Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell 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

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 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 ....

Protocol

1. Large-scale Synchrony - Optimal for Western Blot, Microarray/RNA-Seq, and Other Material Intensive Assays

1. From a freezer stock or a plate, grow a 5 ml O/N culture of strain NA1000 by shaking at 28 °C in PYE medium.
2. Inoculate 0.5 ml of the cells from step 1 in 25 ml of M2G (Tables 1-2) and shake at 28 °C until the culture reaches an OD₆₀₀ between 0.5 and 0.6.
3. Inoculate the cells into 1 L of M2G and shake at 28 °C.
4. Once OD₆₀₀ reaches 0.5 to 0.6, confirm the presence of swarmer cells using liquid mounted phase microscopy. Spot 1 µl of cells on a glass slide, cover with a....

Results

Synchronization typically yields two bands of cells (Figure 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 2). The western blot for the cell c.......

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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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