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Cloning and molecular characterization of a novel rolling-circle replicating plasmid, pK1S-1, from Bacillus thuringiensis subsp. kurstaki K1

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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 Kl which were not found in B. thuringiensis subsp. kurstaki HD-1, and determined the complete nucleotide sequence of plasmid pKlS-1 (5.5 kb). Of the six putative open reading frames (ORF2-ORF7) in pKlS-1, ORF2 (MobKl) 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 pKlS-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 pKlS-1, seven subclones were contructed in the B. thuringiensis ori-negative pHTIK vector and were electroporated into a plasmid cured B. thuringiensis strain. The 1.6 kb region that included the putative ORF3 (ReplK), dso and ORF4, exhibited replication ability. These findings identified pKlS-1 as a new RCR group VII plasmid, and determined its replication region.

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Authors and Affiliations

  1. State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, 430070, P. R. China

    Ming Shun Li, Zi Niu Yu, Zi Duo Liu, Qin Liu & Hong Guang Xu

  2. Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul, 151-742, Republic of Korea

    Jong Yul Roh, Xueying Tao, Hee Jin Shim, Yang-Su Kim, Yong Wang & Yeon Ho Je

  3. Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 151-742, Republic of Korea

    Jae Young Choi

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  1. Ming Shun Li
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Correspondence to Jong Yul Roh.

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Li, M.S., Roh, J.Y., Tao, X. et al. Cloning and molecular characterization of a novel rolling-circle replicating plasmid, pK1S-1, from Bacillus thuringiensis subsp. kurstaki K1. J Microbiol. 47, 466–472 (2009). https://doi.org/10.1007/s12275-009-0020-2

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