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Determination of NO and CSF Levels Produced by Bacillus subtilis
枯草芽孢杆菌生成的NO和CSF水平测定   

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Nature Communications
Jan 2017

Abstract

The cell-to-cell communication and division of labour that occurs inside a beneficial biofilm produce significant differences in gene expression compared with the gene expression pattern of cells grew under planktonic conditions. In this sense, the levels of NO (nitric oxide) and CSF (Competence Sporulation Stimulating Factor) produced in Bacillus subtilis cultures have been measured only under planktonic growth conditions. We sought to determine whether NO and/or CSF production is affected in B. subtilis cells that develop as a biofilm. To measure the production levels of the two prolongevity molecules, we grew B. subtilis cells under planktonic and biofilm supporting condition.

Keywords: Bacillus subtilis (枯草芽孢杆菌), Planktonic growth (浮游生长), Biofilm (生物膜), NO (NO), CSF (CSF)

Background

NO is a key signalling molecule, playing a role in a variety of biological processes in vertebrates (Kerwin et al., 1995). C. elegans is unable to produce its own NO but is able to incorporate the NO produced by B. subtilis (Cabreiro and Gems, 2013; Gusarov et al., 2013; Kim, 2013; Clark and Hodgkin, 2014). Most organisms produce NO through aerobic conversion of L-arginine to L-citrulline in a reaction catalysed by the enzyme NO synthetase encoded by the nos gene (Sudhamsu and Crane, 2009). E. coli strains, several of which are routinely used to feed worms (OP50, HB101) (Cabreiro and Gems, 2013; Kim, 2013; Clark and Hodgkin, 2014), are not proficient in aerobic NO production because they lack a functional copy of nos (Sudhamsu and Crane, 2009). However, E. coli can produce NO under anaerobic/microaerophilic conditions by a series of biochemical reactions associated with the anaerobic respiratory chain of the bacterium (Corker and Poole, 2003). In such case, E. coli might find permissive conditions for NO production in the oxygen-depleted environment of the worm intestine. Bacteria produced-NO in worm gut that freely diffuses through the plasma membrane is oxidized to nitrate and nitrite, and thus, the concentration of nitrate and nitrite are directly proportional to the level of NO production (Gusarov et al., 2013) and can be determined using a colorimetric assay.

Intra- and interspecific quorum sensing (QS) constitutes molecules that bacteria use in nature to communicate with each other and with cells of different kingdoms (Shapiro, 1998; Ben Jacob et al., 2004; Parsek and Greenberg, 2005; Bassler and Losick, 2006). B. subtilis QS pentapeptide CSF (Competence Sporulation Stimulating Factor, also named PhrC) (Lazazzera et al., 1997) was previously reported to contribute to intestinal homeostasis by activating key survival pathways of the host (p38 MAP kinase and protein kinase B) and by inducing cytoprotective heat shock proteins (Hsps) (Fujiya et al., 2007; Willians, 2007). These effects of CSF (Willians, 2007) depend on its uptake by the protein OCTN2, a host cell membrane transporter of organic cations present in the apical face of epithelial cells (Fujiya et al., 2007). To quantify bacteria-produced CSF, promoters are commonly fused to heterologous reporter genes that encode enzymes that can be quantified using highly sensitive assays. Typically, incorporation to B. subtilis of a reporter lacZ gene, encoding β-galactosidase (β-gal) to the promoter region of a gene of interest, can be used to determine the level of CSF produced by this probiotic bacterium. When used in this manner, the Pcsf-lacZ fusion is integrated into the chromosome at the non-essential amyE locus. The basic colorimetric assay described here is the simplest and less expensive assay for quantifying β-gal activity. The cells are lysed and an aliquot of the extract is mixed with the reaction substrate, O-nitrophenyl-β-D-galactopyranoside (ONPG). When the yellow product becomes visible, the optical densities of the samples are determined spectrophotometrically.

