A Spectrofluorophotometrical Method Based on Fura-2-AM Probe to Determine Cytosolic Ca²⁺ Level in Pseudomonas syringae Complex Bacterial Cells

Materials and Reagents

1. Inoculation loops 10 µl (Laboindustria S.P.A., catalog number: 21131 )

2. Petri dish Ø 90 (Laboindustria S.P.A., catalog number: 21050 )

3. Pipette tips (Mettler Toledo, catalog numbers: 17007956, 17007952 )

4. High clarity polypropylene (PP) conical centrifuge tube 50 ml (Falcon, catalog number: 352070 )

5. NIR Quartz SUPRASIL 300 Rectangular Macro Cell with Lid, volume 3.5 ml (PerkinElmer, catalog number: B0631015 )

6. PIREX Media bottles, graduated, Corning, 500 ml (VWR, PIREX, catalog number: 1395-500 )

7. Pseudomonas savastanoi pv. savastanoi (Psav) DAPP-PG 722 strain (Moretti et al., 2014), stored at -80 °C in 15% glycerol

8. Pseudomonas syringae pv. tomato (Pto) DC3000 strain (Gizjen, 2008), stored at -80 °C in 15% glycerol

9. MilliQ double distilled water

10. Tris base (Sigma-Aldrich, catalog number: T1503 ); prepare 0.12 M aqueous solution, adjust pH to 8.0 with 1 M HCl, autoclave and store at room temperature

11. Fura 2-AM (Sigma-Aldrich, catalog number: F0888 ); prepare 2 mM solution in DMSO in aliquots of 50 µl and freeze at -20 °C. Store at -20 °C, wrap it in aluminum foil to avoid photodegradation. Avoid repeated freezing and thawing of the aliquots

12. EGTA (Sigma-Aldrich, catalog number: E3889 ); prepare a 0.5 M aqueous stock solution, adjust pH to 8.0 with 0.5 M NaOH, autoclave and store at room temperature. Prepare a 2 mM aqueous solution from the stock solution

13. Sodium chloride (Sigma-Aldrich, catalog number: S7653 )

14. Tryptone (Sigma-Aldrich, Millipore, catalog number: T7293 )

15. Yeast extract (Sigma-Aldrich, catalog number: Y1625 )

16. Calcium chloride (Sigma-Aldrich, catalog number: 449709 ); prepare a 50 mM aqueous solution and autoclave at 121 °C for 20 min

17. Hank’s Buffered Salt Solution (HBSS) buffer; prepare 1 L aqueous solution with 8.18 g/L NaCl, 0.4 g/L KCl (Sigma-Aldrich, catalog number: P9541 ), 5.96 g/L HEPES (Sigma-Aldrich, catalog number: H3375 ), adjust pH to 7.4 and autoclave at 121 °C for 20 min

18. EDTA (Sigma-Aldrich, catalog number: E9884 ); prepare a 0.5 M aqueous stock solution, adjust pH to 8.0 with NaOH, autoclave at 121 °C for 20 min and store at room temperature. Prepare a 0.1 mM aqueous solution from the stock solution

19. Triton X-100 (Sigma-Aldrich, catalog number: X100 ); prepare a 1% aqueous solution and autoclave at 121 °C for 20 min

20. Luria Bertani (LB) medium (see Recipes)

Equipment

1. 1 L measuring cylinder (DWK Life Sciences, catalog number: 21 390 54 08 )

2. BRAND magnetic stirring bar (Sigma-Aldrich, Aldrich, catalog number: BR137630 )

3. Magnetic stirrer (Heidolph MR 2000, catalog number: 200-505-20000-00 )

4. Eppendorf Research Plus G pippetes (Sigma-Aldrich, Eppendorf, catalog number: EP3123000918 )

5. Autoclave (EXAPro, Lequeux, catalog number: P80602001 )

6. Shaking incubator SI500 (Stuart, catalog number: FV-79520-00 )

7. Eppendorf^® Centrifuge 5804R (Sigma-Aldrich, Sigma, catalog number: EP022628146 )

8. Heratherm^TM Incubator (Thermo Scientific, catalog number: 51028112 )

9. LS-50B Luminescence Spectrometer (PerkinElmer, catalog number: 17931 )

10. Laminar flow cabinet Gelaire BSB 6A (Gelaire)

Software

1. FL WinLab Software, version 3 (PerkinElmer)

2. Prism 8 (GraphPad, https://www.graphpad.com/scientific-software/prism/)

Procedure

1. Bacterial growth (Day 1: ~15 min followed by 16 h incubation)

   Note: Do not use any antibiotics in the growth medium, because it can interfere with the experiments. Do not use more than 10% of maximum tube volume to ensure growth of the bacterium. Conduct all steps under sterile conditions.

