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

• Applicant: Dr. Sandra Schwarz
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms ; Parasitology and Biology of Tropical Infectious Disease Pathogens
• Term: from 2016 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 314744378

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.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1879: Nucleotide Second Messenger Signaling in Bacteria