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Oct 2017

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Bacterial Competition Assay Based on Extracellular D-amino Acid Production
基于细胞外D-氨基酸生成的细菌竞争测定   

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Abstract

Bacteria live in polymicrobial communities under tough competition. To persist in a specific niche many species produce toxic extracellular effectors as a strategy to interfere with the growth of nearby microbes. One of such effectors are the non-canonical D-amino acids. Here we describe a method to test the effect of D-amino acid production in fitness/survival of bacterial subpopulations within a community. Co-cultivation methods usually involve the growth of the competing bacteria in the same container. Therefore, within such mixed cultures the effect on growth caused by extracellular metabolites cannot be distinguished from direct physical interactions between species (e.g., T6SS effectors). However, this problem can be easily solved by using a filtration unit that allows free diffusion of small metabolites, like L- and D-amino acids, while keeping the different subpopulations in independent compartments.

With this method, we have demonstrated that D-arginine is a bactericide effector produced by Vibrio cholerae, which strongly influences survival of diverse microbial subpopulations. Moreover, D-arginine can be used as a cooperative instrument in mixed Vibrio communities to protect non-producing members from competing bacteria.

Keywords: D-amino acid (D-氨基酸), Competition (竞争), Co-cultivation (共培养), Viability (成活力), D-amino acid oxidase (DAAO) assay (D-氨基酸氧化酶(DAAO)测定)

Background

Bacteria live in polymicrobial communities where a great diversity of species coexist and compete for the available resources. One of the many tactics that bacteria have devised to persist in a specific niche is the production of toxic extracellular metabolites as a strategy to interfere with growth and/or viability of other microbes. D-amino acids have been known for a long time to have a powerful effect in cell shape and viability in bacterial cultures (Bopp, 1965; Fox et al., 1944; Kobayashi et al., 1948; Yaw and Kakavas, 1952; Lark and Lark, 1959; Grula, 1960; Tuttle and Gest, 1960). However, it has not been until recently that D-amino acids have gained physiological meaning when it was reported that many taxonomically unrelated bacteria could release millimolar concentrations of non-canonical D-amino acids (NCDAAs) to the extracellular medium (Lam et al., 2009). Vibrio cholerae, the causative agent of the diarrheal disease cholerae, presents a periplasmic broad spectrum racemase called BsrV reported to produce a great variety of D-amino acids, mainly D-Met and D-Leu (Lam et al., 2009; Cava et al., 2011). Further studies demonstrated that the main mode of action of these D-amino acids was through their incorporation into the peptidoglycan polymer, an essential bacterial structure that plays a role in morphology determination and cell integrity (Caparros et al., 1992; Lam et al., 2009; Cava et al., 2011). Peptidoglycan is a macromolecule composed of glycan chains crosslinked by short peptides. Interestingly, NCDAAs can be incorporated into the peptidoglycan into the 4th or the 5th residue of the peptide stem of the muropeptide subunits and this editing has a key role in synchronizing cell wall metabolism with growth arrest (Lam et al., 2009; Cava et al., 2011).

A recent study demonstrated that the cell wall is not the only target of non-canonical D-amino acids (Alvarez et al., 2018). V. cholerae and many other bacteria produce a great variety of D-amino acids which have distinct functions (Lam et al., 2009; Alvarez et al., 2018). D-arginine stands out as a fitness modulator of bacterial subpopulations, since it shows a significantly higher growth inhibitory activity against a wide diversity of bacterial species compared with other D-amino acids. In contrast to D-methionine, which has a major modulatory role in cell wall biosynthesis, D-arginine growth inhibition is suppressed by mutations in the chaperone systems and the phosphate uptake machinery in several model organisms, strongly supporting different roles for NCDAAs in bacterial physiology (Alvarez et al., 2018).

