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Jean Marc Chatel - One of the best experts on this subject based on the ideXlab platform.
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Plasmid Transfer efficiency using Lactoccocus lactis strains depends on invasiveness status but also on Plasmid copy number
FEMS Microbiology Letters, 2017Co-Authors: Denis Mariat, Philippe Langella, Véronique Robert, Jean Marc ChatelAbstract:Lactic acid bacteria as Lactococcus lactis are used as vector for protein but also DNA delivery into intestinal cells in vitro and in vivo. For the Plasmid delivery strategy, the Plasmid copy number per bacteria (PCN) is thus of great importance. The aim of this paper is to determine the physiological conditions when PCN is the highest in the bacteria. PCN was characterized by qPCR in five different recombinant Lactococcus lactis strains, containing one (mono-) or two different Plasmids (biPlasmidic), at exponential or stationary phase. We showed that in all cases but one, PCN is higher at exponential than stationary phase. PCN seems to depend on (i) monoPlasmidic or biPlasmidic strain; (ii) origin of replication of the Plasmid; and (iii) the DNA load of the bacteria. Then we studied Plasmid Transfer in vitro from recombinant L. lactis to eukaryotic COS-7 cells using culture at exponential or stationary phase. We showed that Plasmid Transfer can be improved in vitro by using bacteria at exponential phase.
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Surface display of an anti-DEC-205 single chain Fv fragment in Lactobacillus plantarum increases internalization and Plasmid Transfer to dendritic cells in vitro and in vivo
Microbial Cell Factories, 2015Co-Authors: Christophe Michon, Philippe Langella, Katarzyna Kuczkowska, Vincent G. H. Eijsink, Geir Mathiesen, Jean Marc ChatelAbstract:Background: Lactic acid bacteria (LAB) are promising vehicles for delivery of a variety of medicinal compounds, including antigens and cytokines. It has also been established that Lare able to deliver cDNA to host cells. To increase the efficiency of LAB-driven DNA delivery we have constructed Lactobacillus plantarum strains targeting DEC-205, which is a receptor located at the surface of dendritic cells (DCs). The purpose was to increase uptake of bacterial cells, which could lead to improved cDNA delivery to immune cells. Results: Anti-DEC-205 antibody (aDec) was displayed at the surface of L. plantarum using three different anchoring strategies: (1) covalent anchoring of aDec to the cell membrane (Lipobox domain, Lip); (2) covalent anchoring to the cell wall (LPXTG domain, CWA); (3) non-covalent anchoring to the cell wall (LysM domain, LysM). aDec was successfully expressed in all three strains, but surface location of the antibody could only be demonstrated for the two strains with cell wall anchors (CWA and LysM). Co-incubation of the engineered strains and DCs showed increased uptake when anchoring aDec using the CWA or LysM anchors. In a competition assay, free anti-DEC abolished the increased uptake, showing that the internalization is due to specific interactions between the DEC-205 receptor and aDec. To test Plasmid Transfer, a Plasmid for expression of GFP under control of an eukaryotic promoter was transformed into the aDec expressing strains and GFP expression in DCs was indeed increased when using the strains producing cell-wall anchored aDec. Plasmid Transfer to DCs in the gastro intestinal tract was also detected using a mouse model. Surprisingly, in mice the highest expression of GFP was observed for the strain in which aDec was coupled to the cell membrane. Conclusion: The results show that surface expression of aDec leads to increased internalization of L. plantarum and Plasmid Transfer in DCs and that efficiency depends on the type of anchor used. Interestingly, in vitro data indicates that cell wall anchoring is more effective, whereas in vivo data seem to indicate that anchoring to the cell membrane is preferable. It is likely that the more embedded localization of aDec in the latter case is favorable when cells are exposed to the harsh conditions of the gastro-intestinal tract.
