Grant: $529,048 - National Science Foundation - Jul. 20, 2009
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Award Description: The majority of biological processes such as differentiation, growth, development and transformation are under tight control of cellular signaling systems. But detailed mechanisms of how cells decide when and where to execute these processes remains one of the great mysteries of modern biology. Discovery of general principles underlying signal transduction in a simple unicellular organism is an essential step towards understanding the complexity of genetic and biochemical networks. Sensory behaviors of bacteria therefore serve as powerful models for exploring the molecular basis of cell signaling, differentiation and development. Sporulation in the soil bacterium Bacillus subtilis is one of the best studied model systems but the mechanisms of cell-fate decisions are still far from understood. The sporulation process involves sensing of presently unidentified environmental cues, temporal and spatial control of gene expression, and stochastic but robust intracellular signal processing. The long-term goal of this project is to reveal how bacterial cells process signals to make cell decisions during sporulation. As a step toward that goal, the proposed research will elucidate systemic properties of the sporulation initiating network (SIN, a phosphorelay signaling cascade coupled to transcriptional autoregulatory loops) with a synergistic combination of wet-lab experimentation, computational data analysis, and mathematical modeling. The cornerstones of the approach include: (i) measurement of various biochemical parameters of the phosphorelay with in vivo and in vitro assays; (ii) quantification of cell growth dynamics and expression of early sporulation genes at the single cell level using time-lapse microscopic images in combination with a multicolor fluorescence reporter system; (iii) dissection of the SIN into smaller functional modules using a newly devised artificial B. subtilis strain in which the sporulation signal is known and can be monitored and controlled; (iv) use of statistical models to systematically analyze the experimental data, identifying important factors that bias the sporulation decision; (v) formulation of integrative mathematical models of the phosphorelay to analyze robustness, noise, and temporal variation of gene expression. The proposed methods and approaches will also address fundamental biological questions on the relationship between population heterogeneity, noise, integrated system behavior, and decision making in response to environmental stimuli. The training of undergraduate and graduate students is an integral part of this proposal. The PIs are committed to working closely with trainees. Some of the proposed experiments and computational work can be performed by undergraduate students and comprise a suitable teaching tool to provide broader insight into the biological and scientific questions addressed by the emergent field of systems biology. The project will provide abundant opportunities for the students involved in its experimental and/or mathematical modeling aspects to work together synergistically. The research will thus train scientists capable of effectively bridging the gaps between the biological sciences and quantitative disciplines. Over the last three years, nine undergraduate students with minority status have participated research projects in the Fujita and Igoshin labs (7 and 2, respectively). The PIs will continue their efforts to recruit students from these typically underrepresented groups through the Alliances for Graduate Education in the Professoriate Summer School – an exciting undergraduate research program that is specifically designed to give hands-on research experience to undergraduate students.
Project Description: Our research team has begun to explore the initiation of sporulation in Bacillus subtilis by synergistic cytological, biochemical, and bioinformatic approaches. Sporulation is governed by a cascade of proteins that ultimately determines whether the master response regulator Spo0A is phosphorylated. Phosphorylated Spo0A then activates an array of genes that direct the morphological change from a vegetative growing cell to a spore. As an added complication, Spo0A becomes active only in a subpopulation of genetically identical cells. As a long-term goal, we will determine how a sporulation initiation network (SIN) activates the master regulator Spo0A in the subpopulation. In specific objective 1, we will examine gene expression at the single cell-level. We have devised a time-lapse microscopy system that examines cell fate of live cultures. For this, a variety of CFP and YFP-reporter systems will be used as read-outs of different outputs of the sporulation process. In specific objective 2, we will use quantitative western analysis to measure the number of molecules of the phosphorelay mechanisms and also recapitulate the system in vitro using physiologically-relevant levels to measure the kinetics of phosphorylation/dephosphorylation. In specific objective 3, we will use rigorous statistical methods to analyze the data and use mathematical methods to model the sporulation signaling network. The model will be tested using published data. In this quarter, first, to distinguish between growing and sporulation cells in the population, the abrB promoter (active during growth) and the spoIIG promoter (Spo0A-dependent promoter of spoIIG) were fused to the gene for YFP or CFP, respectively. Second, to quantify the extrinsic and intrinsic noises in sporulation gene expression experimentally, we constructed a strain harboring two reporter genes (CFP and YFP) controlled by identical promoters. We successfully confirmed the expression of the reporter genes in each strain.
Infrastructure Description: n/a
Jobs Summary: The University of Houston values research as one of its top institutional priorities; it is a vital part of the university’s strategic vision. To take our place among the nation’s great metropolitan research institutions, we are building a well-rounded, robust research core group , made up of outstanding faculty and staff. This core group will ensure production of innovative research important to our community and beyond. The ARRA funding is critical to the building of that research core group. The workforce for ARRA funded research projects may include academic faculty and staff with the following titles: Principal Investigator, Co-Principal Investigator, Professor, Associate Professor, Assistant Professor, Research Scientist, Research Professor, Research Associate Professor, Research Assistant Professor, Senior Research Scientist, Graduate Research Assistant, Research Associate, Lab Technician, Research Lab Manager, Post Doctoral Fellow. Each of these titles contributes to the future of discovery — scientific and otherwise. (Total jobs reported: 1)
Project Status: Less Than 50% Completed
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