Oxygen-dependent regulation of a diguanylate cyclase lacking canonical sensory and regulatory domains in Pseudomonas aeruginosa

缺乏典型的感觉和调节结构域的二鸟苷酸环化酶（DGC）广泛存在于变形菌的所有类别中。然而，其监管机制仍然难以捉摸。为了促进我们对c-di-GMP信号传导原理的理解，分析这些DGC的活性是如何控制的是必要的。我们先前表明，在厌氧条件下，膜结合的DGC SadC刺激铜绿假单胞菌中的藻酸盐生产，并且与需氧条件相比，在厌氧条件下表达的SadC的纯化的GGEEF结构域的活性显著更高，这导致几乎检测不到c-di-GMP的水平。虽然SadC不包含典型的感觉和调节模块，我们以前表明，该蛋白质形成了一个预测的水合酶和双加氧酶还原酶的调节模块-作为一个积极的和消极的调节藻酸盐合成，分别。体外酶促测定表明，双加氧酶还原酶，但不是水合酶抑制SadC。本研究的目的是分析铜绿假单胞菌中SadC在氧气和缺氧条件下的变化。为此，我们试图定义的亚细胞定位和相互作用网络的蛋白质在有氧和厌氧条件下使用免疫和时间推移荧光显微镜和体内交联方法等。此外，我们的目标是确定参与氧依赖性调节的SadC区域，并通过LC-MS进一步分析共价修饰的结构域。对于后者，细菌也将在稳定标记的18 O存在下生长，以研究分子氧掺入SadC以控制其DGC活性的可能性。

Diguanylate cyclases (DGC) lacking canonical sensory and regulatory domains are widespread and found in all classes of Proteobacteria. However, the mechanisms underlying their regulation remain elusive. In order to advance our understanding on the principles of c-di-GMP signaling it is essential to analyze how the activity of these DGCs is controlled. We previously showed that the membrane bound DGC SadC stimulates alginate production in Pseudomonas aeruginosa under anaerobic conditions and that the activity of the purified GGEEF domain of SadC expressed under anaerobic is significantly higher as compared with aerobic conditions, which result in barely detectable levels of c-di-GMP. While SadC does not contain canonical sensory and regulatory modules we previously showed that the protein forms a regulatory module with a predicted hydratase and dioxygenase reductase -which act as a positive and negative regulator of alginate synthesis, respectively. In vitro enzymatic assays indicate that the dioxygenase reductase but not the hydratase inhibits SadC. The objective of the study is to dissect the modifications of SadC in P. aeruginosa in response to oxygen and anoxia. To this end, we seek to define the subcellular localization and interaction network of the proteins under aerobic and anaerobic conditions using immuno- and time lapse fluorescence microscopy and in vivo cross linking approaches among others. Furthermore, we aim to identify the region(s) of SadC involved in oxygen-dependent regulation and to further analyze the domains by LC-MS for covalent modifications. For the latter, the bacteria will also be grown in the presence of stably labeled 18O to investigate the possibility that molecular oxygen is incorporated into SadC to control its DGC activity.