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Lars Axelsson - One of the best experts on this subject based on the ideXlab platform.
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high level inducible gene expression in lactobacillus sakei and lactobacillus plantarum using versatile expression vectors
Microbiology, 2005Co-Authors: Kristine Naterstad, Vincent G H Eijsink, Elisabeth Sorvig, Geir Mathiesen, Lars AxelssonAbstract:Vectors have been developed for inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum in which expression of the gene of interest is driven by strong, regulated promoters from bacteriocin operons found in L. sakei strains. The activity of these promoters is controlled via a two-component signal transduction system, which responds to an externally added peptide pheromone. The vectors have a modular design, permitting easy exchange of all essential elements: the inducible promoter, the cognate regulatory system, the gene of interest, the antibiotic resistance marker and the replicon. Various variants of these so-called 'pSIP' vectors were constructed and tested, differing in terms of the bacteriocin regulon from which the regulatory elements were derived (sakacin A or sakacin P), the regulated promoter selected from these regulons, and the replicon (derived from p256 or pSH71). Using beta-glucuronidase (GusA) and aminopeptidase N (PepN) as reporters, it was shown that the best vectors permitted inducible, pheromone-dose-dependent gene expression at very high levels, while displaying moderate basal activities when not induced. The most effective set-up was obtained using a vector containing the pSH71 replicon, the orfX promoter from the sakacin P regulon, and the cognate regulatory genes, in a L. sakei host. GusA levels obtained with this set-up were approximately ten times higher than the levels obtained with prototype pSIP versions, whereas PepN levels amounted to almost 50% of total cellular protein.
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characterization of a new bacteriocin operon in sakacin p producing lactobacillus sakei showing strong translational coupling between the bacteriocin and immunity genes
Applied and Environmental Microbiology, 2005Co-Authors: Geir Mathiesen, Lars Axelsson, Kathrin Huehne, Lothar Kroeckel, Vincent G H EijsinkAbstract:ABSTRACT Previous studies of genes involved in the production of sakacin P by Lactobacillus sakei Lb674 revealed the presence of an inducible promoter downstream of the known spp gene clusters. We show here that this promoter drives the expression of an operon consisting of a bacteriocin gene (sppQ), a cognate immunity gene (spiQ), another gene with an unknown function (orf4), and a pseudoimmunity gene containing a frameshift mutation (orf5). The leader peptide of the new one-peptide bacteriocin sakacin Q contains consensus elements that are typical for so-called “double-glycine” leader peptides. The mature bacteriocin shows weak similarity to the BrcA peptide of the two-peptide bacteriocin brochocin C. Sakacin Q has an antimicrobial spectrum that differs from that of sakacin P, thus expanding the antimicrobial properties of the producer strain. The genes encoding sakacin Q and its cognate immunity protein showed strong translational coupling, which was investigated in detail by analyzing the properties of a series of β-glucuronidase fusions. Our results provide experimental evidence that production of the bacteriocin and production of the cognate immunity protein are tightly coregulated at the translational level.
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Sakacin P non-producing Lactobacillus sakei strains contain homologues of the sakacin P gene cluster
Research in Microbiology, 2005Co-Authors: Trond Møretrø, Monique Zagorec, Stephane Chaillou, Christine Naterstad, Ellen Wang, Inga Aasen, Lars AxelssonAbstract:Some strains of Lactobacillus sakei are known to produce the bacteriocin sakacin P, encoded by the spp gene cluster. In strains unable to produce sakacin P, spp homologues were observed. The analysis of 15 strains not producing sakacin P revealed that all contained a region corresponding to a part of sppKR encoding the regulatory elements for sakacin P production. In some strains homologues of sppE and sppT, responsible for sakacin P transport, and the sakacin P structural gene sppA and its immunity gene spiA, were also present. The sequence of the chromosomal spp-related gene cluster was determined in two non-producing strains: L. sakei Lb790 and L. sakei 23K. The L. sakei Lb790 spp gene cluster encompasses genes homologous to sppK, sppR, sppT and sppE. In L. sakei 23K, only sppK and sppR homologues were present. The sppK homologues appeared non-functional as they contained mutations and/or an insertion element. In addition to the spp homologues, several small putative genes were found in the gene clusters of the two strains. Some were similar in both strains, and their organization suggests a mosaic structure resulting from successive rearrangements. Transcriptional analysis showed that the genes of the L. sakei Lb790 spp cluster were expressed when genes encoding an operative sakacin P regulatory system were introduced in this strain, thus complementing the inactive sppK gene. Expression experiments also suggested that some of the spp homologues maintained their function in non-producing strains.
