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-β-β-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 β-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 β-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.     

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‐β1‐β2‐α2‐coil structure is available and implies that the two β‐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 + A_mut 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 β1 or β2 resulted in Vac^– transformants. A replacement of residues near the β‐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.     

The homologies of gas vesicle proteins.

In addition to GvpA, the main structural protein, an SDS-soluble protein has been found in gas vesicles isolated from six different genera of cyanobacteria. N-terminal sequence analysis of the first 30 to 60 residues of the gel-purified proteins showed that they were homologous to GvpC, a protein that strengthens the gas vesicle in Anabaena flos-aquae. The proteins from some of the organisms showed rather low homology, however, and this may explain why the genes that encode them have not been found by Southern hybridization studies. The gas vesicles of another cyanobacterium, Dactylococcopsis salina, contained two SDS-soluble proteins (M(r) 17,000 and 35,000) that were identical in sequence for the first 24 residues but not thereafter; these two proteins showed no clear homology to GvpC. The sequence of GvpA, the main structural gas vesicle protein, was very similar in each of the organisms investigated. GvpA from the purple bacterium Amoebobacter pendens was different for the first 8 residues but 51 of the next 56 residues were identical to those of the cyanobacterial GvpA. Analysis of the GvpA and GvpC sequences provides support for the idea that the low diversity of GvpA reflects a high degree of conservation rather than a recent origin followed by lateral gene transfer between different bacteria.     

Complexity of gas vesicle biogenesis in Halobacterium sp. strain NRC-1: identification of five new proteins

The genome of Halobacterium sp. strain NRC-1 contains a large gene cluster, gvpMLKJIHGFEDACNO, that is both necessary and sufficient for the production of buoyant gas-filled vesicles. Due to the resistance of gas vesicles to solubilization, only the major gas vesicle protein GvpA and a single minor protein, GvpC, were previously detected. Here, we used immunoblotting analysis to probe for the presence of gas vesicle proteins corresponding to five additional gvp gene products. Polyclonal antisera were raised in rabbits against LacZ-GvpF, -GvpJ, and -GvpM fusion proteins and against synthetic 15-amino-acid peptides from GvpG and -L. Immunoblotting analysis was performed on cell lysates of wild-type Halobacterium sp. strain NRC-1, gas vesicle-deficient mutants, and purified gas vesicles, after purification of LacZ fusion antibodies on protein A and beta-galactosidase affinity columns. Our results show the presence of five new gas vesicle proteins (GvpF, GvpG, GvpJ, GvpL, and GvpM), bringing the total number of proteins identified in the organelles to seven. Two of the new gas vesicle proteins are similar to GvpA (GvpJ and GvpM), and two proteins contain predicted coiled-coil domains (GvpF and GvpL). GvpL exhibited a multiplet ladder on sodium dodecyl sulfate-polyacrylamide gels indicative of oligomerization and self-assembly. We discuss the possible functions of the newly discovered gas vesicle proteins in biogenesis of these unique prokaryotic flotation organelles.     

Functional analysis of the gas vesicle gene cluster of the halophilic archaeon Haloferax mediterranei defines the vac-region boundary and suggests a regulatory role for the gvpD gene or its product

A series of deletions introduced into the gvp gene cluster of Haloferax mediterranei, comprising 14 genes involved in gas vesicle synthesis (mc-vac-region), was investigated by transformation experiments. Gas vesicle production and the expression of the gvpA gene encoding the major gas vesicle protein, GvpA, was monitored in each Haloferax volcanii transformant. Whereas transformants containing the entire mc-vac-region produced gas vesicles (Vac+), various deletions in the region 5' to gvpA (encompassing gvpD-gvpM) or 3' to gvpA (containing gvpC, gvpN and gvpO) revealed Vac- transformants. All these transformants expressed gvpA and contained the 8 kDa GvpA protein as shown by Western analysis. However, transformants containing the gvpA gene by itself indicated a lower level of GvpA than observed with each of the other transformants. None of these transformants containing deletion constructs assembled the GvpA protein into gas vesicles. In contrast, transformants containing a construct carrying a 918 bp deletion internal to gvpD exhibited a tremendous gas vesicle overproduction, suggesting a regulatory role for the gvpD gene or its product. This is the first assignment of a functional role for one of the 13 halobacterial gvp genes found in addition to gvpA that are involved in the synthesis of this unique structure.     

