Abstract
Thermococcus kodakarensis (T. kodakarensis), a hyperthermophilic, genetically accessible model archaeon, encodes two putative restriction modification (R-M) defense systems, TkoI and TkoII. TkoI is encoded by TK1460 while TkoII is encoded by TK1158. Bioinformative analysis suggests both R-M enzymes are large, fused methyltransferase (MTase)-endonuclease polypeptides that contain both restriction endonuclease (REase) activity to degrade foreign invading DNA and MTase activity to methylate host genomic DNA at specific recognition sites. In this work, we demonsrate T. kodakarensis strains deleted for either or both R-M enzymes grow more slowly but display significantly increased competency compared to strains with intact R-M systems, suggesting that both TkoI and TkoII assist in maintenance of genomic integrity in vivo and likely protect against viral- or plasmid-based DNA transfers. Pacific Biosciences single molecule real-time (SMRT) sequencing of T. kodakarensis strains containing both, one or neither R-M systems permitted assignment of the recognition sites for TkoI and TkoII and demonstrated that both R-M enzymes are TypeIIL; TkoI and TkoII methylate the N6 position of adenine on one strand of the recognition sequences GTGAAG and TTCAAG, respectively. Further in vitro biochemical characterization of the REase activities reveal TkoI and TkoII cleave the DNA backbone GTGAAG(N)20/(N)18 and TTCAAG(N)10/(N)8, respectively, away from the recognition sequences, while in vitro characterization of the MTase activities reveal transfer of tritiated S-adenosyl methionine by TkoI and TkoII to their respective recognition sites. Together these results demonstrate TkoI and TkoII restriction systems are important for protecting T. kodakarensis genome integrity from invading foreign DNA.
Highlights
Across all domains of life, organisms have evolved a variety of innate defense systems that provide protection against invading foreign DNA
REBASE, an open source on-line database of DNA MTases, R-M systems and associated proteins identified two putative DNA MTases in the T. kodakarensis genome encoded by genes TK1460 and TK1158 (TkoI and TkoII, respectively) (Figure 1)
Pacific Bioscience (PacBio) single molecule realtime (SMRT) sequencing has emerged as a gold standard for the rapid discovery of new restriction and modification systems due to the ability to quickly identify methylated motifs in a genome sequence that result from host modification MTases
Summary
Across all domains of life, organisms have evolved a variety of innate defense systems that provide protection against invading foreign DNA. The T. kodakarensis genome encodes a variety of putative cellular defense systems including genes annotated as components of the abortive infection/phage exclusion systems (ABI), Bacteriophage Exclusion (BREX), Clustered regularly interspaced short palindromic repeats with cas genes (CRISPRCAS), R-M systems, Defense island system associated with restriction–modification (DISARM), DNA phosphorothioation (DND), and Toxin-Antitoxin, amongst others (Makarova et al, 2013; Zhang et al, 2020) (Supplementary Table 1) The retention of such a diverse range of host defense systems adumbrates their biological value, but to date, the importance and activities of few, if any of these systems have been evaluated with combined genetic and biochemical investigations. These results suggest a critical role for R-M systems in protecting and maintaining the archaeal T. kodakarensis genome, and further highlight the diversity and span of R-M systems across life
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