Co-cultivation is an excellent method to assess the inhibitory effect of D-arginine in mixed bacterial populations. However, when the competing bacteria present very different growth rates (e.g., V. cholerae and Caulobacter crescentus used in this study), relative cell counting can be challenging. Besides, it might be difficult to assess the role of small metabolites in species competition when other mechanisms, such as cell-to-cell dependent interactions (e.g., T6SS), can occur simultaneously. Here we present a method to assess the effect of small metabolites on bacterial populations. The design is based in the compartmentalization of the competing subpopulations in two independent rooms separated by a filter that permits diffusion of small metabolites such as amino acids. Furthermore, this method can be used to demonstrate the metabolic cooperation between producer and non-producer bacteria (e.g., V. cholerae wild-type and ΔbsrV mutant) that share extracellular D-amino acids to outcompete other species in the environment. Finally, we also describe the methodology to determine the total D-amino acid concentration in the media.

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 ScientificTM, model: MaxQTM 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: EONTM, 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

  1. Alvarez, L., Aliashkevich, A., de Pedro, M. A. and Cava, F. (2018). Bacterial secretion of D-arginine controls environmental microbial biodiversity. ISME J 12(2): 438-450.
  2. Bopp, M. (1965). [Inhibition of Agrobacterium tumefaciens by D-amino acids]. Z Naturforsch B 20(9): 899-905.
  3. Caparros, M., Pisabarro, A. G. and de Pedro, M. A. (1992). Effect of D-amino acids on structure and synthesis of peptidoglycan in Escherichia coli. J Bacteriol 174(17): 5549-5559.
  4. Cava, F., de Pedro, M. A., Lam, H., Davis, B. M. and Waldor, M. K. (2011). Distinct pathways for modification of the bacterial cell wall by non-canonical D-amino acids. EMBO J 30(16): 3442-3453.
  5. Espaillat, A., Carrasco-Lopez, C., Bernardo-Garcia, N., Pietrosemoli, N., Otero, L. H., Alvarez, L., de Pedro, M. A., Pazos, F., Davis, B. M., Waldor, M. K., Hermoso, J. A. and Cava, F. (2014). Structural basis for the broad specificity of a new family of amino-acid racemases. Acta Crystallogr D Biol Crystallogr 70(Pt 1): 79-90.
  6. Fox, S., Fling, M. and Bollenback, N. (1944). Inhibition of bacterial growth by D-leucine. J Biol Chem 155: 465-468.
  7. Grula, E. A. (1960). Cell division in a species of Erwinia. I. Inhibition of division by D-amino acids. J Bacteriol 80: 375-385.
  8. Kobayashi, Y., Fling, M. and Fox, S. W. (1948). Antipodal specificity in the inhibition of growth of Escherichia coli by amino acids. J Biol Chem 174(2): 391-398.
  9. Komarova, N. V., Golubev, I. V., Khoronenkova, S. V., Chubar, T. A. and Tishkov, V. I. (2012). Engineering of substrate specificity of D-amino acid oxidase from the yeast Trigonopsis variabilis: directed mutagenesis of Phe258 residue. Biochemistry (Mosc) 77(10): 1181-1189.
  10. Lam, H., Oh, D. C., Cava, F., Takacs, C. N., Clardy, J., de Pedro, M. A. and Waldor, M. K. (2009). D-amino acids govern stationary phase cell wall remodeling in bacteria. Science 325(5947): 1552-1555.
  11. Lark, C. and Lark, K. G. (1959). The effects of D-amino acids on Alcaligenes fecalis. Can J Microbiol 5: 369-379.
  12. Tuttle, A. L. and Gest, H. (1960). Induction of morphological aberrations in Rhodospirillum rubrum by D-amino acids. J Bacteriol 79: 213-216.
  13. Yaw, K. E. and Kakavas, J. C. (1952). Studies on the effects of D-Amino acids on Brucella abortus. J Bacteriol 63(2): 263-268.

