Preparation and Ultrastructure of the Outer Coats

Transcription

1 JOURNAL OF BACTERIOLOGY, June 1969, p Copyright 1969 American Society for Microbiology Vol. 98, No. 3 Printed in U.S.A. Preparation and Ultrastructure of the Outer Coats of Azotobacter vinelandii Cysts1 L. P. LIN AND H. L. SADOFF Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan Received for publication 8 March 1969 Cysts of Azotobacter vinelandii were ruptured with 3.0 mm ethylenediaminetetraacetate in 0.05 M tris(hydroxymethyl)aminomethane buffer (ph 7.8) and fractionated by differential centrifugation in density gradients of sucrose or Ficoll (polysucrose) at 10,000 X g for 2 hr at 4 C. The average densities of the cyst exine, the cyst central body, the vegetative cell, and the intact cyst were found to be 1.13, 1.26, 1.28, and 1.31 g/cm3 in sucrose, and 1.08, 1.12, 1.13, and 1.17 g/cm3 in Ficoll, respectively. These data were utilized to devise procedures for the isolation of cyst components. Cyst exine appeared as a multilayered structure in ultrathin sections. This same sheetlike structure was seen when exines were subjected to detergent treatment, metal-shadowed, and examined in an electron microscope. The exine was lysed by trypsin and was resistant to lysozyme, indicating that protein may be the principal structural material of the outer cyst coat. Cells of Azotobacter vinelandii undergo a morphogenetic process which yields desiccationresistant cysts. Examination by electron microscopy reveals that these resting forms consist of several outer electron-dense layers (the exine), an intermediate electron-transparent region (the intine), and a central body which is a small replica of the vegetative cell (4). To understand the chemical events occurring during encystment, it is necessary to know the chemical and physical properties of cysts and their component parts. Because cyst structures have not been studied, we devised fractionation procedures by which cyst components could be obtained in sufficient quantities for chemical analyses. Socolofsky and Wyss (6) demonstrated that cysts can be ruptured in dilute solutions of ethlenediaminetetraacetic acid (EDTA), and Goldschmidt and Wyss (2, 3) described the ionic conditions for cyst survival in the presence of chelating agents. The fractionation method, based on buoyant density differences among the cyst components, is described as well as buoyant densities and the fine structure of the exine. MATERIALS AND METHODS Preparation of cells. Cysts and vegetative cells of A. vinelandii ATCC were produced as described previously (4). Cysts were ruptured with 3.0 mm EDTA in 0.05 M tris(hydroxymethyl)aminomethane i Published with the approval of the Director of the Michigan Agricultural Experiment Station as Journal Article (Tris) buffer, ph 7.8, and changes of absorbance of cyst suspensions were monitored in a Beckman DU spectrophotometer at 660 nm. Preparation of density gradient solution. Crystalline sucrose (Mallinckrodt Chemical Works, St. Louis, Mo.) or Ficoll (Pharmacia, Uppsala, Sweden) was used for the preparation of gradients. Prior to use, 50 g of Ficoll was dissolved in 450 ml of distilled water, exhaustively dialyzed against several changes of distilled water, and dried by lyophilization. Linear gradients of sucrose (30 to 70%, w/w) or Ficoll (30 to 50%, w/w) were prepared in 0.05 M Tris buffers, ph 7.8, in 50-ml cellulose nitrate centrifuge tubes. The total volume of each gradient solution was 37.5 ml. The gradients were formed at room temperature and cooled to 4 C before use. Centrifugation and sampling. Approximately 50 mg (dry weight) of ruptured cysts in 0.5 ml of 0.05 M Tris buffer (ph 7.8) was layered on the gradient solutions and centrifuged in a model B-60 ultracentrifuge (International Equipment Co., Needham Heights, Mass.) with swinging-bucket rotor SB- 110 at 4 C for 2 hr at 12,000 rev/min. The centrifugal fields at this speed were 10,000 and 20,000 X g, respectively, at the top and bottom of the tubes. The distribution of materials in the gradient was determined by their absorbance at 660 nm with a Beckman DB-G grating spectrophotometer. Fractions of 1.5 ml each were collected by puncturing the bottom of the tube with a device that allowed minimal shaking. The identity of the cells or cell components at the respective densities was established by phase-contrast or electron microscopy. The density of each fraction collected was calculated from its refractive index. No change in the distribution of materials in the gradient was observed when the centrifugation time was increased to 8 hr. 1335