Materials and Reagents

  1. Pipette tips
  2. Petri dishes 60 x 15 mm 500/cs (Fisher Scientific, catalog number: FB0875713A )
  3. Sterile 150 x 20 mm-culture tube (Sigma-Aldrich, catalog number: C1048 )
  4. Sterile 150 x 16 mm-culture tube (Science Lab Supplies, catalog number: 6135-5-012 )
  5. 1.5 ml tube
  6. Amicon Ultra 0.5 ml centrifugal filters MWCO 10 kDa (Sigma-Aldrich, catalog number: Z677108)
    Manufacture: EMD Millipore, catalog number: UFC501096 .
  7. Amicon Ultra centrifugal filter units Ultra-4, MWCO 30 kDa (Sigma-Aldrich, catalog number: Z648035)
    Manufacture: EMD Millipore, catalog number: UFC803024 .
  8. Cryovial (Simport, catalog number: T310-2A )
  9. 96-well solid plate (Colorimetric assay) (Cayman Chemical, catalog number: 400014 )
  10. 96-well cover sheet (Cayman Chemical, catalog number: 400012 )
  11. Derivative of B. subtilis NCIB31610
  12. Bacillus subtilis NCIB3610 and JH642 (Bacillus Genetic Stock Center, catalog numbers: 3A1 and 1A96 )
  13. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  14. Bacto peptone (BD, BactoTM, catalog number: 211677 )
  15. Nitrate/Nitrite Colorimetric Assay Kit (Cayman Chemical, catalog number: 780001 )
  16. Nitrate/Nitrite assay buffer (Cayman Chemical, catalog number: 780022 )
  17. Nitrate Reductase Enzyme Preparation (Cayman Chemical, catalog number: 780010 )
  18. Nitrate Reductase Cofactor Preparation (Cayman Chemical, catalog number: 780012 )
  19. Nitrate Standard (Cayman Chemical, catalog number: 780016 )
  20. Griess Reagent R1 (Cayman Chemical, catalog number: 780018 )
  21. Griess Reagent R2 (Cayman Chemical, catalog number: 780020 )
  22. Luria Bertani broth (Sigma-Aldrich, catalog number: L3522 )
  23. Luria Bertani broth with agar (Sigma-Aldrich, catalog number: L2897 )
  24. Agar (Sigma-Aldrich, catalog number: A1296 )
  25. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P2222 )
  26. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P9791 )
  27. MOPS (Sigma-Aldrich, catalog number: M9381 )
  28. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: M1880 )
  29. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C3881 )
  30. Manganese(II) chloride (MnCl2·2H2O) (EMD Millipore, catalog number: 1059340100 )
  31. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  32. Iron(III) chloride hexahydrate (FeCl3·6H2O) (Sigma-Aldrich, catalog number: 236489 )
  33. Glutamate (Sigma-Aldrich, catalog number: 49621 )
  34. Tryptophan (Sigma-Aldrich, catalog number: T0254 )
  35. Phenylalanine (Sigma-Aldrich, catalog number: P2126 )
  36. Chloramphenicol (Sigma-Aldrich, catalog number: C0378 )
  37. 100% ethanol (Sigma-Aldrich, catalog number: E7023 )
  38. Cholesterol (Sigma-Aldrich, catalog number: C8667 )
  39. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S3264 )
  40. Sodium phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S3139 )
  41. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  42. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  43. Lysozyme from chicken egg white (Sigma-Aldrich, catalog number: L6876 )
  44. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  45. O-nitrophenyl-β-D-galactopyranoside (Sigma-Aldrich, catalog number: N1127 )
  46. Sodium carbonate (Na2CO3) (Sigma-Aldrich, catalog number: 223484 )
  47. 10 N NaOH
  48. Nematode growth medium (NGM) broth (see Recipes)
  49. MSgg medium (see Recipes)
  50. 5 mg/ml chloramphenicol (see Recipes)
  51. 5 mg/ml cholesterol (see Recipes)
  52. 1 M MgSO4 (see Recipes)
  53. 1 M CaCl2 (see Recipes)
  54. Phosphate buffer (see Recipes)
  55. Z buffer (see Recipes)
  56. 10 mg/ml lysozyme solution (see Recipes)
  57. 10% Triton X-100 solution (see Recipes)
  58. O-nitrophenyl-β-D-galactopyranoside (ONPG) 4.5 mg/ml (see Recipes)
  59. 1.2 M Na2CO3 (see Recipes)
  60. 50% glycerol (see Recipes)
  61. 100 mM MOPS pH = 7 (see Recipes)

Equipment

  1. Erlenmeyer flask (Fisher Scientific, catalog number: FB5006000 )
  2. Pipettor (Gilson, catalog number: F167300 )
  3. Glass pipette
  4. Refrigerated incubator (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 51028064 ; Fisher Scientific, catalog number: 37-20 )
    Note: The product “Fisher Scientific, catalog number: 37-20 ” has been discontinued.
  5. Water bath (AQUA® LYTIC incubator 37 °C)
  6. Freezers (-20 °C; So-Low Environmental Equipment) (Siemens, model: So-Low Ultra C85-22 )
  7. Autoclave (Tuttnauer, model: Model 6690 )
  8. Stirring hotplate (Corning, catalog number: 6795-620 )
  9. Centrifuge (Eppendorf, model: 5430 )
  10. Tabletop centrifuge (Eppendorf, model: 5424 )
  11. Molecular Dynamics Model SpectraMAX 340 PC microplate reader (Molecular Devices, model: SpectraMAX 340PC384 )

Procedure

  1. Preparation of B. subtilis strains culture
    1. Using sterile procedures, add 2 ml of sterile MSgg or NGM broth (see Recipes) in a sterile 150 x 20 mm-tube. Inoculate medium with 20 µl of a B. subtilis strains frozen culture (JH642 or NCIB3610) (see Note 3). For NGM broth’s preparation, mix 3 g NaCl and 2.5 g Bacto peptone in an Erlenmeyer flask. Add to 1 L of dH2O. Autoclave at 121 °C for 20 min. Cool flask in 55 °C water bath for 15 min. Add 1 ml 1 M CaCl2, 1 ml 5 mg/ml cholesterol in ethanol, 1 ml 1 M MgSO4 and 25 ml 1 M KPO4 buffer. Swirl to mix well.
    2. Incubate for 16-18 h at 37 °C with shaking (overnight culture).

  2. Preparation of B. subtilis strains culture supporting biofilm formation
    1. Using sterile procedures, add 2 ml of sterile MSgg or NGM broth in a sterile 150 x 16 mm-tube. Inoculate medium with 20 µl of a B. subtilis strain overnight culture.
    2. Incubate at 25 °C for 36 h with (planktonic condition) or without shaking (biofilm-supporting condition) (see Note 4).

  3. Preparation of B. subtilis strains samples for NO determination (according to the kit’s instructions)
    1. Take the tubes and disrupt the biofilm by vortexing until a homogenous suspension is obtained. Take a 100 µl aliquot for CFU (colony-forming units) counting (see Note 1).
    2. Centrifuge at 12,470 x g for 20 min. Transfer the supernatant to a sterile 1.5 ml Eppendorf tube and discard pellet.
    3. Ultrafilter using a 10 or 30 kDa molecular weight cut-off filter using a commercially available centrifuge. The filters should be pre-rinsed with ultrapure water prior ultrafiltration.
    4. Wait for 3 h, the time required for the conversion of nitrate to nitrite.

