Phenotypic and integrated analysis of a comprehensive Pseudomonas aeruginosa PAO1 library of mutants lacking cyclic-di-GMP-related genes.
Phenotypic and integrated analysis of a comprehensive Pseudomonas aeruginosa PAO1 library of mutants lacking cyclic-di-GMP-related genes.
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DOI：
10.3389/fmicb.2022.949597
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发表时间：
2022
影响因子：
5.2
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铜绿假单胞菌是一种革兰氏阴性细菌，能够在许多小生境中生存和适应，并在许多不同的宿主中茁壮成长。这种多功能性在于其庞大的ca基因组。6 Mbp，并严格控制数千个基因的表达。在细菌中广泛存在的调节机制中，循环二GMP信号传导是影响所有控制水平的机制之一。c-di-GMP由二鸟苷酸环化酶产生并由磷酸二酯酶降解，而该分子的细胞内水平驱动表型反应。信号传导涉及在c-di-GMP结合时酶或蛋白质功能的修饰，包括修饰调节剂的活性，这反过来将影响转录组。在铜绿假单胞菌中，有ca。40个编码推定的DGC或PDE的基因。这些酶的组合活性应反映总体c-di-GMP浓度，而特定的表型输出可与给定的一组dgc/pde相关。这种特异性的概念已经在几项研究和不同的铜绿假单胞菌菌株中得到了解决。在这里，我们设计了铜绿假单胞菌PAO 1中41个单独dgc/pde基因的突变体文库。在大多数情况下，我们观察到的显着轻微的变化，在全球c-di-GMP池中的细胞生长的非甾体激素，而几个突变体显示表型的影响，生物膜，包括初始附着和成熟。如果与显著表型影响相关的c-di-GMP水平的微小变化的观察结果似乎是真实的，则其进一步支持局部与全局c-di-GMP合并液的想法。相比之下，对运动性几乎没有影响，这与以前的研究不同。我们的RNA-seq分析表明，所有PAO 1 dgc/pde基因在增殖和生物膜生长条件下均表达，我们的工作表明，需要分别为每种菌株重建c-di-GMP网络，并且不能从一种菌株外推到另一种菌株。
Pseudomonas aeruginosa is a Gram-negative bacterium that is able to survive and adapt in a multitude of niches as well as thrive within many different hosts. This versatility lies within its large genome of ca. 6 Mbp and a tight control in the expression of thousands of genes. Among the regulatory mechanisms widespread in bacteria, cyclic-di-GMP signaling is one which influences all levels of control. c-di-GMP is made by diguanylate cyclases and degraded by phosphodiesterases, while the intracellular level of this molecule drives phenotypic responses. Signaling involves the modification of enzymes’ or proteins’ function upon c-di-GMP binding, including modifying the activity of regulators which in turn will impact the transcriptome. In P. aeruginosa, there are ca. 40 genes encoding putative DGCs or PDEs. The combined activity of those enzymes should reflect the overall c-di-GMP concentration, while specific phenotypic outputs could be correlated to a given set of dgc/pde. This notion of specificity has been addressed in several studies and different strains of P. aeruginosa. Here, we engineered a mutant library for the 41 individual dgc/pde genes in P. aeruginosa PAO1. In most cases, we observed a significant to slight variation in the global c-di-GMP pool of cells grown planktonically, while several mutants display a phenotypic impact on biofilm including initial attachment and maturation. If this observation of minor changes in c-di-GMP level correlating with significant phenotypic impact appears to be true, it further supports the idea of a local vs global c-di-GMP pool. In contrast, there was little to no effect on motility, which differs from previous studies. Our RNA-seq analysis indicated that all PAO1 dgc/pde genes were expressed in both planktonic and biofilm growth conditions and our work suggests that c-di-GMP networks need to be reconstructed for each strain separately and cannot be extrapolated from one to another.