Our long term goal is to define the regulatory pathways that respond to cell envelope stress using Bacillus subtilis as a model system. Exposure of bacteria to antibiotics that interfere with cell wall synthesis or membrane function activates several large regulons coordinated by extracytoplasmic function (ECF) sigma factors and two-component regulatory systems (TCS). These regulons include genes involved in the inactivation or efflux of antibiotics, in remodeling of the cell surface to increase resistance, and in the production of antibiotics. We will pursue three aims directed at understanding the global stress responses elicited by cell envelope stress. First, we will characterize a bacteriocin produced by sporulating B. subtilis cells that selectively kills non-sporulating cells of the same species. This bacteriocin will be structurally characterized, its spectrum and mode of activity defined, and the role of the Sigma-W regulon in defending against bacteriocin activity will be explored. We will also characterize a Sigma-W dependent bacteriocin that is produced in response to antibiotic exposure. Second, we will investigate a TCS that responds to a variety of cell wall active antibiotics that interfere with lipid II function or cycling. This TCS, LiaRS, has an unusual sensor kinase that is representative of a family of intramembrane-sensing kinases and may interact with the LiaF membrane proten. We will explore the nature of signal-sensing by the LiaRS system and define the LiaR regulon. Third, we will use transcriptional profiling to define the stress responses elicited by exposure to antibiotics. This work will compare the stimulons associated with antibiotics with well defined mechanisms of action and closely related structural variants with altered function. In addition, the stimulons elicited by co-culture of B. subtilis with other antibiotic producing bacteria, including Bacilli and Streptomycetes, will be explored. Finally, we will identify and characterize novel antibiotic resistance mechanisms focusing on genes that are strongly induced as part of the cell envelope stress responses. ? ?
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