简介

在激烈的竞争中,细菌生活在多种微生物群落中。为了坚持特定的生态位,许多物种会产生有毒的细胞外效应物作为干扰附近微生物生长的策略。这种效应子之一是非规范的D-氨基酸。在这里我们描述一种方法来测试D-氨基酸生产对社区内细菌亚群的适应/存活的影响。共培养方法通常涉及相同容器中竞争细菌的生长。因此,在这种混合培养物中,细胞外代谢物对生长的影响不能与物种间的直接物理相互作用区分开(例如T6SS效应物)。然而,通过使用允许小分解代谢物(例如L-和D-氨基酸)自由扩散的过滤单元可以容易地解决这个问题,同时将不同亚群保持在独立区室中。

通过这种方法,我们已经证明D-精氨酸是由霍乱弧菌产生的杀菌剂效应物,其强烈影响不同微生物亚群的存活。此外,D-精氨酸可作为混合菌群中的一种协同工具,用于保护非生产成员免受竞争细菌的侵害。

【背景】细菌生活在多种多样的物种共存并争夺现有资源的多种微生物群落中。细菌设计为在特定生态位持续存在的许多策略之一是产生有毒的细胞外代谢物作为干扰其他微生物生长和/或生存力的策略。已知D-氨基酸长时间在细菌培养物中具有细胞形状和活力的强大作用(Bopp,1965; Fox等人,1944; Kobayashi等人, 1948年; Yaw和Kakavas,1952年; Lark和Lark,1959年; Grula,1960年; Tuttle和Gest,1960年)。然而,直到最近,当报道许多分类学上不相关的细菌可以将毫摩尔浓度的非经典D-氨基酸(NCDAAs)释放到细胞外培养基中时,D-氨基酸已经获得了生理意义(Lam等, et al。,2009)。霍乱弧菌是腹泻病霍乱弧菌的致病因子,它提出了一种被称为BsrV的周质广谱消旋酶,据报道其产生多种D-氨基酸,主要是D-Met和D-Leu(Lam <等人,2009; Cava等人,2011年)。进一步的研究表明,这些D-氨基酸的主要作用模式是通过将它们掺入到肽聚糖聚合物中,该肽聚糖聚合物是一种在形态测定和细胞完整性中起作用的基本细菌结构(Caparros等人 ,1992; Lam等人,2009; Cava等人,2011)。肽聚糖是由短肽交联的聚糖链组成的大分子。有趣的是,NCDAAs可以掺入肽聚糖中进入鼠肽亚基肽干的第4或第5 th残基,这种编辑在同步细胞中起关键作用(Lam等人,2009; Cava等人,2011)。

最近的一项研究表明,细胞壁不是非经典D-氨基酸的唯一靶标(Alvarez et al。,2018)。 诉霍乱弧菌和许多其他细菌产生多种具有不同功能的D-氨基酸(Lam等人,2009; Alvarez等人,2018年, )。 D-精氨酸作为细菌亚群的适应性调节剂而脱颖而出,因为与其他D-氨基酸相比,它对多种细菌种类显示出显着更高的生长抑制活性。与在细胞壁生物合成中具有主要调节作用的D-甲硫氨酸相反,D-精氨酸生长抑制被伴侣系统中的突变和几种模式生物中的磷酸盐吸收机制抑制,强烈支持NCDAAs在细菌生理学中的不同作用(Alvarez et al。,2018)。

共培养是评估D-精氨酸在混合细菌种群中的抑制作用的极好方法。然而,当竞争细菌呈现出非常不同的生长速率(例如本研究中使用的例如霍乱弧菌和新月杆杆菌)时,相对细胞计数可能具有挑战性。此外,当其他机制如细胞间依赖性相互作用(例如,T6SS)可以同时发生时,可能难以评估小的代谢物在物种竞争中的作用。在这里我们提出一种评估小代谢物对细菌种群影响的方法。该设计基于两个独立房间中竞争性亚群的划分,所述两个独立的房间由允许小分解代谢物例如氨基酸扩散的过滤器隔开。此外,这种方法可用于证明生产细菌和非生产细菌(如霍乱弧菌,野生型和ΔsrvV)之间的代谢合作,突变体)共享细胞外D-氨基酸以超越环境中的其他物种。最后,我们还介绍了确定培养基中总D-氨基酸浓度的方法。

