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A segregationally stable expression and secretion vector for Saccharomyces cerevisiae, named pYABD01, was constructed by cloning the yeast gene region encoding the mating pheromone alpha-factor 1 secretion signal (MFalpha1(s)) into the S. cerevisiae high-copy-number expression vector pYES2. The structural genes of the two leaderless peptides of enterocin L50 (EntL50A and EntL50B) from Enterococcus faecium L50 were cloned, separately (entL50A or entL50B) and together (entL50AB), into pYABD01 under the control of the galactose-inducible promoter P(GAL1). The generation of recombinant S. cerevisiae strains heterologously expressing and secreting biologically active EntL50A and EntL50B demonstrates the suitability of the MFalpha1(s)-containing vector pYABD01 to direct processing and secretion of these antimicrobial peptides through the S. cerevisiae Sec system.

Chimeras of pediocin PA-1 (PedA-1), a bacteriocin produced by Pediococcus acidilactici PLBH9, fused to the signal peptide of enterocin P (EntP), a sec-dependent bacteriocin produced by Enterococcus faecium P13, permitted the production of PedA-1 in Lactococcus lactis. Chimeric genes encoding the EntP signal peptide (SP(entP)) fused to mature PedA-1 (pedA), with or without its immunity gene (pedB), were cloned into the expression vector pMG36c to generate the recombinant plasmids pMPP9 (SP(entP):pedA) and pMPP14i (SP(entP):pedA + pedB). Transformation of competent L. lactis subsp. lactis IL1403, L. lactis subsp. cremoris NZ9000, and L. lactis subsp. lactis DPC5598 with the recombinant plasmids has permitted the detection and quantitation of PedA-1 and the coproduction of nisin A and PedA-1 in supernatants of producer cells with specific anti-PedA-1 antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay. Recombinant L. lactis hosts carrying pMPP9 or pMPP14i displayed antimicrobial activity, suggesting that mature PedA-1 fused to SP(EntP) is the minimum requirement for the synthesis, processing, and secretion of biologically active PedA-1 in L. lactis. However, the production and antimicrobial activity of the PedA-1 produced by L. lactis was lower than that produced by the P. acidilactici control strains.

Replacement of the leader sequence of enterocin A (EntA), a bacteriocin produced by Enterococcus faecium PLBC21, by the signal peptide of enterocin P (EntP), a sec-dependent bacteriocin produced by E. faecium P13, permitted production of EntA in Lactococcus lactis. Chimeras encoding the EntP signal peptide (SP( entP )) fused to mature EntA (entA), with or without its immunity gene (entiA), were cloned into the expression vector pMG36c to generate the recombinant plasmids, pMPA15 (SP( entP ):entA) and pMPA10i (SP( entP ):entA + entiA). Transformation of competent L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris NZ9000 with the recombinant plasmids permitted production of EntA by the transformed cells, and the co-production of nisin A and EntA by the L. lactis subsp. lactis DPC5598 transformants. Mature EntA fused to SP(EntP) is the minimum requirement for synthesis, processing and secretion of biologically active EntA in L. lactis. The production of EntA by most recombinant L. lactis hosts was larger than in the E. faecium control strains. All L. lactis derivatives showed antimicrobial activity against Listeria spp., and L. lactis (pMPA15) displayed the highest antilisterial effect.

Polyclonal antibodies with specificity for enterocin L50A (EntL50A), enterocin L50B (EntL50B), and enterocin Q (EntQ) produced by Enterococcus faecium L50 have been generated by immunization of rabbits with chemically synthesized peptides derived from the C terminus of EntL50A (LR1) and EntL50B (LR2) and from the complete enterocin Q (EntQ) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and specificity of these antibodies were evaluated by a noncompetitive indirect enzyme-linked immunosorbent assay (NCI-ELISA) and a competitive indirect ELISA (CI-ELISA). The NCI-ELISA was valuable for detecting anti-EntL50A-, anti-EntL50B-, and anti-EntQ-specific antibodies in the sera of the LR1-KLH-, LR2-KLH-, and EntQ-KLH-immunized animals, respectively. Moreover, these antibodies and those specific for enterocin P (EntP) obtained in a previous work (J. Gutiérrez, R. Criado, R. Citti, M. Martín, C. Herranz, M. F. Fernández, L. M. Cintas, and P. E. Hernández, J. Agric. Food Chem. 52:2247-2255, 2004) were used in an NCI-ELISA to detect and quantify the production of EntL50A, EntL50B, EntP, and EntQ by the multiple-bacteriocin producer E. faecium L50 grown at different temperatures (16 to 47 degrees C). Our results show that temperature has a strong influence on bacteriocin production by this strain. EntL50A and EntL50B are synthesized at 16 to 32 degrees C, but production becomes negligible when the growth temperature is above 37 degrees C, whereas EntP and EntQ are synthesized at temperatures ranging from 16 to 47 degrees C. Maximum EntL50A and EntL50B production was detected at 25 degrees C, while EntP and EntQ are maximally produced at 37 and 47 degrees C, respectively. The loss of plasmid pCIZ1 (50 kb) and/or pCIZ2 (7.4 kb), encoding EntL50A and EntL50B as well as EntQ, respectively, resulted in a significant increase in production and stability of the chromosomally encoded EntP.

