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. 1997 Nov;63(11):4321–4330. doi: 10.1128/aem.63.11.4321-4330.1997

Biochemical and genetic characterization of enterocin P, a novel sec-dependent bacteriocin from Enterococcus faecium P13 with a broad antimicrobial spectrum.

L M Cintas 1, P Casaus 1, L S Håvarstein 1, P E Hernández 1, I F Nes 1
PMCID: PMC168752  PMID: 9361419

Abstract

Enterocin P is a new bacteriocin produced by Enterococcus faecium P13 isolated from a Spanish dry-fermented sausage. Enterocin P inhibited most of tested spoilage and food-borne gram-positive pathogenic bacteria, such as Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum. Enterocin P is produced during growth in MRS broth from 16 to 45 degrees C; it is heat resistant (60 min at 100 degrees C; 15 min at 121 degrees C) and can withstand exposure to pH between 2.0 and 11.0, freeze-thawing, lyophilization, and long-term storage at 4 and -20 degrees C. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation, gel filtration, cation-exchange, hydrophobic-interaction, and reverse-phase liquid chromatography. The sequence of 43 amino acids of the N terminus was obtained by Edman degradation. DNA sequencing analysis of a 755-bp region revealed the presence of two consecutive open reading frames (ORFs). The first ORF encodes a 71-amino-acid protein containing a hydrophobic N-terminal sec-dependent leader sequence of 27 amino acids followed by the amino acid sequence corresponding to the purified and sequenced enterocin P. The bacteriocin is apparently synthesized as a prepeptide that is cleaved immediately after the Val-Asp-Ala residues (positions -3 to -1), resulting in the mature bacteriocin consisting of 44 amino acids, and with a theoretical molecular weight of 4,493. A second ORF, encoding a putative immunity protein composed of 88 amino acids with a calculated molecular weight of 9,886, was found immediately downstream of the enterocin P structural gene. Enterocin P shows a strong antilisterial activity and has the consensus sequence found in the pediocin-like bacteriocins; however, enterocin P is processed and secreted by the sec-dependent pathway.

