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Mounir Ferchichi - One of the best experts on this subject based on the ideXlab platform.
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in vitro assessment of the probiotic properties and bacteriocinogenic potential of pediococcus pentosaceus mzf16 isolated from artisanal tunisian meat dried ossban
Frontiers in Microbiology, 2018Co-Authors: Mohamed Zommiti, Emeline Bouffartigues, Olivier Maillot, Magalie Barreau, Sabine Szunerits, Khaled Sebei, Marc Feuilloley, Nathalie Connil, Mounir FerchichiAbstract:Pediococcus pentosaceus MZF16 has been isolated from artisanal Tunisian meat so called “Dried Ossban”, an original ecological niche, and identified by MALDI-TOF mass spectrometry and 16S rDNA sequencing. This bacterium showed a high tolerance to gastric stress conditions, and toward bile salts. P. pentosaceus MZF16 also demonstrated a hydrophobic surface profile (high adhesion to xylene), autoaggregation, and adhesive abilities to the human intestinal Caco-2/TC7 cell line. These properties may help the bacterium colonizing the gut. Furthermore, MZF16 was found to be resistant to gentamycin and chloramphenicol but did not harbor any transferable resistance determinants and/or virulence genes. The data also demonstrated absence of cytotoxicity of this strain. Conversely, P. pentosaceus MZF16 can slightly stimulate the immune system and enhance the intestinal epithelial barrier function. Moreover, this bacterium has been shown to be highly active against Listeria spp. due to bacteriocin production. Characterization of the bacteriocin by PCR amplification, sequencing and bioinformatic analyses revealed that MZF16 produces a bacteriocin 100% identical to Coagulin, a pediocin-like inhibitory substance produced by Bacillus coagulans. To our knowledge, this is the first report that highlights the production of a pediocin 100% identical to Coagulin in a Pediococcus strain. As Coagulin, pediocin MZF16 has the consensus sequence YYGNGVXCXXXXCXVXXXXA (X denotes any amino acid), which confirms its belonging to class IIa bacteriocins, and its suitability to preserve foods from Listeria monocytogenes development. According to these results, P. pentosaceus MZF16 can be proposed as a probiotic and bioprotective agent for fermented foods, including Tunisian dry meat and sausages. Further investigations will aim to study the behavior of this strain in meat products as a component of functional food.
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Image_1_In vitro Assessment of the Probiotic Properties and Bacteriocinogenic Potential of Pediococcus pentosaceus MZF16 Isolated From Artisanal Tunisian Meat “Dried Ossban”.TIF
2018Co-Authors: Mohamed Zommiti, Emeline Bouffartigues, Olivier Maillot, Magalie Barreau, Sabine Szunerits, Khaled Sebei, Marc Feuilloley, Nathalie Connil, Mounir FerchichiAbstract:Pediococcus pentosaceus MZF16 has been isolated from artisanal Tunisian meat so called “Dried Ossban,” an original ecological niche, and identified by MALDI-TOF mass spectrometry and 16S rDNA sequencing. This bacterium showed a high tolerance to gastric stress conditions, and toward bile salts. P. pentosaceus MZF16 also demonstrated a hydrophobic surface profile (high adhesion to xylene), autoaggregation, and adhesive abilities to the human intestinal Caco-2/TC7 cell line. These properties may help the bacterium colonizing the gut. Furthermore, MZF16 was found to be resistant to gentamycin and chloramphenicol but did not harbor any transferable resistance determinants and/or virulence genes. The data also demonstrated absence of cytotoxicity of this strain. Conversely, P. pentosaceus MZF16 can slightly stimulate the immune system and enhance the intestinal epithelial barrier function. Moreover, this bacterium has been shown to be highly active against Listeria spp. due to bacteriocin production. Characterization of the bacteriocin by PCR amplification, sequencing and bioinformatic analyses revealed that MZF16 produces a bacteriocin 100% identical to Coagulin, a pediocin-like inhibitory substance produced by Bacillus coagulans. To our knowledge, this is the first report that highlights the production of a pediocin 100% identical to Coagulin in a Pediococcus strain. As Coagulin, pediocin MZF16 has the consensus sequence YYGNGVXCXXXXCXVXXXXA (X denotes any amino acid), which confirms its belonging to class IIa bacteriocins, and its suitability to preserve foods from Listeria monocytogenes development. According to these results, P. pentosaceus MZF16 can be proposed as a probiotic and bioprotective agent for fermented foods, including Tunisian dry meat and sausages. Further investigations will aim to study the behavior of this strain in meat products as a component of functional food.
