Modulation of Pseudomonas aeruginosa Biofilm Dispersal by a Cyclic-Di-GMP Phosphodiesterase with a Putative Hypoxia-Sensing Domain
Modulation of Pseudomonas aeruginosa Biofilm Dispersal by a Cyclic-Di-GMP Phosphodiesterase with a Putative Hypoxia-Sensing Domain
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DOI：
10.1128/aem.01233-10
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发表时间：
2010-12-01
影响因子：
4.4
通讯作者：
Zhang, Lian-Hui
中科院分区：
文献类型：
--
作者：
An, Shuwen;Wu, Ji'en;Zhang, Lian-Hui
铜绿假单胞菌编码许多酶，这些酶可能与广泛保守的第二信使环二GMP（c-di-GMP）的合成或降解相关。在这项研究中，我们表明，rbdA，它编码的PAS-PAC-GGDEF-EAL多域组成的融合蛋白的突变，导致生物膜分散减少。RbdA含有高度保守的GGDEF结构域和EAL结构域，分别参与c-di-GMP的合成和降解。然而，体内和体外分析表明，全长RbdA蛋白只显示磷酸二酯酶活性，导致c-di-GMP降解。进一步的分析表明，GGDEF结构域的RbdA在激活磷酸二酯酶活性的EAL结构域在GTP的存在下发挥作用。此外，我们发现，PAS结构域的缺失或取代的关键残基参与传感低氧应力废除的功能RbdA。随后的研究表明，RbdA参与细菌运动性和鼠李糖脂的产生的正调节，这与生物膜的分散有关，并参与生物膜形成所需的胞外聚合物的产生的负调节。这些数据表明，c-di-GMP降解调节蛋白RbdA通过其对生物膜发育的双管齐下的作用促进生物膜分散，即，下调生物膜形成和上调与生物膜扩散相关的因子的产生。
Pseudomonas aeruginosa encodes many enzymes that are potentially associated with the synthesis or degradation of the widely conserved second messenger cyclic-di-GMP (c-di-GMP). In this study, we show that mutation of rbdA, which encodes a fusion protein consisting of PAS-PAC-GGDEF-EAL multidomains, results in decreased biofilm dispersal. RbdA contains a highly conserved GGDEF domain and EAL domain, which are involved in the synthesis and degradation of c-di-GMP, respectively. However, in vivo and in vitro analyses show that the full-length RbdA protein only displays phosphodiesterase activity, causing c-di-GMP degradation. Further analysis reveals that the GGDEF domain of RbdA plays a role in activating the phosphodiesterase activity of the EAL domain in the presence of GTP. Moreover, we show that deletion of the PAS domain or substitution of the key residues implicated in sensing low-oxygen stress abrogates the functionality of RbdA. Subsequent study showed that RbdA is involved in positive regulation of bacterial motility and production of rhamnolipids, which are associated with biofilm dispersal, and in negative regulation of production of exopoly-saccharides, which are required for biofilm formation. These data indicate that the c-di-GMP-degrading regulatory protein RbdA promotes biofilm dispersal through its two-pronged effects on biofilm development, i.e., downregulating biofilm formation and upregulating production of the factors associated with biofilm dispersal.