Synkronisering av<em> Caulobacter Crescentus</em> For etterforskning av bakteriecellen Cycle
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 bakterielle cellesyklus styrer både replikasjon av genomet, og delingen av datterceller. Viktigere, som antibiotikaresistens er en voksende trussel mot folkehelsen, presenterer bakterienes cellesyklusen en uutnyttet mål for antibiotika utvikling.
I bakterien Caulobacter crescentus, fører hver celle syklus til en asymmetrisk divisjon har resultert i to datterceller med forskjellige skjebner (Figur 1a) 1,2. En datter celle arver en flagellum og er bevegelige mens den andre datter arver en stilk og er fastsittende. En integrert genetisk krets styrer cellecyklusprogresjonen og celle skjebne ved transkripsjonsregulering, fosfor-signalering, og regulert proteolyse tre. I tillegg kromosomreplikasjon og samtidige segregering avkastning datterceller som inneholder nøyaktig én kopi av kromosom 4. Viktigere, kan disse to celletyper hurtig separeres ved Colloidal silisiumdioksyd partikkeltetthet sentrifugering i synchronizable NA1000 stamme 5-7 som tillater isolering av Swarmer cellene fra resten av befolkningen med høye utbytter (figur 1B). Isolert Swarmer cellene deretter fortsette synkront gjennom asymmetrisk celledeling. Her, vi detalj protokollen som brukes for å synkronisere Caulobacter belastning NA1000. Vi gir protokoller og vanlige feilsøkingstips for både stor og liten skala synkroniseringer. Dette eksperimentelle prosedyren gir et kraftig verktøy for å avhøre den spatiotemporal kontroll av Caulobacter cellesyklusen og celle skjebne.
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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