Essential involvement of the Bacillus subtilis ABC transporter, EcsB, in genetic transformation of purified DNA but not native DNA from protoplast lysates
Involvement of the Bacillus subtilis ABC transporter EcsB in genetic transformation with native DNA from protoplast lysate (LP transformation) was investigated using an ecsB deletion mutant constructed by fusion polymerase chain reaction. In these experiments, the non-transformability phenotype of the ecsB mutant was reversed and high numbers of transformants generated (1.5 × 105/μg DNA). The relative efficiency of transformation (RET) of ecsB to wild type (1.2 × 10− 2) was a thousand times higher using native chromosomal DNA than the RET obtained from purified DNA (< 8.6 × 10− 6). Similar transformation efficiencies were observed using native plasmid DNA. These results rule out a primary role for EcsB as a competence gene regulator. DNA-binding proteins attached to native DNA are not present in purified DNA preparations, and it is possible that such proteins could account for the transformability of the ecsB mutant. Because EcsB may play a role in protein(s) export, we tested exogenous proteins to identify functional replacements. We found that bovine serum albumin (fraction V) partially suppressed the phenotype of the ecsB mutation, leading to transformability with purified DNA. Linkage analysis of the ecsB mutant by LP co-transformation produced a higher co-transformation ratio (42% and 20%) at a distance of 34 kb and 121 kb in the ecsB mutant, compared to the wild-type strain, AYG2 (30.5% and 12.3%). The stimulatory linkage effect observed could be derived from a regulating gene involved in homologous recombination.
Journal: Journal of Bioscience and Bioengineering - Volume 112, Issue 3, September 2011, Pages 209–214