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[70] The isolation of gas vesicles from blue-green algae
678
PREPARATIONSDERIVED FROM UNICELLULAR ORGANISMS
[70]
The supernatant of the EDTA-treated E-0.1 fraction is designated the yellow protein fraction. Its buoyant density in a CsC1 gradient is 1.33 g / c m 3, typical for proteins. The UV-absorption maximum is at 270 nm. In the analytical ultracentrifuge in 0.1 M NaC1, 0.01 M sodium E D T A p H 7.3, a symmetrical peak is observed corresponding to a sedimentation constant s 20,w o = 5.9 × 10 -13 (see) and a diffusion coefficient D 20,w O = 2.2 × 107 (cm2/second). In acrylamide disc gel electrophoresis, a main band is seen after amido black staining which is not identical with a smaller yellow band. The yellow color is apparently due to the flavin and cytochrome pigments in the yellow band. The yield of material in the yellow band may vary considerably from one preparation to the next. The yellow protein fraction may therefore appear colorless. The fractionation of cell envelopes is summarized in Fig. 7. Note Added in Proof: Since the article has been written a function of the purple membrane as a light energy transducing system has been suggested. The reader is referred to the following articles: D. Oesterhelt and B. Hess. Reversible photolysis of the purple complex in the purple membrane of Halobacterium halobium. Eur. J. Biochem. 37, 316-326 (1973). D. Oesterhelt and G. Krippahl. Light inhibition of respiration in Halobacterium halobium. F E B S Letts. 36, 72-76 (1973). D. Oesterhelt and W. Stoeckenius. Functions of a new photoreceptor membrane. Proc. Nat. Acad. Sci. 70, 2853-2857 (1973). E. Racker and W. Stoeckenius. Reconstitution of purple membrane vesicles catalyzing light-driven proton uptake and adenosine triphosphatase formation. J. Biol. Chem. 249, 662 (1974).
[70] The Isolation of Gas Vesicles from Blue-Green By
Algae
A. E. WALSBY
Gas vesicles are hollow, cylindrical, or spindle-shaped structures which, stacked in a regular hexagonal fashion or in loose clusters, comprise the gas vacuoles found only in blue-green algae and bacteria. 1,2 Each gas vesicle consists simply of a rigid wall of protein, 3,4 about 2 nm thick, which entirely surrounds a space devoid of liquid or solid contents. Gases diffuse 'G. 2A. 'D. "D.
Cohen-Bazire, R. Kunisawa, and N. Pfennig, J. Bacteriol. 100, 1049 (1969). E. Walsby, Bacteriol. Rev. 36, 1 (1972). D. Jones and M. Jost, Arch. Mikrobiol. 70, 43 (1970). D. Jones and M. Jost, Planta 100, 277 (1971).
[70]
THE ISOLATION OF GAS VESICLES FROM BLUE-GREEN ALGAE 679
freely to and fro across the wall between the space and the surrounding solution. ~ When subjected to a sufficient pressure, known as the critical pressure [which may vary from less than 200 to more than 600 kN m -2 ( 2 - 6 atm)] the gas vesicles collapse irreversibly to flattened, envelopelike structures, the gas they contained diffusing away into the surrounding solution as this happens2 It is feasible to recover collapsed vesicles by standard, densitygradient centrifugation techniques, 7 but the preparations so obtained are not easily purified 3 and are of limited usefulness. Using special precautions to avoid exposing gas vesicles to pressures in excess of their critical pressure they may be isolated and purified in an intact state. 8 Such precautions are not required in the preparation of any other subcellular structures and particular emphasis is given to this aspect here. Determination of Critical Pressures Since several of the stages in the isolation procedure may involve the generation of pressure, it is first essential to determine the pressure that the gas vesicles will withstand. Their collapse in aqueous suspension is accompanied by a marked decrease in turbidity, and nephelometry or colofimetry may be used to assess the degree of collapse ensuing application of any given pressure2 A test tube of the algal or gas vesicle suspension is placed in a simple, air-tight chamber capable of withstanding about 2 MN m -2 (20 atm) pressure2 The chamber is connected, via high-pressure hose, to a cylinder of compressed air or nitrogen which will deliver gas to 1.4, MN m -2 (about 200 psi). The suspension is subjected to a pressure of 50 kN m -2 for 30 seconds, and the turbidity (or absorbance) is measured. This process is repeated at steps of 50 or 100 kN m -2 up to 1 MN m -2 (or until there is no further turbidity change). The percentage gas vesicle collapse at any pressure is then equal to (x - - y ) / ( x
-- z) X 100
where x is the turbidity of the untreated suspension (with gas vesicles), y of the treated suspension, and z of the suspension in which all the gas vesicles have been collapsed. Pressure-collapse curves are plotted as shown in Fig. 1. As seen in the figure, the mean critical pressure of the isolated vesicles appears greater than that of the vesicles inside algal cells from the same sample. This is 5A. E. Walsby, Proc. Roy. Soc. Ser. B 173, 235 (1969). "A. E. Walsby, Proc. Roy. Soc. Ser. B 178, 301 (1971). ~W. Stoeckenius and W. H. Kunau, J. Cell Biol. 38, 336 (1968). * A. E. Walsby and B. Buckland, Nature (London) 224, 716 (1969).
