ABSTRACT Listeria monocytogenes is a foodborne pathogen capable of surviving under challenging conditions both outside and inside the host. During the transition from exponential to stationary phase it experiences a series of environmental changes that require an appropriate response to maintain cell viability. In this study the autolytic behaviour of a L. monocytogenes strain was investigated by two-dimensional electrophoresis. The study was done at the permissive autolysis temperature, 30℃ and at 20℃, an autolysis non-permissive temperature. An autolytic strain proteome was also compared to a non-autolytic strain at the permissive autolysis temperature. The autolytic strain proteome at 30℃ in comparison to 20℃ evidenced increased synthesis of the P60 autolysin, glycolytic enzymes and proteins related with environmental stress responses. The over-production of P45 autolysin, was observed when the autolytic strain proteome was compared with the non-autolytic strain. The proteomes at the non-permissive temperature and the proteome of the non-autolytic strain were characterized by a diminished synthesis of several stress related proteins. The lack of autolysis seems to be associated to the over-production of proteins linked to fatty acid and amino acid synthesis, transcription regulation and cell morphogenesis as evidenced by the proteome at the non-permissive temperature and the non-autolytic strain. Autolysis proteome evidenced the over-production of P60 autolysins, glycolysis and stress proteins whereas the proteome obtained in conditions of absence of autolysis reveal a completely different group of proteins. Possible targets to activate listerial autolysis were identified.
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E. Pinto, N. Marques, P. W. Andrew and M. Leonor Faleiro, "Over-Production of P60 Family Proteins, Glycolytic and Stress Response Proteins Characterizes the Autolytic Profile of Listeria monocytogenes," Advances in Microbiology, Vol. 2 No. 2, 2012, pp. 181-200. doi: 10.4236/aim.2012.22023.
 E. Jones, G. Shama, D. Jones, I. S. Roberts and P. A. Andrew, “Physiological and Biochemical Studies on Phychrotolerance in Listeria monocytogenes.” Journal of Applied Microbiology, Vol. 83. No. 1, 1997, pp. 31-35.
 L. Pine, G. B. Malcom, J. B. Brooks and M. I Danesshvar, “Physiological Studies on the Growth and Utilization of Sugars by Listeria Species,” Canadian Journal of Microbiology, Vol. 35, No. 2, 1989, pp. 245-254.
 J. V. Holtje, “From Growth to Autolysis: The Murein Hydrolases in Escherichia coli,” Archives of Microbiology, Vol. 164, No. 4, 1995, pp. 243-254.
 A. M. Berry, R. A. Lock, D. Hansman and J. C. Paton, “Contribution of Autolysin to Virulence of Streptococcus pneumoniae,” Infection and Immunity, Vol. 57, No. 8, 1989, pp. 2324-2330.
 N. Mani, L. M. Baddour, D. Q. Offutt, U. Vijaranakul, M. J. Nadakavukaren and R. K. Jayaswal, “Autolysis-Defective Mutant of Staphylococcus aureus: Pathological Considerations, Genetic Mapping and Electron Microscopic Studies,” Infection and Immunity, Vol. 62, No. 4, 1994, pp. 1406-1409.
 E. A. Marcus and D. R. Scott, “Cell Lysis Is Responsible for the Appearance of Extracellular Urease in Helicobacter pylori,” Helicobacter, Vol. 6, No. 2, 2001, pp. 93-99. doi:10.1046/j.1523-5378.2001.00014.x
 A. Bubert, M. Kuhn, W. Goebel and S. Kohler, “Structural and Functional Properties of the p60 Proteins from Different Listeria Species,” Journal of Bacteriology, Vol. 174, No. 24 1992, pp. 8166-8171.
 D. Cabanes, O. Dussurget, P. Dehoux and P. Cossart, “Auto, a Surface Associated Autolysin of Listeria monocytogenes Required for Entry into Eukaryotic cells and Virulence,” Molecular Microbiology, Vol. 51, No. 6, 2004, pp. 1601-1614. doi:10.1111/j.1365-2958.2003.03945
 S. A Carroll, T. Hain, U. Technow, A. Darji, P. Phashalidis, S. W. Joseph and T. Chakraborty, “Identification and Characterization of a Murein Hydrolase, MurA, of Listeria monocytogenes, a Muramidase Needed for Cell Separation.” Journal of Bacteriology, Vol. 185, No. 23, 2003, pp. 6801-6808.
