The ubiquitous bacterial second messenger c-di-GMP is synthesized by diguanylate cyclase (DGC) and degraded by phosphodiesterase (PDE). Pseudomonas putida has dozens of DGC/PDE-encoding genes in its genome, but the phenotypical-genotypical correlation and transcriptional regulation of these genes are largely unknown. Herein, we characterize function and transcriptional regulation of a P. putida c-di-GMP-metabolizing enzyme, GcsA. GcsA consists of two per-ARNT-sim (PAS) domains, followed by a canonical conserved central sequence pattern (GGDEF) domain and a truncated EAL domain. In vitro analysis confirmed the DGC activity of GcsA. The phenotypic observation revealed that GcsA inhibited swimming motility in an FlgZ-dependent manner. In terms of transcriptional regulation, gcsA was found to be cooperatively regulated by c-di-GMP and cAMP via their effectors, FleQ and Crp respectively. The transcription of gcsA was promoted by c-di-GMP and inhibited by cAMP. In vitro binding analysis revealed that FleQ indirectly regulated the transcription of gcsA, while Crp directly regulated the transcription of gcsA by binding to its promoter. Besides, an inverse relationship between the cellular c-di-GMP and cAMP levels in P. putida was confirmed. These findings provide basic knowledge regarding the function and transcriptional regulation of GcsA and demonstrate a crosstalk between c-di-GMP and cAMP in the regulation of the expression of GcsA in P. putida.

中文翻译：

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c-di-GMP和cAMP在调节GcsA（一种参与假单胞菌游泳运动的双鸟苷酸环化酶）的转录中的串扰。

普遍存在的细菌第二信使c-di-GMP由双鸟苷酸环化酶（DGC）合成，并被磷酸二酯酶（PDE）降解。恶臭假单胞菌在其基因组中有数十个DGC / PDE编码基因，但是这些基因的表型-基因型相关性和转录调控在很大程度上尚不清楚。在本文中，我们表征恶臭假单胞菌c-di-GMP代谢酶GcsA的功能和转录调控。GcsA由两个每个ARNT-sim（PAS）域组成，后跟一个规范的保守中心序列模式（GGDEF）域和一个截短的EAL域。体外分析证实了GcsA的DGC活性。表型观察表明，GcsA以FlgZ依赖的方式抑制了游泳运动。在转录调控方面，发现gcsA受c-di-GMP和cAMP分别通过其效应物FleQ和Crp共同调控。c-di-GMP促进了gcsA的转录，而cAMP抑制了gcsA的转录。体外结合分析显示，FleQ间接调节gcsA的转录，而Crp通过结合其启动子直接调节gcsA的转录。此外，证实恶臭假单胞菌的细胞c-di-GMP与cAMP水平呈反比关系。这些发现提供了关于GcsA的功能和转录调控的基础知识，并证明了c-di-GMP和cAMP之间在恶臭假单胞菌中GcsA表达的调控中的串扰。体外结合分析显示，FleQ间接调节gcsA的转录，而Crp通过结合其启动子直接调节gcsA的转录。此外，证实恶臭假单胞菌的细胞c-di-GMP与cAMP水平呈反比关系。这些发现提供了关于GcsA的功能和转录调控的基础知识，并证明了c-di-GMP和cAMP之间在恶臭假单胞菌中GcsA表达的调控中的串扰。体外结合分析显示，FleQ间接调节gcsA的转录，而Crp通过结合其启动子直接调节gcsA的转录。此外，证实恶臭假单胞菌的细胞c-di-GMP与cAMP水平呈反比关系。这些发现提供了关于GcsA的功能和转录调控的基础知识，并证明了c-di-GMP和cAMP之间在恶臭假单胞菌中GcsA表达的调控中的串扰。