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construction of vectors for inducible gene expression in lactobacillus sakei and l plantarum
Fems Microbiology Letters, 2003Co-Authors: Elisabeth Sorvig, Vincent G H Eijsink, Kristine Naterstad, Sonja Gronqvist, Geir Mathiesen, Lars AxelssonAbstract:We have constructed vectors for inducible expression of genes in Lactobacillus sakei and Lactobacillus plantarum. The key elements of these vectors are a regulatable promoter involved in the production of the bacteriocins sakacin A and sakacin P and the genes encoding the cognate histidine protein kinase and response regulator that are necessary to activate this promoter upon induction by a peptide pheromone. The vectors are built up of cassettes that permit easy exchange of all parts through restriction enzyme digestion and ligation. Using β-glucuronidase as a reporter enzyme, variants of these vectors were compared with each other, and with a corresponding system based on genes involved in the production of nisin. Several of the new vectors permitted tightly controlled and efficient expression of β-glucuronidase in both L. sakei and L. plantarum.
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interactions of the bacteriocins sakacin p and nisin with food constituents
International Journal of Food Microbiology, 2003Co-Authors: Inga Marie Aasen, Lars Axelsson, Trond Møretrø, Tone Katla, Sidsel Markussen, Kristine NaterstadAbstract:Abstract Bacteriocins are amphiphilic peptides susceptible to adsorption to food macromolecules and proteolytic degradation. These properties may limit their use as preservation agents. The aim of the present work has been to elucidate the fate of the bacteriocin sakacin P in food. Nisin was used in a few experiments for comparison. Recovery of bacteriocins was studied in homogenates of cold-smoked salmon, chicken cold cuts and raw chicken, with verification of the results in the corresponding food products. More than 80% of the added sakacin P and nisin were quickly adsorbed to proteins in the food matrix. In foods that had not been heat-treated, proteolytic activity caused a rapid degradation of the bacteriocins, with less than 1% of the total activity left after 1 week in cold-smoked salmon, and even less in raw chicken. In heat-treated foods, the bacteriocin activity was stable for more than 4 weeks. The high fat content in salmon compared to chicken had no adverse effect on bacteriocin recovery or activity. However, mixing of triglyceride oils and bacteriocin solutions caused a considerable loss of activity. No principal differences were observed between sakacin P and nisin, but less nisin was adsorbed to muscle proteins at low pH, and the negative effect of oils was less pronounced for nisin. Growth of Listeria monocytogenes was completely inhibited for at least 3 weeks in both chicken cold cuts and cold-smoked salmon by addition of sakacin P (3.5 μg/g), despite the proteolytic degradation in the salmon.
Jon Nissenmeyer - One of the best experts on this subject based on the ideXlab platform.
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mutational analysis and membrane interactions of the β sheet like n terminal domain of the pediocin like antimicrobial peptide sakacin p
Biochimica et Biophysica Acta, 2006Co-Authors: Gunnar Fimland, Jussi Pirneskoski, Jasadee Kaewsrichan, Arimatti Jutila, Per Eugen Kristiansen, Paavo K J Kinnunen, Jon NissenmeyerAbstract:To gain insight into how the N-terminal three-stranded beta-sheet-like domain in pediocin-like antimicrobial peptides positions itself on membranes, residues in the well-conserved (Y)YGNGV-motif in the domain were substituted and the effect of the substitutions on antimicrobial activity and binding of peptides to liposomes was determined. Peptide-liposome interactions were detected by measuring tryptophan-fluorescence upon exposing liposomes to peptides in which a tryptophan residue had been introduced in the N-terminal domain. The results revealed that the N-terminal domain associates readily with anionic liposomes, but not with neutral liposomes. The electrostatic interactions between peptides and liposomes facilitated the penetration of some of the peptide residues into the liposomes. Measuring the antimicrobial activity of the mutated peptides revealed that the Tyr2Leu and Tyr3Leu mutations resulted in about a 10-fold reduction in activity, whereas the Tyr2Trp, Tyr2Phe, Tyr3Trp and Tyr3Phe mutations were tolerated fairly well, especially the mutations in position 3. The Val7Ile mutation did not have a marked detrimental effect on the activity. The Gly6Ala mutation was highly detrimental, consistent with Gly6 being in one of the turns in the beta-sheet-like N-terminal domain, whereas the Gly4Ala mutation was tolerated fairly well. All mutations involving Asn5, including the conservative mutations Asn5Gln and Asn5Asp, were very deleterious. Thus, both the polar amide group on the side chain of Asn5 and its exact position in space were crucial for the peptides to be fully active. Taken together, the results are consistent with Val7 positioning itself in the hydrophobic core of target membranes, thus forcing most of the other residues in the N-terminal domain into the membrane interface region: Tyr3 and Asn5 in the lower half with their side chains pointing downward and approaching the hydrophobic core, Tyr2, Gly4 and His8 and 12 in the upper half, Lys1 near the middle of the interface region, and the side chain of Lys11 pointing out toward the membrane surface.
