Biology subjects

Bruger, E. L.

Publications and source records attributed to Bruger, E. L..

7 recordsLinked to original sources

bioRxiv2026

Adaptation of Methylobacterium extorquens to alternating carbon sources identifies the regulator CstR as an intersectional hub of cellular carbon metabolic dynamics and stress response

Bacteria frequently face challenges adapting to changing environmental conditions, such as shifting resource utilization, to survive and thrive. Methylotrophs capable of growth on reduced single-carbon compounds are prevalent in the phyllosphere (aerial plant surfaces), where they face continual and predictable shifts in the availability of different plant-produced carbon sources. We examined the ability of the methylotroph Methylobacterium extorquens PA1 to adapt to repeated shifts between two different carbon and energy sources: the one-carbon compound methanol and the multi-carbon organic acid succinate, both present in the phyllosphere. Evolved lineages of wild-type cells all increased their capacity for rapid transition between the carbon sources through high frequencies of loss-of-function mutations affecting a previously uncharacterized gene, named cstR for carbon source transition regulator, which encodes an orphan single-domain response receiver. Characterization showed that mutant strains were more competitive bidirectionally in the succinate-methanol transition. Though evolved populations of the {Delta}efgA and {Delta}ttmR strains, which are defective in the succinate-to-methanol transition, experienced similar phenotypic improvements in carbon-source transitions, we did not observe cstR mutations rise to prominence as extensively or frequently in these lineages. Transcriptomic work revealed loss-of-function to cstR impacted expression of genes involved in motility/chemotaxis, energy metabolism, and stress response, among others, suggesting that it coordinates responses to metabolic cues that are prevalent in certain carbon source and growth phase transitions. Loss of cstR function did not compromise exogenous formaldehyde tolerance in the {Delta}efgA and {Delta}ttmR mutants, breaking a previously described tradeoff between these two phenotypes. However, this loss did lead to defects under exposure to certain stressors, including heat, desiccation, oxidative stress agents, and particularly pH stress. Altered levels of NAD+/NADH across conditions, improved growth under acidic pH, and diminished ATP and increased mortality under heightened pH together support a model where CstR is responsible for coordinating cell signaling to manage the balance between growth and maintaining stress resilience. ImportanceThe ability of heterotrophic bacteria to navigate transitions between different carbon sources is critical to their growth and success. Facultative methylotrophs can grow upon multiple types of carbon sources, including reduced single-carbon compounds, and frequently exist in host-beneficial associations on the leaf surfaces of plants. The exposure to the methylotrophic substrate methanol is known to come in temporal bursts for bacteria residing in the phyllosphere. The utilization of methanol is accompanied by obligatory conversion and exposure to the toxic compound formaldehyde. We sought to investigate the capacity of Methylobacterium extorquens to adapt to repetitive transitions between multi-carbon succinate and single-carbon methanol, and in the process identified a novel signaling protein: a single-domain response regulator receiver that mediates cellular growth response to carbon source transitions, at the cost of resistance to certain stressors including oxidative stress and alkaline pH exposure.

microbiology↗

bioRxiv2026

Decreasing peptide deformylase activity is a beneficial strategy for increasing formaldehyde resistance in Methylobacterium extorquens

Formaldehyde is a highly toxic metabolite that can cause extensive damage to DNA and proteins, and strategies to mitigate formaldehyde toxicity are poorly understood. Methylotrophic bacteria, such as Methylobacterium extorquens, thrive on one-carbon compounds as sole sources of carbon and energy. These organisms are excellent models for discovering formaldehyde stress response systems because formaldehyde is an obligate intermediate in their central carbon metabolism. Here, we characterize an evolved def allele (defevo) that increases formaldehyde resistance in M. extorquens. The def gene encodes peptide deformylase (PDF, EC:3.5.1.88), an enzyme that contributes to protein processing by removing the formyl group from N-formylmethionine (fMet) on nascent peptides. The defevoallele has a single missense mutation that decreases PDF activity both in vitro and in vivo. Transcriptomic analysis of the defevo strain indicates there are pleiotropic effects of this mutation and a differential response to formaldehyde stress. We investigate possible mechanisms for the defevo mutants increased resistance to formaldehyde, including mitigation of formaldehyde-induced protein stress and altered membrane physiology. We find that the defevo allele selectively alleviates exogenous, but not endogenous, formaldehyde stress and identify a tradeoff in heat shock resistance. This study reports the first observation of lowered PDF activity benefiting a cellular physiological phenotype. Our work indicates that altered protein metabolism can mitigate the toxic effects of formaldehyde and furthers our understanding of the strategies that can protect cells from formaldehyde-induced damage. ImportanceFormaldehyde is a toxic chemical that can damage essential molecules inside of cells, yet all organisms inevitably produce it during normal metabolism. Despite its ubiquity, our understanding of strategies for how cells navigate formaldehyde toxicity is incomplete. This study focuses on Methylobacterium extorquens, which naturally generates high levels of formaldehyde as part of its growth on simple carbon compounds. We show herein that a single genetic change, which slows down how newly made proteins are processed during translation, can unexpectedly improve the bacteriums ability to resist formaldehyde stress. Further, we show that this single change has numerous effects on the cell, many of which may contribute to formaldehyde resistance.

