Materials and Reagents

Plastic disposable dish (for BG-11 plates)
1.5 ml microtubes
Synechococcus elongatus PCC 7942
Escherichia coli K-12 W3110
Chloramphenicol (Nacalai tesque, catalog number: 06285 )
Glutaraldehyde solution (50% in water) (KANTO KAGAKU, catalog number: 17026-32 )
Tween 20 (KANTO KAGAKU, catalog number: 40350-02 )
Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: D5652 )
Liquid nitrogen
Trisodium citrate dihydrate (KANTO KAGAKU, catalog number: 37150-00 )
SYTOX Green Nucleic Acid Stain (5 mM solution in DMSO) (Thermo Fisher Scientific, catalog number: S7020 )
BG-11 liquid medium (Castenholz, 1988) (see Recipes)
BG-11 plates (see Recipes)
50x BG-11 stock (see Recipes)
1,000x Micro elements A6 (see Recipes)
1,000x K₂HPO₄ (see Recipes)
1,000x CaCl₂ (see Recipes)
1,000x Ammonium Iron(lll) citrate brown (see Recipes)
1,000x Na₂S₂O₃ (see Recipes)
1 M HEPES-KOH (pH 8.2) (see Recipes)
M9 medium (Sambrook et al., 1989) (see Recipes)
10x M9 salt (see Recipes)
PBS buffer (see Recipes)

Equipment

Plant growth chamber (TOMY, model: CLE-405 )
Growth chamber (YAMATO, model: IC602 )
Laboratory shaker (TAITEC, model: PERSONAL11 )
UV/Vis spectrophotometer (Shimadzu, model: UV-1800 )
Pharmaceutical refrigerator (Panasonic, model: MPR-3120CN-PJ )
Rotator (TAITEC, model: RT-50 )
FACS Calibur instrument (BD Biosciences, FACSCalibur^TM)
100 ml test tubes (for culturing)
Microtube centrifuge (TOMY, model: MX-107 )

Software

Analytical software: BD CellQuest Pro (Becton-Dickinson)

Procedure

Preparation of Synechococcus 7942 culture
1. Synechococcus 7942 is streaked and pre-incubated on BG-11 agar plates at 30 °C under continuous illumination (40 µE/m²/s) for 1 week in the plant growth chamber.
2. Cells from the plates are suspended in approximately 1 ml of BG-11 medium with high turbidity and diluted into 80 ml of BG-11 medium at an OD₇₅₀ of 0.05.
3. The culture is incubated at 30 °C under continuous illumination (40 µE/m²/s) with 2% CO₂ bubbling for 10 days.
4. The culture is then diluted to OD₇₅₀ = 0.2 with fresh BG-11 medium. After 18-h incubation under dark conditions at 30 °C with 2% CO₂ bubbling, the culture is transferred to light conditions (40 µE/m²/s) to restart cell growth.
5. A 1-ml aliquot of the cultured cells is used for the following assay (Procedure C, D and E).
Preparation of E. coli culture as a standard
1. E. coli is streaked and pre-incubated on M9 agar plates at 37 °C in the growth chamber.
2. Cells from the plates are suspended in approximately 1 ml of M9 medium with high turbidity and then diluted into 10 ml of M9 medium at an OD₆₀₀ of 0.2.
3. The culture is incubated at 30 °C with shaking for 4 h.
4. 10 µl of 20 mg/ml chloramphenicol is added to the culture (final concentration, 20 µg/ml).
5. After incubation with shaking for 2 h, 1 ml of cell culture is subjected to the following assay.
Fixation
1. To the 1-ml aliquots of cell culture, 10 µl of 0.5% Tween 20 and 20 µl of 50% glutaraldehyde are added (final conc. 0.005% and 1%, respectively), and incubated for 30 min at 4 °C with gentle agitation by a rotator.
2. The cells are centrifuged at 20,000 x g for 1 min and then washed with 1 ml of PBS, and the cell pellets were stored at -30 °C until further analysis.
DNA staining
1. Cell pellets were frozen with liquid N₂, and then thawed at room temperature.
  Note: This step is necessary for the permeation of SYTOX Green.
2. The cell pellets are resuspended in 50 µl of 10 µM SYTOX Green solution, which was diluted with 50 mM trisodium citrate (pH 8.0).
3. The samples are incubated overnight at 4 °C.
FACS analysis
1. The SYTOX Green-stained cells are subjected to FACS analysis. The stained cell suspension (5 µl) is diluted with 250 µl of PBS and immediately analyzed by FACS.
2. The cells could be selected by cell size and internal complexity by using forward (FSC) and side scatter (SSC) values. The SYTOX Green signal corresponds to the DNA amount, and is detected by the FL1 detector (laser: 488 nm, filter: 533/30 nm) (Figures 1 and 2).
  Note: The signal of Synechococcus 7942 cells can be identified based on chlorophyll autofluorescence using FL3 (laser: 488 nm, filter: 610/20 nm).
3. The major peak of the chloramphenicol-treated E. coli sample, which corresponds to 1 copy of the chromosome per E. coli cell, is extracted and used as a standard for estimating the chromosomal copy number.
Estimation of chromosomal copy number
The Synechococcus 7942 chromosome copy number (ca. 2.7 Mbp) is calculated based on the peak location of E. coli (ca. 4.7 Mbp) (Figure 3). The copy number of Synechococcus 7942 changes under our culture conditions (2-4 copies under dark and 3-8 copies under light conditions) (Watanabe et al. 2015) as reported previously (Binder and Chisholm, 1995; Griese et al., 2011).
Equation: Copy number of the Synechococcus 7942 chromosome = FL1 value_{Synechococcus} x 4.7 (Genome size in E. coli)/FL1 value_E.coli x 2.7 (Genome size in Synechococcus)

