Morphogenesis of Stigmatella aurantiaca Fruiting Bodies

Transcription

1 JOURNAL OF BACTERIOLOGY, Aug. 1985, p /85/ $02.00/0 Copyright C) 1985, American Society for Microbiology Vol. 163, No. 2 Morphogenesis of Stigmatella aurantiaca Fruiting Bodies GABRIELA M. VASQUEZ, FRANK QUALLS, AND DAVID WHITE* Microbiology Program, Insdiana University, Bloomington, Indiana Received 27 February 1985/Accepted 30 April 1985 Scanning electron micrographs of intermediate stages of fruiting body formation in the myxobacterium StigmateUa aurantiaca suggest that fruiting body formation can be divided into several stages distinguishable on the basis of the motile behavior of the cells. Aggregates formed at sites where cells glide as groups in circles or spirals. Thus, each aggregate was surrounded by a wide band of cells. Several streams of cells were pointed toward and connected to the wide band of cells at the base of the aggregate, suggesting directed cell movement toward the aggregate. The pattern of cells at the base of taller, more mature aggregates suggested that grqups of cells enter the aggregate from the surrounding band of cells by changing the pitch of their movement, thus creating an ascending spiral. Stalk formation was characterized by a distinctly different pattern, which suggested that single cells emerge from the band of cells and move toward the aggregate, under it, and then vertically to create the stalk. At this stage, the aggregate appeared to be torn from'the substrate as it was lifted off the surface. The cells in the completed stalks were well separated, and most had their long axes pointed in a vertical direction. A great deal of the stalk material appeared to be slime in which the cells were embedded and through which they were presumably movin,g in the live material. Some suggestions regarding factors that may direct the observed morphogenetic movements are discussed. The myxobacteria are gram-negative procaryotes found principally in the soil, on decaying vegetation, and on animal dung (10). They are remarkable for their social behavior which is the most sophisticated among the known procaryotes (14). The organisms live in communities called swarms and feed cooperatively in nature on other microorganisms. Within the swarms, the cells move by gliding in numerous self-made slime trails that intersect, merge, and diverge (2). Upon nutrient deprivation, the cells aggregate into numerous centers and construct multicellular fruiting bodies. During fruiting body formation in several genera of myxobacteria, a portion of the cell population differentiates into desiccationresistant, resting cells called myxospores. In Stigmatella aurantiaca, the final stages of myxospore differentiation are completed after the fruiting body is formed (9). When the fruiting body is transferred to new locations where nutrients are available, the myxospores germinate to give rise to a small swarm. Thus, the fruiting body serves as a unit of dispersal for the organism and as a resting structure resistant to desiccation. A book devoted to the growth and development of myxobacteria was recently published (12). Photographs or movies of aggregation and fruiting body formation generally have not been adequate to resolve the individual cells. This inadequacy is in part due to the high cell densities in the swarms and also to the slime which is produced by the myxobacteria. Hence, there are no detailed descriptions of cell movements during aggregation or construction of the fruiting bodies. However, in the films made by Reichenbach et al. (11) and the scanning electron micrographs by Grilione and Pangbom (7), there is some indication of how aggregation might occur. During fruiting body formation in Chondromyces apiculatus, a spiraling movement of amorphous material, presumably slime and cells, occurs at the site of aggregation (11). In the photographs by Grilione and Pangborn, one can sometimes see a ring of cells at the base of the aggregate formed by S. aurantiaca. This may represent the spiraling movements observed in the films * Corresponding author. 515 (11). Thus, aggregation may begin as a spiraling movement of cells. The formation of the stalk in S. aurantiaca is not understood. It has been suggested that myxobacteria fruiting body stalks are composed of slime and that the cells raise themselves off the surface of the support by secreting a slime stalk (3). Photographs by Grilione and Pangborn (7) and by Stephens and White (15), on the other hand, show that the stalks of S. aurantiaca consist of both slime and cells, suggesting that the fruiting body may be shaped by morphogenetic movements of the cells instead of, or in addition to, slime secretion. It is not known what guides the cells during aggregation and the construction of the fruiting body. Dworkin and Eide (5) reported that they were unable to demonstrate chemotaxis in the myxobacterium Myxococcus xanthus. They also rejected, on theoretical grounds, the notion of chemotaxis by myxobacteria based upon a temporal sensing mechanism because of the slow rate of gliding motility versus the rates of diffusion of molecules. However, one cannot theoretically rule out chemotaxis based upon a spatial sensing mechanism (1, 2). Although chemotaxis cannot be ruled out, especially during stalk formation, we provide a possible explanation for directed cell motility based upon elasticotactic movements on oriented polysaccharide molecules in the slime. MATERIALS AND METHODS Conditions for growth and fruiting body formation. All experiments were carried out with S. aurantiaca DW135, which is a derivative of strain DW4 lacking cohesive system A (6). Cells were grown in a shaking suspension in 1% tryptone (Difco Laboratories, Detroit, Mich.) and 8 mm MgSO4 at 30 C. The cells were washed once with 10 mm N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), ph 7.2, containing 3.5 mm CaCl2, and then suspended to either 4 x 1010 or 1.3 x 1010/ml in the buffer. Then, 5,ul was spotted on a strip of filter paper previously lightly coated with 1.5% Difco agar in the HEPES-CaCl2 buffer and resting on agar of the same composition. The agar in all experiments contained 0.8 mm GMP because that was

