Enhanced catabolism of glycine betaine and derivatives provides improved osmotic stress protection in Methylorubrum extorquens PA1.

Integration of metabolites into the overall metabolic network of a cell requires careful coordination dependent upon the ultimate usage of the metabolite. Different stoichiometric needs, and thus pathway fluxes, must exist for compounds destined for diverse uses, such as carbon sources, nitrogen sources, or stress-protective agents. Herein, we expand upon our previous work that highlighted the nature of glycine betaine (GB) metabolism in Methylobacteria to examine the utilization of GB-derivative compounds dimethylglycine (DMG) and sarcosine into Methylorubrum extorquens in different metabolic capacities, including as sole nitrogen and/or carbon sources. We isolated gain-of-function mutations that allowed M. extorquens PA1 to utilize dimethylglycine as a carbon source and dimethylglycine and sarcosine as nitrogen source. Characterization of mutants demonstrated selection for variants of the AraC-like regulator Mext_3735 that confer constitutive expression of the GB metabolic gene cluster, allowing direct utilization of the downstream GB derivatives. Finally, among the distinct isolates examined, we found that catabolism of the osmoprotectant used for selection (GB or dimethylglycine) enhanced osmotic stress resistance provided in the presence of that particular osmolyte. Thus, access to the carbon and nitrogen and osmoprotective effects of GB and DMG are made readily accessible through adaptive mutations. In M. extorquens PA1, the limitations to exploiting this group of compounds appear to exist predominantly at the levels of gene regulation and functional activity, rather than being constrained by transport or toxicity.IMPORTANCEOsmotic stress is a common challenge for bacteria colonizing the phyllosphere, where glycine betaine (GB) can be found as a prevalent osmoprotectant. Though Methylorubrum extorquens PA1 cannot use GB or its demethylation products, dimethylglycine (DMG) and sarcosine, as a sole carbon source, utilization is highly selectable via single nucleotide changes for both GB and DMG growth. The innate inability to use these compounds is due to limited flux through steps in the pathway and regulatory constraints. Herein, the characterization of the transcriptional regulator, Mext_3735 (GbdR), expands our understanding of the various roles in which GB derivatives can be used in M. extorquens PA1. Interestingly, increased catabolism of GB and derivatives does not interfere with, but rather improves, the ability of cells to thrive under increased salt stress conditions, suggesting that metabolic flux improves stress tolerance rather than providing a distinct tension between uses.

Glycine betaine metabolism is enabled in Methylorubrum extorquens PA1 by alterations to dimethylglycine dehydrogenase.

Low nutrient availability is a key characteristic of the phyllosphere (the aerial surface of plants). Phyllospheric bacteria utilize a wide array of carbon sources generated by plant hosts. Glycine betaine (GB) is a plant-derived compound that can be metabolized by certain members of the phyllosphere microbiota. Metabolism of glycine betaine generates formaldehyde, an intermediate of methylotrophic metabolism, leading us to investigate how the ubiquitous plant colonizing bacterium Methylorubrum extorquens PA1 might metabolize GB encountered in its native environment. M. extorquens PA1 cannot utilize GB as a sole carbon source. Through suppressor mutation analysis, we show that M. extorquens PA1 encodes a conserved GB utilization pathway that can be activated by single point mutations conferring GB utilization as a carbon source. We identified the gene cluster encoding the GB catabolic enzymes and found that gene expression was induced in the presence of GB. We show that utilization of GB is conserved among representative Methylobacterium species and generates the one-carbon metabolism intermediate formaldehyde, which M. extorquens utilizes as a source of energy. Our results support a model where suppressor mutations in Mext_3745 or ftsH (Mext_4840) prevent the degradation of the dimethylglycine dehydrogenase subunit DgcB by the membrane integral protease FtsH, conferring the ability to utilize GB by either (i) restoring stable membrane topology of DgcB or (ii) decreasing FtsH protease activity, respectively. Both mutations alleviate the bottleneck at the second step of GB degradation catalyzed by DgcAB.IMPORTANCEOvercoming low nutrient availability is a challenge many bacteria encounter in the environment. Facultative methylotrophs are able to utilize one-carbon and multi-carbon compounds as carbon and energy sources. The utilization of plant-derived glycine betaine (GB) represents a possible source of multi-carbon and one-carbon substrates. The metabolism of glycine betaine produces formaldehyde and glycine, which may be used simultaneously by facultative methylotrophs. However, the genes required for the utilization of GB in the ubiquitous plant-associated bacterium Methylorubrum extorquens have yet to be identified or described. Our work identifies and validates the genes required for glycine betaine metabolism in M. extorquens and shows that it directly intersects with methylotrophic metabolism through the production of formaldehyde.

