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.     

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 in actinomycetes?

Gas vesicles encoded by gvp genes provide buoyancy in many prokaryotes. In a recent Trends in Microbiology article entitled 'Gas vesicles in actinomycetes: old buoys in novel habitats?' van Keulen et al. documented the occurrence of gvp genes in soil-inhabiting actinomycetes but questioned whether any of them produce gas vesicles. We suggest that the protein encoded by gvpA in actinomycetes might be incompatible with the structure of the standard gas vesicle. Perhaps it has another role associated with the air-water interface.     

The properties and buoyancy-providing role of gas vacuoles in Trichodesmium Ehrenberg

All three species of the marine blue-green alga Trichodesmium collected in the Sargasso and Caribbean seas were found to possess gas vacuoles. The constituent gas vesicles were much stronger than those found in any freshwater blue-green alga, the mean critical collapse pressures being 12 bars in T. erythraeum, 34 bars in T. contortum and 37 bars in T. thiebautii. This great strength is obviously an adaptation to the hydrostatic pressures at the depths to which these organisms occur in the ocean. In each case the gas vesicles are far too strong to be collapsed by rising cell turgor pressure, though gas-vacuolation could be slowly regulated by the differential growth of gas vesicles and cells. Since the vesicles are of a similar shape and size to those in other species, the vesicle wall material must be stronger. The majority of Trichodesmium colonies collected were positively buoyant, and in all cases tested the buoyancy was dependent on the presence of gas vacuoles. The buoyancy is important in increasing the residence time of these slowly growing algae in the euphotic zone and it is responsible for the surface water-blooms which they form.     

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.     

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     

REGULATION OF GAS VESICLE CONTENT AND BUOYANCY IN LIGHT-OR PHOSPHATE-LIMITED CULTURES OF APHANIZOMENON FLOS-AQUAE (CYANOPHYTA)1

Measurements of the gas vesicle space in steady-state light or phosphate-limited cultures of Aphanizomenon flos-aquae Ralfs, strain 7905 showed that gas vesicle content decreased as energy-limited growth rate increased but was the same at several phosphate-limited growth rates. Upon a decrease in growth irradiance, gas vesicle content did increase in phosphate-limited cultures, but the cultures remained nonbuoyant as long as P was limiting. Buoyant, energy-limited cultures lost their buoyancy in less than 2 h when exposed to higher irradiances. The primary mechanism for buoyancy loss was the accumulation of polysaccharide as ballast. Collapse of gas vesicles by turgor pressure played a minor role in the loss of buoyancy. When cultures were exposed to higher irradiances, cells continued to synthesize gas vesicles at the same rate as before the shift for at least 1 generation time. The amount of ballast required to make individual filaments in the population sink varied 4-fold. This variation appears to be due to differences in gas vesicle content among individual filaments.     

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.     

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.     

Solid-State NMR Evidence for Inequivalent GvpA Subunits in Gas Vesicles

Gas vesicles are organelles that provide buoyancy to the aquatic microorganisms that harbor them. The gas vesicle shell consists almost exclusively of the hydrophobic 70-residue gas vesicle protein A, arranged in an ordered array. Solid-state NMR spectra of intact collapsed gas vesicles from the cyanobacterium Anabaena flos-aquae show duplication of certain gas vesicle protein A resonances, indicating that specific sites experience at least two different local environments. Interpretation of these results in terms of an asymmetric dimer repeat unit can reconcile otherwise conflicting features of the primary, secondary, tertiary, and quaternary structures of the gas vesicle protein. In particular, the asymmetric dimer can explain how the hydrogen bonds in the β-sheet portion of the molecule can be oriented optimally for strength while promoting stabilizing aromatic and electrostatic side-chain interactions among highly conserved residues and creating a large hydrophobic surface suitable for preventing water condensation inside the vesicle.     

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.     

Gas vesicles in actinomycetes: old buoys in novel habitats?

Gas vesicles are gas-filled prokaryotic organelles that function as flotation devices. This enables planktonic cyanobacteria and halophilic archaea to position themselves within the water column to make optimal use of light and nutrients. Few terrestrial microbes are known to contain gas vesicles. Genome sequences that have become available recently for many bacteria from non-planktonic habitats reveal gas vesicle gene clusters in members of the actinomycete genera Streptomyces, Frankia and Rhodococcus, which typically live in soils and sediments. Remarkably, there is an additional level of complexity in cluster number and gene content. Here, we discuss whether putative gas vesicle proteins in these actinomycetes might actually be involved in flotation or whether they might fulfil other cellular functions.     

Buoyancy regulation of Microcystis flos-aquae during phosphorus-limited and nitrogen-limited growth

The dominance of gas-vacuolate cyanobacteria is often attributedto their buoyancy and to their ability to regulate buoyancyin response to environmental conditions. Changes in absolutegas vesicles volume, carbohydrate content, protein content andcolony buoyancy of Microcystis flos-aquae were investigatedduring nitrogen-limited, phosphorus-limited and nutrient-repletegrowth. When nutrient-replete, M. flos-aquae cells consistentlyhad excess gas vesicles, which provided sufficient buoyancythat the influence of daily carbohydrate changes on cells uponfloatation was negligible. However, during nitrogen-limitedgrowth, gas vesicle volume per cell decreased significantlywith nitrogen exhaustion. The maximum decrease of gas vesiclevolume was up to 84–88%. At the same time, cellular carbohydratecontent had an accumulation trend. The decrease of gas vesiclebuoyancy together with the daily increase in carbohydrate aresuggested to explain the daily changes in the cell floatation.During phosphorus-limited growth, gas vesicle volume per celldecreased slightly (maximum to 22–32%), and they stillprovided sufficient buoyancy that most cells kept floating eventhough there were significant daily carbohydrate changes. Sincenitrogen limitation caused more significant buoyancy loss thanphosphorus limitation did, surface water blooms may disappearor appear frequently in nitrogen limited water bodies whilethey may persist a longer time in phosphorus limited water bodies.The quantitative analysis in buoyancy change by gas vesicles,carbohydrate and protein suggested that long-term buoyancy regulationwas mainly determined by changes of gas vesicle volume whereasshort-term buoyancy regulation was mainly determined by carbohydrateaccumulation and consumption. Both long-term and short-termbuoyancy regulation were influenced by cell nutrient status.Furthermore, gas vesicle volume per cell and protein contentchanged in the same way in both nitrogen-limited and phosphorus-limitedgrowth, which implied that the decrease of gas vesicles wereassociated with controls of total protein synthesis.     

