| Thuringiensin (Thu) is a small molecular weight adenosine insecticidal toxin produced by some Bacillus thuringiensis. Bacillus thuringiensis CT-43 produces high production of thuringiensin and secretes it to the supernatant. Early study of ours laboratory, we cloned biosynthesis cluster of Thu in CT-43. We derived and verified the biosynthesis’pathway of Thu, further.Type IV secretion systems (T4SSs) are versatile secretion systems existing in gram-negative bacteria wildly and are also found in gram-positive bacteria, wall-less bacteria, Archaea. Type IV secretion systems can secret proteins, protein-DNA and multi-subunit toxins. So far, there is no report about small molecules substance of Type IV secretion systems.Sequence analysis showed that there is a type IV secretion system-like gene cluster containing virB4, virBll and virD4 gene flanking the Thu biosynthesis cluster in CT-43, which may be associated with the secretion of Thu. In this study, by the method of gene knockout, we constructed a homologous double-crossover vector and knocked out the virD4 gene in CT-43 and got a mutant BMB1122. HPLC results showed that the characteristic peak of thuringiensin was not detected in the fermentation supernatant of mutant BMB1122, which showed the knockout of virD4 gene interrupted the synthesis of thuringiensin.The LCMS-IT-TOF result showed that the corresponding m/z with molecular weight of proposed precursors in Thu biosynthesis could be detected in intracellular metabolites of mutant BMB1122, and the corresponding m/z with molecular weight of thuringiensin was not detected. These results suggest that the disruption of virD4 gene kept Thu synthesis staying in the final step. In addition, the derivation pathway of Thu that the last step adding a molecule of phosphoric acid is in the process of across membrane. Therefore, there may be knockout of virD4 gene damage type IV secretion-like system, which lead to the last intermediates of Thu synthesis can not be secreted across the membrane.Some work for virBll gene knockout and functional verification has been carried out. A homologous double-crossover vector has been successfully constructed. The virBllgene functional in the synthesis and secretion of Thu is still follow-up. This study demonstrated virD4 gene was related to Thu. The disruption of virD4 gene kept Thu synthesis staying in the final step before across membrane. This study deepened our understanding of the process of Thu systhesis and secretion. This study also shows that Type IV secretion-like system in Bacillus thuringiensis can secret small molecules similar Thu, which provide new information for Type IV secretion systems in gram-positive bacteria. |