680
PREPARATIONS DERIVED FROM UNICELLULAR ORGANISMS
[70]
0
2o
° u
40
8(
100
I
I 2110
400
600
pressure applied (kN m - 2 )
FIG. 1. Collapse of gas vesicles with pressure. Curve a, vesicles inside turgid cell;
curve b, vesicles isolated from the same cells. The mean distance between the two curves is equivalent to the cell turgor pressure. Figure modified from A. E. Walsby, Proc. Roy. Soc. Ser. B 178, 301 (1971). because those vesicles inside the cells are already subjected to cell turgor pressure (equal to the mean separation between the two curves, a and b).6 The curves shown are fairly typical for blue-green algae, and in the absence of equipment for determining critical collapse pressures, could be used as a rough guide for deciding the acceptable pressure limits in subsequent stages of the procedure. Concentrating Cell Suspensions Concentrated cell suspensions, required for quantitative gas vesicle preparations, may be obtained in the following ways. Flotation. Gas vesicles render algal cells buoyant if they occupy a sufficient proportion of the cell volume. If cultures or samples gathered from water blooms are allowed to stand, the alga forms a cream at the surface which may be drawn off with a fine syringe needle, attached to an evacuated vessel, held in contact with the meniscus. This method may be used to select for ceils of high buoyancy, , usually associated with a high degree of gas vacuolation. 8 Accelerated Flotation. The flotation process may be accelerated by centrifugation as long as the pressure generated (see p. 682) does not exceed the apparent critical pressure of vesicles in the turgid cells (Fig. l, curve a). Gas-vacuolate cells heavier than water may also be recovered, as a pellet, in this way without further loss of gas vesicles. Filtration. Algal slurries may be further concentrated by filtration, col-
[70]
THE ISOLATION OF GAS VESICLES FROM BLUE-GREEN ALGAE
681
lecting the cells or filaments on a sintered-glass filter of wide diameter (100 mm) and small pore size (5-15 /zm). Continuous scraping with a rubber spatula helps to prevent the filter from becoming clogged) Lysing the Algal Cells Techniques often employed in breaking open algal cells, such as ultrasonication and use of a French pressure cell cannot be used because they develop very high pressures on the vesicle walls. Osmotic shock can be used only after weakening the cells to the point that they burst below critical pressure of the gas vesicles. The following methods are recommended for different algae. Osmotic Shrinkage. When placed in strongly hypertonic sucrose solutions, blue-green algal cells lose water and shrink, usually without plasmolyzing. The cell wall, which seems to be firmly attached to the plasmolemma, is placed under tension and tends to rupture. Osmotic shrinkage is particularly effective in lysing algae of the Anabaena and Nostoc type, in which the filaments show a weakness where they are constricted at the septa; it is somewhat less effective with algae of the Oscillatoria type, with filaments of uniform diameter, and only partially effective with unicellular forms, such as Microcystis and Coelosphaerium. The algal slurry should be mixed rapidly with a concentrated solution of sucrose, giving a final concentration of 0.7 M. Rapid mixing is not possible with crystalline sucrose. Equal volumes of alga and 1.4 M sucrose are recommended. Most of the lysis occurs in the first 10-15 minutes, but it may be necessary to leave the mixture stirring for 1 or 2 hours at room temperature to achieve maximum lysis. Osmotic Shock o/Penicillin-Weakened Cells. Penicillin will weaken the walls of algal cells that are actively growing and dividing, by inhibiting the incorporation of the mucopeptides (responsible for tensile strength) into the growing walls) The cells can then be broken by osmotic shock after infiltrating with glycerol, before large turgor pressures are generated, collapsing the gas vesicles. Jones and Jost 3 recommended incubating cells (5 × 107 per milliliter) in the presence of benzylpenicillin (K salt; Sigma, 200-250 units liters -1) and Mg ~÷ (1 mM) for 5 hours, concentrating by filtration, and resuspending in glycerol (1 M) for 15 minutes. The infiltrated cells are then mixed rapidly with 3 volumes of a Tris .HC1 buffer (0.02 M), pH 7.7, and stored for 2 hours at 4 C. This method is more suitable for Microcystis and other unicells than that of osmotic shrinkage, but the sensitivity of cells to penicillin is greatly ~R. Y. Stanier and C. B. van Niel, Arch. Mikrobiol. 42, 17 (1962).
