In many bacterial species, viral DNA (prophage elements) may constitute a considerable fraction of the whole genome and may have detrimental effects on the growth and fitness of industrial strains. In this project, we focused on Corynebacterium glutamicum as one of the most important biotechnological platform organisms and on the fast-growing marine bacterium Vibrio natriegens representing an emerging model host for molecular biology and biotechnology. In RRO_PHAGE we successfully constructed several prophage-free variants of these strains. Final prophage-free strains proved as suitable platform strains for the engineering of small molecule or protein production and are by now frequently used platform strains for biotechnological applications.
Following on the question of how ‘foreign’/viral genetic material is tolerated by the host and how it is integrated into host regulatory networks, we systematically addressed the function of xenogeneic silencing (XS) proteins. In actinobacteria, Lsr2-like nucleoid-associated proteins function as XS of horizontally acquired genomic regions, including viral elements, virulence gene clusters in Mycobacterium tuberculosis, and genes involved in cryptic specialized metabolism in Streptomyces species. Consequently, a detailed mechanistic understanding of Lsr2 binding in vivo is relevant for the use as a potential drug target and for the identification of novel bioactive compounds. In our study, we followed an in vivo approach to investigate the rules underlying xenogeneic silencing and countersilencing of the Lsr2-like XS CgpS from Corynebacterium glutamicum. Our results demonstrated that CgpS distinguishes between self and foreign DNA by recognizing a distinct drop in GC profile in combination with a short, sequence-specific motif at the nucleation site. Following a synthetic counter-silencer approach, we studied the potential and constraints of transcription factors to counteract CgpS silencing, thereby facilitating the integration of new genetic traits into host regulatory networks. We further developed targeted counter-silencing approaches by implementing dCas9-based countersilencing providing an unprecedented opportunity for the targeted activation of XS target genes. The principle of XS was further harnessed for the establishment of inducible expression systems representing key modules in regulatory circuit design and metabolic engineering approaches.
Actinobacteria are well known as producers of a variety of bioactive compounds, including molecules with antibacterial, anti-fungal or anti-cancer activity. During phage isolation efforts conducted in this project, we noticed that viral infectios trigger antibiotic production in Streptomyces species. This led to the hypothesis that bacteria produce and secrete small secondary metabolites involved in the defense against viral infections at the multicellular level. Following this hypotheis, we showed that aminoglycosides, a well-known class of antibiotics produced by Streptomyces, are potent inhibitors of phage infection in widely divergent bacterial hosts. We demonstrated that aminoglycosides block an early step of the viral life cycle, prior to genome replication. Phage inhibition was also achieved using supernatants from natural aminoglycoside producers, indicating a broad physiological significance of the antiviral properties of aminoglycosides. Altogether, our study expands the knowledge of aminoglycoside functions, suggesting that aminoglycosides not only are used by their producers as toxic molecules against their bacterial competitors but also could provide protection against the threat of phage predation at the community level.
