Expanding the diversity of bacterial DNA partitioning: A CTP-independent ParABS system for plasmid partitioning in Streptomyces

The ATP- and CTP-dependent ParA-ParB-parS segrosome is a macromolecular complex that segregates chromosomes/plasmids in most bacterial species. CTP binding and hydrolysis enable ParB to slide along DNA and to bridge and condense DNA, thereby dictating the size and dynamics of the tripartite ParABS complex. Several other evolutionarily distinct systems can also segregate DNA, although the full diversity of bacterial DNA partition systems remains unknown. Here, we identify a CTP-independent ParABS system that maintains the conjugative plasmid SCP2 in the filamentous bacterium Streptomyces coelicolor. We demonstrate that an SCP2 ParB-like protein, ParT, loads onto DNA at an 18-bp parS site and diffuses away to the adjacent DNA despite lacking an apparent CTPase domain and detectable NTPase activity. We further show that parS DNA facilitates ParT transition from loading to a diffusing state, allowing ParT to accumulate on DNA, and that ParT activates the ATPase activity of its cognate partner protein, ParA. Additionally, we identify numerous structural homologs of ParT, suggesting that CTP-independent diffusion on DNA might be more common than previously recognized. Overall, our findings reveal a CTP-independent DNA translocation as an alternative and unexpected mechanism for assembling a bacterial DNA segregation complex and suggest that CTP binding and hydrolysis are not universal features of ParABS-like systems. Chromatin-immunoprecipitation with deep sequencing experiments (ChIP-seq) were performed on Streptomyces coelicolor strains grown in liquid cultures (see the supplementary Materials and Methods for the exact treatment that were applied to each strain).

依赖ATP与CTP的ParA-ParB-parS分离体（segrosome）是多数细菌物种中介导染色体与质粒分离的大分子复合物。CTP的结合与水解可促使ParB沿DNA滑动，并介导DNA的桥联与浓缩，进而调控三方ParABS复合物的规模与动态特性。尽管细菌DNA分配系统的完整多样性仍未完全探明，但已有多种演化起源迥异的其他系统同样可介导DNA分离。本研究鉴定出一种不依赖CTP的ParABS系统，该系统可在丝状细菌天蓝色链霉菌（Streptomyces coelicolor）中维持接合性质粒SCP2的稳定存续。我们证实，一种SCP2编码的ParB类蛋白ParT可在18 bp的parS位点结合于DNA，且尽管缺乏典型的CTP酶结构域与可检测的核苷三磷酸酶活性，仍可扩散至邻近DNA区域。我们进一步发现，parS DNA可推动ParT从结合状态向扩散状态转变，使ParT能够在DNA上富集，同时ParT可激活其同源配对蛋白ParA的ATP酶活性。此外，我们还鉴定出多个ParT的结构同源物，这表明不依赖CTP的DNA扩散机制可能比此前认知的更为普遍。综上，本研究的发现揭示了一种不依赖CTP的DNA转位机制，该机制可作为组装细菌DNA分离复合物的一种新颖且出乎意料的途径，并表明CTP的结合与水解并非ParABS类系统的通用特征。本研究对在液体培养基中培养的天蓝色链霉菌菌株开展了染色质免疫沉淀结合深度测序实验（ChIP-seq），具体处理方案详见补充材料与方法部分。