Bacillus anthracis, the causative agent of anthrax, is poorly transformed with DNA that is methylated on adenine or cytosine. Here we characterize three genetic loci encoding type IV methylation-dependent restriction enzymes that target DNA containing C5-methylcytosine (m5C). Strains in which these genes were inactivated, either singly or collectively, showed increased transformation by methylated DNA. Additionally, a triple mutant with an ~30-kb genomic deletion could be transformed by DNA obtained from Dam(+)Dcm(+)E. coli, although at a low frequency of ~10(-3) transformants/10(6)cfu. This strain of B. anthracis can potentially serve as a preferred host for shuttle vectors that express recombinant proteins, including proteins to be used in vaccines. The gene(s) responsible for the restriction of m6A-containing DNA in B. anthracis remain unidentified, and we suggest that poor transformation by such DNA could in part be a consequence of the inefficient replication of hemimethylated DNA in B. anthracis.
Published by Elsevier B.V.