus plantarum LMG P-26358, proved to be most effective at reducing Listeria numbers and was significantly better than a combination of the three Bacteriocin-producing strains, as the double producer is not inhibited by either of the Class I Bacteriocins. Since the simultaneous use of lacticin and nisin should reduce the emergence of Bacteriocin-tolerant derivatives, this study suggests that a protective starter system produced by Bacteriocin stacking is a worthwhile multihurdle approach for food safety applications.
Pascal Hols - One of the best experts on this subject based on the ideXlab platform.
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the inhibitory spectrum of thermophilin 9 from streptococcus thermophilus lmd 9 depends on the production of multiple peptides and the activity of blpg st a thiol disulfide oxidase
Applied and Environmental Microbiology, 2008Co-Authors: Laetitia Fontaine, Pascal HolsAbstract:The blp(St) cluster of Streptococcus thermophilus LMD-9 was recently shown to contain all the genetic information required for the production of Bacteriocins active against other S. thermophilus strains. In this study, we further investigated the antimicrobial activity of S. thermophilus LMD-9 by testing the susceptibility of 31 bacterial species (87 strains). We showed that LMD-9 displays an inhibitory spectrum targeted toward related gram-positive bacteria, including pathogens such as Listeria monocytogenes. Using deletion mutants, we investigated the contribution of the three putative Bacteriocin-encoding operons blpD(St)-orf2, blpU(St)-orf3, and blpE(St)-blpF(St) (bac(St) operons) and of the blpG(St) gene, which encodes a putative modification protein, to the inhibitory spectrum and immunity of strain LMD-9. Our results present evidence that the blp(St) locus encodes a multipeptide Bacteriocin system called thermophilin 9. Among the four Class II Bacteriocin-like peptides encoded within the bac(St) operons, BlpD(St) alone was sufficient to inhibit the growth of most thermophilin 9-sensitive species. The blpD(St) gene forms an operon with its associated immunity gene(s), and this functional Bacteriocin/immunity module could easily be transferred to Lactococcus lactis. The remaining three Bac(St) peptides, BlpU(St), BlpE(St), and BlpF(St), confer poor antimicrobial activity but act as enhancers of the antagonistic activity of thermophilin 9 by an unknown mechanism. The blpG(St) gene was also shown to be specifically required for the antilisteria activity of thermophilin 9, since its deletion abolished the sensitivities of most Listeria species. By complementation of the motility deficiency of Escherichia coli dsbA, we showed that blpG(St) encodes a functional thiol-disulfide oxidase, suggesting an important role for disulfide bridges within thermophilin 9.
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Quorum-sensing regulation of the production of Blp Bacteriocins in Streptococcus thermophilus
Journal of Bacteriology, 2007Co-Authors: Laetitia Fontaine, Céline Boutry, Eric Guédon, Alain Guillot, Mariam Ibrahim, Benoît Grossiord, Pascal HolsAbstract:The blp gene cluster identified in the genome sequences of Streptococcus thermophilus (blp(St)) LMG18311, CNRZ1066, and LMD-9 displays all the characteristics of a Class II Bacteriocin locus. In the present study, we showed that the blp(St) locus is only fully functional in strain LMD-9 and regulates the production of antimicrobial peptides that inhibit strains LMG18311 and CNRZ1066. The blp(St) cluster of LMD-9 contains 23 genes that are transcriptionally organized in six operons: blpABC(St) (peptide transporter genes and pheromone gene); blpRH(St) (two-component regulatory system genes); blpD(St)-orf1, blpU(St)-orf3, and blpE-F(St) (Bacteriocin precursors and immunity genes); and blpG-X(St) (unknown function). All the operons, except the regulatory unit blpRH(St), were shown to be coregulated at the transcriptional level by a quorum-sensing mechanism involving the mature S. thermophilus pheromone BlpC* (BlpC*(St)), which was extracellularly detected as two active forms (30 and 19 amino acids). These operons are differentially transcribed depending on growth phase and pheromone concentration. They all contain a motif with two imperfect direct repeats in their mapped promoter regions that could serve as binding sites of the response regulator BlpR(St). Through the construction of deletion mutants, the blp(St) locus of strain LMD-9 was shown to encode all the essential functions associated with Bacteriocin production, quorum-sensing regulation, and immunity.
