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

Paper ID Volume ID Publish Year Pages File Format Full-Text
21606 43231 2011 6 PDF Available
Title
Role of ComFA in controlling the DNA uptake rate during transformation of competent Bacillus subtilis
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 × 104 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.

Keywords
Bacillus subtilis; ComFA; DNA uptake; Competent transformation; Brownian ratchet
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Role of ComFA in controlling the DNA uptake rate during transformation of competent Bacillus subtilis
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 111, Issue 6, June 2011, Pages 618–623
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us