Role of ComFA in controlling the DNA uptake rate during transformation of competent Bacillus subtilis

The roles of ComFA and ComEC in DNA uptake by competent Bacillus subtilis were analyzed by transformation with DNA in protoplast lysates (LP transformation). Deletion mutants of comFA and comEC and putative Walker A mutants (K152N, K152Q, K152E) of comFA were constructed by fusion polymerase chain reaction. Transformants of comEC mutant with purified DNA and DNA in protoplast lysate were not obtained, which shows a lack of transformation ability and backwards recombination of the mutant. Transformants of the comFA mutant were obtained by LP transformation (1.8 × 10(4) transformants/μg DNA). Low relative efficiency of transformation (RET) of comFA compared to wild type (4.3 × 10(-4)) showed an important role for comFA in DNA uptake. Walker A mutants showed 1.8-19 × 10(-4) RET, suggesting a dependence on ATPase activity for transformation. Co-transformation between short linkages was only detected in comFA mutants. The results demonstrated that ComFA controlled the DNA uptake rate. The interpretation was further supported by analyzing the plasmid used in LP transformation of the comFA mutant. The RET of comFA compared to the wild type was 2.7 × 10(-2), 60-fold higher than that with chromosomal DNA (4.3 × 10(-4)). Following addition of DNA into comFA culture, transformants were obtained after 15 min, with the number of transformants increasing over time. The kinetics strongly suggested that in comFA mutants, formation of another DNA uptake complex without ComFA would be a lengthy process.     

Role of ComEA in DNA uptake during transformation of competent Bacillus subtilis

The role of the competence protein ComEA in DNA uptake during transformation of competent Bacillus subtilis was analyzed by lysed-protoplast transformation (LP transformation). A comEA deletion mutant was constructed by a fusion polymerase chain reaction. Transformants of the mutant were obtained by LP transformation at a frequency of 1.1 × 10(2) transformants per μg DNA, representing a low relative efficiency of transformation [RET (mutant/wild type)] of 2.7 × 10(-6). This implied an important role of the protein during DNA uptake. When analyzing LP transformation of comEA with a plasmid (5.7 kb), a similar RET (mutant/wild type) of 5.6 × 10(-5) was obtained. Following addition of DNA into the comEA mutant culture, the number of transformants increased at a rate of 0.5 transformants/min, which was very low compared with the wild-type (6.9×10(4) transformants/min). However, even in the comEA mutant, DNA uptake began immediately after addition of DNA. Using co-transformation analysis of the comEA mutant, short linkages at distances of 2-156 kb could be detected, but not long linkages at distances of 671-1662 kb. Taken together, the results indicate that ComEA plays an important role in the transfer of transforming DNA into the DNA channel and in controlling the rate of DNA uptake.