SUPPLEMENTARY INFORMATION
Nitrogen acquisition in Agave tequilana from degradation of endophytic bacteria
Miguel J. Beltran-Garcia1, 3, James F. White Jr2, Fernanda M. Prado3, Katia R. Prieto3,
Lydia F. Yamaguchi4, Monica S. Torres2, Massuo J. Kato4, Marisa H. G. Medeiros3 &
Paolo Di Mascio3
1
Departamento de Química ICET, Universidad Autonoma de Guadalajara, Patria 1201,
Lomas del Valle, Zapopan Jalisco, Mexico.
2
Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ
USA.
3
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo,
05508-000, São Paulo, SP, Brazil.
4
Departamento de Química Fundamental, Instituto de Química, Universidade de São
Paulo, 05508-000, São Paulo, SP, Brazil.
James F. White (white@rci.rutgers.edu) & Paolo Di Mascio (pdmascio@iq.usp.br)
Supplementary Note 1.
Protein extraction from A. tequilana.
Protein extraction from the leaves of A. tequilana was made after six months of
treatments as described above. Freshly collected leaves (90-140.0 mg) were ground to
powder in liquid nitrogen, washed with 1mL of 50 mM phosphate buffer (pH 6.0) and
centrifuged at 12, 000 x gravity for 20 min at room temperature. Proteins were
precipitated adding 2.4M (NH4)2SO4 (600µL), followed by agitation (10 °C during 30
min), and then the solution was centrifuged (10, 000 x gravity, 4 ºC for 10 min). In the
final step 600 µL of 6 M HCl was added, and solution was maintained under agitation
for 24 h at 90 ºC. The solvent was dried in a Speed Vac, after that 500µL of water was
added prior HPLC-MS/MS analysis.
Supplementary Note 2.
HPLC-MS/MS analysis of tryptophan.
HPLC-MS/MS analysis was performed in an Agilent HPLC (1200 series, Agilent
Waldbronn, Germany). The column oven and auto sampler temperatures were set at 25
°C and 4 °C, respectively. For the separation, a C18 column was used (250 mm x 4.6
mm, 5μm particle size, Gemini Phenomenex, Torrance, California, USA) and flow rate
was set at 1.0 mL/min. Gradient elution was carried out with 0.1 % formic acid (A) and
acetonitrile (B). The separation was conducted with 0 % B during first 5 min, 0 to 60 %
B for 15 min, 60 % B for 5 min, 60 to 0 % B for 25.5 min and 0 % B for 30 min.
Samples of 100 µL were injected. For the MS/MS analysis, it was used a 4000 QTRAP
mass spectrometer (Applied Biosystems, Foster City, CA, USA) with an electrospray
ionization source. The samples were analyzed in the positive ion mode by an Enhanced
Mass Spectrum (EMS), Enhanced Product Ion (EPI), and Selected Reaction Monitoring
(SRM) mode. Isotopically [15N]-labeled tryptophan (15N-Trp) was detected and
quantified by SRM mode, using melatonin isotopically D3-labeled (Mel-D3) as internal
standard 24. The SRM detection of the 15N-Trp and Mel-D3 was performed by the mass
transitions m/z 207 189 and 236 174, respectively. A standard calibration curve for
15
N-Trp was prepared using six aliquots of increasing concentrations (0, 0.03, 0.75, 1.5,
3.0 and 6.0 nM) and Mel-D3 as internal standard in the final concentration of 50 nM.
The SRM analyses were performed using collision energy of 17eV for 15N-Trp and
23eV for Mel-D3. Collision excitation potential was 10V and 8V for 15N-Trp and MelD3, respectively. The collision activated dissociation (CAD) gas flow was set as
medium. Each transition was obtained with 5 ms of pause time and a dwell time of 200
ms. The information dependent acquisition (IDA) scan intensity threshold was set at
500, 000 counts per seconds (cps). EMS survey scan was acquired with a scan rate of
1000 amu/s. The CAD gas flow was adjusted as medium and the mass range was set at
190-250 m/z. EPI scan analyses of 15N-Trp (m/z 207) and Mel-D3 (m/z 236) were
performed with CE and collision excitation spread at 15eV and 10 eV, respectively. The
dynamic fill time was used, setting first the linear ion trap (LIT) fill time at 20ms. The
CAD gas flow for EPI experiments was adjusted as low and the mass range was set at
100-240 m/z. The source parameters were set as follows: curtain gas at 10 psi; ion
source at 5500 V; temperature at 700°C; gas 1 and gas 2 at 50 psi; declustering potential
at 51V and entrance potential at 10V.
