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Peptidomics

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Co-expression and characterization of enterocin CRL35 and its mutant in Escherichia coli Rosetta

Emilse Masías
  • Instituto Superior de Investigaciones Biologicas (CONICET-UNT) e Instituto de Quimica Biologica ‘‘Dr. Bernabe Bloj’’, San Miguel de Tucuman, Argentina
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Gianluca Picariello
  • Istituto di Scienze dell’Alimentazione – Consiglio Nazionale delle Ricerche (CNR), Via Roma, 64 – 83100 Avellino, Italy
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  • De Gruyter OnlineGoogle Scholar
/ Leonardo Acuña
  • Instituto Superior de Investigaciones Biologicas (CONICET-UNT) e Instituto de Quimica Biologica ‘‘Dr. Bernabe Bloj’’, San Miguel de Tucuman, Argentina
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Miriam Chalón
  • Instituto Superior de Investigaciones Biologicas (CONICET-UNT) e Instituto de Quimica Biologica ‘‘Dr. Bernabe Bloj’’, San Miguel de Tucuman, Argentina
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  • De Gruyter OnlineGoogle Scholar
/ Fernando Sesma / Roberto Morero
  • Instituto Superior de Investigaciones Biologicas (CONICET-UNT) e Instituto de Quimica Biologica ‘‘Dr. Bernabe Bloj’’, San Miguel de Tucuman, Argentina
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/ Lucila Saavedra / Carlos Minahk
  • Corresponding author
  • Chacabuco 461, S.M. de Tucuman (T4000ILI), Argentina, Fax: +54 0381 4248921, Tel: +54 0381 4248921
  • Email
  • Other articles by this author:
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Published Online: 2014-04-24 | DOI: https://doi.org/10.2478/ped-2014-0003

Abstract

Even though many sequences and structures of bacteriocins from lactic acid bacteria have been fully characterized so far, little information is currently available about bacteriocins heterologously produced by Escherichia coli. For this purpose, the structural gene of enterocin CRL35, munA, was PCR-amplified using specific primers and cloned downstream of PelB sequence in the pET22b (+) expression vector. E. coli Rosetta (DE3) pLysS was chosen as the host for production and enterocin was purified by an easy two-step protocol. The bacteriocin was correctly expressed with the expected intramolecular disulfide bond. Nevertheless, it was found that a variant of the enterocin, differing by 12 Da from the native polypeptide, was co-expressed by E. coli Rosetta in comparable amount. Indeed, the mutant bacteriocin contained two amino acid substitutions that were characterized by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) and HPLCelectrospray (ESI)-Q-TOF tandem mass spectrometry (MS/ MS) sequencing. This is the first report regarding the production of mutants of pediocin-like bacteriocins in the E. coli expression system.

Keywords : bacteriocins; heterologous expression; MALDITOF MS analysis; nanoHPLC-ESI MS/MS analysis

References

  • [1] Cleveland, J., Montville, T. J., Nes, I. F., Chikindas, M. L., Bacteriocins: safe, natural antimicrobials for food preservation, Int. J. Food Microbiol, 71, (2001), 1-20.Google Scholar

  • [2] De Vuyst, L., Leroy, F., Bacteriocins from lactic acid bacteria: production, purification, and food applications, J. Mol.Microbiol. Biotechnol, 13, (2007), 194-199.Google Scholar

  • [3] Acuna, L., Morero, R. D., Bellomio, A., Development of Wide-Spectrum Hybrid Bacteriocins for Food Biopreservation, Food and Bioprocess Technology, 4, (2011), 1029 - 1049.Web of ScienceGoogle Scholar

  • [4] Drider, D., Fimland, G., Hechard, Y., McMullen, L. M., Prevost, H., The continuing story of class IIa bacteriocins, Microbiol.Mol. Biol. Rev., 70, (2006), 564-582.PubMedGoogle Scholar

  • [5] Gillor, O., Etzion, A., Riley, M. A., The dual role of bacteriocins as anti- and probiotics, Appl. Microbiol. Biotechnol., 81, (2008), 591-606.Google Scholar

  • [6] Salvucci, E., Saavedra, L., Hebert, E. M., Haro, C., Sesma, F., Enterocin CRL35 inhibits Listeria monocytogenes in a murine model, Foodborne Pathog. Dis., 9, (2012), 68-74.Web of ScienceCrossrefGoogle Scholar

