Influence of tyrosine on Bacillus cereus ATCC 14579 biofilm formation

Bacillus cereus is a food-borne pathogen capable of producing biofilms. The protective layers of a biofilm increase the resistance of bacterial cells to environmental hazards such as antibiotics and cleaning detergents. Subsequently, these hard to get rid of biofilms can detach and cause recontamination of food products leading to possible quality and safety issues. Transposon screening and subsequent testing of a mutant of B. cereus ATCC 14579 lacking the BC2939 gene (ΔBC2939), encoding the enzyme that mediates the conversion of Prephenate to 4-Hydroxy-phenylpyruvate (4-HPPA), showed decreased biofilm formation. The mutant is thus impaired in the production of tyrosine via the Shikimate pathway, which also has branches resulting in two other aromatic amino acids (AAA): tryptophan and phenylalanine. Upon supplementation of the growth medium with different AAAs, only tyrosine showed a biofilm stimulating effect in ΔBC2939, resulting in WT-like biofilm levels. Additionally, supplementation of the growth medium with 4-HPPA, the precursor of tyrosine, also restored biofilm production in the ΔBC2939 mutant. Determination of total viable counts (TVC) and spore counts in supernatants of cultures without and with added tyrosine, showed no significant difference between WT and ΔBC2939. However, significant differences were found in biofilm TVCs, showing approximately 10-fold lower cell numbers for ΔBC2939 compared to WT. Notably, mutant biofilm cell counts increased to WT numbers with the supplementation of tyrosine, while no effect was observed for WT. Combining all data, this points to a role for tyrosine in B. cereus adhesion and biofilm formation. A combined genotyping and phenotyping analysis was performed including proteome analysis of WT and mutant planktonic and biofilm cells, to elucidate how tyrosine influences B. cereus biofilm formation.