The Experts below are selected from a list of 18 Experts worldwide ranked by ideXlab platform
Eva Valdivia - One of the best experts on this subject based on the ideXlab platform.
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analysis of the gene cluster involved in production and immunity of the peptide Antibiotic as 48 in enterococcus faecalis
Molecular Microbiology, 1998Co-Authors: Manuel Martinezbueno, Antonio Galvez, Eva Valdivia, Jacques Coyette, Mercedes MaquedaAbstract:Instituto de Biotecnologi´a, Universidad de Granada,E-18071 Granada, Spain.SummaryA region of 7.8kb of the plasmid pMB2 from Entero-coccus faecalisS-48 carrying the information neces-sary for production and immunity of the peptideAntibiotic as-48 has been cloned and sequenced. Itcontains the as-48astructural gene plus five openreading frames (as-48B, as-48C, as-48C1, as-48Dandas-48D1). Besides as-48D, all the predicted gene pro-ducts are basic hydrophobic proteins with potentialmembrane-spanning domains (MSDs). None of themshows any homology with protein sequences storedin databanks,exceptfor as-48D, which shows similar-ity to the C-terminal domain of ABC transporters andcontains a highly conserved ATP-binding site. Thegene products of as-48B, as-48C, as-48C1and as-48Dare thought to be involved in as-48 production andsecretion. The only gene able to provide resistanceto as-48 by itself isas-48D1. Immunity also seems tobe enhanced at least by the products of as-48B, as-48C1and as-48Dgenes. Transcription analysis usingprobes derived from the different ORFs revealed twolarge (3.5 and 2.7kb) mRNas, suggesting that thedifferent genes are organized in two constitutiveoperons.IntroductionThe Antibiotic as-48 is a non-lAntibiotic cyclic peptide withunique structure produced by Enterococcus faecalis S-48. It has a broad antimicrobial spectrum against bothGram-positive and Gram-negative bacteria (Ga´lvez et al.,1989a,b) and is encoded by the 68kb pheromone-respon-sive plasmid pMB2 (Marti´nez-Buenoet al., 1990). Thedetermination of the DNA sequence of the as-48astruc-tural gene (Marti´nez-Buenoet al., 1994) and the aminoacid sequence of as-48 (Samynet al., 1994) proved thata post-translational modification should occur to producea cyclic molecule with antibacterial activity, involving atail–head peptide bond formation. This is, therefore, thefirst example of a ribosomally synthesized cyclic peptideAntibiotic. The target of as-48 is the cytoplasmic mem-brane,inwhichit inserts ina voltage-independentmanner,forming pores and rendering the membrane permeable tosmall molecules (Ga´lvez et al., 1991).Many Gram-positive bacteria secrete ribosomallysynthesized polypeptides, which inhibit a range of relatedmicroorganisms(Kolter andMoreno,1992; Klaenhammer,1993). Some of them, and especially those produced bylactic acid bacteria, are particularly interesting and offerpromising prospects for biotechnological applications asnatural food preservatives in the dairy and canning indus-tries. The genes required for expression of these sub-stances are usually organized in a single operon, althoughadditional gene products are needed either for post-trans-lational modifications or for processing and extracellularexport, as well as immunity (Klein et al., 1992; Kolter andMoreno, 1992; Klaenhammer, 1993; Kuipers et al., 1993;Gilmore et al., 1994; Venema et al., 1995).The immunity determinants of most antibacterial pep-tides studied so far are closely linked to the structuralgene. Nevertheless, information concerning the identityand modeofaction of immunity proteins isscarce,althoughit is believed that immunity proteins against pore-formingbacteriocins either prevent pore formation or block thepores once they are formed (Geli and Lazdunski, 1992).In the case of lAntibiotics, two self-protection mechanismshave been described. One of them involves membrane-attached proteins (e.g. SpaI, NisI and PepI), which confera high levelofprotectionagainst thepeptideAntibioticssub-tilin, nisin and Pep5 respectively (Kuipers et al., 1993; Kleinand Entian, 1994; Reis et al., 1994). A second immunitymechanism involves secretion of the mature Antibioticmolecule, mediated by an ABC transporter (Fath and
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determination of the gene sequence and the molecular structure of the enterococcal peptide Antibiotic as 48
Journal of Bacteriology, 1994Co-Authors: Manuel Martinezbueno, Bart Samyn, Mercedes Maqueda, Antonio Galvez, Jacques Coyette, Jozef Van Beeumen, Eva ValdiviaAbstract:The structural gene of the enterococcal peptide Antibiotic as-48 (as-48) has been identified and cloned by using two degenerate 17-mer DNA oligonucleotides on the basis of the amino acid sequences of two peptides obtained by digestion of the Antibiotic with Glu-C endoproteinase. That as-48 gene codes for a 105-amino-acid prepeptide, giving rise to a 70-amino-acid mature protein. Comparative analysis demonstrated that the 16-amino-acid sequence of one of the as-48 Glu-C peptides, designated V8-5, was composed of a 12-amino-acid sequence corresponding to the C-terminal end sequence (from isoleucine +59 to tryptophan +70 [I+59 to W+70]) of the prepeptide and terminated in four residues forming the N terminus (M+1 to E+4) of a putative as-48 propeptide. These data, combined with the characteristics of the gene sequence, strongly suggested that the Antibiotic peptide was a 70-residue cyclic molecule. We propose that the as-48 translated primary product is very likely submitted to a posttranslational modification during secretion (i) by an atypical or a typical signal peptidase that cleaves off a 35-residue or shorter signal peptide, respectively, from the prepeptide molecule and (ii) by the linkage of the methionine residue (M+1) to the C-terminal tryptophan residue (W+70) to obtain the cyclic peptide (a tail-head linkage).
