Department of Horticultural Sciences, Texas A&M University, College Station, TX, United States of America.
Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX, United States of America.
PLoS One. 2018 Feb 16;13(2):e0193063. doi: 10.1371/journal.pone.0193063. eCollection 2018.
Phenazines are bacterial secondary metabolites and play important roles in the antagonistic activity of the biological control strain P. chlororaphis 30-84 against take-all disease of wheat. The expression of the P. chlororaphis 30-84 phenazine biosynthetic operon (phzXYFABCD) is dependent on the PhzR/PhzI quorum sensing system located immediately upstream of the biosynthetic operon as well as other regulatory systems including Gac/Rsm. Bioinformatic analysis of the sequence between the divergently oriented phzR and phzX promoters identified features within the 5'-untranslated region (5'-UTR) of phzX that are conserved only among 2OHPCA producing Pseudomonas. The conserved sequence features are potentially capable of producing secondary structures that negatively modulate one or both promoters. Transcriptional and translational fusion assays revealed that deletion of 90-bp of sequence at the 5'-UTR of phzX led to up to 4-fold greater expression of the reporters with the deletion compared to the controls, which indicated this sequence negatively modulates phenazine gene expression both transcriptionally and translationally. This 90-bp sequence was deleted from the P. chlororaphis 30-84 chromosome, resulting in 30-84Enh, which produces significantly more phenazine than the wild-type while retaining quorum sensing control. The transcriptional expression of phzR/phzI and amount of AHL signal produced by 30-84Enh also were significantly greater than for the wild-type, suggesting this 90-bp sequence also negatively affects expression of the quorum sensing genes. In addition, deletion of the 90-bp partially relieved RsmE-mediated translational repression, indicating a role for Gac/RsmE interaction. Compared to the wild-type, enhanced phenazine production by 30-84Enh resulted in improvement in fungal inhibition, biofilm formation, extracellular DNA release and suppression of take-all disease of wheat in soil without negative consequences on growth or rhizosphere persistence. This work provides greater insight into the regulation of phenazine biosynthesis with potential applications for improved biological control.
吩嗪是细菌的次生代谢产物，在生物防治菌株 P. chlororaphis 30-84 对小麦全蚀病的拮抗活性中发挥重要作用。P. chlororaphis 30-84 吩嗪生物合成操纵子（phzXYFABCD）的表达依赖于位于生物合成操纵子上游的 PhzR/PhzI 群体感应系统以及包括 Gac/Rsm 在内的其他调节系统。对 phzR 和 phzX 启动子反向取向之间的序列进行生物信息学分析，鉴定出 phzX 的 5'-非翻译区（5'-UTR）中仅在产生 2OHPCA 的假单胞菌中保守的特征。保守的序列特征可能产生负调节一个或两个启动子的二级结构。转录和翻译融合测定表明，与对照相比，phzX 的 5'-UTR 缺失 90-bp 的序列导致报告基因的表达增加了 4 倍，这表明该序列负调节吩嗪基因的转录和翻译表达。该 90-bp 序列从 P. chlororaphis 30-84 染色体中缺失，导致 30-84Enh 产生的吩嗪比野生型显著更多，同时保留群体感应控制。30-84Enh 的 phzR/phzI 转录表达和产生的 AHL 信号量也显著大于野生型，表明该 90-bp 序列也负调控群体感应基因的表达。此外，90-bp 的缺失部分解除了 RsmE 介导的翻译抑制，表明 Gac/RsmE 相互作用的作用。与野生型相比，30-84Enh 增强的吩嗪产生导致真菌抑制、生物膜形成、细胞外 DNA 释放和抑制土壤中的小麦全蚀病得到改善，而对生长或根际持久性没有负面影响。这项工作提供了对吩嗪生物合成调控的更深入了解，具有潜在的应用价值，可用于改善生物防治。