关键字:D-氨基酸, 竞争, 共培养, 成活力, D-氨基酸氧化酶(DAAO)测定

材料和试剂

  1. 有线环路或一次性接种环(SARSTEDT,目录号:86.1562.050)
  2. 15毫升试管(SARSTEDT,目录号:62.554.502)
  3. 比色杯(SARSTEDT,目录号:67.742)
  4. 150毫升Stericup过滤装置,0.22微米孔径(Merck,目录号:SCGPU01RE)
  5. 胶带
  6. Parafilm(Sigma-Aldrich,目录号:P7793-1EA)
  7. 针(BD,目录号:302200)
  8. 注射器1毫升,10毫升(BD,产品目录号:303172,307736)
  9. 1.5毫升微管(Eppendorf,目录号:0030120086)
  10. 无菌透明平底96孔盖板(Corning,Falcon ®,产品目录号:353072)
  11. 无菌玻璃珠3毫米(Merck,产品目录号:1040150500)
  12. 培养皿(SARSTEDT,目录号:82.1473)
  13. 过滤器单元,0.22μm孔径(Merck,目录号:SLGS033SB)
  14. 一次性移液管吸头(VWR,产品目录号:613-1083,613-1079,613-1077)
  15. 细菌菌株:霍乱弧菌 N16961
  16. Trigonopsis variabilis DAAO(Jose M. Guisan赠送,西班牙ICP-CSIC催化部门)(Komarova et al。,2012)
  17. L-精氨酸(L-Arg)(Sigma-Aldrich,目录号:A5006-100G)
  18. D-精氨酸(D-Arg)(Sigma-Aldrich,目录号:A2646-5G)
  19. 蒸馏水
  20. MilliQ水
  21. 盐酸发烟37%(HCl)(Merck,目录号:1003171000)
  22. 胰蛋白胨(来自酪蛋白的蛋白胨)(VWR,目录号:84610.0500)
  23. 酵母提取物(VWR,目录号:84601.0500)
  24. 氯化钠(NaCl)(Sigma-Aldrich,目录号:71376-1KG)
  25. 氢氧化钠丸(NaOH)(Merck,目录号:1064821000)
  26. 蛋白胨,肉(动物组织的酶解)(VWR,目录号:84620.0500)
  27. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:M2643-500G)
  28. 氯化钙(CaCl 2 2)(Sigma-Aldrich,目录号:C5670-500G)
  29. 细菌琼脂(VWR,目录号:84609.0500)
  30. 磷酸二氢钠一水合物(NaH 2 PO 4·2H 2 O)(Sigma-Aldrich,目录号:71507-250G) >
  31. 磷酸氢二钠七水合物(Na 2 HPO 4·7H 2 O)(Sigma-Aldrich,目录号:431478-250G) >
  32. 正磷酸85%(Merck,目录号:1005731000)
  33. 黄素腺嘌呤二核苷酸二钠盐水合物(FAD)(Sigma-Aldrich,目录号:F6625-100MG)
  34. - 苯二胺(OPD)(Sigma-Aldrich,目录号:P23938-5G)
  35. 甲醇(VWR,目录号:20847.307)
  36. 辣根过氧化物酶(Sigma-Aldrich,目录号:77332-100MG)
  37. LB培养基(见食谱)
  38. PYE培养基(见食谱)
  39. 琼脂平板(见食谱)
  40. L-和D-氨基酸储备液(见食谱)
  41. 磷酸钠缓冲液500 mM pH 7.5(见食谱)
  42. DAAO反应缓冲液(见食谱)

设备

  1. 层流柜
  2. 本生燃烧器
  3. 移液器(Gilson,产品目录号:F144563,F144565,F144566)
  4. 多道移液器(Gilson,目录号:F14403)
  5. 玻璃器皿:瓶子,量筒,烧杯
  6. pH计(VWR,目录号:662-1422)