Representative data

Figure 1. DNA content profiles of chloramphenicol-treated E. coli cells. After culturing for 2 h in M9 medium with 20 µg/ml chloramphenicol, the cells are fixed and analyzed. A. Profiles of cell size (X axis: FSC-H) and DNA content (Y axis). B. Profiles of DNA content (X axis) and cell number (Y axis). For preparing the standard peak, which corresponds to single copy chromosome in E. coli, the major peak (asterisk) was extracted. The bottom panel represents the profiles obtained after extraction by gating.

Figure 2. The DNA content profiles of cells cultured in the dark (Top) and lag-phase Synechococcus 7942 cells (bottom) cultured in light conditions for 9 h. A. Profiles of cell size (X axis: FSC-H) and DNA content (Y axis). B. Profiles of DNA content (X axis) and cell number (Y axis).

Figure 3. DNA content profiles of cells cultured in the dark (thin line), lag-phase Synechococcus 7942 cells cultured in light conditions for 9 h (bold line), and the chloramphenicol-treated E. coli culture (thin red line). The copy number of the Synechococcus 7942 chromosome (ca. 2.7 Mbp) was calculated based on the peak location of E. coli (ca. 4.7 Mbp).

Recipes

BG-11 liquid medium (pH 8.2)/1 L
50x BG-11 stock 20 ml
1,000x Micro elements A6 1 ml
1,000x K₂HPO₄ 1 ml
1,000x CaCl₂ 1 ml
1,000x Ammonium Iron(lll) citrate brown 1 ml
1 M HEPES-KOH (pH 8.2) 20 ml
H₂O up to 1 L
Autoclave (A. C.) (120 °C, 20 min)
BG-11 plates (pH 8.2)/1 L
1. 2x BG-11 stock
  50x BG-11 stock 20 ml
  1,000x Micro elements A6 1 ml
  1,000x K₂HPO₄ 1 ml
  1,000x CaCl₂ 1 ml
  1,000x Ammonium Iron(lll) citrate brown 1 ml
  1,000x Na₂S₂O₃ 1 ml
  1 M HEPES-KOH (pH 8.2) 20 ml
  H₂O up to 500 ml
  A. C. (120 °C, 20 min)
2. 2x Agar stock
  Bacto agar (Nissui) 15 g
  H₂O 500 ml
  A. C. (120 °C, 20 min)
  After dissolve Recipe 2b, mix with Recipe 2a.
50x BG-11 stock, 1 L
NaNO₃ 75 g
MgSO₄·7H₂O 3.75 g
Citric acid 0.33 g
EDTA-3Na Salt 0.05 g
Na₂CO₃ 1.0 g
H₂O up to 1 L
A. C. (120 °C, 20 min)
1,000x Micro elements A6, 1 L
H₃BO₃ 2.86 g
MnCl₂·4H₂O 1.81 g
ZnSO₄·7H₂O 0.222 g