Ana Rodríguez - One of the best experts on this subject based on the ideXlab platform.
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listeriaphages and Coagulin c23 act synergistically to kill listeria monocytogenes in milk under refrigeration conditions
International Journal of Food Microbiology, 2015Co-Authors: Lorena Rodriguezrubio, Ana Rodríguez, Pilar Garcia, Craig Billington, Andrew J Hudson, Beatriz MartínezAbstract:Abstract Bacteriophages and bacteriocins are promising biocontrol tools in food. In this work, two Listeria bacteriophages, FWLLm1 and FWLLm3, were assessed in combination with the bacteriocin Coagulin C23 to inhibit Listeria monocytogenes. Preliminary results under laboratory conditions demonstrated that both antimicrobials act synergistically when they were applied in suboptimal concentrations. The combined approach was further assessed in milk contaminated with 5 × 104 CFU/ml L. monocytogenes 2000/47 and stored at 4 °C for 10 days. When used alone, phage FWLLm1 added at 5 × 106 PFU/ml, FWLLm3 at 5 × 105 PFU/ml and Coagulin C23 at 584 AU/ml kept L. monocytogenes 2000/47 counts lower than the untreated control throughout storage. However, when used in combination, inhibition was enhanced and in the presence of FWLLm1 and Coagulin C23, L. monocytogenes 2000/47 counts were under the detection limits (less than 10 CFU/ml) from day 4 until the end of the experiment. Resistant mutants towards phages and Coagulin C23 could be obtained, but cross-resistance was not detected. Mutants resistant to FWLLm3 and Coagulin C23 were also recovered from surviving colonies after cold storage in milk which may explain the failure of this combination to inhibit L. monocytogenes. Remarkably, the fraction of resistant mutants isolated from the combined treatment was lower than that from each antimicrobial alone, suggesting that synergy between bacteriocins and phages could be due to a lower rate of resistance development and the absence of cross-resistance.
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growth and bacteriocin production by lactic acid bacteria in vegetable broth and their effectiveness at reducing listeria monocytogenes in vitro and in fresh cut lettuce
Food Microbiology, 2007Co-Authors: Ana Allende, Victoria Selma, Juan E. Suárez, Beatriz Martínez, Ana RodríguezAbstract:Abstract The fresh-cut fruit and vegetable industry is searching for alternatives to replace chemical treatments with biopreservative approaches that ensure the safety of the product and fulfil consumer preferences for minimally processed foods. In this study, the use of bacteriocins produced by lactic acid bacteria has been tested as a substitute for chemical disinfection of fresh-cut iceberg lettuce. First, the ability of several non-plant origin bacteriocinogenic strains (nisin Z + , plantaricin C + , lacticin 481 + , Coagulin + or pediocin PA-1 + ) to grow in a lettuce extract at 4 °C, 10 °C and 32 °C was tested. All strains were able to grow, but bacteriocin production was predominantly detected at 32 °C. Addition of bacteriocinogenic supernatants (nisin + , Coagulin + and a nisin–Coagulin + cocktail) to tryptic-soy agar plates inoculated with Listeria monocytogenes reduced Listeria counts by approximately 1–1.5 log units compared with the control plates without bacteriocin, after 48 h of storage at 4 °C. The effect of washing with bacteriocin-containing solutions on survival and proliferation of Listeria monocytogenes was also evaluated in fresh-cut lettuce packaged in macro-perforated polypropylene bags and stored for 7 days at 4 °C. Washing fresh-cut lettuce with these solutions decreased the viability of Listeria monocytogenes by 1.2–1.6 log units immediately after treatment, but, during storage at 4 °C, bacteriocin treatments only exerted minimal control over the growth of the pathogen. Natural microbiota were little affected by bacteriocins during storage.
