Biology subjects

Lee, J. A.

Publications and source records attributed to Lee, J. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Retrograde trafficking inhibitors allosterically trap Get3 to block tail-anchored protein biogenesis

Retro-1 and Retro-2 are structurally distinct small molecules that protect cells from diverse toxins and viruses by disrupting retrograde trafficking, yet their mechanism of action has remained elusive. We show that both compounds target Get3, the ATPase chaperone of the guided entry of tail-anchored proteins (GET) pathway, which mediates biogenesis of tail-anchored SNARE proteins required for retrograde transport to the ER membrane. Cryo-electron microscopy reveals that Retro compounds bind a cryptic pocket in Get3, allosterically stabilizing Get3 in a stalled complex with upstream pathway components. Our work uncovers the GET pathway as an unsuspected vulnerability in pathogen entry, provides clear routes toward compound optimization, and establishes stabilization of dynamic protein complexes as a therapeutic strategy.

biochemistry↗

bioRxiv2026

Toward resolving gravitational effects on microbial growth with computer simulations

As the duration of space flights increases, so does the need to optimize off-planet microbial growth. Microbes can both be unintentionally brought into space and cause human disease or be intentionally harnessed for on-site bioengineering functions. However, optimizing microbial growth is challenging due to an insufficient understanding of how microbial communities are affected by the extraterrestrial environment. To address this gap, we have modified a previously developed model for cell growth in microgravity. By improving the functional form used for cell growth as well as the code usability, we enable further research into how microbial communities are influenced by gravity. Applying this model to isolate individual effects of gravity on cell growth indicates that a lack of gravity-driven flow decreases cell growth in microgravity, while the absence of sedimentation increases cell growth in microgravity. These opposite effects likely contribute to the system-dependent effects of microgravity observed experimentally.

biophysics↗

bioRxiv2026

Enhanced Bacillus subtilis natural competence enables multiplexed genome and spore engineering

Bacillus subtilis is an important chassis for biotechnology, but its use in multiplex genome engineering is limited by low natural transformation efficiency. Here, we compared inducible promoter systems for synthetic activation of the competence regulator ComK and evaluated their effects on the comG operon competence reporter and transformation efficiency. Xylose- and mannitol-inducible systems outperformed IPTG-based constructs and shifted 96-99% of cells into a reporter-positive competent state. However, reporter activation alone did not predict transformation potential. Optimization of culture density and induction timing increased transformant yield 45-fold relative to the initial protocol and 2800-fold relative to the conventional Spizizen method. Disruption of native competence regulatory genes did not improve performance and often reduced transformation output, highlighting the importance of endogenous regulatory circuitry. Using the optimized strain and protocol, we achieved co-transformation frequencies of 11-18% and constructed multiplex spore-display libraries containing fluorescent protein fusions integrated at multiple loci. Screening identified strong dual-display combinations and showed that cargo loading depends on anchor protein, integration locus, and genetic background. SscA fusions supported the highest display capacity and promoted synergistic co-display. Together, these results show improvements in natural transformation-based genome engineering in B. subtilis and provide insight into the construction of multifunctional engineered spores.

synthetic biology↗

bioRxiv2026

Real-time, in situ fluorescence and optical density measurements of liquid cultures in simulated microgravity

As human space exploration expands to the Moon, Mars, and beyond, there is a growing need to study the effects of altered gravity on the microbial systems that we will bring with us for life support. Because spaceflight experiment opportunities are rare and resource-intensive, most space biology experiments are conducted using ground-based simulators. The most common microgravity simulator for microbial experiments, the rotating wall vessel, can approximate the low-shear and low-turbulence conditions that characterize microgravity. However, current designs do not allow for real-time measurement of growth or metabolic activity during rotation: experiments require destructive sampling or disruption of the microgravity simulation conditions. Here, we describe the development of an in situ spectroscopy system compatible with the Cell Spinpod rotating wall vessel, which enables measurement of both optical absorbance and fluorescence with high temporal resolution, producing growth curves similar to those from an off-the-shelf plate reader. These results are validated using two common microbial hosts: Escherichia coli and Saccharomyces cerevisiae. The Spinpod Optical System has the potential to diversify the types of microbiology experiments possible in simulated microgravity, allowing the measurement of not only growth curve parameters but also metabolic activity, gene expression, or community dynamics. It thus has the potential to improve the quality of experiments seeking to characterize microbial responses to spaceflight conditions.

