ABSTRACT
AIMS: Purification and partial characterization of an extracellular bacteriocin produced by the ruminal isolate Enterococcus faecalis II/1 and determine the frequency of occurrence of enterolysin A structural gene within the ruminal cocci. METHODS AND RESULTS: Bacteriocin produced by E. faecalis II/1 was purified to homogeneity. Purified bacteriocin exhibited a single band on sodium dodecylsulphate polyacrylamide gel electrophoresis with an apparent molecular weight of about 35 kDa. The amino acid sequence of the first 30 amino acids of purified bacteriocin was identical with the enterolysin A sequence. The DNA sequence of the nearly complete E. faecalis II/1 bacteriocin structural gene was identical to the enterolysin A gene sequence, confirming that this bacteriocin is identical to enterolysin A, a cell wall-degrading bacteriocin from E. faecalis LMG 2333. Enterolysin A structural genes were detected in approximately one-sixth of the Gram-positive ruminal cocci examined by PCR using primers targeting the enterolysin A structural gene. CONCLUSIONS: Bacteriocin produced by E. faecalis II/1 is identical to enterolysin A. Enterolysin A structural gene homologues are frequently encountered in rumen enterococcal and streptococcal bacterial strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first evidence of a large heat-labile bacteriocin produced by rumen E. faecalis strain, enlarging the number and types of known anti-bacterial proteins produced by rumen bacteria.
Subject(s)
Bacteriocins/biosynthesis , Enterococcus faecalis/metabolism , Ruminants/microbiology , Amino Acid Sequence , Animals , Bacteriocins/genetics , Bacteriocins/pharmacology , Base Sequence , DNA, Bacterial/analysis , Electrophoresis, Polyacrylamide Gel , Enterococcus faecalis/genetics , Gram-Positive Cocci/genetics , Microbial Sensitivity Tests , Molecular Sequence Data , Sequence HomologyABSTRACT
Enterocin BC25, a bacteriocin produced by Enterococcus faecium BC25 isolated from the rumen of cow was purified to homogeneity and sequenced. Twenty amino acids were identified in the peptide chain (TTHSGKYYGNGVYCT-KNKCT), identical to the N-terminal sequence of enterocin A. The DNA sequence of the enterocin BC25 structural gene and putative immunity protein exhibited high similarity to the entA gene. The occurrence of a 726 bp amplicon containing the enterocin A structural gene was studied among gram-positive ruminal cocci by PCR. Our results showed wide occurrence of the entA structural gene among ruminal enterococcal and streptococcal bacterial strains tested, and indicate variable ability to express bacteriocin production and resistance.
Subject(s)
Bacteriocins/metabolism , Enterococcus faecium/metabolism , Genes, Bacterial , Gram-Positive Cocci/genetics , Amino Acid Sequence , Animals , Bacteriocins/chemistry , Bacteriocins/genetics , Bacteriocins/isolation & purification , Cattle , Enterococcus faecium/genetics , Enterococcus faecium/isolation & purification , Gram-Positive Cocci/isolation & purification , Gram-Positive Cocci/metabolism , Molecular Sequence Data , Polymerase Chain Reaction , Rumen/microbiology , Sequence Analysis, DNAABSTRACT
Similar sequences of distribution of structural genes encoding enterocin A (isolated from the ruminal strain E. faecium BC25) and enterolysin A (isolated from the ruminal amylolytic strain S. bovis II/1) were demonstrated by PCR using oligonucleotide primers specific for these bacteriocins within the ruminal enterococcal and streptococcal strains. Variable occurrence of these bacteriocins was found within the populations of Gram-positive ruminal cocci.
Subject(s)
Bacteriocins/genetics , Bacteriocins/metabolism , Gram-Positive Cocci/genetics , Gram-Positive Cocci/metabolism , Polymerase Chain Reaction , Rumen/microbiology , Sequence Analysis, DNA , Animals , Genes, BacterialABSTRACT
A bacteriocin-like activity produced by Enterococcus faecium BC25, isolated from the the rumen of a cow, was partially purified and characterized. The active substance was prepared by ammonium sulfate and chloroform/methanol precipitation of culture supernatant. The bacteriocin was a protein of molecular mass 17.5 kDa. Activity was inactivated by trypsin and proteinase K. The bacteriocin BC25 inhibited growth of amylolytic ruminal strains of Streptococcus bovis, including S. bovis AO 24/85. Results showed that bacteriocine substance BC25 has a bacteriostatic effect when present in concentrations exceeding 125 AU/ml. Agar overlays and batch culture growth experiments proved that E. faecium BC25 was producing bacteriocin that inhibited the growth of S. bovis.
