Chapter 2. Isolation and taxonomic study of the class Ktedonobacteria
2.2 Materials and Methods
2.2.7 Chemotaxonomic analysis of the novel Ktedonobacteria isolates
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and sucrose. All of the sugars are bought from Kanto Chemical Co., Inc., Tokyo, Japan.
The 10-fold diluted R2A agar medium was autoclaved at 121 °C, 20 min, and then aseptically added each 10 ml of each stock sugar solutions to make the final concentration of 0.5 %. The stock sugar solutions were pre-prepared by filter (Toyo Roshi Kaisha, Ltd.,Tokyo, Japan) sterilize and stored at 4 °C.
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7. Add 1.25 ml of Extraction solvent to the test tube and vortex for 10 min.
8. Pipette out and discard the aqueous (lower) phase.
9. Add 3.0 ml of Wash solvent the organic phase remaining in the tubes and tumble on a sliding rotor (BC-740, Bio Craft,Tokyo, Japan) for 5 min.
10. Pipette the organic (upper) phase into an Eppendorf tube and centrifuge at 15,000 rpm for 5 min.
11. Pipette the supernatant to a new Eppendorf tube and store at -20 °C until use.
12. Cellular fatty acid composition was identified using the Sherlock Microbial Identification System (v 6.0; MIDI) and the TSBA6 database by Techno Suruga Laboratory Co., Ltd., Shizuoka, Japan.
Saponification solvent Sodium hydroxide 15.0 g
Methanol 50 ml
MilliQ water Fill up to 100 ml
Extraction solvent
Hexane 50 ml
Methyl tert-butyl ether (MTBE) 50 ml
2.2.7.2 Analysis of polar lipid composition
Polar lipids were extracted and separated by two-dimensional thin layer chromatography (TLC) according to the method of (Tindall, 1990a; Tindall, 1990b), and visualized by spraying with appropriate detection reagents (Tindall et al., 2007):
A. Polar lipid extraction
Methylation solvent
6M Hydrochloric acid 130 ml
Methanol 110 ml
Wash solvent
Sodium hydroxide 1.2 g
MilliQ water Fill up to 100 ml
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1. Put 100 mg of dried cell pellets into a screw cap glass test tube and add 6.75 ml of chloroform:methanol:0.3% aqueous NaCl (5:10:4, v/v/v) solvent to the test tube. Tumble gently at room temperature overnight.
2. Centrifuge at 4,000 rpm for 5 min.
3. Transfer the supernatant to a new test tube and add 1.75 ml of chloroform and 1.75 ml of 0.3% NaCl solution to the test tube. Tumble gently at room temperature for 1 h.
4. Centrifuge at 4,000 rpm for 5 min.
5. Pipette the organic (lower) phase into a new test tube with Pasteur pipette.
6. Dry the extracted polar lipids with nitrogen gas flow for 10~20 min.
7. Dissolve the dried sample in 200 μl of chloroform:methanol (2:1, v/v) solvent.
Store at -20 °C until use.
B. TLC separation
1. Cut the Silica gel 60 TLC glass plate (10x20 cm, Merck KGaA, Darmstadt, Germany) into square plate (10x10 cm) with a diamond cutter.
2. Use a pencil to gently draw a straight line across the plate approximately 1 cm from the bottom.
3. Spot 10 μl of the extracted polar lipid sample at the across where the two pencil lines are overlapped. Dry the spot with a dryer. Prepare and spot five TLC plates for each sample.
4. Equilibrate the developing chamber with 100 ml of the 1st-dimensional developing solvent (chloroform:methanol:MiliQ water (65:25:4, v/v/v)) for approx.
30 min.
5. Place the plate into the chamber as evenly as possible and lean it against the side. Allow the plate to develop until the solvent rise up to the top of the plate (approx. 25~30 min).
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6. Remove the plate from the chamber and air dry the plate.
7. Equilibrate the developing chamber with 100 ml of the 2nd-dimensional developing solvent (chloroform:Acetic acid:methanol:MiliQ water (85:15:12:4, v/v/v/v)) for approx. 30 min.
8. Place the plate into the chamber as evenly as possible and lean it against the side. Allow the plate to develop until the solvent rise up to the top of the plate (approx. 25~30 min).
9. Remove the plate from the chamber and air dry the plate. Immediately put the plate into a jar containing iodine crystals and mark the visualized spots with a pencil.