  7. 高压灭菌器(CertoClav,目录号:8510174)
  8. 孵化器(Memmert,目录号:IN55)
  9. 摇床培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:MaxQ TM 5000,目录号:SHKE5000)
  10. 带多孔板适配器的Thermomixer(Eppendorf,产品目录号:5355000011,5363000012)
  11. 分光光度计(GE Healthcare,目录号:29003605)
  12. 酶标仪(Biotek,型号:EON TM,目录号:EONC)

程序

  1. 细菌共培养
    注意:所有步骤都需要在无菌条件下进行,通过火焰或层流柜内进行。
    1. 将来自冷冻库的Streak霍乱弧菌细胞加入到LB琼脂平板上并在37℃倒置孵育16小时。 诉霍乱 lacZ + 野生型和 V。 cholerae lacZ- bsrV 可用于评估BsrV在D-氨基酸生产中的作用。
    2. Streak C。将新鲜的crescentus细胞从冷冻库储存到PYE琼脂平板上并在28℃下倒置孵育48小时。
      注:任何细菌组合(理想情况下,其中一个D-氨基酸生产者)可用于本协议,只要两者都能在兼容条件下生长即可。
    3. 用无菌线圈从琼脂平板挑选每个菌株的单菌落,并在15ml试管中接种2ml液体PYE培养基。 28°C过夜培养液体细菌培养物(16-18小时),并以150转/分振荡。
      注意:温度和摇动可能因竞争测定中使用的细菌种类而异。
    4. 确定培养物的OD 600:在900μl新鲜PYE培养基中稀释100μl过夜培养物,转移至比色杯中,并使用分光光度计读取600nm处的吸光度。使用装满1毫升新鲜PYE的比色杯作为空白。
    5. 在10mlPYE培养基中稀释细菌悬浮液至起始浓度OD 600 = 0.01。取决于所使用的细菌菌株,OD 600到CFU ml -1比率将不同并且需要为每个菌株确定:例如,对于V 。霍乱弧菌野生型菌株,OD 600 = 0.01-10×10 7细胞ml -1。
    6. Stericup过滤装置用作共培养室。用100毫升新鲜的PYE培养基填充我们称为隔室A的上部。用200毫升新鲜的PYE培养基填充我们称为隔室B的下部。紧紧地连接两个隔间。使用Parafilm和胶带牢牢密封隔室A的盖子,避免渗漏,并将共培养室水平放置。用热针,在每个隔间顶部打一个小孔,以便接种和收集样品;这个孔可以用胶带密封(图1A)。
      注意:确保共培养室水平放置,孔朝上,以尽量减少样品溢出。在这里,我们使用100ml培养基在隔室A和200ml培养基隔室B中,因为两部分的大小不同;理想情况下,两个隔间应具有相同的尺寸,形状和中等体积。
    7. L-氨基酸补充:用10ml注射器和针头,分别向隔室A和B加入2.5ml和5ml L-精氨酸(200mM无菌原液)(最终L-氨基酸浓度分别为5mM隔间)。
      使用未补充的共培养室来评估不含L-或D-氨基酸的生长情况(图1B)。
    8. 用1ml注射器和针头将细菌接种到共培养室中:将100μl霍乱弧菌(OD 600 = 0.01)接种到隔室A中;将500μl新月香(OD 600 = 0.01)接种到隔室B中。使用未接种的室作为在没有竞争细菌的情况下污染和生长的对照(图1B)。
      注:事先评估竞争细菌的生长速率,包括生长条件(中和温度)相容性。我们使用2.5倍C. crescentus接种物来补偿PYE培养基中霍乱弧菌和C. crescentus的非常不同的生长速率。使用霍乱弧菌lacZ +野生型作为D-氨基酸生产菌。霍乱弧菌lacZ-ΔbsrV用作D-氨基酸非生产者。霍乱弧菌lacZ +野生型和霍乱弧菌lacZ-ΔbsrV的1:1混合物用于证明生产者和非生产者之间的代谢协作能够胜过其他细菌。
    9. 温和搅拌(100 rpm)在28°C温育共培养室。将用于接种的孔放在顶部以最小化样品溢出(图1C)。
      来自两个车厢的样品将在不同的时间点收集:0,24,48和72小时。