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.     

Diel Buoyancy Changes by the Cyanobacterium Aphanizomenon ovalisporum from a Shallow Reservoir

In the summer of 1999, a bloom (11 100 filaments ml^–1)of the gas vacuolate cyanobacteriumAphanizomenon ovalisporumdeveloped in a shallow (1.7 m deep) reservoir containing nutrient-enrichedwater from Lake Kinneret (Israel). During 4 days, A. ovalisporumshowed a marked diel periodicity in buoyancy: the proportionof floating filaments fluctuated between 76–84% from middayto evening and 94–98% at the end of the night, in bothsurface and bottom samples. Buoyant filaments were present throughoutthe water column, presumably due to wind-driven vertical mixing.Aphanizomenonfilaments collected from the reservoir were maintained undermean photon irradiances of 15 (LL), 150 (ML) and 1100 (HL) µmolm^–2 s^–1 in a computer-controlled set-up, which simulatedthe diel light changes at different depths in the reservoir.In the LL cultures, filament buoyancy showed no diel fluctuationpatterns during the 4 days of incubation, but ML and HL culturesshowed regular diel changes, with a higher proportion of filamentsfloating at the end of the night than during midday–evening.There was no evidence for either turgor-driven collapse of gasvesicles or dilution of gas vesicles by cell growth by any ofthe treatments. Gas vesicles of A. ovalisporum had a relativelylow mean critical pressure (p_c of 0.57 MPa), but the daytimerise in turgor pressure was too small to cause gas vesicle collapse.The observed diel buoyancy changes may be explained by accumulationof carbohydrate ballast during the day and decrease during thenight.     

REGULATION OF GAS VESICLE CONTENT AND BUOYANCY IN LIGHT- OR PHOSPHATE-LIMITED CULTURES OF APHANIZOMENON FLOS-AQUAE (CYANOPHYTA)1

Measurements of the gas vesicle space in steady-state light or phosphate-limited cultures of Aphanizomenon flos-aquae Ralfs, strain 7905 showed that gas vesicle content decreased as energy-limited growth rate increased hut was the same at several phosphate-limited growth rates. Upon a decrease in growth irradiance, gas vesicle content did increase in phosphate-limited cultures, hut the cultures remained nonbuoyant as long as P was limiting. Buoyant, energy-limited cultures lost their buoyancy in less than 2 h when exposed to higher irradiances. The primary mechanism for buoyancy loss was the accumulation of polysaccharide as ballast. Collapse of gas vesicles by turgor pressure played a minor role in the loss of buoyancy. When cultures were exposed to higher irradiances, cells continued to synthesize gas vesicles at the same rate as before the shift for at least 1 generation time. The amount of ballast required to make individual filaments in the population sink varied 4-fold. This variation appears to be due to differences in gas vesicle content among individual filaments.     

Molecular genetic and physical analysis of gas vesicles in buoyant enterobacteria

Different modes of bacterial taxis play important roles in environmental adaptation, survival, colonization and dissemination of disease. One mode of taxis is flotation due to the production of gas vesicles. Gas vesicles are proteinaceous intracellular organelles, permeable only to gas, that enable flotation in aquatic niches. Gene clusters for gas vesicle biosynthesis are partially conserved in various archaea, cyanobacteria, and some proteobacteria, such as the enterobacterium, Serratia sp. ATCC 39006 ( S39006 ). Here we present the first systematic analysis of the genes required to produce gas vesicles in S39006 , identifying how this differs from the archaeon Halobacterium salinarum. We define 11 proteins essential for gas vesicle production. Mutation of gvpN or gvpV produced small bicone gas vesicles, suggesting that the cognate proteins are involved in the morphogenetic assembly pathway from bicones to mature cylindrical forms. Using volumetric compression, gas vesicles were shown to comprise 17% of S39006 cells, whereas in Escherichia coli heterologously expressing the gas vesicle cluster in a deregulated environment, gas vesicles can occupy around half of cellular volume. Gas vesicle production in S39006 and E. coli was exploited to calculate the instantaneous turgor pressure within cultured bacterial cells; the first time this has been performed in either strain.     

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。     

The identification of gas vacuoles and their abundance in the hypolimnetic bacteria of Arco Lake,Minnesota

Bright refractile granules in bacterial cells are identified as gas vacuoles if they disappear on application of a few atmospheres pressure. This paper describes a simple method for observing individual cells under the light microscope before and after application of pressure and the use of this method in making a comprehensive survey of gas-vacuolate organisms in a sample. In water samples from the hypolimnion of a stratified lake (Arco Lake) in Northern Minnesota, gas vacuoles were found in nearly 30 different bacteria, representing possibly 60% or more of those present. The pressure sensitivity of gas vacuoles in these organisms is illustrated by micrograph pairs. Gas vacuoles, which are otherwise uncommon in bacteria, are evidently of great selective value in the hypolimnia of stratified lakes, perhaps by regulating cell buoyancy.