Fluorescence in situ Localization of Gene Expression Using a lacZ Reporter in the Heterocyst-forming Cyanobacterium Anabaena variabilis

Materials and Reagents

1. 1.7 ml Avant microtubes (MIDSCI, catalog number: AVSS1700 )
   
2. Aluminum foil
   
3. 0.22 µm filter (Thermo Fisher Scientific, Fisher Scientific, catalog number: 09-720-004 )
   
4. Microscope cover glass (Thermo Fisher Scientific, Fisher Scientific, catalog number: 12-545A )
   
5. Microscope slides (Thermo Fisher Scientific, Fisher Scientific, catalog number: 12-550-A3 )
   
6. BP830, an A. variabilis ATCC 29413 derivative, containing a pcnfR1:lacZ fusion (Pratte and Thiel, 2016)
   
7. Ammonium chloride (NH₄Cl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: A661-500 )
   
8. TES buffer (AG Scientific, catalog number: T-1050 )
   
9. DMSO (Dimethyl sulfoxide) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP231-1 )
   
10. Millipore water
    
11. Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: M63-500 )
    
12. Calcium chloride dihydrate (CaCl₂·2H₂O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP510-500 )
    
13. Sodium chloride (NaCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: S271-1 )
    
14. Potassium phosphate dibasic anhydrous (K₂HPO₄) (Thermo Fisher Scientific, Fisher Scientific, catalog number: P288-500 )
    
15. Manganese chloride tetrahydrate (MnCl₂·4H₂O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: M87-100 )
    
16. Sodium molybdate dihydrate (Na₂MoO₄·2H₂O) (Sigma-Aldrich, catalog number: M1003 )
    
17. Zinc sulfate heptahydrate (ZnSO₄·7H₂O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: Z76-500 )
    
18. Copper(II) sulfate pentahydrate (CuSO₄·5H₂O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP346-500 )
    
19. Boric acid (H₃BO₃) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP168-500 )
    
20. Cobaltous chloride hexahydrate (CoCl₂·6H₂O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: C371-100 )
    
21. Potassium hydroxide (KOH) (Thermo Fisher Scientific, Fisher Scientific, catalog number: P250-500 )
    
22. Ethylenediaminetetraacetic acid (Na₂EDTA·2H₂O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP120-1 )
    
23. Ferrous sulfate heptahydrate (FeSO₄·7H₂O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: I146-500 )
    
24. 25% glutaraldehyde solution (Sigma-Aldrich, catalog number: G5882 )
    
25. ImaGene Green^TM C₁₂FDG lacZ Gene Expression Kit (Thermo Fisher Scientific, catalog number: I2904 )
    
26. p-Phenylenediamine (Sigma-Aldrich, catalog number: P-6001 )
    
27. Glycerol (Thermo Fisher Scientific, catalog number: G33-1 )
    
28. Sodium bicarbonate (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP328-500 )
    
29. Allen and Arnon (AA) medium (Allen and Arnon, 1955): (AA/8 = 8-fold dilution of AA) (see Recipes)
1. AA/8 media
   
2. AA Phosphate stock solution
   
3. K₂HPO₄ stock solution
   
4. Microelements stock solution
   
5. AA FeEDTA solution
   
30. 0.04% glutaraldehyde solution (see Recipes)
    
31. 100 µM 5-dodecanoylaminefluorescein di-β-d galactopyranoside (C12-FDG) in 25% DMSO (see Recipes)
    
32. 0.5 M carbonate buffer (see Recipes)
    
33. Antifade solution (see Recipes)
    

Equipment

1. 125-ml glass flasks (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10-040D )
   
2. Plugs for 125-ml flasks (Thermo Fisher Scientific, Fisher Scientific, catalog number: 1412740C )
   
3. Shaker (set at 170 rpm) (Eppendorf, New Brunswick^TM, model: Innova^® 2100 )
   
4. Centrifuge (Eppendorf, model: 5415D )*
   
5. Incubator (waterbath) (set at 37 °C) (Polyscience, model: 2LS-M )*
   
6. Environmental chamber set at 30 °C with 70% humidity and light
   
7. Spectrophotometer (Bibby Scientific, JENWAY, model: 7300 )
   
8. Zeiss Confocal LSM700 using a Plan-Apochromat 63x/1.4 Oil DIC M27 objective (Carl Zeiss, model: LSM700)
   

*Note: These products have been discontinued.

