Converting Escherichia coli to a Synthetic Methylotroph Growing Solely on Methanol.

Methanol, being electron rich and derivable from methane or CO2, is a potentially renewable one-carbon (C1) feedstock for microorganisms. Although the ribulose monophosphate (RuMP) cycle used by methylotrophs to assimilate methanol differs from the typical sugar metabolism by only three enzymes, turning a non-methylotrophic organism to a synthetic methylotroph that grows to a high cell density has been challenging. Here we reprogrammed E. coli using metabolic robustness criteria followed by laboratory evolution to establish a strain that can efficiently utilize methanol as the sole carbon source. This synthetic methylotroph alleviated a so far uncharacterized hurdle, DNA-protein crosslinking (DPC), by insertion sequence (IS)-mediated copy number variations (CNVs) and balanced the metabolic flux by mutations. Being capable of growing at a rate comparable with natural methylotrophs in a wide range of methanol concentrations, this synthetic methylotrophic strain illustrates genome editing and evolution for microbial tropism changes and expands the scope of biological C1 conversion.

Exquisite exposure: Formaldehyde as a metabolic regulator.

Throughout life, whether through external consumption or internal production, we are exposed to different reactive metabolites considered toxic to the body. Pham et al.1 uncover metabolic regulation by one such harmful metabolite: formaldehyde.

A Surge of DNA Damage Links Transcriptional Reprogramming and Hematopoietic Deficit in Fanconi Anemia.

Impaired DNA crosslink repair leads to Fanconi anemia (FA), characterized by a unique manifestation of bone marrow failure and pancytopenia among diseases caused by DNA damage response defects. As a germline disorder, why the hematopoietic hierarchy is specifically affected is not fully understood. We find that reprogramming transcription during hematopoietic differentiation results in an overload of genotoxic stress, which causes aborted differentiation and depletion of FA mutant progenitor cells. DNA damage onset most likely arises from formaldehyde, an obligate by-product of oxidative protein demethylation during transcription regulation. Our results demonstrate that rapid and extensive transcription reprogramming associated with hematopoietic differentiation poses a major threat to genome stability and cell viability in the absence of the FA pathway. The connection between differentiation and DNA damage accumulation reveals a novel mechanism of genome scarring and is critical to exploring therapies to counteract the aplastic anemia for the treatment of FA patients.

Aldehyde-driven transcriptional stress triggers an anorexic DNA damage response.

Endogenous DNA damage can perturb transcription, triggering a multifaceted cellular response that repairs the damage, degrades RNA polymerase II and shuts down global transcription1-4. This response is absent in the human disease Cockayne syndrome, which is caused by loss of the Cockayne syndrome A (CSA) or CSB proteins5-7. However, the source of endogenous DNA damage and how this leads to the prominent degenerative features of this disease remain unknown. Here we find that endogenous formaldehyde impedes transcription, with marked physiological consequences. Mice deficient in formaldehyde clearance (Adh5-/-) and CSB (Csbm/m; Csb is also known as Ercc6) develop cachexia and neurodegeneration, and succumb to kidney failure, features that resemble human Cockayne syndrome. Using single-cell RNA sequencing, we find that formaldehyde-driven transcriptional stress stimulates the expression of the anorexiogenic peptide GDF15 by a subset of kidney proximal tubule cells. Blocking this response with an anti-GDF15 antibody alleviates cachexia in Adh5-/-Csbm/m mice. Therefore, CSB provides protection to the kidney and brain against DNA damage caused by endogenous formaldehyde, while also suppressing an anorexic endocrine signal. The activation of this signal might contribute to the cachexia observed in Cockayne syndrome as well as chemotherapy-induced anorectic weight loss. A plausible evolutionary purpose for such a response is to ensure aversion to genotoxins in food.

Regulatory mechanism of formaldehyde release in heme degradation catalyzed by Staphylococcus aureus IsdG.

