[Cloning and functional analysis of the stable plasmid pBMB175 in Bacillus thuringiensis subsp. tenebrionis strains YBT-1765].
A 15.2 kb plasmid pBMB175 from Bacillus thuringiensis subsp. tenebrionis strains YBT-1765 was cloned and the restriction map was constructed. The mini-replicating region of pBMB175 was located in a 1151 bp fragment by functional analysis. The sequence of a 4152 bp fragment which contained the mini-replicating region was analyzed and results showed that the fragment had three potential open reading frames (ORF1, ORF2 and ORF3). Sequence comparison and homology search revealed that ORF1 (767AA) has 20% approximately 30% similarity to UvrD-helicase, RecD and RecB family proteins; no homology was found between ORF2 (149AA) and other known proteins; ORF3 shared 34% identification to a potential protein (ORF7) in pGI3. Deletion and sequence analysis presumed that the protein encoded by ORF2 maybe a new replication protein. Above all, pBMB175 likely belongs to a new plasmid family with a new replicon. The recombinant plasmid harboring the mini-replicating region is very stable, even after growth for more than 40 generations without selection, so it might be used as a cloning and expression vector.
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
Dong-Mei
DM
Han
Jun-Yan
JY
Huang
Zi-Niu
ZN
Yu
Ming
M
Sun
D001413:Bacillus thuringiensis / Q000235:genetics*; D003001:Cloning, Molecular; D004261:DNA Replication; D016366:Open Reading Frames; D010957:Plasmids; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein