Optimized Protocol for the Incorporation of FDAA (HADA Labeling) for in situ Labeling of Peptidoglycan

Materials and Reagents

1. Sterile Petri dishes 92 × 16 mm with cams (SARSTEDT, catalog number: 82.1473)
2. Pipette tips 10 µl, 200 µl and 1,000 µl (STARLAB, catalog numbers: S1111-3700, S1113-1700, S1111-6701)
3. 1.5 ml micro-tubes (SARSTEDT, catalog number: 72.690.001)
4. Sterile serological pipettes (5, 10 and 25 ml) (SARSTEDT, catalog numbers: 86.1253.001, 86.1254.001 and 86.1685.001)
5. Sterile 50 ml CELLSTAR tubes with blue screw cap (Greiner BIO-ONE, catalog number: 227261)
6. Concentric Luer-Lok Syringe 50 ml (BD Plastipak, BD Luer Lok, catalog number: 10636531)
7. 0.22 µm syringe filter (PES membrane, sterile) (STARLAB, catalog number: E4780-1226)
8. Diagnostic microscopic slides (12 wells, 5.2 mm numbered) (Thermo Scientific, catalog number: 10028210)
9. Microscope slides SuperFrost plus (25 × 75 × 1 mm) (R. Langenbrinck Labor + Medizintechnik, catalog number: 03-0060)
10. Cover glasses, square (22 × 22 mm, thickness 1.5) (VWR, catalog number: 631-0125)
11. Semi-micro cuvettes (polystyrene, 10 × 4 × 45 mm) (SARSTEDT, catalog number: 67.742)
12. Milli-Q quality water (ddH₂O) and distilled water
13. Peptone from casein, enzymatic digest (Sigma-Aldrich, catalog number: 82303)
14. Peptone from soybean, enzymatic digest (Fluka Analytical, catalog number: 90765)
15. Sodium chloride (NaCl) (VWR, catalog number: 27810.295)
16. Di-Potassium hydrogen phosphate (K₂HPO₄) (VWR, catalog number: 26931.263)
17. D-Glucose (anhydrous) (Melford, catalog number: G1400)
18. Bacto Agar (BD Diagnostics, catalog number: 214030)
19. Bacto Tryptone (BD Diagnostics, catalog number: 211699)
20. Yeast extract powder (Ohly KAT, catalog number: FllOHLYKAT)
21. FDAA 7-hydroxycoumarincarbonylamino-D-alanine (HADA) provided by Michael S. VanNieuwenhze, Indiana University (Kuru et al., 2015) 
22. Dimethylsulfoxide anhydrous (DMSO) (Invitrogen, catalog number: D12345)
23. Citric acid (anhydrous) (Sigma-Aldrich, catalog number: C2404)
24. Sodium hydroxide (NaOH) (VWR, catalog number: 28245.298)
25. 10× PBS buffer with 0.017 M KH₂PO₄, 0.05 M Na₂HPO₄, 1.5 M NaCl, pH 7.4 (phosphate saline) (Lonza Walkersville INC, AccuGENE, catalog number: 51226)
26. 16% Paraformaldehyde (formaldehyde) aqueous solution (Electron Microscopy Science, catalog number: 15710-S)
27. SB Molecular Biology Grade Agarose (Severn Biotech LtD., catalog number: 30 10 50)
28. Tryptic soy broth (TSB) and Tryptic soy agar (TSA) (see Recipes)
29. Miller Luria-Bertani (LB) medium and LB agar (see Recipes)
30. HADA stock solution (see Recipes)
31. 10× Sodium citrate buffer, pH 2.25 (see Recipes)
32. 1× sodium citrate buffer, pH 3.0 (see Recipes)
33. 1× Phosphate buffered saline pH 7.4 (PBS) (see Recipes)
34. 3% Paraformaldehyde solution (see Recipes)
35. Agar slides for microscopic imaging (see Recipes)
    

