Project Summary Dental caries is the most common chronic disease, globally, is associated with a tremendous economic burden, and disproportionally affects populations least able to access dental care and afford treatment. Streptococcus mutans (Smu) is a common constituent of dental plaque, and the primary etiologic agent of dental caries. Smu causes disease by forming robust biofilms on the tooth surface and producing organic acids which dissolve the underlying tooth enamel. To persist in the dental plaque community and cause disease, Smu must be able to directly outcompete commensal organisms. Our research group recently identified the small molecules mutanic acid (MTA), mutanicyclins (MTC) A-C and their biosynthetic gene cluster (mta BGC) in several globally distributed Smu strains, including B04Sm5, which was isolated from a child with severe early childhood caries. B04Sm5, and MTC A specifically, had significant antimicrobial effects on other oral bacteria. In addition, B04Sm5 produced a more acidic local environment than the Smu paradigm strain, UA159, and this phenotype was dependent on a functional mta BGC. The overarching goal of this proposal is to determine the role of MTA and MTCs in Smu ecology and virulence. We propose an interdisciplinary research approach with two specific aims: Aim 1: Functional insights of the mta BGC and its products MTA and MTCs. MTA and MTC(s) production and regulation will be characterized by liquid chromatography mass spectrometry and RNA-seq analysis of deletion mutants of mta BGC genes. Next, the spectrum of activity of MTA and MTCs will be explored utilizing imaging mass spectrometry (IMS) of competition assays pitting B04Sm5, or the ΔmtaD mutant strain, which does not produce MTA or MTCs, against other oral bacteria. IMS will also be used to identify additional small molecules pertinent to the interspecies competitions. Aim 2: Determine the impacts of MTA, MTC A-C, and mta encoding Smu on oral biofilm virulence and community function. The effects of purified MTA, MTCs, as well as MTA/MTC producing strains of Smu, on oral microbial ecology and biofilm virulence will be explored using our established in vitro oral biofilm model, featuring over 100 taxa, and downstream meta-OMICS analyses. Changes in community virulence via acid production and biofilm formation will also be monitored. The proposed study provides a unique opportunity to expand our knowledge of the role of BGCs in the oral microbiome, which is severely lacking, and specifically elucidate a novel virulence pathway in Smu. This research will significantly increase our understanding of the pathogenesis of caries, which represents a tremendous global medical burden, and is likely to guide development of new therapeutics and preventative measures.