  4. Preparation of a standard curve (according to the kit’s instructions)
    1. Standard curve for nitrate and nitrite must be included. If you plan to measure only total NO products (nitrate + nitrite), only the nitrate standard curve is required.
    2. In a clean tube, place 0.9 ml of ‘Nitrate Assay Buffer’ from the kit. To this, add 0.1 ml of reconstituted ‘Nitrate Standard’ and vortex for 20 sec. The concentration of this stock standard is 200 µM.
    3. Use this standard for the preparation of the nitrate standard curve as described below. The standard curve for nitrate is prepared by addition of reagents to the plate wells in the following way (Figure 1):


      Figure 1. Preparation of nitrate standard curve according to the kit’s instructions

  5. Performing the assay (according to the kit’s instructions)
    1. Add 200 µl of water or ‘Nitrate Assay Buffer’ to the blank wells. Do not add any other reagents to these wells.
    2. Add up to 80 µl of sample to the wells in a pattern you choose. The final volume must be adjusted to 80 µl using the ‘Nitrate Assay Buffer’ solution.
    3. Add 10 µl of the ‘Enzyme Cofactor Mixture’ to each of the wells (standards and unknown) (see Note 6).
    4. Add 10 µl of the ‘Nitrate Reductase Mixture’ to each of the wells (standards and unknown).
    5. Cover the plate with the plate cover and incubate at room temperature for 2 h. It is not necessary to shake the plate during incubation.
    6. After incubation time, add 50 µl of the ‘Griess Reagent R1’ to each of the wells (standards and unknown) (see Note 7). Pipette up and down 2-3 times.
    7. After incubation time, add 50 µl of the ‘Griess Reagent R2’ to each of the wells (standards and unknown) (see Note 7). Pipette up and down 2-3 times.
    8. Allow the color to develop for 10 min at room temperature. It is not necessary to cover the plate.
    9. Read the absorbance at 540 nm using a plate reader.

  6. Calculations
    1. Subtract the absorbance value of the blank wells from the absorbance values of all the other wells.
    2. Make a plot of the absorbance at 540 nm as a function of nitrate concentration. The nitrate standard curve is used for determination of total nitrate + nitrite concentration. An example is shown in Figure 2.
    3. Calculate NO concentration as follows: (Nitrate + Nitrite) (µM) = ([A540 - y - intercept)/slope] x (200 µl/volume of sample used (µl)) x dilution
    4. According to the CFU obtained in step C1, normalize NO (µM) to 1 x 108 CFU.


      Figure 2. A typical standard curve (according to the kit’s instructions)

  7. Determination of β-galactosidase activity (Arabolaza et al., 2003) (see Note 5)
    1. The bacterial strain used in this study is a derivative of B. subtilis NCIB31610 containing Pcsf-lacZ fusion integrated into the bacterial chromosome at the non-essential amyE locus.
    2. Introduce 2 ml of MSgg or NGM broth aseptically into previously autoclaved 150 x 16 mm-culture tube and 2 µl of 5 mg/ml chloramphenicol.
    3. Each tube is inoculated with 20 µl of an overnight culture; uninoculated tubes containing only MSgg or NGM broth serve as controls for cross-contamination.
    4. The cultures are placed on a rotating shaker at 130 rpm (planktonic conditions) or without shaking (biofilm-supporting conditions) and incubated at 37 °C.
    5. The tubes are incubated for at least 36 h. To determine the culture density, take the tubes and disrupt the biofilm by vortexing until a homogenous suspension is obtained. Take a 100 µl aliquot for CFU counting (see Note 1). The A525 of the diluted culture (1/10 dilution) is determined using a Molecular Dynamics Model SpectraMAX 340PC microplate reader. As discussed below, dilution of the culture is required because cell density is not proportional to A525 when the values are above 0.8-1.
    6. Two aliquots of 1 ml from the disrupted culture are arrested by the addition of 2 µl of 5 mg/ml chloramphenicol (see Recipes) using a repeater pipettor and the tubes are placed on ice. Then, centrifuge the 1.5 ml Eppendorf tubes, for 5 min at 4 °C at 12,470 x g. Supernatants are removed and pellets are either frozen at -20 °C or assayed immediately (see Note 2) (Figure 3).


      Figure 3. Flowchart image showing the basic steps of culturing bacteria in planktonic or biofilm-supporting conditions and samples collection

    7. For cell permeabilization, 0.73 ml of Z buffer (see Recipes) is dispensed into the pellet.
    8. A reagent blank containing 0.73 ml of Z buffer is run with the unknowns.
    9. Add 10 µl of 10 mg/ml fresh lysozyme solution (see Recipes) to the tubes and incubate for 15 min in a 37 °C water bath.
    10. Transfer the tubes to bench and add 10 µl of 10% Triton X-100 (see Recipes). Permeabilization is accomplished by aspirating and dispensing the mixtures 10 times.
    11. Immediately after permeabilization, at zero time, the assay is initiated by adding 100 µl of 4.5 mg/ml ONPG (see Recipes) to each tube. In the end of the assay, the tubes are incubated at 28 °C for the appropriate length of time, e.g., 15 min, before the reaction is terminated by the addition of 150 µl of 1.2 M Na2CO3 to each tube (see Recipes).
    12. Then, the tubes’ content is transferred to a microplate, which is introduced into the plate reader and the A420 values are determined.
    13. Calculation of β-galactosidase activity expressed as Miller units (M.U.) is performed as follows:

      M.U. = (OD420 x 66.7)/(OD525 x ml used)

      where,
      OD420 is the optical density of β-gal assay,
      OD525 is the optical density of the bacterial culture,
      66.7 is a correction factor,
      ml used is the volume of Z buffer used to resuspend the bacterial pellet (in this case 0.73 ml).
    14. According to the UFC obtained in step C1, normalize β-gal (M.U.) to 1 x 108 UFC.

Data analysis

  1. The absorbance data for A420 and A525 are transferred to a Microsoft Excel spreadsheet and β-galactosidase specific activities in Miller units are calculated.
  2. Each assay should be repeated at least three times for quadruplicate.
  3. Use the Student’s t-test with a significance cut-off level of P < 0.05 for comparisons between two groups.
  4. Use the one-factor (ANOVA) variance analysis and correct by the post hoc Bonferroni test for multiple comparisons.

