Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, School of Biomedical Engineering, Shenzhen Universitygrid.263488.3 Health Science Center, Shenzhen, China.
School of Medicine, Southern University of Science and Technology, Shenzhen, China.
Appl Environ Microbiol. 2022 Jun 28;88(12):e0003922. doi: 10.1128/aem.00039-22. Epub 2022 May 31.
In Pseudomonas aeruginosa PAO1, 41 genes encode proteins predicted to be involved in the production or degradation of c-di-GMP, a ubiquitous secondary messenger that regulates a variety of physiological behaviors closely related to biofilm and aggregate formation. Despite extensive effort, the entire picture of this important signaling network is still unclear, with one-third of these proteins remaining uncharacterized. Here, we show that the deletion of , which produces a protein containing two PAS domains upstream of a GGDEF-EAL tandem, significantly increased the intracellular c-di-GMP level and promoted the formation of aggregates both on surfaces and in planktonic cultures. However, this regulatory effect was not contributed by either of the two classic pathways modulating biofilm formation, exopolysaccharide (EPS) overproduction or motility inhibition. Transcriptome sequencing (RNA-Seq) data revealed that the expression levels of 361 genes were significantly altered in a Δ mutant strain compared to the wild type (WT), indicating the critical role of PipA in PAO1. The most remarkably downregulated genes were located on the Pf4 bacteriophage gene cluster, which corresponded to a 2-log reduction in the Pf4 phage production in the Δ mutant. The sizes of aggregates in Δ cultures were affected by exogenously added Pf4 phage in a concentration-dependent manner, suggesting the quantity of phage plays a part in regulating the formation of aggregates. Further analysis demonstrated that PipA is highly conserved across 83 P. aeruginosa strains. Our work therefore for the first time showed that a c-di-GMP phosphodiesterase can regulate bacteriophage production and provided new insights into the relationship between bacteriophage and bacterial aggregation. The c-di-GMP signaling pathways in P. aeruginosa are highly organized and well coordinated, with different diguanylate cyclases and phosphodiesterases playing distinct roles in a complex network. Understanding the function of each enzyme and the underlying regulatory mechanisms not only is crucial for revealing how bacteria decide the transition between motile and sessile lifestyles, but also greatly facilitates the development of new antibiofilm strategies. This work identified bacteriophage production as a novel phenotypic output controlled transcriptionally by a phosphodiesterase, PipA. Further analysis suggested that the quantity of phage may be important in regulating autoaggregation, as either a lack of phage or overproduction was associated with higher levels of aggregation. Our study therefore extended the scope of c-di-GMP-controlled phenotypes and discovered a potential signaling circuit that can be target for biofilm treatment.
在铜绿假单胞菌 PAO1 中，有 41 个基因编码的蛋白质被预测参与 c-di-GMP 的产生或降解，c-di-GMP 是一种普遍存在的二级信使，它调节着与生物膜和聚集物形成密切相关的多种生理行为。尽管已经进行了广泛的研究，但这个重要信号网络的全貌仍然不清楚，其中三分之一的蛋白质仍然没有被描述。在这里，我们表明，缺失产生一种蛋白质的基因，该蛋白质在上游含有两个 PAS 结构域和一个 GGDEF-EAL 串联结构域，会显著增加细胞内 c-di-GMP 水平，并促进表面和浮游培养物中聚集物的形成。然而，这种调节作用并不是由调节生物膜形成的两种经典途径之一（即多糖过度产生或运动性抑制）所贡献的。转录组测序（RNA-Seq）数据显示，与野生型（WT）相比，Δ突变株中 361 个基因的表达水平显著改变，表明 PipA 在 PAO1 中起着关键作用。最显著下调的基因位于 Pf4 噬菌体基因簇上，这对应于 Pf4 噬菌体在 Δ突变株中的产量降低了 2 个对数级。在 Δ 培养物中，外源性添加 Pf4 噬菌体的浓度依赖性影响了聚集物的大小，表明噬菌体的数量在调节聚集物形成中起着一定的作用。进一步的分析表明，PipA 在 83 株铜绿假单胞菌中高度保守。因此，我们的工作首次表明，一种 c-di-GMP 磷酸二酯酶可以调节噬菌体的产生，并为噬菌体和细菌聚集之间的关系提供了新的见解。铜绿假单胞菌中的 c-di-GMP 信号通路高度组织化且协调良好，不同的双鸟苷酸环化酶和磷酸二酯酶在复杂网络中发挥着不同的作用。了解每种酶的功能和潜在的调控机制不仅对于揭示细菌如何决定从运动到静止生活方式的转变至关重要，而且还极大地促进了新的抗生物膜策略的发展。这项工作确定了噬菌体的产生是一种由磷酸二酯酶 PipA 转录控制的新表型输出。进一步的分析表明，噬菌体的数量可能在调节自动聚集中很重要，因为噬菌体的缺乏或过度产生都与更高水平的聚集有关。因此，我们的研究扩展了 c-di-GMP 控制表型的范围，并发现了一个可能的信号回路，可以作为生物膜治疗的靶点。
