GvpD-induced breakdown of the transcriptional activator GvpE of halophilic archaea requires a functional p-loop and an arginine-rich region of GvpD

The two proteins involved in the regulation of gas vesicle formation in Haloferax mediterranei, mcGvpE (activator) and mcGvpD (repressive function), are able to interact in vitro. It was also found that the respective proteins cGvpE and cGvpD of Halobacterium salinarum and the heterologous pairs mcGvpD-cGvpE and cGvpD-mcGvpE were able to interact. Previously constructed mcGvpD mutants with alterations in regions affecting the repressive function of GvpD (p-loop motif or the two arginine-rich regions bR1 and bR2) were tested for their ability to interact with GvpE, and all still bound GvpE. Even a deletion of or near the p-loop motif in GvpD did not affect this ability to interact. Further deletion variants lacking larger N- or C-terminal portions of mcGvpD yielded that neither the N-terminal region with the p-loop motif nor the C-terminal portion were important for the binding of GvpE, and suggested that the central portion is involved in GvpE binding. The GvpD protein also induces a reduction in the amount of GvpE in Haloferax volcanii transformants expressing both genes under fdx promoter control on a single plasmid. Such DE(ex) transformants contain GvpD, but no detectable GvpE, whereas large amounts of GvpE are found in DeltaDE(ex) transformants that have incurred a deletion within the gvpD gene. A similar reduction was observed in D(ex)+E(ex) transformants harbouring both reading frames under fdx promoter control on two different plasmids. GvpD wild-type and also GvpD mutants were tested, and a significant reduction in the amount of GvpE was obtained in the case of GvpD wild-type and the super-repressor mutant GvpD(3-AAA). In contrast, transformants harbouring GvpD mutants with alterations in the p-loop motif or the bR1 region still contained GvpE. Since the amount of gvpE transcript was not reduced, the reduction occurred at the protein level. These results underlined that a functional p-loop and the arginine-rich region bR1 of GvpD were required for the GvpD-mediated reduction in the amount of GvpE.