Glutaraldehyde treatment of proteinaceous gas vesicles from cyanobacterium Anabaena flos-aquae

As potential gas microcarriers, gas vesicles (GVs) were isolated from cultures of the filamentous cyanobacterium Anabaena flos-aquae and treated with glutaraldehyde. The effects of glutaraldehyde treatment on the stability of GVs, against elevated temperatures (40-121 degrees C) and protein-stripping agents such as urea and sodium dodecyl sulfate (SDS), were then examined with the pressure collapse curves generated using pressure nephelometry. The treatment was very beneficial to GVs against the exposure to SDS and urea; however, it did not make the evolution-optimized vesicle structure stronger or more temperature-resistant. In the presence of these protein-stripping agents, the treated vesicles had higher median (50%) collapse pressures (by > or =1 atm) than the untreated ones, at both room temperature and 40 degrees C. This increase has been presumably attributed to the cross-linking of the large GvpC protein to the ribbed GvpA shell, thereby resisting the stripping of GvpC that provides the primary mechanical strength to the vesicle wall. The glutaraldehyde treatment also restored the strength of GVs weakened by a 5-week storage in a refrigerator and, therefore, is expected to improve the stability of GVs for long-term storage. GVs could not be autoclaved. If necessary for the intended applications, glutaraldehyde treatment may also serve to chemically sterilize the vesicles, with the glutaraldehyde subsequently removed by dialysis.     

Chemical composition of gas vesicles isolated from Anabaena flos-aquae

Summary Chemical analysis of purified preparations of gas vesicles isolated from the filamentous blue-green alga Anabaena flos-aquae has shown that they are similar in composition to those isolated from the unicellular alga Microcystis aeruginosa by Jones and Jost (1970), being proteinaceous structures, free of lipid and carbohydrate. The gas vesicle protein from Anabaena contains the same 14 amino acids, in broadly the same proportions; in addition there is a small proportion of proline. No sulphur-containing amino acids are present. The empirical formula, suggested by the amino acid ratios, indicates a molecule of 15000 MW.     

Flotation characteristics of cyanobacterium Anabaena flos-aquae for gas vesicle production

Cyanobacterium Anabaena flos-aquae was cultivated in photobioreactors for production of intracellular gas vesicles (GVs), as potential oxygen microcarriers. Natural flotation of the buoyant culture was investigated as a potential means of cell harvesting, because filtration and centrifugation tended to destroy the vesicles. Best flotation was found with actively growing culture and when conducted in the dark. The flotation-related cell properties, including the specific GV content, vesicle-collapsed filament density, and intracellular carbohydrate content, were measured to understand the phenomena. During the batch culture, the specific GV content remained relatively constant at 370 microL/(g dry cells) but the filament density (ranging 1.02 to 1.08 g/cm3) showed a decrease-then-increase profile. The increase began when the growth slowed down because of the reduced light availability at high cell concentrations. The dark flotation was studied with both actively growing (mu approximately 0.2 day-1) and stationary-phase cultures. The specific GV content of the stationary-phase culture remained relatively constant while that of the growing culture increased slightly. The intracellular carbohydrate content of the growing culture decreased much faster and more significantly, from 57 to 10 mg/(g dry cells) in 相似文献   

Structure of Anabaena flos-aquae gas vesicles revealed by cryo-ET

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Elemental composition of the cyanobacterium Anabaena flos-aquae collected from different depths within a stratified lake