Correction: Microbial phenotypic heterogeneity in response to a metabolic toxin: Continuous, dynamically shifting distribution of formaldehyde tolerance in Methylobacterium extorquens populations.

[This corrects the article DOI: 10.1371/journal.pgen.1008458.].

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.

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.

Microbial phenotypic heterogeneity in response to a metabolic toxin: Continuous, dynamically shifting distribution of formaldehyde tolerance in Methylobacterium extorquens populations.

While microbiologists often make the simplifying assumption that genotype determines phenotype in a given environment, it is becoming increasingly apparent that phenotypic heterogeneity (in which one genotype generates multiple phenotypes simultaneously even in a uniform environment) is common in many microbial populations. The importance of phenotypic heterogeneity has been demonstrated in a number of model systems involving binary phenotypic states (e.g., growth/non-growth); however, less is known about systems involving phenotype distributions that are continuous across an environmental gradient, and how those distributions change when the environment changes. Here, we describe a novel instance of phenotypic diversity in tolerance to a metabolic toxin within wild-type populations of Methylobacterium extorquens, a ubiquitous phyllosphere methylotroph capable of growing on the methanol periodically released from plant leaves. The first intermediate in methanol metabolism is formaldehyde, a potent cellular toxin that is lethal in high concentrations. We have found that at moderate concentrations, formaldehyde tolerance in M. extorquens is heterogeneous, with a cell's minimum tolerance level ranging between 0 mM and 8 mM. Tolerant cells have a distinct gene expression profile from non-tolerant cells. This form of heterogeneity is continuous in terms of threshold (the formaldehyde concentration where growth ceases), yet binary in outcome (at a given formaldehyde concentration, cells either grow normally or die, with no intermediate phenotype), and it is not associated with any detectable genetic mutations. Moreover, tolerance distributions within the population are dynamic, changing over time in response to growth conditions. We characterized this phenomenon using bulk liquid culture experiments, colony growth tracking, flow cytometry, single-cell time-lapse microscopy, transcriptomics, and genome resequencing. Finally, we used mathematical modeling to better understand the processes by which cells change phenotype, and found evidence for both stochastic, bidirectional phenotypic diversification and responsive, directed phenotypic shifts, depending on the growth substrate and the presence of toxin.

The three-legged stool of understanding metabolism: integrating metabolomics with biochemical genetics and computational modeling.

Traditional biochemical research has resulted in a good understanding of many aspects of metabolism. However, this reductionist approach is time consuming and requires substantial resources, thus raising the question whether modern metabolomics and genomics should take over and replace the targeted experiments of old. We proffer that such a replacement is neither feasible not desirable and propose instead the tight integration of modern, system-wide omics with traditional experimental bench science and dedicated computational approaches. This integration is an important prerequisite toward the optimal acquisition of knowledge regarding metabolism and physiology in health and disease. The commentary describes advantages and drawbacks of current approaches to assessing metabolism and highlights the challenges to be overcome as we strive to achieve a deeper level of metabolic understanding in the future.

Untargeted metabolomics confirms and extends the understanding of the impact of aminoimidazole carboxamide ribotide (AICAR) in the metabolic network of Salmonella enterica.

