Massively parallel transposon mutagenesis identifies temporally essential genes for biofilm formation in Escherichia coli

Biofilms complete a life cycle where cells aggregate, grow and produce a structured community before dispersing to seed biofilms in new environments. Progression through this life cycle requires controlled temporal gene expression to maximise fitness at each stage. Previous studies have focused on the essential genome for the formation of a mature biofilm, but here we present an insight into the genes involved at different stages of biofilm formation. We used TraDIS-Xpress; a massively parallel transposon mutagenesis approach using transposon-located promoters to assay expression of all genes in the genome. We determined how gene essentiality and expression affects the fitness of E. coli growing as a biofilm on glass beads after 12, 24 and 48 hours. A selection of genes identified as important were then validated independently by assaying biofilm biomass, aggregation, curli biosynthesis and adhesion ability of defined mutants. We identified 48 genes that affected biofilm fitness including genes with known roles and those not previously implicated in biofilm formation. Regulation of type 1 fimbriae and motility were important at all time points. Adhesion and motility were important for the early biofilm, whereas matrix production and purine biosynthesis were only important as the biofilm matured. We found strong temporal contributions to biofilm fitness for some genes including some which were both beneficial and detrimental depending on the stage at which they are expressed, including dksA and dsbA. Novel genes implicated in biofilm formation included ychM and truA involved in cell division, crfC and maoP in DNA housekeeping and yigZ and ykgJ of unknown function. This work provides new insights into the requirements for successful biofilm formation through the biofilm life cycle and demonstrates the importance of understanding expression and fitness through time.