[20] Gas vesicles: Chemical and physical properties

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quots are counted in a liquid scintillation counter and assayed for amino acid content so that nanomoles of amino acid could be quantitated from the disintegrations per minute of the supernatant after protease assay. Typical preparations yielded 35 ml of substrate cyanophycin with 3.5 × 103 dpm//zl. Crude Extracts Cells are harvested, resuspended in 20 mM tricine buffer, pH 8.0, and broken in a French pressure cell at 1050 kg/cm 2 followed by centrifugation at 27,000 g for 15 min. Protein concentration of the crude extract should be approximately 20 mg/ml. Procedure To 75 /xl of [14C]cyanophycin (>5 × 105 dpm and >100 dpm/nmol arginine) in a microfuge tube is added 16/zl 1.2 M NaCI, 106/zl 0.8 M Tricine, pH 8, and 75 tzl of crude extract or 20 mM Tricine for the blank. Incubation is at 35°. At zero time and at 20 min, 100/.d is removed and added to 100/zl of cold 10% trichloroacetic acid, followed by centrifugation for 10 min in a microfuge. Triplicate 30-tzl aliquots of the supernatant are counted in a liquid scintillation counter. Increase in radioactivity in the supernatant with time indicates proteinase activity. The product(s) of hydrolysis can be qualitatively determined by paper chromatography as described above.

[20] G a s Vesicles: C h e m i c a l a n d P h y s i c a l P r o p e r t i e s By P. K. HAYES Introduction Gas vesicles occur in a wide variety of cyanobacteria. In planktonic species they are often constitutive cell components; elsewhere their production may be restricted to differentiating hormogonia2 Gas vesicles provide an organism with hollow spaces which bring about a reduction in cell density, and if enough are accumulated within a cell it will float. Planktonic cyanobacteria regulate their buoyancy with gas vesicles. 2 x A. E. Walsby, in "The Prokaryotes" (M. P. Starr, H. Stolp, H. G. Triiper, A. Balows, and H. G. Schlegel, eds.), p. 224. Springer-Verlag, Berlin, 1981. 2 A. E. Walsby, Bacteriol. Rev. 36, 1 (1972).

METHODS IN ENZYMOLOGY, VOL. 167

Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

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TABLE I MEAN CYLINDER DIAMETER AND MEDIAN CRITICAL COLLAPSE PRESSURE FOR GAS VESICLES ISOLATED FROM DIFFERENT CYANOBACTERIA

Species a

Dactylococcopsis salina Anabaena flos-aquae Aphanizomenon flos-aquae Microcystis sp. Oscillatoria agardhii Trichodesmium thiebautii I'

Mean cylinder diameter (nm)

Median critical collapse pressure (MPa)

107 85 79 67

0.50 0.60 0.62 0.76 0.90 3.70

64 45

Strain numbers are given in the legend to Fig. 1. b Observations on material gathered at sea. a

Gas vesicles are hollow, cylindrical structures closed at either end with a conical cap. 2 The cylinder diameter of gas vesicles varies within rather narrow limits in a given species, but marked differences in mean diameter are found when comparisons are made between some species (Table 1). 3 Length varies markedly in all species because the gas vesicle grows by elongation of the central cylinder from a biconical initial. 4 Both the central cylinder and the conical caps are made up of ribs oriented perpendicular to the long axis of the cylinder. X-Ray crystallography has shown that these ribs are 4.57 nm in width and have repeats every 1.15 nm along their length. 5 The average thickness of the gas vesicle wall is 1.8 nm which means that the individual repeats along the rib have a volume of 9.46 nm. 3 A unit cell of this volume will accommodate a protein of Mr 7500. Protein is the sole structural component of isolated gas vesicles.6,7 A protein, GVPa, of Mr 7397, very close to the value estimated from the volume of the crystallographic unit cell, s forms the shell of the structure. 9 Another protein, GVPc, of Mr 21,985, forms a hydrophilic surface on the outside of the structure.l° GVPa accounts for 92% of the mass of the gas vesicle and GVPc the remaining 8%. 3 p. K. Hayes and A. E. Walsby, Br. Phycol. J. 21, 191 (1986). 4 j. R. Waaland and D. Branton, Science 163, 1339 (1969). 5 A. E. Blaurock and A. E. Walsby, J. Mol. Biol. 105, 183 (1976). 6 A. E. Walsby and B. Buckland, Nature (London) 224, 716 (1969). 7 D. D. Jones and M. Jost, Arch. Mikrobiol. 70, 43 (1970). s p. K. Hayes, A. E. Walsby, and J. E. Walker, Biochem. J. 236, 31 (1986). 9 p. K. Hayes, C. M. Lazarus, A. Bees, J. E. Walker, and A. E. Walsby, Mol. Microbiol. (in press). l0 A. E. Walsby and P. K. Hayes, J. Gen. Microbiol. (in press).