      图1.共培养室的制备。 :一种。用适当的介质填充Stericup过滤装置的两个隔室。连接并密封两个部分并添加L-氨基酸至终浓度为5mM。最后用竞争细菌接种隔室。 B.完全竞争实验的培养基和细菌组合,包括对照(C)和测试样品(测试)。 Vc:V。霍乱。抄送: C。新月柄。 C.共培养室。

    10. 在所需的时间点,使用1 ml注射器和针头从每个隔室和共培养室中收集500μl培养样品,并转移至以前标记的微管。
    11. 活细胞计数立即执行(见程序B)。将200μl新鲜收集的样品从隔室A(霍乱弧菌 em>)和B C. crescentus 转移至无菌96孔板。
    12. 在整个实验结束时进行D-氨基酸浓度的测定(参见程序C)。使用微型离心机在室温下将剩余的300μl样品在21,000×gg下离心5分钟。小心地将上清液(300μl)转移到新的微管中,并将样品储存在-20°C直到实验结束。确保没有细胞颗粒转移到新管中。

  2. 活细胞数
    注意:所有步骤都需要在无菌条件下进行,通过火焰或层流柜内进行。
    1. 确定 V的活细胞计数。霍乱和 C。 crescentus ,连续稀释细胞1:10。从先前的稀释液中转移20μl到一个新的孔中并加入180μl新鲜培养基(LB用于 V。cholerae ,PYE用于 C.crescentus ),混合物均匀。按照此程序,稀释培养物9次。确保在每个稀释步骤中更改提示。
    2. 在琼脂平板上(每种稀释液100μl)(对于霍乱弧菌,对于新月体为PYE),用于CFU计数。首先,将6-10个无菌玻璃珠添加到每个平板上,然后小心吸取细菌培养物。然后,关上盖子并搅拌平板以使细胞培养物均匀分布并使其干燥。仔细倾斜平板,取下玻璃珠。玻璃珠可以在去污染,洗涤和消毒后重新使用。对于em-V,在37°C下将CFU计数板颠倒孵育16小时。霍乱弧菌和在28℃下48小时的 C。 crescentus 。
    3. 一旦生长,在琼脂平板上计数菌落并基于所应用的稀释因子计算每毫升存活的CFU(图2)。


      图2.活细胞计数。 :一种。 ℃。 crescentus 生长表示为CFU ml-1 。 B.使用与对照相比的相对增长的替代表示。 - : C。 crescentus 控制,WT:竞争 C。 crescentus vs。 诉霍乱 lacZ + 野生型,Δ bsrV :竞争 C。 crescentus vs. V。霍乱 lacZ- Δ bsrV ,MIX:competition C。 crescentus 与1:1的V混合物。霍乱 lacZ + 野生型和 V。 cholerae lacZ - Δ bsrV 。

  3. 总D-氨基酸浓度的测定:DAAO分析
    注意:不需要无菌条件。在这两步测定中,DAAO产生α-酮酸,NH 3和H 2,从D-氨基酸中分离出来的氨基酸;过氧化物酶减少H 2 0 2 释放自由与OPD反应,导致产生2,3-二氨基吩嗪,这是一种可用分光光度计检测的比色产物(图2)。 Alvarez等,2018; Espaillat等,2014)。
    1. D-氨基酸标准曲线:在MilliQ水中以0.05,0.1,0.25,0.5,1,1.5和2mM浓度制备D-精氨酸稀释液。将20μl的每种稀释液转移至透明的平底96孔板中。转移20μLMilliQ水到另一口井作为空白。为了重复性,准备三重标准曲线和空白。
    2. 上清液样品:将保存在-20℃的上清液样品完全融化(让它们在冰上放置20分钟)。使用MilliQ水在新的微管中制备1:2,1:5和1:10稀释液。将20μl各样品(未稀释,1:2,1:5和1:10)转移至透明平底96孔板。为了重复性,准备三重反应。
    3. 准备DAAO反应缓冲液(见食谱),并使用多道移液器将60μl加入到每个孔中。最终反应体积为80μl。
    4. 关闭96孔板盖以避免蒸发,并在37℃剧烈摇动(400rpm)下孵育1小时。积极的反应会变成黄色。
    5. 使用多道移液器,向每个孔中加入2体积(160μl)的2N HCl以使反应失活。黄色将变成橙色。
    6. 使用酶标仪在492 nm处读取吸光度。

