Molecular Identification of Endophytic Bacteria in Leucojum aestivum In Vitro Culture, NMR-Based Metabolomics Study and LC-MS Analysis Leading to Potential Amaryllidaceae Alkaloid Production
Abstract
:1. Introduction
2. Results and Discussion
2.1. Isolation and Identification of Endophytic Bacteria from Leucojum aestivum In Vitro Bulblets
2.2. Untargeted 1H NMR-Based Metabolomics: Identification and Quantification
2.3. Statistical Analysis with Multivariate Data Analysis
2.4. Quantification by NMR
2.5. Target Research of Amaryllidaceae Alkaloids by 1H NMR, LC-MS and GC-MS Analysis in Bacterial Endophyte Bacillus sp.
3. Materials and Methods
3.1. Chemicals
3.2. Plant Materials, Preparation of In Vitro Culture of L. aestivum
3.3. Isolation of Endophytic Bacteria
3.4. Identification of Endophytic Bacteria and PCR Amplification
3.5. In Silico Analysis
3.6. Preparation of Plants Samples and Endophytic Bacteria for Untargeted NMR-Based Metabolomics
3.7. Preparation of Enriched Endophytic Bacteria Sample for NMR, LC-MS and GC-MS Analysis
3.8. NMR Equipment and Experimental Conditions
3.9. Softwares and the Statistical Analysis
3.10. LC-MS and GC-MS Equipments and Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Glossary
MeOH | Methanol |
CH3CN | Acetonitrile |
HCOOH | Formic acid |
NH4OH | Ammonium hydroxide |
LA | Leucojum aestivum |
MS | Murashige and Skoog |
NA | Nutrient agar |
DW | Dry weight |
SPE | Solid phase extraction |
NMR | Nuclear Magnetic Resonance |
ESI-HRMS | Electrospray Ionization—High Resolution Mass Spectrometry |
MS | Mass spectrometry |
LC | Liquid chromatography |
GC | Gas chromatography |
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Molecules | Molecular Weight | Molecular Formula | Chemical Shifts, Multiplicity (ppm), J (Hz) | Extract from In Vivo L. aestivum Bulb | Extract from In Vitro L. aestivum Bulblet | Extract from Bacillus sp. |
---|---|---|---|---|---|---|
Adenine | 135,113 | C5H5N5 | 8.2 (s), 8.21 (s) | − | − | Y |
Cytosine | 111.1 | C4H5N3O | 7.52 (s), 7.51 (s) | − | − | Y |
Phenylalanine | 165.19 | C9H11NO2 | 7.42–7.33 (m), 3.09 (dd, J = 14.8, 8.3) | − | − | Y |
Tyrosine | 181.19 | C9H11NO3 | 7.16 (d, J = 8.4), 6.83 (d, J = 8.5) | Y | Y | Y |
Tyramine | 137.18 | C8H11NO | 7.11 (d, J = 8.6) 6.71,(d, J = 8.5) | Y | Y | Y |
Gallic acid | 170.12 | C7H6O5 | 7.02 (s) | Y | Y | − |
Lycorine | 287.32 | C16H17NO4 | 7.00 (s), 6.85 (s) | Y | Y | − |
Galanthamine | 287.35 | C17H21NO3 | 6.95 (d, J = 8.5) 6.89 (d, J = 8.5) | Y | − | − |
Sucrose | 342.30 | C12H22O11 | 5.39 (d, J = 3.8) | Y | Y | − |
α-Glucose | 180.16 | C6H12O6 | 5.17 (d, J = 3.8) | Y | Y | − |
β-Glucose | 180.16 | C6H12O6 | 4.57 (d, J = 7.9) | Y | Y | − |
Malate | 134.09 | C4H6O5 | 4.34 (dd, J = 6.6 Hz, 4.7 Hz) | − | − | Y |
Oxaloacetic acid | 132.07 | C4H4O5 | 3.64 (s) | Y | Y | − |