microbiology↗

bioRxiv2024

Advancing protein display on bacterial spores through an extensive survey of coat components

The profound stability of bacterial spores makes them a promising platform for biotechnological applications like biocatalysis, bioremediation, drug delivery, etc. However, though the Bacillus subtilis spore is composed of >40 proteins, only [~]12 have been explored as fusion carriers for protein display. Here, we assessed the suitability of 33 spore proteins (SPs) as enzyme display carriers by direct allele tagging at native genomic loci. Of the 33 SP investigated, 26 formed functional fusions with {beta}-glucuronidase (GUS) - a [~]272 kDa homotetramer. This almost triples the number of SPs assessed for enzyme display and doubles the number of functional fusions documented in the literature. We quantitatively assessed the 1) SP promoter activation dynamics during growth, 2) supported GUS activity on spores, 3) surface availability, 4) protection from thermal and proteolytic degradation, and 5) compatibility of co-expression of many of these fusions. Multi-copy expression and pairwise co-expression of the most promising SP-GUS fusions highlighted the complexity of spore structure/assembly and difficulty in predicting compatibility between different SPs fusions. We also assessed the suitability of engineered spores to degrade PET (polyethylene terephthalate) films and found that surface exposed SPs were most effective. Beyond the broad survey, a key outcome of our work was the identification of SscA (small spore coat assembly protein A) as an effective spore display carrier. SscA supported enzyme activity at least fourfold higher than any other SP, including the well-established and popular anchor, CotY. We attribute this to the biochemical and genetic features of SscA; its promoter demonstrated early and sustained activation relative to other SPs and its small size ([~]3 KDa) likely minimally interferes with enzyme folding, oligomerization, and activity. Although the specific localization of SscA within the spore coat has not been established, its hydrophobic nature and low surface availability suggests that it assembles internally within the spore coat, which makes it highly stabilizing and suitable for many biocatalytic applications. Overall, this work serves as a knowledgebase to advance the biotechnological utility of B. subtilis spores.

synthetic biology↗

bioRxiv2024

Semi-automated Production of Cell-free Biosensors

Cell-free synthetic biology biosensors have potential as effective in vitro diagnostic technologies for the detection of chemical compounds such as toxins and human health biomarkers. They have several advantages over conventional laboratory-based diagnostic approaches, including being able to be assembled, freeze-dried, distributed, and then used at the point-of-need. This makes them an attractive platform for cheap and rapid chemical detection across the globe. Though promising, a major challenge is scaling up biosensor manufacturing to meet the needs of their multiple uses. Currently, cell-free biosensor assembly during lab-scale development is mostly performed manually by the operator, leading to quality control and performance variability issues. Here we explore the use of liquid handling robotics to manufacture cell-free biosensor reactions. We compare both manual and semi-automated reaction assembly approaches using the Opentrons OT-2 liquid handling platform on two different cell-free gene expression assay systems that constitutively produce colorimetric (LacZ) or fluorescent (GFP) signals. We test the designed protocol by constructing an entire 384-well plate of fluoride sensing cell-free biosensors and demonstrate that they perform closely to expected detection outcomes.

synthetic biology↗

bioRxiv2024

Neutralizing antibodies elicited in sequentially immunized macaques recognize V3 residues on altered conformations of HIV-1 Env trimer

Eliciting broadly neutralizing antibodies that protect against diverse HIV-1 strains is a primary goal of AIDS vaccine research. We characterized Ab1456 and Ab1271, two heterologously-neutralizing antibodies elicited in non-human primates by priming with an engineered V3-targeting SOSIP Env immunogen and boosting with increasingly native-like SOSIP Envs derived from different strain backgrounds. Structures of Env trimers in complex with these antibodies revealed V3 targeting, but on conformational states of Env distinct from the typical closed, prefusion trimeric SOSIP structure. Env trimers bound by Ab1456 adopted conformations resembling CD4-bound open Env states in the absence of soluble CD4, whereas trimers bound by Ab1271 exhibited a trimer apex-altered conformation to accommodate antibody binding. The finding that elicited antibodies cross-neutralized by targeting altered, non-closed, prefusion Env trimer conformations provides important information about Env dynamics that is relevant for HIV-1 vaccine design aimed at raising antibodies to desired epitopes on closed pre-fusion Env trimers. Highlights- Sequential immunization regimen elicits V3 antibodies targeting non-closed Envs - Cryo-EM structures reveal recognition of multiple Env conformational states - Neutralization by elicited antibody does not require antibody-virus preincubation Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/605918v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@6210b1org.highwire.dtl.DTLVardef@35f683org.highwire.dtl.DTLVardef@cb2d3corg.highwire.dtl.DTLVardef@106e066_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Missense mutations in CMS22 patients reveal that PREPL has both enzymatic and non-enzymatic functions

