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. 1995 Mar;177(5):1144–1151. doi: 10.1128/jb.177.5.1144-1151.1995

Characterization of the protein conferring immunity to the antimicrobial peptide carnobacteriocin B2 and expression of carnobacteriocins B2 and BM1.

L E Quadri 1, M Sailer 1, M R Terebiznik 1, K L Roy 1, J C Vederas 1, M E Stiles 1
PMCID: PMC176717  PMID: 7868585

Abstract

Cloning of a 16-kb DNA fragment from the 61-kb plasmid of Carnobacterium piscicola LV17B into plasmidless C. piscicola LV17C restores the production of the plasmid-encoded carnobacteriocin B2 and the chromosomally-encoded carnobacteriocin BM1 and restores the immune phenotype. This fragment also has sufficient genetic information to allow the expression of carnobacteriocin B2 and its immunity in a heterologous host. The gene locus (cbiB2) responsible for immunity to carnobacteriocin B2 is located downstream of the structural gene for carnobacteriocin B2 and encodes a protein of 111 amino acids (CbiB2). CbiB2 was expressed in Escherichia coli as a fusion of the maltose-binding protein and CbiB2. The fusion protein was purified on an amylose column and cleaved with factor Xa, and pure CbiB2 was isolated by high-performance liquid chromatography. The N-terminal amino acid sequence and mass spectrometry (molecular weight [mean +/- standard error], 12,662.2 +/- 3.4) of the purified protein agree with the information deduced from the nucleotide sequence of cbiB2. Western blot (immunoblot) analysis indicates that the majority of the intracellular pool of this immunity protein is in the cytoplasm and that a smaller proportion is associated with the membrane. CbiB2 confers immunity to carnobacteriocin B2, but not to carnobacteriocin BM1, when it is expressed in homologous or heterologous hosts. No protective effect is observed for sensitive cells growing in the presence of the bacteriocin when the immunity protein is added to the medium. The purified immunity protein does not show significant binding to microtiter plates coated with carnobacteriocin B2 and is not able to inactivate the bacteriocin in solution.

