Deciphering the Rules Underlying Xenogeneic Silencing and Counter-Silencing of Lsr2-like Proteins Using CgpS of Corynebacterium glutamicum as a Model.

Lsr2-like nucleoid-associated proteins play an important role as xenogeneic silencers (XS) of horizontally acquired genomic regions in actinobacteria. In this study, we systematically analyzed the constraints underlying silencing and counter-silencing of the Lsr2-like protein CgpS in Genome-wide analysis revealed binding of CgpS to regions featuring a distinct drop in GC profile close to the transcription start site (TSS) but also identified an overrepresented motif with multiple A/T steps at the nucleation site of the nucleoprotein complex. Binding of specific transcription factors (TFs) may oppose XS activity, leading to counter-silencing. Following a synthetic counter-silencing approach, target gene activation was realized by inserting operator sites of an effector-responsive TF within various CgpS target promoters, resulting in increased promoter activity upon TF binding. Analysis of reporter constructs revealed maximal counter-silencing when the TF operator site was inserted at the position of maximal CgpS coverage. This principle was implemented in a synthetic toggle switch, which features a robust and reversible response to effector availability, highlighting the potential for biotechnological applications. Together, our results provide comprehensive insights into how Lsr2 silencing and counter-silencing shape evolutionary network expansion in this medically and biotechnologically relevant bacterial phylum. In actinobacteria, Lsr2-like nucleoid-associated proteins function as xenogeneic silencers (XS) of horizontally acquired genomic regions, including viral elements, virulence gene clusters in , and genes involved in cryptic specialized metabolism in species. Consequently, a detailed mechanistic understanding of Lsr2 binding is relevant as a potential drug target and for the identification of novel bioactive compounds. Here, we followed an approach to investigate the rules underlying xenogeneic silencing and counter-silencing of the Lsr2-like XS CgpS from Our results demonstrated that CgpS distinguishes between self and foreign 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.