2 1 336 LIN AND SADOFF J. BACriaoL. Treatment of isolated exine. Samples of purified exines were suspended in 0.05 M Tris buffer (ph 7.8) to achieve an absorbance of 0.5 to 0.6 at 660 nm. One-milliliter amounts of this suspension were mixed with the following reagents: (i) 1 ml of 1% (w/v) sodium dodecyl sulfate (SDS) in Tris buffer (ph 7.8), and incubated for 6 hr with constant shaking; (ii) 1 ml of 0.04% (w/v) trypsin (Nutritional Biochemicals Corp., Cleveland, Ohio) in Tris buffer (ph 7.8), and incubated at 37 C for 8 to 10 hr; (iii) 1 ml of 0.02% (w/v) lysozyme (Armour Pharmaceutical Co., Kankakee, Ill.) in Tris buffer ~~~~60 (ph 7.8) for 30 min. Exines ~~~~~~ Z suspended in Tris buffer were also subjected to sonic z disruption for 5 min in an MSE 100-w ultrasonic disintegrator (Measuring and Scientific 006~~ ~ ~ ~ ~ ~ ~ 2 Equipment, Ltd., London, England). to 0.4 Analytical procedures. The change in viscosity of the suspending medium which occurred during disruption of cysts was measured with an Ostwald _ CYST 0 20 viscosimeter (Fisher Scientific Co., Pittsburgh, Pa.). 0.3 The extent of lysis of central bodies during cyst VEGETATIVE CELL disruption was followed by measuring the absorbance of the suspending medium at 260 and 280 nm and then estimating the nucleic acid content (8). The total nitrogen content of purified exines was determined by the modified micro Kjeldahl procedure (7) and their protein content was measured after partial digestion in 3 N HCI at 110 C for 4 hr, utilizing the Folin phenol reagent (5). Bovine serum albumin (Nutritional Biochemicals Corp.) was used as a protein standard. Electron microscopy. Specimens were mounted on Formvar-coated grids and shadowed lightly with platinum-palladium (80:20) alloy in a Varian vacuum evaporator (Varian Vacuum Division, Palo Alto, Calif.) at an angle of approximately 300. Carbon replicas were prepared (1) and shadowed with platinum-palladium (80:20). Negative staining was done with 1% (w/v) phosphotungstic acid solution in distilled water. The ph of this solution was adjusted to 7.0 with 0.1 M KOH. The specimens were examined in a Hitachi HU-11 electron microscope at 75 kv. Micrographs were taken on Kodak Electron Image Plates, and initial magnifications ranged from 15,000 to 45,000. RESULTS Disruption of cysts with EDTA. A series of experiments was performed to determine the optimal concentration of EDTA for the disruption of cysts (Fig. 1). Suspensions of exponentially growing cells and cysts were prepared in buffered EDTA solutions over the range of 0 to 5.0 mm, and the turbidities were observed. Little lysis occurred when young vegetative cells were exposed to EDTA. In contrast, suspensions of cysts in as little as 0.5 mm EDTA exhibited a sharp drop in their absorbance, indicating the rupture of the cyst coat. This was verified by microscopic examination of the cyst suspensions and the determination of the percentage of cyst disruption. The exposure of cysts to 3.0 mm EDTA brought about greater than 90% disrup CYST ~~~~~VEGETATIVE CELL E J N4-EDTA CONCENTRATION(mM) FIG. 1. Relationship between absorbance reduction and percentage of disruption of cysts or vegetative cells of Azotobacter vinelandii by treatment with increasing concentrations of EDTA. tion after a 15-min treatment and produced an increase in the viscosity of the suspending medium. The initial viscosity of the Tris-EDTA suspending medium was 1.01 centipoises but, after disruption of a cyst suspension [1.18 mg (dry weight)/ml], the viscosity was 1.24 centipoises at 20 C. Its nucleic acid content was 4,g/ml. Density gradient centrifugation. To determine the ultrastructure and properties of cysts and cyst components, fractionation procedures were developed which utilized the disruption of cysts with EDTA and separation of the structural components by gradient centrifugation. Cysts were broken in 3.0 mm EDTA and centrifuged in a 30 to 70% (w/w) sucrose or 30 to 50% (w/w) purified Ficoll gradient. Isopycnic banding occurred in these systems, making possible the recovery of insoluble cyst components from the tubes. Furthermore, the densities of the cyst components could be determined by their positions in the gradients. The exines formed a band near the middle of the sucrose gradient, but central bodies, vegetative cells, and intact cysts were observed near the bottom of the tubes. The average buoyant densities of cells, cysts, and their components are summarized in Table 1. The exine has a lower density than the central body, vegetative cell, or cyst. This difference made possible the isolation of exine from other materials since it floated in 45% sucrose solutions -~~~~~~0

