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Lysozyme expression in Lactococcus lactis

  • Applied Genetics and Regulation
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Summary

Three lysozyme-encoding genes, one of eukaryotic and two of prokaryotic origin, were expressed in Lactococcus lactis subsp. lactis. Hen egg white lysozyme (HEL) could be detected in L. lactis lysates by Western blotting. No lysozyme activity was observed, however, presumably because of the absence of correctly formed disulphide bonds in the L. lactis product. The functionally related lysozymes of the E. coli bacteriophages T4 and λ were produced as biologically active proteins in L. lactis. In both cases, the highest expression levels were obtained using configurations in which the bacteriophage lysozyme genes had been translationally coupled to a short open reading frame of lactococcal origin. Both enzymes, like HEL, may prevent the growth of food-spoilage bacteria.

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References

  • Bienkowska-Szewczyk K, Taylor A (1980) Murein transglycosylase from phage λ lysate; purification and properties. Biochim Biophys Acta 615:489–496

    Google Scholar 

  • Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523

    CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Casadaban MJ, Cohen SN (1980) Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol 138:179–207

    Google Scholar 

  • Chang S, Cohen SN (1979) High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet 168:111–115

    CAS  PubMed  Google Scholar 

  • Chopin A, Chopin MC, Moillo-Batt A, Langella P (1984) Two plasmid-determined restriction systems in Streptococcus lactis. Plasmid 11:260–263

    Google Scholar 

  • Crawford RJM (ed) (1987) The use of lysozyme in the prevention of late blowing in cheese, Bulletin of the International Dairy Federation no. 216. International Dairy Federation, Brussels

    Google Scholar 

  • Edens L, Heslinga L, Klok R, Ledeboer AM, Maat J, Toonen MY, Visser C, Verrips CT (1982) Cloning of cDNA encoding the sweet-tasting plant protein thaumatin and its expression in Escherichia coli. Gene 18:1–12

    CAS  PubMed  Google Scholar 

  • Garrett J, Fusselman R, Hise J, Chiou L, Smith-Grillo D, Schulz J, Young R (1981) Cell lysis by induction of cloned lambda lysis genes. Mol Gen Genet 182:326–331

    Google Scholar 

  • Grütter MG, Weaver LH, Matthews BW (1983) Goose lysozyme structure: an evolutionary link between hen and bacteriophage lysozymes? Nature 303:828–831

    Google Scholar 

  • Guchte M van de, Kodde J, Vossen JMBM van der, Kok J, Venema G (1990) Heterologous gene expression in Lactococcus lactis susp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease. Appl Environ Microbiol 56:2606–2611

    Google Scholar 

  • Guchte M van de, Kok J, Venema G (1991) Distance-dependent translational coupling and interference in Lactococcus lactis. Mol Gen Genet 227:65–71

    Google Scholar 

  • Guchte M van de, Vossen JMBM van der, Kok J, Venema G (1989) Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis. Appl Environ Microbiol 55:224–228

    Google Scholar 

  • Hohn B (1979) In vitro packaging of γ and cosmid DNA. Methods Enzymol 68:299–309

    Google Scholar 

  • Hughey VL, Johnson EA (1987) Antimicrobial activity of lysozyme against bacteria involved in food spoilage and food-borne disease. Appl Environ Microbiol 53:2165–2170

    Google Scholar 

  • Hughey VL, Wilger PA, Johnson EA (1989) Antibacterial activity of hen egg white lysozyme against Lysteria monocytogenes Scott A in foods. Appl Environ Microbiol 55:631–638

    Google Scholar 

  • Imoto T, Yamada H, Yasukochi T, Yamada E, Ito Y, Ueda T, Nagatani H, Miki T, Horiuchi T (1987) Point mutation of alanine (31) to valine prohibits the folding of reduced lysozyme by sulfhydryl-disulfide interchange. Protein Engineering 1:333–338

    Google Scholar 

  • Jollés P, Jollés J (1984) What's new in lysozyme research? Mol Cell Biochem 63:165–189

    Google Scholar 

  • Kok J, Vossen JMBM van der, Venema G (1984) Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli. Appl Environ Microbiol 48:726–731

    Google Scholar 

  • Kok J, Leenhouts KJ, Haandrikman AJ, Ledeboer AM, Venema G (1988) Nucleotide sequence of the cell wall proteinase gene of Streptococcus cremoris Wg2. Appl Environ Microbiol 54:231–238

    Google Scholar 

  • Koteswara Rao GR, Burma DP (1971) Purification and properties of phage P22-induced lysozyme. J Biol Chem 246:6474–6479

