The phagocytosis-like process of engulfment is the hallmark of endospore formation in bacteria from the genera Bacillus and Clostridium, which produce unusually durable endospores that are the infectious agent of Anthrax and Botulism. Engulfment mediates a dramatic change in cellular architecture, rearranging the sporangium from two cells that lie side by side, to an endospore in which one cell (the forespore) lies within the cytoplasm of another (the mother cell). The studies supported by this grant have provided new insights into the mechanisms by which coordinated peptidoglycan synthesis and degradation mediate this dramatic example of the architectural plasticity of bacterial cells, and they are providing insight into the role essential proteins play in this process. We here propose to visualize the dramatic morphological rearrangements of sporulation and the protein complexes that mediate them, by using a new implementation of cryo-electron tomography that uses a focused ion beam to produce thin lamella of bacterial cells, producing images that reveal structures within cells at nanometer resolution. We also propose to pursue preliminary data that has revealed that sporulation entails a dramatic and previously unrecognized metabolic differentiation of the two cells, after which the future spore is completely dependent on the mother cell for the precursors for biosynthesis. This process, which appears to be mediated by a massive forespore-specific proteolysis event, effectively converts the two cells into synthrophic partners, providing an accessible model for studying coupled metabolism, which is prevalent in microbial communities and biofilms. We here propose to study these processes, using genetics, metabolomics, fluorescence and cryo- electron microscopy, integrating these experimental efforts with computational analysis and modeling. These studies will reveal the cellular and metabolic landscape of sporulation in unprecedented detail, capitalizing on the dispensability and streamlined machinery of sporulation to provide insight into conserved processes that are often essential for growth.

Public Health Relevance

Sporulation provides an ideal system for understanding the dynamic organization and metabolic specialization of bacterial cells, processes that are essential for bacterial growth, pathogenesis and development. This developmental pathway depends on essential processes, such as peptidoglycan and membrane biosynthesis, and central metabolism, that must be strictly regulated to maintain viability. These enzymes are attractive targets for novel antibiotics, and our studies might also identify new drug targets in proteins that remodel bacterial membranes, localize proteins or mediate metabolic differentiation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM057045-21
Application #
10085240
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Flicker, Paula F
Project Start
1998-01-01
Project End
2021-12-31
Budget Start
2021-01-01
Budget End
2021-12-31
Support Year
21
Fiscal Year
2021
Total Cost
Indirect Cost
Name
University of California, San Diego
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
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Lopez-Garrido, Javier; Ojkic, Nikola; Khanna, Kanika et al. (2018) Chromosome Translocation Inflates Bacillus Forespores and Impacts Cellular Morphology. Cell 172:758-770.e14
Chaikeeratisak, Vorrapon; Nguyen, Katrina; Khanna, Kanika et al. (2017) Assembly of a nucleus-like structure during viral replication in bacteria. Science 355:194-197
Miller, Marina; Tam, Arvin B; Mueller, James L et al. (2017) Cutting Edge: Targeting Epithelial ORMDL3 Increases, Rather than Reduces, Airway Responsiveness and Is Associated with Increased Sphingosine-1-Phosphate. J Immunol 198:3017-3022
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Piña, Francisco Javier; Fleming, Tinya; Pogliano, Kit et al. (2016) Reticulons Regulate the ER Inheritance Block during ER Stress. Dev Cell 37:279-88
Lamsa, Anne; Lopez-Garrido, Javier; Quach, Diana et al. (2016) Rapid Inhibition Profiling in Bacillus subtilis to Identify the Mechanism of Action of New Antimicrobials. ACS Chem Biol 11:2222-31
Sahonero-Canavesi, Diana X; Sohlenkamp, Christian; Sandoval-Calderón, Mario et al. (2015) Fatty acid-releasing activities in Sinorhizobium meliloti include unusual diacylglycerol lipase. Environ Microbiol 17:3391-406
Yen Shin, Jae; Lopez-Garrido, Javier; Lee, Sang-Hyuk et al. (2015) Visualization and functional dissection of coaxial paired SpoIIIE channels across the sporulation septum. Elife 4:e06474
Ojkic, Nikola; López-Garrido, Javier; Pogliano, Kit et al. (2014) Bistable forespore engulfment in Bacillus subtilis by a zipper mechanism in absence of the cell wall. PLoS Comput Biol 10:e1003912

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