INVIVO METHYLATION IN ESCHERICHIA-COLI BY THE BACILLUS-SUBTILIS PHAGE-PHI-3T-I METHYLTRANSFERASE TO PROTECT PLASMIDS FROM RESTRICTION UPON TRANSFORMATION OF CLOSTRIDIUM-ACETOBUTYLICUM ATCC-824

The restriction endonuclease Cac824I has been shown to be a major barrier to electrotransformation of Clostridium acetobutylicum ATCC 824 (L. D. Mermelstein, N. E. Welker, G. N. Bennett, and E. T. Papoutsakis, Bio/Technology 10:190-195, 1992). Methylation by the phi3T I methyltransferase encoded by Bacillus subtilis phage phi3T was shown to protect plasmid DNA from restriction by Cac824I. Expression in Escherichia coli of the phi3tI gene (which encodes the phi3T I methyltransferase) from pAN1, which replicates via the p15A origin of replication, was sufficient to completely methylate coresident E. coli-C. acetobutylicum shuttle vectors with ColE1 origins of replication. Three shuttle vectors (pIMP1, pSYL2, and pSYL7) methylated in this manner were used to efficiently electrotransform strain ATCC 824. These vectors could not be introduced into strain ATCC 824 when unmethylated because the E. coli portions of the plasmids contain a large number of Cac824I sites. This method obviates the need to use B. subtilis-C. acetobutylicum shuttle vectors with few Cac824I sites to introduce DNA into C. acetobutylicum ATCC 824.