Gas vesicles are strengthened by the outer-surface protein,GvpC

The critical collapse pressure of gas vesicles isolated from Anabaena flos-aquae decreased from 0.557 to 0.190 MPa when GvpC, the hydrophilic 22 kDa protein present on the outer surface of the gas vesicle, was removed by rising in 6 M urea. Recombinant GvpC was purified from inclusion bodies, produced in an E. coli strain containing an expression vector bearing the gene ecoding GvpC from A. flos-aquae, and then solubilised in 6 M urea. This recombinant GvpC became bound to gas vesicles that had been stripped of their native protein, when the urea was removed by dialysis; the amount which bound increased with the concentration of GvpC present. The critical pressure of these reconstituted gas vesicles increased to 0.533 MPa, 96% of the original value. These results indicate that the function of GvpC is to increase the strength of the structure.Non-standard abbreviations SBTI Soy bean trypsin inhibitor - Gvp Gas vesicle protein - SDS Sodium dodecyl sulphate - PAGE Polyacrylamide gel electrophoresis     

GvpCs with reduced numbers of repeating sequence elements bind to and strengthen cyanobacterial gas vesicles

We have previously shown that the gas-vesicle protein GvpC is present on the outer surface of the gas vesicle, can be reversibly removed and rebound to the surface, and increases the critical collapse pressure of the gas vesicle. The GvpC molecule, which contains five partially conserved repeats of 33 amino acids (33-RR) sandwiched between 18 N-terminal and 10 C-terminal amino acids, is present in a ratio of 1:25 with the GvpA molecule, which forms the ribs of the gas vesicle. By using recombinant techniques we have now made modified versions of GvpC that contain only the first two, three or four of the 33-amino-acid repeats. All of these proteins bind to and strengthen gas vesicles that have been stripped of their native GvpC. Recombinant proteins containing three or four repeats bind in amounts that give the same ratio of 33-RR:GvpA (i.e. 1:5) as the native protein, and they restore much of the strength of the gas vesicle; the protein containing only two repeats binds at a lower ratio (1:7.7), however, and restores less of the strength. Ancestral proteins with only two, three or four of the 33-amino-acid repeats would have been functional in strengthening the gas vesicle but the progressive increase in number of repeats would have provided strength with increased efficiency.     

Evaluation of oxygen permeability of gas vesicles from cyanobacterium Anabaena flos-aquae

The enhancement of oxygen permeability in aqueous medium by addition of cyanobacterial gas vesicles (GVs) has been examined. The GVs were isolated from cultures of Anabaena flos-aquae that had been cultivated in photobioreactors and harvested by dark flotation. Prior to the permeability experiments, the collected GVs were treated with glutaraldehyde for improved stability. Measurements of oxygen permeability were made with a polarographic oxygen electrode in suspensions of various GV volume fractions (0-2.1%). The experimental results were compared with the values predicted theoretically (Fricke's equation) assuming different permeability through the GVs (PmGV), ranging from 0 to 8.30 x 10(-4) mol m-1 atm-1 s-1. The former corresponded to impermeable vesicles, the latter to air at 22 degrees C as if there were no vesicle wall. The best-fit value of PmGV was 9.9 x 10(-7) mol m-1 atm-1 s-1, ca. 36-fold higher than that in water. GVs were therefore very permeable to oxygen. However, the value was much lower than that predicted for air, implying the existence of wall resistance.     

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。     

Gas vesicles isolated from Halobacterium cells by lysis in hypotonic solution are structurally weakened

Analysis of pressure-collapse curves of Halobacterium cells containing gas vesicles and of gas vesicles released from such cells by hypotonic lysis shows that the isolated gas vesicles are considerably weaker than those present within the cells: their mean critical collapse pressure was around 0.049-0.058 MPa, as compared to 0.082-0.095 MPa for intact cells. The hypotonic lysis procedure, which is widely used for the isolation of gas vesicles from members of the Halobacteriaceae, thus damages the mechanical properties of the vesicles. The phenomenon can possibly be attributed to the loss of one or more structural gas vesicle proteins such as GvpC, the protein that strengthens the vesicles built of GvpA subunits: Halobacterium GvpC is a highly acidic, typically "halophilic" protein, expected to denature in the absence of molar concentrations of salt.     

The diameter and critical collapse pressure of gas vesicles in Microcystis are correlated with GvpCs of different length

In cyanobacteria the protein on the outside of the gas vesicle, GvpC, is characterised by the presence of a 33 amino acid residue repeat (33RR), which in some genera is highly conserved. The number of 33RRs correlates with the diameter of the gas vesicle and inversely with its strength. Gas vesicles isolated from Microcystis aeruginosa strain PCC 7806 were found to be wider and have a lower critical collapse pressure than those from Microcystis sp. strain BC 8401. The entire gas-vesicle gene cluster of the latter strain was sequenced and compared with the published sequence of the former: the sequences of nine of the ten gvp genes differed by only 1-5% between the two strains; the only substantial difference was in gvpC which in strain BC 8401 lacked a 99-nucleotide section encoding a 33RR. This observation further narrows the correlation of gas vesicle width to the number of 33RRs and suggests how Microcystis strains might be used in experimental manipulation of gas vesicle width and strength.     