682
PREPARATIONSDERIVED FROM UNICELLULAR ORGANISMS
[70]
diminished with alga in the stationary phase of growth, when gas vacuolation is often highest. 1°,11 Lysozyme Treatment. Lysozyme (muramidase) attacks the mucopeptide components of prokaryotic cell walls. In hypotonic solutions lysis follows as a consequence of osmotic swelling) Biggins lz suggested using a final enzyme concentration of 0.05 % ( w / v ) of muramidase (Worthington Biochemical Corporation, Freehold, New Jersey) and incubating at 35 C, (although higher concentrations should be used with dense algal slurries). Incubation times in excess of 3 hours give extensive cell rupture. The method can be combined with subsequent glycerol infiltration and osmotic shock (see above), and is generally effective with cells at all stages of growth. Gas vesicles, whose walls are made only of protein, ~ are not attacked by lysozyme. The gas vesicles released by cells broken in these ways retain their characteristic shape, fine structure, 1'~ and dimensions. 1~ Centrifugally Accelerated Flotation of Gas Vesicles Intact gas vesicles have a density (of about 100 kg m-~) 2 much lower than that of any other cell components, and despite their small size float up more rapidly. The process can be accelerated by centrifugation and, by Stokes' law, the rate of rise will be proportional to a, the acceleration. Pressure Considerations. The acceleration will generate a pressure, p, equal to hpa, where h is the depth below the meniscus of the liquid and p its density. If maximum recovery is required the pressure at the bottom of the centrifuge cup (where it is greatest) should not exceed the critical pressure of the weakest gas vesicles s (determined from curve b in Fig. 1 ). For this reason it is advantageous to employ small values of h; this will, at the same time, reduce the time taken for gas vesicles to rise from the bottom of the cup to the meniscus. On the other hand, it decreases the volume of suspension that can be treated. To counteract this, centrifuge vessels of the widest available cross-sectional area should be used. The ideal solution is a zonal rotor head of wide diameter and large cylinder depth. The maximum acceleration permitted with vesicles of different minimum critical pressure and different values of h, may be computed from the data in Fig. 2. Alternatively, the value may be calculated as follows. Sample Calculation. Aliquots of algal lysate in 0.7 M sucrose, and showing the pressure-collapse characteristics of curve b in Fig. 1, formed ~°R. V. Smith and A. Peat, Arch. Mikrobiol. 58, 117 (1967). aH. Lehmann and M. Jost, Arch. Mikrobiol. 79, 59 (1971). ~J. Biggins, Plant Physiol. 42, 1442 (1967). tSA. E. Walsby and H. H. Eichelberger, Arch. Mikrobiol. 60, 76 (1968). ~4M. Jost and D. D. Jones, Can. J. Microbiol. 16, 159 (1970).
[70]
THE ISOLATION OF GAS VESICLES FROM BLUE-GREEN ALGAE 683
5
4 X
X
i
E 3--
.o_ =
(J
2
1
I 2
I 0
I
I 4
I
I I I 6 8 d e p t h (cm)
I
I 10
I
I 12
0
FIG. 2. The centrifugal acceleration a (given in m sec-~ on the left, and gravities on the right) generating a pressure of 100 kN m -~ (approximately 1 atm) at different depths in a 0.7 M sucrose solution, having a density of 1090 kg m -3. The pressure generated is directly proportional to a. Hence for other values of pressure, p in kN m -~, multiply the acceleration given by p/100. Figure modified from B. A. Buckland, Ph.D. Thesis, University of London (1971).
layers 50 m m deep in the centrifuge bottles. T h e highest p e r m i s s i b l e g is given b y a = p/hp
where h = 50 m m = 50 × 10 -3 m; o = 1.09 g c m -a = 1090 kg m -a ( d e n sity of 0.7 M s u c r o s e ) ; p = 200 × 103 N m -2 at b a s e of cup. a
200 X 103 N m -2 50 X 10-3 m X 1090 kg m -3 = 3670 m sec -2 (390 gravities)
Since the acceleration p r o d u c e d varies linearly with distance from the rotor spindle, this value of a should be applied at the position r' ( e q u a l to r h/2, where r is the distance from the rotor spindle to the base of the centrifuge b o t t l e ) . F o r example, given that r' is 150 m m , the rotor speed, n, generating the required acceleration is given by
684
PREPARATIONS DERIVED F R O M U N I C E L L U L A R ORGANISMS
[70]
a
n =
(2rr) 2rt /
3670 m sec-2 (2r) 2 X 1.15 m
= 25 sec-1 (1500 rpm)
Ancillary Considerations 1. From curve b of Fig. l, it is seen that only 20% of the gas vesicles collapse at 400 kN m -2, twice the minimum critical pressure. This pressure applied at the base of the cup will permit a doubling of a, and the resultant loss of vesicles will be considerably less than 20% (in fact only about 3.2% ) because the pressure generated decreases with both h and g decreasing toward the surface. 2. Despite the fact that it increases h, it is highly advantageous to layer water (to a depth of 5 - 1 0 ram) over the sucrose-containing lysate2 This layer serves to rinse the vesicles free of soluble contaminants as they rise to the surface and allows them to separate from particles rising more slowly from the surface of the lysate. 1~ 3. Both the water layer and the lysate should be buffered if their pH falls outside the range of 6 to 9.5, as this is the region of the vesicles' maximum stability. 1~ 4. As the gas vesicles rise toward the surface, the effective value of h decreases, allowing a to be increased accordingly. The rate of rise depends on the viscosity of the medium and has been measured as 0.85 and 2.04 m sec -1 for a 1.0 m sec -2 field of acceleration in 0.7 M sucrose and water, respectively. 1~ From these figures the rate at which the acceleration can be increased may be calculated (see example given by Buckland and Walsby~). 5. The preparation should not be centrifuged for a time greater than that required to bring all gas vesicles to the surface. 15 This is mainly because with time other particles which are lighter than water may float up and contaminate the vesicle layer. Also gas vesicles left in an unpurified state tend to become weaker on standing, and may collapse. 6. The weakening of gas vesicles, which is apparently due to enzymatic attack, is greatly diminished in the cold (5 C), at which temperature the lysate should be kept in all stages following lysis. Subsequent Centri]ugation Steps. The surface cream of gas vesicles is removed, after completing the first centrifugation, by using a 20 ml-capacity syringe fitted with a fine, square-ended needle held in contact with the 1~B. Buckland and A. E. Walsby, Arch. Mikrobiol. 79, 327 (1971).
