Genetic reductionist approach for dissecting individual roles of GGDEF proteins within the c-di-GMP signaling network in Salmonella
Genetic reductionist approach for dissecting individual roles of GGDEF proteins within the c-di-GMP signaling network in Salmonella
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DOI：
10.1073/pnas.0812573106
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发表时间：
2009-05-12
影响因子：
11.1
通讯作者：
Lasa, Inigo
中科院分区：
文献类型：
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作者：
Solano, Cristina;Garcia, Begona;Lasa, Inigo
细菌已经发展出一种独特的信号转导系统，涉及多个二胍酸环化酶和磷酸二酯酶结构域蛋白（分别为GGDEF和EAL/HD-GYP），它们调节同一扩散分子3'-5'-环二胍酸（c-di-GMP）的水平，以传递信号并获得特定的细胞反应。目前关于c-di-GMP信号传导的知识主要来自重组细菌的分析，这些重组细菌缺乏或过量产生该途径的单个成员，而没有解决它们之间潜在的补偿作用或干扰。在这里，我们通过构建缺乏所有ggdef结构域蛋白的沙门氏菌菌株，然后产生衍生物，每个衍生物恢复1个蛋白，来剖析c-di-GMP信号。我们的分析表明，大多数GGDEF蛋白是组成性表达的，它们的表达水平不是相互依赖的。编码ggdef结构域蛋白的基因的完全缺失会破坏毒力、运动性、长期存活以及纤维素和菌毛的合成。单独恢复发现，沙门氏菌中的4个蛋白和鼠疫菌中的1个蛋白完全以c-di-GMP依赖的方式恢复纤维素合成，这表明不同GGDEF蛋白产生的c-di-GMP可以激活相同的靶标。然而，含有stm4551编码基因的恢复菌株通过独立于c-di-GMP的基因表达调节恢复了所有其他表型。具体而言，通过调节csgD和质粒编码的spvAB mRNA水平，分别恢复了菌毛合成和毒力。本研究提供的证据表明，ggdef结构域蛋白网络的调控发生在两个水平：一个水平严格要求c-di-GMP直接控制酶的活性，在我们的实验条件下仅限于纤维素的合成；另一个水平涉及基因调控，c-di-GMP的合成可以被替代。
Bacteria have developed an exclusive signal transduction system involving multiple diguanylate cyclase and phosphodiesterase domain-containing proteins (GGDEF and EAL/HD-GYP, respectively) that modulate the levels of the same diffusible molecule, 3'-5'-cyclic diguanylic acid (c-di-GMP), to transmit signals and obtain specific cellular responses. Current knowledge about c-di-GMP signaling has been inferred mainly from the analysis of recombinant bacteria that either lack or overproduce individual members of the pathway, without addressing potential compensatory effects or interferences between them. Here, we dissected c-di-GMP signaling by constructing a Salmonella strain lacking all GGDEF-domain proteins and then producing derivatives, each restoring 1 protein. Our analysis showed that most GGDEF proteins are constitutively expressed and that their expression levels are not interdependent. Complete deletion of genes encoding GGDEF-domain proteins abrogated virulence, motility, long-term survival, and cellulose and fimbriae synthesis. Separate restoration revealed that 4 proteins from Salmonella and 1 from Yersinia pestis exclusively restored cellulose synthesis in a c-di-GMP-dependent manner, indicating that c-di-GMP produced by different GGDEF proteins can activate the same target. However, the restored strain containing the STM4551-encoding gene recovered all other phenotypes by means of gene expression modulation independently of c-di-GMP. Specifically, fimbriae synthesis and virulence were recovered through regulation of csgD and the plasmid-encoded spvAB mRNA levels, respectively. This study provides evidence that the regulation of the GGDEF-domain proteins network occurs at 2 levels: a level that strictly requires c-di-GMP to control enzymatic activities directly, restricted to cellulose synthesis in our experimental conditions, and another that involves gene regulation for which c-di-GMP synthesis can be dispensable.