Engineering of Bacillus subtilis Strains To Allow Rapid Characterization of Heterologous Diguanylate Cyclases and Phosphodiesterases
Engineering of Bacillus subtilis Strains To Allow Rapid Characterization of Heterologous Diguanylate Cyclases and Phosphodiesterases
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DOI：
10.1128/aem.01638-14
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发表时间：
2014-10-01
影响因子：
4.4
通讯作者：
Dann, Charles E., III
中科院分区：
文献类型：
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作者：
Gao, Xiaohui;Dong, Xiao;Dann, Charles E., III
微生物过程，包括生物膜的形成，运动性和毒力，通常由环二聚鸟苷一磷酸（c-di-GMP）的可用浓度的变化来调节。通常，高c-di-GMP浓度与运动性降低和生物膜形成增加相关，低c-di-GMP浓度与运动性增加和毒力途径激活相关。然而，c-di-GMP的研究很复杂，因为生物体通常编码数十种多余的酶来合成和水解c-di-GMP、二鸟苷酸环化酶（DGCs）和c-di-GMP磷酸二酯酶（PDEs）;因此，确定任何一种特定酶的贡献都具有挑战性。在努力开发一个简单的系统来研究c-di-GMP代谢酶，我们已经设计了一套枯草芽孢杆菌菌株，以评估个别异源表达的蛋白质对c-di-GMP水平的影响。作为原理的证明，我们使用来自在c-di-GMP控制的核糖开关控制下表达的绿色荧光蛋白（GFP）的群集运动和荧光的平行读数来表征艰难梭菌中编码预测的DGC和PDE的所有37个已知基因。我们发现37个假定的C. difficile 630 c-di-GMP代谢酶具有活性环化酶或磷酸二酯酶活性，我们的运动表型与基于荧光的c-di-GMP报告基因之间一致。最后，我们表明，似乎有一个阈值水平的c-di-GMP需要抑制枯草芽孢杆菌的运动。
Microbial processes, including biofilm formation, motility, and virulence, are often regulated by changes in the available concentration of cyclic dimeric guanosine monophosphate (c-di-GMP). Generally, high c-di-GMP concentrations are correlated with decreased motility and increased biofilm formation and low c-di-GMP concentrations are correlated with an increase in motility and activation of virulence pathways. The study of c-di-GMP is complicated, however, by the fact that organisms often encode dozens of redundant enzymes that synthesize and hydrolyze c-di-GMP, diguanylate cyclases (DGCs), and c-di-GMP phosphodiesterases (PDEs); thus, determining the contribution of any one particular enzyme is challenging. In an effort to develop a facile system to study c-di-GMP metabolic enzymes, we have engineered a suite of Bacillus subtilis strains to assess the effect of individual heterologously expressed proteins on c-di-GMP levels. As a proof of principle, we characterized all 37 known genes encoding predicted DGCs and PDEs in Clostridium difficile using parallel readouts of swarming motility and fluorescence from green fluorescent protein (GFP) expressed under the control of a c-di-GMP-controlled riboswitch. We found that 27 of the 37 putative C. difficile 630 c-di-GMP metabolic enzymes had either active cyclase or phosphodiesterase activity, with agreement between our motility phenotypes and fluorescence-based c-di-GMP reporter. Finally, we show that there appears to be a threshold level of c-di-GMP needed to inhibit motility in Bacillus subtilis.