A p-loop motif and two basic regions in the regulatory protein GvpD are important for the repression of gas vesicle formation in the archaeon Haloferax mediterranei

DeltaD transformants containing all 14 gvp genes of Haloferax mediterranei required for gas vesicle formation except for gvpD are gas vesicle overprod ucers (Vac(++)), whereas DeltaD/D transformants containing the gvpD reading frame under ferredoxin promoter control on a second construct in addition to DeltaD did not form gas vesicles (Vac(-)). The amino acid sequence of Gv pD indicates three interesting regions (a putative nucleotide-binding site called the p-loop motif, and two basic regions); these were altered by muta tion, and the resulting GvpD(mut) proteins tested in DeltaD/D-mut transform ants for their ability to repress gas vesicle formation. The exchange of am ino acids at conserved positions in the p-loop motif resulted in Vac(++) De ltaD/D-mut transformants, indicating that these GvpD(mut) proteins were una ble to repress gas vesicle formation. In contrast, a GvpD(mut) protein with an alteration of a non-conserved proline in the p-loop region (P41A) was s till able to repress. The repressing function of the various GvpD proteins was also investigated at the promoter level of the gvpA gene. This promoter is only activated during the stationary phase, depending on the transcript ional activator protein GvpE. Whereas the Vac(++) DeltaD transformants cont ained very high amounts of gvpA mRNA predominantly in the stationary growth phase, the amount of this transcript was significantly reduced in the Vac( -) transformants DeltaD/D and DeltaD/D-P41A. In contrast, the Vac(++) Delta D/D-mut transformants harbouring GvpD(mut) with mutations at conserved posi tions in the p-loop motif contained large amounts of gvpA mRNA already duri ng exponential growth, suggesting that this motif is important for the GvpD repressor function during this growth phase. The GvpD mutants containing m utations in the two basic regions were mostly defective in the repressing f unction. The GvpD(mut) protein containing an exchange of the three arginine residues 494RRR496 to alanine residues was able to repress gas vesicle for mation. No gvpA mRNA was detectable in this transformant, demonstrating tha t this GvpD protein was acting as a strong repressor. All these results imp ly that the GvpD protein is able to prevent the GvpE-mediated gvpA promoter activation, and that the p-loop motif as well as the two basic regions ave important for this function.