 A. M McLaughlan and S. J. Foster, “Molecular Characterization of an Autolytic Amidase of Listeria monocytogenes EGD,” Microbiology, Vol. 144, No. Pt 5, 1998, pp. 1359-1367.
 E. Milohanic, R. Jonquieres, P. B. Coossart, “The Autolysin Ami Contributes to the Adhesion of Listeria monocytogenes to Eukaryotic Cells via its Cell wall Anchor,” Molecular Microbiology, Vol. 39, No.5, 2001, pp. 1212-1224. doi:10.1111/j.1365-2958.2001.02208.x
 M. Popowska and Z Markiewicz, “Characterization of Listeria monocytogenes Protein Lmo0327 with Murein Hydrolase Activity,” Archives of Microbiology, Vol. 186, No.1, 2006, pp. 69-86. doi:10.1007/s2003-006-0122-8
 K. Schubert, A. M. Bichlmaier, E. Mager, K. Wolff, G. Ruhland, F. Fiedler, “P45, an Extracellular 45 kDa Protein of Listeria monocytogenes with Similarity to Protein p60 and Exhibiting Peptidoglycan Lytic Activity,” Archives of Microbiology, Vol. 173, No. 1, 2000, pp. 21-28.
 S. Pilgrim, A. Kolb-M?urer, I. Gentshev, W. Goebel and M. Kuhn, “Deletion of the Gene Encoding p60 in Listeria monocytogenes Leads to Abnormal Cell Division and Loss of Actin-Based Motility,” Infection and Immunity, Vol. 71, No. 6, 2003, pp. 3473-3484.
 M. D. Wuenscher, S. K?hler, A. Bubert, U. Gerike and W. Goebel, “The iap Gene of Listeria monocytogenes Is Essential for Cell Viability, and its Gene Product, p60, Has Bacteriolytic Activity,” Journal of Bacteriology, Vol. 175, No. 11, 1993, pp. 3491-350.
 R. Gonzalez, A. J. Martinez-Rodriguez and A. V. Carrascosa, “Yeast Autolytic Mutants Potentially Useful for Sparking Wine Production,” International Journal of Food Microbiology, Vol. 84, No. 1, 2003, pp. 21-26.
 T. Emri, Z. Molnár, M. Szilágyi and I. Pócsi, “Regulation of Autolysis in Aspergillus nidulans,” Applied Biochemistry and Biotechnology, Vol. 151, No. 2-3, 2008, pp. 211-220. doi:10.1007/s12010-008-8174-7
 F. C. Neuhaus and J. Baddiley, “A Continuum of Anionic Charge: Structures and Functions of D-Alanyl-teichoic Acids in Gram-Positive Bacteria,” Microbiology and Molecular Biology, Vol. 67, No. 4, 2003, pp. 686-723.
 I. Fedtke, D. Mader, T. Kohler, H. Moll, G. Nicholson, R. Biswas, K. Henseler, F. G?tz, U. Z?hringer and A. Peschel, “A Staphylococcus aureus ypfP Mutant with Strongly Reduced Lipoteichoic Acid (LTA) Content: LTA Governs Bacterial Surface Properties and Autolysin Activity,” Molecular Microbiology, Vol. 65, No. 4, 2007, pp. 1078-1091. doi:10.1111/j.1365-2958.2007.05854.x
 J. R. Scott and T. C. Barnett, “Surface Proteins of Gram-Positive Bacteria and How They Get There,” Annual Reviews of Microbiology, Vol. 60, 2006, pp. 397-423.
 J. Wecke, K Madela and W. Fischer, “The Absence of D-Alanine from Lipoteichoic Acid and Wall Teichoic Acid Alters Surface Charge, Enhances Autolysis and Increases Susceptibility to Methicillin in Bacillus subtilis,” Microbiology, Vol. 143, No. 9, 1997, pp. 2953-2960.
 S. Mesnage, T. Fontaine, M. Mignot, M. Delepierre, M. Mock and A. Fouet, “Bacterial SLH Domain Proteins Are Non-Covalently Anchored to the Cell Surface via a Conserved Mechanism Involving Wall Polysaccharide Pyruvylation,” Vol. 19, No. 17, The EMBO Journal, 2000, pp. 4473-4484. doi:10.1093/emboj/19.17.4473
 L. O. Ingram, “Mechanism of lysis of Escherichia coli by Ethanol and other Chaotropic Agents,” Journal of Bacteriology, Vol. 146, No. 1, 1981, pp. 331-336.
