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. 1996 Nov;62(11):4095–4099. doi: 10.1128/aem.62.11.4095-4099.1996

Expression of the antimicrobial peptide carnobacteriocin B2 by a signal peptide-dependent general secretory pathway.

J K McCormick 1, R W Worobo 1, M E Stiles 1
PMCID: PMC168231  PMID: 8900000

Abstract

Carnobacteriocin B2 is a well-characterized class II bacteriocin produced by a 61-kb plasmid from Carnobacterium piscicola LV17. Export of this bacteriocin is dependent on specific ABC (ATP-binding cassette) secretion proteins. Divergicin A is a strongly hydrophobic narrow-spectrum bacteriocin produced by a 3.4-kb plasmid from Carnobacterium divergens LV13. Predivergicin A contains a signal peptide and utilizes the general secretary pathway for export (R. W. Worobo, M. J. van Belkum, M. Sailer, K. L. Roy, J. C. Vederas, and M. E. Stiles, J. Bacteriol. 177:3143-3149, 1995). Fusion of the carnobacteriocin B2 structural gene (devoid of its natural leader peptide) behind the signal peptide of divergicin A in the expression vector pMG36e permitted production and export of active carnobacteriocin B2 in the absence of the specific secretion genes. N-terminal sequencing of purified carnobacteriocin B2 established that correct processing of the prebacteriocin occurred beyond the Ala-Ser-Ala cleavage site of the signal peptide. Carnobacteriocin B2 was produced by the wild-type strain of C. divergens, LV13, and in C. piscicola LV17C, the nonbacteriocinogenic, plasmidless variant of the original carnobacteriocin B2 producer strain. The corresponding immunity gene was included immediately downstream of the structural gene. Both of the host strains are sensitive to the bacteriocin, and both acquired immunity when they were transformed with the construct. C. divergens LV13 containing the divergicin A signal peptide-carnobacteriocin B2 fusion construct produces both divergicin A and carnobacteriocin B2 and demonstrates the first example of multiple-bacteriocin expression via the general secretory pathway. The small amount of genetic material required for independent bacteriocin expression has implications for the development of a food-grade multiple-bacteriocin expression vector for use in lactic acid bacteria.