Notes

  1. For the LB plates used to seed Bacillus subtilis, we strongly recommend drying plates at 45 °C for 20 min or 37 °C for 40 min upside down before to drop the dilutions onto each plate to avoid the sliding movement of this bacterium in solid surface with water drops from condensation.
  2. Samples should be used immediately or store for 1 month at -20 °C.
  3. Bacteria on an LB agar plate can be stored at 4 °C for a few weeks. However, for long-term storage, it is recommended to make glycerol stocks. The addition of glycerol stabilizes the frozen bacteria, preventing damage to the cell membranes and keeping the cells alive. A glycerol stock of bacteria can be stored stably at -80 °C for many years. For this, prepare an overnight bacteria culture, add 500 μl of the overnight culture to 500 μl of 50% glycerol (see Recipes) in a 2 ml cryovial and gently mix. Freeze the glycerol stock tube at -80 °C. The stock is now stable for years, as long as it is kept at -80 °C. Subsequent freeze and thaw cycles will reduce shelf life. To recover bacteria from your glycerol stock, open the tube and use a sterile loop or pipette tip to scrape some of the frozen bacteria off of the top. Do not let the glycerol stock thaw. Always keep your glycerol stock on ice while procedure is carried out. Streak the bacteria onto an LB agar plate. Grow Bacillus subtilis overnight at 37 °C.
  4. For incubation in planktonic condition, tubes inoculated with the bacterial overnight culture should keep in a 45° position with shaking at 130 rpm. For incubation in a biofilm-supporting condition, tubes inoculated with the bacterial overnight culture should keep in a vertical position without shaking.
  5. The parameters have been worked out with Bacillus subtilis and results obtained with other bacteria may not be accurate.
  6. The ‘Nitrate Reductase Cofactor Mixture’ is light-sensitive. Protect from light. Keep on ice during use. Store at -20 °C when not in use.
  7. The ‘Griess Reagent R1’ and ‘Griess Reagent R2’ are light-sensitive. Protect from light, especially sunlight. Store at 4 °C when not in use.

Recipes

  1. Nematode growth medium (NGM) broth
    1. Dissolve 3 g NaCl and 2.5 g Bacto peptone to 1 L of dH2O
    2. Autoclave
    3. Store at room temperature
  2. MSgg medium
    1. Dissolve 17.4 g K2HPO4, 13.6 g KH2PO4, 20.9 g MOPS, 18.3 g MgSO4 heptahydrate, 11 g CaCl2 dihydrate, 3.1 g MnCl2 dihydrate, 40.2 ml glycerol, 0.6 g FeCl3 hexahydrate, 20 g glutamate, 1 g tryptophan, 1 g phenylalanine to 100 ml of dH2O
    2. Autoclave
    3. Store at room temperature
  3. 5 mg/ml chloramphenicol
    1. Dissolve 0.05 g chloramphenicol to 10 ml of 100% ethanol
    2. Store at 4 °C
  4. 5 mg/ml cholesterol
    1. Dissolve 0.25 g of cholesterol in 50 ml of 100% ethanol
    2. Do not autoclave
    3. Store at room temperature
  5. 1 M MgSO4
    1. Dissolve 6 g MgSO4 heptahydrate in 50 ml of dH2O
    2. Autoclave
    3. Store at room temperature
  6. 1 M CaCl2
    1. Dissolve 5.55 g CaCl2 dihydrate in 50 ml of dH2O
    2. Autoclave
    3. Store at room temperature
  7. Phosphate buffer
    1. Dissolve 10.7 g K2HPO4 and 32.5 g KH2PO4 to 300 ml of dH2O
    2. Adjust pH to 6.0
    3. Autoclave
    4. Store at room temperature
  8. Z buffer
    1. Dissolve 2.55 g Na2HPO4, 1.44 g NaH2PO4, 0.22 g KCl, 0.074 MgSO4 heptahydrate and 1,050 µl β-mercaptoethanol to 300 ml of dH2O
    2. Store at room temperature
  9. 10 mg/ml lysozyme solution
    1. Dissolve 10 mg lysozyme to 1 ml of dH2O
    2. Store at 4 °C
  10. 10% Triton X-100 solution
    1.  Dissolve 1 ml Triton X-100 to 10 ml of dH2O
    2. Store at room temperature
  11. O-nitrophenyl-β-D-galactopyranoside (ONPG) 4.5 mg/ml
    1. Dissolve 0.225 g ONPG to 50 ml of dH2O
    2. Store at -20 °C. Use until a yellow colour develops
  12. 1.2 M Na2CO3
    1. Dissolve 6.36 g Na2CO3 to 50 ml of dH2O
    2. Store at 4 °C
  13. 50% glycerol
    1. Dissolve 25 ml 100% glycerol to 25 ml of dH2O
    2. Autoclave
    3. Store at room temperature
  14. 100 mM MOPS pH = 7
    1. Dissolve 20.93 g MOPS in 80 ml of dH2O
    2. Adjust the pH to the desired value with 10 N NaOH
    3. Bring up the volume to 100 ml with dH2O
    4. Sterilize by filtration

Acknowledgments

This work was supported by CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas) and FONCyT (Fondo para la Investigación Científica y Tecnológica) with the aid of the Pew Latin-American Program in Biological Sciences (Philadelphia, PA, USA), the Fulbright Committee (Washington, DC, USA) and former Fundación Antorchas (Buenos Aires, Argentina). We adapted the determination of β-galactosidase activity from Arabolaza et al. (2003).