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the cyclic structure of the enterococcal peptide Antibiotic as 48
FEBS Letters, 1994Co-Authors: Bart Samyn, Manuel Martinezbueno, Bart Devreese, Mercedes Maqueda, Antonio Galvez, Eva Valdivia, Jacques Coyette, Jozef Van BeeumenAbstract:The complete primary structure of the peptide Antibiotic as-48 produced by Enterococcus faecalis has been determined by chemical degradation analysis. The cyclic nature of this 70 residues containing peptide was demonstrated by plasma desorption mass analysis of the generated peptides and electrospray ionisation mass analysis of the native polypeptide. as far as we know, this is the first example of an Antibiotic protein cyclised by a tail—head peptide bond formation and not by branching of the polypeptide side chains.
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neutralizing antibodies against the peptide Antibiotic as 48 immunocytological studies
Antimicrobial Agents and Chemotherapy, 1993Co-Authors: Mercedes Maqueda, Manuel Martinezbueno, Antonio Galvez, I Guerra, Eva ValdiviaAbstract:Antisera against the broad-spectrum peptide Antibiotic as-48 produced by Enterococcus faecalis were obtained from immunized rabbits. Appreciable antibody titers were obtained only after repeated immunization, suggesting a feeble antigenicity for as-48. Upon incubation with as-48, the antisera neutralized its bacteriolytic action on E. faecalis S-47, although the simultaneous addition of as-48 and serum did not prevent lysis. Crude serum cross-reacted with outer envelope components of enterococci, although specific anti-as-48 antibodies, purified by affinity chromatography, reacted only with as-48-treated cells. Labelling with immunofluorescence and colloidal gold particles was carried out on sensitive and resistant bacterial species to determine the interaction of as-48 with cell structures.
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transformation of enterococcus faecalis ogix with the plasmid pmb2 encoding for the peptide Antibiotic as 48 by protoplast fusion and regeneration on calcium alginate
1992Co-Authors: Manuel Martinezbueno, Mercedes Maqueda, Antonio Galvez, I Guerra, Eva ValdiviaAbstract:The ability to introduce individual molecules of plasmid DNA into bacterial cells by transformation has been of central importance to the recent and rapid advance of plasmid biology and to the development of DNA-cloning methods. Protoplast fusion in the presence of polyethylenglycol (PEG) has proven an efficient method for transformation of yeasts (Svoboda & Ourednicek, 1990) and bacteria such as Bacillus (Chang & Cohen, 1979), Lactococcus (Rondo & McRay, 1982) and Enterococcus (Smith, 1985).
Manuel Martinezbueno - One of the best experts on this subject based on the ideXlab platform.