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three dimensional structure in lipid micelles of the pediocin like antimicrobial peptide sakacin p and a sakacin p variant that is structurally stabilized by an inserted c terminal disulfide bridge
Biochemistry, 2003Co-Authors: Marianne Uteng, Jon Nissenmeyer, Gunnar Fimland, Havard Hildeng Hauge, Phineus R L Markwick, Dimitris Mantzilas, Claudia MuhlegollAbstract:The three-dimensional structures in dodecylphosphocholine (DPC) micelles and in trifluoroethanol (TFE) of the pediocin-like antimicrobial peptide sakacin P and an engineered variant of sakacin P (termed sakP[N24C+44C]) have been determined by use of nuclear magnetic resonance spectroscopy. SakP[N24C+44C] has an inserted non-native activity- and structure-stabilizing C-terminal disulfide bridge that ties the C-terminus to the middle part of the peptide. In the presence of DPC, the cationic N-terminal region (residues 1−17) of both peptides has an S-shaped conformation that is reminiscent of a three-stranded antiparallel β-sheet and that is more pronounced when the peptide was dissolved in TFE instead of DPC. The four positively charged residues located in the N-terminal part are found pointing to the same direction. For both peptides, the N-terminal region is followed by a well-defined central amphiphilic α-helix (residues 18−33), and this in turn is followed by the C-terminal tail (residues 34−43 for saka...
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mutational analysis of the role of charged residues in target cell binding potency and specificity of the pediocin like bacteriocin sakacin p
Microbiology, 2002Co-Authors: Maja Kazazic, Jon Nissenmeyer, Gunnar FimlandAbstract:The significance of charged residues for the target-cell binding, potency and specificity of pediocin-like bacteriocins has been studied by site-directed mutagenesis of sakacin P. Most of the charged residues are located in the N-terminal half, which is thought to mediate the initial binding of these bacteriocins to target cells through electrostatic interaction. All the mutated peptides in which the net positive charge was reduced by one (by replacing a charged residue with threonine) exhibited reduced binding to target cells and a 2–15-fold reduction in potency. The least deleterious of these mutations was the removal of the positive charge in position 8 (H8T). This mutation was, in fact, less deleterious than the conservative His to Lys mutation, indicating that the positive charge in position 8 per se is not of major importance. Somewhat more deleterious was the removal of positive charges at the N- and C-terminal ends (K1T, K43T). Most deleterious was the elimination of the positive charge at positions 11 and (but to a lesser extent) 12, demonstrating the importance of the cationic patch in the middle of the N-terminal half of pediocin-like bacteriocins. Mutated peptides in which the net positive charge was increased by one were also constructed. Some of these exhibited increased cell binding and a potency that was the same as (44K, i.e. an extra positive charge at the C-terminus), or somewhat greater (T20K) than, that of sakacin P, whereas others (0K, i.e. an extra positive charge at the N-terminus) had reduced potency. Sakacin P contains only one negatively charged residue (Asp17). This negative charge and its orientation in space were crucial for activity, since the Asp to Asn mutation and (especially) the conservative Asp to Glu mutation were deleterious. Mutations that made the peptide less cationic had, overall, less effect on the potency toward the Carnobacterium piscicola strain than on the potency toward the three other strains tested, whereas the opposite was the case for mutations that made the peptide more cationic. Thus, charged residues in the N-terminal half may – apparently via the initial electrostatic binding of the bacteriocin to target cells – influence the target-cell specificity.
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a c terminal disulfide bridge in pediocin like bacteriocins renders bacteriocin activity less temperature dependent and is a major determinant of the antimicrobial spectrum
Journal of Bacteriology, 2000Co-Authors: Gunnar Fimland, Ingolf F Nes, Line Johnsen, Lars Axelsson, May Bente Brurberg, Vincent G H Eijsink, Jon NissenmeyerAbstract:Several lactic acid bacteria produce so-called pediocin-like bacteriocins that share sequence characteristics, but differ in activity and target cell specificity. The significance of a C-terminal disulfide bridge present in only a few of these bacteriocins was studied by site-directed mutagenesis of pediocin PA-1 (which naturally contains the bridge) and sakacin P (which lacks the bridge). Introduction of the C-terminal bridge into sakacin P broadened the target cell specificity of this bacteriocin, as illustrated by the fact that the mutants were 10 to 20 times more potent than the wild-type toward certain indicator strains, whereas the potency toward other indicator strains remained essentially unchanged. Like pediocin PA-1, disulfide-containing sakacin P mutants had the same potency at 20 and 37°C, whereas wild-type sakacin P was approximately 10 times less potent at 37°C than at 20°C. Reciprocal effects on target cell specificity and the temperature dependence of potency were observed upon studying the effect of removing the C-terminal disulfide bridge from pediocin PA-1 by Cys→Ser mutations. These results clearly show that a C-terminal disulfide bridge in pediocin-like bacteriocins contributes to widening of the antimicrobial spectrum as well as to higher potency at elevated temperatures. Interestingly, the differences between sakacin P and pediocin PA-1 in terms of the temperature dependency of their activities correlated well with the optimal temperatures for bacteriocin production and growth of the bacteriocin-producing strain.
