C Determination of Number of Myxospores in Myxococcus xanthus fruiting bodies. Guadalupe Virginia Nevarez-Moorillon Biology Department University of North Texas P. o. Box 5218 Denton, Tx. 76203 Myxobacteria are common soil bacteria with unique morphological properties. They are typical Gram negative, rod shaped aerobic bacteria that are distinguished by their gliding motility, developmental characteristics and social interaction. The life cycle ofMyxobacteria includes the vegetative state, and when high cell densities and conditions are appropriate, the formation of a fruiting body, where the transformed vegetative cells are preserved as myxospores. Fruiting body formations is still a unexplored subject. For the study of fruiting body formation, several data are still missing. Although it is easy to recognize Myxobacteria by fruiting bodies morphology, little is known about the biochemical characterization and morphogenesis of the fruiting bodies. An interesting data to get will be the efficiency of packing of myxospores in the fruiting body, generating in this way, a number that can correlate the number of myxospores with the size of fruiting body. An attempt to get this number was made by using four strains ofM xanthus isolated from the Woods Hole area . The myxospores were grown in KJK medium, with rabbit dung pellets embedded on the agar plates, and in TPM starvation medium. The number of myxospores per fruiting body was determined by resuspending a determined number of fruiting bodies in distilled water, sonicate the suspension and count the number of myxospores using the Petrof-Hauser chamber. At the same time, the diameter of the fruiting bodies was measure so it can be transformed to volume. Significant differences were observed in the size of fruiting bodies generated in TPM medium versus K/K medium (dung pellets), being smaller in the former. In both cases, however, a high variance in fruiting body volume as well as .number of myxospores was observed. This variance can be explained by the diversity of fruiting body sizes, having in the same plate 10 fold differences. The number of myxospores per fruiting body and the mean fruiting body volume were significant correlated (r=0.5083, p=O.Ol4) . A number of myxospores per volume fruiting body is expected to be generated from this data. However, more data with respect to At. xanthus as well as other myxobacteria species is required. 0 Determination of Number of Myxospores in Myxococcus xanthus fruiting bodies. Guadalupe Virginia Nevarez-Moorillon Biology Department University of North Texas P. 0. Box 5218 Denton, Tx. 76203 Myxobacteria are common soil bacteria with unique morphological properties. These organisms are a great example of the diversity of strategies that have evolved to persist in the environment. The complex life cycle and the social behavior displayed by Myxobacteria are unique among the prokaryotes and have provided a useful system to study the molecular basis of development and cell-cell interactions. Myxobacteria are typical Gram negative, rod shaped aerobic bacteria that are distinguished by their gliding motility, developmental characteristics and social interaction. The life cycle ofMycobacteria includes the vegetative state, and when high cell densities and conditions are appropriate, the formation of a fruiting body, where the transformed vegetative cells are preserved as myxospores. Myxospores are considered to be the resting state of the organism and are substantially different than bacterial spores, varying in physical properties as well as in formation strategy. Myxospores are similar to Azotobacter cysts. Instead of the vegetative cell producing an endospore, as in Bacillus, the vegetative cell itself converts to the myxospore. The myxospores are resistant to desiccation, moderate heat (60 O C) and considerable mechanical stress, such as ultrasonic treatment. The myxospores are more resistant to ultraviolet radiation than the vegetative cells . All those properties are fundamental for survival in the ecological niches of Myxobacteria. MyxobacteHa usually are found in soil rich in organic matter and decaying material. These environments are sometimes subjected to hostile conditions such as desiccation or high heat for short periods of time. Myxospores are, then, the stage of the Myxobacteria that ensure survival. Myxospores are physiologically almost inactive, as observed by their low respiratory rate. When the conditions are feasible, myxospores will germinate to give rise to vegetative cells. The vegetative cell, will reproduce, show social behavior, move by gliding motility, grow in high densities, and eventually form fruiting bodies. Fruiting body formations is still a unexplored subject. It is known that for a fruiting body formation high density of vegetative cells is required, and is usually tiggered by a depletion of nutrients. Although it is known that vegetative cells inside the fruiting bodies are transformed to myxospores, generation of myxospores does not necessarily require the presence of fruiting bodies. Myxospores can be found in the surroundings