 S. R. Watt and A. J. Clarke, “Role of Autolysins in the EDTA-Induced Lysis of Pseudomonas aeruginosa,” FEMS Microbiology Letters, Vol. 124, No. 1, 1994, pp. 113-120.
 T. Ochiai, “Salt-Sensitive Growth of Staphylococcus aureus: Stimulation of Salt-Induced Autolysis by Multiple Environmental Factors,” Microbiology and Immunology, Vol. 43, No. 7, 1999, pp. 705-709.
 T. L. Trivett and E. A Meyer, “Citrate Cycle and Related Metabolism of Listeria monocytogenes,” Journal of Bacteriology, Vol. 107, No. 3, 1971, pp. 770-779.
 A. Adri?o, M. Vieira, I. Fernandes, M. Barbosa, M. Sol, R. P. Tenreiro, L. Chambel, B. Barata, I. Zilh?o, G. Shama, S. Perni, S. J. Jordan, P. W. Andrew and M. L. Faleiro, “Marked Intra-Strain Variation in Response of Listeria monocytogenes Dairy Isolates to Acid or Salt Stress and the Effect of Acid or Salt Adaptation on Adherence to Abiotic Surfaces,” International Journal of Food Micro- biology, Vol. 123, 2008, pp. 142-150.
 M. L.Faleiro, P. W. Andrew and D. Power, “Stress Response of Listeria monocytogenes Isolated from Cheese and other Foods,” International Journal of Food Microbiology, Vol. 84, No. 2, 2003, pp. 207-221.
 R. Fontana, M. Boaretti, A. Grossato, E. A. Tonin, M. M. Lléo and G. Satta, “Paradoxical Response of Enterococcus faecalis to the Bactericidal Activity of Penicillin is Associated with Reduced Activity of one Autolysin,” Antimicrobial Agents and Chemotherapy, Vol. 34, No. 2, 1990, pp. 314-320.
 C.-Y. Chen, G. W. Nace and P. L. Irwin, “A 6 × 6 Drop Plate Method for Simultaneous Colony Counting and MPN Enumeration of Campylobacter jejuni, Listeria monocytogenes and Escherichia coli,” Journal of Microbiological Methods, Vol. 55, No. 2, 2003, pp. 475-479.
 M. Trost, D. Wehmh?ner, U. K?rst, G. Dieterich, J. Wehland and L. J?nsch, “Comparative Proteome Analysis of Secretory Proteins from Pathogenic and Non-Pathogenic Listeria Species,” Proteomics, Vol. 5, No. 6, 2005, pp. 1544-1557. doi:10.1002/pmic.200401024
 P. Folio, P. Chavant, I. Chafsey, A. Belkorchia, C. Chambon and M Hébraud, “Two-Dimensional Electrophoresis Database of Listeria monocytogenes EGDe Proteome and Proteomic Analysis of Mild-Log and Stationary Growth Phase Cells,” Proteomics, Vol. 4, 2004, pp. 3187-3201.
 O. Duché, F. Tremoulet, P. Glaser and J. Labadie, “Salt Stress Proteins Induced in Listeria monocytogenes,” Applied and Environmental Microbiology, Vol. 68, No. 4 2002, pp. 1491-1498.
 C. Castaldo, R. A. Siciliano, L. Muscariello, R. Marasco and M. Sacco, “CcpA Affects Expression of the groESL and dnaK operons in Lactobacillus plantarum,” Microbial Cell Factories, Vol. 5, 2006, p. 35.
 G. Cacace, M. F. Mazzeo, A. Sorrentino, V. Spada, A. Malorni and R. A Siciliano, “Proteomics for the Elucidation of Cold Adaptation Mechanisms in Listeria monocytogenes,” Journal of Proteomics, Vol. 73, No. 10, 2010, pp. 2021-2030. doi:10.1016/j.prot.2010.06.011
 H. Antelmann, J. Bernhardt, R. Schmid, H. Mach, U. V?lker and M. Hecker, “First Steps from a Two-Dimensional Protein Index towards a Response-Regulation Map for Bacillus subtilis,” Electrophoresis, Vol. 18, No. 8, 1997, pp. 1451-1463. doi:10.1002/elps.1150180820
 P. Graumann, K. Schr?der, R. Schmid and M. A Marahiel, “Cold Shock Stress-Induced Proteins in Bacillus subtilis,” Journal of Bacteriology, Vol. 178, No. 15, 1996, pp. 4611-4619.
