Determination of the Predatory Capability of Bdellovibrio bacteriovorus HD100

Materials and Reagents

1. 0.45 µm sterilization filter (Sartorius, catalog number: 16555-K )
   
2. 0.22 µm sterilization filter (Sartorius, catalog number: 16532-K )
   
3. 10-ml glass test tubes (Fisher Scientific, catalog number: 15175134 )
   
4. Glass microscope slides (76 x 26 mm) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 10143562BEF ) (see Note 1)
   
5. Glass microscope coverslips (22 x 22 mm) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 3306 )
   
6. 10 ml syringe (BD, catalog number: 307736 )
   
7. Bdellovibrio bacteriovorus HD100 (ATCC, catalog number: 15356 ) (Stolp and Starr, 1963)
   
8. Pseudomonas putida KT2440 (ATCC, catalog number: 47054 ) (Nelson et al., 2002)
   
9. Glycerol (EMD Millipore, catalog number: 104094 )
   
10. Bacto tryptone (BD, Bacto^TM, catalog number: 211705 )
    
11. Yeast extract (Conda, catalog number: 1702 )
    
12. Sodium chlorice (NaCl) (EMD Millipore, catalog number: 106404 )
    
13. Sodium hydroxide (NaOH) pellets for analysis (EMD Millipore, catalog number: 106498 )
    
14. Agar (BD, Bacto^TM, catalog number: 214010 )
    
15. Nutrient broth (NB) (BD, Difco^TM, catalog number: 234000 )
    
16. Calcium chloride dihydrate (CaCl₂·2H₂O) (EMD Millipore, catalog number: 102382 )
    
17. Magnesium chloride hexahydrate (MgCl₂·6H₂O) (EMD Millipore, catalog number: 105833 )
    
18. HEPES buffer (Sigma-Aldrich, catalog number: H3375 )
    
19. Lysogeny broth (LB) medium (1 L, pH 7.5) (see Recipes)
    
20. LB, 1.5% (w/v) agar (see Recipes)
    
21. NB medium (1 L) (see Recipes)
    
22. CaCl₂ and MgCl₂ salts (see Recipes)
    
23. Diluted nutrient broth (DNB) medium (1 L, pH 7.4) (see Recipes)
    
24. DNB, 0.7% (w/v) agar (see Recipes)
    
25. DNB, 1.5% (w/v) agar (see Recipes)
    
26. HEPES buffer (see Recipes)
    

Equipment

1. Centrifuge (Eppendorf, model: 5810 R )
   
2. 100 ml flasks
   
3. 30 °C chamber (JP Select, catalog number: 001257 )
   
4. 30 °C shaking incubator (250 rpm) (Eppendorf, New Brunswick^TM, model: Innova^® 44 )
   
5. Water bath (JP Select, catalog number: 6000138 )
   
6. Phase contrast microscopy (Nikon Instruments, model: OPTIFHOT-2 ) (Note 1)
   
7. Spectrophotometer (Shimadzu, model: UV-260 )
   
8. Leica DFC345 FX camera (Leica Microsystems, model: DFC345 FX )
   
9. Autoclave
   

Procedure

1. Culture of Bdellovibrio preying upon a prey (Figures 1A and 1B)
   1. Preparation of prey cell suspension (Figure 1A)
   1. In this example, P. putida KT2440 is used as prey. To obtain cell suspensions, grow the prey in NB medium at 30 °C and 250 rpm for 16 h (see Note 2), centrifuge (30 min, 4000 x g, 4 °C) and resuspend to OD₆₀₀ = 10 in HEPES buffer (see Note 3).
      
   2. Prey cells can be stored at 4 °C for up to 2 weeks. Immediately prior to use, dilute the cells to OD₆₀₀ = 1.
      
   2. Preparation of the preinocule of Bdellovibrio (two-step cultivation) (Figure 1B)
   1. Recover Bdellovibrio from glycerol stocks stored at -80 °C (see Note 4) by adding 50 µl directly to 10 ml of prey cell suspension prepared in DNB medium at OD₆₀₀ = 1. Incubate at 30 °C and 250 rpm.
      
