Characterization of a Novel N4-Methylcytosine Restriction-Modification System in Deinococcus radiodurans
Abstract
:1. Introduction
2. Results
2.1. A m4C Restriction-Modification System DraI Existed in Deinococcus radiodurans
2.2. The m4C R-M System DraI Inhibits Foreign Plasmid and DNA Fragment Acquisition
2.3. R.DraR1 of the DraI System Could Specifically Cleave Unmethylated or m5C-Methylated 5′-CCGC/GG-3′ Sequences In Vitro
2.4. Imbalance in the DraI System Leads to a Significant Increase in Endogenous Cell Death
3. Discussion
4. Materials and Methods
4.1. Bacterial Strains and Culture Conditions
4.2. DNA Manipulation
4.3. Database Search for R-M Systems Which Recognize 5′-CCGCGG-3′ Sequence
4.4. Transformation and Recombination Efficiency Assays
4.5. Expression and Purification of R.DraR1
4.6. In Vitro M.DraR1 Protection and R.DraR1 Cleavage Assays
4.7. Fluorescence Microscopy and FACS Analysis
4.8. Over-Expression of R.DraR1 and β-Galactosidase Assay
4.9. RNA Isolation and Sequencing Analysis (RNA seq)
4.10. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene ID | Function Annotation | ΔR.DraR1 vs. R1.DEG | ΔM.DraR1 vs. R1.DEG | ||
---|---|---|---|---|---|
log2FC | p-Value | log2FC | p-Value | ||
DNA damage response protein | |||||
DR_0423 | Single-stranded DNA-binding protein, ddrA | −1.86 | 6.56 × 10−6 | 6.61 | 1.59 × 10−56 |
DR_0070 | Single-stranded DNA-binding protein, DdrB | −1.47 | 1.56 × 10−4 | 4.66 | 5.89 × 10−27 |
DR_0003 | DNA damage response protein C, ddrC | −2.39 | 3.07 × 10−9 | 4.36 | 9.44 × 10−19 |
DR_0326 | DNA damage response protein D, ddrD | −1.39 | 1.15 × 10−3 | 5.89 | 2.11 × 10−26 |
DR_B0100 | RNA ligase domain-containing protein, DdrP | −1.08 | 2.34 × 10−3 | 3.14 | 3.73 × 10−14 |
DR_2574 | Transcriptional repressor of the RDR regulon, DdrO | −1.28 | 4.14 × 10−4 | 3.57 | 3.47 × 10−27 |
DR_A0346 | DNA repair protein PprA | −1.52 | 1.53 × 10−3 | 3.13 | 6.37 × 10−12 |
DR_C0012 | GerE family transcriptional regulator | 2.37 | 1.40 × 10−8 | 9.35 | 2.05 × 10−7 |
Transporter | |||||
DR_0203 | ABC transporter permease | −1.18 | 1.36 × 10−2 | 1.79 | 6.25 × 10−5 |
DR_1036 | branched-chain amino acid ABC transporter permease | 1.05 | 2.38 × 10−3 | −0.96 | 1.61 × 10−2 |
DR_1142 | protein transporter | −2.18 | 1.91 × 10−10 | 1.22 | 8.28 × 10−3 |
DR_2612 | undecaprenyl phosphate transporter, DedA | −1.31 | 1.09 × 10−2 | 1.57 | 1.18 × 10−4 |
DR_B0083 | Potassium-transporting ATPase subunit B, kdpB | −1.11 | 1.82 × 10−2 | 2.58 | 1.36 × 10−3 |
DR_B0086 | Potassium-transporting ATPase subunit A, kdpA | −1.32 | 1.60 × 10−2 | 2.23 | 4.56 × 10−4 |
DR_A0073 | cation-transporting P-type ATPase | −1.18 | 4.98 × 10−4 | 2.05 | 1.12 × 10−9 |
Energy production | |||||