The elemental composition of cells of Anabaena flos-aquae was investigated by energy-dispersive electron probe X-ray microanalysis (10?kV) of mixed phytoplankton samples obtained from different depths within a stratified eutrophic freshwater lake. Preliminary studies had indicated that at 10?kV, X-ray information was derived mainly from the peripheral region (up to 1–2?μm) of cells. Routinely detectable elements (present in at least 50% of cells) included Mg (overall mean 112?mmol?kg^?1 dry mass), Si (1850), P(313), S (113), Cl (92), K (300) and Ca (56). Peaks of Na and Al were also frequently present. Individual elements showed a wide range of intracellular concentrations at each depth within the lake. Si had a clear bimodal distribution at depths of 1–8?m, indicating both low-Si (mean concentrations 54–290–3800?mmol?Si?kg^?1) and high-Si (2770–3800?mmol?Si?kg^?1) cell subpopulations. Low-Si cells had significantly higher concentrations of other detectable elements compared with high-Si cells, but could not be distinguished from the latter in terms of morphology or stage of cell cycle (comparison of dividing and non-dividing cells). High-Si cells were intermixed with low-Si cells in individual Anabaena colonies and decreased proportionally with depth, ranging from approximately 75% in the surface sample to 10% at 8?m. With the exception of Si, mean elemental concentrations of Anabaena populations were closely similar throughout the epilimnion. These differed from the metalimnion sample, which had significantly higher concentrations of Mg, P, S and K, and a lower concentration of Si. Mean ratios of certain elements (Mg, P, S and K) and ion groups (monovalent/divalent cations) were highly constant throughout the sampled water column. At each depth, most of the cell elements were significantly and positively inter-correlated (Pearson analysis), with Mg, P, S and K typically constituting the major group (factor analysis). Correlations with Si were invariably negative, suggesting a separate (possibly cell wall) location of this element in the cell.     

Crystalline structure of the gas vesicle wall from Anabaena flos-aquae.

The gas vesicles isolated from Anabaena flos-aquae have been studied by X-ray diffraction. Electron microscopy has previously shown that the gas vesicles are elongated shapes, with a thin wall having regular striations (ribs) at right-angles to the long axis. The X-ray diffraction pattern from a specimen of oriented, intact vesicles includes a number of sharp reflections which are attributed to regular structure in the plane of the wall. After correcting for the imperfect alignment of the long axes of the vesicles, the in-plane reflections are all seen to lie on a few, regularly spaced lines parallel to the long axis. This result shows for the first time that there are subunits regularly spaced along each rib, one subunit every 11 Å. The spacing of the in-plane reflections along each line is consistent with a rib periodicity of 46 Å. The 11 Å repeat, together with the 46 Å repeating distance from rib to rib and the average wall thickness of about 20 Å, define a volume for the subunit. Assuming a reasonable value for the density of the protein making up the wall, the molecular weight of the subunit indicated is about 8000 g/mol.The X-ray data also indicate that a large part of the protein is in the β-sheet conformation. In this structure there are parallel, or anti-parallel, polypeptide chains which are hydrogen-bonded to one another in a regular way to form a thin sheet. Assuming the wall contains β-sheet in two layers, one on top of the other and with the chains in each layer tilted at 35 ° to the long axis of the vesicle, we can explain a number of the X-ray observations: (1) oriented arcs with a Bragg spacing of 4.7 Å, which is the distance between the axes of neighbouring chains in each layer; (2) diffraction oriented in the direction of the chains at a spacing of 6 to 7 Å, which is the repeating distance of the dipeptide unit along the chain; (3) the 11 Å repeat, which is the repeating distance of pairs of chains along each rib; and (4) a broad band of diffraction at right-angles to the plane of the wall and centred at a spacing of 10 Å, which is a reasonable value for the distance between the mid-planes of the two sheets. Moreover, we can also find the remaining lattice parameter, the angle relating the centres of the subunits in neighbouring ribs. Thus the shortest line joining the centres makes an angle of 86 ° with the direction of the ribs.     

Three restriction endonucleases from Anabaena flos-aquae

Three site-specific endonucleases, AflI, AflII and AflIII, have been partially purified from the cyanobacterium Anabaena flos-aquae CCAP 1403/13f. Their recognition and cleavage specificities have been determined to be: (formula; see text) AflII and AflIII are new specificities and may be useful in molecular cloning, as well as in the analysis of DNA. The distribution of type II restriction endonucleases in the cyanobacteria is briefly discussed.     