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Erratum to "Surface display of an anti-DEC-205 single chain Fv fragment in Lactobacillus plantarum increases internalization and Plasmid Transfer to dendritic cells in vitro and in vivo
BMC Public Health, 2015Co-Authors: Christophe Michon, Philippe Langella, Katarzyna Kuczkowska, Vincent G. H. Eijsink, Geir Mathiesen, Jean Marc ChatelAbstract:Background: Lactic acid bacteria (LAB) are promising vehicles for delivery of a variety of medicinal compounds, including antigens and cytokines. It has also been established that Lare able to deliver cDNA to host cells. To increase the efficiency of LAB-driven DNA delivery we have constructed Lactobacillus plantarum strains targeting DEC-205, which is a receptor located at the surface of dendritic cells (DCs). The purpose was to increase uptake of bacterial cells, which could lead to improved cDNA delivery to immune cells. Results: Anti-DEC-205 antibody (aDec) was displayed at the surface of L. plantarum using three different anchoring strategies: (1) covalent anchoring of aDec to the cell membrane (Lipobox domain, Lip); (2) covalent anchoring to the cell wall (LPXTG domain, CWA); (3) non-covalent anchoring to the cell wall (LysM domain, LysM). aDec was successfully expressed in all three strains, but surface location of the antibody could only be demonstrated for the two strains with cell wall anchors (CWA and LysM). Co-incubation of the engineered strains and DCs showed increased uptake when anchoring aDec using the CWA or LysM anchors. In a competition assay, free anti-DEC abolished the increased uptake, showing that the internalization is due to specific interactions between the DEC-205 receptor and aDec. To test Plasmid Transfer, a Plasmid for expression of GFP under control of an eukaryotic promoter was transformed into the aDec expressing strains and GFP expression in DCs was indeed increased when using the strains producing cell-wall anchored aDec. Plasmid Transfer to DCs in the gastro intestinal tract was also detected using a mouse model. Surprisingly, in mice the highest expression of GFP was observed for the strain in which aDec was coupled to the cell membrane. Conclusion: The results show that surface expression of aDec leads to increased internalization of L. plantarum and Plasmid Transfer in DCs and that efficiency depends on the type of anchor used. Interestingly, in vitro data indicates that cell wall anchoring is more effective, whereas in vivo data seem to indicate that anchoring to the cell membrane is preferable. It is likely that the more embedded localization of aDec in the latter case is favorable when cells are exposed to the harsh conditions of the gastro-intestinal tract.
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Correlation between fibronectin binding protein A expression level at the surface of recombinant lactococcus lactis and Plasmid Transfer in vitro and in vivo
BMC Microbiology, 2014Co-Authors: Juliana F Almeida, Denis Mariat, Alejandra De Moreno De Leblanc, Silvina Del Carmen, Anderson Miyoshi, Vasco Azevedo, Rebeca San Martin, Jean Guy Leblanc, Philippe Langella, Jean Marc ChatelAbstract:BACKGROUND: Fibronectin Binding Protein A (FnBPA) is an invasin from Staphylococcus aureus that allows this pathogen to internalize into eukaryote cells. It was previously demonstrated that recombinant Lactococcus lactis expressing FnBPA were invasive and able to Transfer a Plasmid to eukaryotic cells in vitro and in vivo. In this study, the invasivity of recombinant strains of Lactococcus lactis that express FnBPA under the control of its constitutive promoter or driven by the strong nisin inducible expression system (NICE) were studied.\n\nRESULTS: It was demonstrated that the nisA promoter allows an increase of FnBPA expression on the surface of Lactococcus lactis surface, as shown by flow cytometry, which subsequently enhanced internalization and Plasmid Transfer properties in vitro in Caco2 cells and Bone Marrow Dendritic Cells. In vivo, the use of nisA promoter increase the Plasmid Transfer in cells of both the small and large intestine of mice.\n\nCONCLUSION: FnBPA expression at the surface of recombinant L. lactis is positively correlated to internalization and DNA Transfer properties. The recombinant strains of L. lactis that expresses FnBPA under the control of the nisin inducible expression system could thus be considered as an improved tool in the field of DNA Transfer.
