Elucidating the assembly of gas vesicles by systematic protein-protein interaction analysis

Gas vesicles (GVs) are gas-filled microbial organelles formed by unique 3-nm thick, amphipathic, force-bearing protein shells, which can withstand multiple atmospheric pressures and maintain a physically stable air bubble with megapascal surface tension. However, the molecular process of GV assembly remains elusive. To begin understanding this process, we have devised a high-throughput in vivo assay to determine the interactions of all 11 proteins in the pNL29 GV operon. Complete or partial deletions of the operon establish interdependent relationships among GV proteins during assembly. We also examine the tolerance of the GV assembly process to protein mutations and the cellular burdens caused by GV proteins. Clusters of GV protein interactions are revealed, proposing plausible protein complexes that are important for GV assembly. We anticipate our findings will set the stage for designing GVs that efficiently assemble in heterologous hosts during biomedical applications. Growing interest in biomedical applications for gas vesicles (GVs) demands an understanding of their structure and assembly, but little is known to date on the roles of individual gas vesicle proteins (Gvps) as assembly factors. This study utilizes modular, parallelized protein-protein interaction screening to elucidate key interactions and interdependencies of Gvps during GV assembly.Out of 66 possible Gvp-to-Gvp interactions, 28 formed a dense protein-protein interaction network.Selective inclusion of other Gvps led to gain or loss of protein-protein interactions, revealing interdependence during GV assembly.Specific subnetworks of Gvps are revealed that may be linked to different stages of GV assembly.A number of Gvps show tolerance for fusion proteins during GV assembly, unveiling a degree of plasticity with implications for GV engineering. Iburg at al develop a high-throughput in vivo screen to construct a comprehensive map of the interactions among all 11 proteins of the pNL29 gas vesicle operon.