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Gas vesicles are highly permeable to gases. TM The outer gas vesicle surface is hydrophilic ~2 and the inner surface hydrophobic. ~3,14They are rigid structures ~5which will collapse irreversibly if exposed to a pressure that exceeds a certain value known as the critical pressure (Pc). The median value of Pc (determined by the methods given elsewhere in this volume [72]) for a population of gas vesicles varies in different species 2 (Table I): it is inversely correlated with the average cylinder diameter of the gas vesicles. 3 Isolation and Purification of Intact Gas Vesicles The various procedures used to isolate pure gas vesicles have been described in a previous volume~6; an updated account follows. It is essential that the gas vesicle preparation should not be exposed to pressures that approach the critical collapse pressure. For the weakest gas vesicles of some halophilic cyanobacteria this pressure may be as low as 0.2 MPa. 17 Once the gas vesicles have collapsed, buoyancy, which forms the basis of the isolation procedure, is lost. Gas-vacuolate cells are harvested from the surface of undisturbed cultures or concentrated either by collection onto 3.0-tzm pore size Nucleopore filters or by centrifugally accelerated flotation at low speed. The speed selected for the latter procedure should not produce a centrifugal acceleration exceeding 980 m sec -2 (100 g) for a liquid layer of 5 cm depth. Higher speeds will result in the generation of pressures in the liquid layer which may collapse the gas vesicles contained within turgid, cyanobacterial cells. Cell lysis is achieved using one of a number of techniques. Filaments of Anabaena, Aphanizomenon, or Nostoc are suspended in 0.7 M sucrose; osmotic shrinkage puts the cell wall under tension, causing it to rupture. Filaments of Oscillatoria and Calothrix and cells of Microcystis and Dactylococcopsis may be broken using lysozyme (EC 3.2.1.17, 0.5 mg ml -~) in a Tris-HCl buffer (pH 8.0) containing 100 mM EDTA. Alternatively, late exponential cultures of Microcystis are harvested after growth for 5 hr in the presence of benzylpenicillin (potassium salt, Sigma, II A. E. Walsby, Proc. R. Soc. London, Ser. B 223, 177 (1984). 12 A. E. Walsby, Proc. R. Soc. London, Ser. B 178, 301 (1971). 13 D. L. Worcester, Brookhaven Symp. Biol. 27, 37 (1975). ~4A. E. Walsby, in "Relations between Structure and Function in the Prokaryotic Cell" (R. Y. Stanier, H. J. Rogers, and J. B. Ward, eds.), Symp. Soc. Gen. Microbiol. No. 28, p. 327. Cambridge Univ. Press, Cambridge, England, 1978. 15 A. E. Walsby, Proc. R. Soc. London, Ser. B 216, 355 (1982). 16 A. E. Walsby, this series, Vol. 31, p. 678. 17 A. E. Walsby, J. van Rijn, and Y. Cohen, Proc. R. Soc. London, Ser. B 217, 417 (1983).