IsdG-type enzymes catalyze the noncanonical degradation of heme to iron, staphylobilin (SB), and formaldehyde (HCHO), presumably by binding heme in an unusually distorted conformation. Their unique mechanism has been elucidated for MhuD from Mycobacterium tuberculosis, revealing an unusual ring opening of hydroxyheme by dioxygenation. A similar mechanism has been postulated for other IsdG enzymes; however, MhuD, which is special as an IsdG-type enzyme, retains a formyl group in the linearized tetrapyrrole. Recent reports on Staphylococcus aureus IsdG have suggested the formation of SB retaining a formyl group (formyl-SB), but its identification is preliminary. Furthermore, the reaction properties of formyl-SB and the mechanism of HCHO release remain unclear. In this study, the complex reaction of S. aureus IsdG was reexamined to elucidate its mechanism, including the identification of reaction products and their control mechanisms. Depending on the reaction conditions, IsdG produced both SB and formyl-SB as the main product, the latter of which was isolated and characterized by MS and NMR measurements. The formyl-SB product was generated upon the reaction between hydroxyheme-IsdG and O2 without reduction, indicating the dioxygenation mechanism as found for MhuD. Under reducing conditions, hydroxyheme-IsdG was converted also to SB and HCHO by activating another O2 molecule. These results provide the first overview of the complicated IsdG reaction. The heme distortion in the IsdG-type enzymes is shown to generally promote ring cleavage by dioxygenation. The presence or absence of HCHO release can be influenced by many factors, and the direct identification of S. aureus heme catabolites is of interest.

Deciphering the molecular mechanism and regulation of formaldehyde detoxification in Mycobacterium smegmatis.

The build-up of formaldehyde, a highly reactive molecule is cytotoxic and must be eliminated for the organism's survival. Formaldehyde detoxification system is found in nearly all organisms including both pathogenic and non-pathogenic mycobacteria. MscR, a formaldehyde dehydrogenase from Mycobacterium smegmatis (Msm), is an indispensable part of this system and forms a bicistronic operon with its downstream uncharacterized gene, fmh. We here show that Fmh, a putative metallo-beta-lactamase, is essential in tolerating higher amounts of formaldehyde when co-overexpressed with mscR in vivo. Our NMR studies indicate that MscR, along with Fmh, enhances formate production through a mycothiol (MSH)-dependent pathway, emphasizing the importance of Fmh in detoxifying formaldehyde. Although another aldehyde dehydrogenase, MSMEG_1543, induces upon formaldehyde addition, it is not involved in its detoxification. We also show that the expression of the mscR operon is constitutive and remains unchanged upon formaldehyde addition, as displayed by the promoter activity of PmscR and by the transcript and protein levels of MscR. Furthermore, we establish the role of a thiol-responsive sigma factor SigH in formaldehyde detoxification. We show that SigH, and not SigE, is crucial for formaldehyde detoxification, even though it does not directly regulate mscR operon expression. In addition, sensitivity to formaldehyde in sigH-knockout could be alleviated by overexpression of mscR. Taken together, our data demonstrate the importance of MSH-dependent pathways in detoxifying formaldehyde in a mycobacterial system. An absence of such MSH-dependent proteins in eukaryotes and its complete conservation in M. tuberculosis, the causative agent of tuberculosis, further unravel new drug targets for this pathogen.IMPORTANCEExtensive research has been done on formaldehyde detoxification in different bacteria. However, our current understanding of the mechanisms underlying this process in mycobacteria remains exceedingly little. We previously showed that MscR, a formaldehyde dehydrogenase from Mycobacterium smegmatis, plays a pivotal role in this detoxification pathway. Here, we present a potential S-formyl-mycothiol hydrolase named Fmh, thought to be a metallo-beta-lactamase, which functions along with mycothiol (MSH) and MscR to enhance formate production within this detoxification pathway. Co-expression of Fmh with MscR significantly enhances the efficiency of formaldehyde detoxification in M. smegmatis. Our experiments establish that Fmh catalyzes the final step of this detoxification pathway. Although an alternative sigma factor SigH was found to be involved in formaldehyde detoxification, it did not directly regulate the expression of mscR. Since formaldehyde detoxification is essential for bacterial survival, we envisage this process to be a potential drug target for M. tuberculosis eradication.