Jon R. Saunders - One of the best experts on this subject based on the ideXlab platform.
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Rule-based modelling of conjugative Plasmid Transfer and incompatibility.
Bio Systems, 2007Co-Authors: Richard Gregory, Jon R. Saunders, Venetia A. SaundersAbstract:COSMIC-rules, an individual-based model for bacterial adaptation and evolution, has been used to study virtual transmission of Plasmids within bacterial populations, in an environment varying between supportive and inhibitory. The simulations demonstrate spread of antibiotic resistance (R) Plasmids, both compatible and incompatible, by the bacterial gene Transfer process of conjugation. This paper describes the behaviour of virtual Plasmids, their modes of exchange within bacterial populations and the impact of antibiotics, together with the rules governing Plasmid Transfer. Three case studies are examined: Transfer of an R Plasmid within an antibiotic-susceptible population, Transfer of two incompatible R Plasmids and Transfer of two compatible R Plasmids. R Plasmid Transfer confers antibiotic resistance on recipients. For incompatible Plasmids, one or other Plasmid could be maintained in bacterial cells and only that portion of the population acquiring the appropriate Plasmid-encoded resistance survives exposure to the antibiotics. By contrast, the compatible Plasmids Transfer and mix freely within the bacterial population that survives in its entirety in the presence of the antibiotics. These studies are intended to inform models for examining adaptive evolution in bacteria. They provide proof of principle in simple systems as a platform for predicting the behaviour of bacterial populations in more complex situations, for example in response to changing environments or in multi-species bacterial assemblages.
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Plasmid Transfer between Bacillus thuringiensis subsp. israelensis strains in laboratory culture, river water, and dipteran larvae.
Applied and Environmental Microbiology, 2001Co-Authors: D. John I. Thomas, John M. Whipps, J. Alun W. Morgan, Jon R. SaundersAbstract:Bacillus thuringiensis is a gram-positive bacterium that produces insecticidal crystal protein toxins during sporulation. B. thuringiensis was first discovered in diseased silkworms in 1901 (19, 31) but has since been isolated from a range of environments, including insects, soil, dust from stored grain, and leaves of coniferous and deciduous trees (7, 24, 25, 28). In 1977, B. thuringiensis subsp. israelensis was isolated from a mosquito-breeding pond in the Negev Desert of Israel and was found to be highly active towards dipteran larvae (16). A number of insecticidal protein toxins that are encoded on a single 72-MDa Plasmid have been identified in B. thuringiensis subsp. israelensis. Movement of Plasmids within B. thuringiensis strains has been proposed to be the main mechanism for generating diversity in toxin genes. In addition to Plasmids carrying insecticidal toxin genes, many other Plasmids, such as pXO11, pXO13, pXO14, pXO15, pXO16, and pAW63, have been detected in B. thuringiensis, and these Plasmids have no known function apart from their conjugative ability (6, 26, 32). Strains of B. thuringiensis can have as few as one Plasmid to more than six Plasmids. Movement of Plasmids between B. thuringiensis strains has been of interest in the construction of strains with novel toxin combinations, the study of the mechanisms of Plasmid Transfer in bacteria, and the study of the evolution of toxin combinations. Furthermore, since B. thuringiensis is closely related to the human pathogens Bacillus anthracis and Bacillus cereus, interest has been focused on the genetic exchange systems of these bacteria in relation to biosafety (22). Plasmid movement between B. thuringiensis strains can be monitored directly by Plasmid profiling analysis of cells. This is possible when Transfer frequencies are high and all recipients in a population become transconjugants. When the Transfer frequency is lower, a shuttle Plasmid with an antibiotic resistance gene can be used