   2. After 24 h of predation, transfer 100-300 µl of the co-culture to 10 ml of prey suspension prepared in HEPES buffer at OD₆₀₀ = 1 (predator-prey ratio of 1:10). Incubate at 30 °C and 250 rpm for 24 h.
      Note: Set up the co-cultures by adding 10 ml of suspension to 100 ml flasks.
      
   3. Isolation of Bdellovibrio cells (Figure 1B). After predation, filter co-cultures twice through a 0.45 µm filter to recover B. bacteriovorus.
      Note: Use a new filter for the second filtration step.
      
   4. Set up the co-cultures of interest: transfer 100-300 µl of Bdellovibrio cells to 10 ml of prey suspension prepared in HEPES buffer at OD₆₀₀ = 1 (predator-prey ratio of 1:10). Incubate at 30 °C and 250 rpm for 24 h.
      
      
      Figure 1. Workflow to set up Bdellovibrio co-cultures. A. Preparation of the prey cell suspension (here, P. putida KT2440). B. Preparation of Bdellovibrio cells. Two-step cultivation of Bdellovibrio is needed to obtain the predatory cells for the experiment. C. Double layer method to quantify the cell number of B. bacteriovorus HD100. D. Development of B. bacteriovorus HD100 on a lawn of prey on DNB agar plates after 2-3 days of incubation at 30 °C. Bdellovibrio cell number can be quantified as plaque-forming-units (pfu/ml).
      
      
2. Calculation of the viability of Bdellovibrio and prey cells (Figure 1C)
   Make serial dilutions of the co-cultures from 10^-1 to 10^-7 (see Note 5) in DNB medium.
   
   1. Determine Bdellovibrio viability using the double layer method (see Figure 1C) (Lambert et al., 2003):
   1. Add 4 ml of DNB 0.7% (w/v) agar into a glass tube and keep at 45-50 °C in a water bath (see Note 6).
      
   2. Add 0.5 ml of prey cell suspension (P. putida KT2440 prepared at OD₆₀₀ = 10 in HEPES buffer; see Figure 1A).
      
   3. Add 0.1 ml of the appropriate dilution from the co-cultures.
      
   4. Vortex the tube gently.
      
   5. Rapidly, pour the mixture onto a DNB 1.5 % (w/v) agar plate.
      
   6. Incubate the plates for 2-3 days at 30 °C. Bdellovibrio growth is monitored as plaque-forming-units per milliliter (pfu/ml) developing on a lawn of the prey (Figure 1D).
      
   2. Calculation of prey cell viability
      Place 10 µl of each dilution on LB agar plates (see Note 5) and incubate them at 30 °C. Prey cells are counted as colony-forming-units per milliliter (cfu/ml).
      

Data analysis

A representative graph of the predatory activity of Bdellovibrio is shown below (Figure 2). Predator and prey cell number at the beginning of the experiment (0 h) and after 24 h of predation upon P. putida KT2440 are represented.


Figure 2. Predation profile to study the predatory capability of B. bacteriovorus HD100 upon P. putida KT2440. A. Schematic representation of the co-culture involving B. bacteriovorus HD100 and the control culture of P. putida without predator. B. Viability of the prey and predator cells at the beginning of the experiment (0 h) and after 24 h of incubation. The purple bars represent the viability of the prey cells and the orange bars correspond to the predator viable cell number. Error bars indicate the standard deviation of the mean (n ≥ 3).

Notes

1. If possible, predation events should be checked using a phase contrast microscope to ensure the success of the following steps (see Figure 3).
   
2. Select the culture medium based on the prey cells used to grow Bdellovibrio.
   
3. To measure OD₆₀₀, prepare a 1/10 dilution of the culture of interest. E.g., Add 100 µl of the culture to 900 µl of saline solution and mix.
   