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growth and bacteriocin production by lactic acid bacteria in vegetable broth and their effectiveness at reducing listeria monocytogenes in vitro and in fresh cut lettuce
Food Microbiology, 2007Co-Authors: Ana Allende, Victoria Selma, Juan E. Suárez, Beatriz Martínez, Maria I Gil, Ana RodríguezAbstract:The fresh-cut fruit and vegetable industry is searching for alternatives to replace chemical treatments with biopreservative approaches that ensure the safety of the product and fulfil consumer preferences for minimally processed foods. In this study, the use of bacteriocins produced by lactic acid bacteria has been tested as a substitute for chemical disinfection of fresh-cut iceberg lettuce. First, the ability of several non-plant origin bacteriocinogenic strains (nisin Z(+), plantaricin C(+), lacticin 481(+), Coagulin(+) or pediocin PA-1(+)) to grow in a lettuce extract at 4 degrees C, 10 degrees C and 32 degrees C was tested. All strains were able to grow, but bacteriocin production was predominantly detected at 32 degrees C. Addition of bacteriocinogenic supernatants (nisin(+), Coagulin(+) and a nisin-Coagulin(+) cocktail) to tryptic-soy agar plates inoculated with Listeria monocytogenes reduced Listeria counts by approximately 1-1.5 log units compared with the control plates without bacteriocin, after 48 h of storage at 4 degrees C. The effect of washing with bacteriocin-containing solutions on survival and proliferation of Listeria monocytogenes was also evaluated in fresh-cut lettuce packaged in macro-perforated polypropylene bags and stored for 7 days at 4 degrees C. Washing fresh-cut lettuce with these solutions decreased the viability of Listeria monocytogenes by 1.2-1.6 log units immediately after treatment, but, during storage at 4 degrees C, bacteriocin treatments only exerted minimal control over the growth of the pathogen. Natural microbiota were little affected by bacteriocins during storage.
Peter B Armstrong - One of the best experts on this subject based on the ideXlab platform.
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Decoration of the extracellular blood clot by lipopolysaccharide (LPS).
2013Co-Authors: Margaret T. Armstrong, Frederick R. Rickles, Peter B ArmstrongAbstract:FITC-LPS (E. coli O55:B5) decorates the fibrin fibrils of the human clot prepared from platelet-depleted plasma (Fig 1A), the plasma lipoprotein clot of the lobster (Fig 1B), and the Coagulin fibrils of the Limulus clot (Fig. 1C). The Limulus clot shown in Fig 1C was also immunostained with a rabbit anti-Coagulin antibody and DyLight 549 Goat anti-rabbit whole IgG second antibody to show the location of the Coagulin fibrils of the blood clot. Figure 1D shows the same field of the Limulus blood clot as is shown in Figure 1C but illuminated with the rhodamine filter set to show the DyLight 549 signal, demonstrating the co-localization of LPS and the Coagulin structural protein of the clot. The lipid A core of LPS is sufficient for binding to the fibrin clot because a form of LPS that lacks carbohydrate, LPS of S. minnesota R595 (Re), also binds to the fibrin clot (Fig. 1E). Here, biodipy-conjugated polymyxin B, a LPS-binding probe, was used as the fluorescent reporter for the localization of LPS. Fibrin clots prepared by thrombin-mediated clotting of pure human fibrinogen (Hematologic Technologies) also decorate with FITC-LPS (E. coli O55:B5) (Fig. 1F). The micrographs were produced with a Zeiss Axioimager microscope with a 40/0.75 objective. These representative micrographs are drawn from as many as 10 replicates (Fig. 1A) to as few as 2 replicates (Fig. 1B). Scale bar, 50 µm.
