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Feb 2020

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A High-throughput Interbacterial Competition Platform
一个高通量细菌间竞争平台   

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Abstract

Contact-dependent interbacterial competition is a common strategy used by bacteria to fight for their ecological niches. Interbacterial competition is monitored by a competition assay involving co-culturing the attacker and the recipient bacterial cells on agar, followed by recovery of the surviving recipient cells. Conventional interbacterial competition assays rely on serial dilution, plate spreading, and colony counting experiments for the readout. The high demand for time and labor in a competition assay limits its use for large-scale screening. However, a high-throughput interbacterial competition screening method is required to screen genetic factors involved in an interbacterial competition. Here, using Agrobacterium tumefaciens as an attacker and Escherichia coli as a recipient, we developed a robust, fast, efficient, and high-throughput type VI secretion system-dependent interbacterial competition screening platform. This system allows for 96 simultaneous competition assays without the need for serial dilution and plate spreading. Data analysis of this system relies on only direct and straightforward colony counting. This platform may be easily adapted to identify novel factors involved in any contact-dependent interbacterial competition systems.

Keywords: Competition assay (竞争分析), High-throughput screening (高通量筛选), Bacterial competition (细菌竞争), Type VI secretion system (VI型分泌系统), Agrobacterium tumefaciens (根癌农杆菌)

Background

Bacteria have evolved multiple strategies to fight other bacteria to gain ecological niche fitness, and contact-dependent interbacterial competition is one of the widely used strategies (Granato et al., 2019). A bacterium can use its type I (T1SS), type IV (T4SS), type V (T5SS), type VI (T6SS), or type VII (T7SS) secretion systems to deliver protein toxins into its competitor cell, the recipient cell, and kill it in a contact-dependent manner (Aoki et al., 2005; Hood et al., 2010; Souza et al., 2015; Cao et al., 2016; García-Bayona et al., 2017). The protein toxin-delivering bacterium is the attacker cell. The attacker cell also expresses the cognate immunity protein to protect itself from self-intoxication. A competition assay can measure the strength of a contact-dependent interbacterial competition. A competition assay can be divided into four steps: (1) culturing the attacker and recipient cells, (2) preparing the competition spot by mixing the two bacterial strains and incubating it on an agar plate, (3) recovery and selective growth of the recipient cells from the competition spot, and (4) counting the colony-forming units (CFUs) of the surviving recipient cells. The attacker with a robust interbacterial competition activity results in low recipient CFU.

Although the competition assay is a straightforward experiment, a high-throughput platform for 96 parallel competition assays is lacking. Several factors limit the capacity of a competition assay. First, each competition spot has to be separated at a distance to avoid cross-contamination during the recovery step. The need for the separation hinders the competition spots from arranging compactly, as in a 96-well format. Second, the recovery step and also the CFU counting step are time-consuming and laborious. In the recovery step, each competition spot is collected by an inoculating loop. Recording the CFU of the recipient cell requires serial dilution and plate spreading.

Here we present a high-throughput interbacterial competition platform that performs 96 competition assays simultaneously and does not require serial dilution and plate spreading. In this platform, the 96 competition mixtures were spotted on an agar plate solidified on a 96-well lid and recovered by a 96-pin microplate replicator instead of an inoculating loop. Using a 96-pin microplate replicator ensures that equal amounts of the bacteria are recovered, which is equivalent to the area of each pin. Because this method minimizes the amount of recovery to the pin area, serial dilution and plate spreading are not required. Meanwhile, the readout of this platform is direct and straightforward by counting the CFU of the recipient cells. This platform has been used to identify the recipient factors that are involved in enhancing the T6SS activity of Agrobacterium tumefaciens with success (Lin et al., 2020). We tested all 3,909 E. coli Keio mutants for their recovery after co-incubation with a T6SS active A. tumefaciens strain within 1 month. This method can be used to screen for both the attacker and recipient mutants with enhanced or reduced interbacterial competition activity mediated by any secretion system.

Materials and Reagents

  1. 250 μl tip for EzMate (KLBiotech, catalog number: FX-250-R )
  2. 96-pin microplate replicator (Violet BioScience, catalog number: VIO-T96 )
  3. Pipet tip box (Labcon, catalog number: 1055-965-018 )
  4. 96-well microplate, flat bottom (Basic Life, catalog number: BL6052 )
  5. 96-well microplate, U bottom (Basic Life, catalog number: BL6031 )
  6. 96-well lid (Basic Life, catalog number: BL6171 )
  7. 2.2-ml 96 deep-well microplate (Basic Life, catalog number: BL6038 )
  8. Test tube, 16 x 125 mm (Pyrex, catalog number: 9820 )
  9. Test tube cap (Kimble, catalog number: KIM-KAP 73660 )
  10. Pipetting reservoir (Basic Life, catalog number: BL6233 )
  11. A. tumefaciens C58 wild type (WT)
  12. Sucrose (Sigma-Aldrich, catalog number: S5016 )
  13. NZ-Case® Plus, casein enzymatic hydrolysate (Sigma-Aldrich, catalog number: N4642 )
  14. BactoTM Yeast extract (BD, catalog number: 212750 )
  15. K2HPO4 (Merck Millipore, catalog number: 7758-11-4 )
  16. MgSO4·7H2O (Merck Millipore, catalog number: 10034-99-8 )
  17. LB broth, Miller (BD, catalog number: 244620 )
  18. NaH2PO4·2H2O (Merck Millipore, catalog number: 10028-24-7 )
  19. NH4Cl (Merck Millipore, catalog number: 017-014-00-8 )
  20. KCl (Sigma-Aldrich, catalog number: P9541 )
  21. MES (Bio Basic, catalog number: 145224-94-8 )
  22. BactoTM Agar (BD, catalog number: 214010 )
  23. 95% ethanol (TaiSugar, catalog number: ethanol-18L )
  24. 6% bleach (LCY Chemical, catalog number: bleach-4L )
  25. Kanamycin sulfate (BioBasic, catalog number: KB0285-5g )
  26. HCl (Honeywell, catalog number: 30721-2.5L-GL )
  27. 523 broth (see Recipes)
  28. LB broth (see Recipes)
  29. LB agar with 20 μg/ml kanamycin (see Recipes)
  30. AK broth (see Recipes)
  31. AK agar, fresh prepare (see Recipes)
  32. 70% ethanol (see Recipes)
  33. 0.6% bleach (see Recipes)

