Journal of Microbiology

Research Support, Non-U.S. Gov't

Cloning and Molecular Characterization of a Novel Rolling-Circle Replicating Plasmid, pK1S-1, from Bacillus thuringiensis subsp. kurstaki K1: Ming Shun Li , Jong Yul Roh , Xueying Tao , Zi Niu Yu , Zi Duo Liu , Qin Liu , Hong Guang Xu , Hee Jin Shim , Yang-Su Kim , Yong Wang , Jae Young Choi , Yeon Ho Je; J. Microbiol. 2009;47(4):466-472. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-009-0020-2

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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 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.

Stable Secretion Vector Derived from the RCR (rolling-circle replication) Plasmid of Bacillus mesentericus: Seung-Soo Lee , Jeong-Sun Han , In Hyung Lee , Young- Yel l Yang , Soon-Kwang Hong , Joo-Won Suh; J. Microbiol. 2002;40(2):140-145.

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Abstract: The 5.8 kb pMMH1, rolling-circle replication (RCR) plasmid of the wild type soil Bacillus mesentericus was developed into a novel secretion vector system in Bacillus subtilis. The pMMH1 turned out to have a replication origin and two open reading frames (ORFs) of the putative [gamma]-GTP and type I signal peptidase (sipP). To characterize the regions necessary for plasmid stability and high copy number, five vectors (pPS, pPP, pEN, pMN, pME) were constructed by disruption or deletion of each region in pMMH1. Like pMMH1, all constructed vectors were stable over 100 generations in a non-selective medium. Since pPS was the smallest (2.3 kb)of all, it was selected for the construction of a novel secretion vector. Using the [alpha]-amylase promoter/signal sequence of B. subtilils, the novel plasmid pJSN was constructed. When [beta]-glucosidase was expressed using pJSN, we found [beta]-glucosidase activity in the medium. This result strongly suggested that plasmid pJSN can be used for the production of bioactive peptides in B. subtilis.

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