Yang F, Qian S, Tian F, Chen H, Hutchins W, Yang C-H, He C
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.
Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI, USA.
J Appl Microbiol. 2016 Jun;120(6):1646-57. doi: 10.1111/jam.13115. Epub 2016 Apr 4.
Cyclic di-GMP (c-di-GMP), a ubiquitous bacterial second messenger that is synthesized by diguanylate cyclase (DGC) with the GGDEF-domain, regulates diverse virulence phenotypes in pathogenic bacteria. Although 11 genes encoding GGDEF-domain proteins have been shown in the genome of Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99(A) , the causal pathogen of bacterial blight of rice, however, little is known about their roles in the c-di-GMP regulation of virulence in the pathogen. GdpX1, one of the GGDEF-domain proteins in Xoo was investigated in this study to reveal its regulatory function of bacterial virulence expression through genetic analysis.
GdpX1 was functionally characterized in virulence expression through deletion and overexpression analysis. Bioinformatics analysis revealed the GGDEF-domain in GdpX1 was well conserved, indicating it is a putative DGC. Deletion of gdpX1 resulted in significant increases in virulence, exopolysaccharide (EPS) production and flagellar motility. In contrast, overexpression of gdpX1 dramatically reduced these virulence phenotypes. qRT-PCR analysis showed genes related to the type III secretion system (T3SS), EPS synthesis, and flagellar motility, were up-regulated in ∆gdpX1 and down-regulated in the gdpX1-overexpressed strains. In addition, overexpression of gdpX1 promoted biofilm formation and xylanase activity.
GdpX1 is the first GGDEF-domain protein functionally characterized in Xoo, which functions as a negative regulator of bacterial virulence via suppression of virulence-related gene transcription.
Identification and functional characterization of GdpX1 provided additional insights into molecular mechanisms of c-di-GMP regulation of bacterial virulence expression.
环二鸟苷酸（c-di-GMP）是一种普遍存在的细菌第二信使，由具有GGDEF结构域的二鸟苷酸环化酶（DGC）合成，可调节病原菌的多种毒力表型。虽然在水稻白叶枯病菌（Xanthomonas oryzae pv. oryzae，Xoo）PXO99（A）菌株（水稻白叶枯病的致病病原菌）的基因组中已显示有11个编码GGDEF结构域蛋白的基因，但对它们在病原菌毒力的c-di-GMP调控中的作用知之甚少。本研究对Xoo中GGDEF结构域蛋白之一的GdpX1进行了研究，以通过遗传分析揭示其对细菌毒力表达的调控功能。
通过缺失和过表达分析对GdpX1在毒力表达中的功能进行了表征。生物信息学分析表明GdpX1中的GGDEF结构域高度保守，表明它是一种推定的DGC。缺失gdpX1导致毒力、胞外多糖（EPS）产生和鞭毛运动显著增加。相反，gdpX1的过表达显著降低了这些毒力表型。qRT-PCR分析表明，与III型分泌系统（T3SS）、EPS合成和鞭毛运动相关的基因在∆gdpX1中上调，而在gdpX1过表达菌株中下调。此外，gdpX1的过表达促进了生物膜形成和木聚糖酶活性。
GdpX1是第一个在Xoo中进行功能表征的GGDEF结构域蛋白，它通过抑制毒力相关基因转录作为细菌毒力的负调控因子。
GdpX1的鉴定和功能表征为c-di-GMP调控细菌毒力表达的分子机制提供了更多见解。
