Dokument

Summary:
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. In this work fluorescent transcriptional reporters were used to quantitatively analyze spatiotemporal 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 sigma S. Our data reveal 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. Furthermore, our results suggest direct interaction between flagella appendages and curli fibers, which can complement each other in trans during biofilm structure development. Interestingly, despite the known sigma S-dependence of curli induction, there was only a partial correlation between the sigma S activity and curli expression, with subpopulations of cells having high sigma 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. We provide strong evidence that flagellar expression in surface-attached biofilm cells is triggered by inhibition of flagellar rotation via flagellar stator MotAB upon surface sensing. Importantly, our results suggest that flagellar expression in surface-attached cells might serve for holding the biofilm structure on the surface, since downregulation of flagellar expression leads to a detachment of E. coli biofilms under prolonged starvation conditions. Further, we show that differentiation into curli expressing and non-expressing cells arises also in isotropic E. coli planktonic cells upon entry into the stationary phase. Our results indicate that this switch might be triggered by variations in growth rate of individual cells under starving conditions, with a subpopulation of slower growing cells expressing curli. Finally, this work revealed important new details of molecular mechanisms and the physiological relevance underlying the bimodal curli expression.

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