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Derivitization of the ε-amino groups of lysine and arginine reduces capture of LPS by the blood clot.
2013Co-Authors: Margaret T. Armstrong, Frederick R. Rickles, Peter B ArmstrongAbstract:Treatment of the fibrin clot with TNBS to derivitize the ε-amino groups of lysine reduces capture of FITC-LPS (Fig. A1, untreated control fibrin clot, fluorescent LPS; Fig. A2, TNBS-treated fibrin clot, fluorescent LPS; Fig A3, same field as Fig A2, phase contrast). Similarly, treatment of the fibrin clot with PGO to derivitize the ε-amino groups of arginine (Fig. B1, untreated control fibrin clot, fluorescent LPS; Fig B2, PGO-treated fibrin clot, fluorescent LPS; Fig. B3, same field as Fig B2, phase contrast) also reduces LPS capture by the clot fibrils. Treatment of the Coagulin clot of Limulus with PGO reduces capture of LPS by the clot (Fig. C1, untreated control Coagulin clot; Fig. C2, PGO-treated Coagulin clot). The PGO-treated specimen shows faint fluorescence of the nuclei of the underlying blood cells but no fluorescence of the overlying Coagulin clot. TNBS treatment causes cytolysis of the Limulus blood cells and separation of the Coagulin clot from the petri dish surface, preventing assessment of its effects on LPS capture in this system.
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blood collection from the american horseshoe crab limulus polyphemus
Journal of Visualized Experiments, 2008Co-Authors: Peter B Armstrong, Mara ConradAbstract:The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and alpha2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, Coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.
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response of the blood clotting system of the american horseshoe crab limulus polyphemus to a novel form of lipopolysaccharide from a green alga
Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, 2006Co-Authors: Mara L Conrad, R L Pardy, Norman R Wainwright, Alice Child, Peter B ArmstrongAbstract:Lipopolysaccharide (LPS, endotoxin) is a component of Gram-negative bacteria and is the principal indicator to the innate immune systems of higher animals of a Gram-negative bacterial invasion. LPS activates the blood clotting system of the American horseshoe crab, Limulus polyphemus. By stimulating blood cell degranulation, LPS triggers the release of the proteins of the clotting system from the cells, and by activating a protease cascade that converts coagulogen, a soluble zymogen, to Coagulin, the structural protein of the clot, LPS triggers the production of the fibrillar Coagulin blood clot. Although originally thought to be restricted to the Gram-negative bacteria and the cyanobacteria, LPS, or a very similar molecule, has recently been described from a eukaryotic green alga, Chlorella. Here we show that, like LPS from Gram-negative bacteria, the algal molecule stimulates exocytosis of the Limulus blood cell and the clotting of Coagulin. The Coagulin clot efficiently entraps the cells of Chlorella in a network of fibrils. Invasion and erosion of the carapace by green algae is an important cause of mortality of Limulus, and it is suggested that the cellular response to aLPS may contribute to defense against this pathogen.
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association of alpha2 macroglobulin with the Coagulin clot in the american horseshoe crab limulus polyphemus a potential role in stabilization from proteolysis
The Biological Bulletin, 2000Co-Authors: Rengasamy Asokan, M T Armstrong, Peter B ArmstrongAbstract:cells such as mammalian erythrocytes, may contribute to this activity. Under normal conditions, the volume of cuticular secretion is low, but it can be detected by immunological assays. Under conditions of challenge by a polluted environment, the volume of the secretion is augmented. In addition to its anti-biological activity, the continuous production of the cuticular secretion can be expected to exert a mechanical action, entrapping and sweeping potential fouling organisms away from the solid surface of the cuticle. Contrary to previous suggestions (2), the cuticular secretion does not contain secretion products of the blood cells. Supported by Grant No. MCB-97-26771 from the National Science Foundation. We thank Ms. Yvonne Coursey for the anticoagulogen antiserum. Literature Cited
Juan E. Suárez - One of the best experts on this subject based on the ideXlab platform.