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analysis of the gene cluster involved in production and immunity of the peptide Antibiotic as 48 in enterococcus faecalis
Molecular Microbiology, 1998Co-Authors: Manuel Martinezbueno, Antonio Galvez, Eva Valdivia, Jacques Coyette, Mercedes MaquedaAbstract:Instituto de Biotecnologi´a, Universidad de Granada,E-18071 Granada, Spain.SummaryA region of 7.8kb of the plasmid pMB2 from Entero-coccus faecalisS-48 carrying the information neces-sary for production and immunity of the peptideAntibiotic as-48 has been cloned and sequenced. Itcontains the as-48astructural gene plus five openreading frames (as-48B, as-48C, as-48C1, as-48Dandas-48D1). Besides as-48D, all the predicted gene pro-ducts are basic hydrophobic proteins with potentialmembrane-spanning domains (MSDs). None of themshows any homology with protein sequences storedin databanks,exceptfor as-48D, which shows similar-ity to the C-terminal domain of ABC transporters andcontains a highly conserved ATP-binding site. Thegene products of as-48B, as-48C, as-48C1and as-48Dare thought to be involved in as-48 production andsecretion. The only gene able to provide resistanceto as-48 by itself isas-48D1. Immunity also seems tobe enhanced at least by the products of as-48B, as-48C1and as-48Dgenes. Transcription analysis usingprobes derived from the different ORFs revealed twolarge (3.5 and 2.7kb) mRNas, suggesting that thedifferent genes are organized in two constitutiveoperons.IntroductionThe Antibiotic as-48 is a non-lAntibiotic cyclic peptide withunique structure produced by Enterococcus faecalis S-48. It has a broad antimicrobial spectrum against bothGram-positive and Gram-negative bacteria (Ga´lvez et al.,1989a,b) and is encoded by the 68kb pheromone-respon-sive plasmid pMB2 (Marti´nez-Buenoet al., 1990). Thedetermination of the DNA sequence of the as-48astruc-tural gene (Marti´nez-Buenoet al., 1994) and the aminoacid sequence of as-48 (Samynet al., 1994) proved thata post-translational modification should occur to producea cyclic molecule with antibacterial activity, involving atail–head peptide bond formation. This is, therefore, thefirst example of a ribosomally synthesized cyclic peptideAntibiotic. The target of as-48 is the cytoplasmic mem-brane,inwhichit inserts ina voltage-independentmanner,forming pores and rendering the membrane permeable tosmall molecules (Ga´lvez et al., 1991).Many Gram-positive bacteria secrete ribosomallysynthesized polypeptides, which inhibit a range of relatedmicroorganisms(Kolter andMoreno,1992; Klaenhammer,1993). Some of them, and especially those produced bylactic acid bacteria, are particularly interesting and offerpromising prospects for biotechnological applications asnatural food preservatives in the dairy and canning indus-tries. The genes required for expression of these sub-stances are usually organized in a single operon, althoughadditional gene products are needed either for post-trans-lational modifications or for processing and extracellularexport, as well as immunity (Klein et al., 1992; Kolter andMoreno, 1992; Klaenhammer, 1993; Kuipers et al., 1993;Gilmore et al., 1994; Venema et al., 1995).The immunity determinants of most antibacterial pep-tides studied so far are closely linked to the structuralgene. Nevertheless, information concerning the identityand modeofaction of immunity proteins isscarce,althoughit is believed that immunity proteins against pore-formingbacteriocins either prevent pore formation or block thepores once they are formed (Geli and Lazdunski, 1992).In the case of lAntibiotics, two self-protection mechanismshave been described. One of them involves membrane-attached proteins (e.g. SpaI, NisI and PepI), which confera high levelofprotectionagainst thepeptideAntibioticssub-tilin, nisin and Pep5 respectively (Kuipers et al., 1993; Kleinand Entian, 1994; Reis et al., 1994). A second immunitymechanism involves secretion of the mature Antibioticmolecule, mediated by an ABC transporter (Fath and
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determination of the gene sequence and the molecular structure of the enterococcal peptide Antibiotic as 48
Journal of Bacteriology, 1994Co-Authors: Manuel Martinezbueno, Bart Samyn, Mercedes Maqueda, Antonio Galvez, Jacques Coyette, Jozef Van Beeumen, Eva ValdiviaAbstract:The structural gene of the enterococcal peptide Antibiotic as-48 (as-48) has been identified and cloned by using two degenerate 17-mer DNA oligonucleotides on the basis of the amino acid sequences of two peptides obtained by digestion of the Antibiotic with Glu-C endoproteinase. That as-48 gene codes for a 105-amino-acid prepeptide, giving rise to a 70-amino-acid mature protein. Comparative analysis demonstrated that the 16-amino-acid sequence of one of the as-48 Glu-C peptides, designated V8-5, was composed of a 12-amino-acid sequence corresponding to the C-terminal end sequence (from isoleucine +59 to tryptophan +70 [I+59 to W+70]) of the prepeptide and terminated in four residues forming the N terminus (M+1 to E+4) of a putative as-48 propeptide. These data, combined with the characteristics of the gene sequence, strongly suggested that the Antibiotic peptide was a 70-residue cyclic molecule. We propose that the as-48 translated primary product is very likely submitted to a posttranslational modification during secretion (i) by an atypical or a typical signal peptidase that cleaves off a 35-residue or shorter signal peptide, respectively, from the prepeptide molecule and (ii) by the linkage of the methionine residue (M+1) to the C-terminal tryptophan residue (W+70) to obtain the cyclic peptide (a tail-head linkage).
