Diversification of Gene Expression during Formation of Static Submerged Biofilms by Escherichia coli

Many bacteria primarily exist in nature as structured multicellular communities, so called biofilms. Biofilm formation is a highly regulated process that includes the transition from the motile planktonic to sessile biofilm lifestyle. Cellular differentiation within a biofilm is a commonly accepted concept but it remains largely unclear when, where and how exactly such differentiation arises. Here we used fluorescent transcriptional reporters to quantitatively analyze spatio-temporal expression patterns of several groups of genes during the formation of submerged; Escherichia coli; biofilms in an open static system. We first confirm that formation of such submerged biofilms as well as pellicles at the liquid-air interface requires the major matrix component, curli, and flagella-mediated motility. We further demonstrate that in this system, diversification of gene expression leads to emergence of at least three distinct subpopulations of; E. coli; , which differ in their levels of curli and flagella expression, and in the activity of the stationary phase sigma factor σ; S; . Our study reveals mutually exclusive expression of curli fibers and flagella at the single cell level, with high curli levels being confined to dense cell aggregates/microcolonies and flagella expression showing an opposite expression pattern. Interestingly, despite the known σ; S; -dependence of curli induction, there was only a partial correlation between the σ; S; activity and curli expression, with subpopulations of cells having high σ; S; activity but low curli expression and; vice versa; . Finally, consistent with different physiology of the observed subpopulations, we show striking differences between the growth rates of cells within and outside of aggregates.