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,
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.