Procedure

1. Culture growth
   1. Starting from colonies on agar plates, inoculate strains of A. variabilis or Anabaena sp. PCC 7120 containing lacZ fusions (constructed as described in Pratte and Thiel, 2016) in AA/8 containing 5 mM NH₄Cl and 10 mM TES, pH 7.2 and antibiotics, when necessary. Shake cultures at 170 rpm at 30 °C in 100-120 μE/m² s light and allow to grow for about 10 generations (3-4 days).
      
   2. Two days prior to nitrogen-stepdown, dilute cultures 1:100 in AA/8 containing 5 mM NH₄Cl and 10 mM TES, pH 7.2 and allow them to continue growing at 30 °C with shaking and light to an OD₇₂₀ of 0.1-0.2. Cyanobacteria should be actively growing for several generations so that they differentiate heterocysts well after the removal of fixed nitrogen. Consistency in growth conditions improves the reproducibility of β-galactosidase production in the cultures.
      
   3. Wash actively growing (step A2) cyanobacterial cultures 3 x in AA/8 to remove nitrogen. Resuspend cultures to an OD₇₂₀ of < 0.1 in 125-ml flask containing 50 ml AA/8 with (+N) or without (-N) 5 mM NH₄Cl and 10 mM TES, pH 7.2 and grow for 24 h with light and shaking. Check cultures for heterocysts prior to starting in situ localization assays.
      
      
2. In situ localization
   
   1. Spin down 2-5 ml culture in 1.7 ml Eppendorf tube for 5 min at 16,000 x g in microfuge.
      
   2. Wash cells twice with 500 µl water to remove growth medium.
      
   3. Fix cells in 500 µl 0.04% glutaraldehyde at 25 °C for 15 min.
      
   4. Centrifuge cells for 1 min at 16,000 x g in microfuge and remove glutaraldehyde solution.
      
   5. Wash pellet twice with 500 µl water to remove residual glutaraldehyde.
      
   6. Resuspend pellet in 30 µl of substrate - 100 µM 5-dodecanoylaminefluorescein di-β-d galactopyranosine (C12-FDG) in 25% DMSO and incubate in the dark at 37 °C for 30 min.
      
   7. Centrifuge cells for 1 min at 16,000 x g in microfuge and remove substrate.
      
   8. Wash pellet twice with 500 µl water to remove residual substrate.
      Note: This step is important to achieve low background fluorescence.
      
   9. Resuspend pellet in 20 µl of antifade solution to keep the fluorescence stable. Keep cells in the dark until visualizing on microscope. Proceed immediately to imaging.
      
   10. Add ~2 µl of prepared cells to microscope slide with cover slip and visualize on a Zeiss Confocal LSM700 using a plan Apochromat 63x/1.4 Oil DIC M27 objective. Expression of lacZ in cells was visualized using excitation (488 nm) and emission (400-557 nm) wavelengths (from an argon ion laser) specific to detect fluorescein fluorescence, while cyanobacterial autofluorescence was visualized using excitation (561 nm) and emission (565-700 nm) wavelengths to detect cyanobacterial phycobiliprotein fluorescence. Typically we observe hundreds of filaments and then choose fields with sufficient representative filaments for imaging.
       

Data analysis

Long, planar cyanobacterial filaments on a glass microscope slide under a cover slip are identified in the samples using bright-field microscopy with a plan Apochromat 63x/1.4 Oil DIC M27 objective. Bright- field images are obtained using the transmitted light channel during a 30 cycle, time-series acquisition. Images of phycobiliprotein autofluorescence of the same filaments are acquired by excitation using 561 nm irradiation from an argon ion laser and visualization at 565-700 nm using a 30 cycle, time-series acquisition. The gain is set at 600 and the focus is adjusted slightly to optimize phycobiliprotein autofluorescence emitted from the cells during a live, continuous fast scan. Typically, vegetative cells show higher levels of phycobiliprotein autofluorescence than heterocysts. Fluorescein, measuring lacZ expression, is excited using 488 nm irradiation from an argon laser and visualized at 400-557 nm using a 30 cycle, time-series acquisition. The gain to detect fluorescein is typically set at 800, but can be adjusted to detect lower levels of fluorescence. Cells emitting lower levels of fluorescence need a higher gain to visualize the fluorescence, whereas higher levels of fluorescence can be seen at lower gains. Images are saved and then converted to TIFF files for final analysis of the level and cell-type specific expression of lacZ (fluorescein) in the experimental versus the control strains. Examples of typical images are shown in Figure 1.
 