Spatial transcriptomics and the anatomical pathologist: Molecular meets morphology.

In recent years anatomical pathology has been revolutionised by the incorporation of molecular findings into routine diagnostic practice, and in some diseases the presence of specific molecular alterations are now essential for diagnosis. Spatial transcriptomics describes a group of technologies that provide up to transcriptome-wide expression profiling while preserving the spatial origin of the data, with many of these technologies able to provide these data using a single tissue section. Spatial transcriptomics allows expression profiling of highly specific areas within a tissue section potentially to subcellular resolution, and allows correlation of expression data with morphology, tissue type and location relative to other structures. While largely still research laboratory-based, several spatial transcriptomics methods have now achieved compatibility with formalin-fixed paraffin-embedded tissue (FFPE), allowing their use in diagnostic tissue samples, and with further development potentially leading to their incorporation in routine anatomical pathology practice. This mini review provides an overview of spatial transcriptomics methods, with an emphasis on platforms compatible with FFPE tissue, approaches to assess the data and potential applications in anatomical pathology practice.

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.

Metabolic engineering of Komagataella phaffii for the efficient utilization of methanol.

BACKGROUND: Komagataella phaffii, a type of methanotrophic yeast, can use methanol, a favorable non-sugar substrate in eco-friendly bio-manufacturing. The dissimilation pathway in K. phaffii leads to the loss of carbon atoms in the form of CO2. However, the ΔFLD strain, engineered to lack formaldehyde dehydrogenase-an essential enzyme in the dissimilation pathway-displayed growth defects when exposed to a methanol-containing medium. RESULTS: Inhibiting the dissimilation pathway triggers an excessive accumulation of formaldehyde and a decline in the intracellular NAD+/NADH ratio. Here, we designed dual-enzyme complex with the alcohol oxidase1/dihydroxyacetone synthase1 (Aox1/Das1), enhancing the regeneration of the formaldehyde receptor xylulose-5-phosphate (Xu5P). This strategy mitigated the harmful effects of formaldehyde accumulation and associated toxicity to cells. Concurrently, we elevated the NAD+/NADH ratio by overexpressing isocitrate dehydrogenase in the TCA cycle, promoting intracellular redox homeostasis. The OD600 of the optimized combination of the above strategies, strain DF02-1, was 4.28 times higher than that of the control strain DF00 (ΔFLD, HIS4+) under 1% methanol. Subsequently, the heterologous expression of methanol oxidase Mox from Hansenula polymorpha in strain DF02-1 resulted in the recombinant strain DF02-4, which displayed a growth at an OD600 4.08 times higher than that the control strain DF00 in medium containing 3% methanol. CONCLUSIONS: The reduction of formaldehyde accumulation, the increase of NAD+/NADH ratio, and the enhancement of methanol oxidation effectively improved the efficient utilization of a high methanol concentration by strain ΔFLD strain lacking formaldehyde dehydrogenase. The modification strategies implemented in this study collectively serve as a foundational framework for advancing the efficient utilization of methanol in K. phaffii.

Effect of propranolol on temporomandibular joint pain in repeatedly stressed rats.

