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oai:docta.ucm.es:20.500.14352/1033322025-03-18T15:26:24Zcom_20.500.14352_14col_20.500.14352_15

Production of Pumilarin and a Novel Circular Bacteriocin, Altitudin A, by Bacillus altitudinis ECC22, a Soil-Derived Bacteriocin Producer Lafuente, Irene Sevillano, Ester Peña Vidal, Nuria Cuartero, Alicia Hernández Cruza, Pablo Elpidio Cintas Izarra, Luis Miguel Muñoz Atienza, Estefanía Borrero Del Pino, Juan 636.09 Circular bacteriocins Antimicrobial peptides Bacillus altitudinis In vitro cell-free protein synthesis (IV-CFPS) Split-intein-mediated ligation (SIML) of peptides Nutrición 3109.06 Nutrición Author Contributions: Conceptualization, E.M.-A., P.E.H. and J.B.; methodology, I.L., E.S., N.P. and A.C.; investigation, I.L., E.S. and N.P.; resources, A.C., L.M.C., P.E.H. and J.B.; writing—original draft preparation, I.L. writing—review and editing, P.E.H. and J.B.; supervision, E.M.-A. and J.B.; project administration, L.M.C., P.E.H., and J.B.; funding acquisition, L.M.C., P.E.H. and J.B. All authors have read and agreed to the published version of the manuscript. The rise of antimicrobial resistance poses a significant global health threat, necessitating urgent efforts to identify novel antimicrobial agents. In this study, we undertook a thorough screening of soil-derived bacterial isolates to identify candidates showing antimicrobial activity against Gram-positive bacteria. A highly active antagonistic isolate was initially identified as Bacillus altitudinis ECC22, being further subjected to whole genome sequencing. A bioinformatic analysis of the B. altitudinis ECC22 genome revealed the presence of two gene clusters responsible for synthesizing two circular bacteriocins: pumilarin and a novel circular bacteriocin named altitudin A, alongside a closticin 574-like bacteriocin (CLB) structural gene. The synthesis and antimicrobial activity of the bacteriocins, pumilarin and altitudin A, were evaluated and validated using an in vitro cell-free protein synthesis (IV-CFPS) protocol coupled to a split-intein-mediated ligation procedure, as well as through their in vivo production by recombinant E. coli cells. However, the IV-CFPS of CLB showed no antimicrobial activity against the bacterial indicators tested. The purification of the bacteriocins produced by B. altitudinis ECC22, and their evaluation by MALDI-TOF MS analysis and LC-MS/MS-derived targeted proteomics identification combined with massive peptide analysis, confirmed the production and circular conformation of pumilarin and altitudin A. Both bacteriocins exhibited a spectrum of activity primarily directed against other Bacillus spp. strains. Structural three-dimensional predictions revealed that pumilarin and altitudin A may adopt a circular conformation with five- and four-α-helices, respectively. Ministerio de Ciencia e Innovación (España) Comunidad de Madrid Depto. de Nutrición y Ciencia de los Alimentos Fac. de Veterinaria TRUE pub 2024-04-22T14:29:09Z 2024-04-22T14:29:09Z 2024-02-07 journal article VoR https://hdl.handle.net/20.500.14352/103332 XXXX-XXXX 10.3390/ijms25042020 eng info:eu-repo/grantAgreement/MCI//PID2019-104808RAI00 info:eu.repo/grantAgreement/CAM/ProgramaAtraccionTalento/[2018-T1/BIO-10158; 2022-5A/BIO-24232] : Lafuente, I.; Sevillano, E.; Peña, N.; Cuartero, A.; Hernández, P.E.; Cintas, L.M.; Muñoz-Atienza, E.; Borrero, J. Production of Pumilarin and a Novel Circular Bacteriocin, Altitudin A, by Bacillus altitudinis ECC22, a Soil-Derived Bacteriocin Producer. Int. J. Mol. Sci. 2024, 25, 2020. https://doi.org/10.3390/ ijms25042020 Attribution 4.0 International http://creativecommons.org/licenses/by/4.0/ open access application/pdf MDPI