Asparagine | 132.11 | C4H8N2O3 | 3.94 (dd, J = 8.0, 4.0) | Y | Y | − |
Aspartate | 133.10 | C4H7NO4 | 2.78 (dd, J = 1.7, 5.3) | Y | Y | − |
Citrate | 192.12 | C6H8O7 | 2.74 (d, J = 17.6 Hz), | − | Y | − |
Alanine | 89.09 | C3H7NO2 | 1.46 (d, J = 7.2) | Y | Y | − |
Threonine | 119.12 | C4H9NO3 | 1.33 (d, J = 6.6) | Y | Y | − |
Valine | 117.15 | C5H11NO2 | 1.00 (d, J = 6.8) 1.05 (d, J = 6.8) | Y | Y | Y |
Isoleucine | 131.17 | C6H13NO2 | 0.96 (t, J = 7.4) 1.03 (d, J = 6.8) | Y | Y | Y |
Leucine | 131.17 | C6H13NO2 | 1.05 (d, J = 7) 1.01 (t, J = 7) | − | − | Y |
Fatty acids (as oleic acid) | 282.5 | C18H34O2 | 0.89 (d, J = 7.1) | Y | Y | Y |
Compounds Name | Retention Time (min) | Molecular Formula | Molecular Mass [M+H]+ | Identification Methods |
---|---|---|---|---|
Methyl-phenylalanine | 1.9 | C10H13NO2 | 180.1025 | b, d, e, f |
Tazettine | 3.2 | C18H21NO5 | 332.1486 | c, e, f |
Deoxyadenosine | 4.6 | C10H13N5O3 | 252.1097 | c, d, e |
Adenosine | 4.9 | C10H13N5O4 | 268.10481 | b, c, d, e |
Lycorine | 6.1 | C16H17NO4 | 288.1231 | a, c, e, f |
Deoxycytidine | 6.4 | C9H13N3O4 | 228.0997 | c, d, e |
Pseudolycorine | 8 | C16H19NO4 | 290.14 | e, f |
Acetyl pseudolycorine | 11.5 | C18H21NO5 | 332.1498 | c, e |
Compound Name | Retention Time (min) | Molecular Mass | Molecular Formula | Retention Index |
---|---|---|---|---|
Tazettine | 12.84 | 331 | C18H21NO5 | 2415 |
1,2-Dihydro-chlidanthine | 15.15 | 289 | C17H23NO3 | 2211 |
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Spina, R.; Saliba, S.; Dupire, F.; Ptak, A.; Hehn, A.; Piutti, S.; Poinsignon, S.; Leclerc, S.; Bouguet-Bonnet, S.; Laurain-Mattar, D. Molecular Identification of Endophytic Bacteria in Leucojum aestivum In Vitro Culture, NMR-Based Metabolomics Study and LC-MS Analysis Leading to Potential Amaryllidaceae Alkaloid Production. Int. J. Mol. Sci. 2021, 22, 1773. https://doi.org/10.3390/ijms22041773
Spina R, Saliba S, Dupire F, Ptak A, Hehn A, Piutti S, Poinsignon S, Leclerc S, Bouguet-Bonnet S, Laurain-Mattar D. Molecular Identification of Endophytic Bacteria in Leucojum aestivum In Vitro Culture, NMR-Based Metabolomics Study and LC-MS Analysis Leading to Potential Amaryllidaceae Alkaloid Production. International Journal of Molecular Sciences. 2021; 22(4):1773. https://doi.org/10.3390/ijms22041773
Chicago/Turabian StyleSpina, Rosella, Sahar Saliba, François Dupire, Agata Ptak, Alain Hehn, Séverine Piutti, Sophie Poinsignon, Sebastien Leclerc, Sabine Bouguet-Bonnet, and Dominique Laurain-Mattar. 2021. "Molecular Identification of Endophytic Bacteria in Leucojum aestivum In Vitro Culture, NMR-Based Metabolomics Study and LC-MS Analysis Leading to Potential Amaryllidaceae Alkaloid Production" International Journal of Molecular Sciences 22, no. 4: 1773. https://doi.org/10.3390/ijms22041773