In Salmonella enterica, aminoimidazole carboxamide ribotide (AICAR) is a purine biosynthetic intermediate and a substrate of the AICAR transformylase/IMP cyclohydrolase (PurH) enzyme. When purH is eliminated in an otherwise wild-type strain, AICAR accumulates and indirectly inhibits synthesis of the essential coenzyme thiamine pyrophosphate (TPP). In this study, untargeted metabolomics approaches were used to i) corroborate previously defined metabolite changes, ii) define the global consequences of AICAR accumulation and iii) investigate the metabolic effects of mutations that restore thiamine prototrophy to a purH mutant. The data showed that AICAR accumulation led to an increase in the global regulator cyclic AMP (cAMP) and that disrupting central carbon metabolism could decrease AICAR and/or cAMP to restore thiamine synthesis. A mutant (icc) blocked in cAMP degradation that accumulated cAMP but had wild-type levels of AICAR was used to identify changes in the purH metabolome that were a direct result of elevated cAMP. Data herein describe the use of metabolomics to identify the metabolic state of mutant strains and probe the underlying mechanisms used by AICAR to inhibit thiamine synthesis. The results obtained provide a cautionary tale of using metabolite concentrations as the only data to define the physiological state of a bacterial cell.

An Unexpected Route to an Essential Cofactor: Escherichia coli Relies on Threonine for Thiamine Biosynthesis.

UNLABELLED: Metabolism consists of biochemical reactions that are combined to generate a robust metabolic network that can respond to perturbations and also adapt to changing environmental conditions. Escherichia coli and Salmonella enterica are closely related enterobacteria that share metabolic components, pathway structures, and regulatory strategies. The synthesis of thiamine in S. enterica has been used to define a node of the metabolic network by analyzing alternative inputs to thiamine synthesis from diverse metabolic pathways. To assess the conservation of metabolic networks in organisms with highly conserved components, metabolic contributions to thiamine synthesis in E. coli were investigated. Unexpectedly, we found that, unlike S. enterica, E. coli does not use the phosphoribosylpyrophosphate (PRPP) amidotransferase (PurF) as the primary enzyme for synthesis of phosphoribosylamine (PRA). In fact, our data showed that up to 50% of the PRA used by E. coli to make thiamine requires the activities of threonine dehydratase (IlvA) and anthranilate synthase component II (TrpD). Significantly, the IlvA- and TrpD-dependent pathway to PRA functions in S. enterica only in the absence of a functional reactive intermediate deaminase (RidA) enzyme, bringing into focus how these closely related bacteria have distinct metabolic networks. IMPORTANCE: In most bacteria, including Salmonella strains and Escherichia coli, synthesis of the pyrimidine moiety of the essential coenzyme, thiamine pyrophosphate (TPP), shares enzymes with the purine biosynthetic pathway. Phosphoribosylpyrophosphate amidotransferase, encoded by the purF gene, generates phosphoribosylamine (PRA) and is considered the first enzyme in the biosynthesis of purines and the pyrimidine moiety of TPP. We show here that, unlike Salmonella, E. coli synthesizes significant thiamine from PRA derived from threonine using enzymes from the isoleucine and tryptophan biosynthetic pathways. These data show that two closely related organisms can have distinct metabolic network structures despite having similar enzyme components, thus emphasizing caveats associated with predicting metabolic potential from genome content.

Metabolic network structure and function in bacteria goes beyond conserved enzyme components.

For decades, experimental work has laid the foundation for our understanding of the linear and branched pathways that are integrated to form the metabolic networks on which life is built. Genetic and biochemical approaches applied in model organisms generate empirical data that correlate genes, gene products and their biological activities. In the post-genomic era, these results have served as the basis for the genome annotation that is routinely used to infer the metabolic capabilities of an organism and mathematically model the presumed metabolic network structure. At large, genome annotation and metabolic network reconstructions have demystified genomic content of non-culturable microorganisms and allowed researchers to explore the breadth of metabolisms in silico. Mis-annotation aside, it is unclear whether in silico reconstructions of metabolic structure from component parts accurately captures the higher levels of network organization and flux distribution. For this approach to provide accurate predictions, one must assume that the conservation of metabolic components leads to conservation of metabolic network architecture and function. This assumption has not been rigorously tested. Here we describe the implications of a recent study (MBio 5;7(1): e01840-15), which demonstrated that conservation of metabolic components was not sufficient to predict network structure and function.