2 516 VASQUEZ ET AL. previously reported to induce fruiting body formation in low cell densities (15). The petri plates were then placed in incandescent light at 30 C for different periods. Preparation of samples for electron microscopy. Samples were fixed and dehydrated by placing the filter paper strips on filter papers or sponges soaked with the appropriate reagents in closed petri dishes. The cells were fixed in undiluted, unbuffered glutaraldehyde for 30 to 120 min at room temperature. They were then dehydrated in an ethanol series and critical-point dried. The dried samples were coated with gold-palladium with a Polaron sputter coater and examined with a Cambridge 250 MkII scanning electron microscope. RESULTS On both growth and fruiting media, S. aurantiaca cells frequently moved in patterns that resemble circles (Fig. 1). Often the circles filled in with cells to form spiral patterns only to disappear as the cells moved away in new or existing slime trails (unpublished data). Spiral patterns formed even in dense populations, i.e., without a prior formation of empty circles. Although these patterns could be found in the absence of aggregate formation, we always observed them in young aggregates (Fig. 2). Figure 2A is an example of a very young aggregate, showing a typical pattern of a ring of cells around the perimeter. Farther away one can see wide streams of cells lined up with their long axes pointing toward the ring of cells. The several streams of cells appear to join the ring of cells at the perimeter of the aggregate. The pattern of cells within the aggregate was consistent with the notion that cells not only circled the aggregate, but also traveled across the center of the aggregate and joined the ring once more on the other side, thus staying within the aggregation site (Fig. 2). Numerous points of contact can be seen between the cells. Mature aggregates also show a circular band of cells around the perimeter (Fig. 3A). A higher magnification of the base of the aggregate shown in Fig. 3A reveals that the cells within the aggregate are positioned at a slight incline with respect to the cells in the perimeter (Fig. 3B). Figure 4 shows a more mature aggregate. Again, one can see the circular band of cells around the base and the cells within the aggregate arranged at an incline. An important point about these early stages is that the transition from the circle into the aggregate is smooth. The cells appear to have simply changed the pitch of their movement when they enter the aggregate. Figure 5 is a top view of a different aggregate. One can see a circular arrangement of cells at the base of the aggregate as well as a circular arrangement at the top Ṗhotographs of the early stalk stage revealed two changes in cell patterns, one of which is discernible in Fig. 6. Figure 6A shows an immature fruiting body at the initiation of stalk formation. A higher magnification (Fig. 6B) of the base shows an area of vertically oriented cells between the circular ring of cells and the cells in the aggregate. Figure 7 shows a later stage in stalk formation. At this stage, the aggregate has been lifted off the surface. A higher magnification of the base (Fig. 7B) reveals again that the entering stalk cells are either pointed toward the aggregate or oriented vertically in the stalk. In addition, the cells appear to have been moving individually rather than in tightly packed sheets as was characteristic of earlier stages (e.g., Fig. 4B). Figure 8 shows a different fruiting body in the early stages of stalk formation. Once again, one can see an area of cells between the circular band and the young fruiting body. The cells in the intermediate area appear disoriented, especially J. BACTERIOL. FIG. 1. Part of a swarm of S. aurantiaca. Cells (6.7 x 107) were placed in a 5-,ul drop on filter paper. near the circular band of cells. A higher magnification of the base reveals cells in the stalk oriented vertically (Fig. 8B). Again, the cells appear not to be organized in sheets in this region, but appear to have been moving as single cells. Possible mechanisms that influence the directions of cell motility are discussed below. Figure 9A shows a later stage in fruiting body development when the stalk is fully formed. A higher magnification reveals that the stalk is a mixture of cells and slime (Fig. 9B). The cells are well separated from each other and are oriented at various angles to the long axis of the stalk. DISCUSSION A plausible model for fruiting body morphogenesis in S. aurantiaca is suggested by the scanning electron micrographs presented here and by previous data. We propose that aggregates form within sites of circular motility. Sometimes the circles were initially empty and were filled by an inward spiraling movement of the cells. In other instances, the spiral formed within a dense field of cells without proceeding through a stage where an empty circle was formed. It is possible that as cells enter the area of circular motility they tend to remain there because there existed a circular pattern of slime trails that were followed and because in more mature aggregates the large streams of incoming cells may block escape from the aggregate. We suggest that either a developmentally specific event induces circular motility and thus initiates aggregation or circular movements occur spontaneously, forming incipient aggregates that are stabilized by a developmentally specific event. A stabilizing event might be the secretion of a chemical at the sites of aggregation, restricting the cells from changing their direction and thus preventing them from leaving the very young aggregate. The streams of cells pointed toward the more mature aggregates (e.g., Fig. 2) suggest that the maturing aggregate is able to recruit new cells. This may be a later event and not necessarily related to the formation of the aggregates. Although it is possible that a chemical gradient attracted cells to the aggregate, one can speculate on alternative possibili-

