Presence of a biofilm beneficiary alters the evolutionary trajectory of a biofilm former.

Biofilm evolution is typically studied in monocultures or in communities displaying mutualistic or exploitative interactions. However, in communities with fluctuating interactions, the influence of biofilm-beneficiary bacteria on the evolution of biofilm-founder bacteria remains less understood. Biofilm-beneficiary bacteria cannot form robust biofilms independently but can incorporate into the biofilm of biofilm-formers, thereby gaining the ability to colonize given niche. In this study, we demonstrate that the biofilm-former Bacillus velezensis SQR9 reproducibly diversified into biofilm-enhanced slimy and biofilm-weakened rough types, both in the presence and absence of a biofilm-beneficiary Stutzerimonas degradans XL272 (formerly Pseudomonas stutzeri), but with variable frequencies under the two conditions. The exopolysaccharide producer slimy types dominated B. velezensis populations in monoevolution, whereas the exploiter rough types, which exploit the exopolysaccharides produced by the slimy types, dominate in coculture evolution. Phenotypic changes in B. velezensis were linked to mutations in specific genes that regulate biofilm formation and sporulation, including ywcC, comA, comP, degS, degQ, and spo0F. A frameshift mutation in the cpsA gene of S. degradans increased its exopolysaccharide production in the dual-species biofilm, which served as shared resources and allow the B. velezensis exploiter (i.e. rough type) to outcompete the producer (i.e. slimy type) during coculture evolution. Additionally, longitudinal population sequencing and "replay" evolution experiments with the S. degradans mutant revealed that the cpsA mutation accelerated the fixation of the rough type within B. velezensis populations. In conclusion, this research demonstrates that interspecific interactions can adaptively favor exploiters within biofilm populations.