J. Mol. Biol. (1976) 106, 871-888
Structure of the Wall of Halobacterium halobium Gas Vesicles A.E. BLAUROCK-~ AND W.WOBER$ Cardiovascular Research Institute University of California at San Francisco San Francisco, Calij’. 94143, U.S.A. (Received 23 December 1975, and in revised form 14 April 1976) The gas vesicles of Halobacterium halo&urn have been studied by recording X-ray diffraction patterns from both intact and collapsed vesicles. The wall is found to be remarkably thin; the average thickness is no more than 20 A. Electron microscopy indicates that the wall consists of ribs, and the X-ray data confirm this. The thickness is therefore greater than 20 A at some points and less at others. The X-ray data also indicate that the ribs on the two sides of the collapsed vesicle are intermeshed. Our data indicate a large amount of p-sheet in the wall. The /?-sheet consists of parallel (or anti-parallel) polypeptide chains which are regularly hydrogenbonded to one another. This bonding locks the presumed subunit proteins into the wall, which is important for its function at the gas-liquid interface. The p-sheet is in two layers, one on top of the other. The two layers together can stiffen the wall and hence strengthen the vesicle against collapse.
1. Introduction The existence of gas-filled vacuoles inside certain bacterial and algal cells has been known for nearly a century (see Walsby, 1972, for a review). In the electron microscope the gas vacuoles are seen to consist of clusters of sub-microscopic structures with a regular shape (Smith & Peat, 1967), called gas vesicles. The gas vesicles produced by Halobacterium halobium are lemon-shaped and are O-3 to 0.4 pm long (Larsen et al., 1967 ; Stoeckenius & Kunau, 1968). The shape is determined by a thin wall of protein. Small amounts of lipid sometimes found with the isolated gas vesicles are believed to be contaminants (Stoeckenius & Kunau, 1968; Krantz & Ballou, 1973). Electron microscopy indicates that the wall consists of parallel ribs which run at right-angles to the long axis of the gas vesicle. The wall is known to be freely permeable to gases (Walsby, 1969), and the composition and pressure of the gas inside the vesicles is therefore determined directly by the gases dissolved in the cytoplasm. Thus the only known function of the wall is to conserve the gas-filled space. For a gas bubble in water, the tension at the gaswater interface will generate a pressure inside the bubble which is inversely proportional to the diameter. Very high pressures can result (30 atmospheres inside a bubble 0.1 pm in diameter; see Walsby, 1971). Since the gases dissolved in the water are at equilibrium with the atmosphere, gas will go from the bubble into solution. Then as the bubble becomes smaller, the pressure inside will rise even higher and the gas will t Present Calif. 91125, $ Present koferstrasse
address : Department of Chemistry, California Institute of Technology, U.S.A. address: Physiologischen Institut d. Universitiit Mtinchen, 8 Miinchen 12, West Germany. 871
Pasadena, 2, Petten-
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A. E. BLAUROCK
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dissolve all the more rapidly. Thus, the gas-vesicle wall conserves a space which would otherwise quickly vanish. The X-ray diffraction experiments reported here indicate for the first time the presence of large amounts of j-sheet in the wall. In the p-sheet the backbones of the polypeptide chains are hydrogen-bonded to one another in a regular way. The extensive hydrogen bonding locks the presumed protein subunits in the wall, making a structure well-suited to the special position at the gas-cytoplasm int’erface. The X-ray data indicate two layers of /?-sheet in the wall, and a tentative profile of the electron density through the wall has the two narrow peaks expected for the electrondense polypeptide backbone in the two layers. The X-ray results confirm that the wall is very thin. The average thickness per wall in a vacuum-dried stack of the collapsed vesicles is not more than 20 A. Assuming the wall consists of ribs, the thickness will be greater than this value at some points and less at others. The data also indicate that the ribs on the two sides of the collapsed vesicle are intermeshed.