   1. Prepare a fresh culture of Psav DAPP-PG 722 or Pto DC3000 strain by inoculating bacterial cells, scraped with a inoculation loop from a 15% glycerol stock at -80 °C, on Petri dish containing LB agar medium and incubate for 16 h at 27 °C.

      
      

2. Bacterial growth (Day 2: ~15 min followed by 16 h incubation)

   Note: Do not use any antibiotics in the growth medium, because it can interfere with the experiments. Do not use more than 10% of maximum tube volume to ensure growth of the bacterium. Conduct all steps under sterile conditions.

   1. Inoculate a loop of bacterial cells into a 50 ml tube containing 5 ml LB broth. Incubate the tubes in the shaking incubator for 16 h at 27 °C and 200 rpm (until the OD₆₆₀ = 0.8).

      
      

3. Cell preparation with Fura 2-AM (Day 3: ~3.5 h)

   Note: Do not centrifuge the bacterial suspension at >16,000 × g to avoid any cell damage. Wrap the tube containing the Fura 2-AM with aluminum foil or work in the dark to avoid photodegradation of the probe.

   1. Collect the bacterial cells from the 5 ml cell culture by centrifugation (15,585 × g, 3 min) at room temperature (RT).

   2. Discard the supernatant and resuspend the cells in 5 ml of sterile 0.12 mM Tris HCl (pH 8.0).

   3. Adjust the bacterial suspension to 1 × 10⁸ CFU ml^-1 by bringing the OD₆₆₀ at 0.06.

   4. Collect again 10 ml of this bacterial suspension by centrifugation (15,585 × g, 3 min) at RT.

   5. Discard the supernatant and resuspend the cell pellet in 5 ml 0.12 mM Tris HCl (pH 8.0) (= 5 × 10⁹ CFU), add 0.5 ml of a 2 mM EGTA solution (pH 8.0), and incubate at 25 °C for 5 min in an air incubator to render the bacteria receptive to the Ca²⁺ probe.

   6. Add 20 µl of 2 mM CaCl₂ solution to quench the EGTA.

   7. Pellet the cells by centrifuging them at 15,585 × g for 3 min at RT.

   8. Discard the supernatant and resuspend the cells in 5 ml HBSS supplemented with 2 µl of 2 mM Fura 2-AM stock solution. Note, add the Fura 2-AM freshly to the HBSS prior to usage from a fresh stock solution.

   9. Incubate this cell suspension in HBSS + Fura2-AM in the incubator without shaking at 25 °C for 2 h.

   10. Pellet the cells by centrifuging them at 15,585 × g for 3 min at RT.

   11. Add 5 ml HBSS (without the dye), resuspend the cells and incubate for 1 min at RT.

       
       

4. Measurement of the Ca²⁺ levels (Day 3: ~1.5 h)

   Note: Set the LS-50B luminescence spectrometer with FL WinLab Software using the User’s Guide.

   1. Pour 1 ml of the cell suspension in the NIR Quartz SUPRASIL 300 Rectangular Macro Cell containing the BRAND magnetic stirring bar which reduces the swirling and guarantees the correct reading of the samples.

   2. Place the Rectangular Macro Cell in the LS-50B luminescence spectrometer, wait until the Traffic Light bottom becomes green to initialize the spectrometer (Figure 3A) and click on it to start the measurement. Similar results could be obtained with analogous spectrometer.

   3. Wait about 50 s (a time useful for the signal stabilization) and add 30-240 µl of a 50 mM CaCl₂ solution to reach a final concentration ranging from 0.5 to 4 mM (Figure 4).

   4. Add 1 ml of 1% Triton X-100 to disrupt the cells, record the time and wait until the signal stabilizes. Triton X-100 is added at the end of the measurement because it allows one to determine the maximum Ca²⁺ level in the system.

   5. Add 1 ml of 0.1 mM EDTA (pH 8.0) to chelate the Ca²⁺ ions in solution and record the exact time appearing in the display as it is important for the final Rmin determination, wait until the signal stabilizes and press the Traffic Light buttom (Figure 3A), make sure it becomes red to acquire the signals and save the value. By adding EDTA the Ca²⁺ ions are chelated allowing one to determine the minimum Ca²⁺ level in the system.

      Note: Triton X-100 and EGTA are added at the end of the determination so the measurement is performed with intact cells. If the cells were not intact, the minimum and maximum Ca²⁺ value could not be determined.