Modeling culture profiles of the heterocystous N2-fixing cyanobacterium Anabaena flos-aquae

Heterocyst differentiation is a unique feature of nitrogen-fixing cyanobacteria, potentially important for photobiological hydrogen production. Despite the significant advances in genetic investigation on heterocyst differentiation, there were no quantitative culture-level models that describe the effects of cellular activities and cultivation conditions on the heterocyst differentiation. Such a model was developed in this study, incorporating photosynthetic growth of vegetative cells, heterocyst differentiation, self-shading effect on light penetration, and nitrogen fixation. The model parameters were determined by fitting experimental results from the growth of the heterocystous cyanobacterium Anabaena flos-aquae CCAP 1403/13f in media without and with different nitrate concentrations and under continuous illumination of white light at different light intensities (2, 5, 10, 17, 20 and 50 microE m-2 s-1). The model describes the experimental profiles well and gives reasonable predictions even for the transition of growth from that on external N source to that via nitrogen fixation, responding to the change in external N concentrations. The significance and implications of the best-fit values of the model parameters are discussed.     

Determination of Cu environments in the cyanobacterium Anabaena flos-aquae by X-ray absorption spectroscopy

Whole cells and peptidoglycan isolated from cell walls of the cyanobacterium Anabaena flos-aquae were lyophilized and used at pH 2 and pH 5 in Cu(II) binding studies. X-ray absorption spectra measured at the Cu K-edge were used to determine the oxidation states and chemical environments of Cu species in the whole-cell and peptidoglycan samples. In the whole-cell samples, most of the Cu retained at both pH values was coordinated by phosphate ligands. The whole-cell fractions contained significant concentrations of Cu(I) as well as Cu(II). An X-ray absorption near-edge spectrum analysis suggested that Cu(I) was coordinated by amine and thiol ligands. An analysis of the peptidoglycan fractions found that more Cu was adsorbed by the peptidoglycan fraction prepared at pH 5, due to increased chelation by amine and carboxyl ligands. The peptidoglycan fractions, also referred to as the cell wall fractions, contained little or no Cu(I). The Cu loading level was 30 times higher in the cell wall sample prepared at pH 5 than in the sample prepared at pH 2. Amine and bidentate carboxyl ligands had similar relative levels of importance in cell wall peptidoglycan samples prepared at both pH values, but phosphate coordination was insignificant.     

The protein encoded by gvpC is a minor component of gas vesicles isolated from the cyanobacteria Anabaena flos-aquae and Microcyctis sp.

The proteins present in gas vesicles of the cyanobacteria Anabaena flos-aquae and Microcystis sp. were separated by SDS-polyacrylamide gel electrophoresis. Each contained a protein of Mr 22K whose N-terminal amino acid sequences showed homology with that of the Calothrix sp. PCC 7601 gvpC gene product. The gvpC gene from A. flos-aquae was cloned and sequenced. The derived amino acid sequence for the gene product indicated a protein, GVPc, of 193 residues and Mr 21985 containing five highly conserved 33 amino acid repeats. The sequence was identical at the N-terminus to that of the Mr 22K protein present in gas vesicles and showed correspondence to seven tryptic peptides isolated from gas vesicles. This establishes that GVPc forms a second protein component of the gas vesicle, in addition to the main constituent, the 70 residue GVPa. Quantitative amino acid analysis of entire gas vesicles reveals that GVPc accounts for only 2.9% of the protein molecules and 8.2% of the mass present: this is insufficient to form the conical end caps of the gas vesicles. It is suggested that GVPc provides the hydrophilic outer surface of the gas vesicle wall; the 33 amino acid repeats may interact with the periodic structure provided by GVPa.     

Average thickness of the gas vesicle wall in Anabaena flos-aquae.

