Project Summary Essential genes represent the most critical components of a bacterial genome and are required for survival. In recent years, we developed technologies that allowed us to identify and study essential genes in the dental caries pathogen Streptococcus mutans. Through these studies we have identified several genes that have unknown functions and yet, are clearly indispensable for the normal physiology of S. mutans. One of these genes, that we are provisionally naming erfR (essential regulatory factor), is annotated as a transcriptional regulator. Silencing of this gene causes severe growth and cell morphology defects, suggesting that we have identified a new and unique regulatory component of S. mutans biology. Notably, this regulatory protein is also present in the genome of other pathogens, including Group A and Group B streptococci. Since its discovery we have made advances that show that ErfR regulates the expression of a putative integrative conjugative element (ICE) known as TnSmu1. These elements participate in the horizontal transfer of genetic material, and can carry desirable traits such as antimicrobial resistance, and virulence-associated genes. ICEs could be an important mechanism driving genotypic and phenotypic diversity of this pathogen. However, there is little to no in-depth characterization of these elements in Sm. To address this knowledge gap we will exploit TnSmu1 and its regulation by ErfR as a model ICE and have developed three Specific Aims: 1) Functional analysis of TnSmu1 essentiality and potential for horizontal transfer; 2) Identify genes and metabolic activities controlled by TnSmu1 induction by transcriptome analysis; 3) Examine the impact of ICEs on Sm host fitness and evolution. Combined results and conclusions from this proposal will lead to a greater understanding of the mechanisms of erfR essentiality, TnSmu1 function and physiological effects on Sm, and the broader distribution of these elements among Sm strains. The outcomes are expected to position erfR/TnSmu1 as an important model for studying horizontal gene transfer by conjugation in oral bacteria. This will fill a void in our understanding of these elements in Sm pathogenesis and their impacts on oral biofilm ecology. The proposal will also contribute to our long-term aspirations in cataloging essential processes in Sm.