Evolution of Listeria monocytogenes to a strong biofilm producer via the overexpression of Lmo1799

Biofilm formation is key in Listeria monocytogenes' transmission and persistence in food processing environments. To further understand the mechanisms contributing to biofilm formation, an experimental evolution system was used to isolate strong biofilm producing strains of L. monocytogenes EGDe (reference strain) and FBR16 (hypermutator food isolate). After cycles of plastic surface colonisation, biofilm formation, dispersal and attachment to new surfaces, evolved variants (EV) strains were isolated and found to produce up to seven-fold more biofilm than their respective ancestral (AN) strains. Phenotypic assays revealed an increase in cell surface hydrophobicity as a shared dominant feature of EGDe and FBR16 EV isolates. Proteomic analysis showed proteins Lmo1798, a predicted glucosyltransferase, and Lmo1799, a putative peptidoglycan binding protein with 226 Ala-Asp tandem repeats, to be the most upregulated proteins in both EV strains compared to the AN strains. Genomic analysis of the EGDe EV strain identified a single-nucleotide insertion in the upstream region of lmo1799 and an in-frame deletion of 42 nucleotides in lmo1799, conceivably resulting in high-level expression of a functional protein with 219 Ala-Asp repeats. To evaluate the impact of Lmo1799 on the EV phenotypes and the overall biofilm capacity of L. monocytogenes, EGDe EV mutants lacking lmo1799 and/or the upstream insertion were constructed. Notably, both constructed mutants showed reduced biofilm formation and lower surface hydrophobicity compared to the EV strain, indicating the importance of these mutations for the strong biofilm capacity. Overall, these observations indicate a critical role of Lmo1799 in L. monocytogenes cell surface characteristics and biofilm formation.