Congenital myasthenic syndrome-22 (CMS22) is a rare genetic disorder caused by mutations in the prolyl endopeptidase-like (PREPL) gene. Since previous reports only described patients with deletions and nonsense mutations in PREPL, nothing is known about the effect of missense mutations in the pathology of CMS22. In this study, we have characterized missense mutations in PREPL in three CMS22 patients, all with hallmark phenotypes. Biochemical evaluation revealed that these missense mutations do not impair hydrolase activity, thereby challenging the conventional diagnostic criteria. Structural analysis shows that the mutations affect regions most likely involved in intra-protein or protein-protein interactions. Indeed, binding to a selected group of known interactors was differentially reduced for the three mutants. The importance of non-hydrolytic functions of PREPL was investigated in catalytically inactive PREPL p.Ser559Ala cell lines which showed that hydrolytic activity of PREPL is needed for normal mitochondrial function but not for regulating AP1-mediated transport in the trans-Golgi network. In conclusion, these studies show that CMS22 can be caused not only by deletion and truncation of PREPL but also by missense mutations that do not necessarily result in a loss of hydrolytic activity of PREPL. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/572145v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@13dc0bborg.highwire.dtl.DTLVardef@13865d6org.highwire.dtl.DTLVardef@1a5ed3borg.highwire.dtl.DTLVardef@79fdf5_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2023

A framework for quantifiable local and global structure preservation in single-cell dimensionality reduction

The ability to explore high-dimensional single-cell transcriptomics data efficiently is crucial in many biological studies. Dimensionality reduction techniques have therefore emerged as a basic building block of analytical workflows. They generate low-dimensional embeddings that capture important structures in the data, and are often used in discovery, quality control, and downstream analysis. However, the trustworthiness of current methods and the rigour of popular evaluation criteria are limited. We tackle this in an empirical study of structure-preserving data embeddings, delivering two tools. First, we introduce ViScore: a robust scoring framework that improves both unsupervised and supervised quality metrics, with emphasis on scalability and fairness. Second, we introduce ViVAE : a deep learning model that achieves better multi-scale structure preservation and is equipped with new tools for interpretability. We demonstrate the potential of these contributions to advance the trustworthiness of single-cell dimensionality reduction in a quantitative comparison and focused case studies.

bioinformatics↗

bioRxiv2023

Rapid spectrophotometric detection for optimized production of landomycins and characterization of their therapeutic potential

Microbial derived natural products remain a major source of structurally diverse bioactive compounds and chemical scaffolds that have potential as new therapeutics to target drug resistant pathogens and cancers. In particular, genome mining has revealed the vast number of cryptic or low yield biosynthetic gene clusters in the genus Streptomyces. Here, we describe our efforts to improve yields of landomycins - angucycline family polyketides under investigation as cancer therapeutics - by a genetically modified Streptomyces cyanogenus 136. After simplifying the extraction process from S. cyanogenus cultures, we identified a wavelength at which the major landomycin products absorb in culture extracts, which we used to systematically explore culture medium compositions to improve total landomycin titers. Through correlational analysis, we simplified the culture optimization process by identifying an alternative wavelength at which culture supernatants absorb yet is representative of total landomycin titers. Using the subsequently improved sample throughput, we explored landomycin production during the culturing process to further increase landomycin yield and reduce culture time. Testing the antimicrobial activity of the isolated landomycins, we report broad inhibition of Gram-positive bacteria, inhibition of fungi by landomycinone, and broad landomycin resistance by Gram-negative bacteria that is likely mediated by exclusion of landomycins by the bacterial membrane. Finally, the anticancer activity of the isolated landomycins against A549 lung carcinoma cells agrees with previous reports on other cell lines that glycan chain length correlates with activity. Given the prevalence of natural products produced by Streptomyces, as well as the light-absorbing moieties common to bioactive natural products and their metabolic precursors, our method is relevant to improving the yields of other natural products of interest.

bioengineering↗

bioRxiv2023

Agent-based Model for Microbial Populations Exposed to Radiation (AMMPER) simulates yeast growth for deep-space experiments

