Effect of Mutations in GvpJ and GvpM on Gas Vesicle Formation of Halobacterium salinarum.

The two haloarchaeal proteins, GvpM and GvpJ, are homologous to GvpA, the major gas vesicle structural protein. All three are hydrophobic and essential for gas vesicle formation. The effect of mutations in GvpJ and GvpM was studied in Haloferax volcanii transformants by complementing the respective mutated gene with the remaining gvp genes and inspecting the cells for the presence of gas vesicles (Vac+). In case of GvpJ, 56 of 66 substitutions analyzed yielded Vac- ΔJ + Jmut transformants, indicating that GvpJ is very sensitive to alterations, whereas ten of the 38 GvpM variants resulted in Vac- ΔM + Mmut transformants. The variants were also tested by split-GFP for their ability to interact with their partner protein GvpL. Some of the alterations leading to a Vac- phenotype affected the J/L or M/L interaction. Also, the interactions J/A and J/M were studied using fragments to exclude an unspecific aggregation of these hydrophobic proteins. Both fragments of GvpJ interacted with the M1-25 and M60-84 fragments of GvpM, and fragment J1-56 of GvpJ interacted with the N-terminal fragment A1-22 of GvpA. A comparison of the results on the three homologous proteins indicates that despite their relatedness, GvpA, GvpJ, and GvpM have unique features and cannot substitute each other.

Mutations in the major gas vesicle protein GvpA and impacts on gas vesicle formation in Haloferax volcanii.

Gas vesicles are proteinaceous, gas-filled nanostructures produced by some bacteria and archaea. The hydrophobic major structural protein GvpA forms the ribbed gas vesicle wall. An in-silico 3D-model of GvpA of the predicted coil-α1-ß1-ß2-α2-coil structure is available and implies that the two ß-chains constitute the hydrophobic interior surface of the gas vesicle wall. To test the importance of individual amino acids in GvpA we performed 85 single substitutions and analyzed these variants in Haloferax volcanii ΔA + Amut transformants for their ability to form gas vesicles (Vac+ phenotype). In most cases, an alanine substitution of a non-polar residue did not abolish gas vesicle formation, but the replacement of single non-polar by charged residues in ß1 or ß2 resulted in Vac- transformants. A replacement of residues near the ß-turn altered the spindle-shape to a cylindrical morphology of the gas vesicles. Vac- transformants were also obtained with alanine substitutions of charged residues of helix α1 suggesting that these amino acids form salt-bridges with another GvpA monomer. In helix α2, only the alanine substitution of His53 or Tyr54, led to Vac- transformants, whereas most other substitutions had no effect. We discuss our results in respect to the GvpA structure and data available from solid-state NMR.