Restriction/modification polypeptides, polynucleotides, and methods
The present invention relates to the discovery of a novel restriction/modification system in Caldicellulosiruptor bescii . The discovered restriction enzyme is a HaeIII-like restriction enzyme that possesses a thermophilic activity profile. The restriction/modification system also includes a methyltransferase, M.CbeI, that methylates at least one cytosine residue in the CbeI recognition sequence to m 4 C. Thus, the invention provides, in various aspects, isolated CbeI or M.CbeI polypeptides, or biologically active fragments thereof; isolated polynucleotides that encode the CbeI or M.CbeI polypeptides or biologically active fragments thereof, including expression vectors that include such polynucleotide sequences; methods of digesting DNA using a CbeI polypeptide; methods of treating a DNA molecule using a M.CbeI polypeptide; and methods of transforming a Caldicellulosiruptor cell.
1. A method of transforming a microbial cell, the method comprising:
treating a DNA molecule comprising at least one 5′-GGCC-3′ sequence with a M.CbeI polypeptide under condition effective for the M.CbeI polypeptide to methylate at least one C residue of the 5′-GGCC-3′ sequence;
introducing the methylated polynucleotide into the microbial cell that comprises a thermophile or a hyperthermophile.
2. The method of claim 1 wherein the microbial cell comprises a Caldicellulosiruptor spp.
3. The method of claim 2 wherein the Caldicellulosiruptor spp. comprises C. bescii.
4. The method of claim 1 wherein the DNA is treated with the M.CbeI in vitro.
5. A genetically modified Caldicellulosiruptor spp. cell comprising a heterologous polynucleotide comprising at least one 5′-GGCC-3′ sequence comprising at least one methylated C residue.
6. The genetically modified cell of claim 5 wherein the heterologous polynucleotide was methylated in vitro by a M.CbeI polypeptide.
7. The genetically modified cell of claim 5 wherein the Caldicellulosiruptor spp. comprises C. bescii.