Stress substantially increases the risk of developing painful temporomandibular disorders (TMDs) by influencing the release of endogenous catecholamines. Propranolol, an antagonist of ß-adrenergic receptors, has shown potential in alleviating TMD-associated pain, particularly when the level of catecholamines is elevated. The aim of this study was to explore whether intra-articular propranolol administration is effective in diminishing temporomandibular joint (TMJ) pain during repeated stress situations. Additionally, we investigated the effect of repeated stress on the expression of genes encoding ß-adrenoceptors in the trigeminal ganglion. In the present study, rats were exposed to a stress protocol induced by sound, then to the administration of formalin in the TMJ (to elicit a nociceptive response), followed immediately afterward by different doses of propranolol, after which the analgesic response to propranolol was evaluated. We also assessed the levels of beta-1 and beta-2 adrenergic receptor mRNAs (Adrb1 and Adrb2, respectively) using reverse transcription-quantitative PCR (RT-qPCR). Our findings revealed that propranolol administration reduces formalin-induced TMJ nociception more effectively in stressed rats than in non-stressed rats. Furthermore, repeated stress decreases the expression of the Adrb2 gene within the trigeminal ganglion. The findings of this study are noteworthy as they suggest that individuals with a chronic stress history might find potential benefits from ß-blockers in TMD treatment.

Biodegradation enhancement of high concentrations formaldehyde waste gas and verification of the metabolic mechanism.

The enhanced effects of formaldehyde biodegradation in a biofilm packing tower are investigated in this study. Three experimental groups were established: a blank control group, a biochar addition group, and a lanthanum addition group. The inlet gas flow rate, the inlet gas concentration, and the structural succession characteristics of the microbial community in the tower were investigated by regular sampling. The intracellular metabolites and key enzymes of the dominant functional bacteria, Pseudomonas P1 and Methylobacterium Q1, in the tower were analyzed. The results indicated that with the biochar addition, the formaldehyde purification efficiency increased significantly from 91.67-94.67 % to 94.12 96.85 %, and the bio-elimination capacity increased with an increase in the inlet gas flow rate from 2.314 to 13.988 mg L-1h-1 to 2.697-15.051 mg L-1h-1. With the addition of lanthanum, the purification efficiency increased significantly from 90.80-93.98 % to 94.36-96.78 %, and the bio-elimination capacity increased with an increase in the inlet gas concentration from 1.099-11.284 mg L-1h-1 to 1.266-11.961 mg L-1h-1. The microbial community structure in the tower changed with system operation, and the formaldehyde degrading functional bacteria formed the dominant bacteria. It was verified that P1 and Q1 metabolized high concentrations of formaldehyde by the serine cycle and the ribulose monophosphate (RuMP) cycle.

OMIP-088: Twenty-target imaging mass cytometry panel for major cell populations in mouse formalin fixed paraffin embedded liver.

The purpose of this 20-target imaging mass cytometry (IMC) panel is to identify the main cell types in formalin fixed paraffin embedded (FFPE) mouse liver tissue with the Hyperion™ mass cytometer from Standard BioTools (formerly Fluidigm). The antibody panel includes markers to identify hepatocytes (E-cadherin, HNF4α (hepatocyte nuclear factor 4 alpha), Arginase-1), liver sinusoidal endothelial cells (LSECs; CD206), Kupffer cells (F4/80, CD206), neutrophils (Ly6G, CD11b), bone marrow derived myeloid cells (BMDMs; CD11b), cholangiocytes (E-cadherin high), endothelial cells (CD31, α-SMA), plasmacytoid dendritic cells (CD317), B cells (CD19), T cells (CD3e, CD4, CD8a), NK cells (CD161) as well markers of cell activation (CD44, CD74), proliferation (Ki-67) and to aid in cell segmentation (Pan-Actin, E-cadherin, histone H3). The panel has been tested in other mouse tissues, namely the spleen, colon and lung, and therefore is likely to work across various mouse FFPE samples of interest. It has not been tested using human samples, frozen samples or in suspension mass cytometry because FFPE treatment profoundly changes epitope conformation. In summary, this panel is a powerful tool for pre-clinical research to determine cellular abundance and spatial distribution within mouse tissues and serves as a scaffold, to which more targets can be added for project specific requirements.

