Biochemical and Genetic Evidence that Enterococcus faecium L50 Produces Enterocins L50A and L50B, the sec -Dependent Enterocin P, and a Novel Bacteriocin Secreted without an N-Terminal Extension Termed Enterocin Q

Abstract

Enterococcus faecium L50 grown at 16 to 32°C produces enterocin L50 (EntL50), consisting of EntL50A and EntL50B, two unmodified non-pediocin-like peptides synthesized without an N-terminal leader sequence or signal peptide. However, the bacteriocin activity found in the cell-free culture supernatants following growth at higher temperatures (37 to 47°C) is not due to EntL50. A purification procedure including cation-exchange, hydrophobic interaction, and reverse-phase liquid chromatography has shown that the antimicrobial activity is due to two different bacteriocins. Amino acid sequences obtained by Edman degradation and DNA sequencing analyses revealed that one is identical to the sec-dependent pediocin-like enterocin P produced by E. faecium P13 (L. M. Cintas, P. Casaus, L. S. Håvarstein, P. E. Hernández, and I. F. Nes, Appl. Environ. Microbiol. 63: 4321-4330, 1997) and the other is a novel unmodified non-pediocin-like bacteriocin termed enterocin Q (EntQ), with a molecular mass of 3,980. DNA sequencing analysis of a 963-bp region of E. faecium L50 containing the enterocin P structural gene (entP) and the putative immunity protein gene (entiP) reveals a genetic organization identical to that previously found in E. faecium P13. DNA sequencing analysis of a 1,448-bp region identified two consecutive but diverging open reading frames (ORFs) of which one, termed entQ, encodes a 34-amino-acid protein whose deduced amino acid sequence was identical to that obtained for EntQ by amino acid sequencing, showing that EntQ, similarly to EntL50A and EntL50B, is synthesized without an N-terminal leader sequence or signal peptide. The second ORF, termed orf2, was located immediately upstream of and in opposite orientation to entQ and encodes a putative immunity protein composed of 221 amino acids. Bacteriocin production by E. faecium L50 showed that EntP and EntQ are produced in the temperature range from 16 to 47°C and maximally detected at 47 and 37 to 47°C, respectively, while EntL50A and EntL50B are maximally synthesized at 16 to 25°C and are not detected at 37°C or above.

Characterization and identification of weissellicin Y and weissellicin M, novel bacteriocins produced by Weissella hellenica QU 13

“…Most LAB bacteriocins were synthesized as prepeptides containing N-terminal extension or leader sequences, while some class II bacteriocins have been reported to be synthesized without a leader sequence. The latter includes mutacin BHT-B (Hyink et al 2005), lacticin Q (Fujita et al 2007), lacticin Z (Iwatani et al 2007), enterocin Q (Cintas et al 2000) and enterocin L50 (Cintas et al 1998;Criado et al 2006a;Izquierdo et al 2008), most of which are class IId bacteriocins.…”

Modulation of the gut microbiota by prebiotic fibres and bacteriocins

“…Amino acid sequences of representative bacteriocins from different classes: (a) nisin from class I [72]; (b) pediocin PA-1 from class IIa; (c) plantaricins EF and JK from class IIb [73]; (d) garvicin ML from class IIc [74]; and (e) enterocin Q from class IId [75]. The sequences of the peptides that form plantaricins EF and JK have been shown in the order of plantaricin E, plantaricin F, plantaricin J and plantaricin K. The red, blue and yellow amino acids represent acidic amino acids, basic amino acids and uncommon amino acids (Dhb, didehydroaminobutyric acid; Dha, didehydroalanine; Abu, 2-aminobutyric acid), respectively.…”

High-level heterologous production and functional expression of the sec-dependent enterocin P from Enterococcus faecium P13 in Lactococcus lactis

“…Chloramphenicol or erythromycin (Sigma) was added to the cultures of L. lactis at 5 μg ml −1 . Antimicrobial activity of individual colonies was examined by the stab-on-agar test, as previously described by Cintas et al (2000). Cell-free culture supernatants were obtained by centrifugation of cultures at 12,000×g, at 4°C for 10 min.…”

“…They were adjusted to pH 6.2 with 1 M NaOH, filtered through 0.2-μm-pore-size filters (Whatman), and stored at −20°C until use. The antimicrobial activity of the supernatants was examined by an agar well diffusion test (ADT) and a microtitre plate assay (MPA), as previously described by Cintas et al (2000), using E. faecium T136 (sensitive to EntP) and L. lactis MG1363 (resistant to EntP) as the indicator microorganisms. With the MPA, growth inhibition of the sensitive culture was measured spectrophotometrically at 620 nm with a microtitre Labsystems iEMS plate reader (Labsystems).…”

“…However, some class II bacteriocins, such as acidocin B (Leer et al 1995), divergicin A (Worobo et al 1995), bacteriocin 31 (Tomita et al 1996), enterocin P (EntP) , lactoccin 972 , propionicin T1 (Faye et al 2000), and enterolysin A (Nilsen et al 2003), contain N-terminal extensions of the so-called sec-type (signal peptide), which are proteolytically cleaved concomitantly with bacteriocin externalization by the general secretory pathway or sec-dependent pathway (van Wely et al 2001;Herranz and Driessen 2005). Several bacteriocins produced by enterococci, such as enterocins L50 (L50A and L50B), enterocin Q, and enterocin EJ97 (Cintas et al 1998(Cintas et al , 2000Sánchez-Hidalgo et al 2003), have been shown to be synthesized without N-terminal leader sequences, and may represent a new class of bacteriocins with a novel secretion mechanism.…”