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new biologically active hybrid bacteriocins constructed by combining regions from various pediocin like bacteriocins the c terminal region is important for determining specificity
Applied and Environmental Microbiology, 1996Co-Authors: Gunnar Fimland, Knut Sletten, O R Blingsmo, Gunther Jung, I F Nes, Jon NissenmeyerAbstract:The pediocin-like bacteriocins, produced by lactic acid bacteria, are bactericidal polypeptides with very similar primary structures. Peptide synthesis followed by reverse-phase and ion-exchange chromatographies yielded biologically active pediocin-like bacteriocins in amounts and with a purity sufficient for characterizing their structure and mode of action. Despite similar primary structures, the pediocin-like bacteriocins, i.e., pediocin PA-1, sakacin P, curvacin A, and leucocin A, differed in their relative toxicities against various bacterial strains. On the basis of the primary structures, the polypeptides of these bacteriocins were divided into two modules: the relatively hydrophilic and well conserved N-terminal region, and the somewhat more diverse and hydrophobic C-terminal region. By peptide synthesis, four new biologically active hybrid bacteriocins were constructed by interchanging corresponding modules from various pediocin-like bacteriocins. All of the new hybrid bacteriocin constructs had bactericidal activity. The relative sensitivity of different bacterial strains to a hybrid bacteriocin was similar to that to the bacteriocin from which the C-terminal module was derived and quite different from that to the bacteriocin from which the N-terminal was derived. Thus, the C-terminal part of the pediocin-like bacteriocins is an important determinant of the target cell specificity. The synthetic bacteriocins were more stable than natural isolates, presumably as a result of the absence of contaminating proteases. However, some of the synthetic bacteriocins lost activity, but this was detectable only after months of storage. Mass spectrometry suggested that this instability was due to oxidation of methionine residues, resulting in a 10- to 100-fold reduction in activity.
Pablo E Hernandez - One of the best experts on this subject based on the ideXlab platform.
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biotechnological potential and in vitro safety assessment of lactobacillus curvatus bcs35 a multibacteriocinogenic strain isolated from dry salted cod gadus morhua
Lwt - Food Science and Technology, 2019Co-Authors: Beatriz Gomezsala, Ingolf F Nes, Dzung B Diep, Estefania Munozatienza, Javier Feito, Rosa Del Campo, Carmen Herranz, Pablo E Hernandez, Luis M CintasAbstract:Abstract The antimicrobial activity of Lactobacillus curvatus BCS35 is due to the production of, at least, two class IIa bacteriocins: sakacin P-H12Y (SakP-H12Y), which is a sakacin P (SakP) variant with tyrosine (Tyr) instead of histidine (His) at position 12, and sakacin X (SakX). Moreover, DNA sequence analysis revealed the presence of the genes encoding the two-peptide bacteriocin sakacin T. In order to compare the specific antimicrobial activities of SakP-H12Y and SakX with other class IIa bacteriocins (SakP from Lactobacillus sakei LTH673 and pediocin PA-1 (PedPA-1) from Pediococcus acidilactici 347), and to determine if the substitution His12/Tyr12 in SakP-H12Y/SakP exerts any effect on the bacteriocin potency and/or specificity, these four bacteriocins were purified and challenged against different indicator strains. Our results showed that (i) PedPA-1 was, in general, the most active peptide, and (ii) SakP-H12Y, which contains one less positively charged residue in the N-terminal half (His12/Tyr12), has a reduced antimicrobial potency against most of the tested indicators compared to SaKP, confirming the importance of the N-terminal cationic patch of SakP-H12Y/SakP in target-cell binding, potency and specificity. The in vitro safety of Lb. curvatus BCS35 was established by antibiotic susceptibility testing concluding that it may be considered as a safe strain.