3 VOL. 163, 1985 FRUITING BODY FORMATION IN S. AURANTIACA 517 FIG. 2. (A) Very young aggregate. Same conditions as for Fig. 1. (B) Higher magnification of the aggregate shown in panel A. The photograph is a composite of four quadrants. ties, ones that need not include chemotaxis toward a diffusible attractant. The argument can be made that, given the proper hydrodynamic properties of polysaccharide chains within the slime, the swirling movement of the cells in the aggregates oriented the slime molecules and thus provided necessary cues to guide cells into the aggregation centers. If one assumes that the polysaccharide in the slime consists of long asymmetric molecules capable of adhering to the cells and of forming intermolecular bonds, then as cells move in circles within the aggregation centers, the polysaccharide chains in the slime will become aligned. One might expect that within the circular trails the slime molecules will become oriented in the direction of cell movement, whereas the polysaccharide chains outside of the aggregate may become oriented so that their long axes face the aggregate. If this were to occur, cells within a short distance of the

4 518 VASQUEZ ET AL. J. BACTERIOL. FIG. 3. (A) More advanced aggregate showing the circular pattern of cells at the base. Cells (2 x 108) were spotted in a 5-,ul drop on the filter paper. (B) Higher magnification of panel A, showing cells moving up the aggregate from the group moving in circles around the base. FIG. 5. Same conditions as for Fig. 3. This is an advanced aggregate viewed from the top. The circular arrangement of cells is seen at the base and as spiral patterns on the aggregate. aggregate might follow the polysaccharide chains and move toward the aggregate, thus creating slime trails. Once the trails form, other cells would simply follow them to the aggregate. If this argument is correct, one would expect that the movement of cells within the trails leading to the aggregates would help to keep the polysaccharide slime molecules aligned, i.e., the trails would be stabilized by the movement of the cells. It is suggested that the postulated following of polysaccharide molecules within slime trails may be similar to the phenomenon of elasticotaxis originally described in the myxobacteria by Stanier (13) and recently suggested by Dworkin to account for tactic movements of M. xanthus toward objects placed on an agar surface (4). Although Sutherland analyzed myxobacteria slime and reported that it consisted of polysaccharides, there are no FIU. 4. (A) More advanced stage of aggregation than shown in Fig. 3. Same conditions as for Fig. 3. (B) Higher magnification of panel A, showing the ring of cells at the base and the cells moving onto the aggregate.

5 VOL. 163, 1985 FRUITING BODY FORMATION IN S. AURANTIACA 519 A.WXMIWUV FIG. 6. (A) Same conditions as for Fig. 1. This sample shows the early beginnings of stalk formation. (B) Higher magnification of panel A. Note the ring of cells near the base and the whorls in the aggregate. Cells immediate to the base have an orientation suggesting that they were moving directly into the aggregate and up. Downloaded from it.ta on December 7, 2018 by guest --f B -e'''t -,",.4,... _.-t. w I - ".4pokmam, -..o ON&A.- V 6 -% -. "i vw-w - lil- i;

6 520 VASQUEZ ET AL. J. BACTERIOL. published reports of its hydrodynamic or flow characteristics (16). Hence, the possible effects of the circular movements of cells at the aggregation centers on the orientation or flow of the slime molecules and the proposed subsequent influence on directional motility of cells at a distance from the aggregate are purely speculative. Downloaded from FIG. 7. (A) Later stage in stalk formation. The aggregate has become more round and is off the surface. Same conditions as for Fig. 1. (B) Higher magnification of panel A. An area of cells can be seen at the base. The cells appear to be entering under the aggregate as it is torn away from the substrate. FIG. 8. (A) Different early stalk stage. Notice the cells moving in a circle at some distance from the base and an area of apparent disorganization near the base. The cells in the area next to the base are presumably the stalk cells preparing to enter the stalk. (B) Higher magnification of panel A. The cells are entering the stalk through material that appears to be slime. As cells accumulated in the aggregate, they appeared to continue to move in a circular fashion but with an altered pitch, enabling them to move up as in a spiral. A smooth transition occurred between the cells that were moving in the circle and those that were actually entering the aggregate (Fig. 3 and 4). The photographs suggest that groups of cells entered the aggregate from different locations at the base and that the pitch varied even within the same aggregate. Numerous connections between the cells were also seen. These resembled slime but one cannot exclude the possibility that some of these connections may function in cell-to-cell signaling. Occasionally, one observes ridge formation or other cellular accumulations that are not spherical, as are the aggregates described here. We have not yet examined the cell patterns in the ridges or elongated aggregates. Thus, our conclusions regarding aggregate formation apply only to spherical aggregates. These are the ones most commonly seen in fruiting swarms of S. aurantiaca. Stalk formation appeared to be due to a different type of on December 7, 2018 by guest

