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 σS. 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 σS-dependence of curli induction, there was only a partial correlation between the σS activity and curli expression, with subpopulations of cells having high σS 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.

Attachment to surfaces is an important early step during bacterial infection and during formation of submerged biofilms. Although flagella‐mediated motility is known to be important for attachment of Escherichia coli and other bacteria, implications of motility regulation by cellular signalling remain to be understood. Here, we show that motility largely promotes attachment of E. coli, including that mediated by type 1 fimbriae, by allowing cells to reach, get hydrodynamically trapped at and explore the surface. Inactivation or inhibition of the chemotaxis signalling pathway improves attachment by suppressing cell reorientations and thereby increasing surface residence times. The attachment is further enhanced by deletion of genes encoding the cyclic diguanosine monophosphate (c‐di‐GMP)‐dependent flagellar brake YcgR or the diguanylate cyclase DgcE. Such increased attachment in absence of c‐di‐GMP signalling is in contrast to its commonly accepted function as a positive regulator of the sessile state. It is apparently due to the increased swimming speed of E. coli in absence of YcgR‐mediated motor control, which strengthens adhesion mediated by the type 1 fimbriae. Thus, both signalling networks that regulate motility of E. coli also control its engagement with both biotic and abiotic surfaces, which has likely implications for infection and biofilm formation.