Evolution of genotypic and phenotypic diversity in multispecies biofilms

Interactions occurring in microbial communities can affect the fitness and adaptability of their individual members when facing changing environmental conditions. This study investigated the impact of interspecies interactions in selecting Bacillus thuringiensis variants emerging in biofilm and planktonic environments. During evolution experiments, a B. thuringiensis distinct phenotypic variant of B. thuringiensis frequently occurred, despite the presence of other species or culture setup. Remarkably, selection of this variant was significantly favored over its ancestor in biofilm settings and when coexisting with other species co-isolated from a wastewater facility, namely Pseudomonas defluvii and Pseudomonas brenneri. Interestingly, the evolved phenotype did not show higher biofilm productivity than its ancestor under any condition, while it was indeed reduced in mixed-species biofilms. Such observation aligned with the reduced abundance of TasA, a major biofilm matrix component in Bacillus species, and SpoVG, a regulator of sporulation, as revealed by matrix proteomics analysis. Furthermore, the variant showed shorter generation time and a lack of sporulation compared to its ancestor, consistent with mutations in key genes for regulating sporulation. Our results indicate that interspecies interactions within biofilms promote B. thuringiensis diversification and alter traits such as biofilm matrix production. Although sporulation is a survival mechanism, this study provides evidence that sporulation does not confer a fitness advantage in in vitro biofilm setting, even within mixed cultures, in the absence of severe stress.