Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)López Pazos, Silvio AlejandroRojas Arias, Adriana CarolinaLandinez Velandia, Sandra Milena2021-02-222021-02-222020-05-29http://repositorio.uan.edu.co/handle/123456789/1605Bacteriocins comprise a large number of peptides synthesized ribosomically by countless numbers of bacteria. Biosynthesis is carried out by structural genes that encode these peptides. The increasing bacterial resistance threatens human and animal health. Bacillus subtilis has been shown to produce a wide range of bacteriocins. The genus Pseudomonas sp. synthesizes different antimicrobial peptides to dominate over other competing organisms. P. extremeustralis species is a non-pathogenic Gram-negative bacterium, isolated from a pond in the Antarctic Peninsula. Twenty-one bacteriocin sequences were identified in silico in the genome of B. Subtilis ATCC 6633 and two associated sequences in the genome of P. extremeustralis CMPUJ U515. The physicochemical properties allowed establishing the molecular weight, the isoelectric point, the positively and negatively charged residues, the extinction coefficient, the stability index, the aliphatic index and the overall average of hydropaticity (GRAVY). The products obtained from PCR, resulted in three products that correspond to the bacteriocins Subtilin, Sublancin 168 and Subtilocin A. Finally, it was desired to establish the possible growth inhibitory effect of the protein extracts of B. subtilis ATCC 6633 and P. extremaustralis CMPUJ U515, for which a qualitative sensidisk test was performed on the strains of Escherichia coli ATCC 25922 and Staphylococcus aureus CMPUJ 080. Unfortunately no activity was found. In this investigation we concluded that Bacillus subtilis ATCC 6633 has biocontrol potential due to its bacteriocins which can be cloned by homologous recombination into yeast, to analyze its biological functionality.Las bacteriocinas abarcan un gran número de péptidos sintetizados ribosómicamente por innumerable cantidad de bacterias. La biosíntesis es llevada a cabo por genes estructurales que codifican estos péptidos. La creciente resistencia bacteriana amenaza la salud humana y animal. Se ha demostrado que Bacillus subtilis produce amplia gama de bacteriocinas. El género Pseudomonas sp. sintetiza diferentes péptidos antimicrobianas para dominar sobre los otros organismos en competencia. La especie P. extremaustralis es una bacteria Gram negativa no patógena, aislada de un estanque de la Península Antártica. Se identificaron in silico 21 secuencias de bacteriocinas en el genoma de B. Subtilis ATCC 6633 y dos secuencias asociadas en el genoma de P. extremaustralis CMPUJ U515. Las propiedades fisicoquímicas permitieron establecer el peso molecular, el punto isoeléctrico, los residuos cargados positiva y negativamente, el coeficiente de extinción, el índice de estabilidad, el índice alifático y el promedio general de hidropaticidad (GRAVY). Los productos obtenidos de PCR, dio como resultado tres productos que corresponden a las bacteriocinas Subtilina, Sublancina 168 y Subtilocina A. Finalmente se deseaba establecer el posible efecto inhibidor de crecimiento de los extractos proteicos de B. subtilis ATCC 6633 y P. extremaustralis CMPUJ U515, para lo cual se realizó una prueba cualitativa con sensidiscos sobre las cepas de Escherichia coli ATCC 25922 y Staphylococcus aureus CMPUJ 080. Desafortunadamente no se encontró actividad. En esta investigación concluimos que Bacillus subtilis ATCC 6633 tiene potencial biocontrolador debido a sus bacteriocinas las cuales pueden clonarse mediante recombinación homologa en levadura, para analizar su funcionalidad biológica.spaAcceso abiertopéptidos codificados ribosomalmente, lantibióticos, Bacillus subtilis, Pseudomonas extremaustralis, Escherichia coli, Staphylococcus aureus.Tesis y disertaciones (Maestría y/o Doctorado)ribosomally coded peptides, lantibiotics, Bacillus subtilis, Pseudomonas extremaustralis, Escherichia coli, Staphylococcus aureusinfo:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Abriouel, H. F. (2011). Diversity and applications of Bacillus bacteriocins. FEMS Microbiology Reviews, 35(1), 201–232. doi:https://doi.org/10.1111/j.1574-6976.2010.00244.xAgudelo Londoño, N.