[70]
THE ISOLATION OF GAS VESICLES FROM BLUE-GREEN ALGAE
685
meniscus. The vesicles are drawn off in the smallest practicable volume. An air space is kept in the syringe and the vesicle suspension is expressed from it with only slight pressure, to avoid any risk of collapse. This supernatant fraction is diluted with water (or buffer as necessary), preferably at least 10-fold, the volume being determined by that which can reasonably be centrifuged at the next stage. The same considerations determine the acceleration used, but less time will be required as the viscosity of the preparation is decreased. The centrifugation procedure is repeated once or twice more, until a fairly white preparation is obtained. Filtration The two principal contaminants left by centrifugation are intact gasvacuolate cells or protoplasts which float up with the vesicles, and soluble substances (mainly sucrose) which diffuse into the surface layers. These contaminants are removed in a two-stage filtration, a5 1. The suspension is put through a membrane filter (Sartorius, or other makes) of 50 mm diameter, having pores of 1.2 ~m diameter. Cells and protoplasts are held back; the gas vesicles pass through. Just before the filter dries, wash with about 5 ml of water, adding drop-by-drop. 2. Transfer the gas vesicle-containing filtrate to a membrane filter of similar diameter, but of 0.05 ~tm pore size. Use only a moderate vacuum ( - 30 kN m 2) to draw the suspending solution through, leaving the gas vesicles on the filter. They may be rinsed several times with water or buffer, but do not allow the filter to dry: the pressures set up by surface tension at the surface of drying films can easily collapse gas vesicles. 6 Final Centrifugation The preparation is improved by a final centrifugation which serves to separate the veslcles from other particulate matter which might have been released from C~lls breaking at the first filtration. The standard method described above (after resuspending in a large volume of water) is satisfactory. Alternatively, Jones and Jost 3 recommend placing the concentrated vesicles in a narrow layer at the surface over concentrated sucrose and centrifuging at ver3~ high accelerations (200,000 g). If this is done, gas vesicles having a criticdl pressure of 500 kN m -2 will survive only in the top 0.25 mm. Accordingly, the rotor should first be spun for sufficient time at a low acceleration to bring the vesicles very close to the meniscus. Efficiency of ~he Above Method Using the ~ethod described above, Buckland and Walsby 15 record recoveries of between 50 and 60% (from turbidity measurements) and a
686
PREPARATIONSDERIVED FROM UNICELLULARORGANISMS
[71]
percentage purity of 97.6 for the final preparation (according to the results of a radioactive labeling experiment). Storing the Gas Vesicles Bacterial growth in crude lysates results in substantial losses over a period of days, apparently due to enzymatic attack on the gas vesicle protein. However, in a highly purified state, aqueous suspensions of intact vesicles may be stored at 5 C for a year or more without appreciable loss. Vesicles can also be frozen, freeze-dried and then kept in a dried state indefinitely. On simply adding water a suspension of intact vesicles may be reconstituted. 1~ Unfortunately, although drying itself has no adverse effects on the vesicles, pressures developed during the freezing process result in substantial weakening and collapse (50% or more). It is known from freeze-etching studies 16,17 that gas vesicles will survive intact when frozen rapidly in liquid Freon held at near liquid nitrogen temperatures. It may be possible to scale up such procedures for the quantitative preparation of frozen and freeze-dried intact vesicles. ~eM. Jost, Arch. Mikrobiol. 50, 211 (1965). "J. R. Waaland and D. Branton, Science 163, 1339 (1969).
[71] T h e I s o l a t i o n of t h e A m o e b a P l a s m a M e m b r a n e a n d t h e Use of L a t e x B e a d s for t h e I s o l a t i o n of P h a g o c y t i c Vacuole (Phagosome) Membranes from Amoebae Including the Culture Techniques for A m o e b a e B y EDWARD D. KORN A c a n t h a m o e b a castellanii I is one of a group of small soil amoebae that can be grown axenically on soluble medium. Because the amoebae are nutritionally dependent on the processes of phagocytosis and pinocytosis, they provide an unusual opportunity for studying the mechanisms of membrane fission and fusion, the relationship between the plasma membrane and the membrane of the phagocytic vacuole (phagosome), and the dynamics and biochemistry of membrane movements.
Culture Techniques A defined culture medium which will support growth has been described, 2 but for most purposes the soluble culture medium3 shown in 'R. J. Neff, J. Protozool. 4, 176 (1957).
