BldD-based bimolecular fluorescence complementation for in vivo detection of the second messenger cyclic di-GMP,Molecular Microbiology

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BldD-based bimolecular fluorescence complementation for in vivo detection of the second messenger cyclic di-GMP
Molecular Microbiology ( IF 2.6 ) Pub Date : 2021-12-27 , DOI: 10.1111/mmi.14876
Manuel Halte ₁ , Mirka E Wörmann ₂ , Maxim Bogisch ₂ , Marc Erhardt _{1,

3} , Natalia Tschowri ₄

Affiliation

The widespread bacterial second messenger bis-(3′-5′)-cyclic diguanosine monophosphate (c-di-GMP) is an important regulator of biofilm formation, virulence and cell differentiation. C-di-GMP-specific biosensors that allow detection and visualization of c-di-GMP levels in living cells are key to our understanding of how c-di-GMP fluctuations drive cellular responses. Here, we describe a novel c-di-GMP biosensor, CensYBL, that is based on c-di-GMP-induced dimerization of the effector protein BldD from Streptomyces resulting in bimolecular fluorescence complementation of split-YPet fusion proteins. As a proof-of-principle, we demonstrate that CensYBL is functional in detecting fluctuations in intracellular c-di-GMP levels in the Gram-negative model bacteria Escherichia coli and Salmonella enterica serovar Typhimurium. Using deletion mutants of c-di-GMP diguanylate cyclases and phosphodiesterases, we show that c-di-GMP dependent dimerization of CBldD-YPet results in fluorescence complementation reflecting intracellular c-di-GMP levels. Overall, we demonstrate that the CensYBL biosensor is a user-friendly and versatile tool that allows to investigate c-di-GMP variations using single-cell and population-wide experimental set-ups.

中文翻译：

基于 BldD 的双分子荧光互补体内检测第二信使环状二 GMP

广泛存在的细菌第二信使双-(3'-5')-环二鸟苷一磷酸 (c-di-GMP) 是生物膜形成、毒力和细胞分化的重要调节剂。允许检测和可视化活细胞中 c-di-GMP 水平的 C-di-GMP 特异性生物传感器是我们了解 c-di-GMP 波动如何驱动细胞反应的关键。在这里，我们描述了一种新型 c-di-GMP 生物传感器 CensYBL，它基于 c-di-GMP 诱导的链霉菌效应蛋白 BldD 二聚化，导致分裂 YPet 融合蛋白的双分子荧光互补。作为原理证明，我们证明 CensYBL 在检测革兰氏阴性模型细菌大肠杆菌和肠沙门氏菌鼠伤寒血清型。使用 c-di-GMP 二鸟苷酸环化酶和磷酸二酯酶的缺失突变体，我们显示 CBldD-YPet 的 c-di-GMP 依赖性二聚化导致反映细胞内 c-di-GMP 水平的荧光互补。总体而言，我们证明 CensYBL 生物传感器是一种用户友好且多功能的工具，允许使用单细胞和全群体实验装置研究 c-di-GMP 变化。

更新日期：2021-12-27

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