Systematic Analysis of Diguanylate Cyclases That Promote Biofilm Formation by Pseudomonas fluorescens Pf0-1
Systematic Analysis of Diguanylate Cyclases That Promote Biofilm Formation by Pseudomonas fluorescens Pf0-1
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DOI：
10.1128/jb.05483-11
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发表时间：
2011-09-01
影响因子：
3.2
通讯作者：
O'Toole, George A.
中科院分区：
文献类型：
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作者：
Newell, Peter D.;Yoshioka, Shiro;O'Toole, George A.
环二GMP（c-di-GMP）是一种广泛保守的细胞内第二信使分子，其调节许多细菌的生物膜形成。c-di-GMP的合成由含有GGDEF结构域的二鸟苷酸环化酶（DGC）催化，而其降解通过EAL和HD-GYP结构域的磷酸二酯酶活性实现。c-di-GMP通过促进大粘附蛋白LapA的细胞表面定位来控制荧光假单胞菌Pf 0 -1的生物膜形成。LapA定位由LapD和LapG组成的c-di-GMP效应子系统在免疫后调节，其感测细胞质c-di-GMP并修饰外膜中的LapA蛋白。尽管荧光假单胞菌Pf 0 -1的生物膜形成明显需要c-di-GMP，但迄今为止尚未表征来自该菌株的DGC。在这项研究中，我们对30个预测的DGC进行了系统的诱变，发现在测试条件下，仅4个DGC中的突变导致荧光假单胞菌Pf 0 -1的生物膜形成减少。这些DGC的特征在于遗传和生化证实的假设，它们的功能，以产生c-di-GMP在体内。分析了DGC基因突变对与生物膜形成相关的表型的影响。一个DGC优先影响LapA定位，另一个DGC主要控制游泳运动，而第三个DGC影响LapA和运动。我们的数据支持这样的结论，即不同的c-di-GMP调节的输出可以由不同的DGC特异性控制。
Cyclic di-GMP (c-di-GMP) is a broadly conserved, intracellular second-messenger molecule that regulates biofilm formation by many bacteria. The synthesis of c-di-GMP is catalyzed by diguanylate cyclases (DGCs) containing the GGDEF domain, while its degradation is achieved through the phosphodiesterase activities of EAL and HD-GYP domains. c-di-GMP controls biofilm formation by Pseudomonas fluorescens Pf0-1 by promoting the cell surface localization of a large adhesive protein, LapA. LapA localization is regulated post-translationally by a c-di-GMP effector system consisting of LapD and LapG, which senses cytoplasmic c-di-GMP and modifies the LapA protein in the outer membrane. Despite the apparent requirement for c-di-GMP for biofilm formation by P. fluorescens Pf0-1, no DGCs from this strain have been characterized to date. In this study, we undertook a systematic mutagenesis of 30 predicted DGCs and found that mutations in just 4 cause reductions in biofilm formation by P. fluorescens Pf0-1 under the conditions tested. These DGCs were characterized genetically and biochemically to corroborate the hypothesis that they function to produce c-di-GMP in vivo. The effects of DGC gene mutations on phenotypes associated with biofilm formation were analyzed. One DGC preferentially affects LapA localization, another DGC mainly controls swimming motility, while a third DGC affects both LapA and motility. Our data support the conclusion that different c-di-GMP-regulated outputs can be specifically controlled by distinct DGCs.