The Restriction-Modification Systems of Clostridium carboxidivorans P7

P7 (DSM 15243) is a bacterium that converts syngas (a mixture of CO, H , and CO ) into hexanol. An optimized and scaled-up industrial process could therefore provide a renewable source of fuels and chemicals while consuming industry waste gases. However, the genetic engineering of this bacterium is hindered by its multiple restriction-modification (RM) systems: the genome of encodes at least ten restriction enzymes and eight methyltransferases (MTases). To gain insight into the complex RM systems of , we analyzed genomic methylation patterns using single-molecule real-time (SMRT) sequencing and bisulfite sequencing. We identified six methylated sequence motifs. To match the methylation sites to the predicted MTases of , we expressed them individually in for functional characterization. Recognition motifs were identified for all three Type I MTases (C YNNNNNCTGC/GC GNNNNNRTG, CC NNNNNNNNTCG/CG NNNNNNNNTGG and GC NNNNNNNTNNCG/CGNN NNNNNNNTGC), two Type II MTases (GATA T and CRAAA R), and a single Type III MTase (GAA T). However, no methylated recognition motif was found for one of the three Type II enzymes. One recognition motif that was methylated in but not in (AGA GC) was matched to the remaining Type III MTase through a process of elimination. Understanding these enzymes and the corresponding recognition sites will facilitate the development of genetic tools for that can accelerate the industrial exploitation of this strain.