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Extraction, Purification and Quantification of Diffusible Signal Factor Family Quorum-sensing Signal Molecules in Xanthomonas oryzae pv. oryzae
水稻黄单胞菌水稻致病变种可扩散信号因子家族中群体感应信号分子的提取、纯化和定量   

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Molecular Plant Microbe Interactions
Mar 2016

Abstract

Bacteria use quorum-sensing (QS) systems to monitor and regulate their population density. Bacterial QS involves small molecules that act as signals for bacterial communication. Many Gram-negative bacterial pathogens use a class of widely conserved molecules, called diffusible signal factor (DSF) family QS signals. The measurement of DSF family signal molecules is essential for understanding DSF metabolic pathways, signaling networks, as well as regulatory roles. Here, we describe a method for the extraction of DSF family signal molecules from Xanthomonas oryzae pv. oryzae (Xoo) cell pellets and Xoo culture supernatant. We determined the levels of DSF family signals using ultra-performance liquid chromatographic system (UPLC) coupled with accurate mass time-of-flight mass spectrometer (TOF-MS). With the aid of UPLC/MS system, the detection limit of DSF was as low as 1 µM, which greatly improves the ability to detect DSF DSF family signal molecules in bacterial cultures and reaction mixtures.

Keywords: Quorum sensing (QS) (群体感应(QS)), Diffusible signal factor (DSF) (可扩散信号因子(DSF)), Xanthomonas oryzae pv. oryzae (水稻黄单胞菌水稻致病变种), Ultraperformance liquid chromatographic system (UPLC) (超高效液相色谱系统(UPLC)), Mass spectrometry (MS) (质谱法(MS)), Purification (纯化), Quantification (定量)

Background

Xanthomonas oryzae pv. oryzae (Xoo) is a causal agent of bacterial blight disease of rice, and produces multiple DSF family QS signals, including cis-11-methy-dodecenoic acid (DSF), cis-2-dodecenoic acid (BDSF), cis-10-methyl-2-dodecenoic acid (IDSF) and cis,cis-11-methyldodeca-2,5-dienoic acid (CDSF), to regulate virulence factor production (Figure 1). The biosynthesis, perception, and turnover of DSF family signals require components of the rpf (regulation of pathogenicity factors) cluster in Xoo. RpfF is a key DSF biosynthase with both acyl-ACP thioesterase and dehydratase activity. The two-component system, comprising the sensor kinase RpfC and the response regulator RpfG, plays an essential role in the perception and transduction of DSF family signals. RpfB has recently been characterized as a fatty acyl-CoA ligase (FCL), which functions in DSF family signal turnover in Xanthomonas (Wang et al., 2016; Zhou et al., 2015b). Deletion of rpfB in Xoo strain PXO99A leads to an over-production of DSF and BDSF and reduced production of extracellular polysaccharide (EPS), extracellular amylase activity. Moreover, attenuated pathogenicity has also been observed (Wang et al., 2016). Therefore, the RpfB-dependent DSF family signal turnover system is considered a naturally occurring signal turnover system in Xanthomonas. Detection and quantification of DSF family signals are very important in understanding the mechanisms of the DSF signaling system. As a result, detection methods for these signals have improved over the past few years. Initially, DSF detection relied on genetically engineered DSF biosensor-based detection systems (Slater et al., 2000; Wang et al., 2004), which provide an indirect way to analyze the activity of DSF family signals without differentiating structurally similar members of this group. Later, a detection method based on high-performance liquid chromatography (HPLC) was developed, which allowed a direct quantification of production levels of DSF family signal molecules by Xanthomonas (Wang et al., 2004; He et al., 2010; Zhou et al., 2015a). Recently, this HPLC-based method was further improved by using ultra performance liquid chromatographic system/ mass spectrometry (UPLC/MS),which offers better sensitivity and accuracy in the measurement of DSF family signals produced by Xoo, which will be presented in detail in this protocol (Zhou et al., 2015b; Wang et al., 2016).


Figure 1. Chromatogram of ethyl acetate extract of the culture supernatant of the DSF hyper-production mutant ΔrpfCΔrpfB of Xoo strain PXO99A. A. Four molecules of the DSF family QS signals are detected in the supernatant of ΔrpfCΔrpfB in nutrient broth. Among them, DSF and BDSF are the predominant signal molecules. B. The chemical structure of the four DSF family signal molecules.

Materials and Reagents

  1. Pipette tips  
    2-100 μl (Eppendorf, catalog number: 0030000.870 )
    50-1,000 μl (Eppendorf, catalog number: 0030000.919 )
    1-10 ml (Eppendorf, catalog number: 0030000.765 )
  2. 2.0 ml microtubes (Corning, Axygen®, catalog number: MCT-200-C )
  3. pH test strips (0.0-6.0 pH) (Sigma-Aldrich, catalog number: P4661 )
  4. 1.5 ml microtubes (Corning, Axygen®, catalog number: MCT-150-C )
  5. 50 ml centrifuge tube (Corning, catalog number: 430829 )
  6. Acrodisc® MS syringe filters (0.2 µm, 13 mm, WWPTFE membrane) (Pall, catalog number: MS-3301 )
  7. BD Tuberculin syringe with detachable needle (1 ml, 27 G x 1/2 in.) (BD, catalog number: 309623 )
  8. 1.5 ml Semi-micro cuvette (AS ONE, catalog number: 1-2855-02 )
  9. HPLC screw cap vials (Agilent Technologies, catalog number: 5182-0714 )
  10. 400 µl polypropylene flat bottom insert (Agilent Technologies, catalog number: 5183-2087 )
  11. Zorbax Eclipse XDB-C18 reverse phase column (Analytical, 4.6 x 150 mm, 5-micron) (Agilent Technologies, catalog number: 993967-902 )
  12. Sterile Petri dishes (90 mm) (Sartorius, catalog number: 14-555-735 )
  13. Xoo strain ΔrpfB, the rpfB deletion mutant of Xoo strain PXO99A, which overproduces DSF family signal molecules (Wang et al., 2016)
  14. Cephalexin (Sigma-Aldrich, catalog number: 1099008 )
  15. DSF (cis-11-methy-dodecenoic acid) (HPLC grade, purity ≥ 90.0%) (Sigma-Aldrich, catalog number: 42052 )
  16. BDSF (cis-2-dodecenoic acid) (HPLC grade, purity ≥ 90.0%) (Sigma-Aldrich, catalog number: 49619 )
  17. 6 N hydrochloric acid solution (HCl) (Sigma-Aldrich, catalog number: 13-1686 )
  18. Ethyl acetate (ACS reagent grade, purity ≥ 99.5%) (Sigma-Aldrich, catalog number: 676810 )
  19. Methanol (HPLC grade) (Fisher Scientific, catalog number: A452-4 )
  20. Phosphate buffered saline (PBS, pH 7.4) (Sigma-Aldrich, catalog number: P5368 )
  21. Glycerol (Sigma-Aldrich, catalog number: 49781 )
  22. Bacto peptone (BD, BactoTM, catalog number: 211677 )
  23. Bacto beef extract (BD, BactoTM, catalog number: 211520 )
  24. Sucrose (VetecTM reagent grade) (Sigma-Aldrich, catalog number: V900116 )
  25. BBL yeast extract (BD, BBL, catalog number: 211931 )
  26. NaOH
  27. Agar (BD, catalog number: 281230 )
  28. Sodium acetate (ACS reagent grade, purity ≥ 99.0%) (Sigma-Aldrich, catalog number: 791741 )
  29. Acetic acid (ACS reagent grade, purity ≥ 99.7%) (Sigma-Aldrich, catalog number: 695092 )
  30. Sterile deionized H2O
  31. Glycerol stock (see Recipes)
  32. Nutrient broth (NB, see Recipes)
  33. Nutrient agar (NA, see Recipes)
  34. 0.2 M sodium acetate solution (pH 8.0) (see Recipes)
  35. 0.2 M acetic acid solution (pH 2.7) (see Recipes)
  36. 0.2 M sodium acetate buffer (pH 3.8) (see Recipes)
  37. Cephalexin stock solution (20 mg/ml, see Recipes)

