EfgA is a conserved formaldehyde sensor that leads to bacterial growth arrest in response to elevated formaldehyde.

Normal cellular processes give rise to toxic metabolites that cells must mitigate. Formaldehyde is a universal stressor and potent metabolic toxin that is generated in organisms from bacteria to humans. Methylotrophic bacteria such as Methylorubrum extorquens face an acute challenge due to their production of formaldehyde as an obligate central intermediate of single-carbon metabolism. Mechanisms to sense and respond to formaldehyde were speculated to exist in methylotrophs for decades but had never been discovered. Here, we identify a member of the DUF336 domain family, named efgA for enhanced formaldehyde growth, that plays an important role in endogenous formaldehyde stress response in M. extorquens PA1 and is found almost exclusively in methylotrophic taxa. Our experimental analyses reveal that EfgA is a formaldehyde sensor that rapidly arrests growth in response to elevated levels of formaldehyde. Heterologous expression of EfgA in Escherichia coli increases formaldehyde resistance, indicating that its interaction partners are widespread and conserved. EfgA represents the first example of a formaldehyde stress response system that does not involve enzymatic detoxification. Thus, EfgA comprises a unique stress response mechanism in bacteria, whereby a single protein directly senses elevated levels of a toxic intracellular metabolite and safeguards cells from potential damage.

Formaldehyde-responsive proteins, TtmR and EfgA, reveal a tradeoff between formaldehyde resistance and efficient transition to methylotrophy in Methylorubrum extorquens.

For bacteria to thrive they must be well-adapted to their environmental niche, which may involve specialized metabolism, timely adaptation to shifting environments, and/or the ability to mitigate numerous stressors. These attributes are highly dependent on cellular machinery that can sense both the external and intracellular environment. Methylorubrum extorquens is an extensively studied facultative methylotroph, an organism that can use single-carbon compounds as their sole source of carbon and energy. In methylotrophic metabolism, carbon flows through formaldehyde as a central metabolite; thus, formaldehyde is both an obligate metabolite and a metabolic stressor. Via the one-carbon dissimilation pathway, free formaldehyde is rapidly incorporated by formaldehyde activating enzyme (Fae), which is constitutively expressed at high levels. In the presence of elevated formaldehyde levels, a recently identified formaldehyde-sensing protein, EfgA, induces growth arrest. Herein, we describe TtmR, a formaldehyde-responsive transcription factor that, like EfgA, modulates formaldehyde resistance. TtmR is a member of the MarR family of transcription factors and impacts the expression of 75 genes distributed throughout the genome, many of which are transcription factors and/or involved in stress response, including efgA Notably, when M. extorquens is adapting its metabolic network during the transition to methylotrophy, efgA and ttmR mutants experience an imbalance in formaldehyde production and a notable growth delay. Although methylotrophy necessitates that M. extorquens maintain a relatively high level of formaldehyde tolerance, this work reveals a tradeoff between formaldehyde resistance and the efficient transition to methylotrophic growth and suggests that TtmR and EfgA play a pivotal role in maintaining this balance.Importance: All organisms produce formaldehyde as a byproduct of enzymatic reactions and as a degradation product of metabolites. The ubiquity of formaldehyde in cellular biology suggests all organisms have evolved mechanisms of mitigating formaldehyde toxicity. However, formaldehyde-sensing is poorly described and prevention of formaldehyde-induced damage is primarily understood in the context of detoxification. Here we use an organism that is regularly exposed to elevated intracellular formaldehyde concentrations through high-flux one-carbon utilization pathways to gain insight into the role of formaldehyde-responsive proteins that modulate formaldehyde resistance. Using a combination of genetic and transcriptomic analyses, we identify dozens of genes putatively involved in formaldehyde resistance, determined the relationship between two different formaldehyde response systems and identified an inherent tradeoff between formaldehyde resistance and optimal transition to methylotrophic metabolism.