تزامن<em> العنيقاء Crescentus</em> للتحقيق في دورة الخلية البكتيرية
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
تسيطر على دورة الخلية البكتيرية كل من تكرار الجينوم وتقسيم الخلايا الوليدة. الأهم من ذلك، كما مقاومة المضادات الحيوية تشكل تهديدا متزايدا على الصحة العامة، تقدم دورة الخلية البكتيرية هدفا غير مستغلة للتنمية المضادات الحيوية.
في بكتيريا العنيقاء crescentus، كل دورة الخلية يؤدي إلى تقسيم غير المتماثلة، مما أسفر عن خليتين ابنة مصائر مختلفة (الشكل 1A) 1،2. خلية ابنة واحدة يرث السوط وهي متحركة في حين أن ابنة أخرى يرث ساق وهي اطئة. دائرة الوراثية متكاملة تسيطر تقدم دورة الخلية ومصير الخلية عن طريق تنظيم النسخي، الفوسفات، مما يشير، والتحلل البروتيني المنظم 3. وبالإضافة إلى ذلك، وتكرار كروموسوم وخلايا ابنة العائد الفصل المتزامنة التي تحتوي بالضبط نسخة واحدة من كروموسوم 4. الأهم من ذلك، هذه أنواع الخلايا اثنين يمكن فصلها بسرعة عن طريق كولوايدال السيليكا الجسيمات كثافة الطرد المركزي في سلالة synchronizable NA1000 5-7 السماح للعزل الخلايا مخابئ عن بقية السكان مع عوائد عالية (الشكل 1B). عزل مخابئ خلايا ثم المضي قدما بشكل متزامن من خلال انقسام الخلايا غير المتماثلة. هنا، ونحن بالتفصيل بروتوكول يستخدم لمزامنة العنيقاء سلالة NA1000. ونحن نقدم البروتوكولات ونصائح استكشاف الأخطاء وإصلاحها المشتركة لكلا الكبيرة والصغيرة الحجم تزامن. يوفر هذا الإجراء التجريبي أداة قوية لاستجواب السيطرة الزمانية المكانية من دورة الخلية العنيقاء ومصير الخلية.
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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