Sincronizzazione di<em> Caulobacter crescentus</em> Per Indagine del ciclo cellulare batterica
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
Il ciclo cellulare batterica controlla sia la replicazione del genoma e la divisione di cellule figlie. È importante sottolineare che, come la resistenza agli antibiotici è una crescente minaccia per la salute pubblica, il ciclo cellulare batterica presenta un obiettivo non sfruttata per lo sviluppo di antibiotici.
In batterio Caulobacter crescentus, ogni ciclo cellulare porta ad una divisione asimmetrica, ottenendo due cellule figlie di sorti diverse (Figura 1A) 1,2. Una cellula figlia eredita un flagello ed è mobile, mentre l'altra figlia eredita un gambo ed è sessile. Un circuito genetico integrato controlla la progressione del ciclo cellulare e destino cellulare da regolazione trascrizionale, fosfo-segnalazione, e proteolisi regolata 3. Inoltre, la replicazione dei cromosomi e cellule figlie rendimento segregazione simultanee che contengono esattamente una copia del cromosoma 4. È importante sottolineare che questi due tipi di cellule possono essere rapidamente separati da colloIdal particella di silice centrifugazione densità nel ceppo sincronizzabile NA1000 5-7 consente l'isolamento delle cellule swarmer dal resto della popolazione con rese elevate (Figura 1B). Isolato swarmer cellule quindi procedere in modo sincrono attraverso la divisione cellulare asimmetrica. Qui, abbiamo dettaglio il protocollo usato per la sincronizzazione Caulobacter ceppo NA1000. Forniamo protocolli e risoluzione dei problemi comuni sia larga e piccola scala sincronizzazioni. Questa procedura sperimentale fornisce un potente strumento per interrogare il controllo spazio-temporale del ciclo cellulare Caulobacter e il destino delle cellule.
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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