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Selected References

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  1. Allison G. E., Ahn C., Stiles M. E., Klaenhammer T. R. Utilization of the leucocin A export system in Leuconostoc gelidum for production of a Lactobacillus bacteriocin. FEMS Microbiol Lett. 1995 Aug 15;131(1):87–93. doi: 10.1016/0378-1097(95)00241-v. [DOI] [PubMed] [Google Scholar]
  2. Allison G. E., Worobo R. W., Stiles M. E., Klaenhammer T. R. Heterologous expression of the lactacin F peptides by Carnobacterium piscicola LV17. Appl Environ Microbiol. 1995 Apr;61(4):1371–1377. doi: 10.1128/aem.61.4.1371-1377.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anderson D. G., McKay L. L. Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl Environ Microbiol. 1983 Sep;46(3):549–552. doi: 10.1128/aem.46.3.549-552.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Axelsson L., Holck A. The genes involved in production of and immunity to sakacin A, a bacteriocin from Lactobacillus sake Lb706. J Bacteriol. 1995 Apr;177(8):2125–2137. doi: 10.1128/jb.177.8.2125-2137.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Aymerich T., Holo H., Håvarstein L. S., Hugas M., Garriga M., Nes I. F. Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins. Appl Environ Microbiol. 1996 May;62(5):1676–1682. doi: 10.1128/aem.62.5.1676-1682.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Casaus P., Nilsen T., Cintas L. M., Nes I. F., Hernández P. E., Holo H. Enterocin B, a new bacteriocin from Enterococcus faecium T136 which can act synergistically with enterocin A. Microbiology. 1997 Jul;143(Pt 7):2287–2294. doi: 10.1099/00221287-143-7-2287. [DOI] [PubMed] [Google Scholar]
  7. Chikindas M. L., García-Garcerá M. J., Driessen A. J., Ledeboer A. M., Nissen-Meyer J., Nes I. F., Abee T., Konings W. N., Venema G. Pediocin PA-1, a bacteriocin from Pediococcus acidilactici PAC1.0, forms hydrophilic pores in the cytoplasmic membrane of target cells. Appl Environ Microbiol. 1993 Nov;59(11):3577–3584. doi: 10.1128/aem.59.11.3577-3584.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cintas L. M., Rodriguez J. M., Fernandez M. F., Sletten K., Nes I. F., Hernandez P. E., Holo H. Isolation and characterization of pediocin L50, a new bacteriocin from Pediococcus acidilactici with a broad inhibitory spectrum. Appl Environ Microbiol. 1995 Jul;61(7):2643–2648. doi: 10.1128/aem.61.7.2643-2648.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cornwell G. G., 3rd, Sletten K., Johansson B., Westermark P. Evidence that the amyloid fibril protein in senile systemic amyloidosis is derived from normal prealbumin. Biochem Biophys Res Commun. 1988 Jul 29;154(2):648–653. doi: 10.1016/0006-291x(88)90188-x. [DOI] [PubMed] [Google Scholar]
  10. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Devriese L. A., Pot B., Collins M. D. Phenotypic identification of the genus Enterococcus and differentiation of phylogenetically distinct enterococcal species and species groups. J Appl Bacteriol. 1993 Nov;75(5):399–408. doi: 10.1111/j.1365-2672.1993.tb02794.x. [DOI] [PubMed] [Google Scholar]
  12. Diep D. B., Håvarstein L. S., Nes I. F. Characterization of the locus responsible for the bacteriocin production in Lactobacillus plantarum C11. J Bacteriol. 1996 Aug;178(15):4472–4483. doi: 10.1128/jb.178.15.4472-4483.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Farías M. E., Farías R. N., de Ruiz Holgado A. P., Sesma F. Purification and N-terminal amino acid sequence of Enterocin CRL 35, a 'pediocin-like' bacteriocin produced by Enterococcus faecium CRL 35. Lett Appl Microbiol. 1996 Jun;22(6):417–419. doi: 10.1111/j.1472-765x.1996.tb01193.x. [DOI] [PubMed] [Google Scholar]
  14. Fath M. J., Zhang L. H., Rush J., Kolter R. Purification and characterization of colicin V from Escherichia coli culture supernatants. Biochemistry. 1994 Jun 7;33(22):6911–6917. doi: 10.1021/bi00188a021. [DOI] [PubMed] [Google Scholar]
  15. Fimland G., Blingsmo O. R., Sletten K., Jung G., Nes I. F., Nissen-Meyer J. New biologically active hybrid bacteriocins constructed by combining regions from various pediocin-like bacteriocins: the C-terminal region is important for determining specificity. Appl Environ Microbiol. 1996 Sep;62(9):3313–3318. doi: 10.1128/aem.62.9.3313-3318.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Franke C. M., Leenhouts K. J., Haandrikman A. J., Kok J., Venema G., Venema K. Topology of LcnD, a protein implicated in the transport of bacteriocins from Lactococcus lactis. J Bacteriol. 1996 Mar;178(6):1766–1769. doi: 10.1128/jb.178.6.1766-1769.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gierasch L. M. Signal sequences. Biochemistry. 1989 Feb 7;28(3):923–930. doi: 10.1021/bi00429a001. [DOI] [PubMed] [Google Scholar]
