Browsing by Subject "Bacillus subtilis"
Now showing 1 - 4 of 4
- Results Per Page
- Sort Options
Item Defining the Constellation of RNA Elements That Associate with Bacillus Subtilis HFQ(2013-01-17) Dambach, Michael David; Winkler, Wade C.Bacteria utilize a wide variety of genetic regulatory strategies in order to sense and respond to various environmental fluctuations in nutrient availability, temperature, salinity, and oxygen among others. As such, bacterial species have evolved highly coordinated and tightly regulated systems as a means of efficiently responding to potentially deleterious changes in environmental conditions. Traditionally DNA binding transcriptions factors were thought to be the primary means by which the cell executes a selective genetic response. However, the advent of microarray and next generation sequencing platforms, coupled with the wealth of sequenced genomes and powerful bioinformatics have revealed that RNA mediated post transcriptional gene regulation is wide spread in bacterial species and may in fact rival protein based regulatory systems in scope and breadth. RNA mediated post transcriptional gene regulation is broadly divided into two categories-those in which the RNA element is transcribed with the mRNA it regulates (cis-acting regulatory RNAs) or those which are transcribed independently from the gene that they regulate (trans-acting regulatory RNAs). In general cis-acting RNA elements are embedded within a 5' UTR of a gene that they regulate and may or may not require a protein cofactor to execute genetic regulation. Whereas, trans-acting regulatory RNAs, also known as sRNAs, function via base pairing with their target mRNA and this usually requires the protein chaperone Hfq. Hfq mediated gene regulation is poorly understood in Gram-positive organism, thus I undertook studies of this protein in the model Gram-positive organism Bacillus subtilis. I used co-immunoprecipitation and deep-sequencing to define the suite of RNA elements that associate with this regulatory protein. In addition I performed global transcriptomic studies on an Hfq deletion mutant in order to identify genes that are regulated via Hfq. These studies identified sRNAs that may be involved with sporulation. This led me to analyze the transcriptomic profile of Bacillus subtilis spores in an attempt to identify new sRNA regulators.Item Dynamics of Cell Fate Decision Making Between Sporulation and Competence in Bacillus Subtilis(2013-01-02) Kuchina, Anna 1985-; Altschuler, Steven J.; Alto, Neal; Graff, Jonathan M.; Süel, Gürol M.During multipotent differentiation cells must reliably make a cell fate decision under a variety of conditions, yet remain sensitive to changes in extracellular environment. It is unclear how the cells reconcile these seemingly contradictory requirements. To complicate the issue, the cells often face a decision between multiple fates mediated by the respective differentiation programs which could become active at once. How cells make a specific cell fate choice when presented with several possibilities is a fundamental, yet poorly resolved question. To study cell-fate decision-making dynamics, I utilized the soil bacterium Bacillus subtilis which under stress can either become competent for DNA uptake or undergo sporulation. The master regulator of sporulation is the transcription factor Spo0A. Single cell measurements of Spo0A dynamics along with activities of stage-specific sporulation reporters Spo0F, SpoIIE and SpoIIR revealed the reversible and noisy progression of sporulation up until the final irreversible decision point. Mathematical modeling suggested that such strategy might be advantageous for coping with unpredictable environment. The alternative cell fate of competence is controlled by the transcription factor ComK. Using time-lapse fluorescence microscopy, I quantitatively measured the activities of Spo0A and ComK, along with other cross-regulatory genes, simultaneously in single B. subtilis cells. I found that, surprisingly, sporulation and competence progressed independently in the same cell without cross-regulation up to the final decision point. This finding was confirmed by the discovery of cells in a conflicted state that progressed to sporulation despite the expression of ComK. Measurements of gene expression dynamics in these cells revealed key differences in the relative timing of differentiation programs. To investigate the importance of relative timing, I altered it by engineering artificial cross-regulatory links between the sporulation and competence genetic circuits. Results favor a simple model for cellular decision-making that does not require intricate cross-regulation prior to the decision. Rather, cell fate choice appears to be the outcome of a "molecular race" between independently progressing differentiation programs. This temporal competition mechanism provides a simple, yet efficient way to generate mutually exclusive cell fates. Investigation of the benefits and limitations of such strategy opens a promising venue for future studies.Item Structural Insights into Sporulation in Bacillus sibtilis(2010-05-14) Lee, James; Gardner, Kevin H.PAS domains are modular domains that providing specific interaction surfaces for a diverse array of ligands, from small organic compounds to intra and intermolecular protein domains. As such, they are ubiquitous throughout signal transduction pathways in all three kingdoms of life: Bacteria, Archae, and Eucarya (Huang, Edery et al. 1993). The significant role played by PAS domains in Bacillus subtilis sporulation is underscored by the finding that the PAS-A domain is necessary for efficient phosphorylation in the sporulation kinase, KinA(Wang, Fabret et al. 2001). This activity is necessary for initiating a phosphorelay that results in the upregulation of genes required for sporulation. In KinA, dimerization is necessary for individual monomers of the histidine kinase domain to transphosphorylate partner subunits. The dimerization of KinA involves interactions in the PAS-A domain, but molecular details regarding PAS-A dimerization and its importance to KinA activity has not been previous characterized. To investigate these interactions within the context of the KinA homodimer, we expressed the N-terminal PAS-A domain and solved the X-ray crystal structure. Conformational variability was implicated through the observation of different orientations of the dimerization interface in two distinct structural models found in the asymmetric unit of the crystal lattice. These models were used to identify key interfacial residues and the roles of these were tested in a variety of ways by site-directed mutagenesis.Item A Synthetic Quorum Sensing System Reveals Interaction Between Extracellular Matrix and Quorum Sensing Molecules(2015-09-29) Zhang, Fang; Ross, Elliott M.; Süel, Gürol M.; Sperandio, Vanessa; Liou, JenEven though bacteria are unicellular organisms, they commonly reside in structured communities known as biofilms. One of the defining characteristics of biofilms is the presence of an extracellular matrix (ECM) that encapsulates all cells within the community and provides the biofilm with structural integrity. The production and degradation of ECM components are often regulated by quorum sensing (QS), a prevailing cell-cell communication method between bacterial cells. Quorum sensing allows bacteria to communicate with each other by secreting and sensing small molecules called quorum signals. The literature suggests that the ECM may affect diffusion of quorum molecules through a physical connection between these processes. However, since QS regulates ECM expression, ECM expression and QS are tightly coupled and cannot be perturbed independently. Here we constructed a synthetic QS system in Bacillus subtilis to overcome this limitation and investigate whether ECM production affects QS signals, by quantitatively measuring the synthetic QS response in biofilm communities and single cells. Specifically, we constructed a synthetic quorum-sensing system with designated "Sender" and "Receiver" cells in Bacillus subtilis. This synthetic QS system allowed us to uncouple and independently investigate ECM production and QS in both biofilms and single cells. Our results showed that ECM-producing cells have a higher gene expression response to QS signals. The enhanced QS response suggests a private benefit for ECM-producing cells, which may indicate another mechanism to balance the cost of ECM production and constrain ECM production cheaters in biofilms.