 R. J. Thompson, H. G. A. Bouwer, D. A. Portnoy and F. R. Frankel, “Pathogenicity and Immunogenicity of a Listeria monocytogenes Strain that Requires D-Alanine for Growth,” Infection and Immunity, Vol. 66, No. 8, 1998, pp. 3552-3561.
 R. Gardan, O. Duché, S. Leroy-Sétrin, European Listeria Genome Consortium and J. Labadie, “Role of ctc from Listeria monocytogenes in Osmotolerance,” Applied and Environmental Microbiology, Vol. 69, No. 1, 2003, pp. 154-61. doi:10.1128/AEM.69.1.154-161.2003
 U. V?lker, S. Engelmann, B. Maul, S. Riethdorf, A. V?lker, R. Schmid, H. Mach and M. Hecker, “Analysis of the Induction of General Stress Proteins of Bacillus subtilis,” Microbiology, Vol. 140, No. Pt 4, 1994, pp. 741-52.
 B. Pieterse, J. Rob, R. J. Leer, F. H. J. Schuren and M. J. van der Werf, “Unravelling the Multiple Effects of Lactic acid stress on Lactobacillus plantarum by Transcription Profiling,” Microbiology, Vol. 151, No. Pt12, 2005, pp. 3881-3894. doi:10.1099/mic.0.28304-0
 A. Hartke, S. Bouché, J.-C. Giard, A. Benachour, P. Boutibonnes and Y. Auffray, “The Lactic Acid Stress Response of Lactococcus lactis subsp. lactis,” Current Microbiology, Vol. 33, No. 3, 1996, pp. 194-199.
 C.-C. Tsou , C. Chiang-Ni, Y.-S. Lin, W.-J. Chuang, M. T. Lin, C. C. Liu and J. J. Wu, “An Iron-Binding Protein, Dpr, Decreases Hydrogen Peroxide Stress and Protects Streptococcus pyogenes Against Multiple Stresses,” Infection and Immunity, Vol. 76, No. 9, 2008, pp. 4038-4045. doi:10.1128/IAI.00477-08
 T. D. Caldas, A. E. Yaagoubi and G. Richarme, “Chaperone Properties of Bacterial Elongation Factor EF-Tu,” The Journal of Biological Chemistry, Vol. 273, No. 19, 1998, pp. 11478-11482.
 T. Hanawa, M. Fukuda, H. Kawakami, H. Hirano, S. Kamiya and T. Yamamoto, “The Listeria monocytogenes DnaK Chaperone Is Required for Stress Tolerance and Efficient Phagocytosis with Macrophages,” Cell Stress Chaperones, Vol. 4, No. 2, 1999, pp. 118-128.
 C. G. M.Gahan, J. O’Mahony and C. Hill, “Characterization of the groESL Operon in Listeria monocytogenes: Utilization of Two Reporter Systems (gfp and hly) for Evaluating in Vivo Expression,” Infection and Immunity, Vol. 69, No. 6, 2001, pp. 3924-3932.
 O. Fayet, T. Ziegelhoffer and C. Georgopoulos, “The groES and groEL Heat Shock Gene Products of Escherichia coli Are Essential for Bacterial Growth at All Temperatures,” Journal of Bacteriology, Vol. 171, No. 3, 1989, pp. 1379-1385.
 M. W. Qoronfleh, J. E. Gustafson and B. J. Wilkinson, “Conditions that Induce Staphylococcus aureus Heat Shock Proteins also Inhibit Autolysis,” FEMS Microbiology Letters, Vol. 66, No. 1, 1998, pp. 103-107.
 J. K. Powell and K. Y. Young, “Lysis of Escherichia coli by Beta-Lactams which bind Penicillin-Binding Proteins 1a and 1b: Inhibition by Heat Shock Proteins,” Journal of Bacteriology, Vol. 173, No. 13, 1991, pp. 4021-4026.
 V. K. Singh, S. Utaida, L. S. Jackson, R. K. Jayaswal, B. J. Wilkinson and N. R. Chamberlain, “Role of dnaK locus in Tolerance of Multiple Stresses in Staphylococcus aureus,” Microbiology, Vol. 153, No. 9, 2007, pp. 3162-3173. doi:10.1099/mic.0.2007/009506-0
 P. A. J. de Boer, R. E. Crossley and L. I. Rothfield, “Central Role for the Escherichia coli minC Gene Product in Two Different Cell Division-Inhibition Systems,” Proceedings of the National Academy of Sciences of USA, Vol. 87, No. 3, 1990, pp. 1129-1133.