Space radiation poses a substantial health risk to humans traveling beyond Earths orbit to the Moon and Mars. As microbes come with us to space as model organisms for studying radiation effects, a computational model simulating those effects on microorganisms could enable us to better design and interpret those experiments. Here we present Agent-based Model for Microbial Populations Exposed to Radiation (AMMPER), which simulates the effects of protons, a major component of deep-space radiation, on budding yeast (Saccharomyces cerevisiae) growth. The model combines radiation track structure data from the RITRACKS package with novel algorithms for cell replication, motion, damage, and repair. We demonstrate that AMMPER qualitatively reproduces the effects of 150 MeV proton radiation on growth rate, but not lag time, of wild type and DNA repair mutant yeast strains. The variance in AMMPERs results is consistent with the variance in experimental results, suggesting that AMMPER can recapitulate the stochasticity of empirical experiments. Finally, we used AMMPER to predict responses to deep space radiation that may be tested in future experiments. A user-friendly, open-source, extendable Python package for studying the relationship between single-particle radiation events and population-level responses, AMMPER can facilitate the basic research necessary to ensure safe and sustainable exploration of deep space.

microbiology↗

bioRxiv2023

Cell-Free Protein Expression in Polymer Materials

While synthetic biology has advanced complex capabilities like sensing and molecule synthesis in aqueous solutions, important applications may also be pursued for biological systems in solid materials. Harsh processing conditions used to produce many synthetic materials such as plastics makes incorporation of biological functionality challenging. One technology that shows promise in circumventing these issues is cell-free protein synthesis (CFPS), where core cellular functionality is reconstituted outside the cell. CFPS enables genetic functions to be implemented without the complications of membrane transport or concerns over cellular viability or release of genetically modified organisms. Here we demonstrate that dried CFPS reactions have remarkable tolerance to heat and organic solvent exposure during the casting processes for polymer materials. We demonstrate the utility of this observation by creating plastics that have spatially patterned genetic functionality, produce antimicrobials in situ, and perform sensing reactions. The resulting materials unlock the potential to deliver DNA-programmable bio-functionality in a ubiquitous class of synthetic materials.

synthetic biology↗

bioRxiv2022

Antibiotic persistence does not cause phenotypic heterogeneity in tolerance of Escherichia coli to formaldehyde stress but can preserve it through time

The phenomenon of phenotypic heterogeneity, where an isogenic population expresses varying phenotypes, has been uncovered for an expanding variety of organisms and traits. This heterogeneity in phenotype can be physiologically relevant, such as the ability for antibiotic persistence to allow populations to survive lethal conditions due to rare cells being in a slow or non-growing state. Recently, it was discovered that Methylorubrum extorquens, a facultative methylotroph, possesses a continuous spectrum of phenotypic states conferring tolerance to formaldehyde. Formaldehyde is a toxin produced by M. extorquens during growth on methanol. The phenotypic tolerance allowed rapid growth on levels of formaldehyde which were lethal to the majority of the population. Transcriptomics indicated this may be due to upregulation of proteins that attenuate oxidative stress and protein damage rather than increasing formaldehyde oxidation to prevent accumulation. These data suggested that heterogeneity to formaldehyde stress may be present even in non-methylotrophic organisms that do not routinely produce large quantities of formaldehyde, and thus widely distributed across bacteria. To investigate this, we tested Escherichia coli for heterogeneity to formaldehyde stress during growth on glucose. Like M. extorquens, E. coli populations have a wide, continuous range of formaldehyde tolerance thresholds and this tolerance was reversible. Several other features, however, were different from what was found for M. extorquens. Most E. coli growth occurred after formaldehyde levels had dropped, suggesting that persistence could be the cause. The dynamics of antibiotic persistence and formaldehyde tolerance were both tracked but found to not be correlated. On the other hand, the persister cell state can maintain formaldehyde tolerance. These data suggest that persistence can preserve phenotypic heterogeneities in other traits, further expanding its potential role in helping cells survive environmental stressors.

microbiology↗

bioRxiv2022

Comprehensive phylogenomics of Methylobacterium reveals four evolutionary distinct groups and underappreciated phyllosphere diversity.