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the cyclic structure of the enterococcal peptide Antibiotic as 48
FEBS Letters, 1994Co-Authors: Bart Samyn, Manuel Martinezbueno, Bart Devreese, Mercedes Maqueda, Antonio Galvez, Eva Valdivia, Jacques Coyette, Jozef Van BeeumenAbstract:The complete primary structure of the peptide Antibiotic as-48 produced by Enterococcus faecalis has been determined by chemical degradation analysis. The cyclic nature of this 70 residues containing peptide was demonstrated by plasma desorption mass analysis of the generated peptides and electrospray ionisation mass analysis of the native polypeptide. as far as we know, this is the first example of an Antibiotic protein cyclised by a tail—head peptide bond formation and not by branching of the polypeptide side chains.
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neutralizing antibodies against the peptide Antibiotic as 48 immunocytological studies
Antimicrobial Agents and Chemotherapy, 1993Co-Authors: Mercedes Maqueda, Manuel Martinezbueno, Antonio Galvez, I Guerra, Eva ValdiviaAbstract:Antisera against the broad-spectrum peptide Antibiotic as-48 produced by Enterococcus faecalis were obtained from immunized rabbits. Appreciable antibody titers were obtained only after repeated immunization, suggesting a feeble antigenicity for as-48. Upon incubation with as-48, the antisera neutralized its bacteriolytic action on E. faecalis S-47, although the simultaneous addition of as-48 and serum did not prevent lysis. Crude serum cross-reacted with outer envelope components of enterococci, although specific anti-as-48 antibodies, purified by affinity chromatography, reacted only with as-48-treated cells. Labelling with immunofluorescence and colloidal gold particles was carried out on sensitive and resistant bacterial species to determine the interaction of as-48 with cell structures.
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transformation of enterococcus faecalis ogix with the plasmid pmb2 encoding for the peptide Antibiotic as 48 by protoplast fusion and regeneration on calcium alginate
1992Co-Authors: Manuel Martinezbueno, Mercedes Maqueda, Antonio Galvez, I Guerra, Eva ValdiviaAbstract:The ability to introduce individual molecules of plasmid DNA into bacterial cells by transformation has been of central importance to the recent and rapid advance of plasmid biology and to the development of DNA-cloning methods. Protoplast fusion in the presence of polyethylenglycol (PEG) has proven an efficient method for transformation of yeasts (Svoboda & Ourednicek, 1990) and bacteria such as Bacillus (Chang & Cohen, 1979), Lactococcus (Rondo & McRay, 1982) and Enterococcus (Smith, 1985).
Mercedes Maqueda - One of the best experts on this subject based on the ideXlab platform.