1st-dimension 2nd-dimension C. Spot visualization
Total lipid material is detected using molybdatophosphoric acid and specific functional groups detected using spray reagents specific for defined functional groups:
TLC plate 1 displays the total lipid profile. It is sprayed with 10 % 12-Molybdo(VI) Phosphoric Acid n-Hydrate aqueous solution (w/v) with a glass sprayer (2-297-01, AS ONE Co., Osaka, Japan), and incubated at 180 °C in an oven (MOV-202F, Sanyo Shokai Ltd.,Tokyo, Japan) to visualize the spots.
TLC plate 2 confirms the presence of aminolipids and phospholipid groups. To confirm the presence of aminolipids groups, the plate is sprayed with the Ninhydrin Spray Reagent (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and
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incubated at 150 °C for 5~15 min. Next, the presence of phospholipids in the spots was confirmed by spraying with Dittmer-Lester reagent at room temperature.
Preparation of Dittmer-Lester reagent:
1. Add 4.01 g of Molybdenum Trioxide to 100 ml of Sulfuric acid in a beaker. Boil gently in a fume hood until the Molybdenum Trioxide is dissolved.
2. Allow the solution to cool down overnight.
3. Recover the supernatant and label as Dittmer-Lester Solution A.
4. Transfer 50 ml of Dittmer-Lester Solution A to a new beaker and add 0.18 g of molybdenum powder. Boil gently for 15 min.
5. Recover the supernatant and label as Dittmer-Lester Solution B.
6. Mix equal volumes of Solutions A and B in a new beaker, and gently add four volumes of MiliQ water. The final solution should be greenish yellow. Too little water will result in a blue solution, while too much will render it yellow.
TLC plate 3 confirms the presence of glycolipids. It is sprayed with the p-Anisaldehyde:sulphuric acid:acetic acid:methanol (5:5:1:90, v/v/v/v) reagent and incubated at 150°C.
TLC plate 4 confirms the presence of unsaturated periodic acid-Schiff (PAS) stainable glycolipids:
1. Spray the plate with 1 % periodic acid aqueous solution in a fume hood and air dry for 10~15 min.
2. Spray the plate with MiliQ water in a fume hood to remove the remaining periodic acid and air dry.
3. Add 3.2 g citric acid, 4.8 g sodium metabisulfite, and appropriate volume of MiliQ water to desiccator to generate sulfurous acid gas.
4. Put the air-dried plate into the desiccator filled with sulfurous acid gas and let the brown color of iodine disappear.
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5. Spray the plate with Cold Schiff's Reagent (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in a fume hood and air dry.
TLC plate 5 confirms the presence of phosphatidylcholine with the spraying of Dragendorff’s spray reagent at room temperature:
Preparation of Dragendorff’s spray reagent:
1. Dissolve 1.7 g of Bismuth subnitrate and 20 ml of Acetic acid in 80 ml of MiliQ water to make Solution A.
2. Dissolve 40 g of Potassium Iodide in 100 ml of MiliQ water to make Solution B.
3. Mix 5 ml of Solution A, 5 ml of Solution B, 20 ml of Acetic acid, 70 ml of MiliQ water to make the final Dragendorff’s spray reagent.
D. Spot identification
Spots visualized by molybdatophosphoric acid on plate 1 are identified according to the positions and colors on other plates as summarized in Table 2.2.7.2-1.
Table 2.2.7.2-1 Spot colors of various polar lipids visualized by detection reagents on different plates
Polar lipids Spot colors on the plates
Plate 1 Plate 2 Plate 3 Plate 4
PI Gray to Black Blue Light blue Ocher
PG Gray to Black Blue Light blue Magenta
DPG Gray to Black Blue Light blue -
GL Gray to Black - Green Magenta
PL Gray to Black Blue - Magenta
PGL Gray to Black Blue Green Magenta
L Gray to Black - - -
Abbreviations: PI, phosphatidylinositol; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; GL, unidentified glycolipid; PL, unidentified phospholipid;
PGL, phosphoglycolipid; L, unidentified lipid.
38 2.2.7.3 Analysis of menaquinone composition
Major menaquinone was isolated by TLC plate and identified with liquid chromatography-mass spectrometry (LC-MS) following a modified method of (Collins et al., 1977):
A. Menaquinone extraction
1. Put 300 mg of dried cell pellets into a screw cap glass test tube and add 15 ml of chloroform:methanol (2:1, v/v) solvent to the test tube.