DR_A0076 | AAA+ ATPase domain-containing protein | −1.72 | 4.25 × 10−3 | 3.48 | 4.34 × 10−5 |
DR_C0016 | Phosphodiester glycosidase domain-containing protein | −5.12 | 7.99 × 10−25 | 4.44 | 2.27 × 10−6 |
DR_C0009 | KAP NTPase domain-containing protein | 2.08 | 1.53 × 10−6 | 1.23 | 1.04 × 10−2 |
DR_C0023 | N-terminal DNA-binding domain-containing protein, KfrA | −1.27 | 2.53 × 10−2 | 7.66 | 3.71 × 10−4 |
Biosynthetic pathway | |||||
DR_0133 | acetyl xylan esterase | −1.00 | 2.84 × 10−2 | 2.00 | 5.51 × 10−7 |
DR_0422 | Trans-aconitate 2-methyltransferase, tam | −1.26 | 7.85 × 10−5 | 5.17 | 3.86 × 10−31 |
DR_0598 | Peptidase M23 domain-containing protein, LytH | −1.05 | 5.65 × 10−3 | 3.26 | 5.56 × 10−3 |
DR_0659 | frnE protein | −1.35 | 2.57 × 10−5 | 3.15 | 2.59 × 10−4 |
DR_A0029 | 4-aminobutyrate aminotransferase, argD | −1.20 | 1.23 × 10−3 | −1.66 | 1.33 × 10−3 |
DR_A0178 | xanthine dehydrogenase C-terminal subunit | 1.10 | 7.00 × 10−3 | −1.17 | 1.03 × 10−3 |
DR_A0179 | xanthine dehydrogenase accessory protein XdhC | 1.39 | 4.09 × 10−5 | −1.15 | 4.02 × 10−3 |
DR_A0180 | guanine deaminase, guaD | 1.14 | 3.86 × 10−4 | −1.12 | 3.02 × 10−3 |
DR_B0060 | Spore coat protein U domain-containing protein | −2.40 | 9.31 × 10−6 | 6.46 | 8.75 × 10−6 |
DR_C0013 | N-acetylmuramoyl-L-alanine amidase | −4.18 | 4.30 × 10−14 | 6.07 | 2.26 × 10−5 |
Nucleoid-associated gene | |||||
DR_0906 | DNA gyrase subunit B, GyrB | −1.07 | 1.61 × 10−2 | 0.89 | 5.65 × 10−3 |
Exonuclease | |||||
DR_C0006 | putative 3′-5′ ssDNA/RNA exonuclease, TatD | 1.56 | 1.69 × 10−5 | 1.42 | 5.19 × 10−3 |
Natural competence regulated gene | |||||
DR_2338 | cinA protein | −1.25 | 1.59 × 10−3 | 2.56 | 2.18 × 10−15 |
Peptidase activity | |||||
DR_A0028 | DUF4274 domain-containing protein | −1.36 | 5.85 × 10−3 | −1.77 | 4.53 × 10−4 |
DR_A0283 | S8 serine protease | 1.23 | 1.39 × 10−2 | −0.96 | 1.06 × 10−2 |
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Shi, C.; Wang, L.; Xu, H.; Zhao, Y.; Tian, B.; Hua, Y. Characterization of a Novel N4-Methylcytosine Restriction-Modification System in Deinococcus radiodurans. Int. J. Mol. Sci. 2024, 25, 1660. https://doi.org/10.3390/ijms25031660
Shi C, Wang L, Xu H, Zhao Y, Tian B, Hua Y. Characterization of a Novel N4-Methylcytosine Restriction-Modification System in Deinococcus radiodurans. International Journal of Molecular Sciences. 2024; 25(3):1660. https://doi.org/10.3390/ijms25031660
Chicago/Turabian StyleShi, Chenxiang, Liangyan Wang, Hong Xu, Ye Zhao, Bing Tian, and Yuejin Hua. 2024. "Characterization of a Novel N4-Methylcytosine Restriction-Modification System in Deinococcus radiodurans" International Journal of Molecular Sciences 25, no. 3: 1660. https://doi.org/10.3390/ijms25031660