Impact and Regulatory Control of the CGP3 Prophage in $\textit{Corynebacterium glutamicum}$

Almost all bacterial genomes contain substantial amounts of viral DNA, which may significantly impact microbial physiology. The spontaneous induction of prophages (SPI) has been reported as a common phenomenon of lysogenic bacterial strains and occurs even in the absence of an external trigger. By sacrificing a small fraction for the good of all, SPI was shown to promote the fitness of bacterial communities and to contribute to horizontal transfer of genetic information. Generally, it is considered that SPI is triggered by sporadic DNA damage activating the host’s SOS response. However, various examples demonstrated that also alternative pathways maycause the activation of prophages. One of them includes specific counteraction of xenogeneicsilencers (XS). XS such as Lsr2 of $\textit{Mycobacterium tuberculosis}$ and H-NS of $\textit{Escherichia coli}$ aresmall nucleoid-associated proteins, which preferentially bind and silence AT-rich foreign DNA. Furthermore, it is suggested that XS mediate the stepwise acquisition of potentially useful genes and enable a mutual adaption of the host and the foreign element. In the present thesis, the SPI of CGP3, a cryptic prophage of the industrial relevant $\textit{Corynebacterium glutamicum}$ strain ATCC 13032, was investigated to examine prophage-host interactions, focusing on the impact of CGP3 on host physiology and the regulatory control of this element. In a first set of experiments, the dynamics of the SOS response and CGP3 induction were monitored by using promoter reporter fusions. Live-cell imaging of reporter strains in the microfluidic environment enabled to follow the fate of SOS and phage positive cells. This approach revealed that in ~63% of the cells an SOS response preceded prophage activation, whereas >30% of the cells displayed an SOS-independent CGP3 activation. SPI and CGP3 inducing experiments demonstrated that activation of CGP3 causes a growth arrest in all cells (or cell death), evincing that a small fraction of a $\textit{C. glutamicum}$ population is continuously lost due to SPI. This fitness burden was proven by a competitive growth experiment, where the prophage-free $\textit{C. glutamicum}$ strain MB001 showed a slight advantage in comparison to the wild type strain. Further on, comparative analysis revealed neither by phenotypic microarrays (>1100 conditions) nor by a long-term adaptive evolution experiment (>600 generations) significant disadvantages of strain MB001 regarding growth, genome stability and mutation frequencies. In previous studies, the prophage-encoded Lsr2-like protein CgpS was fished by a DNA affinity chromatography using an early phage promoter of CGP3. This work revealed that CgpS, in fact, inherits a crucial role as a silencer of cryptic prophage elements in $\ztextit{C. glutamicum}$. In particular, ChAP-Seq experiments and transcriptome analysis confirmed the binding of CgpS to AT-rich DNA regions and repression of phage genes. Beside the similar mode of action and the XS-typical domain organization, CgpS can complement a Δhns phenotype in $\textit{E. coli}$. Remarkably, bioinformatics analysis revealed that orthologues of CgpS/Lsr2 are present in several actinobacteriophages and occur even more likely in (predicted) temperate rather than in virulent phages. This finding emphasizes that XS-like systems may play important and so far overlooked roles in the interaction of bacteria and their phages. Besides maintaining a stable coexistence, they may also be exploited as weapon by phages in the arms-race of bacteria and phages. [...