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 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 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.

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 cover slip, and image by phase microscopy. Confirm the presence of swarmer cells by visualizing rapidly swimming cells in the population.
5. Spin cells for 15 min at 7 k x g at 4 °C in a JA-10 rotor.
6. Discard the supernatant and add 180 ml of cold M2 (Tables 1-2) and gently resuspend all the cells using a serological pipet. Discard loosely pelleted cells; they are predivisional and stalked cells.
7. Add 60 ml of cold Colloidal silica solution (be sure to mix the Colloidal silica suspension well before adding to the cells) and mix the cell suspension well.
8. Pour the cell suspension into eight 30 ml tubes and spin for 30 min at 6.4 k x g at 4 °C in a JA-20 rotor.
   NOTE: One should see two distinct bands; the swarmer band is the lower band while stalked/predivisional cells are in the top band (Figure 1B).
9. Carefully aspirate the top band off and remove the liquid to ~1 cm above the swarmer band (the lower band).
10. (Critical) Using a Pasteur pipet, carefully remove the swarmer band and place into a clean tube. To wash away the Colloidal silica, top the tube off with cold M2 and spin for 10 min at 6.4 k x g at 4 °C in a JA-20 rotor.
11. Carefully discard the supernatant and resuspend the cells in 20 ml of cold M2 and spin for 10 min at 6.4 k x g at 4 °C in a JA-20 rotor.
12. Resuspend all the pellets into 30 ml of cold M2 and measure the OD₆₀₀ using a spectrophotometer and blank using cold M2 medium. Save 1 µl for phase imaging to check for swarmer cells; 90-95% of cells should be swarmers.
13. Spin down the cells for 5 min at 6.4 k x g at 4 °C in a JA-20 rotor. Resuspend cells in 28 °C M2G medium so that the A₆₀₀ is ~0.3-0.4 and begin shaking at 28 °C.
    NOTE: Typical yields are between 30 and 60mL of swarmer cell culture from 1L of unsynchronized cells.
14. Begin taking time points (wild type culture will take approximately 135-140 minutes to divide) ^8,9. At each time point, measure the OD₆₀₀. Check that the OD₆₀₀ after division is approximately 2X the initial OD₆₀₀.
15. For western blot or gene expression assays, remove 1mL aliquots of the culture at the desired time points, spin down at max speed in a tabletop centrifuge for 30 sec, rapidly decant or aspirate the medium, and flash freeze the cell pellet in liquid nitrogen. Store the cells at -80 °C until downstream analysis.

2. Small-scale Synchrony – Optimal for Microscopy

1. From a freezer stock or a plate, grow a 5 ml O/N culture shaking at 28 °C in M2G.
2. Dilute in 15 ml of M2G (Tables 1-2) and grow until mid-log (OD₆₀₀ = 0.5-0.6).
3. Spin at 6.4 k x g for 10 min at 4 °C in a JA-20 rotor, and resuspend in 1 ml cold M2 (Tables 1-2) and transfer to a 2 ml microcentrifuge tube.
4. Spin at 15 k x g for 3 min in a microcentrifuge tube to pellet cells, aspirate off the supernatant, put the pellet on ice, and resuspend in 900 µl of cold M2.
5. Add 900 µl of cold PVP coated colloidal silica and spin for 20 min at 15 k x g at 4 °C in a microcentrifuge tube.
6. (Critical) Aspirate or pipet off the top stalked/predivisional cell band and collect the bottom swarmer band into a new microcentrifuge tube.
7. Wash the swarmer cells two times in 1 ml of cold M2 while centrifuging at 15 k x g for 3 min.
8. Before the final spin, move the cells into a pre-chilled 1 ml glass test tube and measure the OD₆₀₀ of the cells compared to a blank of M2.
9. Resuspend the final cell pellet into 28 °C M2G at an OD between 0.3 – 0.4 and shake/roll cells at 28 °C.
   NOTE: Typical yields are between 2 and 4 ml of swarmer cell culture.
10. For microscopy experiments, at the desired time points place 1 µl of cells onto an M2G agarose pad for imaging.

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