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growth and bacteriocin production by lactic acid bacteria in vegetable broth and their effectiveness at reducing listeria monocytogenes in vitro and in fresh cut lettuce
Food Microbiology, 2007Co-Authors: Ana Allende, Victoria Selma, Juan E. Suárez, Beatriz Martínez, Ana RodríguezAbstract:Abstract The fresh-cut fruit and vegetable industry is searching for alternatives to replace chemical treatments with biopreservative approaches that ensure the safety of the product and fulfil consumer preferences for minimally processed foods. In this study, the use of bacteriocins produced by lactic acid bacteria has been tested as a substitute for chemical disinfection of fresh-cut iceberg lettuce. First, the ability of several non-plant origin bacteriocinogenic strains (nisin Z + , plantaricin C + , lacticin 481 + , Coagulin + or pediocin PA-1 + ) to grow in a lettuce extract at 4 °C, 10 °C and 32 °C was tested. All strains were able to grow, but bacteriocin production was predominantly detected at 32 °C. Addition of bacteriocinogenic supernatants (nisin + , Coagulin + and a nisin–Coagulin + cocktail) to tryptic-soy agar plates inoculated with Listeria monocytogenes reduced Listeria counts by approximately 1–1.5 log units compared with the control plates without bacteriocin, after 48 h of storage at 4 °C. The effect of washing with bacteriocin-containing solutions on survival and proliferation of Listeria monocytogenes was also evaluated in fresh-cut lettuce packaged in macro-perforated polypropylene bags and stored for 7 days at 4 °C. Washing fresh-cut lettuce with these solutions decreased the viability of Listeria monocytogenes by 1.2–1.6 log units immediately after treatment, but, during storage at 4 °C, bacteriocin treatments only exerted minimal control over the growth of the pathogen. Natural microbiota were little affected by bacteriocins during storage.
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growth and bacteriocin production by lactic acid bacteria in vegetable broth and their effectiveness at reducing listeria monocytogenes in vitro and in fresh cut lettuce
Food Microbiology, 2007Co-Authors: Ana Allende, Victoria Selma, Juan E. Suárez, Beatriz Martínez, Maria I Gil, Ana RodríguezAbstract:The fresh-cut fruit and vegetable industry is searching for alternatives to replace chemical treatments with biopreservative approaches that ensure the safety of the product and fulfil consumer preferences for minimally processed foods. In this study, the use of bacteriocins produced by lactic acid bacteria has been tested as a substitute for chemical disinfection of fresh-cut iceberg lettuce. First, the ability of several non-plant origin bacteriocinogenic strains (nisin Z(+), plantaricin C(+), lacticin 481(+), Coagulin(+) or pediocin PA-1(+)) to grow in a lettuce extract at 4 degrees C, 10 degrees C and 32 degrees C was tested. All strains were able to grow, but bacteriocin production was predominantly detected at 32 degrees C. Addition of bacteriocinogenic supernatants (nisin(+), Coagulin(+) and a nisin-Coagulin(+) cocktail) to tryptic-soy agar plates inoculated with Listeria monocytogenes reduced Listeria counts by approximately 1-1.5 log units compared with the control plates without bacteriocin, after 48 h of storage at 4 degrees C. The effect of washing with bacteriocin-containing solutions on survival and proliferation of Listeria monocytogenes was also evaluated in fresh-cut lettuce packaged in macro-perforated polypropylene bags and stored for 7 days at 4 degrees C. Washing fresh-cut lettuce with these solutions decreased the viability of Listeria monocytogenes by 1.2-1.6 log units immediately after treatment, but, during storage at 4 degrees C, bacteriocin treatments only exerted minimal control over the growth of the pathogen. Natural microbiota were little affected by bacteriocins during storage.
Maria C Urdaci - One of the best experts on this subject based on the ideXlab platform.