Equipment

  1. Corning® glass Erlenmeyer flasks with screw cap
    50 ml (Corning, catalog number: 4985-50 )
    250 ml (Corning, catalog number: 4985-250 )
  2. MaxQTM 6000 Incubated/Refrigerated shaker (Thermo Fisher Scientific, Thermo ScientificTM, model: MaxQTM 6000 , catalog number: SHKE6000-8CE)
  3. UV-visible spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: BioMateTM 3S )
  4. Microcentrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: SorvallTM LegendTM Micro 17R )
  5. Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM MultifugeTM X1R )
  6. Vortex mixer (VWR, catalog number: 10153-840 )
  7. CentriVap benchtop concentrator with glass lid (Labconco, catalog number: 7810040 )
  8. Pipettes  
    10-100 μl (Eppendorf, catalog number: 3120000046 )
    100-1,000 μl (Eppendorf, catalog number: 3120000062 )
    1-10 ml (Eppendorf, catalog number: 3120000089 )
  9. Fume hood
  10. Refrigerator (MEILING BIOLOGY & MEDICAL, model: DW-YL270 )
  11. VibracellTM High Intensity Ultrasonic Liquid Processors (Sonics & Materials, model: VCX 500 ) connected with a Tapered Microtip probe (tip diameter: 3 mm) (Sonics & Materials, catalog number: 630-0422 )
  12. Digital precise water bath (DAIHAN Scientific, model: WB-6 )
  13. Pear shaped glass flask, Ts 29/38, 100 ml (Tokyo Rikakikai, EYELA, catalog number: 116150 )
  14. UPLC/MS system
    Ultra-performance liquid chromatographic system (UPLC) (Agilent Technologies, model: Agilent 1290 Infinity LC ) coupled with an accurate mass time-of-flight (TOF) MS (Agilent Technologies, model: Agilent 6230 Accurate-Mass TOF MS ) equipped with an Agilent Jet Stream (AJS) electrospray ionization (ESI) source
  15. Diode array detector (Agilent Technologies, model: G4212A )
  16. pH meter (Mettler Toledo, model: FE20 )
  17. Diaphragm vacuum pump (Labconco, catalog number: 7393001 )
  18. Rotary evaporator (Tokyo Rikakikai, EYELA, model: N-1100 )
  19. Circulation cooling-water system (Tokyo Rikakikai, EYELA, model: CCA-1111 )
  20. Autoclave (Panasonic Healthcare, model: MLS-3781L )

Software

  1. Agilent MassHunter Workstation Data Acquisition Software (revision B.04)

Procedure

  1. Preparation of the pre-culture (to be used for all subsequent culture conditions)
    1. Streak Xoo strain ΔrpfB from -80 °C glycerol stock on NA plate supplemented with cephalexin at a final concentration of 20 µg/ml.
    2. Incubate the plate at 28 °C for 4 days to obtain single colonies.
    3. With a pipette tip, isolate one ΔrpfB colony and inoculate in 10 ml of NB supplemented with cephalexin with a final concentration of 20 µg/ml in a 50 ml Erlenmeyer flask.
    4. Incubate the ΔrpfB culture in the MaxQTM 6000 Incubated/Refrigerated shaker at 28 °C with shaking at 200 rpm for 36 h.

  2. Preparation of Xoo culture
    1. Measure the optical density at 600 nm (OD600) of the 1:4 diluted ΔrpfB pre-culture in a spectrophotometer and calculate the optical density of the pre-culture by multiplying the measured reading by 4 (the dilution ratio).
    2. Adjust the OD600 of the pre-culture to approximately 1.0 using NB.
    3. Add 1 ml of adjusted ΔrpfB pre-culture to 50 ml nutrient broth (1: 50 dilution) in a 250 ml Erlenmeyer flask with vent cap.
    4. Incubate the culture at 28 °C with shaking at 200 rpm for 36-48 h until it reaches early stationary phase (OD600 ≈ 2.8-3.0) for DSF/BDSF extraction.
      Note: If other bacterial strains are assayed, other specific growth conditions should be optimized.

  3. Extracellular DSF/BDSF extraction
    1. Dispense 4 ml of the ΔrpfB culture in two 2 ml centrifuge tubes and centrifuge at 8,000 x g for 15 min to obtain the culture supernatant for extracellular DSF/BDSF extraction.
    2. Transfer the supernatant to two new 2 ml microtubes and adjust its pH to 3.0-3.5 by adding adequate volume (usually 15 to 20 µl) of 6 N hydrochloric acid and monitoring pH changes with test strips.
    3. Dispense the supernatant in eight 2 ml microtubes (0.5 ml per tube), and then, add 1 ml of ethyl acetate into each tube.
    4. Vortex the microtubes at the highest speed for 5 min to extract DSF and BDSF molecules and centrifuge at 8,000 x g for 10 min to separate the ethyl acetate fraction from the aqueous fraction.
    5. Carefully collect the ethyl acetate fractions (upper layer) into four 1.5 ml microtubes by pipetting and evaporate the solvent in a CentriVap benchtop concentrator at 40 °C to complete dryness (approximately 20 min).
    6. Dispense 0.15 ml of methanol in the four 1.5 ml microtubes and vortex the microtubes vigorously for 30 sec, which ensures that all residues are re-dissolved.
    7. Centrifuge the microtubes at 2,000 x g for 1 min, and then transfer the extraction solution from each microtube into a new 1.5 ml microtube using a pipette.
    8. Evaporate the solvent in the CentriVap benchtop concentrator at 40 °C to complete dryness (approximately 40 min).
      Notes:
      1. At this point, the samples may be frozen at -20 °C for later steps or analyzed immediately as described in the following steps.
      2. For protection from inhalation of volatile solvents, steps C3 to C8 in this section should be performed in a fume hood.

