Bacterial Competition Assay Based on Extracellular D-amino Acid Production

Materials and Reagents

1. Wired-loop or disposable inoculation loops (SARSTEDT, catalog number: 86.1562.050 )
   
2. 15 ml test tubes (SARSTEDT, catalog number: 62.554.502 )
   
3. Cuvettes (SARSTEDT, catalog number: 67.742 )
   
4. 150 ml Stericup filtration units, 0.22 µm pore size (Merck, catalog number: SCGPU01RE )
   
5. Adhesive tape
   
6. Parafilm (Sigma-Aldrich, catalog number: P7793-1EA )
   
7. Needles (BD, catalog number: 302200 )
   
8. Syringes 1 ml, 10 ml (BD, catalog numbers: 303172 , 307736 )
   
9. 1.5 ml microtubes (Eppendorf, catalog number: 0030120086 )
   
10. Sterile clear flat-bottom 96-well plates with lid (Corning, Falcon^®, catalog number: 353072 )
    
11. Sterile glass beads 3 mm (Merck, catalog number: 1040150500 )
    
12. Petri dishes (SARSTEDT, catalog number: 82.1473 )
    
13. Filter units, 0.22 µm pore size (Merck, catalog number: SLGS033SB )
    
14. Disposable pipette tips (VWR, catalog numbers: 613-1083 , 613-1079 , 613-1077 )
    
15. Bacterial strains: V. cholerae N16961 lacZ+ wild-type, V. cholerae N16961 lacZ- ΔbsrV, Caulobacter crescentus NA1000
    
16. Trigonopsis variabilis DAAO (gift from Jose M. Guisan, Catalysis Department, ICP – CSIC, Spain) (Komarova et al., 2012)
    
17. L-Arginine (L-Arg) (Sigma-Aldrich, catalog number: A5006-100G )
    
18. D-Arginine (D-Arg) (Sigma-Aldrich, catalog number: A2646-5G )
    
19. Distilled water
    
20. MilliQ water
    
21. Hydrochloric acid fuming 37% (HCl) (Merck, catalog number: 1003171000 )
    
22. Tryptone (Peptone from casein) (VWR, catalog number: 84610.0500 )
    
23. Yeast extract (VWR, catalog number: 84601.0500 )
    
24. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 71376-1KG )
    
25. Sodium hydroxide pellets (NaOH) (Merck, catalog number: 1064821000 )
    
26. Peptone, meat (enzymatic digest of animal tissue) (VWR, catalog number: 84620.0500 )
    
27. Magnesium sulfate (MgSO₄) (Sigma-Aldrich, catalog number: M2643-500G )
    
28. Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: C5670-500G )
    
29. Bacteriological agar (VWR, catalog number: 84609.0500 )
    
30. Sodium phosphate monobasic monohydrate (NaH₂PO₄·H₂O) (Sigma-Aldrich, catalog number: 71507-250G )
    
31. Sodium phosphate dibasic heptahydrate (Na₂HPO₄·7H₂O) (Sigma-Aldrich, catalog number: 431478-250G )
    
32. Ortho-phosphoric acid 85% (Merck, catalog number: 1005731000 )
    
33. Flavin adenine dinucleotide disodium salt hydrate (FAD) (Sigma-Aldrich, catalog number: F6625-100MG )
    
34. o-Phenylenediamine (OPD) (Sigma-Aldrich, catalog number: P23938-5G )
    
35. Methanol (VWR, catalog number: 20847.307 )
    
36. Horseradish peroxidase (Sigma-Aldrich, catalog number: 77332-100MG )
    
37. LB medium (see Recipes)
    
38. PYE medium (see Recipes)
    
39. Agar plates (see Recipes)
    
40. L- and D-amino acid stock solutions (see Recipes)
    
41. Sodium phosphate buffer 500 mM pH 7.5 (see Recipes)
    
42. DAAO reaction buffer (see Recipes)
    

Equipment

1. Laminar flow cabinet
   
2. Bunsen burner
   
3. Pipettes (Gilson, catalog numbers: F144563 , F144565 , F144566 )
   
4. Multichannel pipettes (Gilson, catalog number: F14403 )
   
5. Glassware: bottles, measurement cylinders, beakers
   
6. pH-meter (VWR, catalog number: 662-1422 )
   
