Gut Microbial Metabolite TMAO Enhances Platelet Hyperreactivity and Thrombosis Risk.

Normal platelet function is critical to blood hemostasis and maintenance of a closed circulatory system. Heightened platelet reactivity, however, is associated with cardiometabolic diseases and enhanced potential for thrombotic events. We now show gut microbes, through generation of trimethylamine N-oxide (TMAO), directly contribute to platelet hyperreactivity and enhanced thrombosis potential. Plasma TMAO levels in subjects (n > 4,000) independently predicted incident (3 years) thrombosis (heart attack, stroke) risk. Direct exposure of platelets to TMAO enhanced sub-maximal stimulus-dependent platelet activation from multiple agonists through augmented Ca(2+) release from intracellular stores. Animal model studies employing dietary choline or TMAO, germ-free mice, and microbial transplantation collectively confirm a role for gut microbiota and TMAO in modulating platelet hyperresponsiveness and thrombosis potential and identify microbial taxa associated with plasma TMAO and thrombosis potential. Collectively, the present results reveal a previously unrecognized mechanistic link between specific dietary nutrients, gut microbes, platelet function, and thrombosis risk.

Drug the Bug!

Microbial metabolism of dietary components has been causally linked to cardiovascular disease and atherosclerosis. Now, Wang et al. demonstrate that inhibition of microbial TMA lyases, essential for production of pro-atherogenic trimethylamines, prevents atherosclerosis in vivo.

Non-lethal Inhibition of Gut Microbial Trimethylamine Production for the Treatment of Atherosclerosis.

Trimethylamine (TMA) N-oxide (TMAO), a gut-microbiota-dependent metabolite, both enhances atherosclerosis in animal models and is associated with cardiovascular risks in clinical studies. Here, we investigate the impact of targeted inhibition of the first step in TMAO generation, commensal microbial TMA production, on diet-induced atherosclerosis. A structural analog of choline, 3,3-dimethyl-1-butanol (DMB), is shown to non-lethally inhibit TMA formation from cultured microbes, to inhibit distinct microbial TMA lyases, and to both inhibit TMA production from physiologic polymicrobial cultures (e.g., intestinal contents, human feces) and reduce TMAO levels in mice fed a high-choline or L-carnitine diet. DMB inhibited choline diet-enhanced endogenous macrophage foam cell formation and atherosclerotic lesion development in apolipoprotein e(-/-) mice without alterations in circulating cholesterol levels. The present studies suggest that targeting gut microbial production of TMA specifically and non-lethal microbial inhibitors in general may serve as a potential therapeutic approach for the treatment of cardiometabolic diseases.

Structural basis of lipid head group entry to the Kennedy pathway by FLVCR1.

Phosphatidylcholine and phosphatidylethanolamine, the two most abundant phospholipids in mammalian cells, are synthesized de novo by the Kennedy pathway from choline and ethanolamine, respectively1-6. Despite the essential roles of these lipids, the mechanisms that enable the cellular uptake of choline and ethanolamine remain unknown. Here we show that the protein encoded by FLVCR1, whose mutation leads to the neurodegenerative syndrome posterior column ataxia and retinitis pigmentosa7-9, transports extracellular choline and ethanolamine into cells for phosphorylation by downstream kinases to initiate the Kennedy pathway. Structures of FLVCR1 in the presence of choline and ethanolamine reveal that both metabolites bind to a common binding site comprising aromatic and polar residues. Despite binding to a common site, FLVCR1 interacts in different ways with the larger quaternary amine of choline in and with the primary amine of ethanolamine. Structure-guided mutagenesis identified residues that are crucial for the transport of ethanolamine, but dispensable for choline transport, enabling functional separation of the entry points into the two branches of the Kennedy pathway. Altogether, these studies reveal how FLVCR1 is a high-affinity metabolite transporter that serves as the common origin for phospholipid biosynthesis by two branches of the Kennedy pathway.

Structural and molecular basis of choline uptake into the brain by FLVCR2.

Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain remains unknown1-3. The major facilitator superfamily transporter FLVCR1 (also known as MFSD7B or SLC49A1) was recently determined to be a choline transporter but is not highly expressed at the blood-brain barrier, whereas the related protein FLVCR2 (also known as MFSD7C or SLC49A2) is expressed in endothelial cells at the blood-brain barrier4-7. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus and embryonic lethality, but the physiological role of FLVCR2 is unknown4,5. Here we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in both inward-facing and outward-facing states using cryo-electron microscopy. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of therapeutic agents into the brain.

