SMC progressively aligns chromosomal arms in Caulobacter crescentus but is antagonized by convergent transcription

The Structural Maintenance of Chromosomes (SMC) complex plays an important role in chromosome organization and segregation in most living organisms. In Caulobacter crescentus, SMC is required to align the left and the right arms of the chromosome that run in parallel down the long axis of the cell. However, the mechanism of SMC-mediated alignment of chromosomal arms remains elusive. Here, using genome-wide methods and microscopy of single cells, we show that Caulobacter SMC is recruited to the centromeric parS site and that SMC-mediated arm alignment depends on the chromosome partitioning protein ParB. We provide evidence that SMC likely tethers the parS-proximal regions of the chromosomal arms together, promoting arm alignment. Furthermore, we show that highly-transcribed genes near parS that are oriented against SMC translocation disrupt arm alignment, suggesting that head-on transcription interferes with SMC translocation. Our results demonstrate a tight interdependence of bacterial chromosome organization and global patterns of transcription. Overall design: Caulobacter crescentus were grown as described previously in Skerker et al., 2005 (PMID: 16176121). When appropriate, media were supplemented with antibiotics at the following concentrations (liquid/solid media) [ug/ml]): chloramphenicol (1/2), kanamycin (5/25), oxytetracycline (1/2), spectinomycin (25/100). Synchronizations were performed on mid-exponential phase cells using Percoll (GE Healthcare) and density gradient centrifugation as described previously in Jones et al., 2001 (PMID: 11283290). After synchronization, swarmer cells were fixed formadehyde for chromosome conformation capture and Hi-C analysis. For antibiotic treatments, swarmer cells were incubated with 25 ug/mL rifampicin (final concentration) for 30 minutes before fixing with formadehyde.染色体结构维持（Structural Maintenance of Chromosomes, SMC）复合物在绝大多数活体细胞的染色体组织与分离过程中发挥关键作用。在新月柄杆菌（Caulobacter crescentus）中，SMC复合物负责使沿细胞长轴平行延伸的染色体左右臂完成对齐。然而，SMC介导的染色体臂对齐机制至今仍未阐明。本研究通过全基因组方法与单细胞显微成像技术，证实新月柄杆菌的SMC复合物被招募至着丝粒parS位点，且SMC介导的染色体臂对齐过程依赖于染色体分离蛋白ParB。本研究提供证据表明，SMC复合物或可通过将染色体臂上靠近parS的区域相互锚定，从而促进臂对齐过程。此外，本研究发现，parS位点附近定向与SMC易位方向相反的高转录基因会破坏染色体臂对齐，这提示反向转录会干扰SMC的易位过程。本研究结果证实，细菌染色体组织与全局转录模式之间存在紧密的相互依存关系。 整体实验设计：新月柄杆菌的培养参照Skerker等人2005年发表的方法（PMID: 16176121）。必要时，培养基中需添加抗生素，浓度如下（液体培养基/固体培养基，单位：μg/ml）：氯霉素（1/2）、卡那霉素（5/25）、土霉素（1/2）、壮观霉素（25/100）。细胞同步化采用Percoll（GE Healthcare）密度梯度离心法，操作参照Jones等人2001年发表的方法（PMID: 11283290），同步化对象为指数中期生长期细胞。同步化完成后，游动细胞使用甲醛固定以进行染色体构象捕获与Hi-C分析。对于抗生素处理组，游动细胞需先以终浓度25 μg/ml的利福平孵育30分钟，再使用甲醛固定。