7. Autoclave (CertoClav, catalog number: 8510174 )
   
8. Incubator (Memmert, catalog number: IN55 )
   
9. Shaker incubator (Thermo Fisher Scientific, Thermo Scientific^TM, model: MaxQ^TM 5000, catalog number: SHKE5000 )
   
10. Thermomixer with adapter for multi-well plates (Eppendorf, catalog numbers: 5355000011 , 5363000012 )
    
11. Spectrophotometer (GE Healthcare, catalog number: 29003605 )
    
12. Microplate reader (Biotek, model: EON^TM, catalog number: EONC )
    

Procedure

1. Bacterial co-cultivation
   Note: All steps need to be performed under sterile conditions, working by the flame or inside a laminar flow cabinet.
   1. Streak V. cholerae cells from the freezer stock onto an LB agar plate and incubate upside-down for 16 h at 37 °C. V. cholerae lacZ+ wild-type and V. cholerae lacZ- ΔbsrV can be used to assess the role of BsrV in D-amino acid production.
      
   2. Streak C. crescentus cells from the freezer stock onto a PYE agar plate and incubate upside-down for 48 h at 28 °C.
      Note: Any combination of bacteria (ideally one of them a D-amino acid producer) can be used in this protocol, as long as both can grow in compatible conditions.
      
   3. Pick a single colony of each strain from the agar plates with a sterile wired-loop and inoculate 2 ml liquid PYE medium in a 15 ml test tube. Grow liquid bacterial cultures overnight (16-18 h) at 28 °C, with shaking at 150 rpm.
      Note: Temperature and shaking may vary depending on the bacterial species used in the competition assay.
      
   4. Determine the OD₆₀₀ of the cultures: dilute 100 µl overnight culture in 900 µl fresh PYE medium, transfer to a cuvette and read absorbance at 600 nm using a spectrophotometer. Use a cuvette filled with 1 ml fresh PYE as blank.
      
   5. Dilute the bacterial suspension in 10 ml PYE medium to a starting concentration of OD₆₀₀ = 0.01. Depending on the bacterial strain used, the OD₆₀₀ to CFU ml^-1 ratio will differ and needs to be determined for each strain: for example, for the V. cholerae wild type strain, OD₆₀₀ 0.01 = ~10⁷ cells ml^-1.
      
   6. Stericup filtration units are used as co-cultivation chambers. Fill the upper part, which we called compartment A, with 100 ml fresh PYE medium. Fill the lower part, which we called compartment B, with 200 ml fresh PYE medium. Tightly attach both compartments. Use Parafilm and adhesive tape to firmly seal the lid of compartment A and avoid leakage and place the co-cultivation chamber horizontally. With a hot needle, make a small hole in the top of each compartment to allow inoculation and sample collection; this hole can be sealed with adhesive tape (Figure 1A).
      Note: Make sure the co-cultivation chambers are placed horizontally with the hole facing the top, to minimize sample spilling. Here we use 100 ml medium in compartment A and 200 ml medium in compartment B because the size of both parts is different; ideally both compartments should have the same size, shape and medium volume.
      
   7. L-amino acid supplementation: with a 10 ml syringe and a needle, add 2.5 ml and 5 ml L-arginine (200 mM sterile stock solution) to compartments A and B, respectively (final L-amino acid concentration is 5 mM in each compartment). Use non-supplemented co-cultivation chambers to assess growth in absence of L- or D-amino acids (Figure 1B).
      
   8. With a 1 ml syringe and a needle, inoculate the bacteria into the co-cultivation chambers: 100 µl V. cholerae (OD₆₀₀ = 0.01) is inoculated into compartment A; 500 µl C. crescentus (OD₆₀₀ = 0.01) is inoculated into compartment B. Use non-inoculated chambers as a control of contamination and growth in absence of a competitor bacteria (Figure 1B).
      Note: Determination of the growth rate of the bacteria in competition should be assessed beforehand, including the growing condition (medium and temperature) compatibility. We use 2.5-fold C. crescentus inoculum to compensate for the very different growth rates of V. cholerae and C. crescentus in PYE medium. V. cholerae lacZ+ wild-type is used as D-amino acid producing bacteria. V. cholerae lacZ- ΔbsrV is used as D-amino acid non-producer. A 1:1 mixture of V. cholerae lacZ+ wild-type and V. cholerae lacZ- ΔbsrV is used to demonstrate the metabolic cooperation between producers and non-producers to outcompete other bacteria.
      
   9. Incubate the co-cultivation chambers at 28 °C with mild agitation (100 rpm). Place the holes used for inoculation on the top to minimize sample spilling (Figure 1C). Samples from both compartments will be collected at different time points: 0, 24, 48 and 72 h.
      
