Short-Term (Co-)Adaptation In Biofilms Of Lactococcus Lactis And Leuconostoc Mesenteroides Impacts Growth Parameters And Interspecific Interactions

Background: In nature, bacteria coexist in polymicrobial complex communities dominated by interspecies interactions. This coexistence contributes to a constant emergence of variants, but their establishment depends on eco-evolutionary dynamics. Spatially structured and heterogenous biofilms enable niche differentiation and local interplay, supporting greater diversity. This study examines the adaptation of mono- and co-cultured Lactococcus lactis and Leuconostoc mesenteroides in a biofilm-selecting environment.

Methods: Cultures were passaged every 24 h by bead transfer for 16 days. Evolved variants and ancestors were compared in their growth metrics, competitiveness in reestablished co-cultures, and global proteomes.

Results: Evolution experiments displayed that, in biofilm co-cultures, L. mesenteroides predominated, but both species coexisted. Comparative analyses of L. lactis biofilm variants revealed an increased biofilm formation but, interestingly, increased culture yield and prolonged generation time exclusively in co-evolved variants. Furthermore, the performance of evolved L. lactis variants was evaluated when co-cultured with L. mesenteroides. Biofilm assays showed a higher proportion of evolved L. lactis strains than their ancestor when co-cultivated with ancestral L. mesenteroides. This effect was particularly evident in low L. lactis starting ratios. Interestingly, in co-cultures with evolved L. mesenteroides, L. lactis was strongly reduced. However, evolved L. lactis strains were more persistent than their ancestor in such conditions. 

Combined, this shows that coexistence in biofilms selects for variants adapted to the biofilm life cycle and the other member’s presence, reflected in their enhanced persistence and prevalence in the mixed biofilm. Preliminary proteomic analyses of mono- and co-cultures suggest that proteomes of mono- and co-evolved L. lactis variants are more distinguishable by the selected ancestor (lineage) than the cultivation method.

Conclusions: This study emphasizes spatially structured biofilms’ importance in maintaining species diversity, as interspecies interactions and the biofilm setting were conditional for the emergence of novel phenotypic variants.
The Novo Nordisk Foundation funded this study, grant No. 27620.