内生菌Pseudomonas sp. G5 phzIR基因的克隆与表达

Cloning of phzIR from the endophytic Pseudomonas sp. G5 and its expression in Escherichia coli

摘要:

假单胞菌菌株G5是分离自香菜(Coriandrum sativum L.)茎内的一株内生菌, 经BIOLOG系统分析其底物利用图谱, 初步鉴定为桔黄假单胞菌Pseudomonas aurantiaca。大量研究已表明许多革兰氏阴性细菌应用群体感应系统, 通过感应扩散性小信号分子―乙酰基高丝氨酸内酯(N-acyl homoserine lactones, AHLs), 以种群密度依赖的方式调控基因表达, 控制植物相关细菌的多种表型。本研究组合应用AHLs检测菌株Chromobacterium violaceum CV026 和薄层层析分析, 初步检测出菌株G5 可产生几种可检测水平的AHLs 信号分子, 其中以N-hexanoyl-homoserine lactone (C6-HSL, HHL)为主, 迁移率Rf值为0.4。进一步克隆和测序了该菌株中由PhzI和PhzR组成的群体感应quorum sensing系统的编码基因phzIR, 并在大肠杆菌中异源表达了AHLs信号分子合成酶基因phzI。序列和系统进化分析表明它们与假单胞菌属其他的phzIR基因有高度同源性和进化上的保守性。

Abstract:

We isolated a new strain of endophytic Pseudomonas G5 from the stems of Chinese parsley (Coriandrum sativum L.), and it is tentatively identified as Pseudomonas aurantiaca according to analysis of the entire substrate utilization profiles using BIOLOG MicrostationTM system (BIOLOG, Inc, Hayward CA). An array of evidence established that many Gram-negative bacteria employ Quorum sensing (QS) system to regulate gene expression in response to cell density using small diffusible signal molecules, N-acyl homoserine lactones (AHLs), and control diverse phenotypic traits in plant-associated bacteria. In this study, we showed that Pseudomonas sp. strain G5 can produce several types of AHLs at a detectable level using Thin Layer Chromatography (TLC) analysis combined with bioreporter Chromobacterium violaceum CV026 bioassay, and N-hexanoyl-homoserine lactone (HHL, C6-HSL) with Rf value 0.4 is the major signal molecule. Furthermore, we have identified its quorum sensing system composed of PhzI and PhzR by cloning and sequencing of phzI-phzR. PhzI is responsible for synthesis of AHLs signal molecules, and PhzR is a transcriptional regulator. Finally, we heterologously expressed the recombinant plasmid pMD-phzIR in Escherichia coli JM109 and verified it using C. violaceum CV026 bioassay. The phylogenetic analysis using MEGA4 revealed highly similarities exist among the phzIR homologs, suggesting it is evolutionary well conserved in the genus Pseudomonas.