Redondo-Moreno, S.; Peralta, C.; Palma, L.

The supernatant of Bacillus toyonensis biovar thuringiensis Bto_UNVM-42 exhibits nematicidal activity, although its molecular basis remains unclear. While pesticidal proteins in Bacillus thuringiensis and related species are classically considered to be intracellular and associated with parasporal crystals, their potential presence in the extracellular fraction has been largely unexplored. Here, LC-MS/MS analysis of the secretome from LB-grown cultures revealed the extracellular presence of pesticidal protein homologs related to Cry32-, Cyt1-, and Mpp3-like protein families, together with degradative enzymes including collagenase, chitinase, proteases, and cytolysins. Signal peptide prediction supported classical secretion for several proteins, while others were consistent with non-classical secretion pathways. The consistent detection of these proteins in cell-free supernatants provides strong proteomic evidence for their extracellular localization. These findings challenge the prevailing crystal-centric paradigm of Bt-like pesticidal proteins and support an expanded model in which soluble extracellular components contribute to pathogenicity. This work highlights the value of secretome analysis for the characterization of Bt-like strains and provides new insights into the molecular basis of nematicidal activity in B. toyonensis.