Enterocins L50A and L50B, two novel bacteriocins from Enterococcus faecium L50, are related to staphylococcal hemolysins

Abstract

Enterocin L50 (EntL50), initially referred to as pediocin L50 (L. M. Cintas, J. M. Rodríguez, M. F. Fernández, K. Sletten, I. F. Nes, P. E. Hernández, and H. Holo, Appl. Environ. Microbiol. 61:2643-2648, 1995), is a plasmid-encoded broad-spectrum bacteriocin produced by Enterococcus faecium L50. It has previously been purified from the culture supernatant and partly sequenced by Edman degradation. In the present work, the nucleotide sequence of the EntL50 locus was determined, and several putative open reading frames (ORFs) were identified. Unexpectedly, two ORFs were found to encode EntL50-like peptides. These peptides, termed enterocin L50A (EntL50A) and enterocin L50B (EntL50B), have 72% sequence identity and consist of 44 and 43 amino acids, respectively. Interestingly, a comparison of the deduced sequences of EntL50A and EntL50B with the corresponding sequences obtained by Edman degradation shows that these bacteriocins, in contrast to other peptide bacteriocins, are secreted without an N-terminal leader sequence or signal peptide. Expression in vivo and in vitro transcription/translation experiments demonstrated that entL50A and entL50B are the only genes required to obtain antimicrobial activity, strongly indicating that their bacteriocin products are not posttranslationally modified. Both bacteriocins possess antimicrobial activity on their own, with EntL50A being the most active. In addition, when the two bacteriocins were combined, a considerable synergism was observed, especially with some indicator strains. Even though the enterocins in some respects are similar to class II bacteriocins, several conserved features common to class II bacteriocins are absent from the EntL50 system. The enterocins have more in common with members of a small group of cytolytic peptides secreted by certain staphylococci. We therefore propose that the enterocins L50A and L50B and the staphylococcal cytolysins together constitute a new family of peptide toxins, unrelated to class II bacteriocins, which possess bactericidal and/or hemolytic activity.

FIG. 1

Genetic organization and partial restriction map of the 3.5-kb pCIZ1 fragment containing the enterocin L50A and enterocin L50B structural genes (orfA and orfB) and surrounding ORFs. Candidate ribosome binding sites are indicated by black rectangles. ORFs within the IS1514 are represented by hatched arrows, and inverted repeats (IRR and IRL) are represented by hatched rectangles. Putative ORFs are represented by white arrows.

FIG. 2

Nucleotide sequence of a 466-bp fragment containing the structural genes of EntL50A (entL50A) and EntL50B (entL50B). The deduced amino acid sequences of EntL50A and EntL50B are shown below the DNA sequence. Putative ribosome binding sites (RBS) are overlined.

FIG. 3

Amino acid sequence alignment of EntL50A, EntL50B, the antigonococcal substances (AGS-1, -2, and -3) secreted by S. haemolyticus (57), and the SLUSH peptides (SLUSH-A, -B, and -C) produced by S. lugdunensis (15). The alignment was performed by using the Genetics Computer Group programs Pileup and Prettybox. Identical residues are shown in a black background, and conservative substitutions are shown in a gray background.

FIG. 4

Antimicrobial activity of in vitro-synthesized EntL50A and/or EntL50B against P. acidilactici 347 by a spot-on-agar test. Upper left spot, in vitro-synthesized EntL50A; upper right spot, in vitro-synthesized EntL50B; lower spot, in vitro-cosynthesized EntL50A and EntL50B.