Figure 1. In situ localization of lacZ expression in an A. variabilis FD strain containing a cnfR1:lacZ reporter (BP830) (Pratte and Thiel, 2016) grown aerobically for 24 h after nitrogen depletion (-N) or with fixed nitrogen (+N). Arrows indicate representative heterocysts. Left panels: light micrographs; Middle panels: red fluorescence from photosynthetic pigments in cyanobacteria; Right panels: fluorescein fluorescence from cleavage of 5-dodecanoyl-fluorescein-β-D-galacto-pyranoside by β-galactosidase.

Notes

1. Wash the cells treated with C12-FDG thoroughly to remove as much of the substrate as possible because it can lead to high background fluorescence.
   
2. Protect cells from the light after they have been treated with C12-FDG, including when they are on the microscope slide.
   
3. Use an anti-fade solution to prevent bleaching of the fluorescein by the excitation light.
   
4. If C12-FDG does not work well in your cells, try unmodified FDG or other FDG derivatives: 5-pentafluorobenzoylamino-fluorescein, C8-FDG, or 5-chloromethylfluorescein (http://www.mobitec.de/probes/docs/sections/1002.pdf).
   
5. If you prefer red fluorescence you can try resorufin or C8-resorufin labeled β-d-galactopyranosides (http://www.mobitec.de/probes/docs/sections/1002.pdf).
   

Recipes

1. Allen and Arnon medium (AA) (diluted 8-fold [AA/8])
   Note: Anabaena variabilis is grown in an eight-fold dilution of Allen and Arnon medium (Allen and Arnon, 1955).
1. AA/8 media
   3.1 ml AA-Phosphate stock solution
   0.8 ml K₂HPO₄ stock solution
   996.0 ml Millipore water
   Aliquot 50 ml in 125-ml flasks with plugs and aluminum foil covering, and then autoclave for 30 min at 121 °C
   
2. AA Phosphate stock solution
   500 ml 4% MgSO₄·7H₂O (final concentration 1%)
   500 ml 1.2% CaCl₂·2H₂O (final concentration 0.3%)
   500 ml 3.8% NaCl (final concentration 0.95%)
   500 ml microelements stock solution
   
3. K₂HPO₄ stock solution
   28.0 g K₂HPO₄
   500.0 ml Millipore water
   
4. Microelements stock solution
   160.0 ml AA Fe-EDTA solution
   360 mg MnCl₂·4H₂O
   35 mg Na₂MoO₄·2H₂O
   44.0 mg ZnSO₄·7H₂O
   15.8 mg CuSO₄·5H₂O
   572.0 mg H₃BO₃
   8.0 mg CoCl₂·6H₂O
   1,090.0 ml Millipore water
   
5. AA FeEDTA solution
2. 0.04% glutaraldehyde solution
   40 µl 1% glutaraldehyde solution
   960 µl Millipore water
   (1% glutaraldehyde solution: 40 µl [25% glutaraldehyde solution] added 960 µl [Millipore water])
   
3. 100 µM 5-dodecanoylaminefluorescein di-β-d galactopyranoside (C12-FDG) in 25% DMSO
   5 µl 20 mM C12-FDG (supplied in ImaGene Green^TM C₁₂FDG lacZ Gene Expression Kit)
   250 µl 100% DMSO
   745 µl sterile Millipore water
   
4. 0.5 M carbonate buffer pH 8.0
   5.42  sodium bicarbonate
   9 ml Millipore water
   Adjust pH to 8.0 and bring volume to 10 ml with Millipore water
   
5. Antifade solution
   Dissolve 30 mg p-Phenylenediamine in 4 ml of sterile Millipore water
   Add 6.0 ml glycerol
   Add 1.0 ml 0.5 M carbonate buffer (pH 8.0)
   Filter through a 0.22-µm filter to remove any undissolved chemical and store in 0.5 ml aliquots at -80 °C in the dark
   

Acknowledgments

This protocol was based on an earlier protocol for Bacillus subtilis (Bylund et al., 1994) that was modified for use in cyanobacteria. Support for this research was provided by National Science Foundation grant MCB-1052241.