Equipment

  1. Automated Pipetting System (KLBiotech, model: EzMateTM 401)
  2. Pipettes (Gilson, models: PIPETMAN P2, P20, P200, P1000, 8𝗑20)
  3. -80 °C freezer
  4. Centrifuge (Eppendorf, model: 5810R )
  5. Centrifuge rotor (Eppendorf, model: A-4-62 )
  6. Orbital shaking incubator (TKS, model: OSI-500R )
  7. Autoclave (LUXLEY, model: HL-326 )
  8. Microwave (Panasonic, model: NN-ST651 )
  9. 500 ml PYREX Erlenmeyer flasks (Corning, catalog number: 4980-500 )
  10. Alcohol burner (DG Life, catalog number: D92J-119250 )

Software

  1. EzStarter v4.0.0.87 (Arise Biotech)

Procedure

  1. Day 1: Pre-culture the attacker cells and the recipient cells
    1. Pre-culture the attacker cells.
      Choose a single colony of A. tumefaciens C58 wild type (WT) grown on 523 agar and grow it in a test tube with 5 ml of 523 medium at 25 °C, 220 rpm for overnight.
    2. Pre-culture the control recipient cells.
      Choose a single colony of E. coli BW25113 WT carrying pRL-nptII plasmid and grow it in a test tube with 5 ml of LB medium with 20 μg/ml kanamycin. Grow the cells at 37 °C, 220 rpm overnight.
      Note: The pRL-nptII provides BW25113 WT with kanamycin resistance so that it can be selectively grown after co-incubating with A. tumefaciens.
    3. Sterilize the 96-pin microplate replicator.
      1. Place an alcohol burner on the left. Take a 200-μl tip box, remove the tip holder inside, flatten the box, and then put it on the right of the burner. Fill the left well of the flattened box with 0.6% bleach and the right well with sterilized water (Figure 1).
      2. Fill the 96-pin microplate replicator holder with 70% ethanol and place it on the right of the sterilized water-containing well (Figure 1).
      Note: Do not put the 70% ethanol-containing replicator holder next to the ethanol burner.


      Figure 1. Setup for sterilizing the microplate replicator. From left to right: alcohol burner, 0.6% bleach, sterilized water, and 70% ethanol. Place the alcohol burner distal to the 70% ethanol to avoid accidents.

      1. Immerse the pins of the 96-pin microplate replicator serially in 0.6% bleach, sterilized water, and 70% ethanol. Let the replicator stand for 10 s in each solution.
      2. Sterilize the replicator with an alcohol burner (Figure 2) and cool the replicator for 10 s to room temperature. The sterilized replicator is ready to subculture E. coli mutants.
        Note: Be very careful when handling the alcohol burner.


      Figure 2. Sterilize the microplate replicator by flame. Place the 96-pin microplate replicator after its pins are immersed in 70% ethanol on top of the alcohol burner for sterilization. Move the replicator around the flame to make sure the alcohol on each pin is burned out, then cool the replicator for 10 s at room temperature. The sterilized replicator is ready to subculture E. coli mutants.

    4. Pre-culture the E. coli mutants from the Keio library that serve as recipient cells.
      1. Take a U-bottom 96-well microplate and fill each well with 100 μl LB medium with 20 μg/ml kanamycin.
      2. Take out an E. coli Keio mutant plate, which is stored as glycerol stock in a 2.2-ml 96 deep-well format in a -80 °C refrigerator. Apply the sterilized replicator to the frozen E. coli stocks.
        Note: Make sure each replicator pin contacts the surface of the glycerol stock.
      3. Stamp the bacteria-containing replicator onto the LB medium in each well of the 96-well microplate prepared in Step A4a and agitate the liquid to resuspend the bacteria thoroughly (Figure 3).
        Note: Make sure each replicator pin contacts the LB medium in each well of a 96-well plate.


        Figure 3. Pre-culture the E. coli mutants from the Keio library. Stamp the bacteria-containing replicator onto LB medium in each well of a 96-well microplate and agitate the liquid to resuspend the bacteria thoroughly.

      4. Reseal and return the E. coli Keio mutants plate back to -80 °C and place the replicator in the 0.6% bleach prepared in Step A3a.
      5. Add a 96-well lid to the E. coli-containing U-bottom 96-well microplate and incubate at 37 °C, 220 rpm for 16 h (Figure 4).
        Note: The incubation can be achieved by adding the anti-slip pad on a regular incubator.


        Figure 4. Incubating the E. coli-containing U-bottom 96-well microplate in an incubator. Remove some of the flask holders and replace the space with an anti-slip pad. Fix the anti-slip pad with screws. The anti-slip–containing area is ready for incubating the 96-well microplate.

    5. Sterilize the 96-pin microplate replicator (Steps A3c to A3d).

  2. Day 2: Sub-culture the attacker cells and the recipient cells
    Note: This step incorporates BW25113(pRL-nptII) into the Keio mutant-containing microplate and synchronizes their growth.
    1. Sub-culture the attacker cells.
      Sub-culture 1 ml of the A. tumefaciens C58 WT from Step A1a with 100 ml of 523 medium in a 500-ml flask, prepare two cultures (200 ml in total). Grow the bacteria at 25 °C, 220 rpm, overnight.
    2. Sub-culture the recipient cells.
      1. Take a U-bottom 96-well microplate and fill each well with 100 μl LB medium with 20 μg/ml kanamycin. Use an 8-channel pipette to subculture 2 μl E. coli mutants grown from Step A4e in the LB-containing U-bottom 96-well microplate but leave some wells empty for the control strain, BW25113(pRL-nptII) (Figure 5).
        Note: Remove appropriate tips from the 8-channel pipette while sub-culturing to leave the well empty for the control strain.
      2. Sub-culture 2 μl BW25113(pRL-nptII) from Step A2 into the empty wells in Step B2a (WT label in Figure 5).
      3. Grow the sub-cultured E. coli strains at 37 °C, 220 rpm, for overnight.