Molecular mechanism of choline and ethanolamine transport in humans.

Human feline leukaemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and FLVCR2) are members of the major facilitator superfamily1. Their dysfunction is linked to several clinical disorders, including PCARP, HSAN and Fowler syndrome2-7. Earlier studies concluded that FLVCR1 may function as a haem exporter8-12, whereas FLVCR2 was suggested to act as a haem importer13, yet conclusive biochemical and detailed molecular evidence remained elusive for the function of both transporters14-16. Here, we show that FLVCR1 and FLVCR2 facilitate the transport of choline and ethanolamine across the plasma membrane, using a concentration-driven substrate translocation process. Through structural and computational analyses, we have identified distinct conformational states of FLVCRs and unravelled the coordination chemistry underlying their substrate interactions. Fully conserved tryptophan and tyrosine residues form the binding pocket of both transporters and confer selectivity for choline and ethanolamine through cation-π interactions. Our findings clarify the mechanisms of choline and ethanolamine transport by FLVCR1 and FLVCR2, enhance our comprehension of disease-associated mutations that interfere with these vital processes and shed light on the conformational dynamics of these major facilitator superfamily proteins during the transport cycle.

Single-cell multiregion dissection of Alzheimer's disease.

Alzheimer's disease is the leading cause of dementia worldwide, but the cellular pathways that underlie its pathological progression across brain regions remain poorly understood1-3. Here we report a single-cell transcriptomic atlas of six different brain regions in the aged human brain, covering 1.3 million cells from 283 post-mortem human brain samples across 48 individuals with and without Alzheimer's disease. We identify 76 cell types, including region-specific subtypes of astrocytes and excitatory neurons and an inhibitory interneuron population unique to the thalamus and distinct from canonical inhibitory subclasses. We identify vulnerable populations of excitatory and inhibitory neurons that are depleted in specific brain regions in Alzheimer's disease, and provide evidence that the Reelin signalling pathway is involved in modulating the vulnerability of these neurons. We develop a scalable method for discovering gene modules, which we use to identify cell-type-specific and region-specific modules that are altered in Alzheimer's disease and to annotate transcriptomic differences associated with diverse pathological variables. We identify an astrocyte program that is associated with cognitive resilience to Alzheimer's disease pathology, tying choline metabolism and polyamine biosynthesis in astrocytes to preserved cognitive function late in life. Together, our study develops a regional atlas of the ageing human brain and provides insights into cellular vulnerability, response and resilience to Alzheimer's disease pathology.

Phospholipid imbalance impairs autophagosome completion.

Autophagy, a conserved eukaryotic intracellular catabolic pathway, maintains cell homeostasis by lysosomal degradation of cytosolic material engulfed in double membrane vesicles termed autophagosomes, which form upon sealing of single-membrane cisternae called phagophores. While the role of phosphatidylinositol 3-phosphate (PI3P) and phosphatidylethanolamine (PE) in autophagosome biogenesis is well-studied, the roles of other phospholipids in autophagy remain rather obscure. Here we utilized budding yeast to study the contribution of phosphatidylcholine (PC) to autophagy. We reveal for the first time that genetic loss of PC biosynthesis via the CDP-DAG pathway leads to changes in lipid composition of autophagic membranes, specifically replacement of PC by phosphatidylserine (PS). This impairs closure of the autophagic membrane and autophagic flux. Consequently, we show that choline-dependent recovery of de novo PC biosynthesis via the CDP-choline pathway restores autophagosome formation and autophagic flux in PC-deficient cells. Our findings therefore implicate phospholipid metabolism in autophagosome biogenesis.

Competence remodels the pneumococcal cell wall exposing key surface virulence factors that mediate increased host adherence.

Competence development in the human pathogen Streptococcus pneumoniae controls several features such as genetic transformation, biofilm formation, and virulence. Competent bacteria produce so-called "fratricins" such as CbpD that kill noncompetent siblings by cleaving peptidoglycan (PGN). CbpD is a choline-binding protein (CBP) that binds to phosphorylcholine residues found on wall and lipoteichoic acids (WTA and LTA) that together with PGN are major constituents of the pneumococcal cell wall. Competent pneumococci are protected against fratricide by producing the immunity protein ComM. How competence and fratricide contribute to virulence is unknown. Here, using a genome-wide CRISPRi-seq screen, we show that genes involved in teichoic acid (TA) biosynthesis are essential during competence. We demonstrate that LytR is the major enzyme mediating the final step in WTA formation, and that, together with ComM, is essential for immunity against CbpD. Importantly, we show that key virulence factors PspA and PspC become more surface-exposed at midcell during competence, in a CbpD-dependent manner. Together, our work supports a model in which activation of competence is crucial for host adherence by increased surface exposure of its various CBPs.