Mammals divert endogenous genotoxic formaldehyde into one-carbon metabolism.

The folate-driven one-carbon (1C) cycle is a fundamental metabolic hub in cells that enables the synthesis of nucleotides and amino acids and epigenetic modifications. This cycle might also release formaldehyde, a potent protein and DNA crosslinking agent that organisms produce in substantial quantities. Here we show that supplementation with tetrahydrofolate, the essential cofactor of this cycle, and other oxidation-prone folate derivatives kills human, mouse and chicken cells that cannot detoxify formaldehyde or that lack DNA crosslink repair. Notably, formaldehyde is generated from oxidative decomposition of the folate backbone. Furthermore, we find that formaldehyde detoxification in human cells generates formate, and thereby promotes nucleotide synthesis. This supply of 1C units is sufficient to sustain the growth of cells that are unable to use serine, which is the predominant source of 1C units. These findings identify an unexpected source of formaldehyde and, more generally, indicate that the detoxification of this ubiquitous endogenous genotoxin creates a benign 1C unit that can sustain essential metabolism.

Compartment-related aspects of XoxF protein functionality in Methylorubrum extorquens DM4 analysed using its cytoplasmic targeting.

The impact of periplasmic localisation on the functioning of the XoxF protein was evaluated in the well-studied dichloromethane-utilising methylotroph Methylorubrum extorquens DM4, which harbors only one paralogue of the xoxF gene. It was found that the cytoplasmic targeting of XoxF by expression of the corresponding gene without the sequence encoding the N-terminal signal peptide does not impair the activation and lanthanide-dependent regulation of the MxaFI-methanol dehydrogenase genes. Analysis of the viability of ΔxoxF cells complemented with the full-length and truncated xoxF gene also showed that the expression of cytoplasmically targeted XoxF even increases the resistance to acids. These results contradict the proposed function of the XoxF protein as an extracytoplasmic signal sensor. At the same time, the observed dynamics of growth with methanol, as well as with dichloromethane of strains expressing cytoplasmic-targeted XoxF, indicate the probable enzymatic activity of lanthanide-dependent methanol dehydrogenase in this compartment. Herewith, the only available substrate for this enzyme in cells growing with dichloromethane was formaldehyde, which is produced during the primary metabolism of the mentioned halogenated toxicant directly in the cytosol. These findings suggest that the maturation of XoxF-methanol dehydrogenase may occur already in the cytoplasm, while the factors changing affinity of this enzyme for formaldehyde are apparently absent there. Together with the demonstrated functioning of an enhancer-like upstream activating sequence in the promoter region of the xoxF gene in M. extorquens DM4, the obtained information enriches our understanding of the regulation, synthesis and role of the XoxF protein.

Endogenous Formaldehyde Is a Hematopoietic Stem Cell Genotoxin and Metabolic Carcinogen.

Endogenous formaldehyde is produced by numerous biochemical pathways fundamental to life, and it can crosslink both DNA and proteins. However, the consequences of its accumulation are unclear. Here we show that endogenous formaldehyde is removed by the enzyme alcohol dehydrogenase 5 (ADH5/GSNOR), and Adh5(-/-) mice therefore accumulate formaldehyde adducts in DNA. The repair of this damage is mediated by FANCD2, a DNA crosslink repair protein. Adh5(-/-)Fancd2(-/-) mice reveal an essential requirement for these protection mechanisms in hematopoietic stem cells (HSCs), leading to their depletion and precipitating bone marrow failure. More widespread formaldehyde-induced DNA damage also causes karyomegaly and dysfunction of hepatocytes and nephrons. Bone marrow transplantation not only rescued hematopoiesis but, surprisingly, also preserved nephron function. Nevertheless, all of these animals eventually developed fatal malignancies. Formaldehyde is therefore an important source of endogenous DNA damage that is counteracted in mammals by a conserved protection mechanism.

