TITLE: Spatial Organization of the Oral Microbiome PI: Jessica Mark Welch Project Summary/Abstract Bacteria colonizing the human mouth play important roles not only in oral health, but in systemic health and disease. This proposal addresses the key unsolved question of how spatial organization of the oral microbiota is connected to its function at the micron scale. Our previous work pioneered the imaging of oral microbiome biogeography by developing a Combinatorial Labeling And Spectral Imaging - Fluorescence in situ Hybridization (CLASI-FISH) strategy to analyze the micron-scale architecture of microbial communities at a systems level. In this project we build on these findings to investigate structure-function relationships, assess complexity of consortia at micrometer scales within single human hosts, and develop multiplexed live cell imaging to study the dynamics of multispecies in vitro biofilms at the single-cell level. In Aim 1 we investigate how a key parameter of host-microbiome interaction, salivary nitrate concentration, affects the structure of the bacterial biofilm on the tongue dorsum. The nitrate-reducing capability of bacteria on the tongue plays a major role in the generation of nitric oxide, which impacts vasodilation and control of blood pressure. In turn, the secretion of nitrate into saliva by salivary glands affects the metabolism and composition of the tongue microbiota. Using dietary nitrate supplementation of volunteers, we will quantify the spatial organization of nitrate-metabolizing taxa in tongue biofilm consortia that developed in high and low salivary nitrate concentrations, and we will use whole-genome shotgun sequence data to determine which species and strains are responsive to nitrate. In Aim 2 we address an almost entirely unexplored question: in micron-scale neighborhoods in the oral microbiome, does each species function as a clone or as a complex mixture of strains? The answer to this question is important for learning to manipulate and modulate the oral microbiome and determining whether, for purposes such as microbiome transplants or building an in vitro biofilm model, a single strain is an adequate representative of the community. We will address this question by microdissection of single tongue dorsum consortia 100-200 micrometers in diameter, followed by whole-genome shotgun sequencing and analysis to assess whether natural oral consortia are made up of clones or complex populations. Live imaging is a powerful method of analysis of taxon-taxon interactions, but multiplexed live imaging of bacteria with single-cell resolution has been almost entirely absent from studies of the oral microbiome. The goal of Aim 3 is to develop a live-imaging system, using vital dyes, to differentiate up to 6 taxa within live simplified communities. We will use this system to investigate the dynamics of taxon-taxon interactions in consortia representing components of dental plaque and the tongue biofilm. Our goal is to ...