  4. Intracellular DSF/BDSF extraction
    1. Decant 40 ml ΔrpfB culture in a 50 ml centrifuge tubes and harvest bacterial cells by centrifuging at 8,000 x g for 15 min at 4 °C.
    2. Discard the supernatant and add 40 ml of 1x PBS buffer in the tube to wash the cell pellets by pipetting gently.
    3. Discard the supernatant and harvest the washed bacterial cells by centrifuging at 8,000 x g for 15 min at 4 °C.
    4. Re-suspend the cell pellets in 10 ml of ice-cold sodium acetate buffer (pH 3.8).
    5. Freeze the re-suspension solution in a -20 °C freezer for 1 h and then incubate the bacterial suspension at room temperature for 30 to 60 min to thaw the suspension completely.
    6. Homogenize the cells by pipetting the thawed bacterial suspension gently.
    7. Repeat steps D5 and D6 once more.
    8. Sonicate the cell homogenate for a total of 6 min by sonicating for 3 sec, pausing for 4 sec, and repeating this sonication/pause cycle120 times (amplitude set at 20%) on ice using the Vibra cellTM High Intensity Ultrasonic Liquid Processors connected with a Tapered Microtip probe (3 mm diameter).
    9. Transfer the centrifuge tube containing the processed cell lysate into a water bath for 5 min at 95 °C to denature the total protein.
    10. Separate the soluble lysis solution with bacterial cell debris by centrifuging at 8,000 x g for 15 min at 4 °C.
    11. After centrifugation, immediately transfer the soluble lysis solution (the transparent part) to a 250 ml Erlenmeyer glass flask using a pipette. This should be performed carefully to avoid disturbing the precipitate at the bottom of the tube.
    12. Add 20 ml of ethyl acetate in the flask and close the flask with the cap immediately to avoid evaporation and spillage of the volatile solvent. Incubate the capped flask in the MaxQTM 6000 shaker at 28 °C with shaking at 200 rpm for 10 min to extract DSF and BDSF.
    13. Transfer the extraction mixture to a 50 ml centrifuge tube and centrifuge at 6,000 x g for 10 min to separate the ethyl acetate fraction from the aqueous fraction.
    14. Transfer the ethyl acetate fractions (upper layer) to a 100 ml pear shaped glass flask by pipetting carefully.
    15. Remove the solvent by rotary evaporation at 40 °C to complete dryness (approximately 5 min).
    16. Dispense 1 ml of methanol into the pear shaped glass flask to re-dissolve the residue by pipetting several times.
    17. Transfer the solution to a 1.5 ml microtube and evaporate the solvent in the CentriVap benchtop concentrator at 40 °C to complete dryness.
      Notes:
      1. At this point, the samples may be frozen at -20 °C for later steps or be analyzed immediately as described in the following steps.
      2. For protection from inhalation of volatile solvents, steps D12 to D17 in this section should be performed in a fume hood.

  5. LC/MS sample preparation
    1. Crude extraction samples can be obtained by adding 200 µl of methanol into the 1.5 ml microtube and vortex at the highest speed.
    2. To remove any insoluble particles in the sample, filter the crude sample through a MS syringe filter (0.2 µm) connected with an 1 ml disposable syringe. Collect the filtered sample (approximately 100 µl) in a new 1.5 ml microtube.
    3. Transfer 60 µl of filtered sample into a chromatography vial fitted with a flat bottom insert using a pipette. Cap the vials. Samples are now ready for LC/MS analysis.

  6. LC/MS procedure
    1. Use HPLC grade methanol (eluent B)-water (eluent A) (80:20, v/v) as mobile phase.
    2. Maintain the column temperature at 30 °C and flow rate at 0.4 ml/min.
    3. Equilibrate the column for 15 min or longer until the baseline is stable.
    4. Inject 5 μl aliquots of prepared samples onto an Agilent 1290 Infinity UPLC with a Zorbax Eclipse XDB-C18 column.
    5. Detect DSF and BDSF molecules using a diode array detector (Agilent G4212A) set to a detection wavelength of 220 nm with a band width of 4 nm.
    6. The sample is then injected into an AJS ESI ion-trap mass spectrometer in negative ionization mode.
    7. MS source parameters are as follows:
      1. Gas temperature: 325 °C
      2. Drying gas: 8 L min-1
      3. Nebulizer: 35 psig
      4. Sheath gas temperature: 350 °C
      5. Sheath gas flow: 11 L min-1
      6. Capillary voltage (Vcap): 3,500 V
      7. Nozzle voltage: 200 V
      8. The mass range: m/z 100-1,700

Data analysis

  1. Use the Agilent MassHunter Workstation Data Acquisition Software (revision B.04) to acquire data in centroid mode. The single mass-to-charge (m/z) expansion for the chromatogram is symmetric 20.0 ppm. Typical mass spectra are shown in Figures 2-4.
    1. For [BDSF-H]- detection, set the monoisotopic exact value (m/z) to 197.1547 (Figure 2)
    2. For [CDSF-H]- detection, set the monoisotopic exact value (m/z) to 209.1547 (Figure 3).
    3. For [DSF-H]- and [IDSF-H]- detection, set the monoisotopic exact value (m/z) to 211.1704 (Figure 4).


      Figure 2. Typical mass spectrum of BDSF. A. Extracted ion chromatogram (counts vs. acquisition time) of BDSF in the ethyl acetate extract of the culture supernatant of ΔrpfB dissolved in methanol (20 times concentrated), in which the y-axis indicates the counts (absolute abundance) of ionized molecules and the x-axis indicates retention time. B. MS analysis of BDSF (counts vs. mass-to-charge [m/z]) shows an exact molecular weight (z = 1) of 197.1543 Da for [BDSF-H]- (the major peak), which determines that the exact molecular weight of BDSF is 198.1547. The y-axis indicates the counts (absolute abundance) of ionized molecules, and the x-axis indicates mass-to-charge (m/z) of ionized molecules acquired from the BDSF peak in the upper panel (Panel A).