The locations of the genetic determinants for enterocin L50 (EntL50A and EntL50B), enterocin Q (EntQ), and enterocin P (EntP) in the multiple bacteriocin producer Enterococcus faecium strain L50 were determined. These bacteriocin genes occur at different locations; entL50AB (encoding EntL50A and EntL50B) are on the 50-kb plasmid pCIZ1, entqA (encoding EntQ) is on the 7.4-kb plasmid pCIZ2, and entP (encoding EntP) is on the chromosome. The complete nucleotide sequence of pCIZ2 was determined to be 7,383 bp long and contains 10 putative open reading frames (ORFs) organized in three distinct regions. The first region contains three ORFs: entqA preceded by two divergently oriented genes, entqB and entqC. EntqB shows high levels of similarity to bacterial ATP-binding cassette (ABC) transporters, while EntqC displays no significant similarity to any known protein. The second region encompasses four ORFs (orf4 to orf7), and ORF4 and ORF5 display high levels of similarity to mobilization proteins from E. faecium and Enterococcus faecalis. In addition, features resembling a transfer origin region (oriT) were found in the promoter area of orf4. The third region contains three ORFs (orf8 to orf10), and ORF8 and ORF9 exhibit similarity to the replication initiator protein RepE from E. faecalis and to RepB proteins, respectively. To clarify the minimum requirement for EntQ synthesis, we subcloned and heterologously expressed a 2,371-bp fragment from pCIZ2 that encompasses only the entqA, entqB, and entqC genes in Lactobacillus sakei, and we demonstrated that this fragment is sufficient for EntQ production. Moreover, we also obtained experimental results indicating that EntqB is involved in ABC transporter-mediated EntQ secretion, while EntqC confers immunity to this bacteriocin.

Enterocin P (EntP), a sec-dependent bacteriocin from Enterococcus faecium P13, was produced by Lactococcus lactis. The EntP structural gene (entP) with or without the EntP immunity gene (entiP) was cloned in (1), plasmid pMG36c under control of the lactococcal constitutive promoter P32, (2) in plasmid pNG8048e under control of the inducible PnisA promoter, and (3) in the integration vector pINT29. Introduction of the recombinant vectors in L. lactis resulted in production of biologically active EntP in the supernatants of L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris NZ9000, and the coproduction of nisin A and EntP in L. lactis subsp. lactis DPC5598. The level of production of EntP, detected and quantified by specific anti-EntP antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay, by the recombinant L. lactis strains depended on the host strain, the expression vector, and the presence of the entiP gene in the constructs of the recombinant L. lactis strains. The highest amount of EntP was produced with derivatives containing entP and entiP, for both L. lactis IL1403 and L. lactis NZ9000. These derivatives produced up to five- to six-fold more EntP than E. faecium P13. Mass spectrometry analysis revealed that EntP purified from L. lactis IL1403 (pJP214) has a molecular mass identical to that purified from E. faecium P13, suggesting that the synthesis, processing, and secretion of EntP progresses efficiently in recombinant L. lactis hosts.

Samples of the intestinal content and carcasses of wood pigeons (Columba palumbus) were evaluated for enterococci with antimicrobial activity. Enterococcus faecium comprised the largest enterococcal species with antagonistic activity, followed by Enterococcusfaecalis and Enterococcus columbae. PCR amplification of genes coding bacteriocins and determination of their nucleotide sequence, and the use of specific antipeptide bacteriocin antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay, permitted characterization of enterococci coding that described bacteriocins and their expression. The efaAfm determinant was the only virulence gene detected in E. faecium, whereas E. faecalis showed a larger number of virulence determinants, and E. columbae did not carry any of the virulence genes examined. Although all E. faecalis isolates manifested a potent direct antimicrobial activity, no activity was detected in supernatants of producer cells. Purification of the antagonistic activity of E. columbae PLCH2 showed multiple chromatographic fragments after matrix-assisted laser desorption-ionization time-of-flight mass spectrometry analysis, suggesting the active peptide(s) had not yet purified to homogeneity. Bacteriocinogenic E. faecium and E. columbae isolates may be considered hygienic for production of enterocins and potentially safe due to their low incidence of potential virulence genes and susceptibility of most relevant clinical antibiotics. However, the presence among the enterococci of E. faecalis strains with a potent antagonistic activity and multiple virulence factors is an issue that must be considered further.