4. To store Bdellovibrio at -80 °C, add 0.3 ml of 85 % (w/v) glycerol and 0.7 ml of the co-culture of Bdellovibrio and the prey bacteria to a cryogenic vial, and place the tube directly in the -80 °C freezer. Bdellovibrio cells can be revived by simply scratching the ice of the cryogenic vial with a sterile loop and adding it to the prey suspension. There is no need to de-freeze the cryogenic vial for the inoculation.
   
5. Dilute samples further if a high concentration of cells is expected. E.g., Prepare 1:10 serial dilutions by adding 100 µl of the co-culture to 900 µl of DNB medium in Eppendorf tubes and repeat until the desired concentration is obtained.
   
6. DNB 0.7 % (w/v) agar tubes can be previously prepared and stored at room temperature. Prior to use, melt the tubes in a water bath at 150-200 °C and keep them at 45-50 °C.
   
7. Prepare DNB medium (or HEPES buffer) and the CaCl₂ and MgCl₂ salts separately. Add the salts immediately prior to use.
   
   
   Figure 3. Co-culture of B. bacteriovorus HD100 preying on P. putida KT2440 under the microscope. A. Co-culture at the onset of predation (time zero); B. After 24 h of incubation, only predatory cells can be observed. Cultures are routinely visualized using a 100x phase-contrast objective and images taken with a Leica DFC345 FX camera. 
   

Recipes

1. Lysogeny broth (LB) medium (1 L)
   10 g Bacto tryptone
   5 g yeast extract
   10 g NaCl
   Adjust to pH 7.5 using 1 N NaOH
   Autoclave at 121 °C for 20 min
   
2. LB, 1.5% (w/v) agar (1 L)
   10 g Bacto tryptone
   5 g yeast extract
   10 g NaCl
   Adjust to pH 7.5 using 1 N NaOH
   15 g agar
   Autoclave at 121 °C for 21 min
   
3. NB medium (1 L)
   8 g NB
   Autoclave at 121 °C for 20 min
   
4. CaCl₂ and MgCl₂ salts
   2 mM CaCl₂·2H₂O
   3 mM MgCl₂·3H₂O
   Sterilize by filtration through a 0.22 µm filter into a sterile container
   
5. Diluted nutrient broth (DNB) medium (1 L) (see Note 7)
   0.8 g NB
   Adjust to pH 7.4 using 1 N NaOH
   Autoclave
   Add CaCl₂ and MgCl₂ salts (see Recipe 4)
   
6. DNB, 0.7% (w/v) agar (1 L) (see Note 7)
   0.8 g NB
   Adjust to pH 7.4 using 1 N NaOH
   7 g agar
   Autoclave at 121 °C for 20 min
   Add CaCl₂ and MgCl₂ salts (see Recipe 4)
   
7. DNB, 1.5% (w/v) agar (1 L) (see Note 7)
   0.8 g NB
   Adjust to pH 7.4 using 1 N NaOH
   15 g agar
   Autoclave at 121 °C for 20 min
   Add CaCl₂ and MgCl₂ salts (see Recipe 4)
   
8. HEPES buffer (see Note 7)
   25 mM HEPES buffer
   Adjust to pH 7.8 using NaOH pellets
   Autoclave at 121 °C for 20 min
   Add CaCl₂ and MgCl₂ salts (see Recipe 4)
   

Acknowledgments

This protocol was modified and adapted from a predatory assay previously described (Martínez et al., 2016; Lambert et al., 2003). This work was funded by the EU Seventh Framework Programme under grant agreement No. 311815 (SYNPOL), and by grants from the Comunidad de Madrid (P2013/MIT2807) and the Spanish Ministerio de Economía y Competitividad, (BIO2010-21049, BIO2013-44878-R).

References

1. Atterbury, R. J., Hobley, L., Till, R., Lambert, C., Capeness, M. J., Lerner, T. R., Fenton, A. K., Barrow, P. and Sockett, R. E. (2011). Effects of orally administered Bdellovibrio bacteriovorus on the well-being and Salmonella colonization of young chicks. Appl Environ Microbiol 77(16): 5794-5803.
   