References

  1. Arabolaza, A. L., Nakamura, A., Pedrido, M. E., Martelotto, L., Orsaria, L. and Grau, R. R. (2003). Characterization of a novel inhibitory feedback of the anti-anti-sigma SpoIIAA on Spo0A activation during development in Bacillus subtilis. Mol Microbiol 47(5): 1251-1263.
  2. Bassler, B. L. and Losick, R. (2006). Bacterially speaking. Cell 125(2): 237-246.
  3. Ben Jacob, E., Becker, I., Shapira, Y. and Levine, H. (2004). Bacterial linguistic communication and social intelligence. Trends Microbiol 12(8): 366-372.
  4. Cabreiro, F. and Gems, D. (2013). Worms need microbes too: microbiota, health and aging in Caenorhabditis elegans. EMBO Mol Med 5(9): 1300-1310.
  5. Clark, L. C. and Hodgkin, J. (2014). Commensals, probiotics and pathogens in the Caenorhabditis elegans model. Cell Microbiol 16(1): 27-38.
  6. Corker, H. and Poole, R. K. (2003). Nitric oxide formation by Escherichia coli. Dependence on nitrite reductase, the NO-sensing regulator Fnr, and flavohemoglobin Hmp. J Biol Chem 278(34): 31584-31592.
  7. Fujiya, M., Musch, M. W., Nakagawa, Y., Hu, S., Alverdy, J., Kohgo, Y., Schneewind, O., Jabri, B. and Chang, E. B. (2007). The Bacillus subtilis quorum-sensing molecule CSF contributes to intestinal homeostasis via OCTN2, a host cell membrane transporter. Cell Host Microbe 1(4): 299-308.
  8. Gusarov, I., Gautier, L., Smolentseva, O., Shamovsky, I., Eremina, S., Mironov, A. and Nudler, E. (2013). Bacterial nitric oxide extends the lifespan of C. elegans. Cell 152(4): 818-830.
  9. Kerwin, J. F., Jr., Lancaster, J. R., Jr. and Feldman, P. L. (1995). Nitric oxide: a new paradigm for second messengers. J Med Chem 38(22): 4343-4362.
  10. Kim, D. H. (2013). Bacteria and the aging and longevity of Caenorhabditis elegans. Annu Rev Genet 47: 233-246.
  11. Lazazzera, B. A., Solomon, J. M. and Grossman, A. D. (1997). An exported peptide functions intracellularly to contribute to cell density signaling in B. subtilis. Cell 89(6): 917-925.
  12. Parsek, M. R. and Greenberg, E. P. (2005). Sociomicrobiology: the connections between quorum sensing and biofilms. Trends Microbiol 13(1): 27-33.
  13. Shapiro, J. A. (1998). Thinking about bacterial populations as multicellular organisms. Annu Rev Microbiol 52: 81-104.
  14. Sudhamsu, J. and Crane, B. R. (2009). Bacterial nitric oxide synthases: what are they good for? Trends Microbiol 17(5): 212-218.
  15. Willians, P. (2007). Bacillus subtilis: a shocking message from a probiotic. Cell Host Microbe 1(4): 248-249.

简介

与浮游生物条件下生长的细胞的基因表达模式相比,有益生物膜内发生的细胞间细胞通讯和分裂产生显着的基因表达差异。 在这个意义上,仅在浮游生长条件下测量枯草芽孢杆菌培养物中产生的NO(一氧化氮)和CSF(能力孢子刺激因子)的水平。 我们试图确定是否NO和/或CSF生产受到影响。 枯草芽孢杆菌细胞作为生物膜发展。 为了测量两种长寿命分子的生产水平,我们生长了B。 枯草芽孢杆菌细胞在浮游生物膜支持条件下。
【背景】NO是关键的信号分子,在脊椎动物的各种生物过程中发挥作用(Kerwin等人,1995)。 ℃。 elegans 不能产生自己的NO,但是能够包含由B生产的NO。 (Cabreiro and Gems,2013; Gusarov et al。,2013; Kim,2013; Clark和Hodgkin,2014)。大多数生物体在通过由基因编码的酶NO合成酶催化的反应中,通过L-精氨酸向L-瓜氨酸的有氧转化而产生NO(Sudhamsu和Crane,2009)。 电子。 (OP50,HB101)(Cabreiro和Gems,2013; Kim,2013; Clark和Hodgkin,2014),其中几种常规用于喂食蠕虫(OP50,HB101),因为缺乏有氧NO生产,不太熟练功能副本 nos (Sudhamsu and Crane,2009)。但是,E。大肠杆菌可以通过与细菌的厌氧呼吸链相关的一系列生化反应在厌氧/微需氧条件下产生NO(Corker和Poole,2003)。在这种情况下,大肠杆菌可能在蠕虫肠的耗氧环境中找到NO产生的许可条件。通过质膜自由扩散的蠕虫肠中产生的细菌NO被氧化成硝酸盐和亚硝酸盐,因此硝酸盐和亚硝酸盐的浓度与NO产生的水平成正比(Gusarov等人, ,2013),并且可以使用比色测定来确定。
内部和种间群体感知(QS)构成了细菌在自然界中与不同王国的细胞相互沟通的分子(Shapiro,1998; Ben Jacob et al。,2004; Parsek和Greenberg,2005; Bassler和Losick,2006)。 B中。据报道,QS五肽CSF(能力孢子刺激因子,也称为PhrC)(Lazazzera等人,1997)以通过激活宿主的关键存活途径来促进肠内体内平衡p38 MAP激酶和蛋白激酶B)和诱导细胞保护性热休克蛋白(Hsps)(Fujiya等人,2007; Willians,2007)。 CSF的这些作用(Willians,2007)取决于其被上皮细胞顶端存在的有机阳离子的宿主细胞膜转运蛋白OCTN2的摄取(Fujiya等人,2007)。为了量化细菌产生的CSF,启动子通常与编码可以使用高度敏感性测定定量的酶的异源报道基因融合。通常,结合到B。编码β-半乳糖苷酶(β-gal)的报告基因lacZ基因的枯草芽孢杆菌可以用于感兴趣的基因的启动子区域,以确定该益生菌产生的CSF的水平细菌。当以这种方式使用时,P csf-lacZ 融合在非必需的amyE基因座整合到染色体中。这里描述的基本比色测定法是用于定量β-gal活性的最简单且更便宜的测定法。将细胞裂解,并将提取物的等分试样与反应底物O-硝基苯基-β-D-吡喃半乳糖苷(ONPG)混合。当黄色产品变得可见时,通过分光光度法测定样品的光密度。

