Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, People's Republic of China.
College of Plant Protection, Nanjing Agricultural University, China/Key Laboratory of Integrated Management of Crop Diseases and Pests (Nanjing Agricultural University, Ministry of Education), Nanjing, 210095, People's Republic of China.
Microb Cell Fact. 2017 Nov 14;16(1):202. doi: 10.1186/s12934-017-0818-2.
Heat-stable antifungal factor (HSAF) is a polycyclic tetramate macrolactam secondary metabolite that exhibits broad-spectrum inhibitory activities against filamentous fungal pathogens. The native yield of this chemical is low. It is also a great challenge to synthesize HSAF artificially, due to its complex structure. Understanding the regulatory mechanism underlying HSAF biosynthesis could provide genetic basis for engineering high HSAF-producing strain. The transcription factor Clp is a global regulator that controls bacterial pathogenicity and the expression of one hundred related genes in the phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc). Diffusible signal factor (DSF) chemical signaling is the only well-characterized upstream regulatory pathway that involves downstream Clp regulation in Xcc. Such a regulatory hierarchy between DSF signaling and Clp is also conserved in the Gram-negative biological control agent Lysobacter enzymogenes, where the DSF signaling system controls antifungal antibiotic HSAF biosynthesis via Clp.
Here, using LLysobacter enzymogenes OH11 as a working organism, we examined a novel upstream regulator, LesR, a LuxR solo that controls Clp expression to modulate HSAF biosynthesis as well as cell aggregation. We found that the overexpression of lesR in strain OH11 almost entirely shut down HSAF production and accelerated cell aggregation. These changed phenotypes could be rescued by the introduction of plasmid-borne clp in the lesR overexpression background. Consistent with findings, we further found that overexpression of lesR led to a decrease in the Clp level.
These results collectively have shown that LesR could exert its function, i.e., HSAF biosynthesis, via downstream Clp. These findings were subsequently validated by a comparative transcriptome analysis, where the regulatory action of LesR was found to largely overlap with that of Clp. Therefore, in addition to the well-known DSF signaling system, the present study reveals that LesR functions as a new upstream regulatory factor of Clp in L. enzymogenes. The key factor was important for the production of HSAF. The strains with high HSAF yield can presumably be constructed by deletion of the negative regulators or overexpression of the positive regulators by genetic engineering.
热稳定抗真菌因子（HSAF）是一种多环四肽大环内酯类次级代谢产物，对丝状真菌病原体具有广谱抑制活性。该化学物质的天然产量很低。由于其复杂的结构，人工合成 HSAF 也是一个巨大的挑战。了解 HSAF 生物合成的调控机制可为工程高产 HSAF 菌株提供遗传基础。转录因子 Clp 是一种全局调控因子，可控制病原菌致病性和植物病原菌黄单胞菌 pv 的一百个相关基因的表达。Xanthomonas campestris（Xcc）。可扩散信号因子（DSF）化学信号是唯一经过充分研究的上游调控途径，涉及 Xcc 下游 Clp 的调控。这种 DSF 信号与 Clp 之间的调控层次结构在革兰氏阴性生物防治剂 Lysobacter enzymogenes 中也得到了保守，其中 DSF 信号系统通过 Clp 控制抗真菌抗生素 HSAF 的生物合成。
在这里，我们使用 Lysobacter enzymogenes OH11 作为工作生物体，研究了一种新的上游调节剂 LesR，它是一种单独的 LuxR，可控制 Clp 的表达，从而调节 HSAF 的生物合成以及细胞聚集。我们发现，在菌株 OH11 中过表达 lesR 几乎完全关闭了 HSAF 的产生并加速了细胞聚集。在 lesR 过表达背景下引入带有质粒的 clp 可以挽救这些表型变化。与发现一致，我们进一步发现过表达 lesR 导致 Clp 水平下降。
这些结果共同表明，LesR 可以通过下游 Clp 发挥其功能，即 HSAF 生物合成。这些发现随后通过比较转录组分析得到了验证，其中发现 LesR 的调控作用在很大程度上与 Clp 的调控作用重叠。因此，除了众所周知的 DSF 信号系统外，本研究还表明，LesR 在 L.enzymogenes 中作为 Clp 的新上游调节因子发挥作用。该关键因素对于 HSAF 的产生很重要。通过遗传工程删除负调节因子或过表达正调节因子，可能构建出具有高 HSAF 产量的菌株。