        Figure 5. Sub-culture the E. coli Keio mutants and the BW25113(pRL-nptII). Use an 8-channel pipette to subculture 2 μl of the overnight-cultured E. coli mutants from step A4e into a new plate filled with 100 μl LB medium with 20 μg/ml kanamycin. Do not sub-culture the wells reserved for BW25113(pRL-nptII) controls (e.g., Wells 3C, D8, D10, and F12 in this case). Sub-culture 2 μl overnight-cultured BW25113(pRL-nptII) into the empty wells as controls (e.g., wells labeled WT).

  3. Day 3: Co-incubate the attacker cells and the recipient cells to enable interbacterial competition
    1. Prepare the competition surface.
      Pour 25 ml melted AK agar on a 96-well lid in the laminar flow and allow it to dry for 45-60 min.
      Note: It is crucial to fix the drying time because surface dryness affects competition outcomes.
    2. Adjust the OD600 of the attacker cells.
      1. Centrifuge the 200 ml cultured A. tumefaciens C58 WT at 8,000 x g for 10 min. Discard the supernatant and resuspend the cell pellet with 20 ml of 0.9% NaCl.
      2. Centrifuge the A. tumefaciens cells at 5,000 x g for 5 min. Discard the supernatant and resuspend the cell pellet with 10 ml of 0.9% NaCl.
      3. Measure the OD600 of the washed A. tumefaciens cells and adjust to OD600 = 3.0 with 0.9% NaCl.
    3. Aliquot attacker cells to a 2.2 ml 96 deep-well microplate using the automated pipetting system.
      1. Open the automated pipetting system and the EzStarter software that controls the operation of the EzMate automated pipetting system. Set the software to aliquot 150 μl of the sample in Block R1 into each well of Block A; repeat the procedure once so that each well contains 300 μl liquid (Figure 6A)
      2. Dispense 50 ml of the OD600-adjusted A. tumefaciens to a pipetting reservoir and place the reservoir in Block R1, a sterilized 2.2 ml 96 deep-well microplate in block A, and a box of EzMate 250 μl tip in block D (Figure 6B).
      3. Use the automated pipetting system to aliquot 150 μl A. tumefaciens into each well of the 2.2-ml 96 deep-well microplate. Repeat the cycle once so that each well contains 300 μl A. tumefaciens (Figure 6C).


        Figure 6. Aliquot attacker cells (A. tumefaciens) to a 2.2-ml 96 deep-well microplate. A. Set the EzStarter program for dispensing 150 μl of the liquid in Block R1 in each well of Block A twice. B. Set up the working station by placing the A. tumefaciens-containing pipetting reservoir in Block R1, a 2.2 ml 96 deep-well microplate in Block A, and a box of EzMate 250 μl tip in Block D. C. The automated pipetting system dispenses A. tumefaciens to Block A.

    4. Mix the attacker and recipient cells and subject the mixture to AK agar to start the interbacterial competition.
      1. Set up the automated pipetting system.
        Remove the A. tumefaciens-containing pipetting reservoir from Block R1 and let the 2.2-ml 96 deep-well microplate with 300 μl A. tumefaciens in each well (from Step C3c) remain in Block A. Take out the overnight-subcultured E. coli plate (from Step B2c) and place it in Block C. Place the competition surface (from Step C1) in Block B. Place a box full of 250 μl tip for EzMate in Block D (Figure 7A).
      2. Use the automated pipetting system to add 10 μl overnight-cultured E. coli (Block C) into the A. tumefaciens-containing 2.2-ml 96 deep-well microplate (Block A). Mix the bacterial suspension 10 times, and drop 10 μl mixture onto the AK plate (Block B) (Video 1).


        Video 1. Mix the attacker and recipient cells and subject the mixture to AK agar. Use the automated pipetting system to add 10 μl overnight-cultured (upper right Block C) into the A. tumefaciens-containing 2.2-ml 96 deep-well microplate (upper left Block A). Mix the bacterial suspension 10 times and drop 10 μl mixture onto the AK plate (lower left Block B).

      3. Repeat each column until all 96 wells are mixed and dispensed (Figure 7B).


        Figure 7. Mix the attacker and recipient cells and subject the mixture to the competition surface (AK agar). A. Set up of the working station. Block A contains the 2.2-ml 96 deep-well microplate with 300 μl A. tumefaciens in each well (from Step C3c), Block C on the upper right contains the overnight-subcultured E. coli (from Step B2C), Block B on the lower left contains the competition surface (from Step C1), and Block D contains a full box of 250 μl tip for EzMate. B. Competition surface after dispensing 10 μl attacker/recipient mixture. Make sure all liquid mixture has dried before incubating it at 25 °C for 16 h.

      4. Dry the bacteria-containing AK agar in a laminar flow until all the competition spots are dry (about 15 min). Cover the dried AK agar with another 96-well lid. Place the plate at 25 °C for 16 h to allow competition to take place (Figure 8).
      Note: All liquid must be dried out before being placed in an incubator. The drying time of each competition plate should be the same.


      Figure 8. The competition surface after 16 h of incubation

  4. Day 4: Recover the competition spots
    1. Prepare the recovery plate.
      Pour 25 ml melted LB agar supplied with 20 μg/ml kanamycin on a 96-well lid in the laminar flow and allow it to dry for 45-60 min.
      Note: This procedure is the same for Step C1 but uses a different medium.
    2. Prepare the recovering liquid.
      Fill each well of a U-bottom 96-well plate with 200 μl of 0.9% NaCl.
    3. Sterilize the 96-pin microplate replicator as in Step A3.
    4. Recover bacterial cells from the competition spot.
      1. Place the sterilized replicator onto the competition surface (Figure 9A).
        Note: Make sure each pin contacts one competition spot.
      2. Lift the replicator (Figure 9B) and immerse the pins in the recovery liquid prepared in Step D2. Swirl and agitate the liquid to thoroughly resuspend the bacterial cells (Figure 9C).


        Figure 9. Recover the interbacterial competing bacterial cells from the AK agar. A. Stamp the bacterial mixture by a sterilized microplate replicator. B. The bacteria-containing AK agar should have an apparent hole in each spot after stamping. C. Resuspend the bacterial mixture into 200 μl of 0.9% NaCl.