  • [7] Saavedra, L., Minahk, C., de Ruiz Holgado, A. P., Sesma, F., Enhancement of the enterocin CRL35 activity by a synthetic peptide derived from the NH2-terminal sequence, Antimicrob.Agents Chemother, 48, (2004), 2778-2781.Google Scholar

  • [8] Farias, M. E., De Ruiz Holgado, A. A. P., Sesma, F., Bacteriocin Production by Lactic Acid Bacteria Isolated from Regional Cheeses: Inhibition of Foodborne Pathogens, J Food Protect, 57, (1994), 1013-1015.Google Scholar

  • [9] Wachsman, M. B., Farias, M. E., Takeda, E., Sesma, F., de Ruiz Holgado, A. P., de Torres, R. A., et al., Antiviral activity of enterocin CRL35 against herpesviruses, Int. J. Antimicrob.Agents, 12, (1999), 293-299.CrossrefGoogle Scholar

  • [10] Minahk, C. J., Dupuy, F., Morero, R. D., Enhancement of antibiotic activity by sub-lethal concentrations of enterocin CRL35, J. Antimicrob. Chemother, 53, (2004), 240-246.Google Scholar

  • [11] Salvucci, E., Saavedra, L., Sesma, F., Short peptides derived from the NH2-terminus of subclass IIa bacteriocin enterocin CRL35 show antimicrobial activity, J. Antimicrob. Chemother, 59, (2007), 1102-1108.Web of ScienceGoogle Scholar

  • [12] Jaradat, Z. W., Bhunia, A. K., Glucose and nutrient concentrations affect the expression of a 104-kilodalton Listeria adhesion protein in Listeria monocytogenes, Appl. Environ.Microbiol., 68, (2002), 4876-4883.Google Scholar

  • [13] Sambrook, J., Molecular Cloning: A Laboratory Manual, Third Edition; 3rd ed.; Cold Spring Harbor Laboratory Press, 2001.Google Scholar

  • [14] Fujita, K., Ichimasa, S., Zendo, T., Koga, S., Yoneyama, F., Nakayama, J., et al., Structural analysis and characterization of lacticin Q, a novel bacteriocin belonging to a new family of unmodified bacteriocins of gram-positive bacteria, Appl.Environ. Microbiol., 73, (2007), 2871-2877.Web of ScienceGoogle Scholar

  • [15] Schagger, H., von Jagow, G., Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa, Anal. Biochem., 166, (1987), 368-379.Google Scholar

  • [16] Dyballa, N., Metzger, S., Fast and sensitive colloidal coomassie G-250 staining for proteins in polyacrylamide gels, J Vis Exp, 2009.Google Scholar

  • [17] Acuna, L., Picariello, G., Sesma, F., Morero, R. D., Bellomio, A., A new hybrid bacteriocin, Ent35-MccV, displays antimicrobial activity against pathogenic Gram-positive and Gram-negative bacteria, FEBS Open Bio, 2, (2012), 12-19.Google Scholar

  • [18] Sandkvist, M., Bagdasarian, M., Secretion of recombinant proteins by Gram-negative bacteria, Curr. Opin. Biotechnol., 7, (1996), 505-511.CrossrefGoogle Scholar

  • [19] Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J., Protein measurement with the Folin phenol reagent, J Biol Chem, 193, (1951), 265-75.Google Scholar

  • [20] Miller, K. W., Schamber, R., Chen, Y., Ray, B., Production of active chimeric pediocin AcH in Escherichia coli in the absence of processing and secretion genes from the Pediococcus pap operon, Appl. Environ. Microbiol., 64, (1998), 14-20.Google Scholar

  • [21] Sanchez, J., Diep, D. B., Herranz, C., Nes, I. F., Cintas, L.M., Hernandez, P. E., Amino acid and nucleotide sequence, adjacent genes, and heterologous expression of hiracin JM79, a sec-dependent bacteriocin produced by Enterococcus hirae DCH5, isolated from Mallard ducks (Anas platyrhynchos), FEMS Microbiol. Lett., 270, (2007), 227-236.Web of ScienceGoogle Scholar

  • [22] Worobo, R. W., Van Belkum, M. J., Sailer, M., Roy, K. L., Vederas, J. C., Stiles, M. E., A signal peptide secretion-dependent bacteriocin from Carnobacterium divergens, J. Bacteriol., 177, (1995), 3143-3149.Google Scholar