Supplementary Note 3.
Enzymatic hydrolysis of DNA from A. tequilana.
Two microliters of 3 M sodium acetate buffer (pH 5.0) were added to a solution (50 µL)
containing approximately 15.0 µg/mL of DNA extracted and 0.1 mM of
desferroxamine. DNA was digested with 2.4 units of nuclease P1 (0.4 U/µL) at 37 °C,
300 rpm for 30 min. Then, 2 µL of 3 M Tris-HCl buffer (pH 7.5) and 12 units of
alkaline phosphatase (2 U/µL) were added for additional incubation (1 hour, 37 ºC, 300
x r.p.m). The final volume of solution was adjusted to 50 µL with methanol and 20 µL
of each sample was injected into HPLC-MS/MS system.
Supplementary Note 4.
HPLC-MS analysis DNA from bacteria.
Bacterial DNA was extracted following a short protocol proposed by Wilson, K (22).
DNA was hydrolyzed with 6 M HCl (2 h at 65 C), neutralized with 25 % ammonia
solution and analysed by HPLC-MS. HPLC-MS analyses were carried out in a
Shimadzu HPLC system (Tokyo, Japan) coupled to a Quattro II mass spectrometer
(Micromass, Manchester, UK) with a Z-spray source. Mass spectrometry analyses were
performed in the positive mode with source temperature at 150°C, desolvation
temperature at 200 °C, capillary voltage at 4.0 kV, sample cone voltage and extractor
cone voltage at 30 V and 5 V, respectively. For analytical purposes a Phenomenex
Gemini C-18 column (250 × 4.6mm i.d., 5 μm particle size) was used with the UV
detector set at 260 nm. DNA bases were separated using the mobile phase 2 mM
ammonium formate (A), acetonitrile (B) and flow rate of 0.6 mL/min. The linear
gradient was 0 %B during 5 min, 0 to 20 %B over 15 min, 20 to 100 % B over 5 min,
100 %B for 5 min, returning to 0 % B over 2 min and maintaining 0 %B until 35 min.
The column oven was set at 25 C. Flux directed to the mass spectrometer was 0.135
mL/min and full-scan data were acquired over a mass range of 100–600 m/z.
(15N-Trp
[15N]-labeled Tryptophan
ng/mg of Fresh Tissue)
50
O
OH
40
15
15
N
NH3+
H
30
20
10
0
H2O
14NH Cl
4
15NH Cl
4
Fig. S1 | Quantification of 15N-labeled tryptophan (15N-Trp) in foliar tissue of A.
tequilana using HPLC-MS/MS by SRM mode. The sample groups analyzed were
H2O treated, 14NH4Cl and 15NH4Cl. The quantity of 15N-Trp (ng/mg) represents the
average of two different samples.
207.4
A
7.5e6
m/z 207
O
OH
6.5e6
15
15
205.4
5.5e6
N
NH3+
H
4.5e6
m/z 205
O
3.5e6
208.4
2.5e6
OH
NH3+
N
1.5e6
H
5.0e5
0.0
190 194
1.3e7
198
202
206
210
214
146.0
B
218
m/z 205
1.1e7
Intensity (cps)
222
188.0
m/z 188
O
9.0e6
OH
7.0e6
144.2
NH3+
N
H
5.0e6
170.1
3.0e6
132.2
205.0
159.1
1.0e6
0.0
120
1.3e7
130
C
140
150
160
170
180
190
200
210
189.0
147.0
m/z 207
1.1e7
m/z 189
9.0e6
O
OH
7.0e6
15
15
5.0e6
145.1
H
161.1 171.0
3.0e6
N
NH3+
207.1
1.0e6
0.0
120
130
140
150
160
170
180
190
200
210
m/z
Fig. S2 | HPLC-MS/MS detection of 14N-Trp and 15N-Trp. Injection of 10µL of
5µM 14N-Trp/15N-Trp standard solution, (A) EMS of 14N-Trp and 15N-Trp, (B) EPI
mass spectrum of 14N-Trp (m/z 205) and (C) EPI mass spectrum of 15 N-Trp (m/z 207).