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[20]

200-250 U liter -1) and I mM Mg 2+, and the cells are ruptured, after infiltration with 1 M glycerol for 15 min, by rapid dilution with 3 volumes of 20 mM Tris-HC1 (pH 7.7) and standing at 4 ° for 2 hr. Shallow layers of cell lysate (2-3 cm deep) are overlaid with 0.1 M phosphate buffer (pH 7.0) to a depth of about 0.5 cm. Intact gas vesicles are collected by centrifugation up through the buffer layer at 4 °. The rate of gas vesicle rise varies with both the viscosity of the fluid through which they are traveling and the centrifugal acceleration to which they are exposed. We have measured rates of rise of about 30/zm hr -~ g-1 for gas vesicles in water and of about 18 tzm hr -l g-~ for gas vesicles in the more viscous 0.6 M sucrose. After centrifugation the accumulated surface layer 'of gas vesicles is harvested using a syringe fitted with a narrow-gauge needle. Most impurities are removed by repeated centrifugation through phosphate buffer at 4 °. Any unlysed, buoyant cells contaminating the gas vesicle preparation are removed by filtration through 0.6-/zm pore size Nucleopore membranes. When large numbers of such cells are present it is advisable to remove most of them by centrifuging shallow (<0.5 cm) layers of the impure gas vesicle preparation at about 14,700 m sec -z (1500 g). During this procedure the gas vesicles (which have a density of between 100 and 160 kg m -3) form a well-defined layer above the cells (which have a density of - 9 9 6 kg m-3). Most of the cells can be removed from below the gas vesicle layer using a syringe and needle; those remaining are removed by filtration. Further purification of gas vesicles can often be achieved after a brief exposure to 0.2% (w/v) sodium dodecyl sulfate (SDS). The detergent disrupts contaminating thylakoid membranes which seem to anneal around gas vesicles to form buoyant globules. This treatment weakens the gas vesicles, but the weakening is reversed by dilution of the SDS.18 The SDS concentration should be reduced to less than 0.002% (w/v) by addition of phosphate buffer prior to the final centrifugal concentration step. An SDS wash should only be included in the purification procedure after testing the stability of the gas vesicles in this detergent; for example, the gas vesicles of Oscillatoria and Dactylococcopsis are unstable in SDS, and higher concentrations will remove GVPc from gas vesicles of Anabaena and Microcystis. 1o Measurement of Gas Vesicle Concentration 14 Gas Volume Measurements

The volume of gas vesicle gas space in a suspension of vesicles can be determined by measuring the reduction in the volume of that suspension r8 A. E. Walsby and R. E. Armstrong, J. Mol. Biol. 129, 279 (1979).

[20]

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when the vesicles are collapsed by applying pressure exceeding the maximum Pc. This is best done using the compression tube ~5 described by Oliver and Walsby ([55] in this volume), but specific gravity bottles may also be used as follows. Small ( - 0 . 5 ml) specific gravity bottles are made from 0.9-mm internal diameter glass capillary tubing with a bulb at one end and a neck of about 50 mm length at the other. The volume of the bottle is calculated from the weight of water needed to fill it to a calibration mark scratched close to the open end of the neck. The bottle is filled with a gas vesicle suspension previously degassed by boiling briefly under reduced pressure. The pressure over the bottle is reduced to 0.05 MPa, and any movement in the position of the meniscus, which indicates the presence of bubbles in the bottle, is noted. If no bubbles are present the bottle and contents are weighed. The gas vesicles in the bottle are collapsed by exposure to pressure. Water is added to bring the volume back up to the calibration mark and the bottle is then reweighed. The volume of water added, and hence the volume of gas space destroyed, is calculated from the weight gain. After measurement of gas volume by either of the above methods the contents of the bottle or compression tube, with washings, are transferred to a preweighed aluminum tray and dried to constant weight in vacuo. From these measurements a relationship between gas vesicle-gas space concentration and dry weight concentration is established. For subsequent samples dry weight concentration can be calculated from a measurement of gas space concentration. The relationship also holds for gas space measurements made on gas-vacuolate cell suspensions.

