Isolation of gas vacuole-less mutants of the blue-green alga Anabaenopsis raciborskii

From a bloom forming blue-green alga, Anabaenopsis raciborskii, spontaneous mutants, which had lost the ability to form gas vacuoles have been isolated; the mutant frequency was 4.8×10^-3. The filaments of gas vacuole-less mutants settled at the bottom of flasks in liquid culture media unlike the parent alga. The growth and nitrogen fixation were comparatively poor in the mutants.     

The development and vertical distribution of populations of gas-vacuolate bacteria in a eutrophic,monomictic lake

The role of gas vacuoles in the vertical stratification of planktonic bacteria is analysed. Measurements made with certain gas-vacuolate bacteria in laboratory culture suggest that only colonial forms could sink or float fast enough to form population maxima in lakes by vertical migration from other depths. It is suggested that in the case of individual cells the importance of the buoyancy provided by gas vacuoles is to minimise sinking rates and thereby to increase residence times of the organisms at depths where conditions support their growth.Changes in the vertical distribution of a number of gas-vacuolate bacteria were followed throughout the year in a monomictic, eutrophic lake (Crose Mere, Shropshire). All were restricted to the anaerobic hypolimnion which developed in summer. The various species formed maxima at different depths and times. With some of them (e.g. species of Thiopedia, Pelonema and Brachyarcus) growth was necessary to explain their development. In others (e. g. species of Pelodictyon and two colourless bacteria) vertical migrations might also have contributed to their development.     

The buoyancy-providing role of gas vacuoles in an aerobic bacterium

The heterotrophic, freshwater bacterium Prosthecomicrobium pneumaticum Staley possesses sufficient gas vacuoles to render it buoyant at all stages of growth. Although the cells have a turgor pressure of about 300 kPa, there is no evidence that this pressure is important in causing collapse of the constituent gas vesicles. A mutant of the bacterium, which produced only 0.2% of the amount of gas vacuoles produced by the wild type, was isolated. It always sank in liquid culture. Wild type and mutant bacteria grew at the same rate in shaken culture, but in static culture the wild type, which floated to the liquid surface grew more quickly than the mutant, which sank. Other competition experiments suggested that the advantage gained in floating at the surface was simply that oxygen was more readily available there to this obligate aerobe. Similar advantages may benefit gas vacuolate forms in natural habitats.A second mutant was isolated which produced about 40% fewer gas vacuoles than the wild type in corresponding stages of growth, insufficient to provide buoyancy, and unlikely to be of selective value. The occurrence of this mutant suggests there may be duplication of the gas vacuole gene.Abbreviations T turbidity - PST pressure sensitive turbidity - kPa kilo-Pascals (100 kPa=1 bar)     

The occurrence of gas-vacuolate bacteria in lakes

In order to assess the importance of gas vacuoles in planktonic bacteria we carried out a survey of 35 lakes of different types. At least 39 morphologically distinct species of gas-vacuolate bacteria were encountered. All of the freshwater lakes, and one of two seawater-flooded quarries, which became thermally stratified in summer, contained gas-vacuolate bacteria in their anaerobic hypolimnia; however, with one exception, none was found in isothermally mixed lakes. This pattern of distribution supports the idea that gas vacuoles are important in providing buoyancy, a function which is relevant only in non-turbulent aquatic systems.     

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     

In vivo photoinactivation of ribulose bisphosphate carboxylase in the gas-vacuolate cyanobacteriumMicrocystis aeruginosa

Irradiation of buoyant, gas-vacuolate cells of the cyanobacteriumMicrocystis aeruginosa by 5·10⁴ Wm^–2 of blue light for 1 h caused a 5% loss of extractable ribulose bisphosphate carboxylase activity compared to dark and red-light controls. Ribulose bisphosphate carboxylase activity was unaffected by blue light in similar experiments conducted with cells containing collapsed gas vacuoles.Abbreviations RuBP Ribulose 1,5-bis-phosphate carboxylase     

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     

Trichodesmium thiebautii; structure of a nitrogen-fixing marine blue-green alga (Cyanophyta)

