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Applied and Environmental Microbiology, November 2006, p. 6948-6954, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.00976-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Minireplicon from pBtoxis of Bacillus thuringiensis subsp. israelensis

Department of Entomology, University of California, Riverside, Riverside, California 92521,¹ John A. Mulrennan, Sr., Public Health Entomology Research and Education Center, Florida A&M University, Panama City, Florida 32405,² Interdepartmental Graduate Programs in Genetics and Cell, Molecular and Developmental Biology, University of California, Riverside, Riverside, California 92521³

Received 25 April 2006/ Accepted 7 August 2006

A 2.2-kb fragment containing a replicon from pBtoxis, the large plasmid that encodes the insecticidal endotoxins of Bacillus thuringiensis subsp. israelensis, was identified, cloned, and sequenced. This fragment contains cis elements, including iterons, found in replication origins of other large plasmids and suggests that pBtoxis replicates by a type A theta mechanism. Two genes, pBt156 and pBt157, encoding proteins of 54.4 kDa and 11.8 kDa, respectively, were present in an operon within this minireplicon, and each was shown by deletion analysis to be essential for replication. The deduced amino acid sequences of the 54.4-kDa and 11.8-kDa proteins showed no substantial homology with known replication (Rep) proteins. However, the 54.4-kDa protein contained a conserved FtsZ domain, and the 11.8 kDa protein contained a helix-turn-helix motif. As FtsZ proteins have known functions in bacterial cell division and the helix-turn-helix motif is present in Rep proteins, it is likely that these proteins function in plasmid replication and partitioning. The minireplicon had a copy number of two or three per chromosome equivalent in B. thuringiensis subsp. israelensis but did not replicate in B. cereus, B. megaterium, or B. subtilis. A plasmid constructed to synthesize large quantities of the Cry11A and Cyt1A endotoxins demonstrated that this minireplicon can be used to engineer vectors for cry and cyt gene expression.

Published ahead of print on 25 August 2006.

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