Equipment

1. Sterile, flexible and disposable Inoculating loops (VWR, catalog number: 612935P)
2. Glass beaker, 600 ml and 1,000 ml (PYREX, catalog numbers: 1000/18D and 1000/22D, respectively)
3. Premium single Cell Analytical Balance (Kern, model: ABT 120-4NM)
4. IKA RH basic 2 magnetic stirrer (IKA, catalog number: 0003339002)
5. pH meter (Cole-Parmer, Jenway, model: 3510, catalog number: 351001)
6. Pipettes (Gilson, catalog numbers: F167300 and F167500)
7. Ultra Low Temperature Freezer VIP^TM Series -86 °C (SANYO, catalog number: MDF-U73V)
8. Comfort TP 1410 table-high, under worktop fridge (Liebherr, catalog number: 4016803034339)
9. Vortex (IKA, model: Minishaker MS2)
10. Microwave MS106 0.6 FT (Matsui)
11. 50 L Laboratory Incubator with Digital Control (GenLab incubator: INC/50/DIG)
12. Microcentrifuge accuSpin Micro 17R, refrigerated (Fisher Scientific, catalog number: 11526873)
13. Nikon Eclipse Ti microscope (Nikon Plan Fluor × 100/1.30 Oil Ph3 DLL objective) equipped with a Photometrics/Cool SNAP HQ2CCD camera using the phase contrast and DAPI channel (filter set: Chroma 49000, excitation at 350/50 nm, emission 460/50 nm)
14. Autoclave
    

Software

1. ImageJ (Rasband W.S./U. S. NIH, Bethesda, Maryland, USA/https://imagej.nih.gov/ij/) 
2. MetaMorph^® Version 7.0 (Microscopy Automation and Image Analysis Software/Molecular DevicesLLC/https://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metamorph-microscopy)
   

Procedure

1. Prepare the following solutions in advance
   
   1. TSB (or LB) and TSB (LB) agar plates (see Recipes).
   2. 50 mM HADA stock solution (see Recipes).
   3. 10 and 1x sodium citrate buffers, 1x PBS and 3% paraformaldehyde (see Recipes).
      Note: Filter and cool 10x and 1x sodium citrate buffers, 1x PBS and 3% paraformaldehyde solution on ice prior to use.
      
      
      
2. Growing the cells
   
   1. Defrost glycerol stock solutions of the bacterial strains on ice. Streak the strains BW25113 (Datsenko and Wanner, 2000), D456 (Edwards and Donachie, 1993) and CS703-1 (Meberg et al., 2001) on TSB agar plates using an inoculation loop and incubate the plates overnight at 37 °C. 
   2. Inoculate 25 ml of TSB (or LB) media with a single bacterial colony and incubate overnight at 37 °C. 
   3. Dilute the overnight cultures to an OD₅₇₈ of 0.1 in TSB (or LB) media and grow at 37 °C until they reach an exponential OD₅₇₈ of 0.4.
   
   
3. Labeling the cells
   
   1. Dilute the cells to an OD₅₇₈ of 0.1 in a final volume of 500 μl of pre-warmed (37 °C) TSB (or LB) in a sterile 1.5 ml micro-tube. 
   2. Add 2.5 µl of the 50 mM stock solution of fluorescent derivative of D-alanine (HADA) to the sample to achieve a final concentration of 250 μM and incubate with shaking for 30 min at 37 °C. 
   
   
4. Stop bacterial growth and washing steps to remove the excess of HADA
   
   1. Add one-tenth of the final volume (50 µl) of ice cold 10x sodium citrate buffer (pH 2.25) to the growing cultures. Place the samples on ice until the centrifuge (step 2) is ready to use.
   2. Centrifuge the samples for 2 min at 16,200 x g at 4 °C. 
   3. Remove the supernatant carefully and resuspend the cell pellet in 1.5 ml ice cold 1x sodium citrate buffer (pH 3.0) by gentle pipetting. 
   4. Centrifuge the samples for 2 min at 16,200 x g at 4 °C. Remove the supernatant carefully and resuspend the cells in 1.5 ml ice cold 1x PBS (pH 7.4) by gentle pipetting and centrifuge the samples again. 
   5. Repeat the washing step with ice cold 1x PBS (pH 7.4) for another time. Remove the supernatants of the samples carefully without touching the cell pellets.
      Note: It was reported that HADA is virtually non-fluorescent in acidic pH or non-buffered water and cells imaged at a pH above 7.0 showed the maximum brightness of HADA labeling (Kuru et al., 2015). Therefore, the last two washing steps with 1× PBS (pH 7.4) are crucial.
   
