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Characterization of a novel N4 -methylcytosine restriction-modification system in Deinococcus radiodurans

ABSTRACT: Deinococcus radiodurans is an extremophilic microorganism that possesses a unique DNA damage repair system, conferring strong resistance to radiation, desiccation, oxidative stress, and chemical damage. Recently, we discovered that D. radiodurans possesses a N4-methylation (m4C) methyltransferase called M.DraR1, which recognizes the 5'-CCGCGG-3' sequence and methylates the second cytosine. Here, we revealed its cognate restriction endonuclease R.DraR1 and recognized that it is the only endonuclease specially for non-4C-methylated 5'-CCGCGG-3' sequence so far. We designated the particular m4C R.DraR1-M.DraR1 as DraI R-M system. Bioinformatics searches displayed the rarity of the DraI R-M homologues system. Meanwhile, recombination and transformation efficiency experiments demonstrated the important role of the DraI R-M system in response to oxidative stress. Besides, in vitro activity experiments showed that R.DraR1 could exceptionally cleave DNA substrates with m5C-methlated 5'-CCGCGG-3' sequence besides its routine activity, suggesting that this particular R-M component possesses a broader substrate choice. Furthermore, an imbalance of the DraI R-M system led to cell death through regulating genes involved in the maintenance of cell survival such as genome stability, transporter, energy production. Thus, our research revealed a novel m4C R-M system that plays key roles in maintaining cell viability and defending foreign DNA in D. radiodurans.

ORGANISM(S): Deinococcus radiodurans

PROVIDER: GSE251690 | GEO | 2024/02/14

REPOSITORIES: GEO

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Project description:Abstract Background The extraordinarily resistant bacterium Deinococcus radiodurans withstands harsh environmental conditions present in outer space. Deinococcus radiodurans was exposed for one year outside the International Space Station within Tanpopo orbital mission to investigate microbial survival and space travel. In addition, a ground-based simulation experiment with conditions, mirroring those from Low Earth orbit, was performed. Methods We monitored Deinococcus radiodurans cells during early stage of recovery after Low Earth orbit exposure using electron microscopy tools. Furthermore, proteomic, transcriptomic and metabolomic analyses were performed to identify molecular mechanisms responsible for the survival of Deinococcus radiodurans in Low Earth orbit. Results D. radiodurans cells exposed to low Earth orbit conditions do not exhibit any morphological damage. However, an accumulation of numerous outer-membrane associated vesicles was observed. On levels of proteins and transcripts, a multifaceted response was detected to alleviate cell stress. The UvrABC endonuclease excision repair mechanism was triggered to cope with DNA damage. Defense against reactive oxygen species is mirrored by the increased abundance of catalases and is accompanied by the increased abundance of putrescine which works as scavenging molecule. In addition, several proteins and mRNAs, responsible for regulatory and transporting functions showed increased abundances. The decrease in primary metabolites indicate alternations in the energy status, which is needed to repair damaged molecules. Conclusion Low Earth orbit induced molecular rearrangements trigger multiple components of metabolic stress response and regulatory networks in exposed microbial cells. Presented results show that the non-sporulating bacterium Deinococcus radiodurans survived long-term Low Earth orbit exposure if wavelength below 200 nm are not present, which mirrors the UV spectrum of Mars, where CO2 effectively provides a shield below 190 nm. These results should be considered in the context of planetary protection concerns and the development of new sterilization techniques for future space missions.

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Characterization of a novel N4 -methylcytosine restriction-modification system in Deinococcus radiodurans

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