Anna Scherhag

• Membrane proteins exert a multitude of essential functions in the cell, that frequently require interaction partners. In particular, for signalling with the second messenger c-di-GMP, protein-protein interactions are considered crucial. Also, in P. aeruginosa - a model organism for biofilm formation and infection - the majority of c-di-GMP synthesizing or degrading enzymes are membrane proteins. In the first part of this thesis, membrane proteins in P. aeruginosa PAO1 were investigated in a global approach. A native membrane-protein library was created through the formation of native nanodiscs by using amphiphilic polymers. The library was characterized by testing for reproducible and stable extraction of membrane proteins, coverage of the membrane proteome, and the capability to detect protein–protein interactions within a lipid-bilayer environment as well as membrane-bound protein complexes. In addition, membrane-associated proteins involved in c-di-GMP signalling and their interaction partners were investigated in more detail. To this end, a set of more than 20 described protein complexes was used to validate global analyses for the prediction of novel protein-protein interactions. Although elution profiles indicated preservation of the membrane complexes, the dataset was not feasible for protein interaction prediction. In the second part of the thesis, the membrane-integrated phosphodiesterase NbdA was investigated for its role in the c-di-GMP network. The current model for c-di-GMP signalling suggests the organisation of local signalling supermodules alongside global factors for specific functions in the cell. Thus, the protein NbdA was tested for protein-protein interaction partners in P. aeruginosa, either by a pulldown assay following mass spectrometry for novel candidate identification, or by bacterial adenylate cyclase two hybrid (B2H) assay for candidate verification. Out of more than 400 novel candidate proteins, five candidates were selected for further validation. The interaction with four proteins, PilB, SadC, CzcR and PA4200 was confirmed in a B2H assay. Moreover, the influence of NbdA on the global c-di-GMP level was tested. Therefore, either a markerless nbdA deletion mutant or overexpression strains were tested. To find out more about the role of the individual domains of NbdA, also global c-di-GMP levels of protein variant overproduction mutants was assayed. Although overexpression of full-length nbdA resulted in reduced global c-di-GMP levels indicating a PDE activity in vivo, the overproduction of truncated protein variants of NbdA resulted in unexpected c-di-GMP levels. In previous studies a possible role of the MHYT domain in cell elongation as well as growth on solid media was observed. To further test for the specificity of these observations, other MHYT domain containing proteins were tested for similar phenotypes in P. aeruginosa. Indeed, the phenotypes were specific for the MHYT domain of NbdA. Previous studies suggested an influence of NbdA overexpression on type IV pili (T4P), thus pili formation was tested in a phage assay and by atomic force microscopy (AFM). Further, the co-localisation of NbdA with pili components was tested by confocal laser scanning microscopy (CLSM) to determine whether NbdA is located at the piliated pole. The findings on NbdA support the model of complex but highly specific c-di-GMP signalling in P. aeruginosa.