Optical Measurements

For isolated gas vesicles, but not for suspensions of gas-vacuolate cells, another type of measurement can be made which will allow the calculation of gas vesicle dry weight concentration. The method involves the establishment of a relationship between dry weight concentration, determined as above, and pressure-sensitive optical density (PSOD), that is, the change in light scattering which occurs when a suspension of gas vesicles is subjected to a pressure exceeding the maximum Pc. Gas vesicle suspensions are diluted to give optical density readings, at a wavelength of 500 nm, in the range 0.2-0.6 cm -1 (in a 1-cm path length cuvette). Optical density readings are taken before and after the collapse of the gas vesicles in the cuvette. The difference between the values is the PSOD. The relationship between PSOD and dry weight concentration remains valid only if all readings are made using the same type of spectrophotometer; different arrangements of cuvette and photocell may signifi-

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cantly affect PSOD readings because the light attenuation is due to scattering rather than absorption. Gas vesicles from different species have different sizes and geometries. This means that the amount of gas space enclosed, and hence light scattered by a given amount of wall material, will vary among species. For this reason the ratios of PSOD to dry weight and of gas space to dry weight must be determined separately for each species under study. Chemistry of Gas Vesicles Protein is the sole constituent of isolated gas vesicles. Although the structures remain intact for years when stored in liquid suspension at 4°, they become much weaker and are not amenable to either electrophoretic or protein sequence analysis. Studies of gas vesicle chemistry should be performed only on freshly prepared material (see above). Electrophoresis Samples of gas vesicles are suspended in 1% SDS by boiling for 2 min. Separation of the solubilized proteins is achieved on gels with a discontinuous buffer system.19 Proteins are made visible by staining in Coomassie blue [0.2% (w/v) in 50% methanol and 7% glacial acetic acid] and destaining in 20% methanol, 7% glacial acetic acid. Silver staining procedures, 2° which have been used with other proteins, produce little or no staining reaction with GVPs. GVPc forms a mobile band of Mr 14K to 35K in different species. 9 GVPa is thought to remain at the origin. Protein Sequence Analysis Sequence information for GVP in intact gas vesicles (Fig. 1) and for GVP-derived peptides 8,2L22 (see below) has been obtained by automated amino acid sequencing on either a Beckman spinning-cup sequencer or an Applied Biosystems 470B gas-phase sequencer. Amino acid phenylthiohydantoin derivatives were identified by HPLC. All quantitative amino acid analyses were performed using a Durrum D-500 amino acid analyzer after the samples had been hydrolyzed in vacuo at 105° in 6 M HC1, 0.1% (w/v) phenol. ~9 U. K. Laemmli, Nature (London) 277, 680 (1970). 2o C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert, Science 211, 1437 (1981). 2~ j. E. Walker and A. E. Walsby, Biochem. J. 2119, 809 (1983). 22 j. E. Walker, P. K. Hayes, and A. E. Walsby, J. Gen. Microbiol. 1311, 2709 (1984).

[20]

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219

I I D R I L D K G

I

(a)

A V E K T N S S S S L A E V

IV

I D A W V R V S L V G

(b)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

(c)

. . . . . . . . . . . . . . . . . . . . . . . .

(d)

....

(e)

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

(f)

. . . . . . . . . . .

I

V . . . . . . . . . . .

V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G - - V . . . . . . .

A

. . . . . . .

V - V - L . . . . . . . . .

FIG. 1. Alignment of amino-terminal amino acid sequences for GVPa from (a) Anabaena flos-aquae CCAP 1403/13f, (b) Aphanizomenonflos-aquae CCAP 1401/1, (c) Calothrix sp. PCC 7601, (d) Oscillatoria agardhii PCC 7821, (e) Microcystis sp. BC (Bristol Collection) 84/I, and (f) Dactylococcopsis salina BC 80/4. Dashes indicate identity with the Anabaena sequence. The solid line indicates the part of the sequence used to construct an oligonucleotide probe. [The single-letter system of amino acids has been used. See Eur. J. Biochem. 138, 4 (1984).]

