Analysis of SOS-Induced Spontaneous Prophage Induction in Corynebacterium glutamicum at the Single-Cell Level

The genome of the Gram-positive soil bacterium Corynebacterium glutamicum ATCC 13032 contains three integrated prophage elements (CGP1 to -3). Recently, it was shown that the large lysogenic prophage CGP3 (similar to 187 kbp) is excised spontaneously in a small number of cells. In this study, we provide evidence that a spontaneously induced SOS response is partly responsible for the observed spontaneous CGP3 induction. Whereas previous studies focused mainly on the induction of prophages at the population level, we analyzed the spontaneous CGP3 induction at the single-cell level using promoters of phage genes (P-int2 and P-lysin) fused to reporter genes encoding fluorescent proteins. Flow-cytometric analysis revealed a spontaneous CGP3 activity in about 0.01 to 0.08% of the cells grown in standard minimal medium, which displayed a significantly reduced viability. A P-recA-eyfp promoter fusion revealed that a small fraction of C. glutamicum cells (similar to 0.2%) exhibited a spontaneous induction of the SOS response. Correlation of P-recA to the activity of downstream SOS genes (P-divS and P-recN) confirmed a bona fide induction of this stress response rather than stochastic gene expression. Interestingly, the reporter output of P-recA and CGP3 promoter fusions displayed a positive correlation at the single-cell level (rho = 0.44 to 0.77). Furthermore, analysis of the P-recA-eyfp/P-int2-e2-crimson strain during growth revealed the highest percentage of spontaneous P-recA and P-int2 activity in the early exponential phase, when fast replication occurs. Based on these studies, we postulate that spontaneously occurring DNA damage induces the SOS response, which in turn triggers the induction of lysogenic prophages.