Evolution of genotypic and phenotypic diversity in multispecies biofilms

Abstract The fitness and adaptability of bacteria in microbial communities can be influenced by the interactions among the community partners, as well as specific spatial organisation. Here, we investigated the evolutionary dynamics of Bacillus thuringiensis in response to the presence or absence of spatial structure and bacterial interspecies interactions. During evolution experiments, a distinct phenotypic variant of B. thuringiensis frequently occurred, irrespective of the conditions tested: planktonic vs. biofilm or monospecies vs. mixed species. Remarkably, selection significantly favored this variant of B. thuringiensis over its ancestor in biofilm settings and when coexisting with Pseudomonas defluvii and Pseudomonas brenneri , co-isolated from a wastewater facility. Interestingly, the evolved phenotype displayed lower biomass in mixed species biofilms, shorter generation time, and lacked sporulation compared to the ancestor. The strains displaying this phenotypic variation contained mutations in the regulator spo0A that initiates sporulation but also de-represses expression of matrix determinants in other Bacilli. Consistently, matrix proteomics revealed a reduced abundance of TasA in the phenotypic variant, a major biofilm matrix component in Bacillus species, while higher abundance was found in co-culture with P. brenneri . Our results indicate that interspecies interactions within biofilms not only promote B. thuringiensis diversification but also select for altered traits such as reduced biofilm matrix production. Moreover, P. brenneri was favoured in co-culture biofilm vs. planktonic settings, suggesting that spatial structure and B. thuringiensis diversification facilitate species co-existence. These findings could impact applications where Bacillus and Pseudomonas are utilized in consortia, such as plant growth promoters or biopesticides.