to monitor the movement of conjugative Plasmids (3). The shuttle Plasmids are either mobilizable or oriT bearing, such as pBC16. Differences in the abilities of conjugative Plasmids to move the reporter Plasmids have been demonstrated. Green et al. (17) transposon tagged conjugative Plasmid pXO12 and demonstrated that it Transferred itself and pBC16 at similar rates (10−2 transconjugant per recipient). In contrast, transposon tagging of Plasmid pXO16 showed that it was Transferred to every recipient cell but mobilized pBC16 only at a frequency of approximately 10−3 to 10−4 transconjugant per recipient (1). Very few studies have examined Plasmid Transfer between B. thuringiensis strains and, in particular, between B. thuringiensis subsp. israelensis strains in the environment. Haack et al. (18) reported Transfer of the broad-host-range conjugative transposon Tn916 between Bacillus subtilis and B. thuringiensis subsp. israelensis in nonsterile sandy soil. Significantly, although B. thuringiensis is applied to waterways to control pests such as mosquitoes and blackflies, no studies have examined Plasmid Transfer between B. thuringiensis strains in water yet. In nature B. thuringiensis strains may find themselves in the midgut environment of killed susceptible larvae. This environment has proven to be conducive to gene exchange. Studies have found that Plasmids can move between donor and recipient strains of B. thuringiensis in larvae of the lepidopteran insects Galleria mellonella, Spodoptera littoralis, and Lacanobia oleracea at levels similar to that found in broth culture (20, 30) but not in the midgut environment of the coleopteran insect Phaedon cochleriae. So far, there have been no studies of Transfer between B. thuringiensis strains in dipteran insects. The primary objective of this study was to obtain information regarding mobilization and Transfer of Plasmids between B. thuringiensis subsp. israelensis strains under environmentally realistic conditions. Insecticidal strains of B. thuringiensis subsp. israelensis were constructed with either a mobilizable Plasmid (pBC16) or a transposon-tagged conjugative Plasmid (pXO16::Tn5401) so that gene Transfer in laboratory media, water, and insects could be studied.
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Plasmid Transfer between the Bacillus thuringiensis subspecies kurstaki and tenebrionis in laboratory culture and soil and in lepidopteran and coleopteran larvae.
Applied and Environmental Microbiology, 2000Co-Authors: D. J. I. Thomas, J. A. W. Morgan, John M. Whipps, Jon R. SaundersAbstract:Plasmid Transfer between Bacillus thuringiensis subsp. kurstaki HD1 and B. thuringiensis subsp. tenebrionis donor strains and a streptomycin-resistant B. thuringiensis subsp. kurstaki recipient was studied under environmentally relevant laboratory conditions in vitro, in soil, and in insects. Plasmid Transfer was detected in vitro at temperatures of 5 to 37 degrees C, at pH 5.9 to 9.0, and at water activities of 0.965 to 0.995, and the highest Transfer ratios (up to 10(-1) transconjugant/donor) were detected within 4 h. In contrast, no Plasmid Transfer was detected in nonsterile soil, and rapid formation of spores by the introduced strains probably contributed most to the lack of Plasmid Transfer observed. When a B. thuringiensis subsp. kurstaki strain was used as the donor strain, Plasmid Transfer was detected in killed susceptible lepidopteran insect (Lacanobia oleracea) larvae but not in the nonsusceptible coleopteran insect Phaedon chocleriae. When a B. thuringiensis subsp. tenerbrionis strain was used as the donor strain, no Plasmid Transfer was detected in either of these insects even when they were killed. These results show that in larger susceptible lepidopteran insects there is a greater opportunity for growth of B. thuringiensis strains, and this finding, combined with decreased competition due to a low initial background bacterial population, can provide suitable conditions for efficient Plasmid Transfer in the environment.
Philippe Langella - One of the best experts on this subject based on the ideXlab platform.