2. Cover the test tube tightly with aluminum foil and tumble gently on a sliding rotor at room temperature overnight.
3. Remove the cell pellets by filtration with φ 185 mm filter paper (No. 1, 00011185, Advantec Toyo Co., Tokyo, Japan). The cell pellets are further extracted with appropriate volume of the chloroform:methanol (2:1, v/v) solvent.
4. Evaporate the extract with an eggplant-shaped flask on an evaporator.
5. Add 600 μl of acetone to the dried extract and recover approx. 500 μl of the soluble components to an Eppendorf tube.
6. Store at -20 °C until later use.
B. TLC isolation
1. Use a pencil to gently draw a straight line at approx. 1.5 cm from the bottom of the Silica gel 60 F254 TLC glass plate (20x20 cm, Merck KGaA, Darmstadt, Germany).
2. Spot 20 μl of 10 mg/ml vitamin K2 aceton solution at the left side of the pencil line as a standard and dry with a dryer.
3. Spot the extract horizontally in the center of the pencil line and dry with a dryer.
4. Equilibrate the developing chamber with 100 ml of toluene for approx. 30 min.
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5. Place the plate into the chamber as evenly as possible and lean it against the side. Allow the plate to develop until the solvent rise up to the top of the plate (approx. 1 h).
6. Remove the plate from the chamber and air dry the plate.
7. Detect the menaquinones routinely by brief irradiation with short-wave ultraviolet light (254 nm and 365 nm). Mark the fluorescent band of the major menaquinone with pencil.
8. Isolate the major menaquinone from the TLC plate with a spatula. Collect the scraped silica gel into a small glass test tube.
9. Elute the major menaquinone from silica gel with appropriate volume of acetone.
Store at -20 °C until later use.
C. LC-MS identification
The major menaquinone composition was identified by Techno Suruga Laboratory Co., Ltd., Shizuoka, Japan, using an Agilent 6530 Accurate-Mass Q-TOF LC-MS in positive-ion mode equipped with an electrospray ionisation source using a COSMOSIL 5C18-AR-II packed column (4.66x250 mm) (Nacalai Tesque, Inc., Kyoto, Japan). The collision energy was 80 eV.
2.2.7.4 Preparation of the cell-wall peptidoglycan
For analysis of cell-wall peptidoglycan amino acids and sugars, the cell-wall contents were extracted with sonication in advance following a modified method of (Schleifer & Kandler, 1972):
1. Put 20 mg of dried cell pellets into a 50 ml centrifuge tube and re-suspend with 10 ml of sterilized MiliQ water.
2. Add appropriate volume of glass beads (φ 0.11~0.22 mm) (AS ONE Co., Osaka, Japan) to the tube with a spatula.
3. Place the centrifuge tube in ice water and crush the cell pellets with an ultrasonic generator (US300, Nissei Co.,Ltd., Tokyo, Japan) at 200 μA for 30 min.
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4. Centrifuge at 3,000 rpm for 30 min at room temperature. Transfer the supernatant to a new 50 ml centrifuge tube to remove glass beads and unbroken cell pellets.
5. Centrifuge at 12,100 rpm for 30 min at room temperature. Discard the supernatant carefully to obtain the precipitated cell-wall fraction.
6. Add 5 ml of 4% (w/v) sodium lauryl sulfate (SDS) aqueous solution to re-suspend the precipitated cell-wall fraction.
7. Transfer to solution to a glass tube and incubate at 100 ° C for 30~40 min.
8. Centrifuge at 12,100 rpm for 30 min at room temperature. Discard the supernatant carefully and re-suspend the precipitated cell wall fraction with 5 ml of sterilized MiliQ water.
9. Repeat step 8 for another three times.
10. Discard the supernatant carefully and re-suspend the precipitated cell-wall fraction with 500 μl of acetone.
11. Centrifuge at 12,100 rpm for 30 min at room temperature.
12. Discard the supernatant carefully and re-suspend the precipitated cell-wall fraction with 500 μl of hexane.
13. Centrifuge at 12,100 rpm for 30 min at room temperature.
14. Discard the supernatant carefully and air dry the precipitated cell-wall fraction for later use.
2.2.7.5 Analysis of peptidoglycan amino acid
Peptidoglycan amino acids were obtained by hydrolyzing the cell-wall fraction prepared in section 2.2.7.4 with 6N hydrochloric acid (HCl) at 100 °C overnight and identified by two-dimensional TLC (Harper & Davis, 1979). To identify enantiomers of peptidoglycan amino acids, the Nα-(5-fluoro-2,4-dinitrophenyl)-D-leucinamide derivatives of these substances were analyzed with LC-MS following a method described by (Také et al., 2016).