PREPARATIONSDERIVED FROM UNICELLULAR ORGANISMS
[70]
The supernatant of the EDTA-treated E-0.1 fraction is designated the yellow protein fraction. Its buoyant density in a CsC1 gradient is 1.33 g / c m 3, typical for proteins. The UV-absorption maximum is at 270 nm. In the analytical ultracentrifuge in 0.1 M NaC1, 0.01 M sodium E D T A p H 7.3, a symmetrical peak is observed corresponding to a sedimentation constant s 20,w o = 5.9 × 10 -13 (see) and a diffusion coefficient D 20,w O = 2.2 × 107 (cm2/second). In acrylamide disc gel electrophoresis, a main band is seen after amido black staining which is not identical with a smaller yellow band. The yellow color is apparently due to the flavin and cytochrome pigments in the yellow band. The yield of material in the yellow band may vary considerably from one preparation to the next. The yellow protein fraction may therefore appear colorless. The fractionation of cell envelopes is summarized in Fig. 7. Note Added in Proof: Since the article has been written a function of the purple membrane as a light energy transducing system has been suggested. The reader is referred to the following articles: D. Oesterhelt and B. Hess. Reversible photolysis of the purple complex in the purple membrane of Halobacterium halobium. Eur. J. Biochem. 37, 316-326 (1973). D. Oesterhelt and G. Krippahl. Light inhibition of respiration in Halobacterium halobium. F E B S Letts. 36, 72-76 (1973). D. Oesterhelt and W. Stoeckenius. Functions of a new photoreceptor membrane. Proc. Nat. Acad. Sci. 70, 2853-2857 (1973). E. Racker and W. Stoeckenius. Reconstitution of purple membrane vesicles catalyzing light-driven proton uptake and adenosine triphosphatase formation. J. Biol. Chem. 249, 662 (1974).
[70] The Isolation of Gas Vesicles from Blue-Green By
Algae
A. E. WALSBY
Gas vesicles are hollow, cylindrical, or spindle-shaped structures which, stacked in a regular hexagonal fashion or in loose clusters, comprise the gas vacuoles found only in blue-green algae and bacteria. 1,2 Each gas vesicle consists simply of a rigid wall of protein, 3,4 about 2 nm thick, which entirely surrounds a space devoid of liquid or solid contents. Gases diffuse 'G. 2A. 'D. "D.
Cohen-Bazire, R. Kunisawa, and N. Pfennig, J. Bacteriol. 100, 1049 (1969). E. Walsby, Bacteriol. Rev. 36, 1 (1972). D. Jones and M. Jost, Arch. Mikrobiol. 70, 43 (1970). D. Jones and M. Jost, Planta 100, 277 (1971).
[70]
THE ISOLATION OF GAS VESICLES FROM BLUE-GREEN ALGAE 679
freely to and fro across the wall between the space and the surrounding solution. ~ When subjected to a sufficient pressure, known as the critical pressure [which may vary from less than 200 to more than 600 kN m -2 ( 2 - 6 atm)] the gas vesicles collapse irreversibly to flattened, envelopelike structures, the gas they contained diffusing away into the surrounding solution as this happens2 It is feasible to recover collapsed vesicles by standard, densitygradient centrifugation techniques, 7 but the preparations so obtained are not easily purified 3 and are of limited usefulness. Using special precautions to avoid exposing gas vesicles to pressures in excess of their critical pressure they may be isolated and purified in an intact state. 8 Such precautions are not required in the preparation of any other subcellular structures and particular emphasis is given to this aspect here. Determination of Critical Pressures Since several of the stages in the isolation procedure may involve the generation of pressure, it is first essential to determine the pressure that the gas vesicles will withstand. Their collapse in aqueous suspension is accompanied by a marked decrease in turbidity, and nephelometry or colofimetry may be used to assess the degree of collapse ensuing application of any given pressure2 A test tube of the algal or gas vesicle suspension is placed in a simple, air-tight chamber capable of withstanding about 2 MN m -2 (20 atm) pressure2 The chamber is connected, via high-pressure hose, to a cylinder of compressed air or nitrogen which will deliver gas to 1.4, MN m -2 (about 200 psi). The suspension is subjected to a pressure of 50 kN m -2 for 30 seconds, and the turbidity (or absorbance) is measured. This process is repeated at steps of 50 or 100 kN m -2 up to 1 MN m -2 (or until there is no further turbidity change). The percentage gas vesicle collapse at any pressure is then equal to (x - - y ) / ( x
-- z) X 100
where x is the turbidity of the untreated suspension (with gas vesicles), y of the treated suspension, and z of the suspension in which all the gas vesicles have been collapsed. Pressure-collapse curves are plotted as shown in Fig. 1. As seen in the figure, the mean critical pressure of the isolated vesicles appears greater than that of the vesicles inside algal cells from the same sample. This is 5A. E. Walsby, Proc. Roy. Soc. Ser. B 173, 235 (1969). "A. E. Walsby, Proc. Roy. Soc. Ser. B 178, 301 (1971). ~W. Stoeckenius and W. H. Kunau, J. Cell Biol. 38, 336 (1968). * A. E. Walsby and B. Buckland, Nature (London) 224, 716 (1969).