The accessory gas vesicle protein GvpM of haloarchaea and its interaction partners during gas vesicle formation

Gas vesicles consist predominantly of the hydrophobic GvpA and GvpC, and the accessory proteins GvpF through GvpM are required in minor amounts during formation. GvpM and its putative interaction partners were investigated. GvpM interacted with GvpH, GvpJ and GvpL, but not with GvpG. Interactions were also observed in vivo in Haloferax volcanii transformants using Gvp fusions to the green fluorescent protein smGFP. Cells producing the hydrophobic M_GFP contained a single fluorescent aggregate per cell, whereas cells containing L_GFP or H_GFP were fully fluorescent. The soluble L_GFP formed stable co-aggregates with GvpM in L_GFPM transformants, but the presence of GvpH resulted in the absence of M_GFP foci in HM_GFP transformants. Substitution- and deletion mutants of GvpM determined functionally important amino acids (aa). Substitution of a polar by a non-polar aa in the N-terminal region of GvpM had no effect, whereas a substitution of a non-polar by a polar aa in this region inhibited gas vesicle formation in transformants. Substitutions in region 44–48 of GvpM strongly reduced the number of gas vesicles, and deletions at the N-terminus resulted in Vac^? transformants. Gas vesicle morphology was not affected by any mutation, implying that GvpM is required during initial stages of gas vesicle assembly.     

The sequence of the major gas vesicle protein,GvpA, influences the width and strength of halobacterial gas vesicles

Transformation experiments with Haloferax volcanii show that the amino acid sequence of the gas vesicle protein GvpA influences the morphology and strength of gas vesicles produced by halophilic archaea. A modified expression vector containing p-gvpA was used to complement a Vac(-) strain of Hfx. volcanii that harboured the entire p-vac region (from Halobacterium salinarum PHH1) except for p-gvpA. Replacement of p-gvpA with mc-gvpA (from Haloferax mediterranei) led to the synthesis of gas vesicles that were narrower and stronger. Other gene replacements (using c-gvpA from Hbt. salinarum or mutated p-gvpA sequences) led to a significant but smaller increase in gas vesicle strength, and less marked effects on gas vesicle morphology.     

Solid-State NMR Characterization of Gas Vesicle Structure

Gas vesicles are gas-filled buoyancy organelles with walls that consist almost exclusively of gas vesicle protein A (GvpA). Intact, collapsed gas vesicles from the cyanobacterium Anabaena flos-aquae were studied by solid-state NMR spectroscopy, and most of the GvpA sequence was assigned. Chemical shift analysis indicates a coil-α-β-β-α-coil peptide backbone, consistent with secondary-structure-prediction algorithms, and complementary information about mobility and solvent exposure yields a picture of the overall topology of the vesicle subunit that is consistent with its role in stabilizing an air-water interface.     

An amyloid organelle, solid-state NMR evidence for cross-β assembly of gas vesicles

Functional amyloids have been identified in a wide range of organisms, taking on a variety of biological roles and being controlled by remarkable mechanisms of directed assembly. Here, we report that amyloid fibrils constitute the ribs of the buoyancy organelles of Anabaena flos-aquae. The walls of these gas-filled vesicles are known to comprise a single protein, GvpA, arranged in a low pitch helix. However, the tertiary and quaternary structures have been elusive. Using solid-state NMR correlation spectroscopy we find detailed evidence for an extended cross-β structure. This amyloid assembly helps to account for the strength and amphiphilic properties of the vesicle wall. Buoyancy organelles thus dramatically extend the scope of known functional amyloids.     

Direct observation of protein secondary structure in gas vesicles by atomic force microscopy.

The protein that forms the gas vesicle in the cyanobacterium Anabaena flos-aquae has been imaged by atomic force microscopy (AFM) under liquid at room temperature. The protein constitutes "ribs" which, stacked together, form the hollow cylindrical tube and conical end caps of the gas vesicle. By operating the microscope in deflection mode, it has been possible to achieve sub-nanometer resolution of the rib structure. The lateral spacing of the ribs was found to be 4.6 +/- 0.1 nm. At higher resolution the ribs are observed to consist of pairs of lines at an angle of approximately 55 degrees to the rib axis, with a repeat distance between each line of 0.57 +/- 0.05 nm along the rib axis. These observed dimensions and periodicities are consistent with those determined from previous x-ray diffraction studies, indicating that the protein is arranged in beta-chains crossing the rib at an angle of 55 degrees to the rib axis. The AFM results confirm the x-ray data and represent the first direct images of a beta-sheet protein secondary structure using this technique. The orientation of the GvpA protein component of the structure and the extent of this protein across the ribs have been established for the first time.     