    5. Spot the recovered bacterial mixture onto the LB agar with 20 μg/ml kanamycin.
      1. Set up the automated pipetting system demonstrated in Figure 10.
        Place the bacteria-containing U-shape 96-well plate (from Step D4b) in Block A, the recovery plate (from Step D1) in Block B, and a box filled with 250 μl tip for EzMate in Block D (Figure 10).


        Figure 10. Set up for spotting the recovered bacteria onto LB agar with 20 μg/ml kanamycin. Place the bacteria-containing U-shape 96-well plate (from Step D4b) in Block A, the recovery plate (from Step D1) in Block B, and a box filled with 250 μl tip for EzMate in Block D.

      2. Use the automated pipetting system to mix the recovered bacterial mixture 10 times (Block A) and transfer 10 μl of the recovered bacterial mixture (Block A) to the recovery plate (Block B) (Video 2).


        Video 2. Transfer 10 μl of the recovered bacteria to the recovery plate. Mix the recovered bacterial suspension 10 times (Block A) and transfer 10 μl of the recovered bacterial mixture (Block A) to the recovery plate (Block B). Repeat each column until all 96 wells are mixed and dispensed.

      3. Repeat each column until all the 96 wells are mixed and dispensed.
    6. Selectively grow the recipient cells.
      Dry the plate in a laminar flow until all spots are dry (takes about 15 min). Cover the dried plate with another 96-well lid. Place the plate at 37 °C overnight to allow recipient cells to grow.
      Note: All liquid must be dried out before being placed in an incubator. The drying time of each plate should be the same.

  5. Day 5: Counting the colony-forming unit (CFU) of the surviving recipient cells.
    Take a picture of the plate for data preservation. The wells with BW25113(pRL-nptII) as the recipient cell should yield no or only 1-2 colonies. Thus, the wells with multiple colonies are the E. coli mutant candidates that are less susceptible to A. tumefaciens T6SS attack.

Data analysis

  1. Check the survival rate of E. coli BW25113(pRL-nptII) control strain in each well (green circle, Figure 11), and all control wells should contain no more than 2 colonies.
  2. Record the data by taking a photo of the recovery plate.
  3. Observe the number of surviving colonies directly without the need for any equipment or software. The wells with multiple colonies (orange circle, Figure 11) are the E. coli Keio mutants that are less susceptible to A. tumefaciens antibacterial killing.
    Note: The wells with more than 7 colonies are considered less susceptible candidates and are selected as resistant candidates for further validation.


    Figure 11. Recovery plate readout. The wells with green circles carry control recipient cells (E. coli BW25113 WT carrying pRL-nptII). The wells with orange circles are the candidate strains that are less susceptible to A. tumefaciens antibacterial killing.

Notes

  1. This protocol describes a high-throughput screening system to identify genetic factors of recipient cells affecting the T6SS-mediated interbacterial competition outcome. However, this method can be adapted to any contact-dependent interbacterial competition system.
  2. The automated pipetting system used in this protocol is EzMate401, but any automated pipetting system can be used and even can be adapted by an 8-channel pipette.

Recipes

  1. 523 broth
    1. Dissolve 10 g sucrose, 8 g casein enzymatic hydrolysate, 4 g yeast extract, 3 g K2HPO4, and 0.3 g MgSO4·6H2O in 800 ml distilled water
    2. Adjust pH to 7.0 with HCl then bring the volume to 1,000 ml with distilled water
    3. Autoclave the solution at 121 °C for 20 min
    4. Store the autoclaved 523 broth at room temperature
  2. LB broth
    1. Dissolve 25 g LB powder in 1,000 ml distilled water
    2. Autoclave the solution at 121 °C for 20 min
    3. Store the autoclaved LB broth at room temperature
  3. LB agar with 20 μg/ml kanamycin
    1. Dissolve 1.5 g agar in 100 ml LB broth
    2. Autoclave the solution at 121 °C for 20 min
    3. Cool the autoclaved media and add kanamycin to a final concentration of 20 μg/ml
    4. Take 25 ml of the media using a 25 ml pipette and pour 20 ml into a 96-well cover
    5. Spread the agar evenly and wait until the agar solidifies 
    6. Store the autoclaved LB agar at 4 °C
  4. AK broth
    1. Dissolve 3 g K2HPO4, 1 g NaH2PO4, 1 g NH4Cl, 0.15 g KCl, and 9.76 g MES in 900 ml distilled water
    2. Adjust pH to 5.5 with NaOH then bring the volume to 1,000 ml with distilled water
    3. Autoclave the solution at 121 °C for 20 min
    4. Store the autoclaved AK broth at room temperature
  5. AK agar, fresh prepared
    1. Dissolve 2 g agar in 100 ml AK broth
    2. Melt the agar by microwave to heat the solution until all the agar dissolves
    3. Mix the media well and cool at room temperature for 2 min
    4. Take 25 ml hot media by using a 25-ml pipet and pour 20 ml into a 96-well cover
    5. Spread the agar evenly and wait for 1 h for the agar to solidify and dry the surface
  6. 70% ethanol
    1. Mix 737 ml of 95% ethanol with 263 ml sterile water
    2. Store at room temperature
  7. 0.6% bleach
    1. Mix 10 ml of 6% bleach with 100 ml sterile water
    2. Store at room temperature, avoid light contact

Acknowledgments

The authors thank Jemal Ali, Hagos Mohammedseid Juhar, and Maria Karmella Apaya for critically reading this protocol. This work reports a detailed protocol used to identify the E. coli recipient factors that are involved in enhancing the T6SS activity of A. tumefaciens C58 (Lin et al., 2020). The funding for this project was provided by the Ministry of Science and Technology of Taiwan (MOST) (grant no. 104-2311-B-001-025-MY3) and Academia Sinica Investigator Award (grant no. AS-IA-107-L01) to E-ML. The E. coli Keio collection library was provided by the National BioResource Project (NIG, Japan): E. coli.

Competing interests

The authors declare no conflicts of interest.

