Synchronisation<em> Caulobacter crescentus</em> Für die Untersuchung der Bakterienzellzyklus
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
Die bakterielle Zellzyklus steuert sowohl die Replikation des Genoms und die Teilung der Tochterzellen. Wichtig ist, wie Antibiotika-Resistenz ist eine wachsende Bedrohung der öffentlichen Gesundheit stellt der bakteriellen Zellzyklus eine ungenutzte Ziel für Antibiotika-Entwicklung.
In dem Bakterium Caulobacter crescentus, führt jede Zellzyklus in einer asymmetrischen Teilung, wodurch zwei Tochterzellen unterschiedlicher Schicksal (1A) 1,2. Eine Tochterzelle erbt eine Geißel und ist beweglich, während die andere Tochter erbt einen Stiel und sitzend. Eine integrierte genetische Schaltung steuert Zellzyklus und Zellschicksal von Transkriptionsregulation, Phospho-Signalisierung und regulierte Proteolyse 3. Zusätzlich Chromosomenreplikation und gleichzeitige Trennung Ausbeute Tochterzellen, die genau eine Kopie des Chromosoms 4 enthalten. Wichtig ist, dass diese beiden Zelltypen schnell durch collo getrennt werdenidal Silicateilchen Dichtezentrifugation im synchronisierbar NA1000 Stamm 5-7 ermöglicht die Isolierung der swarmer Zellen aus dem Rest der Population mit hohen Ausbeuten (1B). Isoliert swarmer Zellen dann synchron gehen durch asymmetrische Zellteilung. Hier haben wir ausführlich das Protokoll für die Synchronisierung von Caulobacter Belastung NA1000. Wir bieten Protokolle und gemeinsame Tipps zur Fehlerbehebung für Klein- und angelegte Synchronisationen. Das experimentelle Verfahren bietet ein leistungsfähiges Werkzeug, um die räumlich-zeitliche Kontrolle der Caulobacter Zellzyklus und Zellschicksal zu verhören.
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_…Disclosures
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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