RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messenger Cyclic di-AMP.
RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messenger Cyclic di-AMP.
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DOI：
10.1021/jacs.5b00275
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发表时间：
2015-05-27
影响因子：
15
通讯作者：
Hammond MC
中科院分区：
文献类型：
--
作者：
Kellenberger CA;Chen C;Whiteley AT;Portnoy DA;Hammond MC
环二AMP（cdiA）是一种广泛存在于革兰氏阳性菌、某些革兰氏阴性菌和大肠杆菌中的第二信使。在人类病原体单核细胞增生李斯特菌中，cdiA是调节代谢功能和细胞壁稳态的重要分子，并且cdiA水平降低导致抗生素敏感性增加。我们已经通过将Spinach2适体融合到cdiA核糖开关的配体结合结构域来产生cdiA的荧光生物传感器。该生物传感器用于可视化活L.单核细胞增多症菌株，并确定磷酸二酯酶PdeA的催化结构域。此外，基于该生物传感器的流式细胞术测定用于筛选二腺苷酸环化酶活性，并确认来自艰难梭菌和詹氏甲烷球菌的DisA样蛋白的酶活性。因此，我们已经扩大了基于RNA的生物传感器在革兰氏阳性菌体内代谢物成像的发展，并已验证了第一个二核苷酸环化酶从大肠杆菌。
Cyclic di-AMP (cdiA) is a second messenger predicted to be widespread in Gram-positive bacteria, some Gram-negative bacteria, and Archaea. In the human pathogen Listeria monocytogenes, cdiA is an essential molecule that regulates metabolic function and cell wall homeostasis, and decreased levels of cdiA result in increased antibiotic susceptibility. We have generated fluorescent biosensors for cdiA through fusion of the Spinach2 aptamer to ligand-binding domains of cdiA riboswitches. The biosensor was used to visualize intracellular cdiA levels in live L. monocytogenes strains and to determine the catalytic domain of the phosphodiesterase PdeA. Furthermore, a flow cytometry assay based on this biosensor was used to screen for diadenylate cyclase activity and confirmed the enzymatic activity of DisA-like proteins from Clostridium difficile and Methanocaldococcus jannaschii. Thus, we have expanded the development of RNA-based biosensors for in vivo metabolite imaging in Gram-positive bacteria and have validated the first dinucleotide cyclase from Archaea.