Introduction: Thermus thermophilus is a model organism to study the structure and function of natural transformation systems (1). Previously we identified a hexameric motor ATPase PilF powering the natural transformation machinery and type IV pili assembly in T. thermophilus. Structurally, PilF is particularly remarkable because its N-terminus harbours three successive GSPII domains. Our recent studies revealed that two of these domains bind the second messenger c-di-GMP albeit with significantly different affinities (2,3).
Goals: Next we aimed to elucidate the functional importance of c-di-GMP binding to PilF in vivo and in vitro.
Materials & Methods: The functional importance of c-di-GMP binding to PilF was analysed by site directed mutagenesis, physiological characterization of mutants and biochemical characterization of PilF variants.
Results: Site directed mutagenesis of PilF followed by NMR spectroscopy analysis in collaboration with Jens Wöhnert (BMRZ, Goethe University Frankfurt) unravelled four glutamine residues in PilF essential for c-di-GMP binding. A glutamine quadruple pilF mutant was significantly reduced in adhesion to solid surfaces and completely defect in movement on solid surfaces and natural transformation. Biochemical analyses of PilF variants provided insights into the role of c-di-GMP binding in PilF complex formation and ATPase activity.
Summary: Here we report on the role of c-di-GMP binding to the motor ATPase PilF in adhesion, motility on solid surfaces and natural transformation of T. thermophilus.
[1] Averhoff, B. (2009) FEMS Microbiol Rev 33:611-626
[2] Keller, H., Kruse, K., Averhoff, B., Duchardt-Ferner, E., Wöhnert, J. (2019) Biomol NMR Assign 13:361-366
[3] Neißner, K., Keller, H., Duchardt-Ferner, E., Hacker, C., Kruse, K., Averhoff, B., Wöhnert, J. (2019) Biomol NMR Assign 13:383–390