      Figure 3. Typical mass spectrum of CDSF. A. Extracted ion chromatogram (counts vs. acquisition time) of CDSF in the ethyl acetate extract of the culture supernatant of ΔrpfB dissolved in methanol (20 times concentrated), in which the y-axis indicates the counts (absolute abundance) of ionized molecules and the x-axis indicates retention time. B. MS analysis of CDSF (counts vs. mass-to-charge [m/z]) shows an exact molecular weight (z = 1) of 209.1547 Da for [CDSF-H]- (the major peak), which determines that the exact molecular weight of CDSF is 210.1547. The y-axis indicates the counts (absolute abundance) of ionized molecules, and the x-axis indicates mass-to-charge (m/z) of ionized molecules acquired from the CDSF peak in the upper panel (Panel A).


      Figure 4. Typical mass spectrum of DSF and IDSF. A. Extracted ion chromatogram (counts vs. acquisition time) of DSF and IDSF in the ethyl acetate extract of the culture supernatant of ΔrpfB dissolved in methanol (20 times concentrated), in which the y-axis indicates the counts (absolute abundance) of ionized molecules and the x-axis indicates retention time. B. MS analysis of DSF (counts vs. mass-to-charge [m/z]) shows an exact molecular weight (z = 1) of 211.1700 Da for [DSF-H]- (the major peak), which determines that the exact molecular weight of DSF is 212.1704. The y-axis indicates the counts (absolute abundance) of ionized molecules, and x-axis indicates mass-to-charge (m/z) of ionized molecules acquired from the DSF peak in the top panel (Panel A). C. MS analysis of IDSF (counts vs. mass-to-charge [m/z]) shows an exact molecular weight (z = 1) of 211.1704 Da for [IDSF-H]- (the major peak), which determines that the exact molecular weight of IDSF is 212.1704. The y-axis indicates the counts (absolute abundance) of ionized molecules, and the x-axis indicates mass-to-charge (m/z) of ionized molecules acquired from the IDSF peak in the top panel (Panel A).

  2. Integrate and quantify peak areas.
  3. Create a standard curve for DSF or BDSF by plotting the peak area vs. the known concentrations. Example standard curves are shown in Figure 5, in which the DSF or BDSF standards at the concentrations of 1 µM, 5 µM, 10 µM and 50 µM were used (Zhou et al., 2015b).
    Note: Since IDSF and CDSF are not commercially available, the standard curve for either of these two signal molecules was not created in the previous studies.


    Figure 5. Example standard curves constructed by measuring the peak intensity (PI) of varying concentrations of BDSF (A) and DSF (B) (Zhou et al., 2015b)

  4. Use the slope and y-intercept from the standard curve to calculate the concentration of DSF or BDSF in the samples. Data can be further normalized to cell count to determine the amount of DSF and BDSF per cell if necessary.

Notes

  1. This protocol is optimized for measuring DSF family signal levels in Xoo. For other bacteria, use the appropriate growth medium, antibiotics, and growth conditions.
  2. The production of DSF family QS signals in Xoo is growth phase-dependent. The rpfB gene of Xanthomonas is involved in DSF family signal turnover in vivo in the late stationary phase (Wang et al., 2016; Zhou et al., 2015b). The highest levels of DSF family signals produced by Xoo strains containing functional RpfB are present only in the early stationary phase and decrease rapidly thereafter; while the rpfB deletion mutant accumulates DSF family signals during growth (Wang et al., 2016). As a result, in order to measure DSF family signals in Xoo cells, it is essential to collect Xoo samples during the appropriate growth stage (early stationary phase), especially for those strains with functional RpfB.
  3. As mass spectrometry is a very sensitive technique, a DSF/BDSF deficient strain is recommended as a negative control for DSF/BDSF extraction and quantification in each individual experiment. For example, the DSF/BDSF deficient mutant ΔrpfF of Xoo could be used as a control sample.
  4. Acidification before extraction is a critical step for DSF/BDSF extraction in this protocol. Ensure the culture supernatant is acidified (pH less than 4.0).

Recipes

  1. Glycerol stock for one vial
    150 µl 87% glycerol
    500 µl Xoo overnight culture
  2. Nutrient broth (1 L)
    5 g Bacto peptone
    3 g Bacto beef extract
    10 g sucrose
    1 g BBL yeast extract
    Bring the volume to 1 L with dH2O and adjust the pH to 6.8 with NaOH
    Sterilize the medium by autoclaving for 20 min at 115 °C
  3. Nutrient agar (200 ml)
    Add 3 g of agar to 200 ml of nutrient broth
    Sterilize the medium by autoclaving for 20 min at 115 °C
  4. 0.2 M sodium acetate solution, pH 8.0 (100 ml)
    Dissolve 1.64 g sodium acetate in 100 ml dH2O
  5. 0.2 M acetic acid solution, pH 2.7 (100 ml)
    Dispense 1.15 ml acetic acid in 98.85 ml dH2O
  6. 0.2 M sodium acetate buffer, pH 3.8 (100 ml)
    12 ml 0.2 M sodium acetate solution
    88 ml 0.2 M acetic acid solution
  7. Cephalexin stock solution (1 ml)
    Dissolve 20 mg cephalexin powder in 1 ml of sterile dH2O
    Sterilize by filtration

Acknowledgments

This protocol is adapted from Zhou et al. (2015b) and Wang et al. (2016).
This work was supported by the research grants from the National Key Research and Development Program of China (No. 2016YFE0101000 to ZL) and the National Natural Science Foundation of China (No. 31471743 to HYW, No. 31301634 to ZL).