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analysis of the gene cluster involved in production and immunity of the peptide Antibiotic as 48 in enterococcus faecalis
Molecular Microbiology, 1998Co-Authors: Manuel Martinezbueno, Antonio Galvez, Eva Valdivia, Jacques Coyette, Mercedes MaquedaAbstract:Instituto de Biotecnologi´a, Universidad de Granada,E-18071 Granada, Spain.SummaryA region of 7.8kb of the plasmid pMB2 from Entero-coccus faecalisS-48 carrying the information neces-sary for production and immunity of the peptideAntibiotic as-48 has been cloned and sequenced. Itcontains the as-48astructural gene plus five openreading frames (as-48B, as-48C, as-48C1, as-48Dandas-48D1). Besides as-48D, all the predicted gene pro-ducts are basic hydrophobic proteins with potentialmembrane-spanning domains (MSDs). None of themshows any homology with protein sequences storedin databanks,exceptfor as-48D, which shows similar-ity to the C-terminal domain of ABC transporters andcontains a highly conserved ATP-binding site. Thegene products of as-48B, as-48C, as-48C1and as-48Dare thought to be involved in as-48 production andsecretion. The only gene able to provide resistanceto as-48 by itself isas-48D1. Immunity also seems tobe enhanced at least by the products of as-48B, as-48C1and as-48Dgenes. Transcription analysis usingprobes derived from the different ORFs revealed twolarge (3.5 and 2.7kb) mRNas, suggesting that thedifferent genes are organized in two constitutiveoperons.IntroductionThe Antibiotic as-48 is a non-lAntibiotic cyclic peptide withunique structure produced by Enterococcus faecalis S-48. It has a broad antimicrobial spectrum against bothGram-positive and Gram-negative bacteria (Ga´lvez et al.,1989a,b) and is encoded by the 68kb pheromone-respon-sive plasmid pMB2 (Marti´nez-Buenoet al., 1990). Thedetermination of the DNA sequence of the as-48astruc-tural gene (Marti´nez-Buenoet al., 1994) and the aminoacid sequence of as-48 (Samynet al., 1994) proved thata post-translational modification should occur to producea cyclic molecule with antibacterial activity, involving atail–head peptide bond formation. This is, therefore, thefirst example of a ribosomally synthesized cyclic peptideAntibiotic. The target of as-48 is the cytoplasmic mem-brane,inwhichit inserts ina voltage-independentmanner,forming pores and rendering the membrane permeable tosmall molecules (Ga´lvez et al., 1991).Many Gram-positive bacteria secrete ribosomallysynthesized polypeptides, which inhibit a range of relatedmicroorganisms(Kolter andMoreno,1992; Klaenhammer,1993). Some of them, and especially those produced bylactic acid bacteria, are particularly interesting and offerpromising prospects for biotechnological applications asnatural food preservatives in the dairy and canning indus-tries. The genes required for expression of these sub-stances are usually organized in a single operon, althoughadditional gene products are needed either for post-trans-lational modifications or for processing and extracellularexport, as well as immunity (Klein et al., 1992; Kolter andMoreno, 1992; Klaenhammer, 1993; Kuipers et al., 1993;Gilmore et al., 1994; Venema et al., 1995).The immunity determinants of most antibacterial pep-tides studied so far are closely linked to the structuralgene. Nevertheless, information concerning the identityand modeofaction of immunity proteins isscarce,althoughit is believed that immunity proteins against pore-formingbacteriocins either prevent pore formation or block thepores once they are formed (Geli and Lazdunski, 1992).In the case of lAntibiotics, two self-protection mechanismshave been described. One of them involves membrane-attached proteins (e.g. SpaI, NisI and PepI), which confera high levelofprotectionagainst thepeptideAntibioticssub-tilin, nisin and Pep5 respectively (Kuipers et al., 1993; Kleinand Entian, 1994; Reis et al., 1994). A second immunitymechanism involves secretion of the mature Antibioticmolecule, mediated by an ABC transporter (Fath and
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determination of the gene sequence and the molecular structure of the enterococcal peptide Antibiotic as 48
Journal of Bacteriology, 1994Co-Authors: Manuel Martinezbueno, Bart Samyn, Mercedes Maqueda, Antonio Galvez, Jacques Coyette, Jozef Van Beeumen, Eva ValdiviaAbstract:The structural gene of the enterococcal peptide Antibiotic as-48 (as-48) has been identified and cloned by using two degenerate 17-mer DNA oligonucleotides on the basis of the amino acid sequences of two peptides obtained by digestion of the Antibiotic with Glu-C endoproteinase. That as-48 gene codes for a 105-amino-acid prepeptide, giving rise to a 70-amino-acid mature protein. Comparative analysis demonstrated that the 16-amino-acid sequence of one of the as-48 Glu-C peptides, designated V8-5, was composed of a 12-amino-acid sequence corresponding to the C-terminal end sequence (from isoleucine +59 to tryptophan +70 [I+59 to W+70]) of the prepeptide and terminated in four residues forming the N terminus (M+1 to E+4) of a putative as-48 propeptide. These data, combined with the characteristics of the gene sequence, strongly suggested that the Antibiotic peptide was a 70-residue cyclic molecule. We propose that the as-48 translated primary product is very likely submitted to a posttranslational modification during secretion (i) by an atypical or a typical signal peptidase that cleaves off a 35-residue or shorter signal peptide, respectively, from the prepeptide molecule and (ii) by the linkage of the methionine residue (M+1) to the C-terminal tryptophan residue (W+70) to obtain the cyclic peptide (a tail-head linkage).
