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LysA2, the Lactobacillus casei bacteriophage A2 lysin is an endopeptidase active on a wide spectrum of lactic acid bacteria

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Abstract

The lysin gene (lysA2) of the Lactobacillus casei bacteriophage A2 was cloned and expressed in Escherichia coli. LysA2 is an endopeptidase that hydrolyzes the bond between the terminal d-alanine of the peptidoglycan tetrapeptide and the aspartic acid residue that forms the bridge with the l-lysine of a neighboring peptidoglycan chain, characteristic of Gram-positive bacteria included into the A4 peptidoglycan subgroup. This includes most lactobacilli, Lactococcus lactis, Pediococcus acidilactici, and Pediococcus pentosaceus, the walls of all of which were substrates for the enzyme. Specific binding of LysA2 to the wall of these bacteria is mediated by its C-terminal moiety, does not need the N-terminal catalytic domain for recognition, and is stable: at least 88% of the molecules were still bound to L. casei after 3 days in phosphate buffer at 4°C. The enzyme acts as a monomer, is active at pH values between 4 and 6, and at temperatures ranging between 18°C and 50°C while being independent of divalent cation addition. The enzyme showed strong resistance to incubation at high and low pH values but became progressively inactivated at 50°C and above. LysA2 is bactericidal, the viability of L. casei cultures dropping to 1% in 10 min, under the standard conditions used for the enzymatic assay.

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References

  • Álvarez MA, Herrero M, Suarez JE (1998) The site-specific recombination system of the Lactobacillus species bacteriophage A2 integrates in Gram-positive and Gram-negative bacteria. Virology 250:185–193

    Article  Google Scholar 

  • Billot-Klein DR, Legrand B, Schoot J, van Heijenoort J, Gutmann L (1997) Peptidoglycan structure of Lactobacillus casei, a species highly resistant to glycopeptide antibiotics. J Bacteriol 179:6208–6212

    CAS  Google Scholar 

  • Brussow H, Suarez JE (2006) Lactobacillus phages. In: Calendar R (ed) The bacteriophages, 2nd edn. Plenum, New York, pp 653–666

    Google Scholar 

  • de Ruyter PG, Kuipers OP, Meijer WC, de Vos WM (1997) Food-grade controlled lysis of Lactococcus lactis for accelerated cheese ripening. Nat Biotechnol 15:976–979

    Article  Google Scholar 

  • Deutsch SM, Guezenec S, Piot M, Foster S, Lortal S (2004) Mur-LH, the broad-spectrum endolysin of Lactobacillus helveticus temperate bacteriophage phi-0303. Appl Environ Microbiol 70:96–103

    Article  CAS  Google Scholar 

  • Deveau H, Labrie SJ, Chopin MC, Moineau S (2006) Biodiversity and classification of lactococcal phages. Appl Environ Microbiol 72:4338–4346

    Article  CAS  Google Scholar 

  • Díaz E, López R, García JL (1990) Chimeric phage-bacterial enzymes: a clue to the modular evolution of genes. Proc Natl Acad Sci USA 87(20):8125–8129

    Article  Google Scholar 

  • Ellis KJ, Morrison JF (1982) Buffers of constant ionic strength for studying pH-dependent processes. Methods Enzymol 87:405–426

    Article  CAS  Google Scholar 

  • García P, Ladero V, Suarez JE (2003) Analysis of the morphogenetic cluster and genome of the temperate Lactobacillus casei bacteriophage A2. Arch Virol 148:1051–1070

    Article  Google Scholar 

  • García PB, Martínez J, Obeso M, Rodríguez A (2008) Bacteriophages and their application in food safety. Lett Appl Microbiol 47:479–485

    Article  Google Scholar 

  • Henrich B, Binishofer B, Bläsi U (1995) Primary structure and functional analysis of the lysis genes of Lactobacillus gasseri bacteriophage phi adh. J Bacteriol 177:723–732

    CAS  Google Scholar 

  • Herrero M, de los Reyes-Gavilán CG, Caso JL, Suarez JE (1994) Characterization of 393-A2, a bacteriophage that infects Lactobacillus casei. Microbiology 140:2585–2590

