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 x 102 transformants per pig DNA, representing a low relative efficiency of transformation [RET (mutant/wild type)] of 2.7 x 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 x 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 x 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. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.