   
5. Cell fixation
   
   1. Resuspend the cell pellets in 12.5 µl 1× PBS and 12.5 μl of 3% paraformaldehyde (see Recipes) for cell fixation. 
   2. Mix well by pipetting and store on ice or 4 °C until microscopic analysis.
      Note: Microscopic analysis of the labeled cells should be performed immediately.
   
   
6. Microscopic analysis
   
   1. Pipette 3 μl of the fixed cells in a well of the prepared microscopic agarose slide (see Recipes) and place a square shaped cover glass carefully on top and press gently. 
   2. Analyze the labeled cells with a Nikon Eclipse Ti microscope (Nikon Plan Fluor × 100/1.30 Oil Ph3 DLL objective) equipped with a Photometrics/Cool SNAP HQ2CCD camera using the phase contrast and DAPI channel (filter set: Chroma 49000, excitation at 350/50 nm, emission 460/50 nm). 
   3. Use exposure times of 100 ms for phase contrast and 1 s for fluorescence images.
      Note: Using this optimized protocol, all analyzed cells were completely labeled, a stronger signal at the septum of constricting cells was revealed (see Figure 1). The addition of 10x sodium citrate buffer to the growing cells, the initial wash with 1x sodium citrate buffer at pH 3.0, and the rapid processing of the samples on ice improved the removal of unincorporated HADA and led to a decreased background fluorescence signal and improved signal detection.
      
      
      Figure 1. PG labeling of E. coli cells using the fluorescent D-amino acid HADA. A. BW25113 (Datsenko and Wanner, 2000), D456 (Edwards and Donachie, 1993) and CS703-1 (Meberg et al., 2011) cells were incubated with 250 µM HADA for 30 min (long pulse labeling). One-tenth of the final volume of 10x sodium citrate buffer (pH 2.25) was added to the cell cultures prior to short washing steps performed at 4 °C. First the cells were washed once with 1x sodium citrate buffer (pH 3.0) followed by two washing steps with phosphate buffer saline (PBS, pH 7.4). The cells were fixed with 3% paraformaldehyde and visualized by phase contrast (left) and fluorescence microscopy using the DAPI channel (right). Scale bars, 2 μm. B. Optional

Data analysis

ImageJ was used to crop the images to prepare figures and MetaMorph (v7) was used to quantify the fluorescence signals at the septum and at the sidewalls of ~50 cells from at least two independent experiments. The fluorescence intensity per unit of area at septal and sidewall positions can be quantified, the background subtracted, and the ratio of the sidewall over the septum signal density calculated. The values should be given as mean ± SD; the Student’s t-test should be used to determine the significance of the results (Peters et al., 2018).

Notes

Experiments were performed in TSB and LB media without changing the reproducibility of the results.

Recipes

1. Tryptic soy broth (TSB) and Tryptic soy agar (TSA)
   To prepare 1 L of TSB weigh the following reagents:
   17 g Peptone from casein
   3 g Peptone from soybean
   5 g Sodium chloride
   2.5 g Di-Potassium hydrogen phosphate (K₂HPO₄)
   2.5 g D-Glucose (anhydrous)
   
   1. Dissolve all ingredients in a final volume of 1 L distilled water under gentle stirring using a magnetic stirrer
   2. Adjust the pH to 7.3 and autoclave the solution for 15 min at 121 °C
   3. In order to prepare TSA add 15 g/L of agar to 1 L of TSB before autoclaving, cool the solution to a temperature of 48 °C after autoclaving and pour the agarose plates under sterile conditions
2. Miller Luria-Bertani (LB) medium and LB agar
   To prepare 1 L of LB, weigh the following reagents:
   10 g Bacto Tryptone
   10 g Sodium chloride
   5 g Yeast extract 
   
