Proper chromosome segregation is essential in all living organisms. In Caulobacter crescentus, the ParA–ParB–parS system is required for proper chromosome segregation and cell viability. The bacterial centromere-like parS DNA locus is the first to be segregated following chromosome replication. parS is bound by ParB protein, which in turn interacts with ParA to partition the ParB-parS nucleoprotein complex to each daughter cell. Here, we investigated the genome-wide distribution of ParB on the Caulobacter chromosome using a combination of in vivo chromatin immunoprecipitation (ChIP-seq) and in vitro DNA affinity purification with deep sequencing (IDAP-seq). We confirmed two previously identified parS sites and discovered at least three more sites that cluster ∼8 kb from the origin of replication. We showed that Caulobacter ParB nucleates at parS sites and associates non-specifically with ∼10 kb flanking DNA to form a high-order nucleoprotein complex on the left chromosomal arm. Lastly, using transposon mutagenesis coupled with deep sequencing (Tn-seq), we identified a ∼500 kb region surrounding the native parS cluster that is tolerable to the insertion of a second parS cluster without severely affecting cell viability. Our results demonstrate that the genomic distribution of parS sites is highly restricted and is crucial for chromosome segregation in Caulobacter.

中文翻译：

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新月形杆菌染色体上着丝粒结合蛋白ParB的允许区

正确的染色体分离对于所有活生物都是必不可少的。在柄杆菌新月，对位ParB- PARS需要系统正确的染色体分离和细胞生存力。细菌着丝粒样parS DNA基因座是染色体复制后第一个分离的基因座。parS与ParB蛋白结合，而ParB蛋白又与ParA相互作用，将ParB- parS核蛋白复合物分配给每个子细胞。这里，我们调查ParB的基因组上的，广泛分布柄杆菌使用的组合染色体体内染色质免疫沉淀（ChIP-SEQ）和体外使用深度测序（IDAP-seq）进行DNA亲和纯化。我们确认了两个先前确定的parS位点，并发现了至少三个另外的距复制起点约8 kb的位点。我们发现，柄杆菌在ParB核化PARS与〜10 kb的侧翼DNA位点和同事非特异性，形成高阶核蛋白复合左手臂染色体。最后，使用转座子诱变结合深度测序（Tn-seq），我们确定了围绕自然parS簇的〜500 kb区域，该区域可忍受第二个parS簇的插入而不会严重影响细胞生存力。我们的结果证明了parS的基因组分布位点受到严格限制，对杆状细菌的染色体分离至关重要。