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RpoS negatively regulates type VI secretion system and orfamide A biosynthesis of Pseudomonas protegens FD6

Yongxin Jiao*, Tao Wu*, Yuxin Zhu, Ziyi Zhang, Qingxia Zhang#

College of Plant Protection, Yangzhou University, Yangzhou 225009, China

Highlights

• RpoS binds to CTANNNT motifs in promoters of tssA and tagH to inhibit T6SS function

• RpoS inhibits orfamide A biosynthesis by binding promoter and repression of luxR1

• LuxR1 binds to promoter and activates of ofaABC to promote orfamide A biosynthesis

• Orfamide A impels motility, biofilm formation, colonization and surface tension

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摘要  

生防细菌Pseudomonas protegens FD6能够产生多种抗真菌的次生代谢产物,从而抑制植物病原真菌的生长。前期研究表明,σ因子RpoS负调控抗生素藤黄绿脓菌素(Pyoluteorin, PLT)及2,4-二乙酰基间苯三酚(2,4-diacetylphloroglucinol, 2,4-DAPG)的合成。本研究对rpoS缺失突变体进行了RNA-seq分析,揭示了RpoS在FD6中的全局转录调控功能。结果显示,rpoS缺失增强了由VI型分泌系统(Type VI secretion system, T6SS)介导的接触依赖性杀伤作用,并导致环脂肽(Cyclic lipopeptide, CLP)orfamide A的合成增加。细菌单杂交(Bacterial one-hybrid, B1H)与凝胶阻滞实验(Electrophoretic mobility shift assay, EMSA)证实,RpoS可直接特异性结合T6SS操纵子基因tssA和tagH以及orfamide A生物合成调控基因luxR1启动子区域的CTANNNT基序。此外,LuxR1可结合orfamide A生物合成基因簇ofaABC启动子区的元件,并作为转录激活因子发挥功能。luxR1或ofaA功能缺失损害orfamide A的生物合成,但不影响FD6对植物病原真菌的拮抗能力。ofaA缺失导致细菌运动性、生物膜形成能力及定殖能力减弱,且orfamide A对表面张力维持具有重要作用。综上所述,本研究揭示RpoS对P. protegens FD6中orfamide A生物合成及T6SS功能的多效调控机制,为通过遗传改造提高生防菌株的防治效果奠定基础。



Abstract  

The biological bacterium Pseudomonas protegens FD6 produces a range of antifungal secondary metabolites to inhibit the growth of phytopathogenic fungi. Previous studies showed that the alternative σ factor RpoS negatively regulated the production of antibiotics pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (2,4-DAPG). In this study, we performed RNA-seq analysis on the rpoS deletion mutant, which indicated the global transcriptional regulation in FD6 by RpoS. We revealed that rpoS deletion increased contact‑dependent killing mediated by the type VI secretion system (T6SS) and led to overproduction of cyclic lipopeptide (CLP) orfamide A. Bacterial one-hybrid (B1H) and electrophoretic mobility shift assay (EMSA) confirmed that RpoS directly bound to the CTANNNT motifs in the promoters of the T6SS operon genes tssA and tagH, as well as the orfamide A biosynthesis regulator gene luxR1. LuxR1 bound to the elements in the promoter of orfamide A biosynthesis gene cluster ofaABC, and acts as a transcriptional activator. Loss of function of luxR1 or ofaA impaired orfamide A biosynthesis, while does not affect antagonism activity against phytopathogens in FD6. Deletion of ofaA resulted in reduced motility, biofilm formation, colonization ability, and orfamide A appeared essential for surface tension. Collectively, our results provided new insights into the pleiotropic regulatory function of RpoS on the orfamide A biosynthesis and T6SS function in P. protegens FD6, laying a foundation for the genetic engineering of biocontrol agent to improve the biological control efficacy.

Pseudomonas protegens, RpoS, biocontrol, orfamide A, T6SS

Keywords:  Pseudomonas protegens       RpoS        biocontrol        orfamide A        T6SS  
Accepted: Online: 14 September 2026  
Fund: 

This work was supported by funds from the National Natural Science Foundation (32472628, 32072471) and the Science and Technology Program of Yangzhou City (YZ2023056).

About author:  #Correspondence Qingxia Zhang, E-mail: qxzhang@yzu.edu.cn *These authors contributed equally to this study.

Cite this article: 

Yongxin Jiao, Tao Wu, Yuxin Zhu, Ziyi Zhang, Qingxia Zhang. 2026.

RpoS negatively regulates type VI secretion system and orfamide A biosynthesis of Pseudomonas protegens FD6 . Journal of Integrative Agriculture, Doi:10.1016/j.jia.2026.09.021

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