    Article  Google Scholar 

  • Hu S, Kong J, Kong W, Guo T, Ji M (2010) Characterization of a novel LysM domain from Lactobacillus fermentum bacteriophage endolysin and its use as an anchor to display heterologous proteins on the surfaces of lactic acid bacteria. Appl Environ Microbiol 76:2410–2418

    Article  CAS  Google Scholar 

  • Hudson JA, Billington C, Carey-Smith G, Greening G (2005) Bacteriophages as biocontrol agents in food. J Food Prot 68:426–437

    CAS  Google Scholar 

  • Hungerer KD, Tipper DJ (1969) Cell wall polymers of Bacillus sphaericus 9602. I. Structure of the vegetative peptidoglycan. Biochemistry 8:3577–3587

    Article  CAS  Google Scholar 

  • Kashige N, Nakashima Y, Miake F, Watanabe K (2000) Cloning, sequence analysis, and expression of Lactobacillus casei phage PL-1 lysis genes. Arch Virol 145:1521–1534

    Article  CAS  Google Scholar 

  • Kneen M, Farinas J, Li Y, Verkman AS (1998) Green fluorescent protein as a noninvasive intracellular pH indicator. Biophys J 74:1591–1599

    Article  CAS  Google Scholar 

  • Labrie SJ, Samson JE, Moineau S (2010) Bacteriophage resistance mechanisms. Nat Rev Microbiol 8:317–327

    Article  CAS  Google Scholar 

  • Leuschner RGQ, Robinson TP, Hugas M, Cocconcelli PS, Richard-Forget F, Klein G, Licht TR, Nguyen-Thé C, Querol A, Richardson M, Suarez JE, Thrane U, Vlak JM, von Wright A (2010) Qualified Presumption of Safety (QPS): A generic risk assessment approach for biological agents notified to the European Food Safety Authority (EFSA). Trends Food Sci Technol 21:425–435

    Article  CAS  Google Scholar 

  • Loeffler JM, Nelson D, Fischetti VA (2001) Rapid killing of Streptococcus pneumoniae with a bacteriophage cell wall hydrolase. Science 294:2170–2172

    Article  CAS  Google Scholar 

  • Loessner MJ, Kramer K, Ebel F, Scherer S (2002) C-terminal domains of Listeria monocytogenes bacteriophage murein hydrolases determine specific recognition and high-affinity binding to bacterial cell wall carbohydrates. Mol Microbiol 44:335–349

    Article  CAS  Google Scholar 

  • Matsuda T, Kotani S, Kato K (1968) Structure of the cell walls of Lactobacillus plantarum ATCC 8014. II. Cross linkage between d-alanine and a,a′-diaminopimelic acid in the cell wall peptidoglycans studied with an L-11 enzyme from Flavobacterium sp. Biken J 11:127–138

    CAS  Google Scholar 

  • Mazé A, Boël G, Zúñiga M, Bourand A, Loux V, Yebra MJ, Monedero V, Correia K, Jacques N, Beaufils S, Poncet S, Joyet P, Milohanic E, Casarégola S, Auffray Y, Pérez-Martínez G, Gibrat JF, Zagorec M, Francke C, Hartke A, Deutscher J (2010) Complete genome sequence of the probiotic Lactobacillus casei strain BL23. J Bacteriol 192:2647–2648

    Article  Google Scholar 

  • Mc Grath S, Fitzgerald GF, van Sinderen D (2007) Bacteriophages in dairy products: pros and cons. Biotechnol J 2:450–455

    Article  CAS  Google Scholar 

  • Moscoso M, Suarez JE (2000) Characterization of the DNA replication module of bacteriophage A2 and use of its origin of replication as a defense against infection during milk fermentation by Lactobacillus casei. Virology 273:101–111

    Article  CAS  Google Scholar 

  • O’Flaherty S, Ross RP, Coffey A (2009) Bacteriophage and their lysins for elimination of infectious bacteria. FEMS Microbiol Rev 33:801–819

    Article  Google Scholar 

  • Obeso JM, Martínez B, Rodríguez A, García P (2008) Lytic activity of the recombinant staphylococcal bacteriophage PhiH5 endolysin active against Staphylococcus aureus in milk. Int J Food Microbiol 128:212–218