of fruiting bodies, and can be produced by adding different chemical compounds to vegetative cell cultures. The real role of fruiting body in nature -. / is still unclear. Although it may give a little more protection to the myxospores, it can be speculated that the real flinction of fruiting bodies is to ensure that the newly generated vegetative cells will be in close proximity with other vegetative cells. In this way, the community structure vital for Myxobacteria survival will be ensured. The community structure is important for Myxobacteria, because of their nutritional characteristics. MyxobacteHa usually degrades biopolymers to survive, using exoenzymes for this purpose. The myxobacteria will excrete the enzymes into the environment and will utilize molecules liberated by the enzymatic activity close to the cell Ifthe myxobacteria were living as isolated cells, the chances of molecules coming close to the cell will be minimal. Being in a community, however, increases the chances to get molecules for nutrition, products of degradation either of its own enzymes, or some other cells exoenzymes. For the study of fruiting body formation, several data are still missing. Although it is easy to recognize myxobacteria by fruiting bodies morphology, little is known about the biochemical characterization and morphogenesis of the fruiting bodies. The most simple myxobacterial fruiting bodies are mounds of soft or hardened slime in which the myxospores are embedded. However, little is known about the distribution of the myxospores within the fruiting body. A interesting data to get will be the efficiency of packing of myxospores in the fruiting body. A number that can correlate the number of myxospores with the size of fruiting body will be helpful in the developmental studies with myxobacteria. For the reasons above described, I gave myselfto the work of an intent to determine the number of myxospores in a fruiting body. MATERIALS AND METHODS Samples of soil and bark were collected in the Woods Hole area and let them sit under appropriate conditions that will favor the fruiting body formation of myxobacteria. From there, fruiting bodies were pick up and transferred to culture media that fhvors the grow of myxobacteria, including K/K medium, a water agar medium with dung pellets embedded on the agar plate, CiT a rich medium with peptones and no sugars as source of carbon, and Nutrient Agar diluted 1:10. The plates were incubated at 32°C for several days. To all the media, cyclohexamide was added in a final concentration of lOFzm/ml to prevent flungal growth. The gliding fronts ofMyxobacteria were transferred again to new media plates to obtain pure cultures. To demonstrate that a myxobacterial culture was pure, the culture was inoculated in full strength nutrient agar and incubated under shaking conditions at 37°C overnight. Myxobacteria cannot growth under this conditions, but contaminants can, demonstrating in this way, that the culture is not pure Once pure cultures ofMyxobacterla were obtained, the next step was to obtain fruiting fl bodies. For this purpose, to media were used: K/K medium, in where the fruiting bodies will be formed on the top of the rabbit dung pellets, and in TPM medium, a chemically defined medium low in nutrient concentration, in where the vegetative cells will not divide but will produce fruiting bodies. To count myxospores in the fruiting bodies already form, a number of fruiting bodies were carefully collected and resuspended in I ml. of sterile distilled water. It is worth to note that only fruiting bodies that were spherical in form were selected. This was with the intention to calculate the volume of the fruiting body. The diameter of the fruiting bodies that were picked up was measure at that point. Once a considerable number of fruiting bodies were picked up, the suspension was processed by to sonication to disrupt the myxospores aggregates. The sonication was done as three cycles, 20 seconds each at a 30% output efficiency in a Ultrasonic sonicator. The myxospore suspension was then centrifhged at 15000 rpm for 2 minutes and the supernatant removed. The myxospores were finally resuspended in 0.1 ml. of sterile distilled water and counted in the Petrof Hauser chamber. A total of 20 squares were counted to obtain the average myxospores/square of the suspension. This measurement was repeated four times for each sample. The results are expressed as number of myxospores per average volume of fruiting bodies. For the experiment, several isolates were used, all of them isolated during the course either by myself or by my classmates. RESULTS Strains used: At xanthus (isolated by Minoru Wada) M xanthus (isolated by Paul Egland) M xanthus (isolated by Malema Martinez-Canamero) M xanthus (isolated by Ann West) M virescens (isolated by Carol Barford) M virescens (isolated by Ann West) M virescens (isolated by Lee Hughes) C. coralloides (isolated by Lee Hughes) C. coralloides (isolated by myself) All the strains were grown in dung pellets and in TPM medium, but some of them did not produce fruiting bodies in TPM medium.