Gas vesicles.

The gas vesicle is a hollow structure made of protein. It usually has the form of a cylindrical tube closed by conical end caps. Gas vesicles occur in five phyla of the Bacteria and two groups of the Archaea, but they are mostly restricted to planktonic microorganisms, in which they provide buoyancy. By regulating their relative gas vesicle content aquatic microbes are able to perform vertical migrations. In slowly growing organisms such movements are made more efficiently than by swimming with flagella. The gas vesicle is impermeable to liquid water, but it is highly permeable to gases and is normally filled with air. It is a rigid structure of low compressibility, but it collapses flat under a certain critical pressure and buoyancy is then lost. Gas vesicles in different organisms vary in width, from 45 to > 200 nm; in accordance with engineering principles the narrower ones are stronger (have higher critical pressures) than wide ones, but they contain less gas space per wall volume and are therefore less efficient at providing buoyancy. A survey of gas-vacuolate cyanobacteria reveals that there has been natural selection for gas vesicles of the maximum width permitted by the pressure encountered in the natural environment, which is mainly determined by cell turgor pressure and water depth. Gas vesicle width is genetically determined, perhaps through the amino acid sequence of one of the constituent proteins. Up to 14 genes have been implicated in gas vesicle production, but so far the products of only two have been shown to be present in the gas vesicle: GvpA makes the ribs that form the structure, and GvpC binds to the outside of the ribs and stiffens the structure against collapse. The evolution of the gas vesicle is discussed in relation to the homologies of these proteins.     

Gas vesicles.

The gas vesicle is a hollow structure made of protein. It usually has the form of a cylindrical tube closed by conical end caps. Gas vesicles occur in five phyla of the Bacteria and two groups of the Archaea, but they are mostly restricted to planktonic microorganisms, in which they provide buoyancy. By regulating their relative gas vesicle content aquatic microbes are able to perform vertical migrations. In slowly growing organisms such movements are made more efficiently than by swimming with flagella. The gas vesicle is impermeable to liquid water, but it is highly permeable to gases and is normally filled with air. It is a rigid structure of low compressibility, but it collapses flat under a certain critical pressure and buoyancy is then lost. Gas vesicles in different organisms vary in width, from 45 to > 200 nm; in accordance with engineering principles the narrower ones are stronger (have higher critical pressures) than wide ones, but they contain less gas space per wall volume and are therefore less efficient at providing buoyancy. A survey of gas-vacuolate cyanobacteria reveals that there has been natural selection for gas vesicles of the maximum width permitted by the pressure encountered in the natural environment, which is mainly determined by cell turgor pressure and water depth. Gas vesicle width is genetically determined, perhaps through the amino acid sequence of one of the constituent proteins. Up to 14 genes have been implicated in gas vesicle production, but so far the products of only two have been shown to be present in the gas vesicle: GvpA makes the ribs that form the structure, and GvpC binds to the outside of the ribs and stiffens the structure against collapse. The evolution of the gas vesicle is discussed in relation to the homologies of these proteins.     

The gas vesicle gene cluster from Microcystis aeruginosa and DNA rearrangements that lead to loss of cell buoyancy

Microcystis aeruginosa is a planktonic unicellular cyanobacterium often responsible for seasonal mass occurrences at the surface of freshwater environments. An abundant production of intracellular structures, the gas vesicles, provides cells with buoyancy. A 8.7-kb gene cluster that comprises twelve genes involved in gas vesicle synthesis was identified. Ten of these are organized in two operons, gvpA(I)A(II)A(III)CNJX and gvpKFG, and two, gvpV and gvpW, are individually expressed. In an attempt to elucidate the basis for the frequent occurrence of nonbuoyant mutants in laboratory cultures, four gas vesicle-deficient mutants from two strains of M. aeruginosa, PCC 7806 and PCC 9354, were isolated and characterized. Their molecular analysis unveiled DNA rearrangements due to four different insertion elements that interrupted gvpN, gvpV, or gvpW or led to the deletion of the gvpA(I)-A(III) region. While gvpA, encoding the major gas vesicle structural protein, was expressed in the gvpN, gvpV, and gvpW mutants, immunodetection revealed no corresponding GvpA protein. Moreover, the absence of a gas vesicle structure was confirmed by electron microscopy. This study brings out clues concerning the process driving loss of buoyancy in M. aeruginosa and reveals the requirement for gas vesicle synthesis of two newly described genes, gvpV and gvpW.     