physiology↗

bioRxiv2021

Global transcriptional response of Methylorubrum extorquens to formaldehyde stress expands the role of EfgA and is distinct from antibiotic translational inhibition

The potency and indiscriminate nature of formaldehyde reactivity upon biological molecules make it a universal stressor. However, some organisms such as Methylorubrum extorquens possess means to rapidly and effectively mitigate formaldehyde-induced damage. EfgA is a recently identified formaldehyde sensor predicted to halt translation in response to elevated formaldehyde as a means to protect cells. Herein, we investigate growth and changes in gene expression to understand how M. extorquens responds to formaldehyde with and without the EfgA-formaldehyde-mediated translational response, and how this mechanism compares to antibiotic-mediated translation inhibition. These distinct mechanisms of translation inhibition have notable differences: they each involve different specific players and in addition, formaldehyde also acts as a general, multi-target stressor and a potential carbon source. We present findings demonstrating that in addition to its characterized impact on translation, functional EfgA allows for a rapid and robust transcriptional response to formaldehyde and that removal of EfgA leads to heightened proteotoxic and genotoxic stress in the presence of increased formaldehyde levels. We also found that many downstream consequences of translation inhibition were shared by EfgA-formaldehyde- and kanamycin-mediated translation inhibition. Our work uncovered additional layers of regulatory control enacted by functional EfgA upon experiencing formaldehyde stress, and further demonstrated the importance this protein plays at both transcriptional and translational levels in this model methylotroph.

microbiology↗

bioRxiv2020

Epistatic interactions shape the interplay between beneficial alleles and gain or loss of pathways in the evolution of novel metabolism

Fitness landscapes are often invoked to interpret the effects of allele substitutions and their interactions; however, evolution also includes larger changes like gene loss and acquisition. Previous work with the methylotrophic bacterium Methylorubrum extorquens AM1 identified strongly beneficial mutations in a strain evolved to utilize a novel, Foreign pathway in place of its native central metabolic pathway for growth on methanol. These mutations were consistently beneficial, regardless of the order in which they arose. Here we extend this analysis to consider loss or acquisition of metabolic pathways by examining strains relying upon either the Native pathway, or both ( Dual) pathways present. Unlike in the Foreign pathway context in which they evolved, these alleles were often deleterious in these alternative genetic backgrounds, following patterns that were strongly contingent on the specific pathways and other evolved alleles present. Landscapes for these alternative pathway backgrounds altered which genotypes correspond to local fitness peaks and would restrict the set of accessible evolutionary trajectories. These epistatic interactions negatively impact the probability of maintaining multiple degenerate pathways, making it more difficult for these pathways to coevolve. Together, our results highlight the uncertainty of retaining novel functions acquired via horizontal gene transfer (HGT), and that the potential for cells to either adopt novel functions or to maintain degenerate pathways together in a genome is heavily dependent upon the underlying epistatic interactions between them. Author SummaryThe evolution of physiology in microbes has important impacts ranging from global cycling of elements to the emergence and spread of pathogens and their resistance to antibiotics. While genetic interactions between mutations in evolving lineages of microbes have been investigated, these have not included the acquisition of novel genes on elements like plasmids, and thus how these elements interact with existing alleles. The dynamics of novel gene retention are of interest from both positive (e.g., biotechnology) and negative (e.g., antimicrobial resistance) practical impacts. We find that the patterns of interactions between evolved alleles appear substantially different, and generally much less positive, when moved into novel genetic backgrounds. Additionally, these preexisting alleles were found to have strong impacts on the ability of genotypes to maintain - and in rare cases coevolve with - novel genes and pathways. These results show that even though they evolved separately, the particular alleles in a genetic background, and importantly the physiological impacts they confer, weigh heavily on whether genes for novel metabolic processes are maintained.

