Structural and functional investigations of the Pseudomonas aeruginosa YfiBNR-system

Most bacteria can exist in two fundamentally different life styles, as motile single cells and as sessile, surface-grown communities called biofilms. A key factor triggering the formation and maintenance of biofilms in a multitude of bacterial species is the second messenger bis-(3'-5')cyclic dimeric guanosine (c-di-GMP). While low intracellular levels of c-di-GMP promote planktonic behavior, where cells are generally motile and express virulence factors, increasing concentrations of c-di-GMP promote the expression of adhesive matrix components and result in multicellular behavior, biofilm formation and persistence. The opportunistic pathogen Pseudomonas aeruginosa is responsible for chronic infections in the lungs of cystic fibrosis patients, a process that is eventually accompanied by the formation of small-colony variants (SCVs). The appearance of SCVs is caused by elevated levels of c-di-GMP and correlates with increased persistence of infection and antibiotic resistance. The YfiBNR-system has been previously identified as a key regulator of the SCV phenotype. While the mechanistic principles of interaction between the three proteins have been established by extensive in vivo studies, no structural information was available for any of the three proteins so far. Moreover, in vitro investigations of the effector of the system YfiN and its repressor YfiR were still missing. In this thesis, the structure of YfiR was solved by X-ray crystallography, which revealed a dimeric assembly of the protein. Moreover, YfiR was shown to adopt a novel fold. The core of the protein is made up by a seven-stranded mixed β-sheet, flanked on the convexed side by three helices, and embracing a long N-terminal α-helix with its concave side. The β-sheet topology is 2-3-1-4-5-6-7, where strand 2 and 7 are aligned in an antiparallel fashion in regards to the rest of the sheet. The protein is stabilized by two intramolecular disulfide bonds, mediated by Cys71-110 as well as Cys145-Cys152. SEC-MALLS analysis of wildtype YfiR and a dimeric-interface mutant demonstrated that the protein is dimerizing with a high affinity in solution, via the same interface observed in the crystal structure. In a next step, a functional assay was established using membrane-bound YfiN, which allowed the investigation of YfiN activity and regulation in vitro. Production of c-di-GMP was observed in a time-dependent fashion, indicating that YfiN showed diguanylate cyclase (DGC) activity. Further evidence was provided that YfiN activity is negatively regulated by YfiR and c-di-GMP, which implied that YfiN is undergoing allosteric feedback inhibition. Based on these results, a homology model of the inactive state of YfiN was generated, which gave insight into the presumable mode of feedback inhibition, involving c-di-GMP mediated cross-linking of the GGDEF and HAMP domains. It is therefore proposed that YfiN activity is regulated either by repression upon binding of dimeric YfiR or by non-competitive product inhibition to avoid excessive substrate consumption.

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