Random mutagenesis by recombinational capture of PCR products in Bacillus subtilis and Acinetobacter calcoaceticus

We describe a general method for random mutagenesis of cloned genes by erro r-prone PCR or DNA shuffling that eliminates the need for post-amplificatio n subcloning following each cycle of mutagenesis, This method exploits the highly efficient and recombinogenic nature of DNA uptake during natural tra nsformation in the Gram-positive bacterium Bacillus subtilis and the Gram-n egative bacterium Acinetobacter calcoaceticus. Plasmid systems were designe d that allow capture of PCR-amplified DNA fragments by marker-replacement r ecombination with a structurally similar helper plasmid resident in the tra nsformation recipient, This recombination event simultaneously transfers th e amplified sequences into the helper plasmid and restores the integrity of a drug resistance gene, thereby affording a direct selection for fragment capture. Although this strategy was sufficiently effective to permit recove ry in B. subtilis of up to 10(3) transformants/mu g of PCR product, equival ent plasmid systems were similar to 100 times more efficient in A. calcoace ticus. Acinetobacter calcoaceticus also offers the advantage of essentially constitutive transformation competence in ordinary complex broth, such as LB, in contrast to two-step growth in semisynthetic media required for opti mal transformation of B. subtilis.