References

  1. Aoki, S. K., Pamma, R., Hernday, A. D., Bickham, J. E., Braaten, B. A. and Low, D. A. (2005). Contact-dependent inhibition of growth in Escherichia coli. Science 309(5738): 1245-1248.
  2. Cao, Z., Casabona, M. G., Kneuper, H., Chalmers, J. D. and Palmer, T. (2016). The type VII secretion system of Staphylococcus aureus secretes a nuclease toxin that targets competitor bacteria. Nat Microbiol 2(1): 16183.
  3. García-Bayona, L., Guo, M. S. and Laub, M. T. (2017). Contact-dependent killing by Caulobacter crescentus via cell surface-associated, glycine zipper proteins. eLife 6: 24869.
  4. Granato, E. T., Meiller-Legrand, T. A. and Foster, K. R. (2019). The evolution and ecology of bacterial warfare. Curr Biol 29(11): R521-R537.
  5. Hood, R. D., Singh, P., Hsu, F., Güvener, T., Carl, M. A., Trinidad, R. R., Silverman, J. M., Ohlson, B. B., Hicks, K. G., Plemel, R. L., Li, M., Schwarz, S., Wang, W. Y., Merz, A. J., Goodlett, D. R. and Mougous, J. D. (2010). A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria. Cell Host & Microbe 7(1): 25-37.
  6. Lin, H.-H., Yu, M., Sriramoju, M. K., Hsu, S.-T. D., Liu, C.-T. and Lai, E.-M. (2020). A High-throughput interbacterial competition screen identifies ClpAP in enhancing recipient susceptibility to type VI secretion system-mediated attack by Agrobacterium tumefaciens. Front Microbiol 10(3077).
  7. Souza, D. P., Oka, G. U., Alvarez-Martinez, C. E., Bisson-Filho, A. W., Dunger, G., Hobeika, L., Cavalcante, N. S., Alegria, M. C., Barbosa, L. R. S., Salinas, R. K., Guzzo, C. R. and Farah, C. S. (2015). Bacterial killing via a type IV secretion system. Nat Commun 6(1): 6453.

简介

[摘要 ] 接触依赖性细菌间竞争是细菌争夺生态位的一种常用策略。通过竞争测定法监测细菌间竞争,该竞争测定法包括在琼脂上共同培养攻击者和受体细菌细胞,然后回收存活的受体细胞。常规的细菌竞争分析需要依靠连续稀释,平板铺展和菌落计数实验来进行读数。竞争测定中对时间和劳力的高需求限制了其在大规模筛选中的用途。然而,需要高通量的细菌间竞争筛选方法来筛选参与细菌间竞争的遗传因素。在这里,使用根癌土壤杆菌作为攻击者, 作为大肠埃希菌,我们开发了一种功能强大,快速,高效和高通量的VI型分泌系统依赖性细菌竞争筛选平台。该系统无需进行连续稀释和铺板即可进行96种同时竞争分析。该系统的数据分析仅依赖于直接和直接的菌落计数。该平台可以很容易地用于识别参与任何依赖接触的细菌竞争系统的新因素。

[背景 ] 细菌已经进化出多种策略来对抗其他细菌以获取生态位适应性,而接触依赖性细菌间竞争是广泛使用的策略之一(Granato 等人,2019)。细菌可以使用其I型(T1SS),IV型(T4SS),V型(T5SS),VI型(T6SS)或VII型(T7SS)分泌系统将蛋白毒素输送到其竞争细胞,受体细胞,并以接触依赖的方式杀死它(Aoki 等人,2005; Hood 等人,2010; Souza 等人,2015; Cao 等人,2016;García-Bayona 等人,2017)。传递蛋白毒素的细菌是攻击细胞。Ť ^ h è攻击细胞也表达牛逼他同源免疫蛋白来保护自己从自我中毒。竞争测定法可以测量接触依赖性细菌竞争的强度。竞争分析可分为四个步骤:(1)培养攻击者和受体细胞,(2)通过混合两种细菌菌株并将其在琼脂板上孵育来准备竞争点,(3)细菌的回收和选择性生长(4)计算存活的受体细胞的菌落形成单位(CFU)。具有强大的细菌间竞争活性的攻击者导致受体CFU降低。

尽管竞争测定法是一项简单的实验,但仍缺乏用于96个平行竞争测定法的高通量平台。几个因素限制了竞争测定的能力。首先,每个比赛地点必须相距一定距离,以避免在恢复步骤中发生交叉污染。对分离的需要阻碍了竞争点的紧凑排列,例如96孔格式。其次,恢复步骤以及CFU计数步骤既费时又费力。在恢复步骤中,每个比赛地点都由接种环收集。记录受体细胞的CFU需要连续稀释和平板铺展。

在这里,我们提供了一个高通量的细菌竞争平台,该平台可以同时执行96个竞争测定,并且不需要连续稀释和平板铺展。在该平台上,96个竞争混合物瓦特ERE点样在琼脂平板上固化96 -w ELL 盖子并通过96针复制器微孔板代替接种环回收。使用96针微孔板复制器可确保回收相等量的细菌,这等于每个针的面积。由于此方法可最大程度地减少回收到针区域的量,因此无需连续稀释和平板铺展。同时,通过计算受体细胞的CFU,可以直接,直接地读取该平台。该平台已被用于鉴定参与增强的T6SS活性收件人因素根癌农杆菌成功(林等,2020) 。在与T6SS活性根癌农杆菌菌株共孵育1个月后,我们测试了所有3909个大肠杆菌Keio突变体的恢复情况。该方法可用于筛选由任何分泌系统介导的细菌间竞争活性增强或降低的攻击者突变体和受体突变体。

关键字:竞争分析, 高通量筛选, 细菌竞争, VI型分泌系统, 根癌农杆菌

材料和试剂


 


