ABSTRACT Dental caries, the most common microbial disease, is caused by overgrowth of acidogenic and aciduric bacteria including Streptococcus mutans. Childhood caries incidence in the U.S. is high and there is a clear imperative to better understand caries pathogenesis. Cariogenic organisms thrive in biofilm environments. Amyloid was first identified in the context of pathology but does not always represent a protein mis-folding pathway. Functional amyloid is also recognized. Amyloid aggregates are evolutionarily conserved cross -sheet quaternary structures with common biophysical properties enabling their detection and study. Multiple microorganisms are now known to produce functional amyloids within biofilm environments. Our group was the first to discover Streptococcus mutans amyloids. We have now identified four amyloid-forming proteins in this bacterium. Three of these, P1 (AgI/II), WapA, and Cnm are sortase-localized adhesins whose extracellular truncation derivatives are amyloidogenic. The previously unknown fourth protein, Smu_63, serves as a negative regulator of biofilm cell density and genetic competence. We have provided extensive tertiary and quaternary structural characterization of adhesin P1 and structural characterization of other proteins is in progress. We have provided definitive X-ray fiber diffraction evidence of a classical stacked -sheet amyloid structure for S. mutans amyloids. Furthermore, our work contributes to a new paradigm for multiple streptococcal and staphylococcal amyloids. Naturally-occurring truncation products play two key roles within these organisms' biofilm life cycles. First by promoting adherence to cognate ligands in their monomeric forms via quaternary interactions with the parent adhesins linked to the cell surface, and second by facilitating detachment of biofilm cells and extracellular matrix components from aging biofilms in their amyloid form. The left-handed Z-configuration of extracellular DNA within biofilms was recently associated with biofilm stability whereas right-handed B-DNA disrupted extant biofilms. The amyloid, but not monomeric form of neuropathologic A, drives conversion of Z-DNA to B-DNA. Cardiolipin-rich mitochondrial membranes modulate amyloidogeneis of -synuclein and Htt involved in Parkinson's and Huntingtin Diseases. We have identified cardiolipin as a prevalent lipid in S. mutans cytoplasmic membranes and extracellular membrane vesicles. In this renewal application we will explore relationships between S. mutans amyloid-forming proteins and B- and Z-forms of DNA in vitro and in vivo within adherent and detaching biofilms (Aim 1), determine the impact of membrane lipid composition on S. mutans amyloid formation within aging biofilms and assess interactions of amyloidogenic proteins with specific lipids of interest (Aim 2), and continue to use state of the art methods including solution and solid-state NMR to identify and characterize structural transitions reflective of mo...