The Genes Coding for Enterocin EJ97 Production by Enterococcus faecalis EJ97 Are Located on a Conjugative Plasmid

Abstract

Enterococcus faecalis EJ97 produces a cationic bacteriocin (enterocin EJ97) of low molecular mass (5,327.7 Da). The complete amino acid sequence of enterocin EJ97 was elucidated after automated microsequencing of oligopeptides generated by endoproteinase GluC digestion and cyanogen bromide treatment. Transfer of the 60-kb conjugative plasmid pEJ97 from the bacteriocinogenic strain E. faecalis EJ97 to E. faecalis OG1X conferred bacteriocin production and resistance on the recipient. The genetic determinants of enterocin EJ97 were located in an 11.3-kb EcoRI-BglII DNA fragment of pEJ97. This region was cloned and sequenced. It contains the ej97A structural gene plus three open reading frames (ORFs) (ej97B, ej97C, and ej97D) and three putative ORFs transcribed in the opposite direction (orfA, orfB, and orfC). The gene ej97A translated as a 44-amino-acid residue mature protein lacking a leader peptide with no homology to other bacteriocins described so far. The product of ej97B (Ej97B) shows strong homology in its C-terminal domain to the superfamily of bacterial ATP-binding cassette transporters. The products of ej97C (Ej97C) and ej97D (Ej97D) could be proteins with 71 and 64 residues, respectively, of unknown functions and with no significant similarity to known proteins. There are two additional ORFs (ORF1 and ORF6) flanking the ej97 module, which have been identified as a transposon-like structure (tnp). ORF1 shows similarities to transposase of the Lactococcus lactis element ISS1 and is up to 50% identical to IS1216. This is flanked by two 18-bp inverted repeats (IRs) that are almost identical to those of ISS1 and IS1216. ORF6 (resEJ97) shows strong homology to the resolvase of plasmid pAM373 and up to 40 to 50% homology with the recombinase of several multiresistant plasmids and transposons from Staphylococcus aureus and E. faecalis. These data suggest that EJ97 could represent a new class of bacteriocins with a novel secretion mechanism and that the whole structure could be a composite transposon. Furthermore, two additional gene clusters were found: one cluster is probably related to the region responsible for the replication of plasmid pEJ97, and the second cluster is related to the sex pheromone response. These regions showed a high homology to the corresponding regions of the conjugative plasmids pAM373, pPD1, and pAD1 of E. faecalis, suggesting that they have a common origin.

Bacteriocin Diversity in Streptococcus and Enterococcus

“…The genome sequence of E. faecalis V583, a vancomycin-resistant clinical isolate, has been published, but it is the only complete Enterococcus genome sequence available so far (99). No putative bacteriocin ORFs were initially identified, but a hypothetical protein of 43 amino acids (EFA0015) is almost identical to the recently characterized plasmid-encoded leaderless enterocin EJ97 (112). The translated peptide EFA0015 is 97% identical to enterocin EJ97, as it lacks the threonine in position 14 of enterocin EF97.…”

Complete Sequence of the Enterocin Q-Encoding Plasmid pCIZ2 from the Multiple Bacteriocin Producer Enterococcus faecium L50 and Genetic Characterization of Enterocin Q Production and Immunity

“…The role of ABC transporters in processing and secretion of leader-containing class II bacteriocins is well documented (14,33,36,39,41,52). Furthermore, it has been reported that secretion of the leaderless bacteriocin LsbB from L. lactis BGM-1 (28) and secretion of aureocin A70 from Staphylococcus aureus A70 (42) are mediated by the ABC-type multidrug resistance transporter LmrB and the ABC transporter AurT, respectively; also, it has been suggested that secretion of the leaderless enterocin EJ97 from E. faecalis EJ97 is mediated by the ABC transporter Ej97B (47). Interestingly, EntqB exhibits extensive homology to these ABC-type transporters (Table 4 and Fig.…”

“…Class II bacteriocins are commonly synthesized as biologically inactive precursors containing an N-terminal extension (the so-called double-glycine-type leader sequence or the sec-dependent leader peptide), which is cleaved off concomitant with externalization of the active bacteriocin (30,33,39). Interestingly, a few bacteriocins described to date are synthesized without an N-terminal extension, including enterocin L50 (11), enterocin Q (13), enterocin EJ97 (47), aureocin A70 (42), aureocin A53 (43), and LsbB (28). Production of most subclass IIa bacteriocins relies on a well-conserved genetic organization including at least the following four genes that are often organized in one or two operon-like structures in gene clusters: (i) the structural gene encoding the prebacteriocin; (ii) a gene encoding the immunity protein, which confers producer self-protection against the toxicity of the bacteriocin; (iii) a gene encoding a dedicated ATP-binding cassette (ABC) transporter required for processing and transport of the bacteriocin; and (iv) a gene encoding an accessory protein required for proper bacteriocin externalization (30,39,52).…”

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

“…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.…”