    Article  CAS  Google Scholar 

  • Pennartz A, Généreux C, Parquet C, Mengin-Lecreulx D, Joris B (2009) Substrate-induced inactivation of the Escherichia coli AmiD N-acetylmuramoyl-l-alanine amidase highlights a new strategy to inhibit this class of enzyme. Antimicrob Agents Chemother 53:2991–2997

    Article  CAS  Google Scholar 

  • Pratto F, Cicek A, Weihofen WA, Lurz R, Saenger W, Alonso JC (2008) Streptococcus pyogenes pSM19035 requires dynamic assembly of ATP-bound ParA and ParB on parS DNA during plasmid segregation. Nucleic Acids Res 36:3676–3689

    Article  CAS  Google Scholar 

  • Pritchard DG, Dong S, Baker JR, Engler JA (2004) The bifunctional peptidoglycan lysin of Streptococcus agalactiae bacteriophage B30. Microbiology 150:2079–2087

    Article  CAS  Google Scholar 

  • Riipinen KA, Forsman P, Alatossava T (2011) The genomes and comparative genomics of Lactobacillus delbrueckii phages. Arch Virol 156:1217–1233

    Article  CAS  Google Scholar 

  • Schleifer KH, Kandler O (1972) Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36:407–477

    CAS  Google Scholar 

  • Shearman C, Underwood H, Jury K, Gasson M (1989) Cloning and DNA sequence analysis of a Lactococcus bacteriophage lysin gene. Mol Gen Genet 218:214–221

    Article  CAS  Google Scholar 

  • Shearman CA, Jury K, Gasson M (1992) Autolytic Lactococcus lactis expressing a lactococcal bacteriophage lysin gene. Biotechnol 10:196–199

    Article  CAS  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

    Article  CAS  Google Scholar 

  • Sturino JM, Klaenhammer TR (2006) Engineered bacteriophage-defense systems in bioprocessing. Nat Rev Microbiol 4:395–404

    Article  CAS  Google Scholar 

  • Vasala A, Välkkilä M, Caldentey J, Alatossava T (1995) Genetic and biochemical characterization of the Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H lysin. Appl Environ Microbiol 61:4004–4011

    CAS  Google Scholar 

  • Villion M, Moineau S (2009) Bacteriophages of Lactobacillus. Front Biosci 14:1661–1683

    Article  CAS  Google Scholar 

  • Wessels S, Axelsson L, Hansen EB, de Vuyst L, Laulund S, Lähteenmäki L, Lindgren S, Mollet B, Salminen S, von Wright A (2004) The lactic acid bacteria, the food chain, and their regulation. Trends Food Sci Technol 15:498–505

    Article  CAS  Google Scholar 

  • Young R, Wang IN (2006) Phage lysis. In: Calendar R (ed) The bacteriophages, 2nd edn. Plenum, New York, pp 104–125

    Google Scholar 

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Acknowledgments

This work was supported by the CICYT grants BFU2007-65781 and AGL2010-15097 from the Ministry of Science and Technology (Spain) and the FEDER Plan. P. R. is the holder of a fellowship from Chemital S. A. We gratefully acknowledge the provision of strains by J. Guijarro, A. Margolles, A. Rodriguez, and R. Rodicio and the help of L. Bermudez-Humarán with the FCM experiments.

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Authors and Affiliations

  1. Area de Microbiología, Instituto Universitario de Biotecnología, Universidad de Oviedo, Oviedo, Spain

    Pedro Ribelles, Isabel Rodríguez & Juan Evaristo Suárez

  2. Instituto de Productos Lácteos de Asturias (CSIC), Villaviciosa, Spain

    Juan Evaristo Suárez

  3. Area de Microbiología, Facultad de Medicina, Universidad de Oviedo, Julián Clavería 6, 33006, Oviedo, Spain

    Juan Evaristo Suárez

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  1. Pedro Ribelles
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  2. Isabel Rodríguez
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Correspondence to Juan Evaristo Suárez.

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Ribelles, P., Rodríguez, I. & Suárez, J.E. LysA2, the Lactobacillus casei bacteriophage A2 lysin is an endopeptidase active on a wide spectrum of lactic acid bacteria. Appl Microbiol Biotechnol 94, 101–110 (2012). https://doi.org/10.1007/s00253-011-3588-5

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