Ionic liquid-caged nucleic acids enable active folding-based molecular recognition with hydrolysis resistance.

Beyond storage and transmission of genetic information in cellular life, nucleic acids can perform diverse interesting functions, including specific target recognition and biochemical reaction acceleration; the versatile biopolymers, however, are acutely vulnerable to hydrolysis-driven degradation. Here, we demonstrate that the cage effect of choline dihydrogen phosphate permits active folding of nucleic acids like water, but prevents their phosphodiester hydrolysis unlike water. The choline-based ionic liquid not only serves as a universal inhibitor of nucleases, exceptionally extending half-lives of nucleic acids up to 6 500 000 times, but highly useful tasks of nucleic acids (e.g. mRNA detection of molecular beacons, ligand recognition of aptamers, and transesterification reaction of ribozymes) can be also conducted with well-conserved affinities and specificities. As liberated from the function loss and degradation risk, the presence of undesired and unknown nucleases does not undermine desired molecular functions of nucleic acids without hydrolysis artifacts even in nuclease cocktails and human saliva.

The moonlighting function of glycolytic enzyme enolase-1 promotes choline phospholipid metabolism and tumor cell proliferation.

Aberrantly upregulated choline phospholipid metabolism is a novel emerging hallmark of cancer, and choline kinase α (CHKα), a key enzyme for phosphatidylcholine production, is overexpressed in many types of human cancer through undefined mechanisms. Here, we demonstrate that the expression levels of the glycolytic enzyme enolase-1 (ENO1) are positively correlated with CHKα expression levels in human glioblastoma specimens and that ENO1 tightly governs CHKα expression via posttranslational regulation. Mechanistically, we reveal that both ENO1 and the ubiquitin E3 ligase TRIM25 are associated with CHKα. Highly expressed ENO1 in tumor cells binds to I199/F200 of CHKα, thereby abrogating the interaction between CHKα and TRIM25. This abrogation leads to the inhibition of TRIM25-mediated polyubiquitylation of CHKα at K195, increased stability of CHKα, enhanced choline metabolism in glioblastoma cells, and accelerated brain tumor growth. In addition, the expression levels of both ENO1 and CHKα are associated with poor prognosis in glioblastoma patients. These findings highlight a critical moonlighting function of ENO1 in choline phospholipid metabolism and provide unprecedented insight into the integrated regulation of cancer metabolism by crosstalk between glycolytic and lipidic enzymes.

Developmental programming and lineage branching of early human telencephalon.

The dorsal and ventral human telencephalons contain different neuronal subtypes, including glutamatergic, GABAergic, and cholinergic neurons, and how these neurons are generated during early development is not well understood. Using scRNA-seq and stringent validations, we reveal here a developmental roadmap for human telencephalic neurons. Both dorsal and ventral telencephalic radial glial cells (RGs) differentiate into neurons via dividing intermediate progenitor cells (IPCs_div) and early postmitotic neuroblasts (eNBs). The transcription factor ASCL1 plays a key role in promoting fate transition from RGs to IPCs_div in both regions. RGs from the regionalized neuroectoderm show heterogeneity, with restricted glutamatergic, GABAergic, and cholinergic differentiation potencies. During neurogenesis, IPCs_div gradually exit the cell cycle and branch into sister eNBs to generate distinct neuronal subtypes. Our findings highlight a general RGs-IPCs_div-eNBs developmental scheme for human telencephalic progenitors and support that the major neuronal fates of human telencephalon are predetermined during dorsoventral regionalization with neuronal diversity being further shaped during neurogenesis and neural circuit integration.

Choline metabolism underpins macrophage IL-4 polarization and RELMα up-regulation in helminth infection.

