1887

Abstract

ΔD transformants containing all 14 genes of required for gas vesicle formation except for are gas vesicle overproducers (Vac), whereas ΔD/D transformants containing the reading frame under ferredoxin promoter control on a second construct in addition to ΔD did not form gas vesicles (Vac). The amino acid sequence of GvpD indicates three interesting regions (a putative nucleotide-binding site called the p-loop motif, and two basic regions); these were altered by mutation, and the resulting GvpD proteins tested in ΔD/D transformants for their ability to repress gas vesicle formation. The exchange of amino acids at conserved positions in the p-loop motif resulted in Vac ΔD/D transformants, indicating that these GvpD proteins were unable to repress gas vesicle formation. In contrast, a GvpD protein with an alteration of a non-conserved proline in the p-loop region (P41A) was still able to repress. The repressing function of the various GvpD proteins was also investigated at the promoter level of the gene. This promoter is only activated during the stationary phase, depending on the transcriptional activator protein GvpE. Whereas the Vac ΔD transformants contained very high amounts of mRNA predominantly in the stationary growth phase, the amount of this transcript was significantly reduced in the Vac transformants ΔD/D and ΔD/D. In contrast, the Vac ΔD/D transformants harbouring GvpD with mutations at conserved positions in the p-loop motif contained large amounts of mRNA already during exponential growth, suggesting that this motif is important for the GvpD repressor function during this growth phase. The GvpD mutants containing mutations in the two basic regions were mostly defective in the repressing function. The GvpD protein containing an exchange of the three arginine residues 494RRR496 to alanine residues was able to repress gas vesicle formation. No mRNA was detectable in this transformant, demonstrating that this GvpD protein was acting as a strong repressor. All these results imply that the GvpD protein is able to prevent the GvpE-mediated promoter activation, and that the p-loop motif as well as the two basic regions are important for this function.

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2001-01-01
2020-01-18
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