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Selected References

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  1. Ahn C., Stiles M. E. Plasmid-associated bacteriocin production by a strain of Carnobacterium piscicola from meat. Appl Environ Microbiol. 1990 Aug;56(8):2503–2510. doi: 10.1128/aem.56.8.2503-2510.1990. [DOI] [PMC free article] [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. 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]
  4. Borchert T. V., Nagarajan V. Effect of signal sequence alterations on export of levansucrase in Bacillus subtilis. J Bacteriol. 1991 Jan;173(1):276–282. doi: 10.1128/jb.173.1.276-282.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cao G., Kuhn A., Dalbey R. E. The translocation of negatively charged residues across the membrane is driven by the electrochemical potential: evidence for an electrophoresis-like membrane transfer mechanism. EMBO J. 1995 Mar 1;14(5):866–875. doi: 10.1002/j.1460-2075.1995.tb07068.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  7. Dinh T., Paulsen I. T., Saier M. H., Jr A family of extracytoplasmic proteins that allow transport of large molecules across the outer membranes of gram-negative bacteria. J Bacteriol. 1994 Jul;176(13):3825–3831. doi: 10.1128/jb.176.13.3825-3831.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fath M. J., Kolter R. ABC transporters: bacterial exporters. Microbiol Rev. 1993 Dec;57(4):995–1017. doi: 10.1128/mr.57.4.995-1017.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gilson L., Mahanty H. K., Kolter R. Four plasmid genes are required for colicin V synthesis, export, and immunity. J Bacteriol. 1987 Jun;169(6):2466–2470. doi: 10.1128/jb.169.6.2466-2470.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gilson L., Mahanty H. K., Kolter R. Genetic analysis of an MDR-like export system: the secretion of colicin V. EMBO J. 1990 Dec;9(12):3875–3884. doi: 10.1002/j.1460-2075.1990.tb07606.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. González B., Arca P., Mayo B., Suárez J. E. Detection, purification, and partial characterization of plantaricin C, a bacteriocin produced by a Lactobacillus plantarum strain of dairy origin. Appl Environ Microbiol. 1994 Jun;60(6):2158–2163. doi: 10.1128/aem.60.6.2158-2163.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. 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]
  15. 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]
  16. Joerger M. C., Klaenhammer T. R. Cloning, expression, and nucleotide sequence of the Lactobacillus helveticus 481 gene encoding the bacteriocin helveticin J. J Bacteriol. 1990 Nov;172(11):6339–6347. doi: 10.1128/jb.172.11.6339-6347.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Kuipers O. P., Rollema H. S., de Vos W. M., Siezen R. J. Biosynthesis and secretion of a precursor of nisin Z by Lactococcus lactis, directed by the leader peptide of the homologous lantibiotic subtilin from Bacillus subtilis. FEBS Lett. 1993 Sep 6;330(1):23–27. doi: 10.1016/0014-5793(93)80911-d. [DOI] [PubMed] [Google Scholar]
  19. Lee C., Li P., Inouye H., Brickman E. R., Beckwith J. Genetic studies on the inability of beta-galactosidase to be translocated across the Escherichia coli cytoplasmic membrane. J Bacteriol. 1989 Sep;171(9):4609–4616. doi: 10.1128/jb.171.9.4609-4616.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Li P., Beckwith J., Inouye H. Alteration of the amino terminus of the mature sequence of a periplasmic protein can severely affect protein export in Escherichia coli. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7685–7689. doi: 10.1073/pnas.85.20.7685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Marugg J. D., Gonzalez C. F., Kunka B. S., Ledeboer A. M., Pucci M. J., Toonen M. Y., Walker S. A., Zoetmulder L. C., Vandenbergh P. A. Cloning, expression, and nucleotide sequence of genes involved in production of pediocin PA-1, and bacteriocin from Pediococcus acidilactici PAC1.0. Appl Environ Microbiol. 1992 Aug;58(8):2360–2367. doi: 10.1128/aem.58.8.2360-2367.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Quadri L. E., Sailer M., Terebiznik M. R., Roy K. L., Vederas J. C., Stiles M. E. Characterization of the protein conferring immunity to the antimicrobial peptide carnobacteriocin B2 and expression of carnobacteriocins B2 and BM1. J Bacteriol. 1995 Mar;177(5):1144–1151. doi: 10.1128/jb.177.5.1144-1151.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Randall L. L., Hardy S. J. Correlation of competence for export with lack of tertiary structure of the mature species: a study in vivo of maltose-binding protein in E. coli. Cell. 1986 Sep 12;46(6):921–928. doi: 10.1016/0092-8674(86)90074-7. [DOI] [PubMed] [Google Scholar]
  27. Simonen M., Palva I. Protein secretion in Bacillus species. Microbiol Rev. 1993 Mar;57(1):109–137. doi: 10.1128/mr.57.1.109-137.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Venema K., Kok J., Marugg J. D., Toonen M. Y., Ledeboer A. M., Venema G., Chikindas M. L. Functional analysis of the pediocin operon of Pediococcus acidilactici PAC1.0: PedB is the immunity protein and PedD is the precursor processing enzyme. Mol Microbiol. 1995 Aug;17(3):515–522. doi: 10.1111/j.1365-2958.1995.mmi_17030515.x. [DOI] [PubMed] [Google Scholar]
  29. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Zhang L. H., Fath M. J., Mahanty H. K., Tai P. C., Kolter R. Genetic analysis of the colicin V secretion pathway. Genetics. 1995 Sep;141(1):25–32. doi: 10.1093/genetics/141.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. van de Guchte M., van der Vossen J. M., Kok J., Venema G. Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis. Appl Environ Microbiol. 1989 Jan;55(1):224–228. doi: 10.1128/aem.55.1.224-228.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]

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