Type 2 cytokines like IL-4 are hallmarks of helminth infection and activate macrophages to limit immunopathology and mediate helminth clearance. In addition to cytokines, nutrients and metabolites critically influence macrophage polarization. Choline is an essential nutrient known to support normal macrophage responses to lipopolysaccharide; however, its function in macrophages polarized by type 2 cytokines is unknown. Using murine IL-4-polarized macrophages, targeted lipidomics revealed significantly elevated levels of phosphatidylcholine, with select changes to other choline-containing lipid species. These changes were supported by the coordinated up-regulation of choline transport compared to naïve macrophages. Pharmacological inhibition of choline metabolism significantly suppressed several mitochondrial transcripts and dramatically inhibited select IL-4-responsive transcripts, most notably, Retnla. We further confirmed that blocking choline metabolism diminished IL-4-induced RELMα (encoded by Retnla) protein content and secretion and caused a dramatic reprogramming toward glycolytic metabolism. To better understand the physiological implications of these observations, naïve or mice infected with the intestinal helminth Heligmosomoides polygyrus were treated with the choline kinase α inhibitor, RSM-932A, to limit choline metabolism in vivo. Pharmacological inhibition of choline metabolism lowered RELMα expression across cell-types and tissues and led to the disappearance of peritoneal macrophages and B-1 lymphocytes and an influx of infiltrating monocytes. The impaired macrophage activation was associated with some loss in optimal immunity to H. polygyrus, with increased egg burden. Together, these data demonstrate that choline metabolism is required for macrophage RELMα induction, metabolic programming, and peritoneal immune homeostasis, which could have important implications in the context of other models of infection or cancer immunity.

Exposure to prenatal excess or imbalanced micronutrients leads to long-term perturbations in one-carbon metabolism, trimethylamine-N-oxide and DNA methylation in Wistar rat offspring.

Prenatal multivitamins, including folic acid, are commonly consumed in excess, whereas choline, an essential nutrient and an important source of labile methyl groups, is underconsumed. Here, we characterized profiles of one-carbon metabolism and related pathways and patterns of DNA methylation in offspring exposed to excess or imbalanced micronutrients prenatally. Pregnant Wistar rats were fed either recommended 1× vitamins (RV), high 10× vitamins (HV), high 10× folic acid with recommended choline (HFolRC), or high 10× folic acid with no choline (HFolNC). Offspring were weaned to a high-fat diet for 12 weeks. Circulating metabolites were analyzed with a focus on the hypothalamus, an area known to be under epigenetic regulation. HV, HFolRC, and HFolNC males had higher body weight (BW) and lower plasma choline and methionine consistent with lower hypothalamic S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) and global DNA methylation compared with RV. HV and HFolNC females had higher BW and lower plasma 5-methyltetrahydrofolate and methionine consistent with lower hypothalamic global DNA methylation compared with RV. Plasma dimethylglycine (DMG) and methionine were higher as with hypothalamic SAM:SAH and global DNA methylation in HFolRC females without changes in BW compared with RV. Plasma trimethylamine and trimethylamine-N-oxide were higher in males but lower in females from HFolRC compared with RV. Network modeling revealed a link between the folate-dependent pathway and SAH, with most connections through DMG. Final BW was negatively correlated with choline, DMG, and global DNA methylation. In conclusion, prenatal intake of excess or imbalanced micronutrients induces distinct metabolic and epigenetic perturbations in offspring that reflect long-term nutritional programming of health.

Metabolic disorder and functional disturbance in the central executive network in minimal hepatic encephalopathy.

The aim of this paper is to investigate dynamical functional disturbance in central executive network in minimal hepatic encephalopathy and determine its association with metabolic disorder and cognitive impairment. Data of magnetic resonance spectroscopy and resting-state functional magnetic resonance imaging were obtained from 27 cirrhotic patients without minimal hepatic encephalopathy, 20 minimal hepatic encephalopathy patients, and 24 healthy controls. Central executive network was identified utilizing seed-based correlation approach. Dynamic functional connectivity across central executive network was calculated using sliding-window approach. Functional states were estimated by K-means clustering. Right dorsolateral prefrontal cortex metabolite ratios (i.e. glutamate and glutamine complex/total creatine, myo-inositol / total creatine, and choline / total creatine) were determined. Neurocognitive performance was determined by psychometric hepatic encephalopathy scores. Minimal hepatic encephalopathy patients had decreased myo-inositol / total creatine and choline / total creatine and increased glutamate and glutamine complex / total creatine in right dorsolateral prefrontal cortex (all P ≤ 0.020); decreased static functional connectivity between bilateral dorsolateral prefrontal cortex and between right dorsolateral prefrontal cortex and lateral-inferior temporal cortex (P ≤ 0.001); increased frequency and mean dwell time in state-1 (P ≤ 0.001), which exhibited weakest functional connectivity. Central executive network dynamic functional indices were significantly correlated with right dorsolateral prefrontal cortex metabolic indices and psychometric hepatic encephalopathy scores. Right dorsolateral prefrontal cortex myo-inositol / total creatine and mean dwell time in state-1 yielded best potential for diagnosing minimal hepatic encephalopathy. Dynamic functional disturbance in central executive network may contribute to neurocognitive impairment and could be correlated with metabolic disorder.