      
      Figure 1. Preparation of the co-cultivation chambers. A. Fill both compartments of the Stericup filtration unit with the appropriate media. Attach and seal both parts and add the L-amino acid to a final concentration of 5 mM. Finally inoculate the compartments with the competing bacteria. B. Media and bacteria combinations for a complete competition experiment, including controls (C) and test samples (Test). Vc: V. cholerae. Cc: C. crescentus. C. Co-cultivation chamber.
      
      
   10. At the desired time points, use 1 ml syringes and needles to collect 500 μl culture samples from each compartment and co-cultivation chamber and transfer to previously labeled microtubes.
       
   11. Viable cell count is performed immediately (see Procedure B). Transfer 200 μl of freshly collected samples from compartment A (V. cholerae) and B (C. crescentus) to a sterile 96-well plate.
       
   12. Determination of D-amino acid concentration is performed at the end of the whole experiment (see Procedure C). Centrifuge the remaining 300 μl sample at 21,000 x g for 5 min at room temperature using a microcentrifuge. Carefully transfer the supernatant (300 μl) to new microtubes and store samples at -20 °C until the end of the experiment. Make sure no cell pellet is transferred to the new tube.
       
   
   
2. Viable cell count
   Note: All steps need to be performed under sterile conditions, working by the flame or inside a laminar flow cabinet.
   1. To determine the viable cell count of V. cholerae and C. crescentus, serially dilute cells 1:10. Transfer 20 μl from the previous dilution to a new well and add 180 μl of fresh medium (LB for V. cholerae, PYE for C. crescentus), thoroughly pipetting to make the mixture homogeneous. Following this procedure, dilute the cultures 9 times. Make sure to change the tips in every dilution step.
      
   2. Plate 100 μl of each dilution on agar plates (LB for V. cholerae, PYE for C. crescentus), for CFU count. First, add 6-10 sterile glass beads to each plate and then carefully pipette the bacterial culture. Then, close the lid and agitate the plates to homogeneously distribute the cell culture and let it dry. Remove the glass beads by carefully tilting the plates. Glass beads can be reused after decontamination, washing and sterilization. Incubate CFU count plates upside-down for 16 h at 37 °C for V. cholerae and 48 h at 28 °C for C. crescentus.
      
   3. Once grown, count colonies on the agar plates and calculate the viable CFU per ml based on the dilution factors applied (Figure 2).
      
      
      Figure 2. Viable cell count. A. C. crescentus growth expressed as CFU ml^-1; B. Alternative representation using the relative growth compared to the control. -: C. crescentus control, WT: competition C. crescentus vs. V. cholerae lacZ+ wild-type, ΔbsrV: competition C. crescentus vs. V. cholerae lacZ- ΔbsrV, MIX: competition C. crescentus vs. a 1:1 mixture of V. cholerae lacZ+ wild-type and V. cholerae lacZ- ΔbsrV.
      
      
3. Determination of total D-amino acid concentration: DAAO assay
   Note: No sterile conditions are required. In this two-step assay, DAAO produces α-ketoacid, NH₃ and H₂O₂ from D-amino acids; peroxidase reduces H₂O₂ releasing free O₂ that reacts with OPD, leading to the production of 2,3-diaminophenazine, a colorimetric product that can be detected using a spectrophotometer (Alvarez et al., 2018; Espaillat et al., 2014).
   1. D-amino acid standard curve: prepare D-arginine dilutions at 0.05, 0.1, 0.25, 0.5, 1, 1.5 and 2 mM concentration in MilliQ water. Transfer 20 μl of each dilution to a clear flat-bottom 96-well plate. Transfer 20 µl of MilliQ water to another well to be used as a blank. For reproducibility, prepare triplicate standard curves and blanks.
      
   2. Supernatant samples: completely thaw the supernatant samples kept at -20 °C (let them stand on ice for 20 min). Prepare 1:2, 1:5 and 1:10 dilutions in new microtubes using MilliQ water. Transfer 20 μl of each sample (non-diluted, 1:2, 1:5 and 1:10) to the clear flat-bottom 96-well plate. For reproducibility, prepare triplicate reactions.
      
   3. Prepare the DAAO reaction buffer (see Recipes) and add 60 μl to each well using a multichannel pipette. Final reaction volume will be 80 μl.
      
   4. Close the 96-well plate lid to avoid evaporation and incubate for 1 h at 37 °C with vigorous shaking (400 rpm). Positive reactions will turn yellow.
      
   5. Using a multichannel pipette, add 2 volumes (160 μl) of 2 N HCl to each well to inactivate the reaction. The yellow color will turn orange.
      
