Characterization and Cloning of the Genes Encoding Enterocin 1071A and Enterocin 1071B, Two Antimicrobial Peptides Produced by Enterococcus faecalis BFE 1071

ABSTRACT The pH-neutral cell supernatant of Enterococcus faecalis BFE 1071, isolated from the feces of minipigs in Göttingen, inhibited the growth of Enterococcus spp. and a few other gram-positive bacteria. Ammonium sulfate precipitation and cation-exchange chromatography of the cell supernatant, followed by mass spectrometry analysis, yielded two bacteriocin-like peptides of similar molecular mass: enterocin 1071A (4.285 kDa) and enterocin 1071B (3.899 kDa). Both peptides are always isolated together. The peptides are heat resistant (100°C, 60 min; 50% of activity remained after 15 min at 121°C), remain active after 30 min of incubation at pH 3 to 12, and are sensitive to treatment with proteolytic enzymes. Curing experiments indicated that the genes encoding enterocins 1071A and 1071B are located on a 50-kbp plasmid (pEF1071). Conjugation of plasmid pEF1071 to E. faecalis strains FA2-2 and OGX1 resulted in the expression of two active peptides with sizes identical to those of enterocins 1071A and 1071B. Sequencing of a DNA insert of 9 to 10 kbp revealed two open reading frames, ent1071A andent1071B, which coded for 39- and 34-amino-acid peptides, respectively. The deduced amino acid sequence of the mature Ent1071A and Ent1071B peptides showed 64 and 61% homology with the α and β peptides of lactococcin G, respectively. This is the first report of two new antimicrobial peptides representative of a fourth type ofE. faecalis bacteriocin.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  B. Floriano,et al.  Purification and Genetic Characterization of Enterocin I from Enterococcus faecium 6T1a, a Novel Antilisterial Plasmid-Encoded Bacteriocin Which Does Not Belong to the Pediocin Family of Bacteriocins , 1998, Applied and Environmental Microbiology.

[3]  J. Vederas,et al.  Genetic Characterization and Heterologous Expression of Brochocin-C, an Antibotulinal, Two-Peptide Bacteriocin Produced by Brochothrix campestris ATCC 43754 , 1998, Applied and Environmental Microbiology.

[4]  L. Dicks,et al.  Isolation, purification and partial characterization of plantaricin 423, a bacteriocin produced by Lactobacillus plantarum , 1998, Journal of applied microbiology.

[5]  K. Tanimoto,et al.  Cloning and genetic and sequence analyses of the bacteriocin 21 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pPD1 , 1997, Journal of bacteriology.

[6]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[7]  Trine Nilsen,et al.  Enterocin B, a new bacteriocin from Enterococcus faecium T136 which can act synergistically with enterocin A. , 1997, Microbiology.

[8]  J. V. Van Beeumen,et al.  Purification and characterization of enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA 4 , 1996, Applied and environmental microbiology.

[9]  J. Richard,et al.  Purification, partial characterisation and mode of action of enterococcin EFS2, an antilisterial bacteriocin produced by a strain of Enterococcus faecalis isolated from a cheese. , 1996, International journal of food microbiology.

[10]  K. Tanimoto,et al.  Cloning and genetic organization of the bacteriocin 31 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pYI17 , 1996, Journal of bacteriology.

[11]  I. Nes,et al.  Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins , 1996, Applied and environmental microbiology.

[12]  L. Dicks,et al.  Evaluation of Numerical Analysis of Random Amplified Polymorphic DNA (RAPD)-PCR as a Method to Differentiate Lactobacillus plantarum and Lactobacillus pentosus , 1996, Current Microbiology.

[13]  K. Tanimoto,et al.  Physical mapping of the conjugative bacteriocin plasmid pPD1 of Enterococcus faecalis and identification of the determinant related to the pheromone response , 1995, Journal of bacteriology.

[14]  M. Gilmore,et al.  Genetic structure of the Enterococcus faecalis plasmid pAD1-encoded cytolytic toxin system and its relationship to lantibiotic determinants , 1994, Journal of bacteriology.

[15]  L. Dicks,et al.  TECHNIQUE FOR ISOLATING PLASMIDS FROM EXOPOLYSACCHARIDE PRODUCING LACTOBACILLUS SPP. , 1994 .

[16]  J. V. Van Beeumen,et al.  Determination of the gene sequence and the molecular structure of the enterococcal peptide antibiotic AS-48 , 1994, Journal of bacteriology.