数据分析

为了重现性,生物样本应该一式三份测试。
为了测试细胞活力,计算平板上生长的菌落数并基于所应用的稀释因子和培养物铺板体积(100μl)计算每ml的活CFU(图2)。
例如:用稀释度4(1:10 4 )的平板中的153个新月须菌的菌落 /> (稀释因子)×10(体积校正)= 1.53×10 7 CFU ml -1
相对生长可通过将每个样品和条件的CFU ml -1除以不含L-氨基酸或竞争细菌的对照中的CFU ml -1 -1来计算。
为了确定D-氨基酸的总浓度:

  1. 标准曲线样品的吸光度应符合线性回归模型(图3A)。
    适合度的好处由R 2值表示。
  2. 使用线性回归模型的公式计算每个样品和复制品的总D-氨基酸浓度。考虑稀释因素,如果适用。
    丢弃吸光度值高于1.2单位的所有测量值:在该值以上,模型失去线性,并且D-氨基酸浓度的外推是错误的。
    使用1:2,1:5或1:10稀释度的值。
  3. 总D-氨基酸浓度可以如图3B所示。如果需要,可以从样品值中减去PYE培养基中的基础D-氨基酸浓度。


    图3.D-氨基酸浓度的测定A.代表性D-精氨酸标准曲线; B.共培养室48小时培养基中的总D-氨基酸浓度。 - : C。 crescentus 控制,WT:竞争 C。 crescentus vs. V。霍乱 lacZ + 野生型,Δ bsrV :竞争 crescentus 与 V。霍乱 l acZ - Δ bsrV ,MIX:competition C。 crescentus 与1:1的V混合物。霍乱弧菌lacZ + 野生型和 V。 cholerae lacZ- Δ bsrV 。

食谱

  1. LB媒介
    10.0克L-1胰蛋白胨
    5.0克L -1酵母提取物
    10.0克L -1 NaCl。
    将组分溶于蒸馏水
    使用2 N NaOH调节pH至7.0
    调整最终体积并通过高压灭菌(在121°C和1个大气压下15分钟)
    灭菌 在室温下储存
  2. PYE媒介
    2克L-1蛋白胨
    1.0克L -1酵母提取物
    1毫升的L -1 MgSO 4 4水溶液 0.5毫升L -1 M CaCl 2 2/2 将组分溶于蒸馏水
    调整最终体积并通过高压灭菌(在121°C和1个大气压下15分钟)
    灭菌 在室温下储存
  3. 琼脂平板
    1. 为了制备琼脂平板,将培养基组分溶解在蒸馏水中,加入15g L -1细菌琼脂,然后根据需要调节pH,并且最终体积并且通过高压灭菌(121℃下15分钟灭菌C和1个大气压)
    2. 让培养基冷却至50°C,并在无菌条件下倒入无菌培养皿(每个培养皿约20 ml)。让板在室温下固化。将盘子存放在4°C
  4. L-和D-氨基酸储备液
    将相应量的L-精氨酸或D-精氨酸溶解在MilliQ水中至终浓度为200mM
    使用0.22微米孔径的过滤单元消毒
    在室温下储存
  5. 磷酸钠缓冲液500mM pH 7.5
    12.9克L -1 NaH 2 PO 4 4 H 2 O
    109.1克L -1 Na 2 HPO 4·7H 2 O
    将组分溶于蒸馏水
    用正磷酸25%(v / v)或2N NaOH调节pH值至7.5,并调节最终体积
    在室温下储存
  6. DAAO反应缓冲液
    每次反应,制备60μl含有磷酸钠缓冲液33.3mMpH7.5,FAD8.3μgml -1,新鲜制备的OPD83.3μgml -1的最终体积缓冲液(参见下表) ,辣根过氧化物酶41.7μgml -1和三角酵母DAAO(Komarova等人,2012)33.3μgml-1 1 。
    除非另有说明,所有储备溶液均在MilliQ水中制备。
    在-20°C分装并储存FAD,辣根过氧化物酶和DAAO储液

致谢

这项工作详细报道了以前用于证明霍乱弧菌使用D-精氨酸以使其他细菌失效的细菌竞争测定法(Alvarez et al。 ,2018)。这项工作由Knut和Alice Wallenberg基金会(KAW),瑞典分子感染医学实验室(MIMS),瑞典研究委员会和Kempe基金会资助。作者声明不存在利益冲突或利益冲突。

参考

  1. Alvarez,L.,Aliashkevich,A.,de Pedro,M.A。和Cava,F。(2018)。 D-精氨酸的细菌分泌控制着环境微生物的多样性 ISME J 12(2):438-450。
  2. Bopp,M.(1965)。 [用D-氨基酸抑制根癌土壤杆菌]。 a> Naturforsch B 20(9):899-905。
  3. Caparros,M.,Pisabarro,A.G。和de Pedro,M.A。(1992)。 D-氨基酸对大肠杆菌中肽聚糖的结构和合成的影响 Bacteriol 174(17):5549-5559。
  4. Cava,F.,de Pedro,M.A.,Lam,H.,Davis,B.M。和Waldor,M.K。(2011)。 通过非经典D-氨基酸修饰细菌细胞壁的不同途径 EMBO J 30(16):3442-3453。
  5. Espaillat,A.,Carrasco-Lopez,C.,Bernardo-Garcia,N.,Pietrosemoli,N.,Otero,LH,Alvarez,L.,de Pedro,MA,Pazos,F.,Davis,BM,Waldor,MK ,Hermoso,JA和Cava,F。(2014)。 氨基酸消旋体新家族广泛特异性的结构基础。 Acta Crystallogr D Biol Crystallogr 70(Pt 1):79-90。
  6. Fox,S.,Fling,M.和Bollenback,N.(1944)。 D-亮氨酸抑制细菌生长 J Biol Chem 155:465-468。
  7. Grula,E.A。(1960)。在欧文氏菌种中的细胞分裂。 I. D-氨基酸对分裂的抑制作用 J Bacteriol 80:375-385。
  8. Kobayashi,Y.,Fling,M.和Fox,S.W。(1948)。 氨基酸抑制大肠杆菌生长的对映体特异性。 J Biol Chem 174(2):391-398。
  9. Komarova,N.V.,Golubev,I.V.,Khoronenkova,S.V.,Chubar,T.A。和Tishkov,V.I。(2012)。 从酵母中制备D-氨基酸氧化酶的底物特异性三角酵变:定向诱变Phe258残基。生物化学(Mosc) 77(10):1181-1189。
  10. Lam,H.,Oh,D.C.,Cava,F.,Takacs,C.N。,Clardy,J.,de Pedro,M.A。和Waldor,M.K。(2009)。 D-氨基酸控制细菌的固定相细胞壁重塑。 科学 325(5947):1552-1555。
  11. Lark,C。和Lark,K.G。(1959)。 D-氨基酸对产碱杆菌的影响。 > Can J Microbiol 5:369-379。
  12. Tuttle,A.L。和Gest,H。(1960)。 用D-氨基酸诱导紫红红螺杆菌的形态畸变 / a> J Bacteriol 79:213-216。
  13. Yaw,K.E。和Kakavas,J.C。(1952)。 D-氨基酸对布鲁氏菌流产的影响研究。< / a> J Bacteriol 63(2):263-268。
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引用:Alvarez, L. and Cava, F. (2018). Bacterial Competition Assay Based on Extracellular D-amino Acid Production. Bio-protocol 8(7): e2787. DOI: 10.21769/BioProtoc.2787.
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