Functional Whole Genome Screen of Nutrient-Starved Mycobacterium tuberculosis Identifies Genes Involved in Rifampin Tolerance
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains
2.2. Generation of Deletions
2.3. Media and Buffers
2.4. Generation of Transposon Mutant Library
2.5. Setup for Tn Screen in Nutrient-Rich Broth
2.6. Setup for Tn Screen in Nutrient Starvation
2.7. DNA Extraction
2.8. DNA Library Preparation
2.9. Hypothesis Testing for Antibiotic Hypersusceptibility
2.10. Oligonucleotides and Primers
2.11. Plasmids
2.12. Time-Kill Assays in Nutrient-Rich Broth
2.13. Time-Kill Assays in Nutrient Starvation Conditions
3. Results
3.1. Overview of Isoniazid and Rifampin Screens of Mtb Transposon Mutant Libraries
3.2. Identification of Mtb Genes Contributing to Rifampin Tolerance in Nutrient-Rich Broth and under Nutrient Starvation
3.3. Validation of Specific Mtb Genetic Requirements for Rifampin Hypersusceptibility Using Targeted Mutagenesis
4. Discussion
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Mtb Gene | Mav Gene | Mtb LFC | Mav LFC | Mtb Annotation | Mav Annotation |
---|---|---|---|---|---|
rv0049 | DFS55_00355 | −2.99 | −1.09 | hypothetical protein | hypothetical protein |
rv0819 | DFS55_21365 | 1.33 | 1.23 | mycothiol acetyltransferase | mycothiol synthase |
rv0820 | DFS55_21345 | −1.42 | −1.69 | phosphate ABC transporter ATP-binding protein PhoT | phosphate ABC transporter ATP-binding protein |
rv0929 | DFS55_20215 | −1.23 | −1.87 | phosphate ABC transporter permease PstC | phosphate ABC transporter permease PstC |
rv0930 | DFS55_20210 | −1.16 | −1.00 | phosphate ABC transporter permease PstA | phosphate ABC transporter permease PstA |
rv1836c | DFS55_12730 | −1.45 | −1.44 | hypothetical protein | hypothetical protein |
rv2179c | DFS55_14810 | −2.72 | −1.09 | 3′-5′ exoribonuclease A | hypothetical protein |
rv2224c | DFS55_15065 | −1.80 | −2.17 | carboxylesterase A | alpha/beta hydrolase |
rv3005c | DFS55_07355 | −1.25 | −1.06 | hypothetical protein | hypothetical protein |
Gene | 7H9 6d LFC | PBS 7d LFC | PBS 14d LFC | Annotation |
---|---|---|---|---|
rv0458 | −0.11 | 2.30 | 1.05 | aldehyde dehydrogenase |
rv0819 | 1.33 | −1.44 | −1.67 | mycothiolacetyl transferase |
rv0989c | 0.03 | 2.77 | 1.01 | polyprenyl-diphosphate synthase GrcC |
rv0998 | −0.22 | −1.24 | −1.25 | acetyltransferase Pat |
rv1183 | 0.1 | 0.52 | 1.12 | transmembrane transport protein MmpL10 |
rv1908c | 0.85 | −0.88 | −1.15 | catalase-peroxidase |
rv2051c | −0.19 | −1.33 | −1.23 | polyprenol-monophosphomannose synthase |
rv2199c | −0.5 | 2.53 | 2.42 | cytochrome c oxidase polypeptide 4 |
rv2374c | −0.2 | −1.47 | −1.68 | heat-inducible transcription repressor HrcA |
rv2392 | 0.95 | −1.29 | −1.17 | phosphoadenosine phosphosulfate reductase |
rv2633c | 0.41 | 1.76 | 1.02 | hypothetical protein |
rv2709 | 1.16 | −1.26 | −1.16 | transmembrane protein |
rv2733c | 0.01 | −1.55 | −1.19 | (Dimethylallyl)adenosine tRNA methylthiotransferase |
rv3680 | −0.26 | −1.12 | −1.40 | anion transporter ATPase |
rv3923c | −0.17 | −1.1 | −1.36 | ribonuclease P protein component |
Gene | 7H9 6d LFC | PBS 7d LFC | PBS 14d LFC | Annotation |
---|---|---|---|---|
rv0049 | −2.99 | −1.45 | −1.81 | hypothetical protein |
rv0199 | 1.14 | 4.50 | 2.53 | membrane protein |
rv0200 | 1.61 | 4.69 | 2.56 | transmembrane protein |
rv0655 | 1.32 | 5.03 | 3.01 | ABC transporter ATP-binding protein |
rv0819 | 1.33 | −1.44 | −1.67 | mycothiol acetyltransferase |
rv0994 | −3.25 | −1.27 | −1.01 | molybdopterin molybdenumtransferase 1 |
rv2179c | −2.72 | −1.32 | −1.30 | 3′-5′ exoribonuclease |
rv2690c | 5.56 | 3.26 | 1.63 | integral membrane protein |
rv2709 | 1.16 | −1.26 | −1.16 | transmembrane protein |
rv3723 | 1.18 | 4.94 | 3.04 | transmembrane protein |
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Matern, W.M.; Harris, H.T.; Danchik, C.; McDonald, M.; Patel, G.; Srivastava, A.; Ioerger, T.R.; Bader, J.S.; Karakousis, P.C. Functional Whole Genome Screen of Nutrient-Starved Mycobacterium tuberculosis Identifies Genes Involved in Rifampin Tolerance. Microorganisms 2023, 11, 2269. https://doi.org/10.3390/microorganisms11092269
Matern WM, Harris HT, Danchik C, McDonald M, Patel G, Srivastava A, Ioerger TR, Bader JS, Karakousis PC. Functional Whole Genome Screen of Nutrient-Starved Mycobacterium tuberculosis Identifies Genes Involved in Rifampin Tolerance. Microorganisms. 2023; 11(9):2269. https://doi.org/10.3390/microorganisms11092269
Chicago/Turabian StyleMatern, William M., Harley T. Harris, Carina Danchik, Marissa McDonald, Gopi Patel, Aashish Srivastava, Thomas R. Ioerger, Joel S. Bader, and Petros C. Karakousis. 2023. "Functional Whole Genome Screen of Nutrient-Starved Mycobacterium tuberculosis Identifies Genes Involved in Rifampin Tolerance" Microorganisms 11, no. 9: 2269. https://doi.org/10.3390/microorganisms11092269
APA StyleMatern, W. M., Harris, H. T., Danchik, C., McDonald, M., Patel, G., Srivastava, A., Ioerger, T. R., Bader, J. S., & Karakousis, P. C. (2023). Functional Whole Genome Screen of Nutrient-Starved Mycobacterium tuberculosis Identifies Genes Involved in Rifampin Tolerance. Microorganisms, 11(9), 2269. https://doi.org/10.3390/microorganisms11092269