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the cyclic structure of the enterococcal peptide Antibiotic as 48
FEBS Letters, 1994Co-Authors: Bart Samyn, Manuel Martinezbueno, Bart Devreese, Mercedes Maqueda, Antonio Galvez, Eva Valdivia, Jacques Coyette, Jozef Van BeeumenAbstract:The complete primary structure of the peptide Antibiotic as-48 produced by Enterococcus faecalis has been determined by chemical degradation analysis. The cyclic nature of this 70 residues containing peptide was demonstrated by plasma desorption mass analysis of the generated peptides and electrospray ionisation mass analysis of the native polypeptide. as far as we know, this is the first example of an Antibiotic protein cyclised by a tail—head peptide bond formation and not by branching of the polypeptide side chains.
-
neutralizing antibodies against the peptide Antibiotic as 48 immunocytological studies
Antimicrobial Agents and Chemotherapy, 1993Co-Authors: Mercedes Maqueda, Manuel Martinezbueno, Antonio Galvez, I Guerra, Eva ValdiviaAbstract:Antisera against the broad-spectrum peptide Antibiotic as-48 produced by Enterococcus faecalis were obtained from immunized rabbits. Appreciable antibody titers were obtained only after repeated immunization, suggesting a feeble antigenicity for as-48. Upon incubation with as-48, the antisera neutralized its bacteriolytic action on E. faecalis S-47, although the simultaneous addition of as-48 and serum did not prevent lysis. Crude serum cross-reacted with outer envelope components of enterococci, although specific anti-as-48 antibodies, purified by affinity chromatography, reacted only with as-48-treated cells. Labelling with immunofluorescence and colloidal gold particles was carried out on sensitive and resistant bacterial species to determine the interaction of as-48 with cell structures.
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transformation of enterococcus faecalis ogix with the plasmid pmb2 encoding for the peptide Antibiotic as 48 by protoplast fusion and regeneration on calcium alginate
1992Co-Authors: Manuel Martinezbueno, Mercedes Maqueda, Antonio Galvez, I Guerra, Eva ValdiviaAbstract:The ability to introduce individual molecules of plasmid DNA into bacterial cells by transformation has been of central importance to the recent and rapid advance of plasmid biology and to the development of DNA-cloning methods. Protoplast fusion in the presence of polyethylenglycol (PEG) has proven an efficient method for transformation of yeasts (Svoboda & Ourednicek, 1990) and bacteria such as Bacillus (Chang & Cohen, 1979), Lactococcus (Rondo & McRay, 1982) and Enterococcus (Smith, 1985).
Antonio Galvez - One of the best experts on this subject based on the ideXlab platform.
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analysis of the gene cluster involved in production and immunity of the peptide Antibiotic as 48 in enterococcus faecalis
Molecular Microbiology, 1998Co-Authors: Manuel Martinezbueno, Antonio Galvez, Eva Valdivia, Jacques Coyette, Mercedes MaquedaAbstract:Instituto de Biotecnologi´a, Universidad de Granada,E-18071 Granada, Spain.SummaryA region of 7.8kb of the plasmid pMB2 from Entero-coccus faecalisS-48 carrying the information neces-sary for production and immunity of the peptideAntibiotic as-48 has been cloned and sequenced. Itcontains the as-48astructural gene plus five openreading frames (as-48B, as-48C, as-48C1, as-48Dandas-48D1). Besides as-48D, all the predicted gene pro-ducts are basic hydrophobic proteins with potentialmembrane-spanning domains (MSDs). None of themshows any homology with protein sequences storedin databanks,exceptfor as-48D, which shows similar-ity to the C-terminal domain of ABC transporters andcontains a highly conserved ATP-binding site. Thegene products of as-48B, as-48C, as-48C1and as-48Dare thought to be involved in as-48 production andsecretion. The only gene able to provide resistanceto as-48 by itself isas-48D1. Immunity also seems tobe enhanced at least by the products of as-48B, as-48C1and as-48Dgenes. Transcription analysis usingprobes derived from the different ORFs revealed twolarge (3.5 and 2.7kb) mRNas, suggesting that thedifferent genes are organized in two constitutiveoperons.IntroductionThe Antibiotic as-48 is a non-lAntibiotic cyclic peptide withunique structure produced by Enterococcus faecalis S-48. It has a broad antimicrobial spectrum against bothGram-positive and Gram-negative bacteria (Ga´lvez et al.,1989a,b) and is encoded by the 68kb pheromone-respon-sive plasmid pMB2 (Marti´nez-Buenoet al., 1990). Thedetermination