关键字:枯草芽孢杆菌, 浮游生长, 生物膜, NO, CSF

材料和试剂

  1. 移液器提示
  2. 培养皿60 x 15 mm 500 / cs(Fisher Scientific,目录号:FB0875713A)
  3. 无菌150×20mm培养管(Sigma-Aldrich,目录号:C1048)
  4. 无菌150 x 16毫米培养管(科学实验室用品,目录号:6135-5-012)
  5. 1.5 ml管子
  6. Amicon Ultra 0.5 ml离心过滤器MWCO 10 kDa(Sigma-Aldrich,目录号:Z677108)
    制造商:EMD Millipore,目录号:UFC501096。
  7. Amicon Ultra离心过滤器Ultra-4,MWCO 30 kDa(Sigma-Aldrich,目录号:Z648035)
    制造商:EMD Millipore,目录号:UFC803024。
  8. Cryovial(Simport,目录号:T310-2A)
  9. 96孔固体板(比色法)(Cayman Chemical,目录号:400014)
  10. 96孔盖板(Cayman Chemical,目录号:400012)
  11. B的衍生物。 subtilis NCIB31610
  12. 枯草芽孢杆菌NCIB3610和JH642(基因组库,目录号:3A1和1A96)
  13. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  14. Bacto蛋白胨(BD,Bacto TM ,目录号:211677)
  15. 硝酸盐/亚硝酸盐比色测定试剂盒(Cayman Chemical,目录号:780001)
  16. 硝酸盐/亚硝酸盐测定缓冲液(Cayman Chemical,目录号:780022)
  17. 硝酸还原酶制剂(Cayman Chemical,目录号:780010)
  18. 硝酸还原酶辅助因子制剂(Cayman Chemical,目录号:780012)
  19. 硝酸盐标准品(开曼化学,目录号:780016)
  20. Griess Reagent R1(Cayman Chemical,目录号:780018)
  21. Griess Reagent R2(开曼化学公司,目录号:780020)
  22. Luria Bertani肉汤(Sigma-Aldrich,目录号:L3522)
  23. Luria Bertani肉汤用琼脂(Sigma-Aldrich,目录号:L2897)
  24. 琼脂(Sigma-Aldrich,目录号:A1296)
  25. 磷酸氢二钾(K 2 H 2 HPO 4)(Sigma-Aldrich,目录号:P2222)
  26. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P9791)
  27. MOPS(Sigma-Aldrich,目录号:M9381)
  28. 硫酸镁七水合物(MgSO 4·7H 2 O)(Sigma-Aldrich,目录号:M1880)
  29. 氯化钙二水合物(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C3881)
  30. 氯化锰(II)(MnCl 2·2H 2 O)(EMD Millipore,目录号:1059340100)
  31. 甘油(Sigma-Aldrich,目录号:G5516)
  32. 氯化铁(III)六水合物(FeCl 3·6H 2 O)(Sigma-Aldrich,目录号:236489)
  33. 谷氨酸(Sigma-Aldrich,目录号:49621)
  34. 色氨酸(Sigma-Aldrich,目录号:T0254)
  35. 苯丙氨酸(Sigma-Aldrich,目录号:P2126)
  36. 氯霉素(Sigma-Aldrich,目录号:C0378)
  37. 100%乙醇(Sigma-Aldrich,目录号:E7023)
  38. 胆固醇(Sigma-Aldrich,目录号:C8667)
  39. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S3264)
  40. 磷酸二氢钠(NaH 2 PO 4)(Sigma-Aldrich,目录号:S3139)
  41. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  42. β-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  43. 来自鸡蛋白的溶菌酶(Sigma-Aldrich,目录号:L6876)
  44. Triton X-100(Sigma-Aldrich,目录号:X100)
  45. O-硝基苯基-β-D-吡喃半乳糖苷(Sigma-Aldrich,目录号:N1127)
  46. 碳酸钠(Na 2 CO 3)(Sigma-Aldrich,目录号:223484)
  47. 10 N NaOH
  48. 线虫生长培养基(NGM)肉汤(见食谱)
  49. MSGG媒体(见食谱)
  50. 5 mg / ml氯霉素(见配方)
  51. 5 mg / ml胆固醇(参见食谱)
  52. 1 M MgSO 4(参见食谱)
  53. 1 M CaCl 2 (见配方)
  54. 磷酸盐缓冲液(见配方)
  55. Z缓冲(见配方)
  56. 10 mg / ml溶菌酶溶液(见配方)
  57. 10%Triton X-100溶液(参见食谱)
  58. O-硝基苯基-β-D-吡喃半乳糖苷(ONPG)4.5mg / ml(参见食谱)
  59. 1.2 M Na 2 CO 3(参见食谱)
  60. 50%甘油(参见食谱)
  61. 100mM MOPS pH = 7(参见食谱)

设备

  1. 锥形瓶(Fisher Scientific,目录号:FB5006000)
  2. Pipettor(Gilson,目录号:F167300)
  3. 玻璃移液管
  4. 冷藏培养箱(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:51028064; Fisher Scientific,目录号:37-20)
    注意:产品“Fisher Scientific,目录号:37-20”已停产。
  5. 水浴(AQUA ® LYTIC孵化器37°C)
  6. 冷冻机(-20°C;低环境设备)(西门子,型号:So-Low Ultra C85-22)
  7. 高压灭菌器(Tuttnauer,型号:6690型)
  8. 搅拌电热板(康宁,目录号:6795-620)
  9. 离心机(Eppendorf,型号:5430)
  10. 台式离心机(Eppendorf,型号:5424)
  11. 分子动力学模型SpectraMAX 340 PC酶标仪(Molecular Devices,型号:SpectraMAX 340PC384)

程序

  1. B的准备。枯草芽孢杆菌菌株培养物
    1. 使用无菌程序,在无菌150 x 20 mm管中加入2 ml无菌MSGG或NGM肉汤(见食谱)。接种20微升B的培养基。枯草芽孢杆菌菌株冷冻培养物(JH642或NCIB3610)(见注3)。对于NGM肉汤的制备,将3g NaCl和2.5g细菌蛋白胨混合在锥形瓶中。加入1L dH 2 O。在121℃高压灭菌20分钟。在55℃水浴中冷却烧瓶15分钟。加入1ml 1M CaCl 2,1ml 5mg / ml胆固醇的乙醇溶液,1ml 1M MgSO 4和25ml 1M KPO 4 / sub>缓冲区。旋转混匀。
    2. 在37℃下振荡孵育16-18小时(过夜培养)。

  2. B的准备。枯草芽孢杆菌菌株培养支持生物膜形成
    1. 使用无菌程序,在无菌150 x 16 mm管中加入2 ml无菌MSGG或NGM肉汤。接种20微升B的培养基。枯草芽孢杆菌菌株过夜培养。
    2. 在25℃下孵育36小时(浮游条件)或不摇动(生物膜支持条件)(见注4)。

