Queuosine (Q) is the only tRNA modification that can be salvaged, linking nutrition to translation. In eukaryotes salvage is the only Q synthesis route and the precursor base queuine (q) is provided by the diet or the gut microflora and directly inserted in target tRNAs. Around 50% of the gut microbiota model species are predicted to be auxotrophs and must salvage a Q precursor. In general, bacterial Q salvage enzymes are very poorly characterized, particularly in Gram-positive bacteria that are prevalent in the gut and oral microbiome. This project focuses on discovering and characterizing Q salvage enzymes as well as novel Q synthesis and Q degradation enzymes in bacteria associated with the human host. We will also explore in detail the physiological role of Q in the major oral pathogen Streptococcus mutans as preliminary results suggest a role of Q and/or Q precursors in biofilm formation, competence and virulence. The goal of Aim 1 is to experimentally characterize two main families of transporters predicted to salvage Q precursors: the YhhQ/COG1738 family and the Energy-Coupling Factor (ECF) substrate specificity families (QueT/QtrT). Indeed, these families are not iso-functional, and members seem to diverge, depending on the species, on the ability to transport different Q precursors. Aim 2 focuses on the biochemical characterization of the novel Q synthesis enzyme QueH that catalyzes the last step of biosynthesis through an unprecedented reaction. In both Aims 1 and 2, we will also hunt for “missing” components in Q metabolism such as missing transporters in prevalent gut microbiome bacteria such as Brevibacteria and potential Q catabolism genes. The final aim will explore the role of Q synthesis genes in S. mutans testing the hypothesis that they could have important roles in regulation of competence. The proposed research is significant because the microbiota is a key source for the queuine micronutrient, hence fully characterizing the bacterial Q synthesis, salvage and degradation pathways in human associated bacteria is a requirement to fully understand and model the competition dynamics for this key resource. In addition, because of the importance of S. mutans in dental caries progression, understanding the molecular basis for the observed importance of Q synthesis genes in competence will increase our understanding of key component of how this organism competes with others in its oral microbiome niche.