of the DNA sequence of the as-48astruc-tural gene (Marti´nez-Buenoet al., 1994) and the aminoacid sequence of as-48 (Samynet al., 1994) proved thata post-translational modification should occur to producea cyclic molecule with antibacterial activity, involving atail–head peptide bond formation. This is, therefore, thefirst example of a ribosomally synthesized cyclic peptideAntibiotic. The target of as-48 is the cytoplasmic mem-brane,inwhichit inserts ina voltage-independentmanner,forming pores and rendering the membrane permeable tosmall molecules (Ga´lvez et al., 1991).Many Gram-positive bacteria secrete ribosomallysynthesized polypeptides, which inhibit a range of relatedmicroorganisms(Kolter andMoreno,1992; Klaenhammer,1993). Some of them, and especially those produced bylactic acid bacteria, are particularly interesting and offerpromising prospects for biotechnological applications asnatural food preservatives in the dairy and canning indus-tries. The genes required for expression of these sub-stances are usually organized in a single operon, althoughadditional gene products are needed either for post-trans-lational modifications or for processing and extracellularexport, as well as immunity (Klein et al., 1992; Kolter andMoreno, 1992; Klaenhammer, 1993; Kuipers et al., 1993;Gilmore et al., 1994; Venema et al., 1995).The immunity determinants of most antibacterial pep-tides studied so far are closely linked to the structuralgene. Nevertheless, information concerning the identityand modeofaction of immunity proteins isscarce,althoughit is believed that immunity proteins against pore-formingbacteriocins either prevent pore formation or block thepores once they are formed (Geli and Lazdunski, 1992).In the case of lAntibiotics, two self-protection mechanismshave been described. One of them involves membrane-attached proteins (e.g. SpaI, NisI and PepI), which confera high levelofprotectionagainst thepeptideAntibioticssub-tilin, nisin and Pep5 respectively (Kuipers et al., 1993; Kleinand Entian, 1994; Reis et al., 1994). A second immunitymechanism involves secretion of the mature Antibioticmolecule, mediated by an ABC transporter (Fath and
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determination of the gene sequence and the molecular structure of the enterococcal peptide Antibiotic as 48
Journal of Bacteriology, 1994Co-Authors: Manuel Martinezbueno, Bart Samyn, Mercedes Maqueda, Antonio Galvez, Jacques Coyette, Jozef Van Beeumen, Eva ValdiviaAbstract:The structural gene of the enterococcal peptide Antibiotic as-48 (as-48) has been identified and cloned by using two degenerate 17-mer DNA oligonucleotides on the basis of the amino acid sequences of two peptides obtained by digestion of the Antibiotic with Glu-C endoproteinase. That as-48 gene codes for a 105-amino-acid prepeptide, giving rise to a 70-amino-acid mature protein. Comparative analysis demonstrated that the 16-amino-acid sequence of one of the as-48 Glu-C peptides, designated V8-5, was composed of a 12-amino-acid sequence corresponding to the C-terminal end sequence (from isoleucine +59 to tryptophan +70 [I+59 to W+70]) of the prepeptide and terminated in four residues forming the N terminus (M+1 to E+4) of a putative as-48 propeptide. These data, combined with the characteristics of the gene sequence, strongly suggested that the Antibiotic peptide was a 70-residue cyclic molecule. We propose that the as-48 translated primary product is very likely submitted to a posttranslational modification during secretion (i) by an atypical or a typical signal peptidase that cleaves off a 35-residue or shorter signal peptide, respectively, from the prepeptide molecule and (ii) by the linkage of the methionine residue (M+1) to the C-terminal tryptophan residue (W+70) to obtain the cyclic peptide (a tail-head linkage).
-
the cyclic structure of the enterococcal peptide Antibiotic as 48
FEBS Letters, 1994Co-Authors: Bart Samyn, Manuel Martinezbueno, Bart Devreese, Mercedes Maqueda, Antonio Galvez, Eva Valdivia, Jacques Coyette, Jozef Van BeeumenAbstract:The complete primary structure of the peptide Antibiotic as-48 produced by Enterococcus faecalis has been determined by chemical degradation analysis. The cyclic nature of this 70 residues containing peptide was demonstrated by plasma desorption mass analysis of the generated peptides and electrospray ionisation mass analysis of the native polypeptide. as far as we know, this is the first example of an Antibiotic protein cyclised by a tail—head peptide bond formation and not by branching of the polypeptide side chains.
