Periodic Reporting for period 3 - MetaboDevo (Metabolic Coupling During Bacterial Development)
Periodo di rendicontazione: 2023-03-01 al 2024-08-31
The cells of multicellular organisms are different from each other, but they all derive from identical progenitor cells, which transform into different cell types during the development of the organism. This process of “cellular differentiation” is responsible for the formation of all our body parts, of our bones, our skin, our brain, our blood. Cellular differentiation depends on the production of specific “differentiation” machineries in the cell that mediate these transformations. Cells also need to maintain general “housekeeping” machineries for the metabolic functions that are necessary for survival, such as the acquisition of nutrients, the production of energy, and the maintenance of their genetic material. How “differentiation” machineries and “housekeeping” machineries interact to mediate the transformation of one cell type into another is not well understood. This is due largely to the fact that it is difficult to interfere with housekeeping machineries in complex organisms without killing the cells, so studies have typically focused on differentiation-specific machineries only.
Bacteria provide terrific systems for studying the interplay between housekeeping metabolism and differentiation. They have streamlined and easily manipulated cellular machineries and a minimal number of essential genes. Many bacterial species embark upon relatively simple differentiation processes that culminate in the formation of specific cell types. In this project, we aim to provide a comprehensive analysis of the role of housekeeping metabolism during endospore formation in the bacterium Bacillus subtilis. Sporulation is a simple differentiation process that entails the interaction between two cells arising from a single cell division: the forespore, which becomes a metabolically dormant spore that is virtually immortal, and the mother cell, which dies after sporulation. We have developed a system to eliminate specific housekeeping machineries in only the mother cell or the forespore during sporulation. This system enables us to define which housekeeping functions are important during sporulation, and in which cell. We are using this system, in combination with a variety of cutting-edge technologies, to obtain a detailed understanding of the role of housekeeping metabolism in sporulation. We expect that our results will provide insights into the metabolic processes that underpin cellular differentiation.
Bacteria provide terrific systems for studying the interplay between housekeeping metabolism and differentiation. They have streamlined and easily manipulated cellular machineries and a minimal number of essential genes. Many bacterial species embark upon relatively simple differentiation processes that culminate in the formation of specific cell types. In this project, we aim to provide a comprehensive analysis of the role of housekeeping metabolism during endospore formation in the bacterium Bacillus subtilis. Sporulation is a simple differentiation process that entails the interaction between two cells arising from a single cell division: the forespore, which becomes a metabolically dormant spore that is virtually immortal, and the mother cell, which dies after sporulation. We have developed a system to eliminate specific housekeeping machineries in only the mother cell or the forespore during sporulation. This system enables us to define which housekeeping functions are important during sporulation, and in which cell. We are using this system, in combination with a variety of cutting-edge technologies, to obtain a detailed understanding of the role of housekeeping metabolism in sporulation. We expect that our results will provide insights into the metabolic processes that underpin cellular differentiation.
We have modified several hundred genes so that the housekeeping metabolic functions for which they are responsible can be quickly eliminated from the mother cell or from the forespore during sporulation. So far, we have studied the effect of removing a subset of these functions upon spore formation. We have found that a variety of housekeeping functions are necessary for sporulation, but that some of them are differentially required in the mother cell and in the forespore. For example, the production of energy and the synthesis of building blocks for different part of the spore are required exclusively in the mother cell. However, processes required for the assembly of final spore components, such as the spore envelope, must proceed in both the mother cell and the forespore. Our findings indicate that the mother cell provides the forespore with building blocks and energy for the synthesis of different cellular parts. To test this, we have developed methodologies to determine if functions required in the forespore depend on energy and building blocks produced by the mother cell. We have also developed methodologies to directly monitor the movement of building blocks between the two cells. Our results so far indicate that the forespore depends on the mother cell for some of its activities, and that key building blocks move between the two cells, in both directions. We have identified cellular machineries that mediate the intercellular movement of building blocks, which we are currently characterizing.
This project is providing experimental evidence to justify the deeply entrenched assumption in the sporulation field that the mother cell nurtures the forespore during spore formation. Our results indicate that the forespore turns off central housekeeping functions, and depends on mother cell-derived building blocks for the assembly of key parts of the spore. Our results also show that building blocks can move between mother cell and forespore. This movement seems to be bi-directional, suggesting that the metabolic relationship between the two cells is more complicated than mother cell provides materials to nurture the forespore. In the second half of this project, we will study the effects of removing additional housekeeping functions from the mother cell and from the forespore. This will allow us to identify additional metabolic dependencies and to pinpoint the specific building blocks that are moved between the two cells. We will also keep working on strategies to monitor the intercellular transport of building blocks and to identify and characterize the machineries that mediate this transport. By the end of the project, we expect to have a detailed description of the metabolic relationship between the mother cell and the forespore, including which housekeeping activities are required in each cell, which building blocks are shared between them, and how these building blocks move between the two cells. Bacillus spores are metabolically dormant, meaning that they have no detectable metabolic activities. Metabolic dormancy underlies the remarkable resiliency of spores, which can remain viable for hundreds of years in the absence of nutrients. Our findings so far suggest that dormancy is achieved gradually during formation of the spore. Our project will provide insights into the metabolic transformations that mediate this transition to dormancy.