Genome-wide screening reveals the genetic determinants of an antibiotic insecticide in Bacillus thuringiensis.
Thuringiensin is a thermostable secondary metabolite in Bacillus thuringiensis and has insecticidal activity against a wide range of insects. Until now, the regulatory mechanisms and genetic determinants involved in thuringiensin production have remained unclear. Here, we successfully used heterologous expression-guided screening in an Escherichia coli-Bacillus thuringiensis shuttle bacterial artificial chromosome library, to clone the intact thuringiensin synthesis (thu) cluster. Then the thu cluster was located on a 110-kb endogenous plasmid bearing insecticide crystal protein gene cry1Ba in strain CT-43. Furthermore, the plasmid, named pBMB0558, was indirectly cloned and sequenced. The gene functions on pBMB0558 were annotated by BLAST based on the GenBank(TM) and KEGG databases. The genes on pBMB0558 could be classified into three functional modules: a thuringiensin synthesis cluster, a type IV secretion system-like module, and mobile genetic elements. By HPLC coupling mass spectrometer, atmospheric pressure ionization with ion trap, and TOF technologies, biosynthetic intermediates of thuringiensin were detected. The thuE gene is proved to be responsible for the phosphorylation of thuringiensin at the last step by vivo and vitro activity assays. The thuringiensin biosynthesis pathway was deduced and clarified. We propose that thuringiensin is an adenine nucleoside oligosaccharide rather than an adenine nucleotide analog, as is traditionally believed, based on the predicted functions of the key enzymes, glycosyltransferase (ThuF) and exopolysaccharide polymerization protein (Thu1).
Liu
Xiao-Yan
XY
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
Ruan
Li-Fang
LF
Hu
Zhen-Fei
ZF
Peng
Dong-Hai
DH
Cao
Shi-Yun
SY
Yu
Zi-Niu
ZN
Liu
Yao
Y
Zheng
Jin-Shui
JS
Sun
Ming
M
D000241:Adenosine / Q000031:analogs & derivatives / Q000737:chemistry; D000483:Alleles; D000900:Anti-Bacterial Agents / Q000378:metabolism; D001413:Bacillus thuringiensis / Q000235:genetics*; D001426:Bacterial Proteins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D022202:Chromosomes, Artificial, Bacterial; D005786:Gene Expression Regulation; D016680:Genome, Bacterial; D007306:Insecticides / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation; D010766:Phosphorylation; D013400:Sugar Acids / Q000737:chemistry
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D007306:Insecticides; D013400:Sugar Acids; C068295:thuringiensin; D000241:Adenosine