References

  1. He, Y. W., Wu, J., Cha, J. S. and Zhang, L. H. (2010). Rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae produces multiple DSF-family signals in regulation of virulence factor production. BMC Microbiol 10: 187.
  2. Ryan, R. P., An, S. Q., Allan, J. H., McCarthy, Y. and Dow, J. M. (2015). The DSF family of cell-cell signals: an expanding class of bacterial virulence regulators. PLoS Pathog 11(7): e1004986.
  3. Slater, H., Alvarez-Morales, A., Barber, C. E., Daniels, M. J. and Dow, J. M. (2000). A two-component system involving an HD-GYP domain protein links cell-cell signalling to pathogenicity gene expression in Xanthomonas campestris. Mol Microbiol 38(5): 986-1003.
  4. Wang, L. H., He, Y., Gao, Y., Wu, J. E., Dong, Y. H., He, C., Wang, S. X., Weng, L. X., Xu, J. L., Tay, L., Fang, R. X. and Zhang, L. H. (2004). A bacterial cell-cell communication signal with cross-kingdom structural analogues. Mol Microbiol 51(3): 903-912.
  5. Wang, X. Y., Zhou, L., Yang, J., Ji, G. H. and He, Y. W. (2016). The RpfB-dependent quorum sensing signal turnover system is required for adaptation and virulence in rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae. Mol Plant Microbe Interact 29(3): 220-230.
  6. Zhou, L., Wang, X. Y., Sun, S., Yang, L. C., Jiang, B. L. and He, Y. W. (2015a). Identification and characterization of naturally occurring DSF-family quorum sensing signal turnover system in the phytopathogen Xanthomonas. Environ Microbiol 17(11): 4646-4658.
  7. Zhou, L., Yu, Y., Chen, X., Diab, A. A., Ruan, L., He, J., Wang, H. and He, Y. W. (2015b). The multiple DSF-family QS signals are synthesized from carbohydrate and branched-chain amino acids via the FAS elongation cycle. Sci Rep 5: 13294.

简介

细菌使用群体感知(QS)系统监测和调节其人口密度。细菌QS涉及作为细菌通信信号的小分子。许多革兰氏阴性细菌病原体使用一类广泛保守的分子,称为扩散信号因子(DSF)家族QS信号。 DSF家族信号分子的测量对于了解DSF代谢途径,信号网络以及调节作用至关重要。在这里,我们描述了从Xanthomonas oryzae pv提取DSF家族信号分子的方法。 ( Xoo )细胞沉淀和Xoo 培养上清液。我们使用超高效液相色谱系统(UPLC)与准确的质量飞行时间质谱仪(TOF-MS)联合测定DSF家族信号的水平。在UPLC / MS系统的帮助下,DSF的检测限低至1μM,大大提高了在细菌培养和反应混合物中检测DSF DSF家族信号分子的能力。

背景 黄单胞菌属 oryzae pv。米糠X ae))))))))))y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y十二碳烯酸(DSF),顺式-2-十二碳烯酸(BDSF),顺式-10-甲基-2-十二碳烯酸(IDSF)和顺式<顺式 -11-甲基十二烷-2,5-二烯酸(CDSF),以调节毒力因子产生(图1)。 DSF家族信号的生物合成,感知和周转需要Xoo 中的rpf (致病因子调节)簇的组成部分。 RpfF是具有酰基-ACP硫酯酶和脱水酶活性的关键DSF生物合成酶。包含传感器激酶RpfC和响应调节剂RpfG的双组分系统在DSF家族信号的感知和转导中起重要作用。 RpfB最近被描述为脂肪酰辅酶A连接酶(FCL),其在黄单胞菌中的DSF家族信号转换中起作用(Wang等人,2016; Zhou >等,,2015b)。在Xoo 菌株PXO99A中删除 rpfB 导致DSF和BDSF的过量产生,并减少细胞外多糖(EPS)的产生,细胞外淀粉酶活性。此外,还观察到减毒的致病性(Wang等人,2016)。因此,依赖于RpfB的DSF系列信号周转系统被认为是“黄单胞病毒”中天然存在的信号转换系统。 DSF系列信号的检测和量化在理解DSF信号系统的机制方面非常重要。因此,这些信号的检测方法在过去几年有所改善。最初,DSF检测依赖于基于遗传工程的基于DSF生物传感器的检测系统(Slater等人,2000; Wang等人,2004),其提供了间接方式分析DSF家族信号的活动,而不区分该组的结构相似的成员。随后,开发了基于高效液相色谱(HPLC)的检测方法,其允许通过黄单胞菌(Xanthomonas)直接定量DSF家族信号分子的生产水平(Wang等人,2004; He等人,2010; Zhou等人,2015a)。最近,通过使用超高效液相色谱系统/质谱(UPLC / MS)进一步改进了基于HPLC的方法,该方法在由Xoo生产的DSF家族信号的测量中提供更好的灵敏度和精度,这将在本协议(Zhou等人,2015b; Wang等人,2016)中详细介绍。


图1. XFX菌株PXO99A的DSF超生产突变体ΔrpfCΔrpfB的培养上清液的乙酸乙酯提取物的色谱图A.四分子的DSF家族QS信号在营养肉汤中的ΔrpfCΔrpfB的上清液中检测到。其中,DSF和BDSF是主要信号分子。 B.四种DSF家族信号分子的化学结构。

关键字:群体感应(QS), 可扩散信号因子(DSF), 水稻黄单胞菌水稻致病变种, 超高效液相色谱系统(UPLC), 质谱法(MS), 纯化, 定量

材料和试剂

  1. 移液器提示
    2-100μl(Eppendorf,目录号:0030000.870)
    50-1,000μl(Eppendorf,目录号:0030000.919)
    1-10 ml(Eppendorf,目录号:0030000.765)
  2. 2.0ml微管(Corning,Axygen ,目录号:MCT-200-C)
  3. pH测试条(0.0-6.0 pH)(Sigma-Aldrich,目录号:P4661)
  4. 1.5ml微管(Corning,Axygen ,目录号:MCT-150-C)
  5. 50ml离心管(Corning,目录号:430829)
  6. Acrodisc ® MS注射器过滤器(0.2μm,13 mm,WWPTFE膜)(Pall,目录号:MS-3301)
  7. BD具有可拆卸针头的结核菌素注射器(1ml,27G×1/2英寸)(BD,目录号:309623)
  8. 1.5 ml半微量比色皿(AS ONE,目录号:1-2855-02)
  9. HPLC螺旋盖小瓶(Agilent Technologies,目录号:5182-0714)
  10. 400μl聚丙烯平底插入物(Agilent Technologies,目录号:5183-2087)
  11. Zorbax Eclipse XDB-C18反相柱(Analytical,4.6 x 150 mm,5-micron)(Agilent Technologies,目录号:993967-902)
  12. 无菌培养皿(90毫米)(Sartorius,目录号:14-555-735)
  13. X oo X strain>>>>>> strain which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which which>>>>/em>等,,2016)
  14. 头孢氨苄(Sigma-Aldrich,目录号:1099008)
  15. DSF(顺式-11-甲基十二碳烯酸)(HPLC级,纯度≥90.0%)(Sigma-Aldrich,目录号:42052)
  16. BDSF(顺式-2-十二碳烯酸)(HPLC级,纯度≥90.0%)(Sigma-Aldrich,目录号:49619)
  17. 6N盐酸溶液(HCl)(Sigma-Aldrich,目录号:13-1686)
  18. 乙酸乙酯(ACS试剂级,纯度≥99.5%)(Sigma-Aldrich,目录号:676810)
  19. 甲醇(HPLC级)(Fisher Scientific,目录号:A452-4)
  20. 磷酸盐缓冲盐水(PBS,pH 7.4)(Sigma-Aldrich,目录号:P5368)
  21. 甘油(Sigma-Aldrich,目录号:49781)
  22. 细菌蛋白胨(BD,Bacto TM ,目录号:211677)
  23. Bacto牛肉提取物(BD,Bacto TM ,目录号:211520)
  24. 蔗糖(Vetec TM 试剂级)(Sigma-Aldrich,目录号:V900116)
  25. BBL酵母提取物(BD,BBL,目录号:211931)
  26. NaOH
  27. 琼脂(BD,目录号:281230)
  28. 乙酸钠(ACS试剂级,纯度≥99.0%)(Sigma-Aldrich,目录号:791741)
  29. 乙酸(ACS试剂级,纯度≥99.7%)(Sigma-Aldrich,目录号:695092)
  30. 无菌去离子H 2 O O
  31. 甘油粉(见配方)
  32. 营养汤(NB,见食谱)
  33. 营养琼脂(NA,见食谱)
  34. 0.2 M醋酸钠溶液(pH 8.0)(见配方)
  35. 0.2 M乙酸溶液(pH 2.7)(见配方)
  36. 0.2 M醋酸钠缓冲液(pH 3.8)(见配方)
  37. 头孢氨苄储备液(20 mg/ml,见食谱)

