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.
細菌の細胞周期は、ゲノムの複製と娘細胞の分裂の両方を制御する。抗生物質耐性は、公衆の健康に成長している脅威であるとして重要なのは、細菌の細胞周期は、抗生物質開発のための未開発の目標を提示します。
細菌カウロバクターcrescentusでは、各細胞周期の異なる運命( 図1A)1,2の2つの娘細胞を生じる、非対称分裂に導く。 One娘細胞は鞭毛を継承し、他の娘は茎を継承し、固着されている間運動性である。集積回路の遺伝子は、転写調節、リン酸化シグナル伝達、およびタンパク質分解規制3ことによって、細胞周期の進行および細胞の運命を制御する。また、染色体複製と同時分離は、第4染色体の正確に一つのコピーを含む娘細胞が得られる。重要なことに、これらの2つの細胞型は急速COLLOによって分離することができる高収率( 図1B)を有する集団の残りからswarmer細胞の単離を可能にする同期可能NA1000株5-7 idalシリカ粒子の密度遠心分離。細胞swarmer分離された後、非対称細胞分裂を通じて同期的に進む。ここでは、詳細プロトコルは、 カウロバクター株 NA1000を同期させるために使用される。私たちは、プロトコルおよび大規模および小規模の両方の同期のための一般的なトラブルシューティングのヒントを提供する。この実験手順は、 カウロバクター細胞周期と細胞の運命の時空間制御を調べるための強力なツールを提供します。
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 |