2. Materials and Methods Collapsed gas vesicles were isolated from H. hdobiwn as described previously (Stoeckenius & Kunau, 1968). Intact, i.e. uncollapsed, vesicles were isolated by lysing the cells and floating the vesicles to the surface. (Similar methods have been used by Larsen et al. ( 1967) to isolate tIaZobacteriwra vesicles, and by Walsby & Buckland (1969) to isolate vesicles from Anabaena Jlos-aquue.) The intact vesicles were then concentrated by floating them on water. Samples of the collapsed vesicles were prepared for X-ray exposure by centrifuging a suspension in water for 1 h at 300,000 g. Material from the resulting pellet was drawn into a l-mm thin-wall glass capillary (Pantak; UniMex Caine), which was then flamesealed inside a larger capillary. A specimen of the intact vesicles was prepared similarly. The collapsed vesicles were oriented in two ways. First, some of the suspension was dried on a mica or glass surface. The specimen was then mounted in the camera with the X-ray beam tangent to the surface. Second, some of the suspension was drawn into a l-mm capillary, with the ends left open to allow water to evaporate until a thin septum had formed across the capillary. This capillary was then sealed inside a larger one to prevent’ further drying, and the specimen was mounted vertically in the camera with the septum edge-on to the X-ray beam. All specimens were exposed under vacuum to avoid air scatter. The X-rays were generated in a microfocus generator (Jarrel-Ash Division of Fisher Scientific Co., Waltham, Mass.) with a copper target. Ni foil was used as the window of the tube to attenuate the K/l line from copper. The Franks-type (Franks, 1955) low-angle camera (Blaurock, 1975) produced e beam about 0.1 mm in diameter. The predominant wavelength of the X-rays was h = 1.542 A (CuKu). The distance from specimen to film was either 30 or 40 mm, to record at wider angles and reduce exposure time, or 80 mm, for best resolution toward the center of the pattern. The diffracted X-rays were recorded on Ilford Industrial C film. A stack of 4 films usually was exposed. The optical density of a film was measured with a recording microdensitometer (mark IIIC, Joyce-Loebel & Co., Inc., Burlington, Mass.).
3. Results (a) Previous
electron microscope observations
A variety of useful observations have been made on the H. halobium gas vesicles. In negatively stained preparations of the intact vesicles (Stoeckenius & Kunau, 1968 ; Larsen et al., 1967) there are rather faint striations running more or less at rightangles to the long axes of the vesicles; the periodicity is 40 to 50 A. In freeze-fractured,
shadowed preparations of the collapsed vesicles, there are prominent
striations at
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right-angles to the long axis and having a similar periodicity (Fig. 1). These striations indicate ridges on the inside surface of the wall. The inside surface is unlikely to be smooth. The shadowing material could perhaps move to a line of sites with an affinity for it,, causing the striations, but in this case the striations should be equally visible whether the shadowing direction projected onto the surface is perpendicular or is parallel to them. The striations, in fact, are barely visible when the direction of shadowing is parallel to them. Other electron microscope observations indicate that the ridges are due to a ribbed structure. In cross-section, the wall of intact (Larsen et al., 1967) or collapsed (Stoeckenius & Rowen, 1967) vesicles sometimes shows a regular beading. For a ribbed structure, the wall will look beaded when the plane of the section in fact is at right-angles to the direction of the ribs. Again, the periodicity is 40 to 50 b.
FIG. 1. Electron micrograph of a freeze-fractured, shadowed preparation showing the intact gas vesicles inside a cell of H. halobiuwa ; the bar indicates 1 pm. The concave inner surfaces of the vesicles are exposed by the fracturing, and the shadowing brings out ridges at right-angles to the lnng axes of the vesicles. The periodicity of the ridges, about 40 11, is in fair agreement with the X-ray data. We thank Dr W. Stoeckenius for supplying this print. 57
FIG. 2. Low-angle X-ray diffraction pattern from intact gas vesicles in suspension. fu Figs 2, 4 and 6. the bar indicates 1 cm on the original negative. (a) The intense, sharp ring just outside the diffuse ditfraction is the fist-order rib reflection at a Bragg spacing of 46.4 A. Third fhm in a pack of four. (b) First ti in the paok. The sharp ring in (a) is now obscured by strong diffuse saattsr; if visible, the ring would be greatly over-exposed. The faint, sharp ring just outside the diffuse soatter is the second-order rib reflection at a Bragg spacing of 22.9 A. Other sharp rings are visible on the original negative at larger radii. 103 h exposure; 8 = 38 mm.