设备

  1. Corning ®玻璃锥形瓶带螺帽
    50毫升(康宁,目录号:4985-50)
    250毫升(康宁,目录号:4985-250)
  2. MaxQ TM TM 6000孵育/冷冻振荡器(Thermo Fisher Scientific,Thermo Scientific TM,型号:MaxQ TM,目录号:SHKE6000-8CE)
  3. 紫外可见分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:BioMate TM 3S)
  4. 微量离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Sorvall TM Legend TM Micro 17R)
  5. 离心机(Thermo Fisher Scientific,Thermo Scientific TM 型号:Heraeus TM Multifuge TM X1R)
  6. 涡街搅拌机(VWR,目录号:10153-840)
  7. 带玻璃盖的CentriVap台式浓缩机(Labconco,目录号:7810040)
  8. 移液器
    10-100μl(Eppendorf,目录号:3120000046)
    100-1,000μl(Eppendorf,目录号:3120000062)
    1-10ml(Eppendorf,目录号:3120000089)
  9. 通风柜
  10. 冰箱(MEILING BIOLOGY& MEDICAL,型号:DW-YL270)
  11. 与锥形微尖头探针(尖端直径:3mm)连接的高强度超声波液体处理器(Sonics& Materials,型号:VCX 500)(Sonics& Materials,目录号:630-0422 )
  12. 数字精密水浴(DAIHAN Scientific,型号:WB-6)
  13. 梨形玻璃烧瓶,Ts 29/38,100ml(Tokyo Rikakikai,EYELA,目录号:116150)
  14. UPLC/MS系统
    超精密液相色谱系统(UPLC)(Agilent Technologies,型号:Agilent 1290 Infinity LC)与准确的质量飞行时间(TOF)MS(Agilent Technologies,型号:Agilent 6230 Accurate-Mass TOF MS)配合安捷伦喷射流(AJS)电喷雾离子化(ESI)源
  15. 二极管阵列检测器(Agilent Technologies,型号:G4212A)
  16. pH计(Mettler Toledo,型号:FE20)
  17. 隔膜真空泵(Labconco,目录号:7393001)
  18. 旋转蒸发器(东京Rikakikai,EYELA,型号:N-1100)
  19. 循环冷却水系统(东京Rikakikai,EYELA,型号:CCA-1111)
  20. 高压釜(Panasonic Healthcare,型号:MLS-3781L)

软件

  1. 安捷伦MassHunter工作站数据采集软件(修订版B.04)

程序

  1. 预培养物的制备(用于所有后续培养条件)
    1. 来自-80℃的NA板上的添加有头孢氨苄的终浓度为20μg/ml的甘油原液的菌株ΔxpfB。
    2. 将板在28℃孵育4天以获得单个菌落。
    3. 用移液管吸头,分离一个ΔrpfB菌落,并在50ml锥形瓶中接种10ml补充头孢氨苄的终浓度为20μg/ml的NB。
    4. 在28℃下,在MaxQ<> 6000培养的/冷冻振荡器中以200rpm摇动36小时孵育Δ rpfB 培养物。

  2. 准备文化
    1. 在分光光度计中测量1:4稀释的ΔrpfB预培养物的600nm(OD 600)的光密度,并通过以下方法计算预培养的光密度:将测量的读数乘以4(稀释比)。
    2. 使用NB将预培养物的OD <600>调整到约1.0。
    3. 在50ml排气帽的锥形烧瓶中加入1ml调整后的ΔrpfB预培养液至50ml营养肉汤(1:50稀释)。
    4. 将培养物在28℃下以200rpm摇动孵育36-48h,直至达到DSF/BDSF提取的早期固定相(OD 600> 2.8-3.0)。
      注意:如果检测到其他细菌菌株,则应优化其他特定生长条件。

  3. 细胞外DSF/BDSF提取
    1. 在2ml离心管中分配4mlΔrpfB培养物,并以8,000xg离心15分钟,得到细胞外DSF/BDSF提取的培养上清液。 />
    2. 将上清液转移到两个新的2ml微量管中,通过加入足够的体积(通常为15至20μl)的6N盐酸并用测试条监测pH变化,将其pH调节至3.0-3.5。
    3. 将上清液分配在8个2ml微管(每管0.5ml)中,然后向每个管中加入1ml乙酸乙酯。
    4. 以最高速度旋转微管5分钟以提取DSF和BDSF分子,并以8,000 x g离心10分钟,从乙酸乙酯部分中分离出水分。
    5. 小心地将乙酸乙酯部分(上层)收集到四个1.5ml微量管中,通过移液并在CentriVap台式浓缩器中在40℃下蒸发溶剂以完成干燥(约20分钟)。
    6. 在四个1.5ml微量管中分配0.15ml甲醇,并剧烈旋转微管30秒,这确保所有残余物被再溶解。
    7. 将微管以2,000 x g离心1分钟,然后使用移液管将每个微管的萃取溶液转移到新的1.5 ml微量管中。
    8. 在CentriVap台式浓缩器中在40°C下蒸发溶剂以完成干燥(约40分钟)。
      注意:
      1. 此时,样品可以在-20℃下冷冻以备后续步骤,或者如以下步骤所述立即进行分析。
      2. 为了防止吸入挥发性溶剂,本节中的步骤C3至C8应在通风橱中进行。

