| Title |
Cloning and Molecular Characterization of a Novel Rolling-Circle Replicating Plasmid, pK1S-1, from Bacillus thuringiensis subsp. kurstaki K1 |
| Author |
Ming Shun Li1, Jong Yul Roh2*, Xueying Tao2, Zi Niu Yu1, Zi Duo Liu1, Qin Liu1, Hong Guang Xu1, Hee Jin Shim2, Yang-Su Kim2, Yong Wang2, Jae Young Choi3, and Yeon Ho Je2 |
| Address |
1State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, P. R. China, 2Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-742, Republic of Korea, 3Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-742, Republic of Korea |
| Bibliography |
Journal of Microbiology, 47(4),466-472, 2009,
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| DOI |
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| Key Words |
Bacillus thuringiensis K1, plasmid capture system (PCS), pK1S-1, rolling circle replication, RCR group VII |
| Abstract |
Bacillus thuringiensis, an entomopathogenic bacterium belonging to the B. cereus group, harbors numerous extra-chromosomal DNA molecules whose sizes range from 2 to 250 kb. In this study, we used a plasmid capture system (PCS) to clone three small plasmids from B. thuringiensis subsp. kurstaki K1 which were not found in B. thuringiensis subsp. kurstaki HD-1, and determined the complete nucleotide sequence of plasmid pK1S-1 (5.5 kb). Of the six putative open reading frames (ORF2-ORF7) in pK1S-1, ORF2 (MobK1) showed approximately 90% aa identity with the Mob-proteins of pGI2 and pTX14-2, which are rolling circle replicating group VII (RCR group VII) plasmids from B. thuringiensis. In addition, a putative origin of transfer (oriT) showed 95.8% identity with those of pGI2 and pTX14-2. ORF3 (RepK1) showed relatively low aa identity (17.8~25.2%) with the Rep protein coded by RCR plasmids, however. The putative double- strand origin of replication (dso) and single-strand origin of replication (sso) of pK1S-1 exhibited approximately 70% and 64% identities with those of pGI2 and pTX14-2. ORF6 and 7 showed greater than 50% similarities with alkaline serine protease, which belongs to the subtilase family. The other 2 ORFs were identified as hypothetical proteins. To determine the replicon of pK1S-1, seven subclones were contructed in the B. thuringiensis ori-negative pHT1K vector and were electroporated into a plasmid cured B. thuringiensis strain. The 1.6 kb region that included the putative ORF3 (Rep1K), dso and ORF4, exhibited replication ability. These findings identified pK1S-1 as a new RCR group VII plasmid, and determined its replication region. |