3 VOL. 98, 1969 A. VINELANDII CYSTS 1337 while central bodies, cysts, and vegetative cells formed a pellet. A scheme for the preparation of exine materials is presented in Fig. 2. Cysts were treated with 3.0 mm EDTA in 0.05 M Tris buffer (ph 7.8) and then centrifuged at 1,500 X g for 15 min. This treatment removed intact cysts and large cellular debris. The pellet was resuspended in buffer and then centrifuged again in order to recover more exines. The supernatant fractions were combined and the crude exines were pelleted by centrifugation at higher speed (20,000 x g for 30 min). Isolated crude exine materials were layered on a 45 % (w/w) sucrose solution and subjected to centrifugation at 15,000 X g for 2 hr. The exines were isolated from the top layer of this sucrose solution with a capillary pipette or a syringe, diluted three- to fivefold with distilled water, and centrifuged at 20,000 X g for TABLE 1. Buoyant density of Azotobacter vinelandii cells and their components Component Buoyant density (g/cm') in" Sucrose Ficoll Exines Central bodies Vegetative cells Cysts a The gradients employed were 30 to 70% (w/w) sucrose and 30 to 50% (w/w) purified Ficoll (polysucrose). (cysts, central bodies, coarse cell debris) (discard) Resuspend in buffer; centrifuge 1,500 X g, 15 min (crude exine) 30 min. The exines were examined in the electron microscope, and the absence of poly- -hydroxybutyrate (PHB) particles in the preparation was used as a critesion of purity. Most preparations contained fewer than three PHB particles per 100 exines. Fine structure of exine. The fine structure of purified isolated exine was investigated by procedures which are normally used to study cell walls. These included metal-shadowing, negative staining, carbon replica techniques, and treatment with SDS, trypsin, and lysozyme. Figure 3 shows several platinum-palladium shadowed exines. Superficially, the exine appears to be very similai to the wall of vegetative cells. When shadowed with platinum-palladium alloy and observed under high magnification, the exine appears to be composed of sheetlike structures (Fig. 4). A negatively stained exine is presented in Fig. 5, showing the layered and semirigid structure. The surface appearance of the exine was examined by the carbon replica technique (Fig. 6). Vegetative cells of A. vinelandii have a smooth regular surface (Fig. 6a), but the cyst surface (Fig. 6b) is wrinkled and irregularly folded. The isolated exine is also wrinkled and appears to be somewhat stretched over the surface of the Formvarcoated grid (Fig. 6c). Effect of physical and chemical agents on exine structure. In further investigations of structure, purified exine was exposed to physical and chemical agents known to affect the cell wall structures. When incubated with 0.5% (w/v) SDS with shaking, the exine appeared as a Supernatant fluid (exine) Centrifuge 15,000 X, 2 hr in 45% sucrose solution Top layer (exine) FIG. 2. Scheme for the disruption andfractionation of Azotobacter vinelandii cysts. Cysts Treat with 3.0 mm EDTA in Tris buffer;centrifuge 1,500 X g; 15 min Supernatant fluid (exine, intine, particles) Centrifuge 20,000 X g, 30 min Supernatant fluid (intine, particles)

4 1338 LIN AND SADOFF J. BACTERIOL. q I& 1: :.:' FIG. 3. Electron micrograph of an isolated exine which was prepared by EDTA disruption and differential centrijugation. The dark granule is a co-sedimented poly-13-hydroxybutyrate granule. Platinum-palladium (80:20) alloy shadowed. Marker represents,m. FIG. 4. Enlarged view of an exine, showing the sheetlike structure at the point of rupture by EDTA. Platinum-palladium (80:20) 'alloy shadowed. Marker represents I,um. multilayered sheet structure (Fig. 7). Sonic tieatment of the exine also revealed the layered structures. The nitrogen and protein contents of the purified exine were 4.8 and 28 %, respectively. The shape and surface convolutions of the exine disappeared after digestion in 0.4 mg of trypsin per ml in 0.05 M Tris buffer (ph 7.8) for 16 hr at 37 C. A typical result of enzymatic hydrolysis which is in marked contrast to physical effects of the SDS is shown in Fig. 8. No unique structures FiG. 5. Electron micrograph of an isolated exine negatively stained with 1% phosphotungstic acid. Marker represents I jum. were revealed by tryptic digestion. When intact cysts were incubated with lysozyme and EDTA for 30 min, the cyst coats were broken open and the central bodies of the cysts were lysed by lysozyme (Fig. 9). The intines and contents of the central bodies were lost into the medium. The large structures encased in the exines are lipid granules originally contained in the central bodies. DISCUSSION Isolated exine material has been prepared by disrupting cysts of A. vinelandii in EDTA and separating the components in density gradients. The exine appears as a multilayered structure when seen in electron micrographs of stained and sectioned cysts (4). The laminations may be the basis of the limited rigidity which these cyst coats possess. The overlaid sheetlike structure is also seen when cyst coats are subjected to sonic or detergent treatment and are then metal-shadowed. The exine is partially lysed by trypsin and is resistant to lysozyme. These results and direct chemical analysis are evidence that protein is an important structural material present in this outer cyst coat. In contrast, the envelope surrounding the central body is sensitive to lysozyme and presumably is a peptidoglycan. The chemical composition of isolated exine is under current investigation. The central bodies of cysts remained intact during cyst disruption by EDTA. This was determined both by direct microscopic examination and by observing that only very low levels of nuclear materials (approximately 0.3% of weight