Summary Trichodesmium thiebautii was collected, as floating bundles composed of uniseriate filaments aligned in parallel, from the Kuroshio waters off Shikoku Island, Japan. The ultrastructure of this alga had basically the same general features as the related speciesT. erythraeum first described byvan Baalen andBrown (1969). InT. thiebautii long electron dense fibers and concentrically lamellated bodies were observed which were either not reported previously, or did not occur inT. erythraeum. The peripheral wall layers were generally typical ofOscillatoria-type blue-green algae, but with a distinctive finely striated outer layer. Thylakoids per cell volume were very sparse compared to most other blue-green algae. Phycobilisomes, apparently hemidiscoidal in shape, typically occurred on the stromal side of the thylakoid surface. Large gas vesicle areas occupied the main volume of the cell, including cells which seemed to be actively growing. The gas vesicle areas were distributed throughout the cell, not only in the cell periphery as inT. erythraeum. Considerable complexity was suggested by the apparent cell compartmentation, particularly because the gas vesicle areas were delimited by one to several thylakoids. Only rarely were the gas vesicle areas traversed by thylakoids. Electron dense fibers (ca. 25 nm diameter) were always observed between the gas vesicles and were usually oriented parallel with them, but they were not rigid appearing as were the gas vesicles. The gas vesicles had a smaller diameter (ca. 45 nm) than most blue-greens. Concentrically lamellated bodies (ca. 1.0 m diameter) were observed in cells of some of the bundles. Each concentric layer was ca. 1.3 nm wide. These concentrically lamellated bodies may be characteristic of older cells. Cylindrical bodies were considerably smaller (ca. 120 nm diameter) and less complex than those reported forT. erythraeum.     

Isolation and chemical characterization of gas-vacuole membranes fromMicrocystis aeruginosa Kuetz. emend. Elenkin

Summary A method involving penicillin treatment was developed to osmotically lyse the cells of the blue-green alga,Microcystis aeruginosa Kuetz. emend. Elenkin, and release the pressure-sensitive gas vacuoles intact. The gas vacuoles were purified by liquid-polymer partitioning or by macromolecular sieving and centrifugation. The degree of purification of the gas vacuoles was followed by observation in the electron microscope and by the use of C¹⁴-labeled vacuolated and nonvacuolated strains ofM. aeruginosa. The gas-vacuole membrane is composed of only protein consisting of 10% basic, 18% acidic and 52% non-polar amino acids.Supported by U.S. Atomic Energy Commission, Contract No. AT(11-1)-1338.     

Effect of inhibitors and uncouplers on light-induced potential changes triggering photophobic responses

Light-energy absorption by Microcystis aeruginosa with and without gas vacuoles was observed, respectively by using an integrating sphere photometer. As far as the concentration of cell suspension of the order of 10⁶⁷ cells/ml in this work was concerned, the performance of gas vacuoles to shield incident light was most unlikely. Referring to a correlation secured by the integrating sphere photometer between light absorption and cell concentration of the suspension, a turbidostat culture of the blue-green alga demonstrated that the growth efficiency, Y _kJ defined as g cells harvested per kJ of light energy absorbed by the cells was nearly 0.004. This value of Y _kJ was almost the same as that of Spirulina platensis.Abbreviation vvm volume of air per volume of medium per min     

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.     

GFS9/TT9 contributes to intracellular membrane trafficking and flavonoid accumulation in Arabidopsis thaliana

Flavonoids are the most important pigments for the coloration of flowers and seeds. In plant cells, flavonoids are synthesized by a multi‐enzyme complex located on the cytosolic surface of the endoplasmic reticulum, and they accumulate in vacuoles. Two non‐exclusive pathways have been proposed to mediate flavonoid transport to vacuoles: the membrane transporter‐mediated pathway and the vesicle trafficking‐mediated pathway. No molecules involved in the vesicle trafficking‐mediated pathway have been identified, however. Here, we show that a membrane trafficking factor, GFS9, has a role in flavonoid accumulation in the vacuole. We screened a library of Arabidopsis thaliana mutants with defects in vesicle trafficking, and isolated the gfs9 mutant with abnormal pale tan‐colored seeds caused by low flavonoid accumulation levels. gfs9 is allelic to the unidentified transparent testa mutant tt9. The responsible gene for these phenotypes encodes a previously uncharacterized protein containing a region that is conserved among eukaryotes. GFS9 is a peripheral membrane protein localized at the Golgi apparatus. GFS9 deficiency causes several membrane trafficking defects, including the mis‐sorting of vacuolar proteins, vacuole fragmentation, the aggregation of enlarged vesicles, and the proliferation of autophagosome‐like structures. These results suggest that GFS9 is required for vacuolar development through membrane fusion at vacuoles. Our findings introduce a concept that plants use GFS9‐mediated membrane trafficking machinery for delivery of not only proteins but also phytochemicals, such as flavonoids, to vacuoles.     

An investigation into the possible light-shielding role of gas vacuoles in a planktonic blue-green alga

When the gas vacuoles of Anabaena flos-aquae Bréb. ex Born. et Flah. are collapsed, the optical properties of the alga change. While this may suggest a light-shielding role, photosynthetic measurements indicate that intact gas vacuoles reduce the light falling on the thylakoids by only 4%, or less. Intact gas vacuoles offer no protection against the lethal effects of ultraviolet light. When the alga is grown at high light intensity the gas vacuoles are fewer in number but are oriented peripherally in the cells. However, this does not markedly affect their light shielding efficiency. Spectrophotometric measurements carried out by others indicate a light shielding role by gas vacuoles in a non-planktonic blue-green alga, Nostoc muscorum Kütz., but do not give a quantitative estimate of this effect. In Anabaena no definite evidence of light-shielding is obtained by such a method. All of the experiments described were conducted with dilute algal suspensions to investigate shielding effects in individual cells. Possible self-shading effects in dense suspensions and surface water blooms require further investigation.     

