Abstract
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).
Highlights
Vegetative insecticidal protein [4, 6], secret insecticidal protein [7], thuringiensin [8], zwittermicin A [9], Mtx-like toxin [10], Bin-like toxin [11], etc
We adapted high performance liquid chromatography (HPLC) to selectively detect the characteristic peak of thuringiensin in an Escherichia coli–B. thuringiensis shuttle bacterial artificial chromosome (BAC)3 library of the thuringiensin high production strain, CT-43
Genome-wide Mining for the Gene (Cluster) Responsible for Thuringiensin Synthesis in B. thuringiensis Strain CT-43—The B. thuringiensis strain CT-43 was isolated from Chinese soil by our group
Summary
Vegetative insecticidal protein [4, 6], secret insecticidal protein [7], thuringiensin [8], zwittermicin A [9], Mtx-like toxin [10], Bin-like toxin [11], etc. From the structural formula, thuringiensin is composed of four precursors: adenosine, glucose, a phosphate group, and gluconic diacid [17] This kind of structure is seldom reported in antibiotic compounds. The consensus has been that it is an adenine nucleotide analog [17], like ATP, and this similarity makes it an inhibitor of DNAdependent RNA polymerases [18]. It is a nonspecific antibiotic insecticide and acaricide. Using a B. thuringiensis strain mutant library, Espinasse et al [19] reported that an ABC transporter, which might be related to the secretion of thuringiensin, was essential for thuringiensin production. The thuringiensin biosynthesis pathway was deduced and clarified by LCMS-IT-TOF detection and the identification of key gene thuE
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