  3. B的准备。枯草芽孢杆菌菌株样品进行NO测定(根据试剂盒说明书)
    1. 取管并通过涡旋破坏生物膜,直到获得均匀的悬浮液。对于CFU(集落形成单位)计数取100μl等分试样(见注1)
    2. 以12,470×g离心20分钟。将上清液转移到无菌的1.5ml Eppendorf管中,弃去沉淀。
    3. 使用市售的离心机使用10或30kDa分子量截止滤器的超滤器。过滤器应在超滤前用超纯水进行预漂洗。
    4. 等待3小时,将硝酸盐转化成亚硝酸盐所需的时间。

  4. 制备标准曲线(根据试剂盒的说明)
    1. 必须包括硝酸盐和亚硝酸盐的标准曲线。如果计划只测量总NO产物(硝酸盐+亚硝酸盐),则只需要硝酸盐标准曲线。
    2. 在干净的管子中,从试剂盒中加入0.9ml'硝酸盐分析缓冲液'。加入0.1ml重构的“硝酸盐标准液”,涡旋20秒。该库存标准的浓度为200μM
    3. 使用本标准制备硝酸盐标准曲线,如下所述。通过以下列方式向板孔中加入试剂制备硝酸盐的标准曲线(图1):


      图1.根据试剂盒说明书制备硝酸盐标准曲线

  5. 进行测定(根据试剂盒说明)
    1. 向空白孔中加入200μl水或“硝酸盐分析缓冲液”。不要在这些井中添加任何其他试剂。
    2. 以您选择的图案将最多80μl样品加入孔中。使用“硝酸盐分析缓冲液”解决方案,最终体积必须调至80μl
    3. 向每个孔(标准品和未知)中加入10μl“酶辅因子混合物”(见附注6)。
    4. 向每个孔中加入10μl“硝酸还原酶混合物”(标准品和未知数)。
    5. 用板盖盖板,室温孵育2小时。孵化期间无需摇动板。
    6. 孵育时间后,向每个孔(标准品和未知)中加入50μl“Griess Reagent R1”(见注7)。移动上下2-3次。
    7. 孵育时间后,向每个孔(标准品和未知)中加入50μl“Griess Reagent R2”(见注7)。移动上下2-3次。
    8. 允许颜色在室温下培养10分钟。不需要盖板。
    9. 使用读卡器读取540nm处的吸光度。

  6. 计算
    1. 从所有其他孔的吸光度值中减去空白孔的吸光度值。
    2. 作为硝酸盐浓度的函数,绘制540 nm处的吸光度图。硝酸盐标准曲线用于测定总硝酸盐+亚硝酸盐浓度。一个例子如图2所示
    3. 计算NO浓度如下:(硝酸盐+亚硝酸盐)(μM)=([A 540] -y-截距)/斜率]×(200μl/使用体积(μl))×稀释< br />
    4. 根据在步骤C1中获得的CFU,将NO(μM)归一化为1×10 <8> CFU。


      图2.典型的标准曲线(根据套件说明)

  7. β-半乳糖苷酶活性的测定(Arabolaza et al。,2003)(见附注5)
    1. 本研究中使用的细菌菌株是B型的衍生物。在非必需的amyE基因座上整合到细菌染色体中的含有P csf 融合在一起的NCIB31610。
    2. 将2ml MSGG或NGM肉汤无菌地引入先前高压灭菌的150×16mm培养管和2μl5mg / ml氯霉素中。
    3. 每个管子用20μl的过夜培养物接种;仅含MSGG或NGM肉汤的未接种管作为交叉污染对照。
    4. 将培养物以130rpm(浮游条件)或不摇动(生物膜支持条件)置于旋转振荡器上,并在37℃下温育。
    5. 将管孵育至少36小时。为了确定培养物的密度,取管并通过涡旋破坏生物膜,直至获得均匀的悬浮液。取100μl等份进行CFU计数(见注1)。使用Molecular Dynamics Model SpectraMAX 340PC酶标仪来确定稀释培养物的A 525(1/10稀释度)。如下所述,需要稀释培养物,因为当值高于0.8-1时,细胞密度不与A 525成比例。
    6. 通过加入2μl5mg / ml氯霉素(参见Recipes),使用中间移液器将两个等分的来自破碎培养物的1ml等分试样停止,并将管置于冰上。然后离心1.5ml Eppendorf管,在4℃下以12,470×g离心5分钟。去除上清液并将沉淀物在-20℃下冷冻或立即测定(参见附注2)(图3)。


      图3.流程图图,显示在浮游生物膜支持条件和样品收集中培养细菌的基本步骤

    7. 对于细胞透化,将0.73ml Z缓冲液(参见食谱)分配到沉淀中。
    8. 含有0.73ml Z缓冲液的试剂空白与未知数一起运行。
    9. 加入10μl10 mg / ml新鲜溶菌酶溶液(参见食谱)到管中,并在37°C水浴中孵育15 min。
    10. 将试管转移到试验台上,加入10μl10%Triton X-100(参见食谱)。通过吸入和分配混合物10次来实现渗透性。
    11. 渗透后立即在零时,通过向每个管中加入100μl的4.5mg / ml ONPG(参见食谱)开始测定。在测定结束时,将管在28℃温育适合的时间长度,例如15分钟,然后在反应终止之前,加入150μl的1.2M Na 2 sub> 2 <3> 3 (参见食谱)。
    12. 然后,将管的内容物转移到微孔板中,将微孔板引入读数器,并确定Aβ420值。
    13. 以米勒单位(M.U.)表示的β-半乳糖苷酶活性的计算如下进行:

      亩。 =(OD 420×66.7)/(OD 525×ml使用)

      其中,
      OD 420是β-gal测定的光密度,
      OD 525是细菌培养物的光密度,
      66.7是修正因子,
      ml是用于重悬细菌沉淀的Z缓冲液的体积(在这种情况下为0.73ml)
    14. 根据步骤C1中获得的UFC,将β-gal(M.U.)归一化为1×10 <8> UFC。

数据分析

  1. 将A 420和A 525的吸光度数据转移到Microsoft Excel电子表格,并计算米勒单位中的β-半乳糖苷酶特异活性。
  2. 每次测定应重复至少三次,一式四份。
  3. 使用学生的 t -test具有重要性截止级别 P 0.05用于比较两组之间。
  4. 使用单因素(ANOVA)方差分析,并通过事后Bonferroni检验进行多重比较。

笔记

  1. 对于用于种子枯草芽孢杆菌的LB板,我们强烈建议在45℃下干燥板20分钟或37℃反复40分钟,然后将稀释液放入每个板上以避免这种细菌在固体表面滑动,水滴从冷凝水中脱落
  2. 应立即使用样品或-20°C储存1个月。
  3. LB琼脂平板上的细菌可以在4℃下储存数周。然而,长期储存时,建议制备甘油储备。甘油的加入使冷冻细菌稳定,防止细胞膜损伤并保持细胞活力。甘油储存的细菌可以稳定地储存在-80℃多年。为此,准备过夜的细菌培养物,将500μl的过夜培养物加入到500μl50%甘油(参见食谱)中,在2ml冷冻浴中轻轻混匀。在-80℃下冷冻甘油储备管。现货现货稳定多年,只要保持在-80°C。随后的冻结和解冻周期将降低保质期。要从您的甘油原料中回收细菌,请打开管子,并使用无菌环或移液管尖端刮去顶部的一些冷冻细菌。不要让甘油股解冻。在执行程序时,始终将甘油储存在冰上。将细菌连接到LB琼脂平板上。在37℃下使枯草芽孢杆菌生长过夜。
  4. 为了在浮游条件下孵育,用细菌过夜培养物接种的管应在130rpm下摇动时保持在45°位置。为了在生物膜支持条件下孵育,用细菌过夜培养物接种的管子应保持垂直位置而不摇动。
  5. 已经使用枯草芽孢杆菌制定了这些参数,并且用其他细菌获得的结果可能不准确。
  6. “硝酸还原酶辅因子混合物”对光敏感。防光。使用过程中保持冰。不使用时在-20°C储存。
  7. “Griess试剂R1”和“Griess Reagent R2”对光敏感。防止光线,尤其是阳光。不使用时在4°C储存。

食谱

  1. 线虫生长培养基(NGM)肉汤
    1. 将3g NaCl和2.5g细菌蛋白胨溶解于1L dH 2 O中,
    2. 高压灭菌器
    3. 在室温下存放
  2. MSGG媒体
    1. 溶解17.4g K 2 H 2 HPO 4,13.6g KH 2 PO 4,20.9g MOPS,18.3g MgSO 4七水合物,11g CaCl 2二水合物,3.1g MnCl 2二水合物,40.2ml甘油,0.6g FeCl 3 六水合物,20g谷氨酸盐,1g色氨酸,1g苯丙氨酸加到100ml dH 2 O中,
    2. 高压灭菌器
    3. 在室温下存放
  3. 5毫克/毫升氯霉素
    1. 将0.05克氯霉素溶于10ml 100%乙醇中
    2. 储存于4°C
  4. 5毫克/毫升胆固醇
    1. 将0.25g胆固醇溶于50ml 100%乙醇中
    2. 不要高压
    3. 在室温下存放
  5. 1M MgSO 4
    1. 将6g硫酸镁四水合物溶于50ml dH 2 O中,
    2. 高压灭菌器
    3. 在室温下存放
  6. 1M CaCl 2
    1. 将5.55g CaCl 2二水合物溶于50ml dH 2 O中,
    2. 高压灭菌器
    3. 在室温下存放
  7. 磷酸盐缓冲液
    1. 将10.7g K 2 N 2 HPO 4和32.5g KH 2 PO 4溶解至300ml dH 3 2 O
    2. 调节pH至6.0
    3. 高压灭菌器
    4. 在室温下存放
  8. Z缓冲区
    1. 溶解2.55g Na 2 HPO 4,1.44g NaH 2 PO 4,0.22g KCl,0.074 MgSO 4,亚> 4个七水合物和1,050μlβ-巯基乙醇加到300ml dH 2 O中,
    2. 在室温下存放
  9. 10mg / ml溶菌酶溶液
    1. 将10毫克溶菌酶溶解于1毫升dH 2 O - / -
    2. 储存于4°C
  10. 10%Triton X-100溶液
    1. 将1ml Triton X-100溶解至10ml dH 2 O→/
    2. 在室温下存放
  11. O-硝基苯基-β-D-吡喃半乳糖苷(ONPG)4.5mg / ml
    1. 将0.225g ONPG溶解在50ml的dH 2 O中
    2. 储存于-20°C。使用直到黄色发展为
  12. 1.2M Na 2 CO 3 3
    1. 将6.36g Na 2 CO 3溶解至50ml dH 2 O→
    2. 储存于4°C
  13. 50%甘油
    1. 将25毫升100%甘油溶解于25毫升dH 2 O - / -
    2. 高压灭菌器
    3. 在室温下存放
  14. 100mM MOPS pH = 7
    1. 将20.93g MOPS溶解在80ml dH 2 O中
    2. 用10N NaOH调节pH至所需值
    3. 使用dH <2> O
      将体积增加至100ml
    4. 过滤灭菌

致谢

这项工作得到了CONICET(Consejo Nacional de InvestigacionesCientíficasyTécnicas)和FONCyT(Fondo para laInvestigaciónCientíficayTecnológica)的支持,在拉丁美洲生物科学计划(Philadelphia,PA,USA),富布莱特委员会(华盛顿特区,美国)和前基督教基金会(阿根廷布宜诺斯艾利斯)。我们调整了来自Arabolaza等人的β-半乳糖苷酶活性的测定。 (2003)。

参考

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引用:Cogliati, S., Rodriguez Ayala, F., Bauman, C., Bartolini, M., Leñini, C., Villalba, J. M., Argañaraz, F. and Grau, R. (2017). Determination of NO and CSF Levels Produced by Bacillus subtilis. Bio-protocol 7(13): e2379. DOI: 10.21769/BioProtoc.2379.
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