The transformation frequency of plasmids into Bacillus anthracis is affected by adenine methylation

Plasmids pLTV1 and pHV33, capable of replicating in both Gram+ and Gram- bacterial hosts (shuttle vectors), when derived from the Escherichia coli strain HB101, were inactive in an electro-transformation assay employing the Bacillus anthracis strains delta Ames-1 and delta V1B-1 as recipients. The same plasmids isolated from the DNA methyltransferase (MTase)-deficient E. coli strain GM2929 (dam, dcm), were able to transform the B. anthracis strains at a frequency of 10(2)-10(3) transformants/micrograms of plasmid DNA. Efficient transformation was also obtained when the plasmids were propagated in strains of B. subtilis 168 (10(2)-10(4) transformants/micrograms of plasmid DNA). The B. subtilis strains used are known to harbor restriction/modification systems that recognize cytosine as a target for methylation. In contrast, no adenine methylation activities have been reported for these strains. The data presented indicate that DNA containing methylated adenine residues is restricted in the B. anthracis strains studied here, resulting in decreased plasmid DNA-mediated transformation frequencies. This inhibition could be alleviated by propagating plasmid species in MTase-deficient (dam) strains of E. coli or B. subtilis 168, before their introduction into strains of B. anthracis.