Melanin Pathway Determination in Sclerotium cepivorum Berk Using Spectrophotometric Assays, Inhibition Compound, and Protein Validation
Abstract
:1. Introduction
2. Materials and Methods
2.1. Micro-Organisms and Culture Media
2.2. Sclerotia Melanin Extraction
2.3. Analysis of the Melanin Composition
2.4. Susceptibility of S. cepivorum to Tricyclazole
2.5. Isolation of S. cepivorum DNA, RNA, and cDNA
2.6. Expression of S. cepivorum SDH Protein in P. pastoris (X-33) Cells
2.7. Zymogram Assay for Recombinant SDH Activity
2.8. Cell extract Obtention from Magnaporthe grisea
2.9. Microscopy Images
3. Results
3.1. Spectroscopy-Based Analysis of Pigment Extracted from S. cepivorum
3.2. S. cepivorum Melanization Is Affected by Treatment with Tricyclazole
3.3. Identification and Obtainment of a S. cepivorum Scytalone Dehydratase Gene
3.4. S. cepivorum Scytalone Dehydratase Recombinant Activity
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
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Purified Compound | Direct Sample | ||
---|---|---|---|
Frequency (cm−1) | Signal Generating Group | Frequency (cm−1) | Signal Generating Group |
3291 | -O-H Elongation vibration. Polymeric association. | 3286 | -O-H Elongation vibration. Polymeric association. |
3060 3005 2953 | =C-H Elongation vibration. Aromatic compound. | 2922 | =C-H Elongation vibration. Aromatic compound. |
1631 1547 1531 1453 | -C=C- Deformation (in plane) aromatic compound. | 1622 | -C=C- Deformation (in plane) aromatic compound. |
1409 1378 | -C-O Elongation vibration. Characteristic of phenol and derivatives. | 1403 1373 | -C-O Elongation vibration. Characteristic of phenol and derivatives. |
1242 | -O-H Deformation (in plane). Characteristic of phenol and derivatives. | 1236 | -O-H Deformation (in plane). Characteristic of phenol and derivatives. |
1151 1076 1029 | -C-H Deformation (in plane) and aromatic ring substitution 1:2:3. | 1069 1027 | -C-H Deformation (in plane) and aromatic ring substitution 1:2:3. |
885 765 718 | -C-H Deformation (out plane) and aromatic ring substitution 1:2:3. | 890 828 747 | -C-H Deformation (out plane) and aromatic ring substitution 1:2:3. |
Chemical Shift, ppm | Chemical Grouping | Reference Materials | Literature Refs. |
---|---|---|---|
172.80 | -CONH | Chitin | [31,32] |
160.19 | Aromatic -C-, O-naphthyl | Poly 1,8 DHN | [31] |
135.77–111.32 | Aromatic -C-, -C=C- | Poly 1,8 DHN, likely oleoyl | [31,33] |
103.47, 100.73 | Aromatic –C-, -O-CH-O- | Chitin, glucan | [32] |
84.12 | -CHOH | α-1.3 glucan | [32] |
73.57–71.41 | -CHOH, -CHO- | Chitin, α-1.3 glucan | [32] |
61.54 | -CH2OH | Chitin | [32] |
60.40 | -CH2OH | α-1.3 glucan | [32] |
54.80 | -CH-NH- | Chitin | [32] |
29.08 | -(CH2)- (C4–C7, C2–C16) | Likely oleoyl | [33] |
23.89 | -C(=O)-CH3, -(CH2)- (C3) | Chitin, likely oleoyl | [32,33] |
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Salazar-García, L.M.; Ortega-Cuevas, R.I.; Martínez-Álvarez, J.A.; González-Hernández, S.E.; Martínez-Álvarez, R.A.; Mendoza-Olivares, D.; Vázquez, M.Á.; Flores-Martínez, A.; Ponce-Noyola, P. Melanin Pathway Determination in Sclerotium cepivorum Berk Using Spectrophotometric Assays, Inhibition Compound, and Protein Validation. Microbiol. Res. 2022, 13, 152-166. https://doi.org/10.3390/microbiolres13020013
Salazar-García LM, Ortega-Cuevas RI, Martínez-Álvarez JA, González-Hernández SE, Martínez-Álvarez RA, Mendoza-Olivares D, Vázquez MÁ, Flores-Martínez A, Ponce-Noyola P. Melanin Pathway Determination in Sclerotium cepivorum Berk Using Spectrophotometric Assays, Inhibition Compound, and Protein Validation. Microbiology Research. 2022; 13(2):152-166. https://doi.org/10.3390/microbiolres13020013
Chicago/Turabian StyleSalazar-García, Luis M., Rocío Ivette Ortega-Cuevas, José A. Martínez-Álvarez, Sandra E. González-Hernández, Román Antonio Martínez-Álvarez, Diana Mendoza-Olivares, Miguel Ángel Vázquez, Alberto Flores-Martínez, and Patricia Ponce-Noyola. 2022. "Melanin Pathway Determination in Sclerotium cepivorum Berk Using Spectrophotometric Assays, Inhibition Compound, and Protein Validation" Microbiology Research 13, no. 2: 152-166. https://doi.org/10.3390/microbiolres13020013
APA StyleSalazar-García, L. M., Ortega-Cuevas, R. I., Martínez-Álvarez, J. A., González-Hernández, S. E., Martínez-Álvarez, R. A., Mendoza-Olivares, D., Vázquez, M. Á., Flores-Martínez, A., & Ponce-Noyola, P. (2022). Melanin Pathway Determination in Sclerotium cepivorum Berk Using Spectrophotometric Assays, Inhibition Compound, and Protein Validation. Microbiology Research, 13(2), 152-166. https://doi.org/10.3390/microbiolres13020013