The objectives of the project are to uncover the mechanisms of gene control that turn on, and distinguish between, developmental programs of spore formation and matrix production by Bacillus subtilis. Spore formation has been traditionally viewed as a behavior of free-living cells, but we now understand that differentiation also occurs in the context of structured, multicellular communities (biofilms) consisting of chains of matrix-producing cells as well as spore-forming cells. Indeed, the mechanisms that govern entry into sporulation are intimately interwoven with those that govern matrix production. This proposal addresses important gaps in our understanding of sporulation and multicellularity with four specific aims: (1) We will identify the natural environmental signals that trigger spore formation and multicellularity by two sensor histidine kinases. (2) We will visualize cell fate switching between planktonic and matrix-producing states in real time using a newly devised microfluidic device. We will determine how a double-negative feedback loop controls switching and how cells discriminate between alternative fates of spore formation and matrix production. (3) We will determine how D-amino acids are produced late in the life cycle of the biofilm and how they trigger biofilm disassembly. We will determine the regulatory mechanisms that control the expression of the racemase genes that are responsible for D-amino acid production, how D-amino acids are incorporated into the peptidoglycan and how they trigger the release of an amyloid-fiber component of the matrix. (4) We will determine the full cascade of regulatory events that govern the differentiation of a cell into a spore, including the mechanisms that govern switching from one sigma factor to another. Understanding how D-amino acids cause biofilm disassembly will inform strategies for blocking biofilm formation by pathogenic bacteria. Also, research into gene control by B. subtilis, the principal model organism for Gram-positive bacteria, has provided, and will continue to provide, fundamental insights into the molecular biology of related, pathogenic bacteria, such as Staphylococcus, Enterococcus and B. anthracis.

Public Health Relevance

An important problem in public health is the capacity of pathogenic bacteria to form surface- associated communities known as biofilms, which exhibit high levels of resistance to antibiotics. This research on the bacterium Bacillus subtilis is revealing novel and general approaches to preventing biofilm formation that appear to be applicable to a wide variety of pathogens, including Staphylococcus and Pseudomonas. Also, the research will provide insights into important human pathogens that are close relatives of B. subtilis, such as Staphylococcus, Streptococcus, and Enterococcus and the bioterrorism agent, Bacillus anthracis.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Gaillard, Shawn R
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Harvard University
Schools of Arts and Sciences
United States
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Cabeen, Matthew T; Leiman, Sara A; Losick, Richard (2016) Colony-morphology screening uncovers a role for the Pseudomonas aeruginosa nitrogen-related phosphotransferase system in biofilm formation. Mol Microbiol 99:557-70
Flanagan, Kelly A; Comber, Joseph D; Mearls, Elizabeth et al. (2016) A Membrane-Embedded Amino Acid Couples the SpoIIQ Channel Protein to Anti-Sigma Factor Transcriptional Repression during Bacillus subtilis Sporulation. J Bacteriol 198:1451-63
DeLoughery, Aaron; Dengler, Vanina; Chai, Yunrong et al. (2016) Biofilm formation by Bacillus subtilis requires an endoribonuclease-containing multisubunit complex that controls mRNA levels for the matrix gene repressor SinR. Mol Microbiol 99:425-37
Gao, Tantan; Foulston, Lucy; Chai, Yunrong et al. (2015) Alternative modes of biofilm formation by plant-associated Bacillus cereus. Microbiologyopen 4:452-64
Cabeen, Matthew T; Losick, Richard (2015) Bacterial backstabbing: EF-Tu, brute? Cell 163:537-9
Bradshaw, Niels; Losick, Richard (2015) Asymmetric division triggers cell-specific gene expression through coupled capture and stabilization of a phosphatase. Elife 4:
Leiman, Sara A; Richardson, Charles; Foulston, Lucy et al. (2015) Identification and characterization of mutations conferring resistance to D-amino acids in Bacillus subtilis. J Bacteriol 197:1632-9
Leiman, Sara A; Arboleda, Laura C; Spina, Joseph S et al. (2014) SinR is a mutational target for fine-tuning biofilm formation in laboratory-evolved strains of Bacillus subtilis. BMC Microbiol 14:301
Cabeen, Matthew T (2014) Stationary phase-specific virulence factor overproduction by a lasR mutant of Pseudomonas aeruginosa. PLoS One 9:e88743
Lebar, Matthew D; May, Janine M; Meeske, Alexander J et al. (2014) Reconstitution of peptidoglycan cross-linking leads to improved fluorescent probes of cell wall synthesis. J Am Chem Soc 136:10874-7

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