Enterocin P (EntP), a strong antilisterial pediocin-like bacteriocin from Enterococcus faecium P13, was produced by Methylobacterium extorquens. For heterologous expression of EntP in the methylotrophic bacterium M. extorquens, a recombinant plasmid was constructed. The gene encoding the EntP structural gene (entP) was cloned into the plasmid vector pCM80, under control of the methanol dehydrogenase promoter (P(mxaF)), to generate plasmid pS25. When M. extorquens ATCC 55366 was transformed with pS25, EntP was detected and quantified in supernatants of the recombinant M. extorquens S25 strain by using specific anti-EntP antibodies and a non-competitive indirect enzyme-linked immunosorbent assay (NCI-ELISA). Purification of EntP by hydrophobic adsorption and reverse-phase (RP-FPLC) chromatographies, permitted recovery of active EntP from the supernatants of M. extorquens S25 grown in a synthetic defined medium.

The gene encoding mature enterocin P (EntP), an antimicrobial peptide from Enterococcus faecium P13, was cloned into the pPICZalphaA expression vector to generate plasmid pJC31. This plasmid was integrated into the genome of P. pastoris X-33, and EntP was heterologously secreted from the recombinant P. pastoris X-33t1 derivative at a higher production and antagonistic activity than from E. faecium P13.

Polyclonal antibodies with specificity for enterocin P (EntP) have been generated by immunization of rabbits with two chemically synthesized N-terminal peptides (P1 and P2) and a C-terminal peptide (P3) of this bacteriocin conjugated to the carrier protein KLH. The sensitivity and specificity of the peptide-KLH-generated antibodies were evaluated by a noncompetitive indirect enzyme-linked immunosorbent assay (NCI-ELISA) and a competitive indirect (CI)-ELISA. The NCI-ELISA but not the CI-ELISA was valuable for detecting the existence of EntP specific antibodies in the sera of the P2-KLH and P3-KLH immunized animals and to detect and quantify the EntP in the supernatant of producer strains. The anti-P2-KLH sera cross-reacted with the supernatant of a strain producer of sakacin A, a bacteriocin closely related to EntP. Immunoaffinity chromatography columns with anti-P2-KLH or anti-P3-KLH immunoglobulins retained the EntP from the supernatant of the producer strain. Western blotting of EntP with the anti-P2-KLH-generated antibodies suggests that purified EntP tends to the formation of aggregates with no antimicrobial activity. Monitoring the purification of EntP with antipeptide antibodies suggests that while the performance of the evaluated purification procedures would be reasonably acceptable in terms of recovery of the antimicrobial activity of the bacteriocin, their yield is far from attractive in terms of recovery of the initial concentration of enterocin P.

Samples of the intestinal content and carcasses of wood pigeons (Columba palumbus) were evaluated for enterococci with antimicrobial activity. Enterococcus faecium comprised the largest enterococcal species with antagonistic activity, followed by Enterococcusfaecalis and Enterococcus columbae. PCR amplification of genes coding bacteriocins and determination of their nucleotide sequence, and the use of specific antipeptide bacteriocin antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay, permitted characterization of enterococci coding that described bacteriocins and their expression. The efaAfm determinant was the only virulence gene detected in E. faecium, whereas E. faecalis showed a larger number of virulence determinants, and E. columbae did not carry any of the virulence genes examined. Although all E. faecalis isolates manifested a potent direct antimicrobial activity, no activity was detected in supernatants of producer cells. Purification of the antagonistic activity of E. columbae PLCH2 showed multiple chromatographic fragments after matrix-assisted laser desorption-ionization time-of-flight mass spectrometry analysis, suggesting the active peptide(s) had not yet purified to homogeneity. Bacteriocinogenic E. faecium and E. columbae isolates may be considered hygienic for production of enterocins and potentially safe due to their low incidence of potential virulence genes and susceptibility of most relevant clinical antibiotics. However, the presence among the enterococci of E. faecalis strains with a potent antagonistic activity and multiple virulence factors is an issue that must be considered further. [ABSTRACT FROM AUTHOR]