References

1. Allen, M. B. and Arnon, D. I. (1955). Studies on nitrogen-fixing blue-green algae. I. Growth and nitrogen fixation by Anabaena cylindrica Lemm. Plant Physiol 30(4): 366-372.
   
2. Arlt, M. J., Born, W. and Fuchs, B. (2012). Improved visualization of lung metastases at single cell resolution in mice by combined in-situ perfusion of lung tissue and X-Gal staining of lacZ-tagged tumor cells. J Vis Exp (66): e4162.
   
3. Bylund, J. E., Zhang, L., Haines, M. A., Higgins, M. L. and Piggot, P. J. (1994). Analysis by fluorescence microscopy of the development of compartment-specific gene expression during sporulation of Bacillus subtilis. J Bacteriol 176(10): 2898-2905.
   
4. Chung, J. D., Conner, S. and Stephanopoulos, G. (1995). Flow cytometric study of differentiating cultures of Bacillus subtilis. Cytometry 20(4): 324-333.
   
5. Currier, T. C. and Wolk, C. P. (1979). Characteristics of Anabaena variabilis influencing plaque formation by cyanophage N-1. J Bacteriol 139(1): 88-92.
   
6. Griffith, K. L. and Wolf, R. E. (2002). Measuring β-galactosidase activity in bacteria: cell growth, permeabilization, and enzyme assays in 96-well arrays. Biochem Biophy Res Co 290: 397-402.
   
7. Kumar, K., Mella-Herrera, R. A. and Golden, J. W. (2010). Cyanobacterial heterocysts. Cold Spring Harb Perspect Biol 2(4): a000315.
   
8. Ma, P., Mori, T., Zhao, C., Thiel, T. and Johnson, C. H. (2016). Evolution of KaiC-dependent timekeepers: A proto-circadian timing mechanism confers adaptive fitness in the purple bacterium Rhodopseudomonas palustris. PLoS Genet 12(3): e1005922.
   
9. Maldener, I. and Muro-Pastor, A. M. (2010). Cyanobacterial Heterocysts. eLS. John Wiley & Sons Ltd, Chichester.
   
10. Miao, F., Todd, P. and Kompala, D. S. (1993). A single-cell assay of β-galactosidase in recombinant Escherichia coli using flow cytometry. Biotechnol Bioeng 42: 708-715.
    
11. Plovins, A., Alvarez, A. M., Ibañez, M., Molina, M. and Nombela, C. (1994). Use of fluorescein-di-β-D-galactopyranoside (FDG) and C12-FDG as substrates for β-galactosidase detection by flow cytometry in animal, bacterial, and yeast cells. Appl Environ Microbiol 60: 4638-4641.
    
12. Pratte, B. S. and Thiel, T. (2016). Homologous regulators, CnfR1 and CnfR2, activate expression of two distinct nitrogenase gene clusters in the filamentous cyanobacterium Anabaena variabilis ATCC 29413. Mol Microbiol 100(6): 1096-1109.
    
13. Silhavy, T. J., Casadaban, M. J., Shuman, H. A. and Beckwith, J. R. (1976). Conversion of β-galactosidase to a membrane-bound state by gene fusion. Proc Natl Acad Sci U S A 73(10): 3423-3427.
    
14. Thiel, T., Lyons, E. M., Erker, J. C. and Ernst, A. (1995). A second nitrogenase in vegetative cells of a heterocyst-forming cyanobacterium. Proc Natl Acad Sci U S A 92(20): 9358-9362.
    
15. Zhang, Y. Z., Naleway, J. J., Larison, K. D., Huang, Z. J. and Haugland, R. P. (1991). Detecting lacZ gene expression in living cells with new lipophilic, fluorogenic β-galactosidase substrates. FASEB J 5(15): 3108-3113.