Related Experiment Video
Updated: Jun 3, 2026

Functional Complementation Analysis (FCA): A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways
Published on: June 24, 2016
Role of ComFA in controlling the DNA uptake rate during transformation of competent Bacillus subtilis
Masaomi Takeno1, Hisataka Taguchi, Takashi Akamatsu
1Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan.
Abstract:
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.
Related Concept Videos
Transformation
Bacterial Transformation
Bacterial Transformation
Combinatorial Gene Control
The expression of more than 30,000 genes is controlled by approximately 2000-3000 transcription factors. This is possible because a single transcription factor can recognize more than one regulatory sequence. The specificity in gene...
Mechanism of Conjugation
Coordination of Gene Expression Processes in Bacteria