A
236.2
2.6e6
2.4e6
m/z 236
2.2e6
D
2.0e6
D
H
1.8e6
1.6e6
D
N
235.3
O
1.4e6
H 3C
1.2e6
O
N
1.0e6
H
8.0e5
237.2
6.0e5
Mel-D3
Intensity (cps)
4.0e5
2.0e5
0.0
224 226 228 230 232 234 236 238 240 242 244 246 248 250
m/z 236
B
D
174.0
1.8e6
D
H
D
N
m/z 174
O
1.6e6
H 3C
1.4e6
O
N
1.2e6
H
236.0
1.0e6
8.0e5
130.2
6.0e5
4.0e5 103.2
131.0
159.0
2.0e5
0.0
100 110 120 130 140 150 160 170 180 190 200 210 220 230 240
m/z
Fig. S3 | HPLC-MS/MS detection of Mel-D3. (A) EMS and (B) EPI mass spectrum of
5µM Mel-D3 (m/z 236), injection of 10µL.
A
14N-Trp
13.1
1.4e5
1.2e5
205
188
1.0e5
8.0e4
6.0e4
4.0e4
2.0e4
0.0
13.0
13.4
13.8
14.2
14.6
15.0
B
Intensity (cps)
15N-Trp
13.1
1.19e5
1.00e5
207
189
8.00e4
6.00e4
4.00e4
2.00e4
0.00
13.0
13.4
13.8
14.2
C
14.6
18.7
15.0
Mel-D3
3.5e5
3.0e5
236
2.5e5
174
2.0e5
1.5e5
1.0e5
5.0e4
0.00
15.0
16.0
17.0
18.0
19.0
20.0
Time (min)
Fig. S4 | HPLC-MS/MS detection of 14N-Trp and 15N-Trp.
14
N-Trp (m/z 205188)
(A), 15N-Trp (m/z 207189) (B) and Mel-D3 (m/z 236174) (C) in a mixture standard
solution (5µM final concentration and 10µL injected).
15N-Bteq
A
15N-Trp
Intensity (cps)
207
B
14N-Bteq
C
189
H2O
Time (min)
Fig. S5 | HPLC-MS/MS detection of 15N-Trp. SRM mode (m/z 207189) in the
samples 15N-Bteq (A), 14N-Bteq (B) and H2O (C). Data represents the mean values ±
standard error of the mean from three independent experiments. ()15N-labeled B.
tequilensis data are significantly different when compared to the H2O and unlabeled B.
tequilensis groups (p<0.05; t-test).
15NH Cl
4
A
15N-Trp
Intensity (cps)
207
B
14NH Cl
4
C
189
H2O
Time (min)
Fig. S6 | HPLC-MS/MS detection of 15N-Trp. SRM mode (m/z 207189) in the
samples 15NH4Cl (A), 14NH4Cl (B) and H2O (C).
13.1
1715
1600
1400
15N-Trp
1200
area/Mel-D3 area
A
1000
800
0.04
15N-Trp
0.03
207
0.02
189
y = 0.0059x + 0.0002
R² = 0.9997
0.01
0.00
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0
15N-Trp
(nM)
Intensity (cps)
600
400
200
0
13.0 13.2 13.4 13.6 13.8 14.0 14.2 14.4 14.6 14.8 15.0
B
18.7
1.20e5
Mel-D3
236
1.00e5
174
8.00e4
6.00e4
4.00e4
2.00e4
0.00
15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0 19.5 20.0
Time (min)
Fig. S7 | HPLC-MS/MS detection of 15N-Trp. SRM mode using the mass transition
m/z 207189 for 15N-Trp (A) and m/z 236174 for the internal standard Mel-D3 (B).
Insert: Calibration curve of 15N-Trp with increased concentrations of 15N-Trp (0, 0.03,
0.75, 1.5, 3.0 and 6.0 nM) vs 50 nM Mel-D3. Mel-D3 was synthesized according to
Almeida et al. (28). Mel-D3 was purified by a HPLC system consisting of two LC10ADVP pumps, a SPD-M10AVP photodiode array with wavelength setting at 280nm,
SCL-10AVP system controller, and monitored by Class-VP 5.032 software (Shimadzu,
Kyoto, Japan). A semi-preparative Luna 10 C-18 (2) column (250 x 10 mm, 5µm
particle size) was used with flow rate of 4 mL/min. Water (A) and methanol (B) were
used in mobile phase with linear gradient of 30–70% B for 10 min, 70% B for 10 min,
70-30% B during 1 min and 30% B for 25 min.
