Susanne Zehner (Kaiserslautern / DE), Justin Rehmel (Kaiserslautern / DE), Nina Bäuerle (Kaiserslautern / DE), Anna Scherhag (Kaiserslautern / DE), Michael T. Agbadaola (Kaiserslautern / DE), Mathias Schösser (Kaiserslautern / DE), Nicole Frankenberg-Dinkel (Kaiserslautern / DE), Eugenio Pérez Patallo (Kaiserslautern / DE)
The molecule c-di-GMP is a second messenger that controls various processes such as virulence and biofilm formation in bacteria (1). Synthesis and degradation of c-di-GMP is mediated by two types of enzymes, diguanylate cyclases (DGC) and phosphodiesterases (PDE) (1, 2). Pseudomonas aeruginosa PAO1, an opportunistic pathogen and biofilm model organism, encodes more than 40 c-di-GMP modulating proteins (3). In total, 22 of these proteins are predicted to be membrane bound. Until now, the biochemical characterization of these proteins has been limited mostly to their soluble cytosolic domains (4).The phosphodiesterase NbdA comprises a membrane bound MHYT domain, predicted to be a sensor for NO, CO or O2 (5), and a cytosolic part consisting of a degenerated diguanylate cyclase and a functional phosphodiesterase domain. In vivo assays showed the phosphodiesterase activity of NbdA, that could be confirmed in vitro with the purified cytosolic part of the protein (4). We established the production and purification of the full- length protein from E. coli using the nanodisc forming polymer DIBMA. We obtained pure and enzymatically active NbdA and characterized the binding of heme to the full-length protein. Furthermore, to study putative interaction partners of the full-length protein NbdA, we analyzed Pseudomonas membranes by mass spectrometry and complexome profiling (6). Several identified interaction partners were subsequently confirmed by bacterial two hybrid analysis. With this work we shed light on another part of the complex c-di-GMP signaling network of Pseudomonas aeruginosa.
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(6) Scherhag, A. et al. (2023) microLife. 4:uqad028.