A total of 1467 mutants of the biocontrol bacterium Bacillus pumilus DX01 were obtained by Tn5 insertional mutagenesis and subjected to the determination of antagonistic capabilities. Compared with the wild-type strain DX01, the mutant M25 was identified to have the most significant reduction in antagonistic capability against the phytopathogen Bipolaris maydis and extracellular proteinase activity. The integration site of the exogenous T-DNA in the genome of mutant M25 was revealed in the coding region of malony CoA-ACP transacylase (MCAT) gene (mcat), which belongs to a polyketide synthase (PKS) gene cluster, DX01pks of B. pumilus DX01. Furthermore, the whole DX01pks gene cluster was cloned using Illumina Solexa sequencing technology, and it has a modular framework different from the other two gene clusters involved in polyketide synthesis in B. amyloliquefaciens FZB42 (pks1) and B. subtilis 168 (pksX). Finally, in order to gain more insights into the molecular mechanisms of biocontrol of B. pumilus DX01, the changes in the relative level of expression of total proteins between the original strain DX01 and the mutant M25 were detected by 2-DE-based proteomic analysis. A total of twenty differentially expressed proteins were identified upon the mcat gene transposition mutagenesis. Of these proteins, seven proteins were up-regulated in expression level and the other proteins were down-regulated. Taken together, the results in this study showed that Tn5 transposon mutagenesis of B. pumilus DX01 can lead to a significant change of antiphytopathogen ability, and the DX01pks gene cluster possibly play a potential role in the biocontrol processes of this bacterium.

中文翻译：

---

使用 Tn5 转座子诱变和基于 2-DE 的比较蛋白质组学分析筛选与短小芽孢杆菌 DX01 生物防治机制相关的候选基因

通过Tn5插入诱变共获得1467个生防细菌短小芽孢杆菌DX01突变体，并进行拮抗能力测定。与野生型菌株 DX01 相比，突变体 M25 被鉴定为对植物病原体 Bipolaris maydis 的拮抗能力和细胞外蛋白酶活性具有最显着的降低。外源性T-DNA在突变体M25基因组中的整合位点在丙二酸辅酶A-ACP转酰基酶（MCAT）基因（mcat）的编码区被揭示，该基因属于聚酮合酶（PKS）基因簇，DX01pks B . 短毛 DX01。此外，整个DX01pks基因簇是使用Illumina Solexa测序技术克隆的，它具有不同于其他两个参与B中聚酮化合物合成的基因簇的模块化框架。解淀粉酶 FZB42 (pks1) 和枯草芽孢杆菌 168 (pksX)。最后，为了更深入地了解短小芽孢杆菌 DX01 的生物防治分子机制，通过基于 2-DE 的蛋白质组学分析检测原始菌株 DX01 和突变体 M25 之间总蛋白表达水平的变化. 通过 mcat 基因转座诱变鉴定了总共 20 种差异表达的蛋白质。在这些蛋白质中，七种蛋白质的表达水平上调，而其他蛋白质的表达水平下调。综上所述，本研究结果表明短小芽孢杆菌 DX01 的 Tn5 转座子诱变可导致抗植物病原体能力发生显着变化，DX01pks 基因簇可能在该细菌的生防过程中发挥潜在作用。