AUTHOR=Zatopek Kelly M. , Burkhart Brett W. , Morgan Richard D. , Gehring Alexandra M. , Scott Kristin A. , Santangelo Thomas J. , Gardner Andrew F. 

TITLE=The Hyperthermophilic Restriction-Modification Systems of Thermococcus kodakarensis Protect Genome Integrity

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.657356

DOI=10.3389/fmicb.2021.657356

ISSN=1664-302X

ABSTRACT=<p><italic>Thermococcus kodakarensis</italic> (<italic>T. kodakarensis</italic>), 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 <italic>T. kodakarensis</italic> 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 <italic>in vivo</italic> and likely protect against viral- or plasmid-based DNA transfers. Pacific Biosciences single molecule real-time (SMRT) sequencing of <italic>T. kodakarensis</italic> 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 N<sup>6</sup> position of adenine on one strand of the recognition sequences GTGA<underline>A</underline>G and TTCA<underline>A</underline>G, respectively. Further <italic>in vitro</italic> biochemical characterization of the REase activities reveal TkoI and TkoII cleave the DNA backbone GTGAAG(N)<sub>20</sub>/(N)<sub>18</sub> and TTCAAG(N)<sub>10</sub>/(N)<sub>8</sub>, respectively, away from the recognition sequences, while <italic>in vitro</italic> 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 <italic>T. kodakarensis</italic> genome integrity from invading foreign DNA.</p>