Effect of an overproduction of accessory Gvp proteins on gas vesicle formation in Haloferax volcanii.

Gas vesicle formation in haloarchaea requires the expression of the p-vac region consisting of 14 genes, gvpACNO and gvpDEFGHIJKLM. Expression of gvpFGHIJKLM leads to essential accessory proteins formed in minor amounts. An overexpression of gvpG, gvpH or gvpM in addition to p-vac inhibited gas vesicle formation, whereas large amounts of all other Gvp proteins did not disturb the synthesis. The unbalanced expression and in particular an aggregation of the overproduced Gvp with other accessory Gvp derived from p-vac could be a reason for the inhibition. Western analyses demonstrated that the hydrophobic GvpM (and GvpJ) indeed form multimers. Fluorescent dots of GvpM-GFP were seen in cells in vivo underlining an aggregation of GvpM. In search for proteins neutralizing the inhibitory effect in case of GvpM, p-vac +pGM(ex), +pHM(ex), +pJM(ex), and +pLM(ex) transformants were constructed. The inhibitory effect of GvpM on gas vesicle formation was suppressed by GvpH, GvpJ or GvpL, but not by GvpG. Western analyses demonstrated that pHM(ex) and pJM(ex) transformants contained additional larger protein bands when probed with an antiserum raised against GvpH or GvpJ, implying interactions. The balanced amount of GvpM-GvpH and GvpM-GvpJ appears to be important during gas vesicle genesis.

Structural model of the gas vesicle protein GvpA and analysis of GvpA mutants in vivo.

Gas vesicles are gas-filled protein structures increasing the buoyancy of cells. The gas vesicle envelope is mainly constituted by the 8 kDa protein GvpA forming a wall with a water excluding inner surface. A structure of GvpA is not available; recent solid-state NMR results suggest a coil-α-ß-ß-α-coil fold. We obtained a first structural model of GvpA by high-performance de novo modelling. Attenuated total reflection (ATR)-Fourier transform infrared spectroscopy (FTIR) supported this structure. A dimer of GvpA was derived that could explain the formation of the protein monolayer in the gas vesicle wall. The hydrophobic inner surface is mainly constituted by anti-parallel ß-strands. The proposed structure allows the pinpointing of contact sites that were mutated and tested for the ability to form gas vesicles in haloarchaea. Mutations in α-helix I and α-helix II, but also in the ß-turn affected the gas vesicle formation, whereas other alterations had no effect. All mutants supported the structural features deduced from the model. The proposed GvpA dimers allow the formation of a monolayer protein wall, also consistent with protease treatments of isolated gas vesicles.