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.

ABSTRACTThe SOS response functions as the central regulator of DNA repair and mutagenesis in most bacteria and stands as a paradigm of gene networks controlled by a master transcriptional regulator, LexA. We developed a single-molecule imaging approach to directly monitor the LexA repressor inside live Escherichia coli cells, demonstrating key mechanisms by which DNA-binding and degradation of LexA regulates the SOS response in vivo. Our approach revealed that self-cleavage of LexA occurs frequently during unperturbed growth and causes substantial heterogeneity in LexA abundances across cells. LexA variability underlies SOS gene expression heterogeneity and triggers spontaneous SOS pulses, which enhance bacterial survival in anticipation of stress.

DNA cloned in yeast artificial chromosomes (YACs) is a valuable resource for functional experiments in cell culture as well as whole animal systems. Where the size or chimerism of a YAC clone are limiting factors it may be desirable to generate recombinant YAC clones. One such approach is based on mitotic recombination, and we describe the development of a methodology that allows multiple recombination cycles for serial reconstruction of overlapping YACs. This approach employs retrofitting with standard plasmid vectors, transfer of YACs to a common haploid host by Kar1 mating, and selection for recombination with 5-fluoro-orotic acid.