   1. Dissolve all ingredients in a final volume of 1 L distilled water under gentle stirring using a magnetic stirrer
   2. Adjust the pH to 7.3 and autoclave the solution for 30 min at 121 °C
   3. In order to prepare LB agar add 15 g/L of agar to 1 L of LB medium before autoclaving, cool the solution to a temperature of 48 °C after autoclaving and pour the agarose plates under sterile conditions
3. HADA stock solution
   
   1. Dilute 2.1 mg of powdered HADA (328.7 g/mol) in 127.77 µl DMSO to create a 50 mM stock solution
   2. Mix the stock solution well by pipetting and aliquot it in 10 µl portions in sterile 1.5 ml micro-tubes
   3. Sterilization of stock solutions is not required. Store at -80 °C and protected from light
   4. Thaw the stock solution rapidly at room temperature shortly before use
   5. Avoid excessive cycles of thawing and refreezing, or long exposure at room temperature and light
      Note: Dry powders of FDAAs are stable at -20°C or lower temperature for several months (Kuru et al., 2015). In our studies we stored HADA at -80°C, either as dry powder or as stock solution in DMSO for several months and without signs of decomposition or altered labeling efficacy.
4. 10x Sodium citrate buffer, pH 2.25 (500 ml)
   805 mM citric acid
   410 mM NaOH
   1.19 M NaCl
   
   1. Weigh 77.5 g citric acid (anhydrous) and 34.9 g of sodium chloride
   2. Add 206 ml of NaOH solution (c = 1 mol/L) and top the volume up to 500 ml with MilliQ water
   3. Dissolve completely by gentle stirring and check the pH with a pH meter. It should be 2.25
5. 1x sodium citrate buffer, pH 3.0 (500 ml)
   80.5 mM citric acid
   41 mM NaOH
   119 mM NaCl
   
   1. Weigh 7.74 g citric acid (anhydrous) and 3.49 g sodium chloride
   2. Add 206 ml NaOH solution (c = 0.1 mol/L) and top the volume up to 500 ml with MilliQ water
   3. Dissolve completely by gentle stirring and check the pH with a pH meter. It should be 3.0
6. 1x Phosphate buffered saline pH 7.4 (PBS)
   To prepare 50 ml of 1x PBS:
   
   1. Pipette 5 ml of commercially available 10x PBS solution into a sterile Greiner tube
   2. Add 45 ml of Milli Q water and mix well
      Note: Filter the prepared solutions (10x and 1x sodium citrate buffer and the 1× PBS) into sterile 50 ml Greiner tubes. Use sterile single use 50 ml syringes and 0.22 µm syringe filters (PES membrane) for filtration. Cool the solutions on ice prior to use.
7. 3% Paraformaldehyde solution
   To prepare 1,000 µl of 3% paraformaldehyde for cell fixation:
   
   1. Pipette 187.5 µl of 16% paraformaldehyde solution in a sterile 1.5 ml micro-tube and add 812.5 µl filtered 1× PBS solution
   2. Vortex and cool down on ice prior use
8. Agar slides for microscopic imaging
   To prepare an agarose solution with a final concentration of 1% w/v:
   
   1. Dissolve 1 g agarose in 100 ml 1× PBS by microwaving
   2. Bring the temperature of the solution to ~60 °C
   3. Spot ~150 μl agarose solution in every second of the 12 wells on a diagnostic microscopic slide
   4. Place a microscope slide on top and press gently down in order to get a flat, even and thin agarose pad
   5. Let the pad solidify before you carefully remove the top microscope slide
   6. Air-dry the agarose pad for ~1 min prior to use
   Note: The cells do not settle on the agarose pad, if the agarose slide is not dried properly. This can make imaging of the cells more difficult.
   

Acknowledgments

This work reports some of the methods used to demonstrate that copper inhibits peptidoglycan LD-transpeptidases suppressing β-lactam resistance due to bypass of penicillin-binding proteins (Peters et al., 2018). This work was funded by Wellcome Trust Grant 101824/Z/13/Z; the United Kingdom Medical Research Council within the Antimicrobial Resistance Cross-Council Initiative Collaborative Grant MR/N002679/1 (to W.V.) and NIH Grant GM113172 (to M.S.V.).

Competing interests

The authors declare that they have no conflict of interest or competing interest.

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