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Quorum-Sensing Regulation of the Production of Blp Bacteriocins in Streptococcus thermophilus†
2007Co-Authors: Benoît Grossiord, Pascal HolsAbstract:The blp gene cluster identified in the genome sequences of Streptococcus thermophilus (blpSt) LMG18311, CNRZ1066, and LMD-9 displays all the characteristics of a Class II Bacteriocin locus. In the present study, we showed that the blpSt locus is only fully functional in strain LMD-9 and regulates the production of antimi-crobial peptides that inhibit strains LMG18311 and CNRZ1066. The blpSt cluster of LMD-9 contains 23 genes that are transcriptionally organized in six operons: blpABCSt (peptide transporter genes and pheromone gene); blpRHSt (two-component regulatory system genes); blpDSt-orf1, blpUSt-orf3, and blpE-FSt (Bacteriocin precur-sors and immunity genes); and blpG-XSt (unknown function). All the operons, except the regulatory unit blpRHSt, were shown to be coregulated at the transcriptional level by a quorum-sensing mechanism involving the mature S. thermophilus pheromone BlpC * (BlpC*St), which was extracellularly detected as two active forms (30 and 19 amino acids). These operons are differentially transcribed depending on growth phase and pheromone concentration. They all contain a motif with two imperfect direct repeats in their mapped promoter regions that could serve as binding sites of the response regulator BlpRSt. Through the construction of deletion mutants, the blpSt locus of strain LMD-9 was shown to encode all the essential functions associated with Bacteriocin production, quorum-sensing regulation, and immunity. Many lactic acid bacteria (LAB) secrete antimicrobial pep
Yanath Belguesmia - One of the best experts on this subject based on the ideXlab platform.
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Heterologous Biosynthesis of Five New Class II Bacteriocins From Lactobacillus paracasei CNCM I-5369 With Antagonistic Activity Against Pathogenic Escherichia coli Strains
Frontiers in Microbiology, 2020Co-Authors: Yanath Belguesmia, Kamel Bendjeddou, Isabelle Kempf, Rabah Boukherroub, Djamel DriderAbstract:Lactobacillus paracasei CNCM I-5369 isolated from a traditional Algerian dairy product produces extracellular inhibitory substances, namely, Bacteriocins, which are active against a panel of pathogenic Escherichia coli strains. This activity was observed only at a narrow pH 4.5-5, and resulted to be heat stable and sensitive to the action of proteolytic enzymes, which indicate a proteinaceous nature. This new strain has a genome of 2,752,975 bp, with a 46.6% G + C ratio and contains at least 2664 coding sequences. The Bagel software analysis identified five open reading frames (ORFs) that are translated to new Class II Bacteriocin. Each ORF was cloned in frame with a His-tag tail and expressed in E. coli BL21 (DE3) (pLysS) strain. Of note, each fusion protein carrying any of these ORFs at the C- or N-terminal position resulted to be active against E. coli 184 strain used as target organism. This manuscript reports the first multi-Bacteriocinogenic strain producing five new Class II Bacteriocins with activity against Gram-negative bacilli (GNB), namely, E. coli. Heterologous expression and activity of each new Class II Bacteriocin were demonstrated.
