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Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Apr;175(7):2087–2096. doi: 10.1128/jb.175.7.2087-2096.1993

Cloning and sequencing of the gene for a lactococcal endopeptidase, an enzyme with sequence similarity to mammalian enkephalinase.

I Mierau 1, P S Tan 1, A J Haandrikman 1, B Mayo 1, J Kok 1, K J Leenhouts 1, W N Konings 1, G Venema 1
PMCID: PMC204311  PMID: 8458851

Abstract

The gene specifying an endopeptidase of Lactococcus lactis, named pepO, was cloned from a genomic library of L. lactis subsp. cremoris P8-2-47 in lambda EMBL3 and was subsequently sequenced. pepO is probably the last gene of an operon encoding the binding-protein-dependent oligopeptide transport system of L. lactis. The inferred amino acid sequence of PepO showed that the lactococcal endopeptidase has a marked similarity to the mammalian neutral endopeptidase EC 3.4.24.11 (enkephalinase), whereas no obvious sequence similarity with any bacterial enzyme was found. By means of gene disruption, a pepO-negative mutant was constructed. Growth and acid production of the mutant strain in milk were not affected, indicating that the endopeptidase is not essential for growth of L. lactis in milk.

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  1. Alloing G., Trombe M. C., Claverys J. P. The ami locus of the gram-positive bacterium Streptococcus pneumoniae is similar to binding protein-dependent transport operons of gram-negative bacteria. Mol Microbiol. 1990 Apr;4(4):633–644. doi: 10.1111/j.1365-2958.1990.tb00632.x. [DOI] [PubMed] [Google Scholar]
  2. Beaumont A., Barbe B., Le Moual H., Boileau G., Crine P., Fournié-Zaluski M. C., Roques B. P. Charge polarity reversal inverses the specificity of neutral endopeptidase-24.11. J Biol Chem. 1992 Feb 5;267(4):2138–2141. [PubMed] [Google Scholar]
  3. Chapot-Chartier M. P., Nardi M., Chopin M. C., Chopin A., Gripon J. C. Cloning and sequencing of pepC, a cysteine aminopeptidase gene from Lactococcus lactis subsp. cremoris AM2. Appl Environ Microbiol. 1993 Jan;59(1):330–333. doi: 10.1128/aem.59.1.330-333.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chomczynski P., Qasba P. K. Alkaline transfer of DNA to plastic membrane. Biochem Biophys Res Commun. 1984 Jul 18;122(1):340–344. doi: 10.1016/0006-291x(84)90480-7. [DOI] [PubMed] [Google Scholar]
  5. D'Adamio L., Shipp M. A., Masteller E. L., Reinherz E. L. Organization of the gene encoding common acute lymphoblastic leukemia antigen (neutral endopeptidase 24.11): multiple miniexons and separate 5' untranslated regions. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7103–7107. doi: 10.1073/pnas.86.18.7103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Devault A., Lazure C., Nault C., Le Moual H., Seidah N. G., Chrétien M., Kahn P., Powell J., Mallet J., Beaumont A. Amino acid sequence of rabbit kidney neutral endopeptidase 24.11 (enkephalinase) deduced from a complementary DNA. EMBO J. 1987 May;6(5):1317–1322. doi: 10.1002/j.1460-2075.1987.tb02370.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Frischauf A. M., Lehrach H., Poustka A., Murray N. Lambda replacement vectors carrying polylinker sequences. J Mol Biol. 1983 Nov 15;170(4):827–842. doi: 10.1016/s0022-2836(83)80190-9. [DOI] [PubMed] [Google Scholar]
  8. Gasson M. J., Davies F. L. High-frequency conjugation associated with Streptococcus lactis donor cell aggregation. J Bacteriol. 1980 Sep;143(3):1260–1264. doi: 10.1128/jb.143.3.1260-1264.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gasson M. J. Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol. 1983 Apr;154(1):1–9. doi: 10.1128/jb.154.1.1-9.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Haandrikman A. J., Meesters R., Laan H., Konings W. N., Kok J., Venema G. Processing of the lactococcal extracellular serine proteinase. Appl Environ Microbiol. 1991 Jul;57(7):1899–1904. doi: 10.1128/aem.57.7.1899-1904.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hiles I. D., Gallagher M. P., Jamieson D. J., Higgins C. F. Molecular characterization of the oligopeptide permease of Salmonella typhimurium. J Mol Biol. 1987 May 5;195(1):125–142. doi: 10.1016/0022-2836(87)90332-9. [DOI] [PubMed] [Google Scholar]
