Name: synchronization of caulobacter crescentus for investigation of the bacterial cell cycle
Uploaded: 2015-04-08T15:00:00
Description: Watch this Scientific Journal Video about Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle at JoVE.com

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.

1. Large-scale Synchrony - Optimal for Western Blot, Microarray/RNA-Seq, and Other Material Intensive Assays
<ol>
	<li>From a freezer stock or a plate, grow a 5 ml&#160;O/N culture of strain NA1000 by shaking at 28 &#176;C in PYE medium.</li>
	<li>Inoculate 0.5 ml of the cells from step 1&#160;in 25 ml of M2G (Tables 1-2) and shake at 28 &#176;C until the culture reaches an OD600 between 0.5 and 0.6.</li>
	<li>Inoculate the cells into 1 L of M2G and shake at 28 &#176;C.</li>
	<li>Once OD600 reache...

     

<ol>
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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_...

The bacterial cell cycle controls both the replication of the genome and the division of daughter cells. Importantly, as antibiotic resistance is a growing threat to public health, the bacterial cell cycle presents an untapped target for antibiotic development.
In the bacterium Caulobacter crescentus, each cell cycle leads to an asymmetric division, yielding two daughter cells of different fates (Figure&#160;1A) 1,2. One daughter cell inherits a flagellum and is motile while the other daughter inherits a stalk and is sessile. An integrated genetic circuit controls cell cycle progression and cell fate by transcriptional regulation, phospho-signaling, and regulated proteolysis 3. In addition, chromosome replication and concurrent segregation yield daughter cells that contain exactly one copy of the chromosome 4. Importantly, these two cell types can be rapidly separated by colloidal silica particle density centrifugation in the synchronizable NA1000 strain 5-7 allowing the isolation of the swarmer cells from the rest of the population with high yields (Figure&#160;1B). Isolated swarmer cells then proceed synchronously through asymmetric cell division. Here, we detail the protocol used for synchronizing Caulobacter strain NA1000. We provide protocols and common troubleshooting tips for both large- and small-scale synchronizations. This experimental procedure provides a powerful tool to interrogate the spatiotemporal control of the Caulobacter cell cycle and cell fate.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PVP Coated Colloidal Silica (Percoll)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4937</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Colloidal Silica (Ludox AS-40)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">420840</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">JA10 Rotor</td>
			<td style="width:71px;">Beckman-Coulter</td>
			<td style="width:119px;">369687</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">JA20 Rotor</td>
			<td style="width:71px;">Beckman-Coulter</td>
			<td style="width:119px;">334831</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ferrous Sulfate Chelate Solution</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>F0518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">30 mL Centrifuge Tubes</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">8445</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Na2HPO4</td>
			<td style="width:71px;">EMD</td>
			<td style="width:119px;">SX0720-1</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">KH2PO4</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">BDH9268-500G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">NH4Cl</td>
			<td style="width:71px;">Amresco</td>
			<td style="width:119px;">0621-500g</td>
			<td></td>
		</tr>
	</tbody>
</table>

synchronization of caulobacter crescentus for investigation of the bacterial cell cycle

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.

Synchronization typically yields two bands of cells (Figure&#160;1B): the swarmer band, which has a higher density, and a stalked/predivisional cell band of lower density. To ensure efficient synchronization common controls include monitoring the OD600 and measuring the levels of CtrA protein by western blot at distinct cell cycle time points. The OD600 should increase by approximately 2 fold during the course of the cell cycle (Figure&#160;2). The western blot for ...

Watch this Scientific Journal Video about Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle at JoVE.com

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

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.

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

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 ...

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