Синхронизация<em> Caulobacter Crescentus</em> По расследованию бактериального клеточного цикла
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
Бактериальный клеточный цикл контролирует как репликация генома и деление дочерних клеток. Важно отметить, что, как устойчивость к антибиотикам является растущей угрозой для здоровья населения, бактериальный клеточный цикл представляет собой нетронутый цель для развития антибиотиков.
В бактерией Caulobacter crescentus, каждый клеточного цикла приводит к асимметричному разделения, получая две дочерние клетки разными судьбами (рис 1А) в 1,2 раза. Один дочерняя клетка наследует жгутик и подвижны, а другая дочь наследует стебель и сидячие. Интегрированный генетический контур регулирует прогрессию клеточного цикла и клеточной судьбы по регуляции транскрипции, фосфо-сигнализации, и регулируемого протеолиза 3. Кроме того, репликация хромосом и одновременно дают сегрегация дочерние клетки, которые содержат ровно одну копию хромосомы 4. Важно, что эти два типа клеток может быть быстро отделен от да КоллоIdal частиц кремнезема плотность центрифугирования в синхронизируемый NA1000 штамма 5-7, позволяющей изоляцию Swarmer клеток от остальной части населения с высокими выходами (рис 1б). Изолированные Swarmer клеток затем приступить синхронно через асимметричных клеточных делений. Здесь мы подробно протокол, используемый для синхронизации Caulobacter деформации NA1000. Мы предоставляем протоколы и общие советы по устранению неполадок для большого и малого масштаба синхронизации. Это экспериментальная процедура представляет собой мощный инструмент допросить пространственно-временной контроль клеточного цикла Caulobacter и судьбы клеток.
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 |
References
- McAdams, H. H., Shapiro, L. System-level design of bacterial cell cycle control. FEBS Lett. 583, 3984-3991 (2009).
- McAdams, H. H., Shapiro, L. The architecture and conservation pattern of whole-cell control circuitry. J. Mol. Biol. 409, 28-35 (2011).
- McAdams, H. H., Shapiro, L. A bacterial cell-cycle regulatory network operating in time and space. Science. 301, 1874-1877 (2003).
- Ptacin, J. L., Shapiro, L. Initiating bacterial mitosis: understanding the mechanism of ParA-mediated chromosome segregation. Cell Cycle. 9, 4033-4034 (2010).
- Evinger, M., Agabian, N. Envelope-associated nucleoid from Caulobacter crescentus stalked and swarmer cells. J. Bacteriol. 132, 294-301 (1977).
- Tsai, J. W., Alley, M. R. Proteolysis of the Caulobacter McpA chemoreceptor is cell cycle regulated by a ClpX-dependent pathway. J. Bacteriol. 183, 5001-5007 (2001).
- Marks, M. E., et al. The genetic basis of laboratory adaptation in Caulobacter crescentus. J. Bacteriol. 192, 3678-3688 (2010).
- Ely, B. Genetics of Caulobacter crescentus. Methods Enzymol. 204, 372-384 (1991).
- Williams, B., Bhat, N., Chien, P., Shapiro, L. ClpXP and ClpAP proteolytic activity on divisome substrates is differentially regulated following the Caulobacter asymmetric cell division. Mol. Microbiol. 93, 853-866 (2014).
- Quon, K. C., Yang, B., Domian, I. J., Shapiro, L., Marczynski, G. T. Negative control of bacterial DNA replication by a cell cycle regulatory protein that binds at the chromosome origin. Proc. Natl. Acad. Sci. U. S. A. 95, 120-125 (1998).
- Laub, M. T., Chen, S. L., Shapiro, L., McAdams, H. H. Genes directly controlled by CtrA, a master regulator of the Caulobacter cell cycle. Proc. Natl. Acad. Sci. U. S. A. 99, 4632-4637 (2002).
- Quon, K. C., Marczynski, G. T., Shapiro, L. Cell cycle control by an essential bacterial two-component signal transduction protein. Cell. 84, 83-93 (1996).
- Ferullo, D. J., Cooper, D. L., Moore, H. R., Lovett, S. T. Cell cycle synchronization of Escherichia coli using the stringent response, with fluorescence labeling assays for DNA content and replication. Methods. 48, 8-13 (2009).
- Degnen, S. T., Newton, A. Chromosome replication during development in Caulobacter crescentus. J. Mol. Biol. 64, 671-680 (1972).
- Bates, D., et al. The Escherichia coli baby cell column: a novel cell synchronization method provides new insight into the bacterial cell cycle. Mol. Microbiol. 57, 380-391 (2005).
- Abel, S., et al. Bi-modal distribution of the second messenger c-di-GMP controls cell fate and asymmetry during the caulobacter cell cycle. PLoS Genet. 9, e1003744 (2013).
- Johnson, R. C., Ely, B. Isolation of spontaneously derived mutants of Caulobacter crescentus. Genetics. 86, 25-32 (1977).
- Britos, L., et al. Regulatory response to carbon starvation in Caulobacter crescentus. PLoS One. 6, e18179 (2011).
- Boutte, C. C., Crosson, S. The complex logic of stringent response regulation in Caulobacter crescentus: starvation signalling in an oligotrophic environment. Mol. Microbiol. 80, 695-714 (2011).
