Stable mutants of restriction-deficient/modification-proficient Bacillus subtilis 168: hub strains for giant DNA engineering

Bacillus subtilis 168 has been explored as a platform for the synthesis and transmission of large DNA. Two inherent DNA incorporation systems, natural transformation and pLS20-based conjugation transfer, enable rapid handling of target DNA. Both systems are affected by the Bsu restriction–modification system that recognizes and cleaves unmethylated XhoI sites, limiting the choice of target DNA. We constructed B. subtilis 168 with stable mutation for restriction-deficient and modification-proficient (r−m+). It was demonstrated that the r−m+ strains can incorporate and transfer synthesized DNA with multiple XhoI sites. These should be of value as hub strains to integrate and disseminate giant DNA between B. subtilis 168 derivatives.

Roles of Active Site Residues and the HUH Motif of the F Plasmid TraI Relaxase

Bacterial conjugation, transfer of a single strand of a conjugative plasmid between bacteria, requires sequence-specific single-stranded DNA endonucleases called relaxases or nickases. Relaxases contain an HUH (His-hydrophobe-His) motif, part of a three-His cluster that binds a divalent cation required for the cleavage reaction. Crystal structures of the F plasmid TraI relaxase domain, with and without bound single-stranded DNA, revealed an extensive network of interactions involving HUH and other residues. Here we study the roles of these residues in TraI function. Whereas substitutions for the three His residues alter metal-binding properties of the protein, the same substitution at each position elicits different effects, indicating that the residues contribute asymmetrically to metal binding. Substitutions for a conserved Asp that interacts with one HUH His demonstrate that the Asp modulates metal affinity despite its distance from the metal. The bound metal enhances binding of ssDNA to the protein, consistent with a role for the metal in positioning the scissile phosphate for cleavage. Most substitutions tested caused significantly reduced in vitro cleavage activities and in vivo transfer efficiencies. In summary, the results suggest that the metal-binding His cluster in TraI is a finely tuned structure that achieves a sufficient affinity for metal while avoiding the unfavorable electrostatics that would result from placing an acidic residue near the scissile phosphate of the bound ssDNA.