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Plasmid Transfer efficiency using Lactoccocus lactis strains depends on invasiveness status but also on Plasmid copy number
FEMS Microbiology Letters, 2017Co-Authors: Denis Mariat, Philippe Langella, Véronique Robert, Jean Marc ChatelAbstract:Lactic acid bacteria as Lactococcus lactis are used as vector for protein but also DNA delivery into intestinal cells in vitro and in vivo. For the Plasmid delivery strategy, the Plasmid copy number per bacteria (PCN) is thus of great importance. The aim of this paper is to determine the physiological conditions when PCN is the highest in the bacteria. PCN was characterized by qPCR in five different recombinant Lactococcus lactis strains, containing one (mono-) or two different Plasmids (biPlasmidic), at exponential or stationary phase. We showed that in all cases but one, PCN is higher at exponential than stationary phase. PCN seems to depend on (i) monoPlasmidic or biPlasmidic strain; (ii) origin of replication of the Plasmid; and (iii) the DNA load of the bacteria. Then we studied Plasmid Transfer in vitro from recombinant L. lactis to eukaryotic COS-7 cells using culture at exponential or stationary phase. We showed that Plasmid Transfer can be improved in vitro by using bacteria at exponential phase.
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Surface display of an anti-DEC-205 single chain Fv fragment in Lactobacillus plantarum increases internalization and Plasmid Transfer to dendritic cells in vitro and in vivo
Microbial Cell Factories, 2015Co-Authors: Christophe Michon, Philippe Langella, Katarzyna Kuczkowska, Vincent G. H. Eijsink, Geir Mathiesen, Jean Marc ChatelAbstract:Background: Lactic acid bacteria (LAB) are promising vehicles for delivery of a variety of medicinal compounds, including antigens and cytokines. It has also been established that Lare able to deliver cDNA to host cells. To increase the efficiency of LAB-driven DNA delivery we have constructed Lactobacillus plantarum strains targeting DEC-205, which is a receptor located at the surface of dendritic cells (DCs). The purpose was to increase uptake of bacterial cells, which could lead to improved cDNA delivery to immune cells. Results: Anti-DEC-205 antibody (aDec) was displayed at the surface of L. plantarum using three different anchoring strategies: (1) covalent anchoring of aDec to the cell membrane (Lipobox domain, Lip); (2) covalent anchoring to the cell wall (LPXTG domain, CWA); (3) non-covalent anchoring to the cell wall (LysM domain, LysM). aDec was successfully expressed in all three strains, but surface location of the antibody could only be demonstrated for the two strains with cell wall anchors (CWA and LysM). Co-incubation of the engineered strains and DCs showed increased uptake when anchoring aDec using the CWA or LysM anchors. In a competition assay, free anti-DEC abolished the increased uptake, showing that the internalization is due to specific interactions between the DEC-205 receptor and aDec. To test Plasmid Transfer, a Plasmid for expression of GFP under control of an eukaryotic promoter was transformed into the aDec expressing strains and GFP expression in DCs was indeed increased when using the strains producing cell-wall anchored aDec. Plasmid Transfer to DCs in the gastro intestinal tract was also detected using a mouse model. Surprisingly, in mice the highest expression of GFP was observed for the strain in which aDec was coupled to the cell membrane. Conclusion: The results show that surface expression of aDec leads to increased internalization of L. plantarum and Plasmid Transfer in DCs and that efficiency depends on the type of anchor used. Interestingly, in vitro data indicates that cell wall anchoring is more effective, whereas in vivo data seem to indicate that anchoring to the cell membrane is preferable. It is likely that the more embedded localization of aDec in the latter case is favorable when cells are exposed to the harsh conditions of the gastro-intestinal tract.