250 微升尖端EzMate (KLBiotech ,目录号:FX-250-R)
96针微孔板复制器(Violet BioScience ,目录号:VIO-T96)
移液器吸头盒(Labcon ,目录号1055-965-018)
96孔微孔板,平底(Basic Life,目录号:BL6052)
96孔微孔板,U底(基本寿命,目录号:BL6031)
96孔盖(Basic Life,货号:BL6171)
2.2毫升96 深孔微孔板(基本寿命,目录号:BL6038)
试管,16 x 125 mm(派热克斯(Pyrex),目录号:9820)
试管盖(Kimble,目录号:KIM-KAP 73660)
移液器(基本寿命,目录号:BL6233)
根癌农杆菌C58野生型(WT)
蔗糖(Sigma-Aldrich,目录号:S5016)
NZ-案例®另外,酪蛋白酶水解物(Sigma-Aldrich公司,目录号:N4642)
Bacto TM 酵母提取物(BD,目录号:212750)
K 2 HPO 4 (默克密理博(Merck Millipore),目录号:7758-11-4)
MgSO 4 ·7H 2 O(Merck Millipore,目录号:10034-99-8)
LB汤,米勒(BD,目录号:244620)
NaH 2 PO 4 ·2H 2 O (默克密理博,目录号:10028-24-7)
NH 4 Cl(默克密理博,目录号:017-014-00-8)
氯化钾(Sigma-Aldrich,目录号:P9541)
MES(Bio Basic,目录号:145224-94-8)
Bacto TM 琼脂(BD,目录号214010)
95%乙醇(TaiSugar ,目录号:18L乙醇)
6%漂白剂(LCY Chemical,目录号:bleach-4L)
硫酸卡那霉素(BioBasic ,目录号:KB0285-5g)
HCl(Honeywell,目录号:30721-2.5L-GL)
523汤(见食谱)
LB汤(请参阅食谱)
含20μg / ml卡那霉素的LB琼脂(请参见食谱)
AK肉汤(请参阅食谱)
AK琼脂,新鲜准备(请参阅食谱)
70%ë THANOL(见配方)
0.6%漂白剂(请参阅食谱)
 


设备


 


自动移液系统(KLBiotech ,型号:EzMate TM 401)
移液器(吉尔森,型号:移液器P2,P20,P200,P1000,8 𝗑 20 )
-80 °C 冷冻室
离心机(Eppendorf,型号:5810R)
离心转子(Eppendorf,型号:A-4-62 )
轨道振动培养箱(TKS,型号:OSI-500R)
高压灭菌器(LUXLEY,型号:HL-326)
微波炉(Panasonic,型号:NN-ST651)
500 ml PYREX锥形瓶(Corning,目录号:4980-500)
酒精燃烧器(DG Life,目录号:D92J-119250)
 


软件


 


EzStarter v4.0.0.87(Arise Biotech)
 


程序


 


第一天:预培养攻击细胞和受体细胞
预培养攻击细胞。
选择的一个单菌落根癌农杆菌生长在523琼脂C58野生型(WT)和生长它在一个测试在25用5ml 523培养基的管℃下,220rpm下过夜。
预培养对照受体细胞。
选择的单个菌落的大肠杆菌BW25113 WT携带PRL-的nptII 质粒和生长它在一个测试用5ml的LB培养基中20管微克/ ml卡那霉素。在37 °C ,220 rpm下培养细胞过夜。


注意:pRL-nptII 为BW25113 WT提供了卡那霉素抗性,因此在与根癌农杆菌共孵育后可以选择性生长。


消毒的96针米icroplate复制器。
将酒精燃烧器放在左侧。拿一个200μl的烙铁头盒,拆下烙铁头支架内部,压平烙铁头盒,然后将其放在燃烧器的右侧。用0.6%的漂白剂填充扁平盒的左孔,并用无菌水填充右孔(图1)。
用70%的乙醇填充96针微孔板复制器支架,并将其放在无菌含水孔的右侧(图1)。
注意:请勿将70%含乙醇的复制器支架放在乙醇燃烧器旁边。


 


 


图1. 用于对微孔板复制器进行灭菌的设置。从左至右:酒精燃烧器,0.6%的漂白剂,无菌水和70%的乙醇。将酒精燃烧器放在70%乙醇的远端,以免发生事故。


 


将96针mi 作物复制器的针依次浸入0.6%的漂白剂,无菌水和70%的乙醇中。让复制器在每个解决方案中停留10 s。
用酒精燃烧器消毒复制器(图2),并将复制器冷却10 s至室温。灭菌的复制子已准备好传代大肠杆菌突变体。
              注意:处理酒精燃烧器时要非常小心。


 


 


图2. 用火焰消毒微孔板复制器。将96针微孔板复制器浸入酒精燃烧器顶部的70%乙醇中后进行灭菌,然后将其放置。将复制器绕着火焰移动,以确保每个针脚上的酒精都已耗尽,然后在室温下将复制器冷却10 s。灭菌的复制子已准备好传代大肠杆菌突变体。


 


从Keio文库中预培养用作受体细胞的大肠杆菌突变体。
成U形底96孔微量培养板和填充每个孔用100 微升含20 LB培养基微克/ ml卡那霉素。
取出的大肠杆菌庆应义塾突变体板,其被存储为甘油贮存在一个2.2毫升的96深孔格式在- 80 ℃的冰箱中。将灭菌的复制子应用于冷冻的大肠杆菌原种。
注意:确保每个复制器销钉都接触甘油原液的表面。


在步骤A4a中准备的96孔微孔板的每个孔中,将含细菌的复制子标记到LB培养基上,并搅动液体,使细菌彻底重悬浮(图3)。
注意:确保每个复制器引脚都与9个6孔板的每个孔中的LB介质接触。


 


 


图3. 预培养的大肠杆菌中号从庆应义塾库utants。将含细菌的复制子盖在96孔微孔板的每个孔中的LB培养基上,并搅动液体以彻底重悬细菌。


 


再密封并返回大肠杆菌庆应义塾突变体板回- 80℃并放置复制在步骤A3A制备的0.6%的漂白剂。
在装有大肠杆菌的U型底96孔微孔板上添加96孔盖,并在37°C,220 rpm下孵育16小时(图4)。
注意:可通过在常规培养箱中添加防滑垫来实现培养。


 


 


图4.孵育所E. 大肠杆菌含U型底96孔微量培养板在培养箱中。取下一些烧瓶架,并用防滑垫替换空间。用螺钉固定防滑垫。包含防滑液的区域已准备就绪,可以孵育96孔微孔板。


 


消毒9 6针微孔板replicato R(步骤小号A3C 到A3D)。
 


第2天:传代攻击细胞和受体细胞的继代培养
注意:此步骤将BW25113(pRL-nptII )掺入含Keio突变体的微孔板中,并使它们的生长同步。


传代攻击细胞。
在500 ml烧瓶中将1 ml 来自步骤A1a的根癌农杆菌C58 WT与100 ml 523培养基进行亚培养,准备两次培养(总共200 ml)。使细菌在25 °C ,220 rpm下过夜培养。


