Structural And Functional Insights Into The Gas Vesicle Protein GvpF Of Microcystis Aeruginosa PCC7806

Microcystis aeruginosa was investigated and identified as a planktonic unicellular cyanobacterium which is responsible for seasonal mass occurrences at the surface of freshwater environments. For example, in Lake Taihu and Lake Chaohu in China, it is the most dominant algae species which can cause the breakout of blue-green algae water bloom. In Microcystis cells, an abundant production of intracellular structures, the gas vesicle, an inert, hollow and gas-filled structure, formed solely from proteins, provides cells with buoyancy by lowering the density of cells and regulates their vertical distribution in natural waters. Bacteria could float taxis toward air-liquid interfaces of water environments, enabling themselves to position under optimal light and oxygen conditions for growth and subsequent niche colonization. Therefore, it is important to understand the characteristics of gas vesicle for cyanobacteria water-bloom control.Gas vesicle formation involves8-14different gas vesicle proteins (Gvp), which are encoded by a gvp gene cluster that has been found in a variety of bacteria and archaea. It was identified that there is an8.7-kb gene cluster that comprises twelve genes involved in gas vesicle synthesis in M. aeruginosa PCC7806. We knew that ten of these are organized in two operons, gvpAⅠAⅡAⅢCNJX and gvpKFG, and the other two, gvpV and gvpW, are expressed individually. Gvp A and GvpC which were studied well form the main mass of the structure of the gas vesicle. The7-8kDa small hydrophobic protein, Gvp A, is the dominant structural component of the gas vesicle wall and forms the ribs of the main structure, and the larger, more hydrophilic protein, GvpC, located on the outer surface of gas vesicle walls, stabilizes the structure by interacting with GvpA. Apart from the gvpA and gvpC genes encoding the structural proteins, several other gvp genes and Gvp proteins may also involve in the gas vesicle formation in cyanobacteria.In this study, we solved the crystal structure of GvpF from M. aeruginosa PCC7806at2.7A resolution using the method of SAD (Single-wavelength Anomalous Diffraction), representing the first structure of Gvp proteins. GvpF is composed of two structurally distinct domains (namely N-and C-domain), both of which display an a+P class overall structure. The N-domain adopts a novel fold whereas the C-domain has a modified ferredoxin fold with an apparent variation due to an extension region consisting of three sequential helices. The two domains pack against each other via interactions with a C-terminal tail that is conserved among cyanobacteria. The outputs of DALI search performed using the overall structure of GvpF did not enable us to identify any proteins similar to GvpF. Taken together, we propose that the overall architecture of GvpF presents a novel fold.The purified gas vesicles with high purity isolated from M. aeruginosa PCC7806are intact as shown in electron micrographs. They also take a cylinder shape with conical end caps. The gas vesicles have a similar diameter of about120nm and the variable lengths from500to1500nm. The outer wall represents regularly spaced ribs running nearly perpendicular to the vesicle long axis. The major structural component GvpC of purified gas vesicles could be detected by SDS-PAGE and further confirmed by mass spectrometry. Moreover, we prove that GvpF is most likely a structural protein localized at the inner surface of the gas vesicle by immunoblotting and immunogold labeling-based tomography.In order to get structural and functional information of other Gvp proteins, we firstly expressed and purified all these Gvp proteins except for GvpA, which was expressed as inclusion body. Furthermore we obtained the crystals of GvpC and GvpW. But the crystals of GvpC diffracted poorly and it was difficult to repeat their growth process. The crystals of GvpW were diffracted at about10A. Crystal optimization was still in progress. We also proved that GvpN was an ATPase. We are doing our best to get the crystals of these Gvp proteins, and we also hope to explore and verify their function by analyzing solved crystal structures.