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Erratum to "Surface display of an anti-DEC-205 single chain Fv fragment in Lactobacillus plantarum increases internalization and Plasmid Transfer to dendritic cells in vitro and in vivo
BMC Public Health, 2015Co-Authors: Christophe Michon, Philippe Langella, Katarzyna Kuczkowska, Vincent G. H. Eijsink, Geir Mathiesen, Jean Marc ChatelAbstract:Background: Lactic acid bacteria (LAB) are promising vehicles for delivery of a variety of medicinal compounds, including antigens and cytokines. It has also been established that Lare able to deliver cDNA to host cells. To increase the efficiency of LAB-driven DNA delivery we have constructed Lactobacillus plantarum strains targeting DEC-205, which is a receptor located at the surface of dendritic cells (DCs). The purpose was to increase uptake of bacterial cells, which could lead to improved cDNA delivery to immune cells. Results: Anti-DEC-205 antibody (aDec) was displayed at the surface of L. plantarum using three different anchoring strategies: (1) covalent anchoring of aDec to the cell membrane (Lipobox domain, Lip); (2) covalent anchoring to the cell wall (LPXTG domain, CWA); (3) non-covalent anchoring to the cell wall (LysM domain, LysM). aDec was successfully expressed in all three strains, but surface location of the antibody could only be demonstrated for the two strains with cell wall anchors (CWA and LysM). Co-incubation of the engineered strains and DCs showed increased uptake when anchoring aDec using the CWA or LysM anchors. In a competition assay, free anti-DEC abolished the increased uptake, showing that the internalization is due to specific interactions between the DEC-205 receptor and aDec. To test Plasmid Transfer, a Plasmid for expression of GFP under control of an eukaryotic promoter was transformed into the aDec expressing strains and GFP expression in DCs was indeed increased when using the strains producing cell-wall anchored aDec. Plasmid Transfer to DCs in the gastro intestinal tract was also detected using a mouse model. Surprisingly, in mice the highest expression of GFP was observed for the strain in which aDec was coupled to the cell membrane. Conclusion: The results show that surface expression of aDec leads to increased internalization of L. plantarum and Plasmid Transfer in DCs and that efficiency depends on the type of anchor used. Interestingly, in vitro data indicates that cell wall anchoring is more effective, whereas in vivo data seem to indicate that anchoring to the cell membrane is preferable. It is likely that the more embedded localization of aDec in the latter case is favorable when cells are exposed to the harsh conditions of the gastro-intestinal tract.
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Correlation between fibronectin binding protein A expression level at the surface of recombinant lactococcus lactis and Plasmid Transfer in vitro and in vivo
BMC Microbiology, 2014Co-Authors: Juliana F Almeida, Denis Mariat, Alejandra De Moreno De Leblanc, Silvina Del Carmen, Anderson Miyoshi, Vasco Azevedo, Rebeca San Martin, Jean Guy Leblanc, Philippe Langella, Jean Marc ChatelAbstract:BACKGROUND: Fibronectin Binding Protein A (FnBPA) is an invasin from Staphylococcus aureus that allows this pathogen to internalize into eukaryote cells. It was previously demonstrated that recombinant Lactococcus lactis expressing FnBPA were invasive and able to Transfer a Plasmid to eukaryotic cells in vitro and in vivo. In this study, the invasivity of recombinant strains of Lactococcus lactis that express FnBPA under the control of its constitutive promoter or driven by the strong nisin inducible expression system (NICE) were studied.\n\nRESULTS: It was demonstrated that the nisA promoter allows an increase of FnBPA expression on the surface of Lactococcus lactis surface, as shown by flow cytometry, which subsequently enhanced internalization and Plasmid Transfer properties in vitro in Caco2 cells and Bone Marrow Dendritic Cells. In vivo, the use of nisA promoter increase the Plasmid Transfer in cells of both the small and large intestine of mice.\n\nCONCLUSION: FnBPA expression at the surface of recombinant L. lactis is positively correlated to internalization and DNA Transfer properties. The recombinant strains of L. lactis that expresses FnBPA under the control of the nisin inducible expression system could thus be considered as an improved tool in the field of DNA Transfer.
Isabel Barcina - One of the best experts on this subject based on the ideXlab platform.