Methylobacterium is a group of methylotrophic microbes associated with soil, fresh water, and particularly the phyllosphere, the aerial part of plants that has been well-studied in terms of physiology but whose evolutionary history and taxonomy are unclear. Recent work has suggested that Methylobacterium is much more diverse than thought previously, questioning its status as an ecologically and phylogenetically coherent taxonomic genus. However, taxonomic and evolutionary studies of Methylobacterium have mostly been restricted to model species, often isolated from habitats other than the phyllosphere, and have yet to utilize comprehensive phylogenomic methods to examine gene trees, gene content, or synteny. By analyzing 189 Methylobacterium genomes from a wide range of habitats, including the phyllosphere, we inferred a robust phylogenetic tree while explicitly accounting for the impact of horizontal gene transfers. We showed that Methylobacterium contains four evolutionary distinct groups of bacteria (namely A, B, C, D), characterized by different genome size, GC content, gene content and genome architecture, revealing the dynamic nature of Methylobacterium genomes. In addition of recovering 59 described species, we identified 45 candidate species, mostly phyllosphere-associated, stressing the significance of plants as a reservoir of Methylobacterium diversity. We inferred an ancient transition from a free-living lifestyle to association with plant roots in Methylobacteriaceae ancestor, followed by phyllosphere association of three of the major groups (A, B, D), which early branching in Methylobacterium history was heavily obscured by HGT. Together, our work lays the foundations for a thorough redefinition of Methylobacterium taxonomy, beginning with the abandon of Methylorubrum.

genomics↗

bioRxiv2021

Methylothon: a versatile course-based high school research experience in microbiology and bioinformatics-- with pink bacteria

Methylothon is an inquiry-based high school learning module in microbial ecology, molecular biology, and bioinformatics that centers around pink-pigmented plant-associated methylotrophic bacteria. Here we present an overview of the modules learning goals, describe course resources (available for public use on http://methylothon.com), and relate lessons learned from adapting Methylothon for remote learning during the pandemic in spring of 2021. The original in-person version of the module allows students to isolate their own strains of methylotrophic bacteria from plants they sample from the environment, to identify these using PCR, sequencing, and phylogenetic analysis, and to contribute their strains to original research in a university lab. The adapted version strengthens the focus on bioinformatics and increases its flexibility and accessibility by making the lab portion optional and adopting free web-based tools. Student feedback and graded assignments from Spring 2021 revealed that the lesson was especially effective at introducing the concepts of BLAST and phylogenetic trees, and that students valued and felt inspired by the opportunity to conduct hands-on work and to participate in community science.

scientific communication and education↗

bioRxiv2021

Myo-inositol Oxygenase Overexpression Rescues Vitamin C Deficient Arabidopsis (vtc) Mutants

Biosynthesis of O_SCPLOWLC_SCPLOW-ascorbate (AsA) in plants is carried out by a complex metabolic network, which involves O_SCPLOWDC_SCPLOW-mannose/O_SCPLOWLC_SCPLOW-galactose, O_SCPLOWDC_SCPLOW-galacturonate, O_SCPLOWLC_SCPLOW-gulose, and myo-inositol as main precursors. Arabidopsis lines over-expressing enzymes in the myo-inositol pathway have elevated AsA, accumulate more biomass of both aerial and root tissues, and are tolerant to abiotic stresses as shown by manual and digital phenotyping. We crossed myo-inositol oxygenase (MIOX4) over-expressers with two low-vitamin C mutants (vtc1-1 and vtc2-1) encoding enzymes involved in O_SCPLOWDC_SCPLOW-mannose/O_SCPLOWLC_SCPLOW-galactose route. The purpose of developing these crosses was to test MIOX4s ability to restore the low AsA phenotype in mutants, and to assess the contribution of individual biosynthetic pathways to abiotic stress tolerance. We used a powerful high-throughput phenotyping platform for detailed phenotypic characterization of the Arabidopsis crosses with visible, fluorescence, near-infrared and infrared sensors. We combined digital phenotyping with photosynthetic parameters and soil water potential measurements. Our results show that MIOX4 is able to restore the AsA content of the mutants and the restored lines (vtc+MIOX4) show high AsA, enhanced growth rate, accumulate more biomass, and display healthier chlorophyll fluorescence and water content profiles compared to controls. HighlightsConstitutive expression of a myo-inositol oxygenase restored vitamin C deficient (vtc mutants). The restored lines have elevated ascorbate content and are tolerant to abiotic stresses. Under normal and abiotic stress conditions, the restored lines have enhanced biomass and increased water retention.

biochemistry↗

bioRxiv2020

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. ImportanceAll 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.

microbiology↗

bioRxiv2020

EfgA is a conserved formaldehyde sensor that halts bacterial translation 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 inhibits translation 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 and may include translational machinery. 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 modulates translational activity.

microbiology↗