7 VOL. 163, 1985 FRUITING BODY FORMATION IN S. AURANTIACA 521 PlFI. 9. (A) same conditions as tor Fig. 3. [he stalk is complete. (B) Higher magnification of panel A, showing cells in the stalk oriented in an upward direction but often at an angle. A great deal of slime appears to be present. movement. Cells left the circular ring surrounding the aggregate and moved directly into or under the aggregate to form the stalk. This could have been due to a chemoattractant produced by the aggregate. Alternatively, the aggregate might have produced slime in which the polysaccharide chains were oriented appropriately to guide cells directly into the stalk area and upward. The scanning electron micrographs suggest that the complex process of fruiting body formation in S. aurantiaca can be divided into stages distinguished by changes in motility patterns. These patterns include circular or spiral movements by groups of cells during aggregate formation, apparent directed motility as wide streams of cells converge from several different directions on aggregates, and what may be directed single-cell motility during stalk formation. The relative importance of chemical signaling and the orientation or flow of slime molecules remains to be determined. Certainly the physiochemical properties of the slime should be investigated. The experimental use of mutants, especially those defective in single-cell or group motility may aid in determining whether they in fact play separate roles in fruiting body formation. Although these mutants are known for M. xanthus, they have not yet been described for S. aurantiaca (8). ACKNOWLEDGMENTS This research was supported by Biomedical Sciences Support grant PHS S07 RR 7031F. LITERATURE CITED 1. Berg, H. C., and E. M. Purcell Physics of chemoreception. Biophys. J. 20: Burchard, R. P Gliding motility and taxes. In E. Rosenberg (ed.), Myxobacteria: development and cell interactions, p Springer-Verlag, New York. 3. Dworkin, M Cell-cell interactions in the myxobacteria, p In J. M. Ashworth and J. E. Smith (ed.), Microbial differentiation. Society for General Microbiology, Symposium no. 23. Cambridge University Press, Cambridge. 4. Dworkin, M Tactic behavior of Myxococcus xanthus. J. Bacteriol. 154: Dworkin, M., and D. Eide Myxococcus xanthus does not respond chemotactically to moderate concentration gradients. J. Bacteriol. 154: Gilmore, D. F., and D. White Energy-dependent cell cohesion in myxobacteria. J. Bacteriol. 161: Grilione, P. L., and J. Pangborn Scanning electron microscopy of fruiting body formation by myxobacteria. J. Bacteriol. 124: Hodgkin, J., and D. Kaiser Genetics of gliding motility in Myxococcus xanthus (Myxobacterales): two gene systems control movement. Mol. Gen. Genet. 171: Inouye, S., D. White, and M. Inouye Development of Stigmatella aurantiaca: effects of light and gene expression. J. Bacteriol. 141: Reichenbach, H., and M. Dworkin The order Myxobacterales. In M. P. Starr, H. G. Stolp, A. Truper, and H. G. Balows (ed.), Prokaryotes. A handbook on habitats, isolation, and identification of bacteria, vol. 1, p Springer- Verlag, Berlin. 11. Reichenbach, H., H. H. Heunert, and H. Kuczka Chondromyces apiculatus (Myxobacteriales)-Schwarmentwicklung und Morphogenese, film E.779. Institut fur den Wissenschlaftlichen Film, Gottingen, Federal Republic of Germany. 12. Rosenberg, E. (ed.) Myxobacteria: development and cell interactions. Springer-Verlag, New York. 13. Stanier, R. Y A note on elasticotaxis in myxobacteria. J. Bacteriol. 44: Stephens, K., G. D. Hegeman, and D. White Pheromone produced by the myxobacterium Stigmatella aurantiaca. J. Bacteriol. 149: Stephens, K., and D. White Scanning electron micrographs of fruiting bodies of the myxobacterium Stigmatella aurantiaca lacking a coat and revealing a cellular stalk. FEMS Microbiol. Lett. 9: Sutherland, I. W Polysaccharides produced by Cystobacter, Archangium, Sorangium and Stigmatella species. J. Gen. Microbiol. 111: