Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.
The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.
细菌细胞周期控制的基因组中的两个复制和子细胞的分裂。重要的是,抗生素耐药性是一个日益严重的威胁到公众健康,细菌细胞周期呈现抗生素发展的尚未开发的目标。
在细菌柄杆菌新月 ,每个细胞周期导致一个不对称分裂,产生不同的命运( 图1A)1,2的两个子细胞。一个子细胞继承了鞭毛是能动的,而另一个女儿继承柄和无柄是。一个集成的基因电路控制细胞周期进程和细胞命运的转录调控,磷酸化信号,和监管蛋白水解3。此外,染色体复制和并发分流率的子细胞包含4号染色体只有一个拷贝。重要的是,这两种类型的细胞可迅速地通过胶体分离在可同步NA1000菌株5-7允许swarmer细胞从具有高产率( 图1B)的人口的其余部分隔离IDAL二氧化硅粒子密度离心。隔离swarmer细胞,然后通过不对称细胞分裂进行同步。在这里,我们详细介绍了协议用 于同步柄杆菌菌株NA1000。我们提供的协议,并为大规模和小规模的同步常见故障排除技巧。这个实验的过程提供了一个功能强大的工具来询问柄杆菌细胞周期和细胞命运的时空控制。
The bacterial cell cycle is a fundamental process in life and is important for the study of growth and as a target for next generation antibiotics. Here, we detailed the rapid synchronization procedures for C. crescentus NA1000, a model organism for the study of the bacterial cell cycle and asymmetric cell division. This method is amendable to western blot, gene expression profiling, and fluorescence microscopy assays to investigate the spatiotemporal regulation of the bacterial cell cycle.
<p class='jove_…The authors have nothing to disclose.
The authors thank members of the Shapiro lab and Erin Schrader for comments on the manuscript. The authors acknowledge financial support from: NIH postdoctoral fellowship F32 GM100732 to JMS and NIH grants R01 GM51426 and R01 GM32506 to LS.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
PVP Coated Colloidal Silica (Percoll) | Sigma-Aldrich | P4937 | |
Colloidal Silica (Ludox AS-40) | Sigma-Aldrich | 420840 | |
JA10 Rotor | Beckman-Coulter | 369687 | |
JA20 Rotor | Beckman-Coulter | 334831 | |
Ferrous Sulfate Chelate Solution | Sigma-Aldrich | F0518 | |
30 mL Centrifuge Tubes | Corning | 8445 | |
Na2HPO4 | EMD | SX0720-1 | |
KH2PO4 | VWR | BDH9268-500G | |
NH4Cl | Amresco | 0621-500g |