evolutionary biology↗

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

Quorum sensing provides a molecular mechanism for evolution to tune and maintain investment in cooperation

As selection frequently favors non-cooperating defectors in mixed populations with cooperators, mechanisms that promote cooperation stability clearly exist. One potential mechanism is bacterial cell-to-cell communication, quorum sensing (QS), which can allow cooperators to prevent invasion by defectors. However, the impact of QS on widespread maintenance of cooperation in well-mixed conditions has not been experimentally demonstrated over extended evolutionary timescales. Here, we use wild-type ( WT) Vibrio harveyi that regulates cooperation with QS and an unconditionally cooperating ( UC) mutant to examine the evolutionary origins and subsequent dynamics of novel defectors during a long-term evolution experiment. We found that UC lineages were completely outcompeted by defectors, whereas functioning QS enabled the maintenance of cooperative variants in most WT populations. Sequencing of evolved populations revealed multiple luxR mutations that swept the UC lineages. However, the evolution of mutant lineages with reduced levels of bioluminescence ( dims) occurred in many WT lineages. These dim variants also decreased other cooperative phenotypes regulated by QS, such as protease production, indicating they result from changes to QS regulation. This diminished investment phenotype optimizes a trade-off between cooperative input and growth output, allowing cooperation to be maintained under QS control even in the presence of evolved defectors.

evolutionary biology↗

bioRxiv2017

Reciprocal control of motility and biofilm formation by the PdhS2 two-component sensor kinase of Agrobacterium tumefaciens

A core regulatory pathway that directs developmental transitions and cellular asymmetries in Agrobacterium tumefaciens involves two overlapping, integrated phosphorelays. One of these phosphorelays putatively includes four histidine sensor kinase homologues, DivJ, PleC, PdhS1, and PdhS2, and two response regulators, DivK and PleD. In several different alphaproteobacteria, this pathway influences a conserved downstream phosphorelay that ultimately controls the phosphorylation state of the CtrA master response regulator. The PdhS2 sensor kinase reciprocally regulates biofilm formation and swimming motility. In the current study the mechanisms by which the A. tumefaciens sensor kinase PdhS2 directs this regulation are delineated. PdhS2 lacking a key residue for phosphatase activity is markedly deficient in proper control of attachment and motility phenotypes, whereas a kinase-deficient PdhS2 mutant is only modestly affected. A genetic interaction between DivK and PdhS2 is revealed, unmasking one of several connections between PdhS2-dependent phenotypes and transcriptional control by CtrA. Epistasis experiments suggest that PdhS2 can function independently of the CckA sensor kinase, the cognate sensor kinase for CtrA which is inhibited by DivK. PdhS2 dynamically localizes to the daughter cell pole in dividing cells. Global expression analysis of the pdhS2 mutant reveals a restricted regulon, functioning through CtrA to separately control motility and regulate levels of the intracellular signal cyclic diguanylate monophosphate (cdGMP), thereby affecting production of adhesive polysaccharides and attachment. We hypothesize that in A. tumefaciens the CtrA regulatory circuit has expanded to include additional inputs through addition of PdhS-type sensor kinases, likely fine-tuning the response of this organism to the soil microenvironment.\n\nIMPORTANCEBacterial developmental processes, such as morphological transformations and behavioral transitions, are tightly regulated. In many alphaproteobacteria cell division and development are coordinated by a specific suite of conserved histidine kinases and their partnered regulatory proteins. Here we describe how the histidine kinase PdhS2 of Agrobacterium tumefaciens regulates complex phenotypes including biofilm formation and motility. PdhS2 genetically interacts with a single-domain response regulator, DivK, and the intracellular signal cyclic diguanylate monophosphate. PdhS2 dynamically localizes to the new pole of recently divided cells, contributing to the regulatory processes that dictate whether these cells remain motile or initiate biofilm formation. These findings expand our understanding of the complex network that integrates cell division and developmental control in A. tumefaciens and related alphaproteobacteria.

microbiology↗