680
PREPARATIONS DERIVED FROM UNICELLULAR ORGANISMS
[70]
0
2o
° u
40
8(
100
I
I 2110
400
600
pressure applied (kN m - 2 )
FIG. 1. Collapse of gas vesicles with pressure. Curve a, vesicles inside turgid cell;
curve b, vesicles isolated from the same cells. The mean distance between the two curves is equivalent to the cell turgor pressure. Figure modified from A. E. Walsby, Proc. Roy. Soc. Ser. B 178, 301 (1971). because those vesicles inside the cells are already subjected to cell turgor pressure (equal to the mean separation between the two curves, a and b).6 The curves shown are fairly typical for blue-green algae, and in the absence of equipment for determining critical collapse pressures, could be used as a rough guide for deciding the acceptable pressure limits in subsequent stages of the procedure. Concentrating Cell Suspensions Concentrated cell suspensions, required for quantitative gas vesicle preparations, may be obtained in the following ways. Flotation. Gas vesicles render algal cells buoyant if they occupy a sufficient proportion of the cell volume. If cultures or samples gathered from water blooms are allowed to stand, the alga forms a cream at the surface which may be drawn off with a fine syringe needle, attached to an evacuated vessel, held in contact with the meniscus. This method may be used to select for ceils of high buoyancy, , usually associated with a high degree of gas vacuolation. 8 Accelerated Flotation. The flotation process may be accelerated by centrifugation as long as the pressure generated (see p. 682) does not exceed the apparent critical pressure of vesicles in the turgid cells (Fig. l, curve a). Gas-vacuolate cells heavier than water may also be recovered, as a pellet, in this way without further loss of gas vesicles. Filtration. Algal slurries may be further concentrated by filtration, col-
[70]
THE ISOLATION OF GAS VESICLES FROM BLUE-GREEN ALGAE
681
lecting the cells or filaments on a sintered-glass filter of wide diameter (100 mm) and small pore size (5-15 /zm). Continuous scraping with a rubber spatula helps to prevent the filter from becoming clogged) Lysing the Algal Cells Techniques often employed in breaking open algal cells, such as ultrasonication and use of a French pressure cell cannot be used because they develop very high pressures on the vesicle walls. Osmotic shock can be used only after weakening the cells to the point that they burst below critical pressure of the gas vesicles. The following methods are recommended for different algae. Osmotic Shrinkage. When placed in strongly hypertonic sucrose solutions, blue-green algal cells lose water and shrink, usually without plasmolyzing. The cell wall, which seems to be firmly attached to the plasmolemma, is placed under tension and tends to rupture. Osmotic shrinkage is particularly effective in lysing algae of the Anabaena and Nostoc type, in which the filaments show a weakness where they are constricted at the septa; it is somewhat less effective with algae of the Oscillatoria type, with filaments of uniform diameter, and only partially effective with unicellular forms, such as Microcystis and Coelosphaerium. The algal slurry should be mixed rapidly with a concentrated solution of sucrose, giving a final concentration of 0.7 M. Rapid mixing is not possible with crystalline sucrose. Equal volumes of alga and 1.4 M sucrose are recommended. Most of the lysis occurs in the first 10-15 minutes, but it may be necessary to leave the mixture stirring for 1 or 2 hours at room temperature to achieve maximum lysis. Osmotic Shock o/Penicillin-Weakened Cells. Penicillin will weaken the walls of algal cells that are actively growing and dividing, by inhibiting the incorporation of the mucopeptides (responsible for tensile strength) into the growing walls) The cells can then be broken by osmotic shock after infiltrating with glycerol, before large turgor pressures are generated, collapsing the gas vesicles. Jones and Jost 3 recommended incubating cells (5 × 107 per milliliter) in the presence of benzylpenicillin (K salt; Sigma, 200-250 units liters -1) and Mg ~÷ (1 mM) for 5 hours, concentrating by filtration, and resuspending in glycerol (1 M) for 15 minutes. The infiltrated cells are then mixed rapidly with 3 volumes of a Tris .HC1 buffer (0.02 M), pH 7.7, and stored for 2 hours at 4 C. This method is more suitable for Microcystis and other unicells than that of osmotic shrinkage, but the sensitivity of cells to penicillin is greatly ~R. Y. Stanier and C. B. van Niel, Arch. Mikrobiol. 42, 17 (1962).