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purification and characterization of the Bacteriocin produced by lactobacillus sakei mbsa1 isolated from brazilian salami
Journal of Applied Microbiology, 2014Co-Authors: Matheus De Souza Barbosa, Svetoslav Dimitrov Todorov, Jean-marc Chobert, Thomas Haertlé, Iskra Ivanova, Yanath Belguesmia, Yvan Choiset, Hanitra Rabesona, B D G M FrancoAbstract:Aims The study aimed at determining the biochemical characteristics of the Bacteriocin produced by Lactobacillus sakei MBSa1, isolated from salami, correlating the results with the genetic features of the producer strain. Methods and Results Identification of strain MBSa1 was performed by 16S rDNA sequencing. The Bacteriocin was tested for spectrum of activity, heat and pH stability, mechanism of action, molecular mass and amino acid sequence when purified by cation-exchange and reversed-phase HPLC. Genomic DNA was tested for Bacteriocin genes commonly present in Lact. sakei. Bacteriocin MBSa1 was heat-stable, unaffected by pH 2·0 to 6·0 and active against all tested Listeria monocytogenes strains. Maximal production of Bacteriocin MBSa1 (1600 AU ml−1) in MRS broth occurred after 20 h at 25°C. The molecular mass of produced Bacteriocin was 4303·3 Da, and the molecule contained the SIIGGMISGWAASGLAG sequence, also present in sakacin A. The strain contained the sakacin A and curvacin A genes but was negative for other tested sakacin genes (sakacins T-α, T-β, X, P, G and Q). Conclusions In the studied conditions, Lact. sakei MBSa1 produced sakacin A, a Class II Bacteriocin, with anti-Listeria activity. Significance and Impact of the Study The study covers the purification and characterization of the Bacteriocin produced by a lactic acid bacteria isolated from salami (Lact. sakei MBSa1), linking genetic and expression information. Its heat-resistance, pH stability in acid conditions (pH 2·0–6·0) and activity against L. monocytogenes food isolates bring up a potential technological application to improve food safety.
Susan Mills - One of the best experts on this subject based on the ideXlab platform.
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a multiBacteriocin cheese starter system comprising nisin and lacticin 3147 in lactococcus lactis in combination with plantaricin from lactobacillus plantarum
Applied and Environmental Microbiology, 2017Co-Authors: Susan Mills, Colin Hill, Wilco C Meijer, Carmel Griffin, Paula M Oconnor, L M Serrano, R.p. RossAbstract:Functional starter cultures demonstrating superior technological and food safety properties are advantageous to the food fermentation industry. We evaluated the efficacies of single- and double-Bacteriocin-producing starters of Lactococcus lactis capable of producing the Class I Bacteriocins nisin A and/or lacticin 3147 in terms of starter performance. Single producers were generated by mobilizing the conjugative bacteriophage resistance plasmid pMRC01, carrying lacticin genetic determinants, or the conjugative transposon Tn5276, carrying nisin genetic determinants, to the commercial starter L. lactis CSK2775. The effect of Bacteriocin coproduction was examined by superimposing pMRC01 into the newly constructed nisin transconjugant. Transconjugants were improved with regard to antimicrobial activity and bacteriophage insensitivity compared to the recipient strain, and the double producer was immune to both Bacteriocins. Bacteriocin production in the starter was stable, although the recipient strain proved to be a more efficient acidifier than transconjugant derivatives. Overall, combinations of Class I Bacteriocins (the double producer or a combination of single producers) proved to be as effective as individual Bacteriocins for controlling Listeria innocua growth in laboratory-scale cheeses. However, using the double producer in combination with the Class II Bacteriocin producer Lactobacillus plantarum or using the lacticin producer with the Class II producer proved to be most effective for reducing bacterial load. As emergence of Bacteriocin tolerance was reduced 10-fold in the presence of nisin and lacticin, we suggest that the double producer in conjunction with the Class II producer could serve as a protective culture providing a food-grade, multihurdle approach to control pathogenic growth in a variety of industrial applications.IMPORTANCE We generated a suite of single- and double-Bacteriocin-producing starter cultures capable of generating the Class I Bacteriocin lacticin 3147 or nisin or both Bacteriocins simultaneously via conjugation. The transconjugants exhibited improved bacteriophage resistance and antimicrobial activity. The single producers proved to be as effective as the double-Bacteriocin producer at reducing Listeria numbers in laboratory-scale cheese. However, combining the double producer or the lacticin-producing starter with a Class II Bacteriocin producer, Lactobacillus plantarum LMG P-26358, proved to be most effective at reducing Listeria numbers and was significantly better than a combination of the three Bacteriocin-producing strains, as the double producer is not inhibited by either of the Class I Bacteriocins. Since the simultaneous use of lacticin and nisin should reduce the emergence of Bacteriocin-tolerant derivatives, this study suggests that a protective starter system produced by Bacteriocin stacking is a worthwhile multihurdle approach for food safety applications.