   6. To automatically determine the Ca²⁺ concentration according to the formula of Grynkiewicz et al. (1985) [see also Data analysis], set the calibration values Rmin and Rmax (Figure 3B) in the Calibration Tab layout of the FL WinLab Software, as reported in the User’s Guide.

      Note: Without this software it is necessary to determine the Rmin and Rmax for each sample as indicated in point 1 of Data analysis.

   7. Press the Convert to [ion] button (Figure 3B) to convert the raw dataset of the signals into Ca²⁺ ion concentration dataset using the calibration values set before and save the generated file. Without FL WinLab Software Ca²⁺ ion concentration has to be calculated using the formula of Grynkiewicz et al. (1985).

      
      

      
      Figure 3. FL WinLab Software. A. The Traffic light button allows to start and stop the analysis and describes the status of the instrument. B. The Calibration Tab layout permits to manage the raw data and the Convert to [ion] button to visualize the data (from the FL WinLab User’s Guide, PerkinElmer, Inc., UK).

      
      

   8. Calculate the difference between the Ca²⁺ ions present before and after CaCl₂ addition (Figure 4).

      
      

      
      Figure 4. Example of a plot generated by FL WinLab Software used to calculate Δ[Ca²⁺]_c before and after CaCl₂ addition. The presence of the peaks in the trace is due to the change of the wavelength that LS-50B Luminescence Spectrometer shifts every two seconds. Therefore, the calculation of the Ca²⁺ concentration has to be performed considering the baseline (dotted line). After about 50 s, when the signal is stabilized, the CaCl₂ is added (red arrow) and the Δ[Ca²⁺]_c is calculated between the highest Ca²⁺ concentration value reached in the baseline (blue arrow) and the value when the CaCl₂ is added (red arrow).

      
      

   9. Incubate the cells in HBSS buffer supplemented with Fura 2-AM (basal condition) or in HBSS buffer supplemented with 2 mM Fura 2-AM and different carbon sources (e.g., 5 mM glucose, fructose or sucrose), 50 μM ATP, or other conditions of interest. Of note, one should optimally add these compounds only after 50s from the start of the measurement (Figure 5).

Data analysis

1. The free intracellular Ca²⁺ concentration was calculated using the formula of Grynkiewicz et al. (1985):

   
   

   

   
   

   K_d: Dissociation constant for the Ca-Fura-2 complex

   β: Fluorescence intensity ratio with an excitation wavelength of 380 nm, with and without Ca²⁺

   R: Ratio of fluorescence intensities obtained

   R_min and R_max: Fluorescence intensity ratio excited at 340 and 380 nm, in the absence (min) and in the presence (max) of Ca²⁺, respectively

2. To validate the results, multiple biological replicates are necessary. Here, ten independent experiments were carried out and the data points and error bars (Figure 5) represent the mean and standard error, respectively. In Psav DAPP-PG 722 cells it has been observed that under basal conditions (i.e., HBSS buffer) an increase in external Ca²⁺ results in an increase in the cytosolic Ca²⁺ concentrations. The cytosolic Ca²⁺ concentrations rapidly increase in response to external Ca²⁺ concentration in the medium (Figure 5). This trend was suppressed when different carbon sources (glucose, fructose or sucrose) or ATP were added in a combination with Ca²⁺ (Figure 5).

   
   

   
   Figure 5. An increase of cytosolic Ca²⁺ levels in Pseudomonas savastanoi pv. savastanoi (Psav) DAPP-PG 722. Psav bacterial cells incubated in HBSS medium alone (basal condition, closed squares) or in the presence of glucose, fructose, sucrose, indole 3 acetic acid (IAA) or tryptophan (open circles) over a concentration range of extracellular calcium chloride. Each point represents the mean of 10 independent experiments ± SE. From Moretti et al. (2019).

Recipe

1. Luria Bertani (LB) medium (1 L)

   1. Add 800 ml of MilliQ water into a 1 L measuring cylinder

   2. Put the measuring cylinder on magnetic stirrer with a magnetic rod

   3. Add 10 g of tryptone, 5 g of yeast extract and 5 g of NaCl

   4. Add MilliQ water to 1 L

   5. Pour 250 ml medium into four 500 ml PYREX^® glass bottles

   6. Autoclave at 121 °C for 20 min

Acknowledgments

This work was financially supported by DSA3 research funds “Fondo di base” to the co-authors CM, RB and CAP. This method has been used in Moretti et al. (2019).

Competing interests

The authors declare no conflict-of-interest and have no competing financial interests.

Informed consent was obtained from all individual participants included in the study.

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