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The Potential of Class II Bacteriocins to Modify Gut Microbiota to Improve Host Health
PLOS ONE, 2016Co-Authors: Özgün C. O. Umu, Pablo E Hernandez, Christine Bäuerl, Marije Oostindjer, Phillip B. Pope, Gaspar Pérez-martínez, Dzung B DiepAbstract:Production of bacteriocins is a potential probiotic feature of many lactic acid bacteria (LAB) as it can help prevent the growth of pathogens in gut environments. However, knowledge on bacteriocin producers in situ and their function in the gut of healthy animals is still limited. In this study, we investigated five bacteriocin-producing strains of LAB and their isogenic non-producing mutants for probiotic values. The LAB bacteriocins, sakacin A (SakA), pediocin PA-1 (PedPA-1), enterocins P, Q and L50 (enterocins), plantaricins EF and JK (plantaricins) and garvicin ML (GarML), are all class II bacteriocins, but they differ greatly from each other in terms of inhibition spectrum and physicochemical properties. The strains were supplemented to mice through drinking water and changes on the gut microbiota composition were interpreted using 16S rRNA gene analysis. In general, we observed that overall structure of the gut microbiota remained largely unaffected by the treatments. However, at lower taxonomic levels, some transient but advantageous changes were observed. Some potentially problematic bacteria were inhibited (e.g., Staphylococcus by enterocins, Enterococcaceae by GarML, and Clostridium by plantaricins) and the proportion of LAB was increased in the presence of SakA-, plantaricins- and GarML-producing bacteria. Moreover, the treatment with GarML-producing bacteria co-occurred with decreased triglyceride levels in the host mice. Taken together, our results indicate that several of these bacteriocin producers have potential probiotic properties at diverse levels as they promote favorable changes in the host without major disturbance in gut microbiota, which is important for normal gut functioning.
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Cloning, production, and functional expression of the bacteriocin sakacin A (SakA) and two SakA-derived chimeras in lactic acid bacteria (LAB) and the yeasts Pichia pastoris and Kluyveromyces lactis
Journal of Industrial Microbiology & Biotechnology, 2013Co-Authors: Juan J. Jiménez, Dzung B Diep, Carmen Herranz, Luis M Cintas, Juan Borrero, Loreto Gútiez, Pablo E HernandezAbstract:Mature sakacin A (SakA, encoded by sapA ) and its cognate immunity protein (SakI, encoded by sapiA ), and two SakA-derived chimeras mimicking the N-terminal end of mature enterocin P (EntP/SakA) and mature enterocin A (EntA/SakA) together with SakI, were fused to different signal peptides (SP) and cloned into the protein expression vectors pNZ8048 and pMG36c for evaluation of their production and functional expression by different lactic acid bacteria. The amount, antimicrobial activity, and specific antimicrobial activity of SakA and its chimeras produced by Lactococcus lactis subsp. cremoris NZ9000 depended on the SP and the expression vector. Only L. lactis NZ9000 (pNUPS), producing EntP/SakA, showed higher bacteriocin production and antimicrobial activity than the natural SakA-producer Lactobacillus sakei Lb706. The lower antimicrobial activity of the SakA-producer L. lactis NZ9000 (pNUS) and that of the EntA/SakA-producer L. lactis NZ9000 (pNUAS) could be ascribed to secretion of truncated bacteriocins. On the other hand, of the Lb. sakei Lb706 cultures transformed with the pMG36c-derived vectors only Lb. sakei Lb706 (pGUS) overproducing SakA showed a higher antimicrobial activity than Lb. sakei Lb706. Finally, cloning of SakA and EntP/SakA into pPICZαA and pKLAC2 permitted the production of SakA and EntP/SakA by recombinant Pichia pastoris X-33 and Kluyveromyces lactis GG799 derivatives although their antimicrobial activity was lower than expected from their production.
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heterologous coproduction of enterocin a and pediocin pa 1 by lactococcus lactis detection by specific peptide directed antibodies
Applied and Environmental Microbiology, 2000Co-Authors: J M Martinez, Jan Kok, Jan Willem Sanders, Pablo E HernandezAbstract:Antibodies against enterocin A were obtained by immunization of rabbits with synthetic peptides PH4 and PH5 designed, respectively, on the N- and C-terminal amino acid sequences of enterocin A and conjugated to the carrier protein KLH. Anti-PH4-KLH antibodies not only recognized enterocin A but also pediocin PA-1, enterocin P, and sakacin A, three bacteriocins which share the N-terminal class IIa consensus motif (YGNGVXC) that is contained in the sequence of the peptide PH4. In contrast, anti-PH5-KLH antibodies only reacted with enterocin A because the amino acid sequences of the C-terminal parts of class IIa bacteriocins are highly variable. Enterocin A and/or pediocin PA-1 structural and immunity genes were introduced in Lactococcus lactis IL1403 to achieve (co)production of the bacteriocins. The level of production of the two bacteriocins was significantly lower than that obtained by the wild-type producers, a fact that suggests a low efficiency of transport and/or maturation of these bacteriocins by the chromosomally encoded bacteriocin translocation machinery of IL1403. Despite the low production levels, both bacteriocins could be specifically detected and quantified with the anti-PH5-KLH (anti-enterocin A) antibodies isolated in this study and the anti-PH2-KLH (anti-pediocin PA-1) antibodies previously generated (J. M. Martinez, M. I. Martinez, A. M. Suarez, C. Herranz, P. Casaus, L. M. Cintas, J. M. Rodriguez, and P. E. Hernandez, Appl. Environ. Microbiol. 64:4536–4545, 1998). In this work, the availability of antibodies for the specific detection and quantification of enterocin A and pediocin PA-1 was crucial to demonstrate coproduction of both bacteriocins by L. lactis IL1403(pJM04), because indicator strains that are selectively inhibited by each bacteriocin are not available.
