Measurement of SOS expression in individual Escherichia coli K‐12 cells using fluorescence microscopy

Abstract

SummaryMany recombination, DNA repair and DNA replication mutants have high basal levels of SOS expression as determined by a sulAp-lacZ reporter gene system on a population of cells. Two opposing models to explain how the SOS expression is distributed in these cells are: (i) the 'Uniform Expression Model (UEM)' where expression is evenly distributed in all cells or (ii) the 'Two Population Model (TPM)' where some cells are highly induced while others are not at all. To distinguish between these two models, a method to quantify SOS expression in individual bacterial cells was developed by fusing an SOS promoter ( sulAp ) to the green fluorescent protein ( gfp ) reporter gene and inserting it at att l l l l on the Escherichia coli chromosome. It is shown that the fluorescence in sulAp-gfp cells is regulated by RecA and LexA. This system was then used to distinguish between the two models for several mutants. The patterns displayed by priA , dnaT , recG , uvrD , dam , ftsK , rnhA , polA and xerC mutants were explained best by the TPM while only lexA (def) , lexA3 (ind -) and recA defective mutants were explained best by the UEM. These results are discussed in a context of how the processes of DNA replication and recombination may affect cells in a population differentially.

Spontaneous DNA breakage in single living Escherichia coli cells

“…In one positive control, lexA(Def) (SOS repressor-null) cultures fluoresce green quantitatively (Fig. 1a) 6 , and two negative controls, SOS-uninducible lexA(Ind − ) or ΔrecA cells, show 2 × 10 −4 ± 0.1 × 10 −4 and 7 × 10 −4 ± 4 × 10 −4 green cells (mean ± s.e.m. ), respectively ( Fig.…”

“…RecA bound to ssDNA promotes proteolysis of the LexA transcriptional repressor, increasing expression of more than 40 SOS damage response genes4. We engineered wild-type E. coli to carry a chromosomally located gfp gene encoding green fluorescent protein (GFP) controlled by the SOS-inducible sulA promoter 6 , which causes cells with SOS-inducing DNA damage to fluoresce green (that is, these cells are GFP + ). Because it is not plasmid borne and does not inactivate any gene (including sulA), damage is detected without perturbing levels of proteins that affect DNA damage processing (Supplementary Methods online).…”

The role of the hok/sok locus in bacterial response to stressful growth conditions

“…The SS996 has a mutation in ftsZ that makes the protein insensitive to the SOS-induced cell division inhibitor SulA, thus allowing continued growth of the cells in conditions that would otherwise induce the SOS response and inhibit cell growth [22,23]. It has been reported that β-lactams and fluoroquinolones induce SOS response that temporarily inhibit cell division and help bacteria survive their lethal effects [24,25].…”

Biology of Three ICE Families: SXT/R391, ICE Bs1 , and ICE St1 /ICE St3

“…The reasons for this striking difference of regulation are unknown but could be the result of a difference in the fraction of the cell population spontaneously expressing the SOS response between V. cholerae and E. coli (57). Indeed, exposure to DNA-damaging agents that are known to induce the SOS response (UV light, mitomycin C, ciprofloxacin) have been shown to stimulate SXT conjugative transfer at frequencies up to 10 -2 per donor (58).…”