Digestion of GVP with Proteolytic Enzymes 8 Gas vesicles remain intact when incubated with trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), V8 protease from Staphylococcus aureus (EC 3.4.21.19, serine proteinase), proteinase K (EC 3.4.21.14), or pronase (EC 3.4.24.4). Chaotropic agents such as 6 M guanidine hydrochloride or 8 M urea also fail to disrupt the structure of intact gas vesicles. In each case the treatment results in a significant reduction in gas vesicle strength. Limited digestion of GVP with trypsin [tosylphenylalanylchloromethane (TPCK) treated, Sigma] can be obtained after denaturation of GVP with formic acid. Freeze-dried gas vesicles are dissolved in 99% formic acid and then dialyzed at 4 °, first against distilled water and then against 2 M urea (deionized in Amberlite MB1 and MB3 ion-exchange resins) in 50 mM ammonium hydrogen carbonate. The denatured protein is digested with trypsin (stock, 1 mg ml-~ in 1 mM HC1) at a substrate-toenzyme ratio of 40 : 1 (w/w) for periods of up to 24 hr at 37°. Reaction products are initially separated on a Sephadex G-50 column (100 x 2 cm) in 50 mM ammonium hydrogen carbonate. Peptide-containing fractions,

220

ULTRASTRUCTURE, INCLUSIONS, AND DIFFERENTIATION

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identified by their absorbance at 225 nm, are further fractionated by HPLC. We use a CI8 reversed-phase column (Macherey, Nagel and Co.) eluted with a linear gradient from 0.1% (v/v) trifluoroacetic acid to 100% acetonitrile. To digest with pepsin (EC 3.4.23.1) freeze-dried gas vesicles are dissolved in 99% formic acid and diluted with 40 volumes of an enzyme stock solution prepared in 1 mM HC1 to give a final substrate-to-enzyme ratio of about 50 : 1 (w/w). The digestion is carried out at room temperature for up to 24 hr. Saturated ammonium hydrogen carbonate is then added to give a pH of about 7.5, and the soluble reaction products separated as above. Succinylated (3-carboxypropionylated) proteins when digested with trypsin are cleaved only after arginine residues and not after the modified lysine residues. Succinylated GVP is prepared as follows. Formic aciddenatured GVP is dialyzed against distilled water. Guanidine hydrochloride is dissolved in the solution of denatured protein to give a final concentration of 6 M. A 50-fold molar excess of succinic anhydride is slowly stirred into the solution while holding the pH between 8.5 and 9.5 with small additions of 0.5 M NaOH. The reaction products are dialyzed first against 0.5% acetic acid, then against distilled water, and finally against 50 mM ammonium hydrogen carbonate. The succinylated protein can be digested with trypsin as above.

Gene Isolation Application of standard techniques has allowed the isolation and sequencing of structural genes which code for GVPs in Calothrix PCC 7601, 23,24 an organism which produces gas vesicles only under special conditions, and Anabaena.9 Recombinant clones carrying the genes were identified using a synthetic oligonucleotide (29-mer) made to code for a 10-amino acid section of the published protein sequence (Fig. 1). 22 Using similar techniques, we have failed to isolate the corresponding gene from Anabaenaflos-aquae CCAP 1403/13f, an organism which unlike Calothrix PCC 7601 produces gas vesicles constitutively, but we have obtained a clone containing DNA from Anabaenaflos-aquae CCAP 1403/13d (a mutant derived from CCAP 1403/13f which does not express GVP or produce gas vesicles 25) which carries closely linked gyp genes encoding the GVPa and GVPc proteins. 23 N. Tandeau de Marsac, D. Mazel, D. A. Bryant, and J. Houmard, Nucleic Acids Res. 13, 7223 (1985). 24 T. Damerval, J. Houmard, G. Guglielmi, K. Csiszar, and N. Tandeau de Marsac, Gene 54, 83 (1987). 25 A. E. Walsby, Arch. Microbiol. 114, 167 (1977).