Vincent G H Eijsink - One of the best experts on this subject based on the ideXlab platform.
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high level expression of recombinant beta galactosidases in lactobacillus plantarum and lactobacillus sakei using a sakacin p based expression system
Journal of Agricultural and Food Chemistry, 2008Co-Authors: Elisabeth Halbmayr, Vincent G H Eijsink, Geir Mathiesen, Thuha Nguyen, Thomas Maischberger, Clemens K Peterbauer, Dietmar HaltrichAbstract:This work presents the cloning and expression of the genes encoding heterodimeric beta-galactosidases from Lactobacillus reuteri L103, Lactobacillus acidophilus R22, Lactobacillus plantarum WCFS1, and Lactobacillus sakei Lb790. These enzymes consist of two subunits of approximately 73 and 35 kDa, which are encoded by two overlapping genes, lacL and lacM, respectively. We have cloned these genes into the lactobacillal expression vectors pSIP403 and pSIP409, which are based on the sakacin P operon of L. sakei ( Sorvig et al. Microbiology 2005, 151, 2439- 2449 ), and expressed them in the host strains L. plantarum WCFS1 and L. sakei Lb790. Results varied considerably, ranging from 2.23 to 61.1 U/mg of beta-galactosidase activity, depending on the origin of the lacLM genes, the host strain, and the expression vector used. Highest expression levels were obtained in a laboratory cultivation of L. plantarum WCFS1 harboring the plasmid pEH3R containing the lacLM gene from L. reuteri L103. These cultivations yielded approximately 23 000 U of beta-galactosidase activity per liter, corresponding to the formation of roughly 100 mg of recombinant protein per liter of fermentation medium, and beta-galactosidase levels amounted to 55% of the total intracellular protein of the host organism. To further verify the suitability of this expression system, recombinant beta-galactosidase from L. reuteri was purified to apparent homogeneity. The properties of the purified enzyme were essentially identical with the properties of purified native beta-galactosidase from L. reuteri L103. The presented results lead the way to efficient overproduction of beta-galactosidase in a food-grade expression system, which is of high interest for applications in food industry.
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high level inducible gene expression in lactobacillus sakei and lactobacillus plantarum using versatile expression vectors
Microbiology, 2005Co-Authors: Kristine Naterstad, Vincent G H Eijsink, Elisabeth Sorvig, Geir Mathiesen, Lars AxelssonAbstract:Vectors have been developed for inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum in which expression of the gene of interest is driven by strong, regulated promoters from bacteriocin operons found in L. sakei strains. The activity of these promoters is controlled via a two-component signal transduction system, which responds to an externally added peptide pheromone. The vectors have a modular design, permitting easy exchange of all essential elements: the inducible promoter, the cognate regulatory system, the gene of interest, the antibiotic resistance marker and the replicon. Various variants of these so-called 'pSIP' vectors were constructed and tested, differing in terms of the bacteriocin regulon from which the regulatory elements were derived (sakacin A or sakacin P), the regulated promoter selected from these regulons, and the replicon (derived from p256 or pSH71). Using beta-glucuronidase (GusA) and aminopeptidase N (PepN) as reporters, it was shown that the best vectors permitted inducible, pheromone-dose-dependent gene expression at very high levels, while displaying moderate basal activities when not induced. The most effective set-up was obtained using a vector containing the pSH71 replicon, the orfX promoter from the sakacin P regulon, and the cognate regulatory genes, in a L. sakei host. GusA levels obtained with this set-up were approximately ten times higher than the levels obtained with prototype pSIP versions, whereas PepN levels amounted to almost 50% of total cellular protein.
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characterization of a new bacteriocin operon in sakacin p producing lactobacillus sakei showing strong translational coupling between the bacteriocin and immunity genes
Applied and Environmental Microbiology, 2005Co-Authors: Geir Mathiesen, Lars Axelsson, Kathrin Huehne, Lothar Kroeckel, Vincent G H EijsinkAbstract:ABSTRACT Previous studies of genes involved in the production of sakacin P by Lactobacillus sakei Lb674 revealed the presence of an inducible promoter downstream of the known spp gene clusters. We show here that this promoter drives the expression of an operon consisting of a bacteriocin gene (sppQ), a cognate immunity gene (spiQ), another gene with an unknown function (orf4), and a pseudoimmunity gene containing a frameshift mutation (orf5). The leader peptide of the new one-peptide bacteriocin sakacin Q contains consensus elements that are typical for so-called “double-glycine” leader peptides. The mature bacteriocin shows weak similarity to the BrcA peptide of the two-peptide bacteriocin brochocin C. Sakacin Q has an antimicrobial spectrum that differs from that of sakacin P, thus expanding the antimicrobial properties of the producer strain. The genes encoding sakacin Q and its cognate immunity protein showed strong translational coupling, which was investigated in detail by analyzing the properties of a series of β-glucuronidase fusions. Our results provide experimental evidence that production of the bacteriocin and production of the cognate immunity protein are tightly coregulated at the translational level.
