The Signaling Pathway That cGAMP Riboswitches Found: Analysis and Application of Riboswitches to Study cGAMP Signaling in Geobacter sulfurreducens
Abstract
:1. Introduction
2. Results
2.1. Riboswitch Analysis: Development of cGAMP Riboswitch Affinity Capture Method
2.2. Riboswitch Analysis: Transcriptome Affinity Capture and RNA-Seq Analysis
2.3. Riboswitch Application: Analysis of a Second Hypr GGDEF in Geobacter sulfurreducens
2.4. Riboswitch Application: Screening for New Conditions and Genes That Affect cGAMP Levels in Geobacter sulfurreducens
3. Materials and Methods
3.1. Preparation of cGAMP-Sepharose Capture Compound
3.2. RNA Labeling with Fluorescein
3.3. Binding of RNA Samples to cGAMP-Sepharose
3.4. Total RNA Isolation and Quality Check
3.5. rRNA Depletion
3.6. RNAseq
3.7. Growth and Transposon Library Generation
3.8. Deletion of GSU1937
3.9. Nanoluciferase (Nluc) Reporter Assay
3.10. Fluorescent Biosensor Assay
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Strain or Transposon ID | Relative Luminescence 1 | Disrupted Gene, Transposon Location and Description |
---|---|---|
WT | 100 ± 27.9 | |
ΔgacA | 26.6 ± 6.5 | GSU1658; Hypr GGDEF |
ΔesnD | 154.1 ± 32.8 | GSU3376; Diguanylate cyclase |
empty | 1.3 ± 1.3 | |
6G9 | 27.3 ± 11.6 | GSU1508; xapK; 463/1119 nt |
3F4 | 35.7 ± 4.1 | GSU1508; xapK; 730/1119 nt |
2G8 | 41.7 ± 15 | GSU1512; Hypothetical |
2F9 | 46.2 ± 44.4 | GSU1497; pilA-C |
1C4 | 56.4 ± 11.3 | 11 bp deletion in GSU0946 (Sensor Diguanylate cyclase/Phosphodiesterase EAL, HAMP-; PAS-containing also Intergenic insertion, negative strand; GSU0538 (hspA-1 ATP dependent chaperone)—GSU0539 (hypothetical). |
4F11 | 189.5 ± 107.6 | GSU1491: pilB; 424/1707 nt |
9C1 | 204.1 ± 52.5 | GSU2220; esnB; CheW-like protein |
4E6 | 266.5 ± 41.4 | GSU1491; pilB; 1522/1707 nt |
Gene Name | Catalytic Domain | Known Cache-Ligand Structures | |||||
---|---|---|---|---|---|---|---|
- | - | PctA: AA | Mlp24: AA | Tlp3: AA | CitA: | DcuS: | TlpB: |
Citrate | Malate | Pyruvate | |||||
GSU0400 | MCP | 0.17 | 0.18 | 0.22 | 0.26 | 0.29 | 0.28 |
GUS0962 | HisK | 0.25 | 0.28 | 0.24 | 0.23 | 0.22 | 0.21 |
GSU1030 | MCP | 0.6 | 0.63 | 0.79 | 0.82 | 0.92 | 0.91 |
GSU1033 | MCP | 0.61 | 0.64 | 0.82 | 0.84 | 0.85 | 0.84 |
GSU1303 | MCP | 0.62 | 0.66 | 0.80 | 0.81 | 0.90 | 0.89 |
GSU2297 | HisK | 0.5 | 0.52 | 0.54 | 0.56 | 0.57 | 0.57 |
GSU2388 | HisK | 0.7 | 0.79 | 0.99 | 0.99 | 0.89 | 0.89 |
GSU2507 | HisK | 0.73 | 0.79 | 0.94 | 0.98 | 0.91 | 0.92 |
GSU3356 | GGDEF | 0.76 | 0.8 | 0.99 | 0.98 | 0.89 | 0.97 |
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Tan, Z.; Chan, C.H.; Maleska, M.; Banuelos Jara, B.; Lohman, B.K.; Ricks, N.J.; Bond, D.R.; Hammond, M.C. The Signaling Pathway That cGAMP Riboswitches Found: Analysis and Application of Riboswitches to Study cGAMP Signaling in Geobacter sulfurreducens. Int. J. Mol. Sci. 2022, 23, 1183. https://doi.org/10.3390/ijms23031183
Tan Z, Chan CH, Maleska M, Banuelos Jara B, Lohman BK, Ricks NJ, Bond DR, Hammond MC. The Signaling Pathway That cGAMP Riboswitches Found: Analysis and Application of Riboswitches to Study cGAMP Signaling in Geobacter sulfurreducens. International Journal of Molecular Sciences. 2022; 23(3):1183. https://doi.org/10.3390/ijms23031183
Chicago/Turabian StyleTan, Zhesen, Chi Ho Chan, Michael Maleska, Bryan Banuelos Jara, Brian K. Lohman, Nathan J. Ricks, Daniel R. Bond, and Ming C. Hammond. 2022. "The Signaling Pathway That cGAMP Riboswitches Found: Analysis and Application of Riboswitches to Study cGAMP Signaling in Geobacter sulfurreducens" International Journal of Molecular Sciences 23, no. 3: 1183. https://doi.org/10.3390/ijms23031183