title: Cloning and molecular characterization of a novel rolling-circle replicating plasmid, pK1S-1, from Bacillus thuringiensis subsp. kurstaki K1.
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.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, P. R. China.
authors.forename: Ming Shun
authors.initials: MS
authors.lastname: Li
authors.forename: Jong Yul
authors.initials: JY
authors.lastname: Roh
authors.forename: Xueying
authors.initials: X
authors.lastname: Tao
authors.forename: Zi Niu
authors.initials: ZN
authors.lastname: Yu
authors.forename: Zi Duo
authors.initials: ZD
authors.lastname: Liu
authors.forename: Qin
authors.initials: Q
authors.lastname: Liu
authors.forename: Hong Guang
authors.initials: HG
authors.lastname: Xu
authors.forename: Hee Jin
authors.initials: HJ
authors.lastname: Shim
authors.forename: Yang-Su
authors.initials: YS
authors.lastname: Kim
authors.forename: Yong
authors.initials: Y
authors.lastname: Wang
authors.forename: Jae Young
authors.initials: JY
authors.lastname: Choi
authors.forename: Yeon Ho
authors.initials: YH
authors.lastname: Je
mesh_terms: D001413:Bacillus thuringiensis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004261:DNA Replication; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids / Q000235:genetics*; D018741:Replication Origin; D016415:Sequence Alignment