Adaptation and phenotypic diversification of Bacillus thuringiensis biofilm are accompanied by fuzzy spreader morphotypes.

Bacillus cereus group (Bacillus cereus sensu lato) has a diverse ecology, including various species that produce biofilms on abiotic and biotic surfaces. While genetic and morphological diversification enables the adaptation of multicellular communities, this area remains largely unknown in the Bacillus cereus group. In this work, we dissected the experimental evolution of Bacillus thuringiensis 407 Cry- during continuous recolonization of plastic beads. We observed the evolution of a distinct colony morphotype that we named fuzzy spreader (FS) variant. Most multicellular traits of the FS variant displayed higher competitive ability versus the ancestral strain, suggesting an important role for diversification in the adaptation of B. thuringiensis to the biofilm lifestyle. Further genetic characterization of FS variant revealed the disruption of a guanylyltransferase gene by an insertion sequence (IS) element, which could be similarly observed in the genome of a natural isolate. The evolved FS and the deletion mutant in the guanylyltransferase gene (Bt407ΔrfbM) displayed similarly altered aggregation and hydrophobicity compared to the ancestor strain, suggesting that the adaptation process highly depends on the physical adhesive forces.