  • [23] Leer, R. J., van der Vossen, J. M., van Giezen, M., van Noort, J. M., Pouwels, P. H., Genetic analysis of acidocin B, a novel bacteriocin produced by Lactobacillus acidophilus, Microbiology (Reading, Engl.), 141 ( Pt 7), (1995), 1629-1635.Google Scholar

  • [24] Cintas, L. M., Casaus, P., Havarstein, L. S., Hernandez, P. E., Nes, I. F., Biochemical and genetic characterization of enterocin P, a novel sec-dependent bacteriocin from Enterococcus faecium P13 with a broad antimicrobial spectrum, Appl.Environ. Microbiol, 63, (1997), 4321-4330.Google Scholar

  • [25] Gutierrez, J., Criado, R., Citti, R., Martin, M., Herranz, C., Nes, I. F., et al., Cloning, production and functional expression of enterocin P, a sec-dependent bacteriocin produced by Enterococcus faecium P13, in Escherichia coli, Int. J. Food Microbiol., 103, (2005), 239-250.Google Scholar

  • [26] Yildirim, S., Konrad, D., Calvez, S., Drider, D., Prevost, H., Lacroix, C., Production of recombinant bacteriocin divercin V41 by high cell density Escherichia coli batch and fed-batch cultures, Appl. Microbiol. Biotechnol, 77, (2007), 525-531.Google Scholar

  • [27] Beaulieu, L., Tolkatchev, D., Jette, J.-F., Groleau, D., Subirade, M., Production of active pediocin PA-1 in Escherichia coli using a thioredoxin gene fusion expression approach: cloning, expression, purification, and characterization, Can. J.Microbiol., 53, (2007), 1246-1258.Google Scholar

  • [28] Liu, S., Han, Y., Zhou, Z., Fusion expression of pedA gene to obtain biologically active pediocin PA-1 in Escherichia coli, J Zhejiang Univ Sci B, 12, (2011), 65-71.Google Scholar

  • [29] Jasniewski, J., Cailliez-Grimal, C., Gelhaye, E., Revol-Junelles, A.-M., Optimization of the production and purification processes of carnobacteriocins Cbn BM1 and Cbn B2 from Carnobacterium maltaromaticum CP5 by heterologous expression in Escherichia coli, J. Microbiol. Methods, 73, (2008), 41-48.Web of ScienceGoogle Scholar

  • [30] Klocke, M., Mundt, K., Idler, F., Jung, S., Backhausen, J. E., Heterologous expression of enterocin A, a bacteriocin from Enterococcus faecium, fused to a cellulose-binding domain in Escherichia coli results in a functional protein with inhibitory activity against Listeria, Appl. Microbiol. Biotechnol., 67, (2005), 532-538.Google Scholar

  • [31] Ingham, A. B., Sproat, K. W., Tizard, M. L. V., Moore, R. J., A versatile system for the expression of nonmodified bacteriocins in Escherichia coli, J. Appl. Microbiol., 98, (2005), 676-683.Google Scholar

  • [32] Chen, H., Tian, F., Li, S., Xie, Y., Zhang, H., Chen, W., Cloning and heterologous expression of a bacteriocin sakacin P from Lactobacillus sakei in Escherichia coli, Appl. Microbiol.Biotechnol., 94, (2012), 1061-1068.Google Scholar

  • [33] Fregeau Gallagher, N. L., Sailer, M., Niemczura, W. P., Nakashima, T. T., Stiles, M. E., Vederas, J. C., Threedimensional structure of leucocin A in trifluoroethanol and dodecylphosphocholine micelles: spatial location of residues critical for biological activity in type IIa bacteriocins from lactic acid bacteria, Biochemistry, 36, (1997), 15062-15072.Google Scholar

  • [34] Wang, Y., Henz, M. E., Gallagher, N. L., Chai, S., Gibbs, A. C., Yan, L. Z., et al., Solution structure of carnobacteriocin B2 and implications for structure-activity relationships among type IIa bacteriocins from lactic acid bacteria, Biochemistry, 38, (1999), 15438-15447. Google Scholar

About the article

Received: 2013-07-26

Accepted: 2013-12-06

Published Online: 2014-04-24

Published in Print: 2014-01-01


Citation Information: Peptidomics, Volume 1, Issue 1, ISSN (Online) 2084-7203, DOI: https://doi.org/10.2478/ped-2014-0003.

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© 2014 Emilse Masías et al.. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0

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