Plasmid transformation of competent Bacillus subtilis by lysed protoplast DNA(GENETICS, MOLECULAR BIOLOGY, AND GENE ENGINEERING)

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Transformation of competent Bacillus subtilis with DNA obtained from lysed protoplasts (LP transformation) was analyzed using several different plasmid vectors: pC194, pUB110, pCB1 (consisting of pC194 and pBluescript II SK+), and pAC32R2 (consisting of pUB110 derivative and pUC19). LP transformation of B. subtilis QB936 with pCB1 was 6500-fold higher than that achieved using conventional transformation with purified DNA. Greater transformation efficiencies were also obtained using pAC32R2. However, transformation frequencies using both protoplast-derived and purified pC194 were very low (1.4-2.0×10^2 transformants per μg DNA). Hence, the efficiency of transformation depends on the nucleotide sequence of the donor plasmid. The LP transformation frequency using pC194 obtained from an add5 mutant was remarkably enhanced (1.6×10^8 transformants per μg DNA), indicating that this unique form of high molecular weight DNA is likely responsible for part of the stimulatory effect. Chromosomal DNA inhibited plasmid transformation using pC194 and pUB110, but had little effect on pCB1 transformation. Conversely, pCB1 DNA did not inhibit transformation with protoplast-derived chromosomal DNA. Competence proteins under the control of transcription factor ComK were likely required for LP plasmid transformation. The DNA concentration-dependence of plasmid transformation was first order and the slope value was one.