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.     

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.     

Heterologous expression of cyanobacterial gas vesicle proteins in Saccharomyces cerevisiae

Given the potential applications of gas vesicles (GVs) in multiple fields including antigen-displaying and imaging, heterologous reconstitution of synthetic GVs is an attractive and interesting study that has translational potential. Here, we attempted to express and assemble GV proteins (GVPs) into GVs using the model eukaryotic organism Saccharomyces cerevisiae. We first selected and expressed two core structural proteins, GvpA and GvpC from cyanobacteria Anabaena flos-aquae and Planktothrix rubescens, respectively. We then optimized the protein production conditions and validated GV assembly in the context of GV shapes. We found that when two copies of anaA were integrated into the genome, the chromosomal expression of AnaA resulted in GV production regardless of GvpC expression. Next, we co-expressed chaperone-RFP with the GFP-AnaA to aid the AnaA aggregation. The co-expression of individual chaperones (Hsp42, Sis1, Hsp104, and GvpN) with AnaA led to the formation of larger inclusions and enhanced the sequestration of AnaA into the perivacuolar site. To our knowledge, this represents the first study on reconstitution of GVs in S. cerevisiae. Our results could provide insights into optimizing conditions for heterologous protein production as well as the reconstitution of other synthetic microcompartments in yeast.     

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.     

Isolation and characterization of gas vesicles from Microcyclus aquaticus

Intact gas vesicles of Microcyclus aquaticus S1 were isolated by using centrifugally accelerated flotation of vesicles and molecular sieve chromatography. Isolated gas vesicles were cylindrical organelles with biconical ends and measured 250×100 nm. The gas vesicle membrane was composed almost entirely of protein; neither lipid nor carbohydrate was detected, although one mole of phosphate per mole of protein was found. Amino acid analysis indicated that the protein contained 54.6% hydrophobic amino acid residues, lacked sulfur-containing amino acids, and had a low aromatic amino acid content. The protein subunit composition of the vesicles was determined by gel electrophoresis in (i) 0.1% sodium dodecyl sulfate at pH 9.0 and (ii) 5 M urea at pH 2.0. The membrane appeared to consist of one protein subunit of MW 50 000 daltons. Charge isomers of this subunit were not detected on urea gels. Antiserum prepared against purified gas vesicles of M. aquaticus S1 cross-reacted with the gas vesicles of all other gas vacuolate strains of M. aquaticus, as well as those of Prosthecomicrobium pneumaticum, Nostoc muscorum, and Anabaena flos-aquae, indicating that the gas vesicles of these widely divergent organisms have some antigenic determinants in common.Abbreviations SDS sodium dodecyl sulfate - MW molecular weight - Tris tris(hydroxymethyl)aminomethane - EDTA disodium ethylenediaminetetraacetic acid - BSA bovine serum albumin - TCA trichloroacetic acid - P _c pressure necessary to collapse gas vesicles     

Antibodies to the N-terminal sequence of GVPa bind to the ends of gas vesicles.

Antibodies were raised against intact gas vesicles of Anabaena flos-aquae, and against a synthetic peptide (GVPaNT) whose sequence is identical to the N-terminal region of the main gas vesicle protein, GVPa. A two-stage centrifugation procedure is described for separating gold-labelled antibodies bound to gas vesicles from unbound antibodies. The GVPaNT antibody bound to gas vesicles that had been previously rinsed with SDS to remove the outer gas vesicle protein, GVPc. Treatment with this antibody caused the gas vesicles to aggregate together end-to-end rather than side-by-side. The binding of the anti-GVPaNT-immunogold particles to the gas vesicle was restricted to the conical ends of the structure. These observations indicate that the sequence to which the GVPaNT antibodies were raised, residues 1 to 13 of the GVPa molecule, is exposed only at the outer surface of the cones and that it is normally obscured by GVPc. As GVPa forms both the conical ends and the cylindrical midsection of the gas vesicle, exposure of the N-terminal sequence only in the cones must be due to differences in the contact between adjacent GVPa molecules in the central cylinders and end-cones.     

Antigen presentation using novel particulate organelles from halophilic archaea

A presentation vehicle was developed based on particulate gas vesicles produced by halophilic archaea. Gas vesicle epitope displays were prepared using standard coupling methods or recombinant DNA technology. When presented in the context of gas vesicle preparations, either the hapten, TNP, or a model six amino acid recombinant insert in the outer gas vesicle protein, GvpC was rendered immunogenic. Assays to quantify humoral responses indicated that each preparation elicited strong antibody responses in the absence of exogenous adjuvant. Thus, each preparation elicited a humoral response when injected into mice and this response was long lived and exhibited immunologic memory. Recombinant gas vesicle preparations therefore constitute a new, self-adjuvanting carrier/display vehicle for presentation of an array of peptidyl epitopes.     

