Thus the rate of LexA dissociation from operators controls the precise timing of SOS gene expression following induction. Consequently genes with lower affinity LexA target sites are expressed prior to genes with high affinity operators [1, 5]. To follow up on these results, we used SPR to study interaction of the chip-immobilized C. difficile RecA* with LexA interacting with either specific or non-specific DNA. We showed that as in E. coli, the C. difficile LexA Daporinad manufacturer repressor interaction with RecA* is prevented by binding to specific DNA targets (Figure 4). In addition, we showed that the key SOS players of E. coli

and C. difficile can cross-react in vitro (Figure 4). Hence, our data indicated that the mode of regulation

of the C. difficile SOS response resembles the one described for E. coli. Nevertheless, in contrast to the E. coli SOS system, we observed among the investigated C. difficile genes, a slowest LexA dissociation from operators of the core SOS genes, recA, lexA and uvrB (Figure 3A and B, Table 2), implying that these are the last genes upregulated upon SOS induction. For instance, LexA dissociation from the E. coli recA operator is more than ALK activation 20-times faster than from C. difficile with regard to the dissociation constants of 4.8 ± 2.1 × 10−3 s−1 (21) and 1.7 ± 0.5 × 10−4 s−1, respectively. Figure 4 Specific DNA precludes C. difficile RecA*-LexA interaction. Interaction of C. difficile LexA repressor (2.6 μM) incubated with specific, 22-bp recA operator (A) or with non-specific DNA fragment, recA operator with modified six nucleotides (B), with the chip-immobilized C. difficile RecA* (~2000 response units). The used DNA interacting with repressor was in 1.4 μM (black line), 2.7 μM (red line), 4.0 μM (green line), 5.4 μM (blue line), 8.1 μM (pink line) concentration. The cyan line presents sensorgram of the free DNA at 8.1 μM concentration SPTLC1 interacting with the RecA*. (C) In vitro repressor cleavage pattern exhibits that AR-13324 purchase purified E. coli and C. difficile key SOS players

can cross-react. C. difficile proteins are marked as RecA* (CD), LexA (CD) and E. coli proteins as RecA* (EC) and LexA (EC), respectively. Time course (min) of either C. difficile or E. coli RecA*-induced inactivation of LexA (CD) or LexA (EC) repressor. Quantification of LexA is presented on the gel above the respective band as the ratio (%) of the protein density value of the initial sample (0 min) relative to the density value obtained from the proteins after indicated time points after addition of RecA*, shown with standard deviation. Table 2 Target DNA sequences of the putative SOS genes of the R20291 strain used for the SPR analysis GENE Function Product Putative LexA operator (R20291 strain) (5`- -3`) Distance from CDS lexA SOS response Transcriptional regulator.