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

Abstract

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 (ϳ187 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 (ϳ0.2%) 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 ( ‫؍‬ 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. G enome sequencing projects have revealed a large amount of prophage DNA in bacterial genomes. Although not all prophage DNA accounts for functional prophages, because it includes degenerated phage remnants, this DNA can have a marked impact on bacterial physiology (1). The biotechnological platform organism Corynebacterium glutamicum is a Gram-positive, biotin-auxotroph soil bacterium that is used for the industrial production of more than four million tons of L-glutamate and L-lysine per year (2, 3). As revealed by whole-genome sequencing, C. glutamicum ATCC 13032 possesses three prophages that are integrated into its genome (CGP1 to -3), of which CGP1 and CGP2 are probably degenerated phage remnants (4-6). Previous studies showed that the large prophage CGP3 (187 kb) retains the ability to be excised from the genome and exist as a circular DNA molecule. Interestingly, a small number of wild-type cells showed a much higher copy number of circular phage DNA per cell than the average of the population (5).Recent studies in Shewanella oneidensis (7) and Streptococcus pneumoniae (8) have provided evidence that sacrificing a small number of cells by spontaneous prophage-induced lysis is beneficial to the remainder of the population. For these species, genomic DNA released into the extracellular space following cell lysis s...

Impact of Spontaneous Prophage Induction on the Fitness of Bacterial Populations and Host-Microbe Interactions

“…Key to research questions centering on the phenotypic heterogeneity of populations is the advancement of the single-cell analytic platforms which have become common in modern microbiology and which will see an even broader use in the upcoming years (11,14,96,97). Recent studies have shown the power of flow cytometry coupled with the use of fluorescent reporters and live-cell imaging enabled by microfluidic devices in elucidating mechanisms governing the activity of foreign DNA within bacterial genomes (12,13). Combined with classical molecular biology approaches, these recent advances in single-cell analytics shed new light on the dynamics of microbial populations and host-microbe interaction.…”

“…During growth, ongoing (multifork) replication has been shown to cause sporadic DNA damage resulting in the derepression of the SOS genes (16). Recent single-cell studies revealed a small fraction of SOS-induced cells in clonal populations grown under standard conditions, and it is reasonable to infer that a prolonged induction will also lead to the activation of resident prophages (13,17,18).…”

“…This spontaneous induction of the SOS response is sufficient to induce key promoters within prophage CGP3, among them the genes encoding a putative integrase and lysin (13). Mutant strains lacking recA showed only a basal level of spontaneous CGP3 induction, which might be due to stochastic effects (see below) or mutations of the prophage.…”

“…Recently, it was shown that single cells of Corynebacterium glutamicum undergo spontaneous SOS induction under standard cultivation conditions (13). This spontaneous induction of the SOS response is sufficient to induce key promoters within prophage CGP3, among them the genes encoding a putative integrase and lysin (13).…”

“…The work performed in these studies was further supported by the advancement of single-cell analytics such as flow cytometry and time-lapse microscopy in microfluidic cultivation chambers, allowing the analysis of triggers for bacteriophages and mobile genetic elements working in a subset of bacterial populations (11)(12)(13)(14)(15). In this review, we set the focus on the triggering of spontaneous activity of prophages or phage remnants and the physiological consequences that this process can have for microbial populations.…”