  4. 细胞内DSF/BDSF提取
    1. 在50ml离心管中倾倒40ml△rpfB培养物,并通过在4℃以8,000×g离心15分钟收获细菌细胞。
    2. 丢弃上清液,并在管中加入40 ml的1x PBS缓冲液,轻轻移液以洗涤细胞沉淀
    3. 弃去上清液并通过在4℃以8,000 x g离心15分钟收集洗涤的细菌细胞。
    4. 将细胞沉淀重新悬浮在10ml冰冷的乙酸钠缓冲液(pH 3.8)中
    5. 将再悬浮溶液在-20°C冷冻箱中冷冻1小时,然后将细菌悬浮液在室温下孵育30至60分钟,以完全解冻悬浮液。
    6. 轻轻移取解冻的细菌悬浮液使细胞匀浆。
    7. 再次重复步骤D5和D6。
    8. 超声处理细胞匀浆6分钟,超声处理3秒,暂停4秒,并使用Vibra细胞 TM在冰上重复该超声/暂停循环120次(振幅设定在20%)。高强度超声波液体处理器与锥形微尖头探针(直径3mm)连接。
    9. 将含有经处理的细胞裂解物的离心管转移到95℃的水浴中5分钟以使总蛋白质变性。
    10. 通过在4℃以8,000 x g离心15分钟将可溶性裂解液与细菌细胞碎片分离。
    11. 离心后,立即用可移液管将可溶性裂解液(透明部分)转移到250ml锥形玻璃烧瓶中。应仔细执行此操作,以避免干扰管底部的沉淀物
    12. 在烧瓶中加入20ml乙酸乙酯,并立即用盖子关闭烧瓶,以避免挥发性溶剂的蒸发和溢出。在MaxQ< sup><>                      
    13. 将提取混合物转移到50ml离心管中,并以6,000 x g离心10分钟,从乙酸乙酯部分中分离出来。
    14. 通过仔细移液将乙酸乙酯部分(上层)转移到100ml梨形玻璃烧瓶中
    15. 通过40°C旋转蒸发除去溶剂,以完成干燥(约5分钟)
    16. 将1ml甲醇分配到梨形玻璃烧瓶中,通过移液多次重新溶解残留物
    17. 将溶液转移到1.5 ml微量管中,并将CentriVap台式浓缩器中的溶剂在40°C下蒸发,以完成干燥。
      注意:
      1. 此时,样品可以在-20°C下冷冻以备后续步骤,或按照以下步骤所述进行立即分析。
      2. 为了防止吸入挥发性溶剂,本节中的步骤D12至D17应在通风橱中进行。

  5. LC/MS样品制备
    1. 粗提取样品可以通过以最高速度向1.5ml微管风扇中加入200μl甲醇而得到。
    2. 为了去除样品中的任何不溶性颗粒,通过与1 ml一次性注射器连接的MS注射器过滤器(0.2μm)过滤粗样品。在一个新的1.5 ml微管中收集过滤的样品(约100μl)
    3. 使用移液管将60μl过滤的样品转移到装有平底插入物的色谱柱中。盖上小瓶。样品现在可以进行LC/MS分析。

  6. LC/MS程序
    1. 使用HPLC级甲醇(洗脱剂B) - 水(洗脱液A)(80:20,v/v)作为流动相。
    2. 将柱温保持在30℃,流速为0.4ml/min
    3. 平衡色谱柱15分钟或更长时间,直到基线稳定。
    4. 使用Zorbax Eclipse XDB-C18色谱柱将5μl等分试样的样品注入Agilent 1290 Infinity UPLC。
    5. 使用二极管阵列检测器(Agilent G4212A)检测DSF和BDSF分子,其设置为波长为220 nm,带宽为4 nm。
    6. 然后将样品以负电离模式注入AJS ESI离子阱陷阱质谱仪。
    7. MS源参数如下:
      1. 气体温度:325°C
      2. 干燥气体:8L min -1
      3. 雾化器:35 psig
      4. 护套气体温度:350°C
      5. 护套气流:11 L min -1
      6. 毛细管电压(Vcap):3,500 V
      7. 喷嘴电压:200 V
      8. 质量范围:m/z 100-1,700

数据分析

  1. 使用Agilent MassHunter工作站数据采集软件(版本B.04)以质心模式获取数据。色谱图的单个质量(m/z)扩展为20.0 ppm对称。典型的质谱如图2-4所示
    1. 对于[BDSF-H] - 检测,将单同位素精确值( m/z )设置为197.1547(图2)
    2. 对于[CDSF-H] - 检测,将单同位素精确值( m/z )设置为209.1547(图3)。
    3. 对于[DSF-H] - 和[IDSF-H] - 检测,将单同位素精确值( m/z )设置为211.1704图4)。


      图2. BDSF的典型质谱。A.溶解ΔrpBB培养上清液的乙酸乙酯提取物中BDSF的提取离子色谱图(计数与采集时间)在甲醇(20倍浓缩)中,y轴表示电离分子的计数(绝对丰度),x轴表示保留时间。 BDSF(计数与质荷比[m/z]]的MS分析显示[BDSF-H] 的197.1543Da的精确分子量(z = 1) - (主峰),其确定BDSF的确切分子量为198.1547。 y轴表示电离分子的计数(绝对丰度),x轴表示从上图中的BDSF峰获得的离子化分子的质量 - 电荷(m/z) (图A)

      图3. CDSF的典型质谱图A.溶解ΔrpBB培养物上清液的乙酸乙酯提取物中CDSF的提取离子色谱图(计数与获取时间)在甲醇(20倍浓缩)中,y轴表示电离分子的计数(绝对丰度),x轴表示保留时间。 CDSF(计数与质荷比[m/z]]的MS分析显示[CDSF-H] 的209.1547Da的精确分子量(z = 1) - (主要峰),其确定CDSF的确切分子量为210.1547。 y轴表示电离分子的计数(绝对丰度),x轴表示从上图中的CDSF峰获得的离子化分子的质量 - 电荷(m/z) (图A)