  12. Hogarth B. G., Higgins C. F. Genetic organization of the oligopeptide permease (opp) locus of Salmonella typhimurium and Escherichia coli. J Bacteriol. 1983 Mar;153(3):1548–1551. doi: 10.1128/jb.153.3.1548-1551.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Holo H., Nes I. F. High-Frequency Transformation, by Electroporation, of Lactococcus lactis subsp. cremoris Grown with Glycine in Osmotically Stabilized Media. Appl Environ Microbiol. 1989 Dec;55(12):3119–3123. doi: 10.1128/aem.55.12.3119-3123.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jongeneel C. V., Bouvier J., Bairoch A. A unique signature identifies a family of zinc-dependent metallopeptidases. FEBS Lett. 1989 Jan 2;242(2):211–214. doi: 10.1016/0014-5793(89)80471-5. [DOI] [PubMed] [Google Scholar]
  15. Kim Y. A., Shriver B., Quay T., Hersh L. B. Analysis of the importance of arginine 102 in neutral endopeptidase (enkephalinase) catalysis. J Biol Chem. 1992 Jun 15;267(17):12330–12335. [PubMed] [Google Scholar]
  16. Kok J. Genetics of the proteolytic system of lactic acid bacteria. FEMS Microbiol Rev. 1990 Sep;7(1-2):15–42. doi: 10.1111/j.1574-6968.1990.tb04877.x. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Lazdunski A. M. Peptidases and proteases of Escherichia coli and Salmonella typhimurium. FEMS Microbiol Rev. 1989 Sep;5(3):265–276. doi: 10.1016/0168-6445(89)90035-1. [DOI] [PubMed] [Google Scholar]
  19. Le Moual H., Devault A., Roques B. P., Crine P., Boileau G. Identification of glutamic acid 646 as a zinc-coordinating residue in endopeptidase-24.11. J Biol Chem. 1991 Aug 25;266(24):15670–15674. [PubMed] [Google Scholar]
  20. Leenhouts K. J., Kok J., Venema G. Campbell-like integration of heterologous plasmid DNA into the chromosome of Lactococcus lactis subsp. lactis. Appl Environ Microbiol. 1989 Feb;55(2):394–400. doi: 10.1128/aem.55.2.394-400.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Leenhouts K. J., Kok J., Venema G. Lactococcal plasmid pWV01 as an integration vector for lactococci. Appl Environ Microbiol. 1991 Sep;57(9):2562–2567. doi: 10.1128/aem.57.9.2562-2567.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Leenhouts K. J., Kok J., Venema G. Stability of Integrated Plasmids in the Chromosome of Lactococcus lactis. Appl Environ Microbiol. 1990 Sep;56(9):2726–2735. doi: 10.1128/aem.56.9.2726-2735.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lennarz W. J., Strittmatter W. J. Cellular functions of metallo-endoproteinases. Biochim Biophys Acta. 1991 Jul 22;1071(2):149–158. doi: 10.1016/0304-4157(91)90022-o. [DOI] [PubMed] [Google Scholar]
  24. Letarte M., Vera S., Tran R., Addis J. B., Onizuka R. J., Quackenbush E. J., Jongeneel C. V., McInnes R. R. Common acute lymphocytic leukemia antigen is identical to neutral endopeptidase. J Exp Med. 1988 Oct 1;168(4):1247–1253. doi: 10.1084/jem.168.4.1247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ludwig W., Seewaldt E., Kilpper-Bälz R., Schleifer K. H., Magrum L., Woese C. R., Fox G. E., Stackebrandt E. The phylogenetic position of Streptococcus and Enterococcus. J Gen Microbiol. 1985 Mar;131(3):543–551. doi: 10.1099/00221287-131-3-543. [DOI] [PubMed] [Google Scholar]
  26. Malfroy B., Kuang W. J., Seeburg P. H., Mason A. J., Schofield P. R. Molecular cloning and amino acid sequence of human enkephalinase (neutral endopeptidase). FEBS Lett. 1988 Feb 29;229(1):206–210. doi: 10.1016/0014-5793(88)80828-7. [DOI] [PubMed] [Google Scholar]