The average thickness of the layer of protein which forms the wall of the gas vesicles in Anabaena flos-aquae was estimated from measurements of their density and geometry. The volume of the gas space in a purified gas vesicle suspension was determined from the contraction which occurred when the gas vesicles were collapsed by pressure. The volume of the protein in the same sample was calculated from its dry weight and density. From knowledge of the geometry of the average gas vesicle the thickness of the protein layer, 1.54 nm, was then calculated. By a similar method the thickness of the Microcystis gas vesicle wall, 1.62 nm, was calculated from data published by others. The average thickness of the protein layer is, as expected, slightly less than the stacking periodicity of collapsed gas vesicle walls indicated by X-ray diffraction studies.Anabaena gas vesicles with a mean length of 494 nm have an average density of 0.119 mg μl^?1 1 mg of protein is present in gas vesicles having a, total volume of 8.43 μl and a gas space of 7.67 μl. Suspensions of isolated gas vesicles with a gas space concentration of 1 μl ml^?1 give a pressure-sensitive optical density, E_1cm (500 nm) of 2.72, but gas vacuoles in cells give a smaller value.     

Molecular weight of gas-vesicle protein from the planktonic cyanobacterium Anabaena flos-aquae and implications for structure of the vesicle

The gas vesicle of the planktonic cyanobacterium Anabaena flos-aquae is a cylindrical shell made of protein enclosing a gas-filled space. Protein sequence analysis shows that the vesicle is made from a single protein. By gel electrophoresis and amino acid analysis its molecular weight was estimated to be 20 600. Taken with previously obtained X-ray data, a simple interpretation of its molecular structure is of the polypeptide snaking in six pairs of antiparallel chains, three in each layer. The molecule would repeat along the ribs of the vesicle at intervals of 3.4 nm.     

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     

Chemical analysis of the phenol-water-extractable materials from Anabaena flos-aquae.

The high molecular-weight carbohydrate substances extracted in the aqueous and phenol phases by phenol-water extraction of Anabaena flos-aquae A-37 were found to be polysaccharides without lipid attached.     

一种水华蓝藻气囊的分离纯化及气囊结构蛋白的鉴定

【目的】从水华蓝藻铜绿微囊藻(Microcystis aeruginosa PCC 7806)的细胞中分离纯化出高纯度且完整的气囊,并对气囊的结构组成蛋白进行鉴定。【方法】采用渗透冲击与溶菌酶处理相结合的方法,进行多次低速离心提纯气囊,纯化所得气囊用负染色透射电镜观察气囊的纯度、完整度和形态。将纯化得到的气囊溶解后进行SDS-PAGE电泳,电泳后的蛋白条带运用LC-MS质谱法完成鉴定。【结果】从气囊的电镜照片可以看出提纯后的气囊纯度高,完整性好。气囊是两端呈锥状的圆柱体状,各气囊的直径大小一致,约120 nm,但长度不同,从约500 nm到1 500 nm不等。SDS-PAGE电泳和质谱法鉴定出气囊的两种主要结构组成蛋白Gvp A和Gvp C。【结论】这些研究对后续揭示气囊的精细结构和深入研究水华蓝藻浮力调节机制具有重要的意义。     

Composition of a Photosystem I chlorophyll protein complex from Anabaena flos-aquae

