The Restriction-Modification Systems of Clostridium carboxidivorans P7.

Clostridium carboxidivorans P7 (DSM 15243) is a bacterium that converts syngas (a mixture of CO, H 2 , and CO 2 ) 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 C. carboxidivorans encodes at least ten restriction enzymes and eight methyltransferases (MTases). To gain insight into the complex RM systems of C. carboxidivorans , 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 C. carboxidivorans , we expressed them individually in Escherichia coli for functional characterization. Recognition motifs were identified for all three Type I MTases (C A YNNNNNCTGC/GC A GNNNNNRTG, CC A NNNNNNNNTCG/CG A NNNNNNNNTGG and GC A NNNNNNNTNNCG/CGNN A NNNNNNNTGC), two Type II MTases (GATA A T and CRAAA A R), and a single Type III MTase (GAA A T). However, no methylated recognition motif was found for one of the three Type II enzymes. One recognition motif that was methylated in C. carboxidivorans but not in E. coli (AGA A 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 C. carboxidivorans that can accelerate the industrial exploitation of this strain.