  27. Malfroy B., Schofield P. R., Kuang W. J., Seeburg P. H., Mason A. J., Henzel W. J. Molecular cloning and amino acid sequence of rat enkephalinase. Biochem Biophys Res Commun. 1987 Apr 14;144(1):59–66. doi: 10.1016/s0006-291x(87)80475-8. [DOI] [PubMed] [Google Scholar]
  28. Mayo B., Kok J., Venema K., Bockelmann W., Teuber M., Reinke H., Venema G. Molecular cloning and sequence analysis of the X-prolyl dipeptidyl aminopeptidase gene from Lactococcus lactis subsp. cremoris. Appl Environ Microbiol. 1991 Jan;57(1):38–44. doi: 10.1128/aem.57.1.38-44.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nardi M., Chopin M. C., Chopin A., Cals M. M., Gripon J. C. Cloning and DNA sequence analysis of an X-prolyl dipeptidyl aminopeptidase gene from Lactococcus lactis subsp. lactis NCDO 763. Appl Environ Microbiol. 1991 Jan;57(1):45–50. doi: 10.1128/aem.57.1.45-50.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Otto R., de Vos W. M., Gavrieli J. Plasmid DNA in Streptococcus cremoris Wg2: Influence of pH on Selection in Chemostats of a Variant Lacking a Protease Plasmid. Appl Environ Microbiol. 1982 Jun;43(6):1272–1277. doi: 10.1128/aem.43.6.1272-1277.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Reid J. R., Ng K. H., Moore C. H., Coolbear T., Pritchard G. G. Comparison of bovine beta-casein hydrolysis by PI and PIII-type proteinases from Lactococcus lactis subsp. cremoris [corrected]. Appl Microbiol Biotechnol. 1991 Dec;36(3):344–351. doi: 10.1007/BF00208154. [DOI] [PubMed] [Google Scholar]
  33. Rottländer E., Trautner T. A. Genetic and transfection studies with B, subtilis phage SP 50. I. Phage mutants with restricted growth on B. subtilis strain 168. Mol Gen Genet. 1970;108(1):47–60. doi: 10.1007/BF00343184. [DOI] [PubMed] [Google Scholar]
  34. Rudner D. Z., LeDeaux J. R., Ireton K., Grossman A. D. The spo0K locus of Bacillus subtilis is homologous to the oligopeptide permease locus and is required for sporulation and competence. J Bacteriol. 1991 Feb;173(4):1388–1398. doi: 10.1128/jb.173.4.1388-1398.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sales N., Dutriez I., Maziere B., Ottaviani M., Roques B. P. Neutral endopeptidase 24.11 in rat peripheral tissues: comparative localization by 'ex vivo' and 'in vitro' autoradiography. Regul Pept. 1991 Apr 25;33(2):209–222. doi: 10.1016/0167-0115(91)90215-3. [DOI] [PubMed] [Google Scholar]
  36. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shipp M. A., Richardson N. E., Sayre P. H., Brown N. R., Masteller E. L., Clayton L. K., Ritz J., Reinherz E. L. Molecular cloning of the common acute lymphoblastic leukemia antigen (CALLA) identifies a type II integral membrane protein. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4819–4823. doi: 10.1073/pnas.85.13.4819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Smid E. J., Poolman B., Konings W. N. Casein utilization by lactococci. Appl Environ Microbiol. 1991 Sep;57(9):2447–2452. doi: 10.1128/aem.57.9.2447-2452.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Strøman P. Sequence of a gene (lap) encoding a 95.3-kDa aminopeptidase from Lactococcus lactis ssp. cremoris Wg2. Gene. 1992 Apr 1;113(1):107–112. doi: 10.1016/0378-1119(92)90676-g. [DOI] [PubMed] [Google Scholar]
  40. Tam L. T., Engelbrecht S., Talent J. M., Gracy R. W., Erdös E. G. The importance of disulfide bridges in human endopeptidase (enkephalinase) after proteolytic cleavage. Biochem Biophys Res Commun. 1985 Dec 31;133(3):1187–1192. doi: 10.1016/0006-291x(85)91262-8. [DOI] [PubMed] [Google Scholar]
