Exploring Innovative Approaches to Isolate a One-Component c-di-GMP Transducer: A Pilot Study.

Environmental nutrients control bacterial biofilm homeostasis, by regulating the intracellular levels of c-di-GMP. One component transducers can sense different classes of small molecules through a periplasmic domain; the nutrient recognition triggers the subsequent regulation of the downstream cytosolic diguanylate cyclase (GGDEF) or phosphodiesterase (EAL) domains, via transmembrane helix(ces), to finally change c-di-GMP levels.Protein studies on such transducers have been mainly carried out on isolated domains due to the presence of the transmembrane portion. Nevertheless, the cleavage of GGDEF and EAL-containing proteins could be detrimental since both tertiary and quaternary structures could be allosterically controlled; to by-pass this limitation, studies on the corresponding full-length proteins are highly desired.We have in silico selected a GGDEF-EAL transducer from Dyella thiooxydans (ann. A0A160N0B7), whose periplasmic binding domain was predicted to bind to arginine, a nutrient often associated with chronic infections and biofilm. This protein has been used as an in vitro tool for the identification of the best approach for its isolation, including (i) protein engineering to produce a water-soluble version via QTY (Glutamine, Threonine, and Tyrosine) code or (ii) nanodiscs assembly. The results on this "prototype" may represent the proof-of-concept for future isolation of other transmembrane proteins sharing the same architecture, including more complex nutrient-based transducers controlling c-di-GMP levels.