-T. M.-L. (2015). Bacteriocinas Producidas Por Bacterias Ácido Lácticas y Su Aplicación En La Industria De Alimentos. Alimentos Hoy, 23(36), 186-205. Obtenido de http://www.alimentoshoy.acta.org.co/index.php/hoy/article/view/356Ahmad, V. K. (2017). Antimicrobial potential of bacteriocins: in therapy, agriculture and food preservation. International Journal of Antimicrobial Agents, 49(1), 1-11. doi:https://doi.org/10.1016/j.ijantimicag.2016.08.016Arumugam, T. D. (2019). Inhibition of Methicillin Resistant Staphylococcus aureus by Bacteriocin Producing Pseudomonas aeruginosa. International Journal of Peptide Research and Therapeutics, 25(1), 339-348. doi:https://doi.org/10.1007/s10989-018-9676-yAylon, Y. y. (2004). New insights into the mechanism of homologous recombination in yeast. Elsevier, 566(3), 231-248. doi:https://doi.org/10.1016/j.mrrev.2003.10.001Bédard, F. y. (2018). Recent progress in the chemical synthesis of class II and S-glycosylated bacteriocins. Frontiers in Microbiology, 9, 1-14. doi: https://doi.org/10.3389/fmicb.2018.01048Ben Lagha, A. H. (2017). Antimicrobial potential of bacteriocins in poultry and swine production. Veterinary Research, 48(1), 1-12. doi:https://doi.org/10.1186/s13567-017-0425-6Benforte, F. C. (2018). Novel essential role of ethanol oxidation genes at low temperature revealed by transcriptome analysis in the antarctic bacterium pseudomonas extremaustralis. PLoS ONE, 13(2), 1-18. doi:https://doi.org/10.1371/journal.pone.0192559Benforte, F. C. (2018). Novel role of the LPS core glycosyltransferase WapH for cold adaptation in the Antarctic bacterium Pseudomonas extremaustralis. PLoS ONE, 13(2), 1–18. doi:https://doi.org/10.1371/journal.pone.0192559Bengtsson-Palme, J. K. (2018). Environmental factors influencing the development and spread of antibiotic resistance. FEMS Microbiology Reviews, 42(1), 68–80. doi:https://doi.org/10.1093/femsre/fux053Caulier, S. N. (2019). Overview of the Antimicrobial Compounds Produced by Members of the Bacillus subtilis Group. Frontiers in Microbiology, 10, 1–19. doi: https://doi.org/10.3389/fmicb.2019.00302Chikindas, M. L. (2018). Functions and emerging applications of bacteriocins. Current Opinion in Biotechnology, 49, 23-28. doi:https://doi.org/10.1016/j.copbio.2017.07.011Cooper y Hausman, .. (2011). La CÉLULA. Marban.Daza Pérez, R. M. (1998). Resistencia bacteriana a antimicrobianos: su importancia en la toma de decisiones en la práctica diaria. InformaciónTerapeutica Del Sistema Nacional de Salud, 22(3), 57-67. Obtenido de http://www.msc.es/fr/biblioPublic/publicaciones/docs/bacterias.pdfDobson, A. C. (2012). Bacteriocin production: A probiotic trait? Applied and Environmental Microbiology, 78(1), 1–6. doi:https://doi.org/10.1128/AEM.05576-11Dorosky, R. J. (2017). Pseudomonas chlororaphis Produces Two Distinct R-Tailocins That Contribute to Bacterial Competition in Biofilms and on Roots. Applied and Environmental Microbiology, 83(15), 1–16. doi:https://doi.org/10.1128/aem.00706-17Dubois, J. Y. (2009). Immunity to the bacteriocin sublancin 168 is determined by the SunI (YolF) protein of Bacillus subtilis. Antimicrobial Agents and Chemotherapy, 53(2), 651-661. doi: https://doi.org/10.1128/AAC.01189-08Egan, K. F. (2016). Bacteriocins: Novel solutions to age old spore-related problems? Frontiers in Microbiology, 7, 1-21. doi:https://doi.org/10.3389/fmicb.2016.00461Ennahar, S. S. (2000). Class IIa bacteriocins: Biosynthesis, structure and activity. FEMS Microbiology Reviews, 24(1), 85-106. doi:https://doi.org/10.1016/S0168-6445(99)00031-5Felix, P. (2018). Homologous Recombination : To Fork and Beyond. Genes, 9, 603. doi:https://doi.org/10.3390/genes9120603Gabrielsen, C. B. (2014). Circular bacteriocins: Biosynthesis and mode of action. Applied and Environmental Microbiology, 80(22), 6854-6862. doi:https://doi.org/10.1128/AEM.02284-14Gasteiger, E. H. (2005). Protein Identification and Analysis Tools on the ExPASy Server. The Proteomics Protocols Handbook, 571-608. doi:https://doi.org/10.1385/1592598900Ghequire, M. G. (2014). Ribosomally encoded antibacterial proteins and peptides from Pseudomonas. FEMS Microbiology Reviews, 38(4), 