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Survival and Plasmid Transfer Ability of Escherichia Coli in Wastewater
Water Air and Soil Pollution, 2001Co-Authors: Inés Arana, A. Muela, J.i. Justo, Isabel BarcinaAbstract:No differences were observed in the survival of Plasmid-bearingand Plasmid-free Escherichia coli strains in the course of a long-term survival process in wastewater, under both illuminated and non-illuminated conditions. While the CFU counts and the number of active cells decreased, the numberof nucleoid-containing cells remained constant throughout the 30 days of experimentation. Visible light efficiently contributed to the reduction in culturability, and T90 values were very different under illuminated and nonilluminatedconditions. In the latter case the time necessary to reduce theculturability of a bacterial population by 90% was 27 days, while in the former it was only 1 day. Plasmid Transfer was abundant, while the survival of donor and recipient cells was extensive. After 24 hr of survival in wastewater, Transfer frequency values ranged from 5.92 × 10-5 to 1.12 × 10-2, depending on mating conditions. In the absence of illumination, the potential Transfer abilitiesremained for survival periods of at least 20 days. Transfer assays between free and adhered cells were carried out by means of dialysis bags and submerged membrane diffusion chambers. Transfer frequency for adhered cells was greater than for free cells (2.80 × 10-2 as opposed to 2.39 × 10-3).
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Effect of growth phase and parental cell survival in river water on Plasmid Transfer between Escherichia coli strains.
Applied and environmental microbiology, 1994Co-Authors: A. Muela, Juan Iriberri, Inés Arana, J.i. Justo, M Pocino, Isabel BarcinaAbstract:We evaluated the Transfer to and from Escherichia coli of endogenously isolated Plasmid material from the River Butron during the growth of three donor strains and two recipient strains as well as after the survival of these parental cells in river water. Transfer frequency varied greatly during the growth of donor cells, with minimum values in the exponential phase; frequency remained constant, however, during the growth of recipient strains. After survival in river water, donor cells lost their ability for Plasmid Transfer before any other physiological variations in the cells caused by environmental stress were detected. Under the same conditions and during equal periods, however, no variation in the ability of recipient cells to receive and express Plasmid material was observed.
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Biotic and abiotic factors affecting Plasmid Transfer in Escherichia coli strains.
Applied and environmental microbiology, 1992Co-Authors: Aurora Fernandez-astorga, A. Muela, R Cisterna, Juan Iriberri, Isabel BarcinaAbstract:The influence of biotic and abiotic factors on Plasmid Transfer between Escherichia coli strains in terms of the variation in the number of transconjugants formed and the variation in Transfer frequency was investigated. The density of parent cells affected the number of transconjugants, reaching a maximum when the cell density was on the order of 10(8) CFU ml-1. As the donor-to-recipient ratios varied from 10(-4) to 10(4), the number of transconjugants varied significantly (P less than 0.001), reaching a maximum with donor-to-recipient ratios between 1 and 10. The concentration of total organic carbon in the mating medium affects both the number of transconjugants and the Transfer frequency, being significantly higher (P less than 0.001) when the total organic carbon concentration was higher than 1,139 mg of C liter-1. However, the transconjugants were detected even with less than 1 mg of C liter-1. Linear regression of log10 transconjugants versus mating temperature showed a highly significant regression line (P less than 0.001). Neither the Transfer frequency nor the transconjugant number varied significantly in the range of pHs assayed. We can conclude that Plasmid Transfer by conjugation can take place within a wide range of conditions, even in such adverse conditions as the absence of nutrients and low temperatures.
Gary M. Dunny - One of the best experts on this subject based on the ideXlab platform.