G
A
100
A
15.82
C
2.00
4.00
6.00
8.00
UV (260 nm)
17.81
16.73
8.24
0
0.00
T
10.00
12.00
14.00
16.00
18.00
20.00
22.00
+H+
O
B
15
N
15
NH
157
100
15 N
15
H
15
N
NH2
m/z 157
158
152
Intensity (cps)
24.00
Time (min)
0
15
+H+
NH2
C
15
N
115
100
15 N
O
H
112
116
m/z 115
0
15
D
15
141
100
15
15 N
15
H
+H+
N
N
m/z 141
142
136
NH2
N
0
O
E
+H+
15
NH2
129
100
15 N
H
O
m/z 129
0
100
105
110
115
120
125
130
135
140
145
150
155
160
165
170
175
180
185
190
195
200
m/z
Fig. S8 | HPLC-MS analysis of DNA. B. tequilensis cultivated in a medium with
15
NH4Cl. UV chromatogram at 260nm (A). Mass spectrum obtained from peak at 15.82
(15N-labeled guanine) (B), 8.24 (15N-labeled cytosine) (C), 17.81 (15N-labeled adenine)
(D) and 16.73 minutes (15N-labeled thymine) (E).
A
B
O
9.11
15
N
15
N
5000
NH
15
N
N
6000
15
N
O
5000
dR
4000
273157
3000
15
NH2
dR
4000
NH2
3.56
15
15
15
231115
3000
2000
Intensity (cps)
2000
1000
0
1000
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34
0
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34
15
NH2
C
8.40
15
N
N
7870
7000
15
N
O
D
10.20
15
N
15
NH2
N
O
1.2e6
dR
5000
4000
8.0e5
3000
6.0e5
2000
4.0e5
1000
2.0e5
2
4
6
dR
1.0e6
257141
0
15
1.6e6
1.4e6
15
6000
H3C
8 10 12 14 16 18 20 22 24 26 28 30 32 34
0
245129
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34
Time (min)
Fig. S9 | HPLC-MS/MS analysis of 15N-labeled 2´-deoxynucleosides from A.
tequilana supplemented with 15NH4Cl. Using the SRM mode, 2´-deoxynucleosides
were detected by the loss of 2-deoxyribose moiety: 15N5-dG, m/z 273157 (A), 15N3dC, m/z 231115 (B), 15N5-dA, m/z 257141 (C) and 15N2-dT, m/z 245129 (D).
15
A
NH 2
H 3C
15
N
a
15
400
NH 2
H 3C
6.68
350
15
300
250
15
15
N
N
N
O
dR
245129
O
200
dR
Intensity (cps)
150
245129
100
50
1.0
3.0
5.0
7.0
B
9.0
11.0
13.0
15.0
15
b
1200
15
NH2
N
15
9.70
1000
15
800
N
600
dR
15
N
CH3
N
271155
400
200
1.0
3.0
5.0
7.0
9.0
11.0
13.0
15.0
Time (min)
Fig. S10 | HPLC-MS/MS analysis of 15N-labeled 2´-deoxynucleosides from A.
tequilana supplemented with 15N-labeled B. tequilensis. Using SRM mode, 2´deoxynucleosides were detected by the loss of 2-deoxyribose moiety: 15N3- MedC, m/z
245129 (A) and 15N5-MedAdo, m/z 271155 (B). Peaks at a and b correspond to
15
N2-dT and 15N5-dG, respectively.
A
a
15
NH 2
H 3C
1800
15
6.41
1400
15
N
N
O
1000
Intensity (cps)
dR
600
245129
200
0
1.0
3.0
5.0
7.0
B
9.0
11.0
13.0
15.0
15
b
15
800
10.0
600
15
NH2
N
15
N
15
N
CH3
N
dR
400
271155
200
1.0
3.0
5.0
7.0
9.0
11
13
15
Time (min)
Fig. S11 | HPLC-MS/MS analysis of 15N-labeled 2´-deoxynucleosides from A.
tequilana supplemented with 15NH4Cl. Using SRM mode, 2´-deoxynucleosides were
detected by the loss of 2-deoxyribose moiety: 15N3- MedC, m/z 245129 (A) and 15N5MedAdo, m/z 271155 (B). Peaks at a and b correspond to 15N2-dT and 15N5-dG,
respectively.