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construction of vectors for inducible gene expression in lactobacillus sakei and l plantarum
Fems Microbiology Letters, 2003Co-Authors: Elisabeth Sorvig, Vincent G H Eijsink, Kristine Naterstad, Sonja Gronqvist, Geir Mathiesen, Lars AxelssonAbstract:We have constructed vectors for inducible expression of genes in Lactobacillus sakei and Lactobacillus plantarum. The key elements of these vectors are a regulatable promoter involved in the production of the bacteriocins sakacin A and sakacin P and the genes encoding the cognate histidine protein kinase and response regulator that are necessary to activate this promoter upon induction by a peptide pheromone. The vectors are built up of cassettes that permit easy exchange of all parts through restriction enzyme digestion and ligation. Using β-glucuronidase as a reporter enzyme, variants of these vectors were compared with each other, and with a corresponding system based on genes involved in the production of nisin. Several of the new vectors permitted tightly controlled and efficient expression of β-glucuronidase in both L. sakei and L. plantarum.
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functional characterization of a composite bacteriocin locus from malt isolate lactobacillus sakei 5
Applied and Environmental Microbiology, 2003Co-Authors: Anne Vaughan, Vincent G H Eijsink, Douwe Van SinderenAbstract:Lactobacillus sakei 5, isolated from malted barley, produces three bacteriocins. Genetic and functional analysis of the purified bacteriocins showed that this strain produces a plasmid-encoded bacteriocin that is identical to sakacin P, as well as two novel, chromosomally encoded bacteriocins, which were designated sakacin T and sakacin X. The structural genes specifying sakacin T and sakacin X are part of the sakacin TX locus, which consists of two adjacent but divergently oriented gene clusters. The first gene cluster includes stxP, stxR, stxK, and stxT, which, based on functional and comparative sequence analysis, are believed to encode an inducing peptide and proteins involved in regulation and secretion of these bacteriocins. The second gene cluster includes the structural and immunity genes for sakacin T, a class IIb two-peptide bacteriocin composed of SakTα and SakTβ, and sakacin X, a class IIa bacteriocin. Interestingly, a so-called transport accessory protein was absent from the locus, and based on our results it appears that a dedicated accessory protein is not required for processing and transport of sakacin T and sakacin X.
Dzung B Diep - One of the best experts on this subject based on the ideXlab platform.
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biotechnological potential and in vitro safety assessment of lactobacillus curvatus bcs35 a multibacteriocinogenic strain isolated from dry salted cod gadus morhua
Lwt - Food Science and Technology, 2019Co-Authors: Beatriz Gomezsala, Ingolf F Nes, Dzung B Diep, Estefania Munozatienza, Javier Feito, Rosa Del Campo, Carmen Herranz, Pablo E Hernandez, Luis M CintasAbstract:Abstract The antimicrobial activity of Lactobacillus curvatus BCS35 is due to the production of, at least, two class IIa bacteriocins: sakacin P-H12Y (SakP-H12Y), which is a sakacin P (SakP) variant with tyrosine (Tyr) instead of histidine (His) at position 12, and sakacin X (SakX). Moreover, DNA sequence analysis revealed the presence of the genes encoding the two-peptide bacteriocin sakacin T. In order to compare the specific antimicrobial activities of SakP-H12Y and SakX with other class IIa bacteriocins (SakP from Lactobacillus sakei LTH673 and pediocin PA-1 (PedPA-1) from Pediococcus acidilactici 347), and to determine if the substitution His12/Tyr12 in SakP-H12Y/SakP exerts any effect on the bacteriocin potency and/or specificity, these four bacteriocins were purified and challenged against different indicator strains. Our results showed that (i) PedPA-1 was, in general, the most active peptide, and (ii) SakP-H12Y, which contains one less positively charged residue in the N-terminal half (His12/Tyr12), has a reduced antimicrobial potency against most of the tested indicators compared to SaKP, confirming the importance of the N-terminal cationic patch of SakP-H12Y/SakP in target-cell binding, potency and specificity. The in vitro safety of Lb. curvatus BCS35 was established by antibiotic susceptibility testing concluding that it may be considered as a safe strain.