 P. A. J. de Boer, R. E. Crossley and L. I. Rothfield, “A Division Inhibitor and a Topological Specificity Factor Coded for by the Minicell Locus Determine Proper Placement of the Division Septum in E. coli,” Cell, Vol. 56, No. 4, 1989, pp. 641-649.
 J. P. Claverys, M. Prudhomme and B. Martin, “Induction of Competence Regulons as General Stress Responses in Gram-positive Bacteria,” Annual Reviews in Microbiology, Vol. 60, 2006, pp. 451-475.
 G. E. Pi?as, P. R. Cortes, A. G. A. Orio and J. Echenique, “Acidic Stress Induces Autolysis by a CSP-Independent ComE Pathway in Streptococcus pneumoniae,” Microbiology, Vol. 154, No. Pt 5, 2008, pp. 1300-1308.
 R. R. Draheim, A. F. Bormans, R. Z Lai and M. D. Manson, “Tryptophan Residues Flanking the Second Transmembrane helix (TM2) Set the Signalling State of the Tar Chemoreceptor,” Biochemistry, Vol. 44, No. 4, 2005, pp. 1268-1277. doi:10.1021/bi048969d
 A. S. Miller and J. J. Falke, “Side Chains at the Membrane-Water Interface Modulate the Signalling State of a Transmembrane Receptor,” Biochemistry, Vol. 43, No. 7, 2004, pp. 1763-1770. doi:10.1021/bi0360206
 R. Carballido-López and A. Formstone, “Shape Determination in Bacillus subtilis,” Current Opinion in Microbiology, Vol. 10, 2007, pp. 611-616.
 R. Carballido-López and A. Formstone, Y. Li, S. D. Herlich, P. Noirot and J. Errington, “Actin Homolog MreBH Governs Cell Morphogenesis by Localization of the Cell Wall Hydrolase LytE,” Developmental Cell, Vol. 11, No. 3, 2006, pp. 399-409. doi:10.1016/j.devcel.2006.07.017
 D. Brekasis and M. Paget, “A Novel Sensor of NADH/ NAD Redox Poise in Streptomyces coelicolor A3(2),” The EMBO Journal, Vol. 22, No. 18, 2003, pp. 4856-4865. doi:10.1093/emboj/cdg453
 M. Schau, Y. Chen and F. M. Hulett, “Bacillus subtilis YdiH is a Direct Negative Regulator of the cydABCD Operon,” Journal of Bacteriology, Vol. 186, No. 14, 2004, pp. 4585-4595. doi:10.1128/JB.186.14.4585-4595.2004
 M. Pagels, S. Fuchs, J. Pané-Farré, C. Kohler, L. Men- schner, M. Hecker, P. J. McNamarra, M. C. Bauer, C. von Wachenfeldt, M. Liebeke, M. Lalk, G. Sander, C. von Eiff, R. A. Proctor and S. Engelmann, “Redox Sensing by a Rex-family Repressor is Involved in the Regulation of Anaerobic Gene Expression in Staphylococcus aureus,” Molecular Microbiology, Vol. 76, No. 5, 2010, pp. 1142-1161. doi:10.1111/j.1365-2958.2010.07105.x
 Y. Boucher and W. F. Doolittle, “The Role of Lateral Gene Transfer in the Evolution of Isoprenoid Biosynthesis Pathways,” Molecular Microbiology, Vol. 37, No. 4, 2000, pp. 703-716.
 R. S. Putra, A. Disch, J. M. Bravo and M. Rohmer, “Distribution of Mevalonate and Glyceraldehyde 3-Phosphate/Pyruvate Routes for Isoprenoid Biosynthesis in some Gram-Negative Bacteria and Mycobacteria,” FEMS Microbiology Letters, Vol. 164, No. 1, 1998, pp. 169-175.
 S. M. C. Newton, P. E. Klebba, C. Raynaud, Y. Shao, X. Jiang, I. Dubail, C. Archer, C. Frehel and A. Charbit, “The svpA-srtB Locus of Listeria monocytogenes: Furmediated Iron Regulation and Effect on Virulence,” Molecular Microbiology, Vol. 55, No. 3, 2005, pp. 927-940.