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685

    PubMed  Google Scholar 

  • Lelie D van der, Vossen JMBM van der, Venema G (1988) Effect of plasmid incompatibility on DNA transfer to Streptococcus cremoris. Appl Environ Microbiol 54:865–871

    Google Scholar 

  • Mandel M, Higa A (1970) Calcium-dependent bacteriophage DNA infection. J Mol Biol 53:159–162

    Google Scholar 

  • Matthews BW, Grütter MG, Anderson WF, Remington SJ (1981a) Common precursor of lysozymes of hen egg-white and bacteriophage T4. Nature 290:334–335

    Google Scholar 

  • Matthews BW, Remington SJ, Grütter MG, Anderson WF (1981b) Relation between hen egg white lysozyme and bacteriophage T4 lysozyme: evolutionary implications. J Mol Biol 147:545–558

    Google Scholar 

  • McKay LL, Baldwin KA (1990) Applications for biotechnology: present and future improvements in lactic acid bacteria. FEMS Microbiol Rev 87:3–14

    Google Scholar 

  • Ostroff GR, Péne JJ (1983) Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis: isolation of a spontaneous mutant of Bacillus subtilis with enhanced transformability for Escherichia coli-propagated chimeric plasmid DNA. J Bacteriol 156:934–936

    Google Scholar 

  • Owen JE, Schultz DW, Taylor A, Smith GR (1983) Nucleotide sequence of the lysozyme gene of bacteriophage T4; analysis of mutations involving repeated sequences. J Mol Biol 165:229–248

    Google Scholar 

  • Perry LJ, Heyneker HL, Wetzel R (1985) Non-toxic expression in Escherichia coli of a plasmid-encoded gene for phage T4 lysozyme. Gene 38:259–264

    Google Scholar 

  • Rottlander E, Trautner TA (1970) Genetic and transfection studies with Bacillus subtilis phage SP50. J Mol Biol 108:47–60

    Google Scholar 

  • Simon D, Chopin A (1988) Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis. Biochimie 70:559–566

    Google Scholar 

  • Stanssen P, Opsomer C, Mckeown YM, Kramer W, Zabeau M, Fritz HJ (1989) Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers. Nucleic Acids Res 17:4441–4454

    Google Scholar 

  • Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130

    CAS  PubMed  Google Scholar 

  • Tabor S, Richardson CC (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci USA 82:1074–1078

    Google Scholar 

  • Tabor S, Richardson CC (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci USA 84:4767–4771

    Google Scholar 

  • Takahashi H, Saito H, Ikeda Y (1978) Viable T4 bacteriophage containing cytosine-sbustituted DNA (T4dC phage). I. Behavior towards the restriction-modification systems of Escherichia coli and derivation of a new T4 phage strain (T4dC) having the complete T4 genome. J Gen Appl Microbiol 24:297–306

    Google Scholar 

  • Taylor A, Das BC, Heijenoort J van (1975) Bacterial-cell-wall peptidoglycan fragments produced by phage λ or Vi II endolysin and containing 1,6-anhydro-N-acetylmuramic acid. Eur J Biochem 53:47–54

    Google Scholar 

  • Terzaghi BE, Sandine WE (1975) Improved medium for lactic streptococci and their bacteriophages. Appl Microbiol 29:807–813

    Google Scholar 

  • Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354

    CAS  PubMed  Google Scholar 

  • Vos WM de (1987) Gene cloning and expression in lactic streptococci. FEMS Microbiol Rev 46:281–295

    Google Scholar 

  • Vos WM de, Vos P, Simons G, David S (1989) Gene organization and expression in mesophilic lactic acid bacteria. J Dairy Sci 72:3398–3405

    Google Scholar 

  • Vossen JMBM van der, Lelie D van der, Venema G (1987) Isolation and characterization of Streptococcus cremoris Wg2-specific promoters. Appl Environ Microbiol 53:2452–2457

    Google Scholar 

  • Vossen JMBM van der, Kok J, Lelie D van der, Venema G (1988) Liposome-enhanced transformation of Lactococcus lactis and plasmid transfer by intergeneric protoplast fusion of Streptococcus lactis and Bacillus subtilis. FEMS Microbiol Lett 49:323–329

    Google Scholar 

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van de Guchte, M., van der Wal, F.J., Kok, J. et al. Lysozyme expression in Lactococcus lactis . Appl Microbiol Biotechnol 37, 216–224 (1992). https://doi.org/10.1007/BF00178174

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