[17]  T. Klaenhammer,et al.  High- and low-copy-number Lactococcus shuttle cloning vectors with features for clone screening. , 1993, Gene.

[18]  T. Klaenhammer,et al.  Genetics of bacteriocins produced by lactic acid bacteria. , 1993, FEMS microbiology reviews.

[19]  T. Abee,et al.  Mode of Action of Lactococcin B, a Thiol-Activated Bacteriocin from Lactococcus lactis , 1993, Applied and environmental microbiology.

[20]  F. Villani,et al.  Enterocin 226NWC, a bacteriocin produced by Enterococcus faecalis 226, active against Listeria monocytogenes. , 1993, The Journal of applied bacteriology.

[21]  D. Clewell,et al.  Evidence that the hemolysin/bacteriocin phenotype of Enterococcus faecalis subsp. zymogenes can be determined by plasmids in different incompatibility groups as well as by the chromosome , 1992, Journal of bacteriology.

[22]  L. Axelsson,et al.  Purification and amino acid sequence of sakacin A, a bacteriocin from Lactobacillus sake Lb706. , 1992, Journal of general microbiology.

[23]  K. Sletten,et al.  A novel lactococcal bacteriocin whose activity depends on the complementary action of two peptides , 1992, Journal of bacteriology.

[24]  M. Pucci,et al.  Cloning, expression, and nucleotide sequence of genes involved in production of pediocin PA-1, and bacteriocin from Pediococcus acidilactici PAC1.0 , 1992, Applied and environmental microbiology.

[25]  J. Piard,et al.  Plasmid profiles and curing of plasmids in Lactobacillus plantarum strains isolated from green olive fermentations. , 1991, The Journal of applied bacteriology.

[26]  K. Sletten,et al.  Purification and amino acid sequence of lactocin S, a bacteriocin produced by Lactobacillus sake L45 , 1991, Applied and environmental microbiology.

[27]  I. Nes,et al.  Lactococcin A, a new bacteriocin from Lactococcus lactis subsp. cremoris: isolation and characterization of the protein and its gene , 1991, Journal of bacteriology.

[28]  G. Venemâ,et al.  Organization and nucleotide sequences of two lactococcal bacteriocin operons , 1991, Applied and environmental microbiology.

[29]  M. Martínez-Bueno,et al.  A transferable plasmid associated with AS-48 production in Enterococcus faecalis , 1990, Journal of bacteriology.

[30]  M. Gilmore,et al.  Genetic analysis of the pAD1 hemolysin/bacteriocin determinant in Enterococcus faecalis: Tn917 insertional mutagenesis and cloning , 1990, Journal of bacteriology.

[31]  A. Bhunia,et al.  Purification, characterization and antimicrobial spectrum of a bacteriocin produced by Pediococcus acidilactici. , 1988, The Journal of applied bacteriology.

[32]  H. Schägger,et al.  Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. , 1987, Analytical biochemistry.

[33]  K. Shimada,et al.  Effect of alternating current exposure on the resistivity of resting Escherichia coli B cells to crystal violet and other basic dyes. , 1987, The Journal of applied bacteriology.

[34]  J. Kennes,et al.  Co-transfer of two plasmids determining bacteriocin production and sucrose utilization in Streptococcus faecium , 1984 .

[35]  D. Clewell,et al.  Mapping of Streptococcus faecalis plasmids pAD1 and pAD2 and studies relating to transposition of Tn917 , 1982, Journal of bacteriology.

[36]  G. Dunny,et al.  Transmissible toxin (hemolysin) plasmid in Streptococcus faecalis and its mobilization of a noninfectious drug resistance plasmid , 1975, Journal of bacteriology.

[37]  H. Sahl,et al.  Lantibiotics: biosynthesis and biological activities of uniquely modified peptides from gram-positive bacteria. , 1998, Annual review of microbiology.

[38]  J. Tagg,et al.  Copyright � 1995, American Society for Microbiology Bacteriocins of Gram-Positive Bacteria , 1995 .

[39]  P. Vandamme,et al.  Analysis of electrophoretic whole organism protein fingerprints , 1994 .

[40]  M. Goodfellow,et al.  Chemical methods in prokaryotic systematics , 1994 .

[41]  J. D. Young,et al.  Cytolytic pore-forming proteins and peptides: is there a common structural motif? , 1991, Trends in biochemical sciences.

[42]  M. Martínez-Bueno,et al.  Bactericidal and bacteriolytic action of peptide antibiotic AS-48 against gram-positive and gram-negative bacteria and other organisms. , 1989, Research in microbiology.