Production of Pumilarin and a Novel Circular Bacteriocin, Altitudin A, by Bacillus altitudinis ECC22, a Soil-Derived Bacteriocin Producer
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2024
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: 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
Abstract
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.
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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.