The role of various factors in the process of biofilm formation by bacilli

The strategy of existence of microorganisms in the structure of biofilms determines their adaptation to specific environmental conditions. Bacteria of the genus Bacillus are actively used as models for studying the processes of regulation and formation of biofilms. This work aims to assess the effect of proteases secreted by bacilli on the formation of biofilms, as well as on the resistance of recombinant strains to environmental stress factors. We compared the dynamics of biofilm formation by the wild strain B. subtilis 168 and the following strains with altered expression of extracellular proteases: non-protease strains (B. subtilis BG2036, B. subtilis BRB8, and B. subtilis BRB14) and strains with increased protease secretion (B. subtilis (aprBp), B. subtilis (gseBp), and B. subtilis (mprBp)). We also investigated the effect of ethanol and sodium chloride on the ability of the B. subtilis strains to form biofilms in a liquid medium. The bacterial strains were cultured at pH 7.4 and temperature 37 ?C in a synthetic E-medium in U-shaped 96-well plates. Biofilm formation was identified by incubation with crystal violet (CV). We found that the level of biofilm formation is higher (by an average of 10%) in the protease-deficient strains of B. subtilis. The recombinant strains expressing genes of serine proteases form biofilms with a reduced level (by an average of 40%). A strain with the expression of metalloendopeptidase is characterized by an increased level of biofilm formation (up to 10%), which is possibly due to the functional in vivo role of this new enzyme. The wild and protease-deficient strains of B. subtilis respond similarly to ethanol stress: biofilm formation is reduced in both of them. The wild strain exhibits a greater sensitivity to osmotic stress than the protease-deficient one