Factors Limiting SOS Expression in Log-Phase Cells of Escherichia coli

Abstract

In Escherichia coli, RecA-single-stranded DNA (RecA-ssDNA) filaments catalyze DNA repair, recombination, and induction of the SOS response. It has been shown that, while many (15 to 25%) log-phase cells have RecA filaments, few (about 1%) are induced for SOS. It is hypothesized that RecA's ability to induce SOS expression in log-phase cells is repressed because of the potentially detrimental effects of SOS mutagenesis. To test this, mutations were sought to produce a population where the number of cells with SOS expression more closely equaled the number of RecA filaments. Here, it is shown that deleting radA (important for resolution of recombination structures) and increasing recA transcription 2-to 3-fold with a recAo1403 operator mutation act independently to minimally satisfy this condition. This allows 24% of mutant cells to have elevated levels of SOS expression, a percentage similar to that of cells with RecA-green fluorescent protein (RecA-GFP) foci. In an xthA (exonuclease III gene) mutant where there are 3-fold more RecA loading events, recX (a destabilizer of RecA filaments) must be additionally deleted to achieve a population of cells where the percentage having elevated SOS expression (91%) nearly equals the percentage with at least one RecA-GFP focus (83%). It is proposed that, in the xthA mutant, there are three independent mechanisms that repress SOS expression in log-phase cells. These are the rapid processing of RecA filaments by RadA, maintaining the concentration of RecA below a critical level, and the destabilizing of RecA filaments by RecX. Only the first two mechanisms operate independently in a wild-type cell.

Genetic analysis of Escherichia coli RadA: functional motifs and genetic interactions

“…Sandler et al . have also shown that loss of RadA leads to stronger SOS induction in vivo when combined with an SOS‐constitutive recA4142 mutation and have hypothesized that RadA processes RecA filaments to maintain a level below the critical level for SOS induction (Massoni et al ., ). Using microscopic analyses of sulA ::GFP fluorescence, these authors observed a modest, 1.5‐fold elevation of expression caused by loss of RadA in an otherwise wild‐type strain.…”

DNA Damage Responses in Prokaryotes: Regulating Gene Expression, Modulating Growth Patterns, and Manipulating Replication Forks

“…These results parallel sensitivity results because SOS-noninducible E. coli are hypersensitive to ionizing radiation, but noninducible B. subtilis are unaffected. From all of these results, it is clear that B. subtilis has substantial and efficient repair pathways without SOS induction, and that RecA focus formation is mechanistically distinct from SOS induction (also see Centore et al 2008;Massoni et al 2012).…”

Specificity in suppression of SOS expression by recA4162 and uvrD303

“…sulA is an SOS gene and this reporter system has been used extensively and described in several other studies [31, 34, 35]. Thus, the strategy used here was to introduce the DNA replication mutations into strains having the sulAp-gfp reporter system with different alleles of recA and uvrD to test the ability of recA4162 or uvrD303 to observe if SOS expression occurred normally in these mutants or was lowered.…”

“…The data is presented in two ways. First, the level of SOS expression of the population of cells is reported in the tables by measuring and computing the average Relative Fluorescence Intensity (RFI) (compared to a wild type population with no sulAp-gfp ) of the cells and by the percentage of the population having nine-fold or greater expression than the average cell in a wild type population (the reason why this level has been chosen is explained elsewhere [31]). Second, the SOS expression of cells across the population is shown by graphing the percentage of cells in the population (y-axis) with a particular level of SOS expression (x-axis) (Figure 1B).…”

“…Although RecA participates in repair of these types of lesions through its ability to form a RecA-DNA filament, it is clear that the SOS response is not usually induced. This is best demonstrated by the observations that about 15–25% of a population of log phase cells have recombination structures at any one time [31–33], yet less than 1% are induced for SOS [34, 35]. Recently, it has been shown that radA , the amount of RecA in the cell, and in some cases recX , prevent these RecA filaments from inducing the SOS response when presumably fixing housekeeping types of damage [31].…”