41 A. Hydrolysis of cell-wall peptidoglycan
1. Put 5 mg of cell-wall fraction into a glass microtube (φ 8x50 mm, AS ONE Co., Osaka, Japan) and add 200 μl of 6N hydrochloric acid aqueous solution.
2. Seal the glass microtube with a gas burner.
3. Hydrolysis at 100 ° C overnight in a heat block.
4. Transfer the hydrolysate into an Eppendorf tube and centrifuge at 15,000 rpm for 5 min at room temperature.
5. Transfer the supernatant into a new Eppendorf tube and evaporate with an air pump (MAS-1, AS ONE Co., Osaka, Japan).
6. Dissolve the precipitates with 50 μl of sterilized MiliQ water to make the peptidoglycan amino acid samples. Store at -20 °C until later use.
7. Prepare 50 mM amino acid standard solution by dissolving β-alanine, D-α-alanine, L-D-α-alanine, D-glutamic acid, glycine, L-serine, L-ornithine in 0.1N hydrochloric acid aqueous solution, respectively. Store at -20 °C until later use.
B. TLC identification
1. Cut the TLC cellulose plate (20x20 cm, Merck KGaA, Darmstadt, Germany) into square plate (10x10 cm) with a utility knife.
2. Use a pencil to gently draw a straight line across the plate approximately 1 cm from the bottom.
3. Spot the prepared peptidoglycan amino acid samples at the across where the two pencil lines are overlapped. Dry the spot with a dryer. Similarly, spot 2~5 μl of the amino acid standard solution.
4. Equilibrate the developing chamber with 100 ml of the 1st-dimensional developing solvent (isopropanol:acetic acid:MiliQ water (75:10:15, v/v/v)) for approx. 30 min.
42
5. Place the plate into the chamber as evenly as possible and lean it against the side. Allow the plate to develop until the solvent rise up to the top of the plate (approx. 4 h).
6. Remove the plate from the chamber and air dry the plate.
7. Equilibrate the developing chamber with 100 ml of the 2nd-dimensional developing solvent (methanol:pyridine:10M Hydrochloric acid:MiliQ water (64:8:2:4, v/v/v/v)) for approx. 30 min.
8. Place the plate into the chamber as evenly as possible and lean it against the side. Allow the plate to develop until the solvent rise up to the top of the plate (approx. 4 h).
9. Remove the plate from the chamber and air dry the plate.
10. Spray the plate with Ninhydrine spray reagent (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and air dry the plate.
11. Incubate at 130 °C in an oven to visualize the spots.
12. Identify the displayed spots by referring to the plate of amino acid standard solution.
C. LC-MS analysis
1. Mix 50 ul of the peptidoglycan amino acid sample with 50 ul of 1M sodium hydrogen carbonate aqueous solution and 50 ul of 1% (w/v) Nα -(5-fluoro-2,4-dinitrophenyl)-D-leucinamide (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) acetone solution.
2. Incubate at 37 °C for 1 h in a heat block.
3. Concentrate on a centrifugal evaporator (CC-105, Tomy Seiko Co, Ltd., Tokyo, Japan) for overnight.
4. Dissolve the dried sample with 2 mM ammonium formate aqueous solution for LC-MS analysis.
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LC-MS analysis condition
Machine Agilent 6530 Accurate-Mass Q-TOF (Agilent Technologies, Palo Alto, USA)
Column Inertsil ODS-4 column (5 µm × 4.6 mm × 250 mm) (GL Sciences Inc., Tokyo, Japan)
Flow 0.5 ml/min
Ion source +ESI (Electrospray ionization), positive mode Sample injection 2 ul
Mobile phase A 2 mM ammonium formate aqueous solution Mobile phase B 2 mM ammonium formate methanol solution
Gradient
Time (min) Mobile phase A (%) Mobile phase B (%)
5 95 5
5~30 95~0 5~100
30~35 0 100
2.2.7.6 Analysis of cell wall sugar
Peptidoglycan sugars were obtained by hydrolyzing the cell-wall fraction prepared in section 2.2.7.4 with 2N hydrochloric acid (HCl) at 100 °C and identified by cellulose TLC. Alditol acetate derivatives of these substances were analyzed with GC-MS (Hasegawa, 1983).