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Selected References

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  1. Abee T., Klaenhammer T. R., Letellier L. Kinetic studies of the action of lactacin F, a bacteriocin produced by Lactobacillus johnsonii that forms poration complexes in the cytoplasmic membrane. Appl Environ Microbiol. 1994 Mar;60(3):1006–1013. doi: 10.1128/aem.60.3.1006-1013.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ahn C., Collins-Thompson D., Duncan C., Stiles M. E. Mobilization and location of the genetic determinant of chloramphenicol resistance from Lactobacillus plantarum caTC2R. Plasmid. 1992 May;27(3):169–176. doi: 10.1016/0147-619x(92)90018-6. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Axelsson L., Holck A., Birkeland S. E., Aukrust T., Blom H. Cloning and nucleotide sequence of a gene from Lactobacillus sake Lb706 necessary for sakacin A production and immunity. Appl Environ Microbiol. 1993 Sep;59(9):2868–2875. doi: 10.1128/aem.59.9.2868-2875.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blight M. A., Holland I. B. Structure and function of haemolysin B,P-glycoprotein and other members of a novel family of membrane translocators. Mol Microbiol. 1990 Jun;4(6):873–880. doi: 10.1111/j.1365-2958.1990.tb00660.x. [DOI] [PubMed] [Google Scholar]
  6. Bukhtiyarova M., Yang R., Ray B. Analysis of the pediocin AcH gene cluster from plasmid pSMB74 and its expression in a pediocin-negative Pediococcus acidilactici strain. Appl Environ Microbiol. 1994 Sep;60(9):3405–3408. doi: 10.1128/aem.60.9.3405-3408.1994. [DOI] [PMC free article] [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. Diep D. B., Håvarstein L. S., Nissen-Meyer J., Nes I. F. The gene encoding plantaricin A, a bacteriocin from Lactobacillus plantarum C11, is located on the same transcription unit as an agr-like regulatory system. Appl Environ Microbiol. 1994 Jan;60(1):160–166. doi: 10.1128/aem.60.1.160-166.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dunn S. D. Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. Anal Biochem. 1986 Aug 15;157(1):144–153. doi: 10.1016/0003-2697(86)90207-1. [DOI] [PubMed] [Google Scholar]
  10. Engelke G., Gutowski-Eckel Z., Kiesau P., Siegers K., Hammelmann M., Entian K. D. Regulation of nisin biosynthesis and immunity in Lactococcus lactis 6F3. Appl Environ Microbiol. 1994 Mar;60(3):814–825. doi: 10.1128/aem.60.3.814-825.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gao F. H., Abee T., Konings W. N. Mechanism of action of the peptide antibiotic nisin in liposomes and cytochrome c oxidase-containing proteoliposomes. Appl Environ Microbiol. 1991 Aug;57(8):2164–2170. doi: 10.1128/aem.57.8.2164-2170.1991. [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. 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]
  14. 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]
  15. Jacobs E., Clad A. Electroelution of fixed and stained membrane proteins from preparative sodium dodecyl sulfate-polyacrylamide gels into a membrane trap. Anal Biochem. 1986 May 1;154(2):583–589. doi: 10.1016/0003-2697(86)90033-3. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Kuipers O. P., Beerthuyzen M. M., Siezen R. J., De Vos W. M. Characterization of the nisin gene cluster nisABTCIPR of Lactococcus lactis. Requirement of expression of the nisA and nisI genes for development of immunity. Eur J Biochem. 1993 Aug 15;216(1):281–291. doi: 10.1111/j.1432-1033.1993.tb18143.x. [DOI] [PubMed] [Google Scholar]
  18. London J. Uncommon pathways of metabolism among lactic acid bacteria. FEMS Microbiol Rev. 1990 Sep;7(1-2):103–111. doi: 10.1111/j.1574-6968.1990.tb04882.x. [DOI] [PubMed] [Google Scholar]
  19. Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978 Jun 15;87(1):206–210. doi: 10.1016/0003-2697(78)90586-9. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. McMullen L. M., Stiles M. E. Microbial ecology of fresh pork stored under modified atmosphere at -1, 4.4 and 10 degrees C. Int J Food Microbiol. 1993 Mar;18(1):1–14. doi: 10.1016/0168-1605(93)90002-x. [DOI] [PubMed] [Google Scholar]
  22. Nissen-Meyer J., Håvarstein L. S., Holo H., Sletten K., Nes I. F. Association of the lactococcin A immunity factor with the cell membrane: purification and characterization of the immunity factor. J Gen Microbiol. 1993 Jul;139(7):1503–1509. doi: 10.1099/00221287-139-7-1503. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Sailer M., Helms G. L., Henkel T., Niemczura W. P., Stiles M. E., Vederas J. C. 15N- and 13C-labeled media from Anabaena sp. for universal isotopic labeling of bacteriocins: NMR resonance assignments of leucocin A from Leuconostoc gelidum and nisin A from Lactococcus lactis. Biochemistry. 1993 Jan 12;32(1):310–318. doi: 10.1021/bi00052a039. [DOI] [PubMed] [Google Scholar]
  25. Stock J. B., Ninfa A. J., Stock A. M. Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev. 1989 Dec;53(4):450–490. doi: 10.1128/mr.53.4.450-490.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stoddard G. W., Petzel J. P., van Belkum M. J., Kok J., McKay L. L. Molecular analyses of the lactococcin A gene cluster from Lactococcus lactis subsp. lactis biovar diacetylactis WM4. Appl Environ Microbiol. 1992 Jun;58(6):1952–1961. doi: 10.1128/aem.58.6.1952-1961.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Stults N. L., Asta L. M., Lee Y. C. Immobilization of proteins on oxidized crosslinked Sepharose preparations by reductive amination. Anal Biochem. 1989 Jul;180(1):114–119. doi: 10.1016/0003-2697(89)90097-3. [DOI] [PubMed] [Google Scholar]
  28. Venema K., Abee T., Haandrikman A. J., Leenhouts K. J., Kok J., Konings W. N., Venema G. Mode of Action of Lactococcin B, a Thiol-Activated Bacteriocin from Lactococcus lactis. Appl Environ Microbiol. 1993 Apr;59(4):1041–1048. doi: 10.1128/aem.59.4.1041-1048.1993. [DOI] [PMC free article] [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. van Belkum M. J., Hayema B. J., Jeeninga R. E., Kok J., Venema G. Organization and nucleotide sequences of two lactococcal bacteriocin operons. Appl Environ Microbiol. 1991 Feb;57(2):492–498. doi: 10.1128/aem.57.2.492-498.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. van Belkum M. J., Kok J., Venema G. Cloning, sequencing, and expression in Escherichia coli of lcnB, a third bacteriocin determinant from the lactococcal bacteriocin plasmid p9B4-6. Appl Environ Microbiol. 1992 Feb;58(2):572–577. doi: 10.1128/aem.58.2.572-577.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. van Belkum M. J., Kok J., Venema G., Holo H., Nes I. F., Konings W. N., Abee T. The bacteriocin lactococcin A specifically increases permeability of lactococcal cytoplasmic membranes in a voltage-independent, protein-mediated manner. J Bacteriol. 1991 Dec;173(24):7934–7941. doi: 10.1128/jb.173.24.7934-7941.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. van der Meer J. R., Polman J., Beerthuyzen M. M., Siezen R. J., Kuipers O. P., De Vos W. M. Characterization of the Lactococcus lactis nisin A operon genes nisP, encoding a subtilisin-like serine protease involved in precursor processing, and nisR, encoding a regulatory protein involved in nisin biosynthesis. J Bacteriol. 1993 May;175(9):2578–2588. doi: 10.1128/jb.175.9.2578-2588.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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