GacA对假单胞菌株M18两个phz基因簇和菌群传感的调控

Regulation of GacA on two phz gene clusters and quorum sensing in Pseudomonas sp.M18

【目的】假单胞菌株M18(Pseudomonassp.M18)是从甜瓜根际土壤中分离获得的一株对多种植物病原菌具有显著拮抗作用的菌株,在菌群传感(quorumsensing)系统的调控下,能分泌吩嗪-1-羧酸(PCA)以及多种吩嗪(phz)类衍生物的抗真菌物质。全局性因子GacA是M18菌株吩嗪类物质的合成与菌群传感系统的重要调控因子,本文将就GacA对上述两者的调控做进一步研究。【方法】PCR基因扩增和测序研究M18菌株中PCA合成基因簇,运用RT-PCR及构建phzA-lacZ转录融合手段研究M18菌株中两个phz基因簇各自的转录特征以及受全局性因子GacA的调控作用,运用翻译融合手段进一步研究M18菌株中GacA对菌群传感系统的调控作用。【结果】M18菌株的染色体中存在着两个PCA合成基因簇phzA1-G1和phzA2-G2,与铜绿假单胞菌株(P.aeruginosa)PAO1的一致性达99%。但是,M18菌株phzA2-G2基因簇下游的非编码区与PAO1菌株不同,存在着三段单位长度为144 bp的重复序列;在野生菌株M18中,phzA1-G1的转录水平明显高于phzA2-G2,GacA促进phzA1-G1转录,抑制phzA2-G2转录,区别性地调控两个phz基因簇的转录,GacA在整体上抑制phz基因簇的转录,与PAO1菌株中,GacA对phz的调控方式相反;gacA基因突变对菌群传感系统中lux家族基因中的lasI表达量无显著影响,但正调控las系统下游的rhlI表达量,GacA对phz基因簇表达的调控部分通过菌群传感系统实现。【结论】在M18菌株和PAO1菌株中,GacA对吩嗪类产物合成的调控方式相反,对菌群传感系统的调控也存在着差异性,这些差异可能是两个菌株在各自不同生境下长期进化的结果。

[Objective] The regulatory function investigation of two component system gacS/gacA on two phz gene clusters expression and quorum sensing system（QS） in Pseudomonas.sp.M18.[Methods] The two phz gene clusters were sequenced and the expressional features by GacA were analyzed using RT-PCR and phzA-lacZ transcriptional fusions,the regulation of GacA over QS system was studied also in P.sp.M18.[Results] Two phenazine sturctural clusters,namely,phzA1-G1 and phzA2-G2 in P.sp.M18 shared 99% identities with those in P. aeruginosa PAO1. However, in the non-coding region downstreamthe phzA2-G2, P. sp. M18 has a three-144 bp-repeat sequence which does not exist in P. aeruginosa PAO1. PhzA1-G1 expressed at a higher level than phzA2-G2 in the wide type M18 strain. GacA functioned differently over these two phz gene clusters but negatively regulating the two clusters expression at the whole level, which was opposite to that in P. aeruginosa PAO1. The inactivation of gacA gene can reduce rhll expression while has no effect on lasl expression, indicating that the phz gene expression regulated by GacA via QS was a minor part and the major phz expression was regulated by GacA through an unknown pathway instead of QS in P. sp. M18. [ Conclusion] The different regulation of GacA activity on secondary metabolites and Qs in P. sp. M18 and P. aeruginosa PAO1 may be the results imposed by the environmental selective pressure during evolution pathway.