Aminoimidazole Carboxamide Ribotide Exerts Opposing Effects on Thiamine Synthesis in Salmonella enterica.

UNLABELLED: In Salmonella enterica, the thiamine biosynthetic intermediate 5-aminoimidazole ribotide (AIR) can be synthesized de novo independently of the early purine biosynthetic reactions. This secondary route to AIR synthesis is dependent on (i) 5-amino-4-imidazolecarboxamide ribotide (AICAR) accumulation, (ii) a functional phosphoribosylaminoimidazole-succinocarboxamide (SAICAR) synthetase (PurC; EC 6.3.2.6), and (iii) methionine and lysine in the growth medium. Studies presented here show that AICAR is a direct precursor to AIR in vivo. PurC-dependent conversion of AICAR to AIR was recreated in vitro. Physiological studies showed that exogenous nutrients (e.g., methionine and lysine) antagonize the inhibitory effects of AICAR on the ThiC reaction and decreased the cellular thiamine requirement. Finally, genetic results identified multiple loci that impacted the effect of AICAR on thiamine synthesis and implicated cellular aspartate levels in AICAR-dependent AIR synthesis. Together, the data here clarify the mechanism that allows conditional growth of a strain lacking the first five biosynthetic enzymes, and they provide additional insights into the complexity of the metabolic network and its plasticity. IMPORTANCE: In bacteria, the pyrimidine moiety of thiamine is derived from aminoimidazole ribotide (AIR), an intermediate in purine biosynthesis. A previous study described conditions under which AIR synthesis is independent of purine biosynthesis. This work is an extension of that previous study and describes a new synthetic pathway to thiamine that depends on a novel thiamine precursor and a secondary activity of the biosynthetic enzyme PurC. These findings provide mechanistic details of redundancy in the synthesis of a metabolite that is essential for nucleotide and coenzyme biosynthesis. Metabolic modifications that allow the new pathway to function or enhance it are also described.

Amino-4-imidazolecarboxamide ribotide directly inhibits coenzyme A biosynthesis in Salmonella enterica.

Aminoimidazole carboxamide ribotide (AICAR) is a purine biosynthetic intermediate and a by-product of histidine biosynthesis. In bacteria, yeast, and humans, accumulation of AICAR has been shown to affect an array of cellular processes by both direct and indirect mechanisms. In purine biosynthesis, AICAR is the substrate of the bifunctional protein phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase (PurH, EC 2.1.2.3/3.5.4.10). Strains lacking PurH accumulate AICAR and have a defect in the synthesis of the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) moiety of thiamine. The formation of HMP is also compromised in vivo when coenzyme A (CoA) levels are reduced. Our results show that the in vivo accumulation of AICAR decreased total CoA pools and, further, that AICAR inhibited the activity of pantoate ß-alanine ligase in vitro (PanC, EC 6.3.2.1). These results demonstrated a mechanism of AICAR action and provide new insights into the metabolic consequences of disrupting purine metabolism.

Plasticity in the purine-thiamine metabolic network of Salmonella.

In Salmonella enterica, 5-aminoimidazole ribonucleotide (AIR) is the precursor of the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) pyrophosphate moiety of thiamine and the last intermediate in the common HMP/purine biosynthetic pathway. AIR is synthesized de novo via five reactions catalyzed by the purF, -D, -T, -G, and -I gene products. In vivo genetic analysis demonstrated that in the absence of these gene products AIR can be generated if (i) methionine and lysine are in the growth medium, (ii) PurC is functional, and (iii) 5-amino-4-imidazolecarboxamide ribotide (AICAR) has accumulated. This study provides evidence that the five steps of the common HMP/purine biosynthetic pathway can be bypassed in the synthesis of AIR and thus demonstrates that thiamine synthesis can be uncoupled from the early purine biosynthetic pathway in bacteria.