Abstract
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 in vivo constraints underlying silencing and counter-silencing of the Lsr2-like protein CgpS in Corynebacterium glutamicum 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.IMPORTANCE In actinobacteria, Lsr2-like nucleoid-associated proteins function as xenogeneic silencers (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 as a potential drug target and for the identification of novel bioactive compounds. Here, we followed an in vivo approach to investigate the rules underlying xenogeneic silencing and counter-silencing of the Lsr2-like XS CgpS from Corynebacterium glutamicum 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.
Highlights
Lsr2-like nucleoid-associated proteins play an important role as xenogeneic silencers (XS) of horizontally acquired genomic regions in actinobacteria
Recent genome-wide profiling studies revealed that the Lsr2-like xenogeneic silencer CgpS preferentially binds to AT-rich DNA sequences in the genome of C. glutamicum ATCC 13032 [4]
An overlay of the GC profiles of all 35 CgpS target promoters located within the prophage element CGP3 revealed a high degree of similarity with a distinct drop in GC content matching the position of maximal CgpS coverage (Fig. 1A)
Summary
Lsr2-like nucleoid-associated proteins play an important role as xenogeneic silencers (XS) of horizontally acquired genomic regions in actinobacteria. 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. CRISPR-Cas and restriction modification systems are nuclease-based defense mechanisms enabling the recognition and targeted degradation of invading DNA [10,11,12] In contrast to these destructive immune systems, xenogeneic silencing enables the tolerance of foreign DNA and fosters the acquisition of novel genetic material into the host chromosome [13]. The recent study by Will et al emphasizes that the principle of H-NS xenogeneic silencing and counter-silencing provides a certain degree of flexibility, fostering evolutionary network expansion [33]
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