Obtainment and Inoculation of Acinetobacter pittii Strain JJ-2, and Combined Action with Plants for Formaldehyde and CO2 Removal: A Research Study.

A formaldehyde-degrading bacterium JJ-2 was isolated from the rhizosphere of Chlorophytum and identified as Acinetobacter pittii by colony morphology and 16S rDNA sequence analysis. Further studies showed that under optimal conditions, JJ-2 could maintain activity for six cycles at an initial formaldehyde concentration of 450 mg L-1. At the same time, the complete degradation time was shortened from 12 to 6 h. When the JJ-2 strain was inoculated into sterile soil, the surface spray method had the best effect, and the removal efficiency of 5 ppm formaldehyde increased by 22.63%. In an actual potted plants system colonized with strain JJ-2, the first and second fumigations (without re-inoculation) increased removal by 1.36 times and 0.92 times during the day and 1.27 times and 2.07 times at night. In addition, in the second fumigation, the plant-bacteria combined system was 693.63 ppm and the plant system was 715.34 ppm, effectively reducing the CO2 concentration. This study provides an economical, ecological, and efficient approach to improve the combined system of plants and bacteria to remove gaseous formaldehyde from indoor air, with a positive impact on carbon neutrality.

A novel approach to diagnosing crystal-storing histiocytosis: utility of scanning electron microscopy for formalin-fixed paraffin-embedded tissue specimens.

Crystal-storing histiocytosis (CSH) is a rare disorder that shows infiltration of histiocytes with an aberrant cytoplasmic accumulation of crystalline structures and is often accompanied by lymphoproliferative-plasma cell disorders (LP-PCD) as background diseases. The diagnosis of CSH requires identification of crystalline structures that accumulate in the infiltrating histiocytes, which may be challenging by optical microscopy alone. In this case report, we describe an atypical course of systemic CSH with multifocal fibrosclerosis of an unknown background disease that was diagnosed by ultrastructural observation, including transmission electron microscopy (TEM) and scanning electron microscopy (SEM), in pathological autopsy. In addition, crystalline structures were successfully identified by scanning electron microscopic observations using formalin-fixed and paraffin-embedded (FFPE) tissue from biopsy specimens taken before death. Since CSH was identified by SEM in a tiny biopsy specimen, observation of histiocytic infiltrative lesions by SEM using FFPE tissue may lead to early detection of and initiation of treatment for CSH.

Feed intake and milk yield and composition of lactating dairy goats in response to partial substitution of soybean meal for formaldehyde-treated sesame meal in the diet.

This study was conducted to evaluate the effect of substitution of soybean meal (SBM) for formaldehyde-treated sesame meal (FTSM) on nutrient intake and digestibility, ruminal and blood parameters and milk production and composition in lactating Murciano-Granadina goats. Forty lactating goats were randomly assigned to one of the following four treatments: (1) diet with 16.5% CP, containing SBM (CON); (2) diet with 16.5% CP, containing untreated SM (USM); (3) diet with 16.5% CP, containing FTSM (FT); and (4) diet with 14.5% CP containing FTSM (LPFT). The results showed that nutrient intake was highest in the FT group (p < 0.001), while it was similar between the CON and LPFT groups, except for the intake of CP, which was higher in the CON group. The FT and LPFT had lower ruminal pH compared to CON and USM groups (p < 0.001), with goats in group FT having the highest volatile fatty acids (VFA) production (p < 0.001). The highest propionate concentration was observed in the LPFT treatment (p < 0.001), followed by the FT, CON, and USM treatments. Goats offered USM and LPFT treatments presented the highest and lowest acetate: propionate values, respectively, among the experimental groups (p < 0.001). The results also showed that LPFT goats had the lowest blood urea nitrogen (BUN) level (p = 0.004), while FT goats presented a lower non-esterified FA (NEFA) level compared with CON and LPFT goats (p = 0.01). Goats offered the FT diet had the highest milk yield (p = 0.002) and energy-corrected milk yield (p < 0.001) among all dietary groups. The highest milk fat (p < 0.001), protein (p = 0.001), lactose (p = 0.007), total solids (p = 0.003), and solids-not-fat (SNF) (p = 0.003) contents were observed in FT goats, which didn't differ from USM goats. The inclusion of formaldehyde-treated SM increased the percentage of C18:3 (p < 0.001) and C20:1 (p = 0.04) FAs compared with USM and CON treatments. Milk from USM, FT, and LPFT goats had lower levels of saturated (p < 0.001) and medium-chain FAs (p = 0.014) compared with CON goats, whereas milk from CON goats had lower levels of unsaturated, monounsaturated, and long-chain FAs compared to other groups (p < 0.001). The lowest and the highest concentrations of polyunsaturated FAs were observed in CON and LPFT goats, respectively (p = 0.001). It can be concluded that SBM can be advantageously replaced by formaldehyde-treated SM in the diet as a feasible alternative to improve feed intake and production performance of dairy goats.