Buoyancy regulation in light-limited continuous cultures of Microcystis aeruginosa

Microcystis aeruginosa was grown in light-limited continuouscultures at different growth rates on light-dark cycles at variousphotopenods. Due to the strength of the gas vesicles the organismwas not able to collapse its gas vesicles by turgor pressure.Below the maximal growth rate, the organism was buoyant dueto its high gas vesicle content. The results suggested thatthe rate of gas vesicle synthesis was not regulated. Upon atransition to high irradiance it took several hours before thecells lost their buoyancy due to polyglucan accumulation. Theresults are interpreted in an ecological context and it is suggestedthat Microcystis is an epilimnetic species due to its buoyancyregulation.     

Comparative study of enzyme activity and stability of bovine and human plasmins in electrophoretic reagents, beta-mercaptoethanol, DTT, SDS, Triton X-100, and urea

Effects of common electrophoretic reagents, reducing agents (beta-mercaptoethanol [BME] and DTT), denaturants (SDS and urea), and non-ionic detergent (Triton X-100), on the activity and stability of bovine plasmin (b-pln) and human plasmin (h-pln) were compared. In the presence of 0.1% SDS (w/v), all reagents completely inhibited two plns, whereas SDS (1%) and urea (1 M) denatured plns recovered their activities after removal of SDS by treatment of 2.5% Triton X-100 (v/v). However, reducing agents (0.1 M of BME and DTT) treated plns did not restore their activities. Based on a fibrin zymogram gel, five (from b-pln) and four (from h-pln) active fragments were resolved. Two plns exhibited unusual stability in concentrated SDS and Triton X-100 (final 10%) and urea (final 6 M) solutions. Two bands, heavy chain-2 (HC-2) and cleaved heavy chain-2 (CHC-2), of b-pln were completely inhibited in 0.5% SDS or 3 M urea, whereas no significant difference was found in h-pln. Interestingly, 50 kDa (cleaved heavy chain-1, CHC-1) of b-pln and two fragments, 26 kDa (light chain, LC) and 29 kDa (microplasmin, MP), of h-pln were increased by SDS in a concentration dependent manner. We also found that the inhibition of SDS against both plns was reversible.     

气囊在细菌中的生理功能、生物合成及应用研究进展

气囊是在水生细菌中广泛存在的一种具有刚性中空蛋白结构的特殊细胞器,不仅为水生细菌提供浮力,还对其在不利环境或应激条件下的生存至关重要。近期研究发现在其他非水生细菌如沙雷氏菌和链霉菌中也存在气囊结构,而且表现出不同的生理功能。来源于不同种属细菌的气囊生物合成基因簇具有各自鲜明的特征,其生物合成和调控机制也有所不同。本综述将介绍和总结不同细菌中气囊的基本生理功能和生物合成及调控机制,以及气囊的生物技术应用,并对气囊在链霉菌中的生物合成研究以及人工重组气囊的潜在应用进行展望。     

Cassette-based presentation of SIV epitopes with recombinant gas vesicles from halophilic archaea

In earlier studies we demonstrated recombinant gas vesicles from Halobacterium sp. NRC-1, expressing a model six amino acid insert, or native vesicles displaying chemically coupled TNP, each were immunogenic, and antigenic. Long-lived responses displaying immunologic memory were elicited without exogenous adjuvant. Here we report the generation and expression of cassettes containing SIV derived DNA. The results indicate a cassette-based display/delivery system derived from recombinant halobacterial gas vesicle genes is highly feasible. Data specifically support four conclusions: (i) Recombinants carrying up to 705 bp of SIV DNA inserted into the gvpC gene form functional gas vesicles; (ii) SIV peptides contained as part of the expressed recombinant, surface exposed GvpC protein are recognized by antibody elicited in monkeys exposed to native SIV in vivo; (iii) in the absence of adjuvant, mice immunized with the recombinant gas vesicle (r-GV) preparations mount a solid, titratable antibody response to the test SIV insert that is long lived and exhibits immunologic memory; (iv) recombinant organelles, created through the generation of cassettes encoding epitopes inserted into the gvpC DNA, can be used to construct a multiepitope display (MED) library, a potentially cost effective vehicle to express and deliver peptides of SIV, HIV or other pathogens.     

Cross-linked stem bromelain: A more stabilized active preparation

Cross-linking of the protease stem bromelain (bromelain) with 0.25 and 1.25% glutaraldehyde (GTA) results in the formation of a large molecular mass, multimeric and soluble aggregate having comparable activity to the unmodified bromelain. Both 0.25 and 1.25% GTA cross-linked (CL) bromelain preparations were more stable against urea, guanidine hydrochloride (GdnHCl) and temperature-induced inactivation, and exhibited slightly better storage stability compared to the unmodified protease. Such a high molecular weight, soluble, active and stable preparation may be useful in industry, i.e. in the textile industry for improving the properties of a fabric without loss of fabric strength and shape.