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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM018568-42
Application #
8611923
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Gaillard, Shawn R
Project Start
1976-02-01
Project End
2016-02-29
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
42
Fiscal Year
2014
Total Cost
$760,596
Indirect Cost
$301,976
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
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Wang Erickson, Anna F; Deighan, Padraig; Garcia, Cinthia P et al. (2017) An Amino Acid Substitution in RNA Polymerase That Inhibits the Utilization of an Alternative Sigma Factor. J Bacteriol 199:
Russell, Jonathan R; Cabeen, Matthew T; Wiggins, Paul A et al. (2017) Noise in a phosphorelay drives stochastic entry into sporulation in Bacillus subtilis. EMBO J 36:2856-2869
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
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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

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