Structural diversity of the coenzyme methylofuran and identification of enzymes for the biosynthesis of its polyglutamate side chain.

Methylofuran (MYFR) is a formyl-carrying coenzyme essential for the oxidation of formaldehyde in most methylotrophic bacteria. In Methylorubrum extorquens, MYFR contains a large and branched polyglutamate side chain of up to 24 glutamates. These glutamates play an essential role in interfacing the coenzyme with the formyltransferase/hydrolase complex, an enzyme that generates formate. To date, MYFR has not been identified in other methylotrophs, and it is unknown whether its structural features are conserved. Here, we examined nine bacterial strains for the presence and structure of MYFR using high-resolution liquid chromatography-mass spectrometry (LC-MS). Two of the strains produced MYFR as present in M. extorquens, while a modified MYFR containing tyramine instead of tyrosine in its core structure was detected in six strains. When M. extorquens was grown in the presence of tyramine, the compound was readily incorporated into MYFR, indicating that the biosynthetic enzymes are unable to discriminate tyrosine from tyramine. Using gene deletions in combination with LC-MS analyses, we identified three genes, orf5, orfY, and orf17 that are essential for MYFR biosynthesis. Notably, the orfY and orf5 mutants accumulated short MYFR intermediates with only one and two glutamates, respectively, suggesting that these enzymes catalyze glutamate addition. Upon homologous overexpression of orf5, a drastic increase in the number of glutamates in MYFR was observed (up to 40 glutamates), further corroborating the function of Orf5 as a glutamate ligase. We thus renamed OrfY and Orf5 to MyfA and MyfB to highlight that these enzymes are specifically involved in MYFR biosynthesis.

Metabolic methanol: molecular pathways and physiological roles.

Methanol has been historically considered an exogenous product that leads only to pathological changes in the human body when consumed. However, in normal, healthy individuals, methanol and its short-lived oxidized product, formaldehyde, are naturally occurring compounds whose functions and origins have received limited attention. There are several sources of human physiological methanol. Fruits, vegetables, and alcoholic beverages are likely the main sources of exogenous methanol in the healthy human body. Metabolic methanol may occur as a result of fermentation by gut bacteria and metabolic processes involving S-adenosyl methionine. Regardless of its source, low levels of methanol in the body are maintained by physiological and metabolic clearance mechanisms. Although human blood contains small amounts of methanol and formaldehyde, the content of these molecules increases sharply after receiving even methanol-free ethanol, indicating an endogenous source of the metabolic methanol present at low levels in the blood regulated by a cluster of genes. Recent studies of the pathogenesis of neurological disorders indicate metabolic formaldehyde as a putative causative agent. The detection of increased formaldehyde content in the blood of both neurological patients and the elderly indicates the important role of genetic and biochemical mechanisms of maintaining low levels of methanol and formaldehyde.