Sincronização de<em> Caulobacter crescentus</em> Para Investigação do Ciclo Celular Bacteriana
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
O ciclo celular bacteriana controla a replicação do genoma e a divisão de células filhas. É importante salientar, como a resistência aos antibióticos é uma ameaça crescente para a saúde pública, o ciclo celular bacteriana apresenta um alvo inexplorado para o desenvolvimento de antibióticos.
No bactéria Caulobacter crescentus, cada ciclo celular conduz a uma divisão assimétrica, obtendo-se duas células filhas de destinos diferentes (Figura 1A) 1,2. Uma célula filha herda um flagelo e é móvel, enquanto a outra filha herda uma haste e é séssil. Um circuito genético integrado controla a progressão do ciclo celular e destino celular por regulação da transcrição, fosfo-sinalização, e proteólise regulamentado 3. Além disso, a replicação do cromossoma e células filhas segregação de rendimento concomitantes que contêm uma cópia exacta do cromossoma 4. É importante notar que estes dois tipos de células podem ser rapidamente separados por colloidal de partículas de sílica de centrifugação de densidade na estirpe sincronizável NA1000 5-7 permitindo o isolamento das células swarmer do resto da população com rendimentos elevados (Figura 1B). Isolado swarmer células em seguida, proceder de forma síncrona através da divisão celular assimétrica. Aqui, nós detalhe o protocolo utilizado para a sincronização de Caulobacter NA1000 estirpe. Nós fornecemos protocolos e dicas de solução de problemas comuns para ambos de grande e pequena escala sincronizações. Este procedimento experimental proporciona uma ferramenta poderosa para interrogar o controlo espacial e temporal da Caulobacter ciclo celular e o destino celular.
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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