Morphogenesis and mechanical properties of Bacillus amyloliquefaciens biofilms: a comparative study of rough and smooth morphotypes

<i>Bacillus amyloliquefaciens</i>, a Gram-positive, endospore-forming bacterium, is ubiquitously found in diverse environments. Its resilience in harsh conditions and biofilm-forming capabilities make it a promising candidate for various agricultural applications. <i>B. amyloliquefaciens</i> can also colonize the air-liquid interface by producing pellicles. Despite its potential, the characterization of <i>B. amyloliquefaciens</i> biofilms remains underexplored. We investigated colony expansion and pellicle formation kinetic, followed by an analysis of the mechanical properties of the latter. This work focused on <i>B. amyloliquefaciens</i> strain L-17, which produced two morphotypes on agar plate: 1) a <b>rough</b> (R) morphotype exhibiting quick colony expansion and 2) a <b>smooth</b> (S) morphotype with a slower but extended colony expansion. The morphogenesis model of pellicle formation proposed in the literature for <i>Bacillus subtilis</i> was applicable to <i>B. amyloliquefaciens</i>, with few modifications for the smooth morphotype, which needed a longer period to spread over the surface. Mature pellicles were visually homogeneous and heterogeneous for R and S, respectively. Surprisingly, (i) their deformability by stretching was heterogeneous for the former and homogeneous for the latter; (ii) R produced a stiff pellicle whereas the entire smooth pellicle showed an elastic response. Taken together, results of this study confirmed that the existence of two distinct morphotypes within the same strain offers a unique opportunity to study the diversity of behaviors in <i>B. amyloliquefaciens</i>, providing insights into its potential applications in agriculture and beyond. Moreover, they led to the hypothesis that surfactant production and flagella expression are differentiating factors between both morphotypes.