  41. Tan P. S., Chapot-Chartier M. P., Pos K. M., Rousseau M., Boquien C. Y., Gripon J. C., Konings W. N. Localization of peptidases in lactococci. Appl Environ Microbiol. 1992 Jan;58(1):285–290. doi: 10.1128/aem.58.1.285-290.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tan P. S., Pos K. M., Konings W. N. Purification and characterization of an endopeptidase from Lactococcus lactis subsp. cremoris Wg2. Appl Environ Microbiol. 1991 Dec;57(12):3593–3599. doi: 10.1128/aem.57.12.3593-3599.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tan P. S., van Alen-Boerrigter I. J., Poolman B., Siezen R. J., de Vos W. M., Konings W. N. Characterization of the Lactococcus lactis pepN gene encoding an aminopeptidase homologous to mammalian aminopeptidase N. FEBS Lett. 1992 Jul 13;306(1):9–16. doi: 10.1016/0014-5793(92)80827-4. [DOI] [PubMed] [Google Scholar]
  44. Terzaghi B. E., Sandine W. E. Improved medium for lactic streptococci and their bacteriophages. Appl Microbiol. 1975 Jun;29(6):807–813. doi: 10.1128/am.29.6.807-813.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  46. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tynkkynen S., von Wright A., Syväoja E. L. Peptide Utilization Encoded by Lactococcus lactis subsp. lactis SSL135 Chromosomal DNA. Appl Environ Microbiol. 1989 Oct;55(10):2690–2695. doi: 10.1128/aem.55.10.2690-2695.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Vijayaraghavan J., Kim Y. A., Jackson D., Orlowski M., Hersh L. B. Use of site-directed mutagenesis to identify valine-573 in the S'1 binding site of rat neutral endopeptidase 24.11 (enkephalinase). Biochemistry. 1990 Sep 4;29(35):8052–8056. doi: 10.1021/bi00487a009. [DOI] [PubMed] [Google Scholar]
  49. Visser S., Exterkate F. A., Slangen C. J., de Veer G. J. Comparative Study of Action of Cell Wall Proteinases from Various Strains of Streptococcus cremoris on Bovine alpha(s1)-, beta-, and kappa-Casein. Appl Environ Microbiol. 1986 Nov;52(5):1162–1166. doi: 10.1128/aem.52.5.1162-1166.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Visser S., Robben A. J., Slangen C. J. Specificity of a cell-envelope-located proteinase (PIII-type) from Lactococcus lactis subsp. cremoris AM1 in its action on bovine beta-casein. Appl Microbiol Biotechnol. 1991 Jul;35(4):477–483. doi: 10.1007/BF00169753. [DOI] [PubMed] [Google Scholar]
  51. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  52. Zabarovsky E. R., Turina O. V. Rapid isolation of lambda phage DNA in micro- and macro-variants. Nucleic Acids Res. 1988 Nov 25;16(22):10925–10925. doi: 10.1093/nar/16.22.10925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zabarovsky E. R., Winberg G. High efficiency electroporation of ligated DNA into bacteria. Nucleic Acids Res. 1990 Oct 11;18(19):5912–5912. doi: 10.1093/nar/18.19.5912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. van Alen-Boerrigter I. J., Baankreis R., de Vos W. M. Characterization and overexpression of the Lactococcus lactis pepN gene and localization of its product, aminopeptidase N. Appl Environ Microbiol. 1991 Sep;57(9):2555–2561. doi: 10.1128/aem.57.9.2555-2561.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. van de Guchte M., Kodde J., van der Vossen J. M., Kok J., Venema G. Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease. Appl Environ Microbiol. 1990 Sep;56(9):2606–2611. doi: 10.1128/aem.56.9.2606-2611.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. van de Guchte M., Kok J., Venema G. Gene expression in Lactococcus lactis. FEMS Microbiol Rev. 1992 Feb;8(2):73–92. doi: 10.1111/j.1574-6968.1992.tb04958.x. [DOI] [PubMed] [Google Scholar]
  57. 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]
  58. van der Vossen J. M., van der Lelie D., Venema G. Isolation and characterization of Streptococcus cremoris Wg2-specific promoters. Appl Environ Microbiol. 1987 Oct;53(10):2452–2457. doi: 10.1128/aem.53.10.2452-2457.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]

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