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221

Electron Microscopy Isolated gas vesicles can be seen in the transmission electron microscope after negative staining, 2 but for size measurements they should be shadowed. 3,26 For shadowing, gas vesicles are suspended in 33% (v/v) glycerol to a final concentration of 0.5 mg ml -~ and then collapsed by application of pressure. The suspension is sprayed through an atomizer (Sigma spray) onto freshly cleaved mica and dried in a vacuum desiccator. Dried specimens are shadowed with platinum/carbon from an angle of 45 ° and coated with carbon from above. We use an Edwards 12E6/178 shadowing unit fitted with Cressington EH5 electron guns connected to a Cressington E6602 PC power supply. For accurate measurements of gas vesicle dimensions it is essential to have a reliable estimate of microscope magnification. The nominal magnification reading taken from the microscope may be in error by as much as 15% or more. For shadowed specimens we mix latex spheres of standard diameter (Agar Aids) with the gas vesicle suspension to act as internal size standards. Where a microscope specimen holder has positions for more than one grid a better estimate of microscope magnification can be obtained using a carbon replica of a diffraction grating. 3,26 After photographing each gas vesicle specimen a grid holding the diffraction grating replica is moved into the electron beam. The image is focused using the vertical adjustment on the specimen stage and not by the lens focus controls as any alteration in lens current will affect the final magnification. After focusing a photograph is taken. The magnification on the negative is calculated by measuring the spacing of the rulings on the diffraction grating. The grating periodicity should be calibrated as the manufacturers values may be in error by up to 3%. (We have calibrated gratings by measuring the images of an identifiable grating grid square on light and electron micrographs and measuring the distance between 100 grating rulings on the latter.) Elastic Compressibility of Gas Vesicles Measurements of gas vesicle elastic compressibility are relevant to the theory of critical pressures and the discussion of buoyancy at depth. They are made using a compression tube apparatus is (see Fig. 1 in [55] in this volume). The inner tube, volume Vi, is filled with a concentrated suspension of gas vesicles. The position of the meniscus in the capillary is noted, and its movement is measured using a vernier microscope when the outer tube is pressurized. The pressure is not allowed to reach a value at which 26 M. Jost and D. D. Jones, Can. J, Microbiol. 16, 159 (1970).

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any collapse of gas vesicles occurs. The volumetric compression of the gas vesicle suspension is calculated as the area of capillary cross section multiplied by the change in meniscus position when the apparatus was placed under pressure. The tube is filled with water and the above procedure repeated. The compression of the gas vesicles (dVg) per 0.1 MPa is calculated from the equation

dVg = dVt

-

-

dVw + dV~

where dVt is the total compression per 0.1 MPa for the tube filled with the gas vesicle suspension, dVw the compression due to water, and dVi the compression in the tube itself. The last two terms are estimated from the measurements made with the tube filled with water; dV~ is calculated equal to -PVi/K, where K is the elastic modulus of glass (46 GPa) and P the applied pressure.15 Dividing dVg by the volume of the gas vesicles in the suspension TM gives the elastic bulk modulus for the intact gas vesicle. The value for Anabaena gas vesicles is 64.5 MPa. From this value the elastic modulus of GVP can be calculated, 2.7 GPa.~5 Measurements are now needed on gas vesicles from other species.

[21] C e l l u l a r D i f f e r e n t i a t i o n : A k i n e t e s By M I C H A E L HERDMAN

Although often referred to as "spores," particularly in the early literature, there is now general agreement that the word akinete (Greek: akinetos, motionless) is suitable to distinguish this differentiated resistant cell from the spores, cysts, endospores, and exospores of other microorganisms and from the baeocytes ~of pleurocapsalean cyanobacteria. Akinetes are the subject of several recent reviews, 2-4 and this chapter describes only some practical aspects of their identification, production in pure culture; isolation, and properties. Although the nomenclature of cyanoM. Herdman and R. Rippka, this volume [22]. 2 j. M. Nichols and N. G. Carr, in " S p o r e s " (C. H. Chambliss and J. C. Vary, eds.), Vol. 7, p. 335. Am. Soc. Microbiol., Washington, D.C., 1978. 3 j. M. Nichols and D. G. Adams, in "The Biology of Cyanobacteria" (N. G. Cart and B. A. Whitton, eds.), p. 387. Blackwell, Oxford, England, 1982. 4 M. Herdman, in "The Cyanobacteria: A Comprehensive Review" (P. Fay and C. Van Baalen, eds.), p. 227. Elsevier, Amsterdam, 1987.

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