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Enterococcus faecalis sex pheromone cCF10 enhances conjugative Plasmid Transfer in vivo
mBio, 2018Co-Authors: Helmut Hirt, Kerryl E. Greenwood-quaintance, Melissa J. Karau, Lisa M. Till, Purna C. Kashyap, Robin Patel, Gary M. DunnyAbstract:ABSTRACT Cell-cell communication mediated by peptide pheromones (cCF10 [CF]) is essential for high-frequency Plasmid Transfer in vitro in Enterococcus faecalis. To examine the role of pheromone signaling in vivo, we established either a CF-producing (CF+) recipient or a recipient producing a biologically inactive variant of CF (CF− recipient) in a germfree mouse model 3 days before donor inoculation and determined Transfer frequencies of the pheromone-inducible Plasmid pCF10. Plasmid Transfer was detected in the upper and middle sections of the intestinal tract 5 h after donor inoculation and was highly efficient in the absence of antibiotic selection. The transconjugant/donor ratio reached a maximum level approaching 1 on day 4 in the upper intestinal tract. Plasmid Transfer was significantly lower with the CF− recipient. While rescue of the CF− mating defect by coculture with CF+ recipients is easily accomplished in vitro, no extracellular complementation occurred in vivo. This suggests that most pheromone signaling in the gut occurs between recipient and donor cells in very close proximity. Plasmid-bearing cells (donors plus transconjugants) steadily increased in the population from 0.1% after donor inoculation to about 10% at the conclusion of the experiments. This suggests a selective advantage of pCF10 carriage distinct from antibiotic resistance or bacteriocin production. Our results demonstrate that pheromone signaling is required for efficient pCF10 Transfer in vivo. In the absence of CF+ recipients, a low level of Transfer to CF− recipients occurred in the gut. This may result from low-level host-mediated induction of the donors in the gastrointestinal (GI) tract, similar to that previously observed in serum. IMPORTANCE Horizontal gene Transfer is a major factor in the biology of Enterococcus faecalis, an important nosocomial pathogen. Previous studies showing efficient conjugative Plasmid Transfer in the gastrointestinal (GI) tracts of experimental animals did not examine how the enterococcal sex pheromone response impacts the efficiency of Transfer. Our study demonstrates for the first time pheromone-enhanced, high-frequency Plasmid Transfer of E. faecalis Plasmid pCF10 in a mouse model in the absence of antibiotic or bacteriocin selection. Pheromone production by recipients dramatically increased Plasmid Transfer in germfree mice colonized initially with recipients, followed by donors. The presence of a coresident community of common gut microbes did not significantly reduce in vivo Plasmid Transfer between enterococcal donors and recipients. In mice colonized with enterococcal recipients, we detected Plasmid Transfer in the intestinal tract within 5 h of addition of donors, before transconjugants could be cultured from feces. Surprisingly, pCF10 carriage provided a competitive fitness advantage unrelated to antibiotic resistance or bacteriocin production.
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enterococcus faecalis pcf10 encoded surface proteins prga prgb aggregation substance and prgc contribute to Plasmid Transfer biofilm formation and virulence
Molecular Microbiology, 2015Co-Authors: Minny Bhatty, Gary M. Dunny, Melissa R Cruz, Kristi L Frank, Jenny Laverde A Gomez, Fernando Andrade, Danielle A Garsin, Heidi B Kaplan, Peter J ChristieAbstract:Enterococcus faecalis pCF10 Transfers at high frequencies upon pheromone induction of the prgQ Transfer operon. This operon codes for three cell-wall-anchored proteins - PrgA, PrgB (aggregation substance), and PrgC - and a type IV secretion system through which the Plasmid is delivered to recipient cells. Here, we defined the contributions of the Prg surface proteins to Plasmid Transfer, biofilm formation, and virulence using the Caenorhabditis elegans infection model. We report that a combination of PrgB and extracellular DNA (eDNA), but not PrgA or PrgC, was required for extensive cellular aggregation and pCF10 Transfer at wild-type frequencies. In addition to PrgB and eDNA, production of PrgA was necessary for extensive binding of enterococci to abiotic surfaces and development of robust biofilms. However, although PrgB is a known virulence factor in mammalian infection models, we determined that PrgA and PrgC, but not PrgB, were required for efficient killing in the worm infection model. We propose that the pheromone-responsive, conjugative Plasmids of E. faecalis have retained Prg-like surface functions over evolutionary time for attachment, colonization and robust biofilm development. In natural settings, these biofilms are polymicrobial in composition and constitute optimal environments for signal exchange, mating pair formation, and widespread lateral gene Transfer.