2. Davidov, Y. and Jurkevitch, E. (2009). Predation between prokaryotes and the origin of eukaryotes. Bioessays 31(7): 748-757.
   
3. Gophna, U., Charlebois, R. L. and Doolittle, W. F. (2006). Ancient lateral gene transfer in the evolution of Bdellovibrio bacteriovorus. Trends Microbiol 14(2): 64-69.
   
4. Jurkevitch, E. and Davidov, Y. (2007). Phylogenetic diversity and evolution of predatory prokaryotes. In: Jurkevitch, E. (Ed.). Microbiol Monogr Predatory Prokaryotes Heidelberg. Springer, pp: 11-56.
   
5. Jurkevitch, E., Minz, D., Ramati, B. and Barel, G. (2000). Prey range characterization, ribotyping, and diversity of soil and rhizosphere Bdellovibrio spp. isolated on phytopathogenic bacteria. Appl Environ Microbiol 66(6): 2365-2371.
   
6. Lambert, C., Smith, M. C. and Sockett, R. E. (2003). A novel assay to monitor predator-prey interactions for Bdellovibrio bacteriovorus 109 J reveals a role for methyl-accepting chemotaxis proteins in predation. Environ Microbiol 5(2): 127-132.
   
7. Lambert, C. and Sockett, R. E. (2008). Laboratory maintenance of Bdellovibrio. Curr Protoc Microbiol 9:7B.2.1-7B.2.13.
   
8. Mahmoud, K. K., McNeely, D., Elwood, C. and Koval, S. F. (2007). Design and performance of a 16S rRNA-targeted oligonucleotide probe for detection of members of the genus Bdellovibrio by fluorescence in situ hybridization. Appl Environ Microbiol 73(22):7488.
   
9. Martínez, V., de la Peña, F., García-Hidalgo, J., Mata, I., García, J. L., and Prieto, M. A. (2012). Identification and biochemical evidence of a medium-chain-length polyhydroxyalkanoate depolymerase in the Bdellovibrio bacteriovorus predatory hydrolytic arsenal. Appl Environ Microbiol 78: 6017-6026.
   
10. Martínez, V., Herencias, C., Jurkevitch, E. and Prieto, M. A. (2016). Engineering a predatory bacterium as a proficient killer agent for intracellular bio-products recovery: the case of the polyhydroxyalkanoates. Sci Rep 6: 24381.
    
11. Nelson, K. E., Weinel, C., Paulsen, I. T., Dodson, R. J., Hilbert, H., Martins dos Santos, V. A., Fouts, D. E., Gill, S. R., Pop, M., Holmes, M., Brinkac, L., Beanan, M., DeBoy, R. T., Daugherty, S., Kolonay, J., Madupu, R., Nelson, W., White, O., Peterson, J., Khouri, H., Hance, I., Chris Lee, P., Holtzapple, E., Scanlan, D., Tran, K., Moazzez, A., Utterback, T., Rizzo, M., Lee, K., Kosack, D., Moestl, D., Wedler, H., Lauber, J., Stjepandic, D., Hoheisel, J., Straetz, M., Heim, S., Kiewitz, C., Eisen, J. A., Timmis, K. N., Dusterhoft, A., Tummler, B. and Fraser, C. M. (2002). Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol 4(12): 799-808.
    
12. Sockett, R. E. (2009). Predatory lifestyle of Bdellovibrio bacteriovorus. Annu Rev Microbiol 63: 523-539.
    
13. Stolp, H. and Starr, M. P. (1963). Bdellovibrio Bacteriovorus gen. et sp. n., a predatory, ectoparasitic, and bacteriolytic microorganism. Antonie Van Leeuwenhoek 29: 217-248.
    
14. Van Essche, M., Sliepen, I., Loozen, G., Van Eldere, J., Quirynen, M., Davidov, Y., Jurkevitch, E., Boon, N and Teughels, T. (2009). Development and performance of a quantitative PCR for the enumeration of Bdellovibrionaceae. Environ Microbiol Rep 1(4):228-33.