-
neutralizing antibodies against the peptide Antibiotic as 48 immunocytological studies
Antimicrobial Agents and Chemotherapy, 1993Co-Authors: Mercedes Maqueda, Manuel Martinezbueno, Antonio Galvez, I Guerra, Eva ValdiviaAbstract:Antisera against the broad-spectrum peptide Antibiotic as-48 produced by Enterococcus faecalis were obtained from immunized rabbits. Appreciable antibody titers were obtained only after repeated immunization, suggesting a feeble antigenicity for as-48. Upon incubation with as-48, the antisera neutralized its bacteriolytic action on E. faecalis S-47, although the simultaneous addition of as-48 and serum did not prevent lysis. Crude serum cross-reacted with outer envelope components of enterococci, although specific anti-as-48 antibodies, purified by affinity chromatography, reacted only with as-48-treated cells. Labelling with immunofluorescence and colloidal gold particles was carried out on sensitive and resistant bacterial species to determine the interaction of as-48 with cell structures.
-
transformation of enterococcus faecalis ogix with the plasmid pmb2 encoding for the peptide Antibiotic as 48 by protoplast fusion and regeneration on calcium alginate
1992Co-Authors: Manuel Martinezbueno, Mercedes Maqueda, Antonio Galvez, I Guerra, Eva ValdiviaAbstract:The ability to introduce individual molecules of plasmid DNA into bacterial cells by transformation has been of central importance to the recent and rapid advance of plasmid biology and to the development of DNA-cloning methods. Protoplast fusion in the presence of polyethylenglycol (PEG) has proven an efficient method for transformation of yeasts (Svoboda & Ourednicek, 1990) and bacteria such as Bacillus (Chang & Cohen, 1979), Lactococcus (Rondo & McRay, 1982) and Enterococcus (Smith, 1985).
Jozef Van Beeumen - One of the best experts on this subject based on the ideXlab platform.
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determination of the gene sequence and the molecular structure of the enterococcal peptide Antibiotic as 48
Journal of Bacteriology, 1994Co-Authors: Manuel Martinezbueno, Bart Samyn, Mercedes Maqueda, Antonio Galvez, Jacques Coyette, Jozef Van Beeumen, Eva ValdiviaAbstract:The structural gene of the enterococcal peptide Antibiotic as-48 (as-48) has been identified and cloned by using two degenerate 17-mer DNA oligonucleotides on the basis of the amino acid sequences of two peptides obtained by digestion of the Antibiotic with Glu-C endoproteinase. That as-48 gene codes for a 105-amino-acid prepeptide, giving rise to a 70-amino-acid mature protein. Comparative analysis demonstrated that the 16-amino-acid sequence of one of the as-48 Glu-C peptides, designated V8-5, was composed of a 12-amino-acid sequence corresponding to the C-terminal end sequence (from isoleucine +59 to tryptophan +70 [I+59 to W+70]) of the prepeptide and terminated in four residues forming the N terminus (M+1 to E+4) of a putative as-48 propeptide. These data, combined with the characteristics of the gene sequence, strongly suggested that the Antibiotic peptide was a 70-residue cyclic molecule. We propose that the as-48 translated primary product is very likely submitted to a posttranslational modification during secretion (i) by an atypical or a typical signal peptidase that cleaves off a 35-residue or shorter signal peptide, respectively, from the prepeptide molecule and (ii) by the linkage of the methionine residue (M+1) to the C-terminal tryptophan residue (W+70) to obtain the cyclic peptide (a tail-head linkage).
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the cyclic structure of the enterococcal peptide Antibiotic as 48
FEBS Letters, 1994Co-Authors: Bart Samyn, Manuel Martinezbueno, Bart Devreese, Mercedes Maqueda, Antonio Galvez, Eva Valdivia, Jacques Coyette, Jozef Van BeeumenAbstract:The complete primary structure of the peptide Antibiotic as-48 produced by Enterococcus faecalis has been determined by chemical degradation analysis. The cyclic nature of this 70 residues containing peptide was demonstrated by plasma desorption mass analysis of the generated peptides and electrospray ionisation mass analysis of the native polypeptide. as far as we know, this is the first example of an Antibiotic protein cyclised by a tail—head peptide bond formation and not by branching of the polypeptide side chains.