PROJECT SUMMARY Formaldehyde is a naturally occurring metabolite found in all cell types. Although it has been implicated in human disease including dementia and diabetes, it has also shown to have critical roles in beneficial processes such as memory formation and purine biosynthesis. In methylotrophic bacteria, one-carbon metabolites such as methanol can serve as growth substrates in pathways where formaldehyde is an obligate central intermediate. Due to its high chemical reactivity, formaldehyde balance in these organisms is critical; however, their formaldehyde stress response systems have remained elusive. EfgA and TtmR are central players of two distinct systems that modulate formaldehyde resistance and disrupt formaldehyde homeostasis in the methylotroph Methylorubrum (formerly Methylobacterium) extorquens. EfgA is a newly identified conserved formaldehyde sensor that halts growth and translation in response to elevated formaldehyde levels. TtmR is a MarR-family transcription factor that regulates many genes involved in regulation, signaling, and stress response, including efgA. Our work will characterize the EfgA and TtmR homeostasis systems to understand how cells sense and respond to formaldehyde levels to prevent otherwise inevitable cellular damage. Specifically, we will employ unbiased sequencing-based approaches and experimental evolution to home in on the mechanisms of these systems and define their regulation. Formaldehyde-mediated cellular damage is a readout of the status of formaldehyde homeostasis; however, the in vivo reactivity of formaldehyde is poorly understood. Our data suggests that protein damage is the predominant cause of cytotoxicity in M. extorquens. We will use proteomics approaches to define the impact of formaldehyde on the proteome and identify cellular strategies for counteracting formaldehyde-induced protein damage. Through this work, we will leverage a model bacterium that is well adapted to maintain formaldehyde homeostasis to explore the burgeoning field of formaldehyde regulatory biology. The results from this work will define essential cellular processes and has implications for analogous homeostasis systems for toxic metabolites. We envision this work will have substantial impacts on the understanding of how cells sense and regulate formaldehyde levels, how cells navigate and avoid accumulation of toxic metabolites generally, and how metabolite-specific and global systems of stress response intersect to provide balanced cellular metabolism and growth.