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biochemical and genetic characterization of Coagulin a new antilisterial bacteriocin in the pediocin family of bacteriocins produced by bacillus coagulans i4
Applied and Environmental Microbiology, 2000Co-Authors: Claire Le Marrec, Bertrand Hyronimus, Philippe Bressollier, Bernard Verneuil, Maria C UrdaciAbstract:A plasmid-linked antimicrobial peptide, named Coagulin, produced by Bacillus coagulans I4 has recently been reported (B. Hyronimus, C. Le Marrec and M. C. Urdaci, J. Appl. Microbiol. 85:42–50, 1998). In the present study, the complete, unambiguous primary amino acid sequence of the peptide was obtained by a combination of both N-terminal sequencing of purified peptide and the complete sequence deduced from the structural gene harbored by plasmid I4. Data revealed that this peptide of 44 residues has an amino acid sequence similar to that described for pediocins AcH and PA-1, produced by different Pediococcus acidilactici strains and 100% identical. Coagulin and pediocin differed only by a single amino acid at their C terminus. Analysis of the genetic determinants revealed the presence, on the pI4 DNA, of the entire 3.5-kb operon of four genes described for pediocin AcH and PA-1 production. No extended homology was observed between pSMB74 from P. acidilactici and pI4 when analyzing the regions upstream and downstream of the operon. An oppositely oriented gene immediately dowstream of the bacteriocin operon specifies a 474-amino-acid protein which shows homology to Mob-Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from gram-positive bacteria. This is the first report of a pediocin-like peptide appearing naturally in a non-lactic acid bacterium genus.
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Coagulin a bacteriocin like inhibitory substance produced by bacillus coagulans i4
Journal of Applied Microbiology, 1998Co-Authors: Bertrand Hyronimus, Le C Marrec, Maria C UrdaciAbstract:A protease-sensitive antibacterial substance produced by Bacillus coagulans I4 strain, isolated from cattle faeces, was classified as a bacteriocin-like inhibitory substance and named Coagulin. The inhibitory spectrum included B. coagulans and unrelated bacteria such as Enterococcus, Leuconostoc, Oenococcus, Listeria and Pediococcus. Coagulin was stable at 60 °C for 90 min, at a pH ranging from 4 to 8 and appeared to be unaffected by α-amylase, lipase or organic solvents (10% v/v). Coagulin exhibited a bactericidal and a bacteriolytic mode of action against indicator cells. The apparent molecular mass was estimated to be about 3–4 kDa by SDS-PAGE. The B. coagulans I4 strain harbours a plasmid, pI4, approximately 14 kb in size. Novobiocin curing experiments yielded two derivatives that no longer produced the bacteriocin-like inhibitory substance. Plasmid content of these two derivatives showed that one had lost pI4,whereas the second harboured a deleted form of this plasmid, thus suggesting a plasmid location for the genes for Coagulin production.
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Coagulin a bacteriocin like inhibitory substance produced by bacillus coagulans i4
Journal of Applied Microbiology, 1998Co-Authors: Bertrand Hyronimus, Le C Marrec, Maria C UrdaciAbstract:A protease-sensitive antibacterial substance produced by Bacillus coagulans I4 strain, isolated from cattle faeces, was classified as a bacteriocin-like inhibitory substance and named Coagulin. The inhibitory spectrum included B. coagulans and unrelated bacteria such as Enterococcus, Leuconostoc, Oenococcus, Listeria and Pediococcus. Coagulin was stable at 60 degrees C for 90 min, at a pH ranging from 4 to 8 and appeared to be unaffected by alpha-amylase, lipase or organic solvents (10% v/v). Coagulin exhibited a bactericidal and a bacteriolytic mode of action against indicator cells. The apparent molecular mass was estimated to be about 3-4 kDa by SDS-PAGE. The B. coagulans I4 strain harbours a plasmid, pI4, approximately 14 kb in size. Novobiocin curing experiments yielded two derivatives that no longer produced the bacteriocin-like inhibitory substance. Plasmid content of these two derivatives showed that one had lost pI4, whereas the second harboured a deleted form of this plasmid, thus suggesting a plasmid location for the genes for Coagulin production.