Distribution of 2-kb miniplasmid pBMB2062 from Bacillus thuringiensis kurstaki YBT-1520 strain in Bacillus species
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ORIGINAL ARTICLE
Distribution of 2-kb miniplasmid pBMB2062 from Bacillus thuringiensis kurstaki YBT-1520 strain in Bacillus species Xiao-Jin Liu & Li-Fang Ruan & Xiao-Yan Lin & Chang-Ming Zhao & Chun-Ying Zhong & Ming Sun
Received: 7 September 2012 / Accepted: 1 March 2013 # Springer-Verlag Berlin Heidelberg and the University of Milan 2013
Abstract A multi-copy and small plasmid pBMB2062 from Bacillus thuringiensis kurstaki YBT-1520 strain was cloned and characterized and its distribution was analyzed using dot-blot analysis with the ORF1 fragment as a probe. Bacillus species of 84 serotypes were evaluated. The pBMB2062 plasmid was found to be present in commercial B. thuringiensis kurstaki (H3abc) and aizawai (H7) insecticides of various serotypes, and one Bacillus cereus UW85 strain (produced Zwittermicin fungicide and Cry toxin synergist). The sequences of 7 pBMB2062-like plasmids from randomly selected Bacillus species (positive signal in the dot-blot analysis) were highly conserved. Two open reading frames (ORFs), ORF1 and ORF2, were present in this plasmid. ORF1 was found to be necessary for plasmid replication, whereas ORF2 did not play a role in replication or stability. Based on its sequence homology, ORF2 was a putative solitary antitoxin. Furthermore, the copy number of the replicon of pBMB2062 was higher than those of ori1030 and ori44 based on the thermogenic data, and ori2062 could drive the stable replication of a recombinant plasmid (11 kb total size) in B. thuringiensis. Keywords Bacillus thuringiensis . Multicopy plasmid . Plasmid replication . Replicon
X.-J. Liu : L.-F. Ruan : X.-Y. Lin : C.-M. Zhao : C.-Y. Zhong : M. Sun (*) State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, 430070, Wuhan, People’s Republic of China e-mail: [email protected]
Introduction Bacillus thuringiensis of various subspecies are well-known insecticides that produce Cry toxins specifically active against lepidopteran, dipteran or coleopteran insects (Schnepf et al. 1998). Bacillus thuringiensis strains generally harbor many plasmids whose numbers vary from 2 to 12 and sizes from 2 to 600 kb (Tang et al. 2006), and the genes encoding the Cry proteins are often located on large conjugative plasmids that are larger than 45-kb (Sylvain et al. 2004). Thus far, 10 small B. thuringiensis plasmids (30 kb) and three small plasmids (70 amino acids) were compared with sequences in the National Center for Biotechnology Information (NCBI) database using the ORF FINDER software, BLASTN and BLASTP algorithms from the NCBI website (http://www.ncbi. nlm.nih.gov/). Construction of recombinant plasmids The plasmid pBMB2062 was gel-purified, digested with HincII (the fragment was named ori2062), and then inserted into the SmaI site of pDG780 to generate the plasmid pBMB0401. Orf1 and orf2 derivatives were constructed as follows. The fragment (1.8-kb) containing an ORF2 gene deletion was amplified from pBMB2062 by using t h e p r i m e r s P 1 a n d P 2 ( P 1 : 5 ′ T T G A A G TA A C GGTTATGTTT 3′
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