传代受体细胞。
成U形底96孔微量培养板和填充每个孔用100 微升含20 LB培养基微克/ ml卡那霉素。使用8通道移液器在含有LB的U底96孔微孔板中传代培养自步骤A4e的2μl 大肠杆菌突变体,但将空白孔留作对照菌株BW25113(pRL-nptII )(图5) 。
注意:在进行亚培养时,请从8道移液器上取下适当的吸头,以使孔留空以用于对照菌株。


子培养2 微升BW25113(PRL-的nptII )从步骤A2到步骤B2a的空孔中(在图5中WT标签)。
生长的子CU ltured 大肠杆菌菌株在37 ℃下,220rpm下,过夜。
 


 


图5.传代大肠杆菌Keio突变体和BW25113(pRL-nptII )。使用8通道移液器将2μl 从步骤A4e 过夜培养的大肠杆菌突变体传代到装有100μl 含20μg / ml卡那霉素的LB培养基的新板中。不要亚文化的BW25113(预留井PRL-的nptII )的控制(例如,那么小号3C,D8,D10,而在这种情况下,F12)。子培养2 微升过夜培养BW25113(PRL-的nptII )插入所述空孔作为对照(例如,孔标记的WT)。


 


第3天:共同孵育攻击细胞和受体细胞以实现细菌间竞争
准备比赛场地。
将25毫升融化的AK琼脂倒在层流中的96孔盖上,并使其干燥45-60分钟。
注意:固定干燥时间至关重要,因为表面干燥会影响比赛结果。


调整攻击细胞的OD 600 。
将200 ml培养的根癌农杆菌C58 WT以8,000 xg离心10分钟。丢弃上清液,并用20 ml的0.9%NaCl重悬细胞沉淀。
以5,000 xg 离心根癌农杆菌细胞5分钟。丢弃上清液,并用10 ml的0.9%NaCl重悬细胞沉淀。
测量洗涤过的根癌农杆菌细胞的OD 600 ,并用0.9%NaCl 调节至OD 600 = 3.0 。
甲liquot攻击者的细胞到2.2毫升96深孔微孔板使用自动移液系统。
打开自动移液系统和控制EzMate 自动移液系统操作的EzStarter 软件。软件设置为等分试样150 微升的样品中的块R1到A座的每个孔的; 重复该过程一次,因此,每个孔中含有300 微升的液体(图6A)。
分配将50ml的OD 600 -adjusted 根瘤农杆菌到移液贮存器,并放置在块R1中的贮存器,灭菌2.2毫升96深孔微孔板中嵌段A,和一箱EzMate 250 微升在块d尖端(图6B)。
使用自动移液系统,以等分试样150个微升根瘤农杆菌到每个孔中的2.2毫升的96深孔微孔板的。重复循环一次,以便使每孔含有300个微升根瘤农杆菌(图6C)。
 


 


图6.将攻击者细胞(根癌农杆菌)分装到2.2 ml 96深孔微孔板中。A. 设置EzStarter 程序用于分配150 微升每孔A座在块R1的液体的两倍。B. 设立的工作站通过将根癌农杆菌在块R1含移液贮存器,一个2.2毫升96深孔微孔板中块A,和一箱EzMate 250 微升尖端块D. C. 该自动移液系统将根癌农杆菌分配给A 区。


 


混合攻击者细胞和受体细胞,并将混合物置于AK琼脂中,开始细菌间竞争。
设置自动移液系统。
取出根癌农杆菌从块R1含移液贮存器,并让用300个2.2毫升96深孔微孔板微升根瘤农杆菌在每个孔(来自步骤体C3c)留在块A.取出overnight- 传代培养E.大肠杆菌板(来自步骤B2C)在块B.将一箱充满并将其放置在块C.将竞争表面(来自步骤C1)250 微升尖端EzMate (图7A)在块d。
使用自动移液系统将10μl 过夜培养的大肠杆菌(C区)添加到含有2.2 ml 96孔96孔微孔板的A. tumefaciens中。将细菌悬浮液混合10次,然后将10μl 混合物滴到AK板上(B块)(视频1)。
 


 


视频1 。混合攻击者细胞和受体细胞,并将混合物置于AK琼脂上。使用自动移液系统将10μl 过夜培养的大肠杆菌(右上块C)添加到含有2.2 ml 96孔96孔微孔板(左上块A)的根癌农杆菌中。将细菌悬浮液混合10次,然后将10μl 混合物滴到AK板上(左下方的方框B)。


 


重复每一列,直到混合并分配所有96孔(图7B)。
 


 


图7.混合攻击细胞和受体细胞,并将混合物置于竞争表面(AK琼脂)上。A. 设置工作站。嵌段A包含用300个2.2毫升96深孔微孔板微升根瘤农杆菌在每个孔(来自步骤体C3c),块C对右上包含overnight- 传代培养的大肠杆菌(来自步骤B2C),B座上左下包含竞争表面(来自步骤C1),和块d包含的完整框250 微升尖端EzMate 。B. 分配10μl 进攻者/接受者混合物后的比赛表面。确保所有液体混合物均已干燥,然后在25 °C下孵育16小时。


 


以层流干燥含细菌的AK琼脂,直到所有竞争点都干燥(约15 m 英寸)。用另一个96孔盖覆盖干燥的AK琼脂。将板在25°C下放置16 h,以进行竞争(图8)。
注意:所有液体在放入培养箱之前必须先干燥。每个比赛板的干燥时间应相同。


 


 


图8.孵育16小时后的竞争面


 


第四天:恢复比赛点
准备回收板。在层流中将
装有20μg / ml卡那霉素的25 ml熔化的LB琼脂倒入96孔盖中的层流中,使其干燥45-60分钟。
注意:此过程与步骤C1相同,但使用的介质不同。


准备回收液。
用200填充每个孔的U底96孔板的微升0.9%NaCl中的。
与步骤A3一样,对96针微孔板复制器进行灭菌。
从竞争点恢复细菌细胞。
将灭菌的复制器放在比赛表面上(图9A)。
注意:确保每个引脚都接触一个竞争点。


提起复制器(图9B),并将销钉浸入在步骤D2中准备的回收液中。旋转并搅动液体,使细菌细胞完全重悬浮(图9C)。
 


 