The use of Triton X-100 to solubilize membrane fragments from Anabaena flos-aquae in conjunction with DEAE cellulose chromatography allows the separation of three green fractions. Fraction 1 is detergent-solubilized chlorophyll, and Fraction 2 contains one polypeptide in the 15 kdalton area. Fraction 3, which contains most of the chlorophyll and shows P-700 and photosystem I activity, shows by SDS gel electrophoresis varying polypeptide profiles which reflect the presence of four fundamental bands as well as varying amounts of other polypeptides which appear to be aggregates containing the 15 kdalton polypeptide. The four fundamental bands are designated Band I at 120, Band II at 52, Band III at 46, and Band IV at 15 kdaltons. Band I obtained using 0.1% SDS contains chlorophyll and P-700 associated with it. When this band is cut out and rerun, the 120 kdalton band is lost, but significant increases occur in the intensities of Bands II, III, and IV as well as other polypeptides in the 20–30 kdalton range.The use of 1% Triton X-100 coupled with sucrose density gradient centrifugation allows the separation of three green bands at 10, 25 and 40% sucrose. The 10% layer contains a major polypeptide which appears to be Band IV. The 25 and 40% layers show essentially similar polypeptide profiles, resembling Fraction 3 in this regard, except that the 40% layer shows a marked decrease in Band III. Treatment of the material layering at the 40% sucrose level with a higher (4%) concentration of Triton X-100 causes a loss (disaggregation) of the polypeptides occurring in the 60–80 kdalton region and an increase in the lower molecular weight polypeptides. Thus, aggregation of the lower molecular weight polypeptides accounts for the variability seen in the electrophoresis patterns. Possible relations of the principal polypeptides to the known photochemical functions in the original membrane are discussed.     

Flavodoxin from the nitrogen-fixing cyanobacterium Anabaena PCC 7119

Flavodoxin has been isolated and purified from cultures of the cyanobacterium Anabaena cultivated in a low-iron medium. This flavoprotein has a molecular weight of 20,000 and contains 1 molecule of flavin mononucleotide per mol of protein. Various biochemical characteristics are reported including amino-acid composition, isoelectric point and the fluorescence properties of the apoprotein. The extinction coefficients and isosbestic points were determined for the oxidized and semiquinone forms of flavodoxin. The electron paramagnetic resonance spectrum of the semiquinone exhibited a spectral linewidth of 23 G, which is typical for a neutral flavoprotein semiquinone. Kinetic measurements give a rate constant of 9.6×10⁷ (M^-1 min^-1) for the reduction of flavodoxin in the photosynthetic electron-transport chain by the photosystem I and 6.6×10⁶ for the reaction in which flavodoxin is reduced by ferredoxin-NADP⁺ oxidoreductase. The Michaelis constant for electron donation to nitrogenase by reduced flavodoxin is 8.5 M.Abbreviations FMN flavin mononucleotide - FNR ferredoxin-NADP⁺ oxidoreductase - PSI photosystem I     

Composition of a photosystem I chlorophyll protein complex from Anabaena flos-aquae.

The use of Triton X-100 to solubilize membrane fragments from Anabaena flos-aquae in conjunction with DEAE cellulose chromatography allows the separation of three green fractions. Fraction 1 is detergent-solubilized chlorophyll, and Fraction 2 contains one polypeptide in the 15 kdalton area. Fraction 3, which contains most of the chlorophyll and shows P-700 and photosystem I activity, shows by SDS gel electrophoresis varying polypeptide profiles which reflect the presence of four fundamental bands as well as varying amounts of other polypeptides which appear to be aggregates containing the 15 kdalton polypeptide. The four fundamental bands are designated Band I at 120, Band II at 52, Band III at 46, and Band IV at 15 kdaltons. Band I obtained using 0.1% SDS contains chlorophyll and P-700 associated with it. When this band is cut out and rerun, the 120 kdalton band is lost, but significant increases occur in the intensities of Bands II, III, and IV as well as other polypeptides in the 20-30 kdalton range. The use of 1% Triton X-100 coupled with sucrose density gradient centrifugation allows the separation of three green bands at 10, 25 and 40% sucrose. The 10% layer contains a major polypeptide which appears to be Band IV. The 25 and 40% layers show essentially similar polypeptide profiles, resembling Fraction 3 in this regard, except that the 40% layer shows a marked decrease in Band III. Treatment of the material layering at the 40% sucrose level with a higher (4%) concentration of Triton X-100 causes a loss (disaggregation) of the polypeptides occurring in the 60-80 kdalton region and in increase in the lower molecular weight polypeptides. Thus, aggregation of the lower molecular weight polypeptides accounts for the variability seen in the electrophoresis patterns. Possible relations of the principal polypeptides to the known photochemical functions in the original membrane are discussed.