  18. Hastings J. W., Sailer M., Johnson K., Roy K. L., Vederas J. C., Stiles M. E. Characterization of leucocin A-UAL 187 and cloning of the bacteriocin gene from Leuconostoc gelidum. J Bacteriol. 1991 Dec;173(23):7491–7500. doi: 10.1128/jb.173.23.7491-7500.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Henderson J. T., Chopko A. L., van Wassenaar P. D. Purification and primary structure of pediocin PA-1 produced by Pediococcus acidilactici PAC-1.0. Arch Biochem Biophys. 1992 May 15;295(1):5–12. doi: 10.1016/0003-9861(92)90480-k. [DOI] [PubMed] [Google Scholar]
  20. Holck A. L., Axelsson L., Schillinger U. Purification and cloning of piscicolin 61, a bacteriocin from Carnobacterium piscicola LV61. Curr Microbiol. 1994 Aug;29(2):63–68. doi: 10.1007/BF01575750. [DOI] [PubMed] [Google Scholar]
  21. Holck A., Axelsson L., Birkeland S. E., Aukrust T., Blom H. Purification and amino acid sequence of sakacin A, a bacteriocin from Lactobacillus sake Lb706. J Gen Microbiol. 1992 Dec;138(12):2715–2720. doi: 10.1099/00221287-138-12-2715. [DOI] [PubMed] [Google Scholar]
  22. Holo H., Nilssen O., Nes I. F. Lactococcin A, a new bacteriocin from Lactococcus lactis subsp. cremoris: isolation and characterization of the protein and its gene. J Bacteriol. 1991 Jun;173(12):3879–3887. doi: 10.1128/jb.173.12.3879-3887.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Håvarstein L. S., Diep D. B., Nes I. F. A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitant with export. Mol Microbiol. 1995 Apr;16(2):229–240. doi: 10.1111/j.1365-2958.1995.tb02295.x. [DOI] [PubMed] [Google Scholar]
  24. Håvarstein L. S., Holo H., Nes I. F. The leader peptide of colicin V shares consensus sequences with leader peptides that are common among peptide bacteriocins produced by gram-positive bacteria. Microbiology. 1994 Sep;140(Pt 9):2383–2389. doi: 10.1099/13500872-140-9-2383. [DOI] [PubMed] [Google Scholar]
  25. Héchard Y., Dérijard B., Letellier F., Cenatiempo Y. Characterization and purification of mesentericin Y105, an anti-Listeria bacteriocin from Leuconostoc mesenteroides. J Gen Microbiol. 1992 Dec;138(12):2725–2731. doi: 10.1099/00221287-138-12-2725. [DOI] [PubMed] [Google Scholar]
  26. Izard J. W., Kendall D. A. Signal peptides: exquisitely designed transport promoters. Mol Microbiol. 1994 Sep;13(5):765–773. doi: 10.1111/j.1365-2958.1994.tb00469.x. [DOI] [PubMed] [Google Scholar]
  27. Jack R. W., Tagg J. R., Ray B. Bacteriocins of gram-positive bacteria. Microbiol Rev. 1995 Jun;59(2):171–200. doi: 10.1128/mr.59.2.171-200.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kersters K., De Ley J. Identification and grouping of bacteria by numerical analysis of their electrophoretic protein patterns. J Gen Microbiol. 1975 Apr;87(2):333–342. doi: 10.1099/00221287-87-2-333. [DOI] [PubMed] [Google Scholar]
  29. Klaenhammer T. R. Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol Rev. 1993 Sep;12(1-3):39–85. doi: 10.1111/j.1574-6976.1993.tb00012.x. [DOI] [PubMed] [Google Scholar]
  30. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  31. Larsen A. G., Vogensen F. K., Josephsen J. Antimicrobial activity of lactic acid bacteria isolated from sour doughs: purification and characterization of bavaricin A, a bacteriocin produced by Lactobacillus bavaricus MI401. J Appl Bacteriol. 1993 Aug;75(2):113–122. doi: 10.1111/j.1365-2672.1993.tb02755.x. [DOI] [PubMed] [Google Scholar]
  32. Leer R. J., van der Vossen J. M., van Giezen M., van Noort J. M., Pouwels P. H. Genetic analysis of acidocin B, a novel bacteriocin produced by Lactobacillus acidophilus. Microbiology. 1995 Jul;141(Pt 7):1629–1635. doi: 10.1099/13500872-141-7-1629. [DOI] [PubMed] [Google Scholar]
  33. McCormick J. K., Worobo R. W., Stiles M. E. Expression of the antimicrobial peptide carnobacteriocin B2 by a signal peptide-dependent general secretory pathway. Appl Environ Microbiol. 1996 Nov;62(11):4095–4099. doi: 10.1128/aem.62.11.4095-4099.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nes I. F., Diep D. B., Håvarstein L. S., Brurberg M. B., Eijsink V., Holo H. Biosynthesis of bacteriocins in lactic acid bacteria. Antonie Van Leeuwenhoek. 1996 Oct;70(2-4):113–128. doi: 10.1007/BF00395929. [DOI] [PubMed] [Google Scholar]