图9.从AK琼脂中回收细菌竞争的细菌细胞。A. 通过灭菌的酶标仪复制细菌混合物。B.冲压后,含细菌的AK琼脂在每个斑点上应有一个明显的孔。C.将细菌混合物重悬于200μl 的0.9%NaCl中。


 


小号罐回收的细菌混合物到LB琼脂用20 微克/ ml卡那霉素。
设置自动移液系统,如图10所示。
将包含细菌的U型96孔板(来自步骤D4b)放置在A块中,将回收板(来自D1步骤)放置在B块中,并用250 图D中EzMate的微升针尖(图10)。
 


 


图10.设置用于将回收的细菌点到含有20μg / ml卡那霉素的LB琼脂上。放置含细菌马蹄形96孔板(来自步骤D4B)B1中OCK 在块B a,回收板(来自步骤D1),并填充的盒与250 微升尖端EzMate 在块D.


 


使用自动移液系统将回收的细菌混合物混合10次(方框A),然后将10μl 的回收细菌混合物(方框A)转移到回收板(方框B)中(视频2)。
 


 


视频2.将10μl 的回收细菌转移到回收板上。将回收的细菌悬浮液混合10次(方框A),然后将10 µl 的回收细菌混合物(方框A)转移到回收板中(方框B)。重复每列,直到所有96孔混合并分配为止。


 


重复每一列,直到所有96 孔混合并分配为止。
选择性生长受体细胞。
层流干燥板,直到所有斑点干燥(大约需要15分钟)。用另一个96孔盖覆盖干燥的板。将板置于37 °C 过夜,以使受体细胞生长。
注意:所有液体在放入培养箱之前必须先干燥。每块板的干燥时间应相同。


 


第5天:计数存活的受体细胞的集落形成单位(CFU)。
拍摄平板以保存数据。以BW25113(pRL-nptII )作为受体细胞的孔应不产生或仅产生1-2个菌落。因此,具有多个菌落的孔是不易受根癌农杆菌T6SS攻击的大肠杆菌突变体候选物。


 


数据分析


 


检查每个孔中大肠杆菌BW25113 (pRL-nptII )对照菌株的存活率(绿色圆圈,图11 ),并且所有对照孔中的菌落不得超过2个。
通过拍摄回收板的照片来记录数据。
无需任何设备或软件即可直接观察存活的菌落数量。具有多个菌落的孔(橙色圆圈,图11 )是大肠杆菌Keio突变体,对突变根癌农杆菌的杀菌作用较不敏感。
注意:菌落超过7个的孔被认为是较不敏感的候选物,并被选作抗性候选物以进行进一步验证。


 


 


图11 。回收板读数。带有绿色圆圈的孔带有对照受体细胞(带有pRL-nptII的大肠杆菌BW25113 WT )。带有橙色圆圈的孔是对根癌农杆菌抗菌杀伤较不敏感的候选菌株。


 


笔记


 


该协议描述了一种高通量的筛选系统,以鉴定影响T6SS介导的细菌竞争结果的受体细胞的遗传因素。但是,该方法可以适用于任何依赖接触的细菌竞争系统。
该协议中使用的自动移液系统为EzMate401,但可以使用任何自动移液系统,甚至可以通过8通道移液器进行调整。
 


菜谱


 


523 汤
溶解10克蔗糖,将8g酪蛋白酶水解物,4克酵母提取物,3gķ 2 HPO 4 和0.3g硫酸镁4 · 6H 2 ö在800毫升蒸馏水水
用HCl将pH调节至7.0,然后用蒸馏水将体积调至1,000 ml
在121°C下高压灭菌溶液20分钟
将灭菌过的523肉汤在室温下保存
LB肉汤
将25克LB粉溶解在1000毫升蒸馏水中
在121°C下高压灭菌溶液20分钟
在室温下保存高压灭菌的LB肉汤
LB琼脂含20μg / ml 纳霉素
将1.5 g琼脂溶解在100 ml LB肉汤中
              在121°C下高压灭菌溶液20分钟
冷却高压灭菌的培养基,并添加卡那霉素至终浓度为20μg / ml
需要25毫升培养基的使用25毫升PIP ETTE和倾20毫升成96 - 孔盖
均匀分散琼脂,等待琼脂凝固
              将高压灭菌的LB琼脂保存在4°C
AK汤
将3 g K 2 HPO 4,1 g NaH 2 PO 4,1 g NH 4 Cl,0.15 g KCl 和9.76 g MES溶解在900 ml蒸馏水中
用NaOH调节pH值到5.5,然后用蒸馏水调节到1,000 ml
在121°C下高压灭菌溶液20分钟
在室温下保存高压灭菌的AK肉汤
AK琼脂,新鲜准备
将2 g琼脂溶于100 ml AK肉汤中
微波融化琼脂以加热溶液,直到所有琼脂溶解
混合均匀,在室温下冷却2分钟
使用25毫升移液器吸取25毫升热介质,然后将20毫升倒入96孔盖中
均匀地分散琼脂,等待1小时,琼脂凝固并干燥表面
70%Ë THANOL
将737毫升95%的乙醇与263毫升无菌水混合
室温保存
0.6%漂白剂
将10毫升的6%漂白剂与100毫升的无菌水混合
存放于室温下,避免光线接触
 


致谢


 


作者感谢Jem al Ali,Hagos Mohammedseid Juhar 和Maria Karmella Apaya认真阅读了此协议。这项工作报告了一个详细的协议,该协议用于识别与增强根癌农杆菌C58 的T6SS活性有关的大肠杆菌受体因子(Lin 等人,2020)。该项目的资金由台湾科学技术部提供的(MOST)(批准号:104-2311-B-001-025-MY3)和中国科学院报研究者奖(批准号:AS-IA-107- L01)到E-ML。该大肠杆菌庆应义塾集合库通过提供国家生物资源:项目(NIG,日本)的大肠杆菌。


 


利益争夺


 


作者宣称没有利益冲突。


 


参考文献


 


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引用:Lin, H. and Lai, E. (2020). A High-throughput Interbacterial Competition Platform. Bio-protocol 10(17): e3736. DOI: 10.21769/BioProtoc.3736.
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