Recent Advances in the Study of Gas Vesicle Proteins and Application of Gas Vesicles in Biomedical Research

Abstract

The formation of gas vesicles has been investigated in bacteria and haloarchaea for more than 50 years. These air-filled nanostructures allow cells to stay at a certain height optimal for growth in their watery environment. Several gvp genes are involved and have been studied in Halobacterium salinarum, cyanobacteria, Bacillus megaterium, and Serratia sp. ATCC39006 in more detail. GvpA and GvpC form the gas vesicle shell, and additional Gvp are required as minor structural proteins, chaperones, an ATP-hydrolyzing enzyme, or as gene regulators. We analyzed the Gvp proteins of Hbt. salinarum with respect to their protein-protein interactions, and developed a model for the formation of these nanostructures. Gas vesicles are also used in biomedical research. Since they scatter waves and produce ultrasound contrast, they could serve as novel contrast agent for ultrasound or magnetic resonance imaging. Additionally, gas vesicles were engineered as acoustic biosensors to determine enzyme activities in cells. These applications are based on modifications of the surface protein GvpC that alter the mechanical properties of the gas vesicles. In addition, gas vesicles have been decorated with GvpC proteins fused to peptides of bacterial or viral pathogens and are used as tools for vaccine development.

Keywords: Halobacterium salinarum; acoustic biosensors; gas vesicles; protein nanostructures; vaccine development.

Figure 1

Inspection of Hbt. salinarum cells containing gas vesicles by transmission electron microscopy. Gas vesicles are seen as white bodies inside the cells. (A,B) Younger cells derived from a liquid culture grown to an optical density of 0.78. The red box in (B) places the enlarged image at the left side in the image on the right. (C) Old cells derived from a surface layer of a liquid culture left standing on the bench for five months (Faist and Pfeifer, TU Darmstadt).

Figure 2

Comparison of GvpA, GvpJ, and GvpM of Hbt. salinarum. (A) Alignment of the three aa sequences highlighting the conserved regions (marked by a bar on top). Polar aa are indicated in red (K,R) or purple (D,E) and nonpolar aa in blue. The putative secondary structure is marked by arrows (α-helices α1–α4, and β-sheets β1 and β2). (B) Comparison of the results on the different ∆X + X_mut transformants (X = A, J, or M) with respect to the Vac phenotype. Vac⁺ transformants are shaded in green (spindle-shaped wild type gas vesicles in light green, cylinder-shaped gas vesicles in dark green), Vac^± transformants in orange, and Vac⁻ transformants in red. Amino acid substitutions in GvpJ, leading to unstable gas vesicles, are marked in yellow. Residues not shaded are not explored. (*) and (:) below the alignment highlight conserved positions (after [61]).

Figure 3

Gas vesicles obtained in Hfx. volcanii ∆C and ∆C + C_mut transformants. (A) Genetic map of the p-vac region derived from Hbt. salinarum. Arrows indicate the direction of the transcription. The ∆C lacks the gvpC reading frame, and the C construct contains gvpC inserted in pJAS35 for the expression. (B) Different versions of GvpC lacking 3, 4, or 7 of the aa repeats labeled 1–7. The Nterm and Cterm fragments of GvpC used for protein–protein interaction studies are marked on top. (C) Transmission electron micrographs of Hfx. volcanii cells (upper lane) and isolated gas vesicles (lower lane) derived from the respective transformants (Faist and Pfeifer, TU Darmstadt).

Figure 4

Hfx. volcanii ∆C + C∆4–7 transformants inspected by transmission electron microscopy after 32 d of growth on solid media (Faist and Pfeifer, TU Darmstadt).

Figure 5

Model for the assembly of gas vesicles. The interacting GvpF–GvpA (F–A) and the complex formation of FGHIJKLM, as well as N–O, are shown. GvpC is presented as a rod able to form dimers or multimers by Nterm/Cterm and Cterm/Cterm interactions. The first gas-filled structure seen in cells by transmission electron microscopy is a small bicone that is enlarged to a spindle-shaped structure. The further addition of GvpA and GvpC yields the cylinder-shaped nanostructure; the formation of the cylinder shape depends on the presence of GvpC. The cryo-electron microscopy of Hbt. salinarum gas vesicles was performed by Daniel Bollschweiler and Harald Engelhardt, Max-Planck-Institute for Biochemistry, Martinsried, Germany.