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In the negatively stained preparations, the intact vesicles invariably look partly collapsed. There are creases which tend to run parallel to the striations, indicating greater rigidity along the striations than at right-angles. Fully collapsed vesicles look even more irregular (Stoeckenius & Rowen, 1967; Stoeckenius & Kunau, 1968). Apparently the vesicles cannot collapse perfectly flat. The wrinkles and folds which occur will interfere with orderly stacking of the collapsed vesicles. Further, although the two sides of a collapsed, dried vesicle will touch because of van der Waals’ forces, the spacing between the two sides is likely to vary at the wrinkles. There is evidence of both kinds of disorder in the X-ray patterns to be described. (b) The &&&on
patterns
The X-ray diffraction patterns from the various specimens all show the sharp reflections expected from the ribbed structure indicated by the electron microscopy. The periodicity of the ribs varied by about 2 Angstrom units, depending on whether the vesicles were intact or collapsed, hydrated or dry. Thus only very minor changes in the structure of the wall are indicated, and useful comparisons have been made between the different patterns by assuming that the structure of the wall does not change. Marked changes in the intensities of some of these reflections (the first order in particular is much waaker after collapse) are attributed to intermeshing of the ribs on the two sides of the collapsed vesicle (see below). The diffraction pattern from the intact, unoriented gas vesicles in suspension shows sharp rings superimposed on strong diffuse diffraction (Figs 2 and 3). The first three sharp rings have Bragg spacings of 45.4 A, 22.9 A and 11.5 A ; the three together indicate a periodicity of 45.7 A f 0.3 A (45.7 812 = 22.9 A and 45.7 A/4 = 11.4 A). In terms of the ribbed structure, there is one rib every 45.7 A. The diffuse diffraction
1
,
h=l “p c ;;
0
0.01
0.03
0.02
0.04
0.015
RWl FIG. vesicles and 2) ground density The profile vesicles
3. Corrected densitometer tracing of the low-angle diffraction from unoriented, intact in suspension; R is the inverse of the Bragg spacing. The narrow peaks at 40 A/h (h = 1 are attributed to the ribbed structure indicated by electron microscopy. A constant backhas been subtracted from the original tracing at each radius R, which is the average over a broad, uniform region well away from the center. R2 correction (Wilkins et al., 1971) would be appropriate to diffraction arising from the of the wall. However, the diffraction is dominated by the contrast of the gas inside the with the wall and the water outside.
FIG. 4. Low-angle patterns from the collapsed, hydrated vesicles. (a) In suspension. Comparing this exposure with Fig. 2 shows that the first-order rib reflection and the diffuse scatt,er are both greatly reduced when the vesicle collspses. The diffuse ring near the center is attributed to profile diffraction (see text). 50 h exposure; s = 39 mm, (b) Oriented in a septum inside a capillary. The plane of the septum was horizontal. On the vertical (profile) axis are the 2 reflections indicating stacking, one vesicle every 63 A. Beyond them are the diffuse bands attributed to cross-interference between the 2 sides of the vesicle; the first of these is at a Bragg spacing of 14 A. On the horizontal (in-plane) axis are the sharp reflections attributed to st.ruoture in the plane of the wall. The reflections all occur in pairs, one t,o each side of center; the Bragg spacing is given for one reflection of each pair. The arcs on the in-plane axis at. 4.7 and 4.9 p1 indicate &sheet. 21 h exposure; s = 39 mm.
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is attributed mainly to contrast of the gas inside the vesicle (