CONICET | Buscador de Institutos y Recursos Humanos

Bacterial biofilms formation involves a complex molecular and physiological mechanism, not yet entirely known. Biofilm development seems to be mediated by the environmental composition and bacterial genetics. The epigenetic DNA methylation may be an important molecular regulator on biofilms formation. However, this mechanism on biofilm formation has been poorly studied. The DNA adenine methyltransferase (Dam) enzyme adds a methyl group to the GATC DNA motif and has several functions, including the regulation of gene expression. The objective of our study was to evaluate the participation of Dam enzyme on biofilm formation in E. coli K-12 strain MG1655. To this purpose we constructed a dam-deficient mutant by site-specific mutagenesis. The absence of Dam activity was confirmed by DNA differential digestion assay using Sau3AI, DpnI and MboI restriction enzymes. We evaluated the ability to generate biofilm on polystyrene after 72 hs of incubation by quantitative assay using crystal violet staining. Wells were inoculated with nutrient medium supplemented with different environmental conditions (glucose 500 mM, dilution in 1/10 of the nutrient media, ethanol 1%, methionine 5 µM, surfactants and EDTA). Our results showed that deletion of the dam gene caused the complete depletion of the DNA methylation in GATC motif. We found that the capacity to develop biofilm on polystyrene of dam mutant increased compared with the parental strain in minimal M9 (0 units for wild type vs. 3 units for dam mutant) and nutrient media (0 units for wild type vs. 1 units for dammutant). The dam mutant biofilms formation was stimulated by the dilution in 1/10 of the nutrient media and ethanol 1%. The rest of the environmental conditions settings tested diminished it. Using congo red agar assay the curli and cellulose production, the main components of E. coli biofilm extracellular matrix, were determined. As expected, the wild type strain showed a pdar morphotype indicating that curli production is affected whereas cellulose production is not. Interestingly, the dam mutant displayed rdarmorphotype, which reflects the co-expression of curli fimbriae and cellulose. Altogether our results indicate that Dam enzyme represses biofilm formation, as demonstrated by the curli-like fibers synthesis, in E. coli K-12 strain MG1655. These data could explain, in part, the reason why this strain is classified as a non-biofilm forming. Further studies are needed to elucidate how DNA methylation by Dam may affect the regulation of biofilm formation in E. coli.