Counting of gas vacuoles by electron microscopy in lysates and purified fractions ofmicrocystis aeruginosa

Summary A microdroplet spray method is described to determine quantitatively the number of gas vacuoles in purified fractions and in total lysates of the blue-green alga,Microcystis aeruginosa.It is found that 6.45×10¹² gas vacuoles of 370 nm length make up one mg of protein. This supports the idea that the entire organelle is made up by a single layer of protein only. The gas vacuole number per cell varies with the stage of growth between 3,700 and 11,000.     

Autophagy delivers misfolded secretory proteins accumulated in endoplasmic reticulum to vacuoles in the filamentous fungus Aspergillus oryzae

Autophagy is a conserved intracellular degradation process of eukaryotic cells. In filamentous fungi, although autophagy has been reported to have multiple physiological roles, it is not clear whether autophagy is involved in the degradation of misfolded proteins. Here, we investigated the role of autophagy in the degradation of misfolded secretory proteins accumulated in endoplasmic reticulum (ER) in the filamentous fungus Aspergillus oryzae. In late-phase cultures, a disulfide bond-deleted mutant of the secretory protein α-amylase AmyB fused with mDsRed that had accumulated in the ER was subsequently delivered to vacuoles, whereas wild-type AmyB-mDsRed was predominantly located at cell walls and septa. To examine the involvement of autophagy in the delivery of mutant AmyB to vacuoles, mutant AmyB-EGFP was expressed in an A. oryzae autophagy-deficient strain (ΔAoatg8). Microscopic examination revealed that the protein delivery to vacuoles did not occur in the absence of autophagic activity, with mutant AmyB-mDsRed forming large spherical structures surrounded by ER membrane. Hence, we conclude that autophagy is responsible for the delivery of misfolded secretory proteins accumulated in the ER to vacuoles for degradation during late-growth phase in A. oryzae. This is the first study to provide evidence that autophagy plays a role in the degradation of misfolded secretory proteins in filamentous fungi.     

Symbiotic Chlorella sp. of the ciliate Paramecium bursaria do not prevent acidification and lysosomal fusion of host digestive vacuoles during infection

Summary. Each symbiotic Chlorella sp. of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole, and thereby the alga is protected from digestion by lysosomal fusion. Algae-free cells can be reinfected with algae isolated from algae-bearing cells by ingestion into digestive vacuoles. To examine the timing of acidification and lysosomal fusion of the digestive vacuoles and of algal escape from the digestive vacuole, algae-free cells were mixed with isolated algae or yeast cells stained with pH indicator dyes at 25 ± 1 °C for 1.5 min, washed, chased, and fixed at various time points. Acidification of the vacuoles and digestion of Chlorella sp. began at 0.5 and 2 min after mixing, respectively. All single green Chlorella sp. that had been present in the host cytoplasm before 0.5 h after mixing were digested by 0.5 h. At 1 h after mixing, however, single green algae reappeared in the host cytoplasm, arising from those digestive vacuoles containing both nondigested and partially digested algae, and the percentage of such cells increased to about 40% at 3 h. At 48 h, the single green algae began to multiply by cell division, indicating that these algae had succeeded in establishing endosymbiosis. In contrast to previously published studies, our data show that an alga can successfully escape from the host’s digestive vacuole after acidosomal and lysosomal fusion with the vacuole has occurred, in order to produce endosymbiosis. Correspondence and reprints: Biological Institute, Faculty of Science, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan.     

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.     

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.     

Kinetics of the assembly of gas vacuoles in the blue-green alga Microcystis aeruginosa Kuetz. emend. Elekin.

Summary In exponentially growing cultures of the blue-green alga Microcystis aeruginosa the number of gas vacuoles per cell decreases and reaches a value of 4.5×10³ at 1.2×10⁷ cells per ml. The assembly of gas vacuoles with respect to number and length was followed after the organelles were caused to collapse by a pulse of ultrasound. The change in the number N of gas vacuoles per cell is N=224.8×t ^0.757, 0<t<24 h. After 24 h 50% of the value of non-sonicated cultures is reached. The changes in the length L of the organelles is expressed by L=87.06 ×t ^0.4084, 0<t<24 h. After 24 h 85% of the control value is reached.Abbreviations used EDTA ethylene diamine tetraacetate - BSA bovine serum albumine - P probability - N number of gas vacuoles - L length of gas vacuoles in nm - t time in hours     

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.