523–568. doi:https://doi.org/10.1111/1574-6976.12079Ghequire, M. G. (2017). Novel immunity proteins associated with colicin M-like bacteriocins exhibit promiscuous protection in pseudomonas. Frontiers in Microbiology, 8, 1–9. doi:https://doi.org/10.3389/fmicb.2017.00093Heng, N. T. (2006). What's in a name? Class distinction for bacteriocins. Nat Rev Microbiol, 4, 160. doi:https://doi.org/10.1038/nrmicro1273-c1Ibarra, J. G. (2014). Genome sequence analysis of Pseudomonas extremaustralis provides new insights into environmental adaptability and extreme conditions resistance. Springer, 19(1), 2017-220. doi:https://doi.org/10.1007/s00792-014-0700-7INS. (2017). Resultados del Programa de Informe de Resultados de la Vigilancia por Laboratorio de Resistencia antimicrobiana en Infecciones Asociadas a la Atención en Salud (IAAS) 2017. Bogotá: Instituto Nacional de Salud.INS. (2018). Resistencia Bacteriana A Los Antimicrobianos En El Ámbito Hospitalario. Bogotá: Instituto Nacional de Salud.INS. (2019). Informe De Resultados De La Vigilancia Por Laboratorio De Resistencia Antimicrobiana En Infecciones Asociadas A La Atención En Salud (Iaas) 2018. Bogotá: Instituto Nacional de Salud.Kaškonienė, V. S.-S. (2017). Current state of purification, isolation and analysis of bacteriocins produced by lactic acid bacteria. Springer, 101(4), 1323-1335. doi: https://doi.org/10.1007/s00253-017-8088-9Krogh, B. O. (2004). Recombination Proteins in Yeast. Annual Review of Genetics, 38(1), 233-271. doi:https://doi.org/10.1146/annurev.genet.38.072902.091500Kumariya, R. G. (2019). Bacteriocins: Classification, synthesis, mechanism of action and resistance development in food spoilage causing bacteria. Microbial Pathogenesis, 128, 171-177. doi:https://doi.org/10.1016/j.micpath.2019.01.002Kunst, F. O. (1997). The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature, 390, 249-256. doi:https://doi.org/10.1038/36786Kyte, J. y. (1982). A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology, 157(1), 105-132. doi:https://doi.org/10.1016/0022-2836(82)90515-0Lu, Z. G. (2018). Isolation, identification and characterization of novel Bacillus subtilis. Journal of Veterinary Medical Science, 80(3), 427-433. doi: doi:10.1292/jvms.16-0572Matsui, H. S. (1993). Regulation of pyocin genes in Pseudomonas aeruginosa by positive (prtN) and negative (prtR) regulatory genes. Journal of Bacteriology, 175(5), 1257-1263. doi:https://doi.org/10.1128/jb.175.5.1257-1263.1993McCaughey, L. C. (2016). Discovery, characterization and in vivo activity of pyocin SD2, a protein antibiotic from Pseudomonas aeruginosa. Biochemical Journal, 473(15), 2345–2358. doi:doi:10.1042/bcj20160470Michel-Briand, Y. y. (2002). The pyocins of Pseudomonas aeruginosa. Biochimie, 84, 499–510. doi:https://doi.org/10.1016/S0300-9084(02)01422-0Minsalud, M. d. (2018). Plan Nacional de Respuesta a la Resistencia a los Antimicrobianos - plan estratégico. Bogotá: Ministerio de Salud y Protección Social.Montiel, D. K.-P. (2015). Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters. Proceedings of the National Academy of Sciences of the United States of America, 112(29), 8953-8958. doi:https://doi.org/10.1073/pnas.1507606112Nonejuie, P. T. (2016). Application of bacterial cytological profiling to crude natural product extracts reveals the antibacterial arsenal of Bacillus subtilis. The Journal of antibiotics, 69(5), 353–361. doi:doi:10.1038/ja.2015.116O’Neill, J. (2016). Book review: Tackling drug-resistant infections globally. WHO. Archives of Pharmacy Practice. doi:https://doi.org/10.4103/2045-080x.186181Oluyombo, O. P. (2019). Competition in Biofilms between Cystic Fibrosis Isolates of Pseudomonas aeruginosa Is Shaped by R-Pyocins. mBio, 10, e01828-18. doi: doi:10.1128/mbio.01828-18Orr Weaver, T. L. (1983). Yeast recombination: The association between double-strand gap repair and crossing-over. Proceedings of the National Academy of Sciences of the United States of America, 80, 4417-4421. doi:https://doi.org/10.1073/pnas.80.14.4417Peix, A. R.