[Objective] Pseudomonas fluorescens 2P24 is an effective biocontrol agent for soil-borne plant diseases caused by microbial pathogens. The PcoI/PcoR quorum-sensing system, which influences the colonization ability of 2P24 on wheat rhizosphere, is an important factor for disease suppression. In this study we performed random mutagenesis to screen novel regulators of the pcol gene, a biosynthase gene responsible for iV-acyl-homoserine lactone (AHL) production. [ Methods ] A gacA gene mutant carrying a pcoI-lacZ fusion was employed as the reporter strain and subjected to a random mini-Tn5 insertion mutagenesis. Expression of pcol kept at a low level under the gacA- negative background. The Tn5-mutants with increased pcol transcription were selected. [Results] Two mutants with significantly increased pcol expression were identified from ~ 10000 Tn5-inserted colonies. The interrupted locus in the mutants was identified as the mvaT gene, a global regulator belonging to the H-NS family. A homolog of the mvaT gene, named mvaV, was also found in the genome draft sequence of 2P24. Genetic inactivation of mvaT or mvaV gene resulted in increased transcription of pcol and the production of AHL molecules. Further qutitification by HPLC showed that the 2,4-diacetylphloroglucinol (2, 4-DAPG) levels in culture supernatant of the mvaT and mvaV mutants were significantly lower than that of the wild type strain. Furthermore, the mvaT or mvaV mutation drastically improved biofilm formation in 2P24. [Conclusion] MvaT and MvaV may function as an important regulatory complex controlling biocontrol capacity of P. fluorescens 2P24.%[目的] 自小麦全蚀病自然衰退土壤分离得到的荧光假单胞菌(Pseudomonas fluorescens)2P24,可防治多种由植物病原菌引起的土传病害.菌株2P24具有群体感应(quorum-sensing,QS)系统PcoI/PcoR,该系统影响生防菌2P24生物膜的形成以及其在小麦根围的定殖能力,从而影响2P24的生防能力.本文利用遗传学方法进一步研究了2P24中QS系统的调控途径.[方法] 将QS系统信号合成基因pcoI的转录报告质粒p970Gm-pcoIp转入gacA基因突变菌株PM201中,再利用Tn5转座子对该菌株进行随机突变,筛选影响pcoI 基因表达的调控因子.[结果] 根据菌落颜色的变化筛选到2株突变菌株.Tn5插入位点和基因序列分析表明这2个突变体中Tn5破坏了同一个基因mvaT;设计引物利用PCR方法从2P24基因组中获得mvaT基因及其同源基因mvaV.转录融合报告实验表明:与野生菌株2P24相比,mvaT及mvaV突变体中pcoI基因的表达和N-乙酰高丝氨酸内酯的产量显著提高；HPLC试验表明mvaT和mvaV基因影响抗生素2,4-二乙酰基间苯三酚的合成.细菌双杂交试验证实,MvaT蛋白和MvaV蛋白在体内发生自身互作,这两个蛋白也可相互作用.[结论] 以上结果表明mvaT和mvaV参与调控生防假单胞菌2P24的PcoI/PcoR群体感应系统,并可能影响其生防功能基因的表达.
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