  35. Olasupo N. A., Schillinger U., Franz C. M., Holzapfel W. H. Bacteriocin production by Enterococcus faecium NA01 from 'wara'--a fermented skimmed cow milk product from west Africa. Lett Appl Microbiol. 1994 Dec;19(6):438–441. doi: 10.1111/j.1472-765x.1994.tb00976.x. [DOI] [PubMed] [Google Scholar]
  36. Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Quadri L. E., Sailer M., Roy K. L., Vederas J. C., Stiles M. E. Chemical and genetic characterization of bacteriocins produced by Carnobacterium piscicola LV17B. J Biol Chem. 1994 Apr 22;269(16):12204–12211. [PubMed] [Google Scholar]
  38. Rodríguez J. M., Cintas L. M., Casaus P., Horn N., Dodd H. M., Hernández P. E., Gasson M. J. Isolation of nisin-producing Lactococcus lactis strains from dry fermented sausages. J Appl Bacteriol. 1995 Feb;78(2):109–115. doi: 10.1111/j.1365-2672.1995.tb02830.x. [DOI] [PubMed] [Google Scholar]
  39. Rodríguez J. M., Cintas L. M., Casaus P., Suárez A., Hernández P. E. PCR detection of the lactocin S structural gene in bacteriocin-producing lactobacilli from meat. Appl Environ Microbiol. 1995 Jul;61(7):2802–2805. doi: 10.1128/aem.61.7.2802-2805.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schillinger U., Lücke F. K. Antibacterial activity of Lactobacillus sake isolated from meat. Appl Environ Microbiol. 1989 Aug;55(8):1901–1906. doi: 10.1128/aem.55.8.1901-1906.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Siragusa G. R. Production of bacteriocin inhibitory to Listeria species by Enterococcus hirae. Appl Environ Microbiol. 1992 Nov;58(11):3508–3513. doi: 10.1128/aem.58.11.3508-3513.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sobrino O. J., Rodríguez J. M., Moreira W. L., Fernández M. F., Sanz B., Hernández P. E. Antibacterial activity of Lactobacillus sake isolated from dry fermented sausages. Int J Food Microbiol. 1991 May;13(1):1–10. doi: 10.1016/0168-1605(91)90130-h. [DOI] [PubMed] [Google Scholar]
  43. Tomita H., Fujimoto S., Tanimoto K., Ike Y. Cloning and genetic organization of the bacteriocin 31 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pYI17. J Bacteriol. 1996 Jun;178(12):3585–3593. doi: 10.1128/jb.178.12.3585-3593.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Villani F., Salzano G., Sorrentino E., Pepe O., Marino P., Coppola S. Enterocin 226NWC, a bacteriocin produced by Enterococcus faecalis 226, active against Listeria monocytogenes. J Appl Bacteriol. 1993 Apr;74(4):380–387. doi: 10.1111/j.1365-2672.1993.tb05142.x. [DOI] [PubMed] [Google Scholar]
  45. Vlaemynck G., Herman L., Coudijzer K. Isolation and characterization of two bacteriocins produced by Enterococcus faecium strains inhibitory to Listeria monocytogenes. Int J Food Microbiol. 1994 Dec;24(1-2):211–225. doi: 10.1016/0168-1605(94)90120-1. [DOI] [PubMed] [Google Scholar]
  46. Williams D. R., Thomas C. M. Active partitioning of bacterial plasmids. J Gen Microbiol. 1992 Jan;138(1):1–16. doi: 10.1099/00221287-138-1-1. [DOI] [PubMed] [Google Scholar]
  47. Worobo R. W., Henkel T., Sailer M., Roy K. L., Vederas J. C., Stiles M. E. Characteristics and genetic determinant of a hydrophobic peptide bacteriocin, carnobacteriocin A, produced by Carnobacterium piscicola LV17A. Microbiology. 1994 Mar;140(Pt 3):517–526. doi: 10.1099/00221287-140-3-517. [DOI] [PubMed] [Google Scholar]
  48. Worobo R. W., Van Belkum M. J., Sailer M., Roy K. L., Vederas J. C., Stiles M. E. A signal peptide secretion-dependent bacteriocin from Carnobacterium divergens. J Bacteriol. 1995 Jun;177(11):3143–3149. doi: 10.1128/jb.177.11.3143-3149.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. van Belkum M. J., Stiles M. E. Molecular characterization of genes involved in the production of the bacteriocin leucocin A from Leuconostoc gelidum. Appl Environ Microbiol. 1995 Oct;61(10):3573–3579. doi: 10.1128/aem.61.10.3573-3579.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. van Belkum M. J., Worobo R. W., Stiles M. E. Double-glycine-type leader peptides direct secretion of bacteriocins by ABC transporters: colicin V secretion in Lactococcus lactis. Mol Microbiol. 1997 Mar;23(6):1293–1301. doi: 10.1046/j.1365-2958.1997.3111677.x. [DOI] [PubMed] [Google Scholar]
  51. von Heijne G. Net N-C charge imbalance may be important for signal sequence function in bacteria. J Mol Biol. 1986 Nov 20;192(2):287–290. doi: 10.1016/0022-2836(86)90365-7. [DOI] [PubMed] [Google Scholar]
  52. von Heijne G. Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem. 1983 Jun 1;133(1):17–21. doi: 10.1111/j.1432-1033.1983.tb07424.x. [DOI] [PubMed] [Google Scholar]

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