Na₂MoO₄·2H₂O 0.391 g
Co(NO₃)₂·6H₂O 0.0494 g
CuSO₄·5H₂O 0.08 g
H₂O up to 1 L
A. C. (120 °C, 20 min)
1,000x K₂HPO₄, 100 ml
K₂HPO₄ 3.0 g
H₂O up to 100 ml
A. C. (120 °C, 20 min)
1,000x CaCl₂, 100 ml
CaCl₂·2H₂O 3.6 g
H₂O up to 100 ml
A. C. (120 °C, 20 min)
1,000x Ammonium Iron(lll) citrate brown, 100 ml
Ammonium Iron(lll) citrate brown 0.6 g
H₂O up to 100 ml
A. C. (120 °C, 20 min)
1,000x Na₂S₂O₃, 100 ml
Na₂S₂O₃ 15.81 g
H₂O up to 100 ml
A. C. (120 °C, 20 min)
1 M HEPES-KOH (pH 8.2), 1 L
HEPES 238.3 g
pH adjusted to 8.2 with KOH
A. C. (120 °C, 20 min)
M9 medium, 1 L
H₂O (A. C.) 895 ml
10x M9 salt 100 ml
1 M MgSO₄ (A. C.) 1 ml
40% Glucose (A. C., 120 °C, 15 min) 5 ml
1% Thiamine-HCl (A. C.)1 ml
1M CaCl₂ (A. C.) 100 µl
10x M9 salt, 1 L
Na₂HPO₄ 60 g
KH₂PO₄ 30 g
NaCl 5 g
NH₄Cl 10 g
H₂O up to 1 L
A. C. (120 °C, 20 min)
PBS buffer
PBS powder 9.6 g
H₂O up to 1 L
A. C. (120 °C, 20 min)

Acknowledgments

This protocol was adapted from Watanabe et al. (2012). This work was supported in part by a Grants-in-Aid 25850056 from the Ministry of Education, Culture, Sports, Science and Technology of Japan to S. W.

References

Binder, B. J. and Chisholm, S. W. (1995). Cell cycle regulation in marine Synechococcus sp. strains. Appl Environ Microbiol 61(2): 708-717.
Castenholz, R. W. (1988). Culturing methods for cyanobacteria. Meth Enzymol 167: 68-93.
Griese, M., Lange, C. and Soppa, J. (2011). Ploidy in cyanobacteria. FEMS Microbiol Lett 323(2): 124-131.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Bacterial media, antibiotics, and bacterial strains. In: Molecular cloning. A laboratory manual. Cold Spring Harbor, A.1-13.
Watanabe, S., Ohbayashi, R., Shiwa, Y., Noda, A., Kanesaki, Y., Chibazakura, T. and Yoshikawa, H. (2012). Light-dependent and asynchronous replication of cyanobacterial multi-copy chromosomes.Mol Microbiol 83(4): 856-865.
Watanabe, S., Ohbayashi, R., Kanesaki, Y., Saito, N., Chibazakura, T., Soga, T. and Yoshikawa, H. (2015). Intensive DNA replication and metabolism during the lag phase in cyanobacteria. PLoS One 10(9): e0136800.