Single-molecule imaging of LexA degradation in Escherichia coli elucidates regulatory mechanisms and heterogeneity of the SOS response

The bacterial SOS response stands as a paradigm of gene networks controlled by a master transcriptional regulator. Self-cleavage of the SOS repressor, LexA, induces a wide range of cell functions that are critical for survival and adaptation when bacteria experience stress conditions(1), including DNA repair(2), mutagenesis(3,4), horizontal gene transfer(5–7), filamentous growth, and the induction of bacterial toxins(8–12), toxin-antitoxin systems(13), virulence factors(6,14), and prophages(15–17). SOS induction is also implicated in biofilm formation and antibiotic persistence(11,18–20). Considering the fitness burden of these functions, it is surprising that the expression of LexA-regulated genes is highly variable across cells(10,21–23) and that cell subpopulations induce the SOS response spontaneously even in the absence of stress exposure(9,11,12,16,24,25). Whether this reflects a population survival strategy or a regulatory inaccuracy is unclear, as are the mechanisms underlying SOS heterogeneity. Here, we developed a single-molecule imaging approach based on a HaloTag fusion to directly monitor LexA inside live Escherichia coli cells, demonstrating the existence of 3 main states of LexA: DNA-bound stationary molecules, free LexA and degraded LexA species. These analyses elucidate the mechanisms by which DNA-binding and degradation of LexA regulate the SOS response in vivo. We show that self-cleavage of LexA occurs frequently throughout the population during unperturbed growth, rather than being restricted to a subpopulation of cells, which causes substantial cell-to-cell variation in LexA abundances. LexA variability underlies SOS gene expression heterogeneity and triggers spontaneous SOS pulses, which enhance bacterial survival in anticipation of stress.