荧光假单胞菌2P24中opgGH基因影响抗生素2,4-二乙酰基间苯三酚的产生

The opgGH genes regulate 2,4-diacetylphloroglucinol production in Pseudomonas fluorescens 2P24

摘要:

【目的】 抗生素2,4-二乙酰基间苯三酚(2,4-diacetylphloroglucinol, 2,4-DAPG)是生防菌株荧光假单胞菌(Pseudomonas fluorescens) 2P24防治植物病害的关键因子,然而对2,4-DAPG生物合成的调控通路并未完全解析。【方法】 前期利用Tn5随机突变的方法获得一株对棉花立枯丝核菌(Rhizoctonia solani)拮抗能力完全丧失的突变菌株W3,本研究利用基因互补等方法研究该突变体中被破坏的基因对菌株2P24分泌2,4-DAPG和其他生防相关性状的影响。【结果】 Tn5插入位点及其序列分析表明突变菌株W3中Tn5破坏了opgG基因。鉴于opgG和opgH基因组成操纵子,利用同源重组技术构建了opgGH内缺失突变菌株。与野生菌株2P24相比,opgGH突变菌株中2,4-DAPG的产量显著降低。对其他生防相关性状的检测发现,突变opgGH基因并不影响群体感应系统(quorum sensing, QS)信号分子的产生、氢氰酸的产生以及生物膜的形成,但可抑制菌株2P24的游动性。转录融合实验进一步表明opgGH基因并不调控gacA基因及其调控的小RNA分子编码基因的表达,这些结果表明opgGH基因可能不通过Gac/Rsm信号通路影响2,4-DAPG的产生。另外,高渗透势条件下,菌株2P24可通过opgGH基因调控2,4-DAPG的产生。【结论】 菌株2P24中opgGH基因正调控抗生素2,4-DAPG的产生、游动性以及抑菌能力,是该菌株中一个与生防性状相关的重要调控因子。

Abstract:

[Objective] The polyketide antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) is essential for the biocontrol effect of Pseudomonas fluorescens 2P24. However, the genetic elements that modulates 2,4-DAPG production remains to be ascertained. [Methods] Previously, Tn5 transposon mutagenesis was employed to get P. fluorescens W3 with the complete loss of antagonism to Rhizoctonia solani, and the strain W3 was then used to screen out the novel gene that influencing the production of 2,4-DAPG. In this study, the effect of the mutated gene in W3 on the 2,4-DAPG production and biocontrol effect of 2P24 was explored. [Results] One of the insertion mutations, exhibiting no activity against R. solani, was mapped to opgG, which encodes the protein for the synthesis of osmoregulated periplasmic glucan (OPG). The deletion of opgGH impaired the production of 2,4-DAPG and bacterial swimming motility, while it did not affect the production of quorum sensing (QS) signals, the generation of hydrocyanic acid, or the biofilm formation. The transcriptional fusion assay indicated that the expression levels of rsmY, rsmX1, and gacA were not changed in the opgGH mutant compared with the wild type. The data suggested that opgGH regulated the production of 2,4-DAPG in a Gac/Rsm-independent manner. [Conclusion] The opgGH operon is essential for the regulation of 2,4-DAPG production, swimming mobility, and antifungal effect, serving as a key regulator for the biocontrol capacity of P. fluorescens 2P24.