682
PREPARATIONSDERIVED FROM UNICELLULAR ORGANISMS
[70]
diminished with alga in the stationary phase of growth, when gas vacuolation is often highest. 1°,11 Lysozyme Treatment. Lysozyme (muramidase) attacks the mucopeptide components of prokaryotic cell walls. In hypotonic solutions lysis follows as a consequence of osmotic swelling) Biggins lz suggested using a final enzyme concentration of 0.05 % ( w / v ) of muramidase (Worthington Biochemical Corporation, Freehold, New Jersey) and incubating at 35 C, (although higher concentrations should be used with dense algal slurries). Incubation times in excess of 3 hours give extensive cell rupture. The method can be combined with subsequent glycerol infiltration and osmotic shock (see above), and is generally effective with cells at all stages of growth. Gas vesicles, whose walls are made only of protein, ~ are not attacked by lysozyme. The gas vesicles released by cells broken in these ways retain their characteristic shape, fine structure, 1'~ and dimensions. 1~ Centrifugally Accelerated Flotation of Gas Vesicles Intact gas vesicles have a density (of about 100 kg m-~) 2 much lower than that of any other cell components, and despite their small size float up more rapidly. The process can be accelerated by centrifugation and, by Stokes' law, the rate of rise will be proportional to a, the acceleration. Pressure Considerations. The acceleration will generate a pressure, p, equal to hpa, where h is the depth below the meniscus of the liquid and p its density. If maximum recovery is required the pressure at the bottom of the centrifuge cup (where it is greatest) should not exceed the critical pressure of the weakest gas vesicles s (determined from curve b in Fig. 1 ). For this reason it is advantageous to employ small values of h; this will, at the same time, reduce the time taken for gas vesicles to rise from the bottom of the cup to the meniscus. On the other hand, it decreases the volume of suspension that can be treated. To counteract this, centrifuge vessels of the widest available cross-sectional area should be used. The ideal solution is a zonal rotor head of wide diameter and large cylinder depth. The maximum acceleration permitted with vesicles of different minimum critical pressure and different values of h, may be computed from the data in Fig. 2. Alternatively, the value may be calculated as follows. Sample Calculation. Aliquots of algal lysate in 0.7 M sucrose, and showing the pressure-collapse characteristics of curve b in Fig. 1, formed ~°R. V. Smith and A. Peat, Arch. Mikrobiol. 58, 117 (1967). aH. Lehmann and M. Jost, Arch. Mikrobiol. 79, 59 (1971). ~J. Biggins, Plant Physiol. 42, 1442 (1967). tSA. E. Walsby and H. H. Eichelberger, Arch. Mikrobiol. 60, 76 (1968). ~4M. Jost and D. D. Jones, Can. J. Microbiol. 16, 159 (1970).
[70]
THE ISOLATION OF GAS VESICLES FROM BLUE-GREEN ALGAE 683
5
4 X
X
i
E 3--
.o_ =
(J
2
1
I 2
I 0
I
I 4
I
I I I 6 8 d e p t h (cm)
I
I 10
I
I 12
0
FIG. 2. The centrifugal acceleration a (given in m sec-~ on the left, and gravities on the right) generating a pressure of 100 kN m -~ (approximately 1 atm) at different depths in a 0.7 M sucrose solution, having a density of 1090 kg m -3. The pressure generated is directly proportional to a. Hence for other values of pressure, p in kN m -~, multiply the acceleration given by p/100. Figure modified from B. A. Buckland, Ph.D. Thesis, University of London (1971).
layers 50 m m deep in the centrifuge bottles. T h e highest p e r m i s s i b l e g is given b y a = p/hp
where h = 50 m m = 50 × 10 -3 m; o = 1.09 g c m -a = 1090 kg m -a ( d e n sity of 0.7 M s u c r o s e ) ; p = 200 × 103 N m -2 at b a s e of cup. a
200 X 103 N m -2 50 X 10-3 m X 1090 kg m -3 = 3670 m sec -2 (390 gravities)
Since the acceleration p r o d u c e d varies linearly with distance from the rotor spindle, this value of a should be applied at the position r' ( e q u a l to r h/2, where r is the distance from the rotor spindle to the base of the centrifuge b o t t l e ) . F o r example, given that r' is 150 m m , the rotor speed, n, generating the required acceleration is given by
684
PREPARATIONS DERIVED F R O M U N I C E L L U L A R ORGANISMS
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a
n =
(2rr) 2rt /
3670 m sec-2 (2r) 2 X 1.15 m
= 25 sec-1 (1500 rpm)
Ancillary Considerations 1. From curve b of Fig. l, it is seen that only 20% of the gas vesicles collapse at 400 kN m -2, twice the minimum critical pressure. This pressure applied at the base of the cup will permit a doubling of a, and the resultant loss of vesicles will be considerably less than 20% (in fact only about 3.2% ) because the pressure generated decreases with both h and g decreasing toward the surface. 2. Despite the fact that it increases h, it is highly advantageous to layer water (to a depth of 5 - 1 0 ram) over the sucrose-containing lysate2 This layer serves to rinse the vesicles free of soluble contaminants as they rise to the surface and allows them to separate from particles rising more slowly from the surface of the lysate. 1~ 3. Both the water layer and the lysate should be buffered if their pH falls outside the range of 6 to 9.5, as this is the region of the vesicles' maximum stability. 1~ 4. As the gas vesicles rise toward the surface, the effective value of h decreases, allowing a to be increased accordingly. The rate of rise depends on the viscosity of the medium and has been measured as 0.85 and 2.04 m sec -1 for a 1.0 m sec -2 field of acceleration in 0.7 M sucrose and water, respectively. 1~ From these figures the rate at which the acceleration can be increased may be calculated (see example given by Buckland and Walsby~). 5. The preparation should not be centrifuged for a time greater than that required to bring all gas vesicles to the surface. 15 This is mainly because with time other particles which are lighter than water may float up and contaminate the vesicle layer. Also gas vesicles left in an unpurified state tend to become weaker on standing, and may collapse. 6. The weakening of gas vesicles, which is apparently due to enzymatic attack, is greatly diminished in the cold (5 C), at which temperature the lysate should be kept in all stages following lysis. Subsequent Centri]ugation Steps. The surface cream of gas vesicles is removed, after completing the first centrifugation, by using a 20 ml-capacity syringe fitted with a fine, square-ended needle held in contact with the 1~B. Buckland and A. E. Walsby, Arch. Mikrobiol. 79, 327 (1971).
