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 degrees C, 60 min; 50% of activity remained after 15 min at 121 degrees 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 and ent1071B, 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 alpha and beta peptides of lactococcin G, respectively. This is the first report of two new antimicrobial peptides representative of a fourth type of E. faecalis bacteriocin.

FIG. 1

Effect of enterocins 1071A and 1071B on the growth of E. faecalis LMG 13566. Counts were made in the absence of enterocins 1071A and -B (◊) and in the presence of enterocins 1071A and -B added at the beginning of the lag phase (□) and after 6 h of growth (▵). Turbidity was determined for cells growing in the absence of enterocins 1071A and -B (⧫) and cells growing in the presence of enterocins 1071A and -B added at the beginning of the lag phase (■) and after 6 h of growth (▴).

FIG. 2

Purification of enterocins 1071A and 1071B with ion-exchange chromatography (SP-Sepharose Fast Flow matrix). The values 0.1 to 0.8 refer to the NH₄COOH gradient used, in moles per liter.

FIG. 3

Separation of enterocins 1071A and 1071B by tricine-SDS-PAGE. Lane 1, Rainbow protein size markers; lane 2, enterocins 1071A and 1071B stained with Coomassie brilliant blue R250; lane 3, enterocins 1071A and 1071B overlaid with cells of E. faecalis LMG 13566 embedded in MRS agar (0.7% agar, wt/vol). The active peptide band is indicated by an arrow.

FIG. 4

Molecular masses of enterocins 1071A (A) and 1071B (B), calculated from the electrospray ionization-mass spectroscopy multiple charged spectra.

FIG. 5

Plasmid profiles of E. faecalis BFE 1071 before and after plasmid curing. (a) Agarose gel electrophoresis of plasmids of E. coli V517 (lane 1), E. faecalis BFE 1071 (lane 2), mutant 1071/78 (lane 3), and mutant 1071/79 (lane 4). The numbers on the left indicate the sizes of the plasmids isolated from E. coli V517. (b) The same gel after Southern blotting onto a MagnaGraph nylon transfer membrane (MSI, Westboro, Mass.) and hybridized with a digoxigenin-labeled cloned EcoRV fragment of plasmid pEF1071 containing the genes encoding enterocins 1071A and 1071B.

FIG. 6

Nucleotide sequence of the regions encoding enterocin 1071A and enterocin 1071B of E. faecalis BFE 1071 and the deduced amino acid sequences. The putative promoter region and transcription initiation sites are indicated in bold, and the ribosome binding sites (RBS) are in italics and underlined. The arrows indicate the processing sites of the peptides.

FIG. 7

Alignment of the peptides of enterocin 1071A and lactococcin G α and of enterocin 1071B and lactococcin G β. Identical amino acids are boxed.

FIG. 8

Edmundson α-helical wheel representation of the amphiphilic regions in enterocin 1071A (A) and enterocin 1071B (B). For enterocin 1071A, the amphiphilic region starts with residue 4 and ends with residue 27; for enterocin 1071B, the amphiphilic region starts with residue 8 and ends with residue 25. The letters with shaded and white backgrounds indicate polar and nonpolar residues, respectively.