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The Potential of Class II Bacteriocins to Modify Gut Microbiota to Improve Host Health
PLOS ONE, 2016Co-Authors: Özgün C. O. Umu, Pablo E Hernandez, Christine Bäuerl, Marije Oostindjer, Phillip B. Pope, Gaspar Pérez-martínez, Dzung B DiepAbstract:Production of bacteriocins is a potential probiotic feature of many lactic acid bacteria (LAB) as it can help prevent the growth of pathogens in gut environments. However, knowledge on bacteriocin producers in situ and their function in the gut of healthy animals is still limited. In this study, we investigated five bacteriocin-producing strains of LAB and their isogenic non-producing mutants for probiotic values. The LAB bacteriocins, sakacin A (SakA), pediocin PA-1 (PedPA-1), enterocins P, Q and L50 (enterocins), plantaricins EF and JK (plantaricins) and garvicin ML (GarML), are all class II bacteriocins, but they differ greatly from each other in terms of inhibition spectrum and physicochemical properties. The strains were supplemented to mice through drinking water and changes on the gut microbiota composition were interpreted using 16S rRNA gene analysis. In general, we observed that overall structure of the gut microbiota remained largely unaffected by the treatments. However, at lower taxonomic levels, some transient but advantageous changes were observed. Some potentially problematic bacteria were inhibited (e.g., Staphylococcus by enterocins, Enterococcaceae by GarML, and Clostridium by plantaricins) and the proportion of LAB was increased in the presence of SakA-, plantaricins- and GarML-producing bacteria. Moreover, the treatment with GarML-producing bacteria co-occurred with decreased triglyceride levels in the host mice. Taken together, our results indicate that several of these bacteriocin producers have potential probiotic properties at diverse levels as they promote favorable changes in the host without major disturbance in gut microbiota, which is important for normal gut functioning.
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Cloning, production, and functional expression of the bacteriocin sakacin A (SakA) and two SakA-derived chimeras in lactic acid bacteria (LAB) and the yeasts Pichia pastoris and Kluyveromyces lactis
Journal of Industrial Microbiology & Biotechnology, 2013Co-Authors: Juan J. Jiménez, Dzung B Diep, Carmen Herranz, Luis M Cintas, Juan Borrero, Loreto Gútiez, Pablo E HernandezAbstract:Mature sakacin A (SakA, encoded by sapA ) and its cognate immunity protein (SakI, encoded by sapiA ), and two SakA-derived chimeras mimicking the N-terminal end of mature enterocin P (EntP/SakA) and mature enterocin A (EntA/SakA) together with SakI, were fused to different signal peptides (SP) and cloned into the protein expression vectors pNZ8048 and pMG36c for evaluation of their production and functional expression by different lactic acid bacteria. The amount, antimicrobial activity, and specific antimicrobial activity of SakA and its chimeras produced by Lactococcus lactis subsp. cremoris NZ9000 depended on the SP and the expression vector. Only L. lactis NZ9000 (pNUPS), producing EntP/SakA, showed higher bacteriocin production and antimicrobial activity than the natural SakA-producer Lactobacillus sakei Lb706. The lower antimicrobial activity of the SakA-producer L. lactis NZ9000 (pNUS) and that of the EntA/SakA-producer L. lactis NZ9000 (pNUAS) could be ascribed to secretion of truncated bacteriocins. On the other hand, of the Lb. sakei Lb706 cultures transformed with the pMG36c-derived vectors only Lb. sakei Lb706 (pGUS) overproducing SakA showed a higher antimicrobial activity than Lb. sakei Lb706. Finally, cloning of SakA and EntP/SakA into pPICZαA and pKLAC2 permitted the production of SakA and EntP/SakA by recombinant Pichia pastoris X-33 and Kluyveromyces lactis GG799 derivatives although their antimicrobial activity was lower than expected from their production.
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the synthesis of the bacteriocin sakacin a is a temperature sensitive process regulated by a pheromone peptide through a three component regulatory system
Microbiology, 2000Co-Authors: Dzung B Diep, Lars Axelsson, Camilla Grefsli, Ingolf F NesAbstract:Sakacin A is a bacteriocin produced by Lactobacillus sakei Lb706. The gene cluster (sap) encompasses a regulatory unit composed of three consecutive genes, orf4 and sapKR. sapKR encode a histidine protein kinase and a response regulator, while orf4 encodes the putative precursor of a 23-amino-acid cationic peptide (termed Sap-Ph). The authors show that Sap-Ph serves as a pheromone regulating bacteriocin production. Lb706 produced bacteriocin when the growth temperature was kept at 25 or 30 °C, but production was reduced or absent at higher temperatures (33·5–35 °C). Production was restored by lowering the growth temperature to 30 °C, but at temperatures of 33–34 °C also by adding exogenous Sap-Ph to the growth medium. A knock-out mutation in orf4 abolished sakacin A production. Exogenously added Sap-Ph complemented this mutation, unambiguously showing the essential role of this peptide for bacteriocin production. Another sakacin A producer, Lactobacillus curvatus LTH1174, had a similar response to temperature and exogenously added Sap-Ph.