   6. Read the absorbance at 492 nm using a microplate reader.
      

Data analysis

For reproducibility, biological samples should be tested in triplicate.
For testing cell viability, count the number of colonies grown on the plates and calculate the viable CFU per ml based on the dilution factors applied and the volume of culture plated (100 µl) (Figure 2).
Example: 153 colonies of C. crescentus in the plate with dilution 4 (1:10⁴)
153 x 10⁴ (dilution factor) x 10 (volume correction) = 1.53 x 10⁷ CFU ml^-1
The relative growth can be calculated by dividing the CFU ml^-1 of every sample and condition by the CFU ml^-1 in the control without L-amino acid or competitor bacteria.
To determine the total concentration of D-amino acid:

1. Absorbance from the standard curve samples should fit to a linear regression model (Figure 3A). The goodness of the fit is represented by the R² value.
   
2. Use the equation of the linear regression model to calculate the total D-amino acid concentration of every sample and replica. Consider the dilution factors, if applied.
   Discard all measurements with absorbance values above 1.2 units: above this value the model loses linearity and the extrapolation of the D-amino acid concentration is wrong. Use the values of the 1:2, 1:5 or 1:10 dilutions instead.
   
3. Total D-amino acid concentration can be represented as in Figure 3B. If needed, the basal D-amino acid concentration in PYE medium can be subtracted from the sample values.
   
   
   Figure 3. Determination of the D-amino acid concentration. A. Representative D-arginine standard curve; B. Total D-amino acid concentration in the media from co-cultivation chambers at 48 h. -: C. crescentus control, WT: competition C. crescentus vs. V. cholerae lacZ+ wild-type, ΔbsrV: competition C. crescentus vs. V. cholerae lacZ- ΔbsrV, MIX: competition C. crescentus vs. a 1:1 mixture of V. cholerae lacZ+ wild-type and V. cholerae lacZ- ΔbsrV.
   

Recipes

1. LB medium
   10.0 g L^-1 tryptone
   5.0 g L^-1 yeast extract
   10.0 g L^-1 NaCl
   Dissolve components in distilled water
   Adjust the pH to 7.0 using 2 N NaOH
   Adjust the final volume and sterilize by autoclaving (15 min at 121 °C and 1 atm)
   Store at room temperature
   
2. PYE medium
   2 g L^-1 peptone
   1.0 g L^-1 yeast extract
   1 ml L^-1 1 M MgSO₄
   0.5 ml L^-1 1 M CaCl₂
   Dissolve components in distilled water
   Adjust the final volume and sterilize by autoclaving (15 min at 121 °C and 1 atm)
   Store at room temperature
   
3. Agar plates
   1. For preparation of agar plates, dissolve the medium components in distilled water, add 15 g L^-1 bacteriological agar, then adjust pH if needed and the final volume and finally sterilize by autoclaving (15 min at 121 °C and 1 atm)
      
   2. Let the medium cool down to 50 °C and pour in sterile Petri dishes under sterile conditions (approximately 20 ml per plate). Let the plates solidify at room temperature. Store plates at 4 °C
      
4. L- and D-amino acid stock solutions
   Dissolve the corresponding amount of L- or D-arginine in MilliQ water to a final concentration of 200 mM
   Sterilize using 0.22 µm pore size filter units
   Store at room temperature
   
5. Sodium phosphate buffer 500 mM pH 7.5
   12.9 g L^-1 NaH₂PO₄·H₂O
   109.1 g L^-1 Na₂HPO₄·7H₂O
   Dissolve components in distilled water
   Adjust the pH to 7.5 using ortho-phosphoric acid 25% (v/v) or 2 N NaOH, and adjust the final volume
   Store at room temperature
   
6. DAAO reaction buffer
   Per reaction, prepare 60 µl final volume buffer (see table below) containing sodium phosphate buffer 33.3 mM pH 7.5, FAD 8.3 µg ml^-1, freshly prepared OPD 83.3 µg ml^-1, horseradish peroxidase 41.7 µg ml^-1 and Trigonopsis variabilis DAAO (Komarova et al., 2012) 33.3 µg ml^-1.
   All stock solutions are prepared in MilliQ water unless otherwise specified. Aliquot and store FAD, horseradish peroxidase and DAAO stock solutions at -20 °C
   
   

Acknowledgments

This work reports in detail the bacterial competition assay previously used to demonstrate the use of D-arginine by V. cholerae to outcompete other bacteria (Alvarez et al., 2018). This work was funded by The Knut and Alice Wallenberg Foundation (KAW), The Laboratory of Molecular Infection Medicine Sweden (MIMS), the Swedish Research Council and the Kempe Foundation. The authors declare no conflict of interest or competing interest.

References

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