Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich, Jülich, Germany.
Institut für Molekulare Physiologie, Mikrobiologie und Weinforschung, Johannes-Gutenberg-Universität Mainz, Mainz, Germany.
mBio. 2020 Feb 4;11(1):e02273-19. doi: 10.1128/mBio.02273-19.
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.
Lsr2 样核相关蛋白在放线菌中作为水平获得的基因组区域的异种沉默（XS）发挥着重要作用。在这项研究中，我们系统地分析了 Lsr2 样蛋白 CgpS 沉默和反沉默的约束条件。
全基因组分析表明，CgpS 与转录起始位点（TSS）附近 GC 特征明显下降的区域结合，但也确定了一个核蛋白复合物成核位点具有多个 A/T 步的代表性 motif。特定转录因子（TFs）的结合可能会反对 XS 活性，导致反沉默。通过在各种 CgpS 靶启动子中插入效应子响应 TF 的操纵子位点，采用合成反沉默方法，实现了靶基因的激活，从而在 TF 结合时增加启动子活性。报告基因构建体的分析表明，当 TF 操纵子位点插入到 CgpS 最大覆盖位置时，反沉默最大。该原理在合成翘板开关中得到了实施，该开关对效应子可用性具有稳健且可逆的响应，突出了生物技术应用的潜力。
总之，我们的研究结果提供了全面的见解，说明 Lsr2 沉默和反沉默如何塑造这个在医学和生物技术上相关的细菌门的进化网络扩展。在放线菌中，Lsr2 样核相关蛋白作为水平获得的基因组区域的异种沉默（XS），包括病毒元件、毒力基因簇和隐匿特殊代谢基因在物种中发挥作用。因此，Lsr2 结合的详细机制理解作为潜在的药物靶点和识别新的生物活性化合物是相关的。在这里，我们采用了一种系统生物学方法来研究 Lsr2 样 XS CgpS 的异种沉默和反沉默的规则，该方法来自。我们的结果表明，CgpS 通过识别与核蛋白复合物成核位点的短序列特异性 motif 相结合的 GC 特征明显下降来区分自身和外来物。通过合成反沉默方法，我们研究了转录因子对抗 CgpS 沉默的潜力和限制，从而促进了新遗传特征整合到宿主调控网络中。