[70]
THE ISOLATION OF GAS VESICLES FROM BLUE-GREEN ALGAE
685
meniscus. The vesicles are drawn off in the smallest practicable volume. An air space is kept in the syringe and the vesicle suspension is expressed from it with only slight pressure, to avoid any risk of collapse. This supernatant fraction is diluted with water (or buffer as necessary), preferably at least 10-fold, the volume being determined by that which can reasonably be centrifuged at the next stage. The same considerations determine the acceleration used, but less time will be required as the viscosity of the preparation is decreased. The centrifugation procedure is repeated once or twice more, until a fairly white preparation is obtained. Filtration The two principal contaminants left by centrifugation are intact gasvacuolate cells or protoplasts which float up with the vesicles, and soluble substances (mainly sucrose) which diffuse into the surface layers. These contaminants are removed in a two-stage filtration, a5 1. The suspension is put through a membrane filter (Sartorius, or other makes) of 50 mm diameter, having pores of 1.2 ~m diameter. Cells and protoplasts are held back; the gas vesicles pass through. Just before the filter dries, wash with about 5 ml of water, adding drop-by-drop. 2. Transfer the gas vesicle-containing filtrate to a membrane filter of similar diameter, but of 0.05 ~tm pore size. Use only a moderate vacuum ( - 30 kN m 2) to draw the suspending solution through, leaving the gas vesicles on the filter. They may be rinsed several times with water or buffer, but do not allow the filter to dry: the pressures set up by surface tension at the surface of drying films can easily collapse gas vesicles. 6 Final Centrifugation The preparation is improved by a final centrifugation which serves to separate the veslcles from other particulate matter which might have been released from C~lls breaking at the first filtration. The standard method described above (after resuspending in a large volume of water) is satisfactory. Alternatively, Jones and Jost 3 recommend placing the concentrated vesicles in a narrow layer at the surface over concentrated sucrose and centrifuging at ver3~ high accelerations (200,000 g). If this is done, gas vesicles having a criticdl pressure of 500 kN m -2 will survive only in the top 0.25 mm. Accordingly, the rotor should first be spun for sufficient time at a low acceleration to bring the vesicles very close to the meniscus. Efficiency of ~he Above Method Using the ~ethod described above, Buckland and Walsby 15 record recoveries of between 50 and 60% (from turbidity measurements) and a
686
PREPARATIONSDERIVED FROM UNICELLULARORGANISMS
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percentage purity of 97.6 for the final preparation (according to the results of a radioactive labeling experiment). Storing the Gas Vesicles Bacterial growth in crude lysates results in substantial losses over a period of days, apparently due to enzymatic attack on the gas vesicle protein. However, in a highly purified state, aqueous suspensions of intact vesicles may be stored at 5 C for a year or more without appreciable loss. Vesicles can also be frozen, freeze-dried and then kept in a dried state indefinitely. On simply adding water a suspension of intact vesicles may be reconstituted. 1~ Unfortunately, although drying itself has no adverse effects on the vesicles, pressures developed during the freezing process result in substantial weakening and collapse (50% or more). It is known from freeze-etching studies 16,17 that gas vesicles will survive intact when frozen rapidly in liquid Freon held at near liquid nitrogen temperatures. It may be possible to scale up such procedures for the quantitative preparation of frozen and freeze-dried intact vesicles. ~eM. Jost, Arch. Mikrobiol. 50, 211 (1965). "J. R. Waaland and D. Branton, Science 163, 1339 (1969).
[71] T h e I s o l a t i o n of t h e A m o e b a P l a s m a M e m b r a n e a n d t h e Use of L a t e x B e a d s for t h e I s o l a t i o n of P h a g o c y t i c Vacuole (Phagosome) Membranes from Amoebae Including the Culture Techniques for A m o e b a e B y EDWARD D. KORN A c a n t h a m o e b a castellanii I is one of a group of small soil amoebae that can be grown axenically on soluble medium. Because the amoebae are nutritionally dependent on the processes of phagocytosis and pinocytosis, they provide an unusual opportunity for studying the mechanisms of membrane fission and fusion, the relationship between the plasma membrane and the membrane of the phagocytic vacuole (phagosome), and the dynamics and biochemistry of membrane movements.
Culture Techniques A defined culture medium which will support growth has been described, 2 but for most purposes the soluble culture medium3 shown in 'R. J. Neff, J. Protozool. 4, 176 (1957).