      图4. DSF和IDSF的典型质谱图A.在ΔrpBB培养上清液的乙酸乙酯提取物中提取的DSF和IDSF的离子色谱图(计数与获取时间)/em>溶解在甲醇(20倍浓缩)中,其中y轴表示电离分子的计数(绝对丰度),x轴表示保留时间。 DSF(计数与质荷比[m/z]]的MS分析显示[DSF-H] 的211.1700Da的精确分子量(z = 1) - (主要峰),这决定了DSF的确切分子量是212.1704。 y轴表示电离分子的计数(绝对丰度),x轴表示从顶板中的DSF峰获得的离子化分子的质荷比(m/z )图A)。 C. IDSF的MS分析(计数与质量/质量比)显示[IDSF-H] 的211.1704Da的精确分子量(z = 1) - (主峰),其确定IDSF的确切分子量为212.1704。 y轴表示电离分子的计数(绝对丰度),x轴表示从顶板中的IDSF峰获得的离子化分子的质荷比(m/z ) (图A)
  2. 整合和量化峰值区域。
  3. 通过绘制峰面积与已知浓度来绘制DSF或BDSF的标准曲线。示例标准曲线如图5所示,其中使用浓度为1μM,5μM,10μM和50μM的DSF或BDSF标准( Zhou 等,。,2015b )。 /> 注意:由于IDSF和CDSF不能在市场上销售,所以在以前的研究中没有创建这两种信号分子之一的标准曲线。


    图5.通过测量不同浓度的BDSF(A)和DSF(B)的峰强度(PI)构建的示例标准曲线( Zhou等等。,2015b )< br />
  4. 使用标准曲线的斜率和y截距来计算样品中DSF或BDSF的浓度。数据可以进一步标准化为细胞计数,以确定必要时每个细胞的DSF和BDSF的量

笔记

  1. 该协议经过优化,用于测量Xoo 中的DSF系列信号电平。对于其他细菌,使用适当的生长培养基,抗生素和生长条件
  2. 在Xoo中生产DSF系列QS信号是增长相位依赖的。 Xanthomonas 的 rpfB 基因在晚期固定期内参与体内DSF家族信号转换(Wang等人 >,2016; Zhou 等人,,2015b)。含有功能性RpfB的Xoo 菌株产生的最高水平的DSF家族信号仅存在于早期固定期,此后迅速降低;而缺失突变体突变体在生长期间累积DSF家族信号(Wang等人,2016)。因此,为了测量Xoo 细胞中的DSF家族信号,必须在适当的生长阶段(早期固定阶段)收集Xoo样品,特别是对于那些具有功能性RpfB的菌株。
  3. 由于质谱是非常敏感的技术,建议DSF/BDSF缺陷菌株作为DSF/BDSF提取和定量在每个实验中的阴性对照。例如,可以将Xoo 的DSF/BDSF缺陷型突变体ΔxpfF用作对照样品。
  4. 提取前的酸化是本协议中DSF/BDSF提取的关键步骤。确保培养上清液酸化(pH小于4.0)

食谱

  1. 甘油储备一个小瓶
    150μl87%甘油 500微升Xoo 过夜文化
  2. 营养肉汤(1升)
    5克Bacto蛋白胨
    3克Bacto牛肉提取物
    10g蔗糖
    1克BBL酵母提取物
    使体积达到1 L,dH <2> O,并用NaOH调节pH至6.8
    通过在115℃下高压灭菌20分钟来消毒培养基
  3. 营养琼脂(200毫升)
    将3克琼脂加入200毫升营养液中 通过在115℃下高压灭菌20分钟来消毒培养基
  4. 0.2M乙酸钠溶液,pH 8.0(100ml)
    将1.64g乙酸钠溶解在100ml dH 2 O中,//
  5. 0.2M乙酸溶液,pH 2.7(100ml) 在98.85 ml dH 2 O中分配1.15 ml乙酸
  6. 0.2M乙酸钠缓冲液,pH 3.8(100ml)
    12ml 0.2M乙酸钠溶液
    88ml 0.2M乙酸溶液
  7. 头孢氨苄储备溶液(1 ml)
    将20mg头孢氨苄粉末溶解在1ml无菌dH 2 O中, 过滤灭菌

致谢

该协议适用于Zhou等人。 (2015b)和Wang等人。 (2016)。
这项工作得到了中国国家重点研究发展计划(2016YFE0101000至ZL)和国家自然科学基金(中国国家自然科学基金资助项目31471743号,第3034344号至ZL)的研究资助。

参考文献

  1. 他,YW,Wu,J.,Cha,JS和Zhang,LH(2010)。  水稻细菌性枯萎病原菌xanthomonas oryzae pv。微生物在调节毒力因子产生时产生多个DSF家族信号。 BMC Microbiol 10:187.
  2. Ryan,RP,An,SQ,Allan,JH,McCarthy,Y.和Dow,JM(2015)。 DSF家族的细胞细胞信号:扩大类细菌毒力调节因子。 11(7):e1004986。 br />
  3. Slater,H.,Alvarez-Morales,A.,Barber,CE,Daniels,MJ和Dow,JM(2000)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm .nih.gov/pubmed/11123673"target ="_ blank">涉及HD-GYP结构域蛋白的双组分系统将细胞 - 细胞信号传导与野油菜黄单胞菌中的致病基因表达相结合。 Mol Microbiol 38(5):986-1003。
  4. Wang,LH,He,Y.,Gao,Y.,Wu,JE,Dong,YH,He,C.,Wang,SX,Weng,LX,Xu,JL,Tay,L.,Fang,RX and Zhang, LH(2004)。细菌细胞通讯信号与跨王国的结构类似物。 Mol Microbiol 51(3):903-912。
  5. Wang,XY,Zhou,L.,Yang,J.,Ji,GH和He,YW(2016)。  oryzae Mol Plant Microbe Interact 29(3):220-230。
  6. Zhou,L.,Wang,XY,Sun,S.,Yang,LC,Jiang,BL and He,YW(2015a)。  鉴定和表征植物病原体Xanthomonas中天然存在的DSF家族群体感应信号转换系统。环境微生物 17(11):4646-4658。
  7. Zhou,L.,Yu,Y.,Chen,X.,Diab,AA,Ruan,L.,He,J.,Wang,H。和He,YW(2015b)。< a class = insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26289160"target ="_ blank">多个DSF家族QS信号通过FAS伸长从碳水化合物和支链氨基酸合成循环。 Sci Rep 5:13294.
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhou, L., Wang, X., Zhang, W., Sun, S. and He, Y. (2017). Extraction, Purification and Quantification of Diffusible Signal Factor Family Quorum-sensing Signal Molecules in Xanthomonas oryzae pv. oryzae. Bio-protocol 7(6): e2190. DOI: 10.21769/BioProtoc.2190.
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