-B. (2018). The current status on the taxonomy of Pseudomonas revisited: An update. Infection, Genetics and Evolution, 57, 106-116. doi:https://doi.org/10.1016/j.meegid.2017.10.026Perez, R. H. (2014). Novel bacteriocins from lactic acid bacteria (LAB): Various structures and applications. Microbial Cell Factories, 13((Suppl 1):S3), (Suppl 1):S3. doi:https://doi.org/10.1186/1475-2859-13-S1-S3Pérez, R. H. (2018). Circular and leaderless bacteriocins: Biosynthesis, mode of action, applications, and prospects. Frontiers in Microbiology, 9, 1-18. doi:https://doi.org/10.3389/fmicb.2018.02085Plessis, A. y. (1993). Multiple tandem integrations of transforming DNA sequences in yeast chromosomes suggest a mechanism for integrative transformation by homologous recombination. Gene, 134(1), 41-50. doi:https://doi.org/10.1016/0378-1119(93)90172-YRegev, A. K. (1996). Synergistic activity of a Bacillus thuringiensis δ-endotoxin and a bacterial endochitinase against Spodoptera littoralis larvae. Applied and Environmental Microbiology, 62(10), 3581–3586.Restrepo, M. A. (2003). Enfermedades Infecciosas. Medellin, Colombia: Corporación para Investigaciones Biologicas.Saati-Santamaría Z, L.-M. R.-G.-M.-F. (2018). Discovery of phloeophagus beetles as a source of pseudomonas strains that produce potentially new bioactive substances and description of pseudomonas bohemica sp. nov. Frontiers in Microbiology, 9. doi:https://doi.org/10.3389/fmicb.2018.00913Sambrook, J. (2001). Molecular cloning: a laboratory manual. Cold Spring Harb Lab Press Cold Spring Harb NY, 999.Sasaki, M. L. (2010). Genome destabilization by homologous recombination in the germ line. Nature, 11, 182-195. doi:https://doi.org/10.1038/nrm2849.Schroeder, M. B. (2017). The complex relationship between virulence and antibiotic resistance. Genes, 39. doi:https://doi.org/10.3390/genes8010039Sharma, G. D. (2018). Antibacterial Activity, Cytotoxicity, and the Mechanism of Action of Bacteriocin from Bacillus subtilis GAS101. Medical Principles and Practice, 27(2), 186-192Stein, T. (2005). Bacillus subtilis antibiotics: Structures, syntheses and specific functions. Molecular Microbiology, 56(4), 845–857. doi:https://doi.org/10.1111/j.1365-2958.2005.04587.xSung, P. M. (2000). Recombination factors of Saccharomyces cerevisiae. Elsevier, 451, 257-275. doi:https://doi.org/10.1016/S0027-5107(00)00054-3Symington, L. S. (2014). Mechanisms and Regulation of Mitotic Recombination in Saccharomyces cerevisiae. Genetics, 198, 795–835. doi:https://doi.org/10.1534/genetics.114.166140Tribelli, P. S.-L. (2015). Novel essential role of ethanol oxidation genes at low temperature revealed by transcriptome analysis in the antarctic bacterium pseudomonas extremaustralis. PLoS ONE, 10(12), 1-19. doi:https://doi.org/10.1371/journal.pone.0145353Tribelli, P. y. (2018). Reporting Key Features in Cold-Adapted Bacteria. life, 8(1), 8. doi:https://doi.org/10.3390/life8010008van Leeuwen, J. A. (2015). Rapid and Efficient Plasmid Construction by Homologous Recombination in Yeast. Cold Spring Harbor Protocols, 853-862. doi:https://doi.org/10.1101/pdb.prot085100Vasilchenko, A. S. (2018). Pore-forming bacteriocins: structural–functional relationships. Springer, 201(2), 147-154. doi:https://doi.org/10.1007/s00203-018-1610-3WHO. (2001). Estrategia mundial de la OMS para contener la resistencia a los antimicrobianos. Revista Panamericana de Salud Publica/Pan American Journal of Public Health, 10(4), 284–294. doi:https://doi.org/10.1590/s1020-49892001001000014WHO. (2017). Global Antimicrobial Resistance Surveillance System (GLASS) Report. Geneva: Organization World Health. doi:https://doi.org/ISBN 978-92-4-151344-9Winn, W. C., Allen, S. D., Janda, W. M., Koneman, E. W., Procop, G. W., Schrenckenberger, P. C., & Woods, G. L. (2008). Koneman. Diagnóstico microbiológico. Panamericana.Zheng, S. y. (2018). Diversified transporters and pathways for bacteriocin secretion in gram-positive bacteria. Springer, 102(10), 4243–4253. doi:https://doi.org/10.1007/s00253-018-8917-5Zor, T. y. (1996). Linearization of the Bradford Protein Assay Increases Its Sensitivity: Theoretical and Experimental Studies. Analytical Biochemistry, 236, 302-308. doi:doi:10.1006/abio.1996.0171Zou, J. J. (2018). Strategies for screening, purification and characterization of bacteriocins. International Journal of Biological Macromolecules, 117, 781-789. doi:https://doi.org/10.1016/j.ijbiomac.2018.05.233