A. Hydrolysis of cell-wall peptidoglycan
1. Put 5 mg of cell-wall fraction into a glass microtube (φ 8x50 mm, AS ONE Co., Osaka, Japan) and add 200 μl of 6N hydrochloric acid aqueous solution.
2. Seal the glass microtube with a gas burner.
3. Hydrolysis at 100 ° C for 3 h in a heat block.
4. Transfer the hydrolysate into an Eppendorf tube and centrifuge at 15,000 rpm for 5 min at room temperature.
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5. Transfer the supernatant into a new Eppendorf tube and evaporate with an air pump (MAS-1, AS ONE Co., Osaka, Japan).
6. Dissolve the precipitates with 200 μl of sterilized MiliQ water to make the peptidoglycan sugar samples. Store at -20 °C until later use.
7. Prepare 10 % (w/v) sugar standard solution by dissolving galactose, D-glucose, L-arabinose, D-mannose, D-xylose, D-fructose, D-ribose, L-rhamnose in sterilized MiliQ water, respectively. Store at -20 °C until later use.
B. TLC identification
1. Use a pencil to gently draw a straight line at the TLC cellulose plate (20x20 cm, Merck KGaA, Darmstadt, Germany) approximately 1.5 cm from the bottom.
2. Spot 2~5 μl of the prepared 10 % (w/v) sugar standard solution on the left and 2~5 μl of the prepared peptidoglycan sugar samples on the right with an interval of 1 cm on the line. Dry the spot with a dryer.
3. Equilibrate the developing chamber with 100 ml of the developing solvent (1-butanol:MilliQ water:pyridine:toluene (10:6:6:1, v/v/v/v)) for approx. 30 min.
4. Place the plate into the chamber as evenly as possible and lean it against the side. Allow the plate to develop until the solvent rise up to the top of the plate (approx. 8 h).
5. Remove the plate from the chamber and air dry the plate.
6. Repeat step 4.
7. Remove the plate from the chamber and dry the plate with a dryer for approx.
30 min.
8. Spray the plate with Aniline phthalate spray solution *.
9. Incubate the plate immediately at 120 °C in an oven to visualize the spots.
10. Identify the displayed spots by referring to the spots of sugar standard solutions.
* Preparation of the Aniline phthalate spray solution
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1. Mix equivalent volume of 1-butanol and MilliQ water thoroughly in a separatory funnel. Stand overnight at room temperature.
2. Collect the organic (upper) phase and label as Water-saturated 1-butanol.
3. Dissolve 3.25 g of phthalic acid and 2 ml of aniline in 100 ml Water-saturated 1-butanol to make the Aniline phthalate spray solution.
C. GC-MS analysis
1. Transfer 100 μl of the peptidoglycan sugar samples into an Eppendorf tube and add 2 drops of ammonia aqueous solution (Nacalai Tesque, Inc., Kyoto, Japan) to adjust the pH to >9.0.
2. Add excess amount of Sodium borohydride (Kanto Chemical Co., Inc., Tokyo, Japan) in small portions gradually to the tube.
3. Incubate at room temperature for 2 h to let it foam.
4. Add Amberlite® IR-120B (H) ion exchange resin (Rohm & Haas Company, Philadelphia, USA) in small portions gradually to the tube to stop foamimg.
5. Transfer the liquid to a new Eppendorf tube and evaporate with an air pump.
6. Add 500 μl of acetic anhydride and 500 μl of pyridine to the tube and stand overnight at room temperature for acetylation.
7. Add 500 μl of methanol to the tube to decompose the remaining acetic acid.
8. Add small amount of toluene to the tube and concentrate on a centrifugal evaporator for several times until the odor of pyridine disappear.
9. Dissolve the dried sample in small amount of acetone for GC-MS analysis. Store at -20 °C until use.
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GC-MS analysis condition Machine Agilent Technologies 5970A
(Agilent Technologies, Palo Alto, USA) Column DB-1 (60 m × 0.25 mm × 0.50 µm)
(Agilent Technologies, Palo Alto, USA)
Pressure 111.22 kpa
Injection port temp. 250 °C Sample injection 2 ul Carrier gas Helium
Mode Split (10:1)
Oven temp.
& Gradient
Time (min) Gradient (°C/min) Temperature (°C)
2 - 160
5 20 200
12 20 245
5 12.5 270
2.2.8 Whole genome-based phylogenomic analysis of the novel Ktedonobacteria