Myxococcus xanthus provides an attractive model to study how cells build multicellular structures and adopt alternative fates. Upon starvation, cells send signals to each other and coordinate their movement to construct uniform mounds. Mounds mature into fruiting bodies as rods inside mounds differentiate into round spores. Other cells undergo lysis or remain outside of fruiting bodies as peripheral rods. To investigate the response of developing rods to nutrients, I added a two-fold dilution series of nutrients at 18 h post-starvation (PS), when cells were not yet committed to form spores (Chapter 2). I discovered an ultrasensitive response to 12.5% vs. 25% nutrient medium addition. This two-fold difference in nutrient addition led to a 30-fold difference in the number of sonication-resistant spores. By systematically measuring transcript and protein levels after nutrient addition, I found that the transcript and protein levels of a key transcription factor, MrpC, correlated best with the sporulation response. The MrpC protein level decreased in the first three hours after nutrient addition, then recovered better after 12.5% vs. 25% nutrient add-back, suggesting that a threshold level of MrpC must be achieved for mounds to persist and spores to form.To visualize cellular shape change in situ, I stained cells with the membrane dye FM 4-64 and visualized them by confocal microscopy (Chapter 3). At 18 h PS, cells were still rods. Rods began transitioning in shape at 24 h PS and many became spores by 42 h PS. Transitioning cells were irregularly-shaped. A second method involving mixtures of fluorescently labeled and unlabeled cells showed consistent results with FM 4-64 staining. Transitioning cells and spores were more abundant close to the radial center of nascent fruiting bodies from the bottom to several cell layers up, and C-signal-dependent gene expression was also greatest near the center. Interestingly, when developing cells were stained with the DNA-binding dye DAPI, the nucleoids condensed and segregated to form two loci as cells were changing shape within nascent fruiting bodies (Chapter 5). Nucleoid segregation was also supported by two other methods, both involving binding of a fluorescent protein to DNA in situ.Development requires not only nutrient scarcity, but also high cell density. C-signal, a short-range signal mediated by the CsgA protein, provides a mechanism for cells to communicate their spatial information. To investigate the effects of C-signaling on the developmental process, I co-developed wild-type and csgA mutant cells that were differentially labeled with constitutively-produced fluorescent proteins (Chapter 4). I found that wild-type cells rescued mound formation and sporulation of the cgsA mutant over a narrow range of ratios. A ratio of 1 wild-type cell to 4 csgA mutant cells (1:4) showed no mound formation or sporulation, at 1:2 mounds formed but not spores, and at 1:1 both mounds and spores formed. These observations indicate that threshold levels of C-signaling are required for mound formation and sporulation.Results from my dissertation suggest that the developmental process of M. xanthus responds ultrasensitively to both nutrients and C-signaling, leading to alternative cell fates. My work has advanced understanding of how cells can integrate multiple signals and respond appropriately to changes in their abiotic and biotic environments.
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