Synkronisering av<em> Caulobacter Crescentus</em> För utredning av bakteriecellcykeln
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
Den bakteriecellcykeln styr både replikation av genomet och uppdelningen av dotterceller. Viktigt eftersom antibiotikaresistens är ett växande hot mot folkhälsan, presenbakteriecellcykeln en outnyttjad mål för antibiotikautveckling.
I bakterien Caulobacter crescentus, leder varje cellcykel till en asymmetrisk delning, vilket gav två dotterceller av olika öden (Figur 1A) 1,2. Ett dottercell ärver en flagellum och är rörliga, medan den andra dottern ärver en stjälk och är oskaftade. En integrerad genetiska kretsen styr cellcykelprogression och cell öde genom transkriptionsreglering, fosfor-signalering, och reglerad proteolys 3. Dessutom kromosomreplikation och samtidiga segregering avkastningsdotterceller som innehåller exakt en kopia av kromosom 4. Viktigt kan dessa två celltyper snabbt separeras genom ColloIdal kiseldioxid partikeltätheten centrifugering i synchronizable NA1000 stam 5-7 tillåter isolering av swarmer cellerna från resten av befolkningen med hög avkastning (Figur 1B). Isolerad swarmer celler sedan fortsätta synkront genom asymmetrisk celldelning. Här, vi detalj protokollet som används för att synkronisera Caulobacter stam NA1000. Vi tillhandahåller protokoll och gemensamma felsökningstips för både grov- och småskaliga synkroniseringar. Denna experimentella procedur ger ett kraftfullt verktyg för att förhöra den Spatiotemporal kontroll av Caulobacter cellcykeln och cell öde.
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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