New structural proteins of Halobacterium salinarum gas vesicle revealed by comparative proteomics analysis

The Halobacterium salinarum gas vesicle (GV) is an extremely stable intracellular organelle with air trapped inside a proteinaceous membrane. Reported here is a comparative proteomics analysis of GV and GV depleted lysate (GVD) to reveal the membrane structural proteins. Ten proteins encoded by gvp-1 (gvpMLKJIHGFED-1 and gvpACNO-1) and five proteins encoded by gvp-2 (gvpMLKJIHGFED-2 and gvpACNO-2) gene clusters for the biogenesis of spindle- and cylindrical-, respectively, shaped GV were identified by LC-MS/MS. The peptides of GvpA1, I1, J1, A2, and J2 were exclusively identified in purified GV, GvpD1, H1, L1, and F2 only in GVD, and GvpC1, N1, O1, F1, H2, and O2 in both samples. The identification of GvpA1, C1, F1, J1, and A2 in GV is in agreement with their previously known structural function. In addition, the detection of GvpI1, N1, O1, H2, J2, and O2 in GV suggested they are new structural proteins. Among these, the structural role of GvpI1 and N1 in GV was further validated by immuno-detection of protein A-tagged GvpI1 and N1 fusion proteins in purified GV. Thus, LC-MS/MS could reveal at least a half dozen gas vesicle structural proteins in the predominant spindle-shaped GV that may be helpful for studying its biogenesis.     

蓝藻伪空胞的特性及浮力调节机制

伪空胞为蓝藻在水体中提供浮力,使其获得适宜的生长条件,最终导致蓝藻水华暴发,了解伪空胞的特征对控制蓝藻水华暴发有重要意义。文章简要回顾了蓝藻伪空胞自1865年被Klebahn发现到1965年被正式命名的研究历程,目前已发现150多种原核生物中含有伪空胞;伪空胞是两末端呈圆锥状的中空圆柱体,伪空胞半径与临界压强遵循方程:Pc=275(r/nm)-1.67MPa;伪空胞气体含量可根据不同原理,利用Walsby伪空胞测定装置、压力浊度计和细胞流式仪测得。总结了伪空胞组成的化学特性,评述了伪空胞gvp基因丛结构功能和GvpA、GvpC的蛋白空间结构。GvpA是伪空胞合成的主要成分,gvpA在伪空胞内存在多个拷贝,其功能仍不清楚;GvpC由33个氨基酸重复单位组成,重复单位越多,伪空胞越不易破裂;概述了伪空胞3种浮力调节机制:镇重物的改变、伪空胞的合成、伪空胞的破裂;归纳了环境因子(光照、温度、氮、磷、钾)参与伪空胞浮力网络调控的途径。提出了目前伪空胞研究面临的困难和问题,对伪空胞的未来研究方向提出探索性的建议。     

Distribution, formation and regulation of gas vesicles

A range of bacteria and archaea produce intracellular gas-filled proteinaceous structures that function as flotation devices in order to maintain a suitable depth in the aqueous environment. The wall of these gas vesicles is freely permeable to gas molecules and is composed of a small hydrophobic protein, GvpA, which forms a single-layer wall. In addition, several minor structural, accessory or regulatory proteins are required for gas vesicle formation. In different organisms, 8-14 genes encoding gas vesicle proteins have been identified, and their expression has been shown to be regulated by environmental factors. In this Review, I describe the basic properties of gas vesicles, the genes that encode them and how their production is regulated. I also discuss the function of these vesicles and the initial attempts to exploit them for biotechnological purposes.     

Absence of gas vesicle protein in a mutant of Anabaena flos-aquae

Filaments without gas vacuoles arose spontaneously in the gas-vacuolate alga Anabaena flos-aquae. The non-vacuolate mutant was enriched by repeated sedimentation and subsequently cloned by microsyringe transfer. No revertants have been observed. In the gas-vacuolate wild-type alga the gas vesicle protein was clearly distinguished by gel electrophoresis as one of the ten most abundant protein species present in whole cell extracts. Electrophoresis indicated that the mutant had lost the ability to synthesize the gas vesicle protein. A second mutant partially defective in production of gas vacuoles and gas vesicle protein has been isolated.Abbreviations gv gas vesicle protein - pb phycobilin - TCA trichloracetic acid