5 W _:.,' A:.. $$e......,. :#'.. ; VOL. 98, 1969 A. VINELANDII CYSTS 1339 <... :' :e.: S:.- :. t:.. ::e: j t,i 4t,<4 _; w ::.: R.: :;: :. ::+ S) : w-vx *. ::: dr ::.: f e j I j ^,. j ' s g~~~~~~~p. 1, ~ ~......,,.,to..,...,4 "..S',, :, :' '_. ',',. }> #X;'.';X; '''.'.s,.:' QS '..;...-,_ee ;B.<... ;-^ ;, ^ ;w;r-x 1 ' X 5t ^ 6 W r m j. '.,l ' v.r ^,l.: j.st ~ ~ ~ ~ A I ;{j>#/ #.'- w~~~~~~s a;ft2f f V~~~~ Sc A FIG. 6. Surface structure of Azotobacter vinelandii cells, cyst, and exine. Fine structure was shown by the carbon replica technique. Note the rod shape and smooth, regular surface of the vegetative cell (Fig. 6a), and the wrinkled and irregularly folded cyst surface (Fig. 6b). The isolated exine is also wrinkled (Fig. 6c). Platilnum-palladium (80:20) alloy shadowed. Marker represents I jum.

6 .. L 8 E... *. ''' o''.e, s.::. o+# *:0r. e... +.S,.,,.t : >..... _*.f.. ee LIN AND SADOFF J. BAcTERIOL. K 0 > e; ^ - * *... '^0*i Xf %a... _ W: 8 9 *:.... I. ".A.!:. X; FIG. 7. Portion of an exine treated with 0.5% SDS. The multilayered sheet structures (arrow) are evident. The dark dense granule is a poly-j3-hydroxybutyrate granule. Platinum-palladium (80:20) alloy shadowed. Marker represents I A.m. FIG. 8. Portion of an exine treated with 0.04% trypsin. Platinum-palladium (80:20) alloy shadowed. Marker represents 1 Am. FIG. 9. Cyst concomitantly treated with 3.0 mm EDTA and 0.02% lysozyme. The dense bodies are lipid granules (1g). Platinum-palladium (80:20) alloy shadowed. Marker represents I,um.

7 VOL. 98, 1969 A. VINELANDII CYSTS 1341 of cysts) were present in the suspending medium after disruption. The increase in viscosity which occurred may have been due to the solubilization of the intine structure. ACKNOWLEDGMENT This investigation was supported by Public Health Service research grant AI from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Bradley, D. E., and D. J. Williams An electron microscope study of the spores of some species of the genus Bacillus using carbon replicas. J. Gen. Microbiol. 17: Goldschmnidt, M. C., and 0. Wyss Chelation effects on Azotobacter cells and cysts. J. Bacteriol. 91: Goldschmidt, M. C., and 0. Wyss Effect of ion concentration on the rupture and survival of Azotobacter cysts. Appl. Microbiol. 16: Lin, L. P., and H. L. Sadoff Encystment and polymer production by Azotobacter vinelandii in the presence of,- hydroxybutyrate. J. Bacteriol. 95: Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall P-rotein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Socolofsky, M. D., and 0. Wyss Cysts of azotobacter. J. Bacteriol. 81: Umbreit, W. W., T. H. Burris, and J. F. Stauffer Manometric techniques. Burgess Publishing Co., Minneapolis. 8. Warburg, O., and W. Christian Isolierung und Kristallisation des Garungsferments Enolase. Biochem. Z. 310: Downloaded from on October 19, 2018 by guest