Senkronizasyon<em> Caulobacter crescentus</em> Bakteriyel Hücre Döngüsü Soruşturma
Summary
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.
Abstract
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.
Introduction
bakteriyel hücre döngüsü genomunun çoğaltma ve kızı hücrelerinin bölünmesini kontrol eder. Antibiyotik direnci halk sağlığı için büyüyen bir tehdit olarak önemlisi, bakteri hücre döngüsü antibiyotik gelişimi için bir hedef kullanılmayan sunuyor.
Bakteri Caulobacter crescentus, her bir hücre döngüsü Farklı kaderi (Şekil 1A) 1,2 iki kız hücre sağlar, asimetrik bölünmesi yol açar. Bir yavru hücre, bir kamçı miras ve diğer kızı sap miras ve sapsız ise hareketlidir. Entegre devre genetik transkripsiyon regülasyonu, fosfor-sinyalizasyon ve düzenlenmiş proteoliz 3 hücre döngüsü ilerlemesini ve hücre kaderi kontrol eder. Buna ek olarak, kromozom replikasyonu ve eşzamanlı ayrımcılık verim kızı hücreleri kromozom 4 tam bir kopyasını içeren. Önemli bir şekilde, bu iki hücre tipi hızla collo ile ayrılabiliryüksek verim (Şekil 1B) ile nüfusun geri kalanından swarmer hücrelerinin izolasyonu sağlayan synchronizable NA1000 suşu 5-7 idal silika parçacık yoğunluğu santrifüj. Hücrelerin swarmer İzole sonra asimetrik hücre bölünmesi yoluyla eşzamanlı devam edin. Burada, biz detay protokolü Caulobacter suşu NA1000 senkronize etmek için kullanılır. Biz protokolleri ve büyük ve küçük ölçekli her iki eşitleme için ortak sorun giderme ipuçları sağlar. Bu deneysel prosedür Caulobacter hücre döngüsü ve hücre kaderinin kontrolünü zamanmekansal sorgulamak için güçlü bir araç sağlar.
Protocol
Representative Results
Discussion
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_…Divulgations
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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 |
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