The developmental trajectory of 1H-MRS brain metabolites from childhood to adulthood.

Human brain development is ongoing throughout childhood, with for example, myelination of nerve fibers and refinement of synaptic connections continuing until early adulthood. 1H-Magnetic Resonance Spectroscopy (1H-MRS) can be used to quantify the concentrations of endogenous metabolites (e.g. glutamate and γ -aminobutyric acid (GABA)) in the human brain in vivo and so can provide valuable, tractable insight into the biochemical processes that support postnatal neurodevelopment. This can feasibly provide new insight into and aid the management of neurodevelopmental disorders by providing chemical markers of atypical development. This study aims to characterize the normative developmental trajectory of various brain metabolites, as measured by 1H-MRS from a midline posterior parietal voxel. We find significant non-linear trajectories for GABA+ (GABA plus macromolecules), Glx (glutamate + glutamine), total choline (tCho) and total creatine (tCr) concentrations. Glx and GABA+ concentrations steeply decrease across childhood, with more stable trajectories across early adulthood. tCr and tCho concentrations increase from childhood to early adulthood. Total N-acetyl aspartate (tNAA) and Myo-Inositol (mI) concentrations are relatively stable across development. Trajectories likely reflect fundamental neurodevelopmental processes (including local circuit refinement) which occur from childhood to early adulthood and can be associated with cognitive development; we find GABA+ concentrations significantly positively correlate with recognition memory scores.

Structure and kinase activity of bacterial cell cycle regulator CcrZ.

CcrZ is a recently discovered cell cycle regulator that connects DNA replication initiation with cell division in pneumococci and may have a similar function in related bacteria. CcrZ is also annotated as a putative kinase, suggesting that CcrZ homologs could represent a novel family of bacterial kinase-dependent cell cycle regulators. Here, we investigate the CcrZ homolog in Bacillus subtilis and show that cells lacking ccrZ are sensitive to a broad range of DNA damage. We demonstrate that increased expression of ccrZ results in over-initiation of DNA replication. In addition, increased expression of CcrZ activates the DNA damage response. Using sensitivity to DNA damage as a proxy, we show that the negative regulator for replication initiation (yabA) and ccrZ function in the same pathway. We show that CcrZ interacts with replication initiation proteins DnaA and DnaB, further suggesting that CcrZ is important for replication timing. To understand how CcrZ functions, we solved the crystal structure bound to AMP-PNP to 2.6 Å resolution. The CcrZ structure most closely resembles choline kinases, consisting of a bilobal structure with a cleft between the two lobes for binding ATP and substrate. Inspection of the structure reveals a major restructuring of the substrate-binding site of CcrZ relative to the choline-binding pocket of choline kinases, consistent with our inability to detect activity with choline for this protein. Instead, CcrZ shows activity on D-ribose and 2-deoxy-D-ribose, indicating adaptation of the choline kinase fold in CcrZ to phosphorylate a novel substrate. We show that integrity of the kinase active site is required for ATPase activity in vitro and for function in vivo. This work provides structural, biochemical, and functional insight into a newly identified, and conserved group of bacterial kinases that regulate DNA replication initiation.

An experimental test of the nicotinic hypothesis of COVID-19.

The pathophysiological mechanisms underlying the constellation of symptoms that characterize COVID-19 are only incompletely understood. In an effort to fill these gaps, a "nicotinic hypothesis," which posits that nicotinic acetylcholine receptors (AChRs) act as additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptors, has recently been put forth. A key feature of the proposal (with potential clinical ramifications) is the suggested competition between the virus' spike protein and small-molecule cholinergic ligands for the receptor's orthosteric binding sites. This notion is reminiscent of the well-established role of the muscle AChR during rabies virus infection. To address this hypothesis directly, we performed equilibrium-type ligand-binding competition assays using the homomeric human α7-AChR (expressed on intact cells) as the receptor, and radio-labeled α-bungarotoxin (α-BgTx) as the orthosteric-site competing ligand. We tested different SARS-CoV-2 spike protein peptides, the S1 domain, and the entire S1-S2 ectodomain, and found that none of them appreciably outcompete [125I]-α-BgTx in a specific manner. Furthermore, patch-clamp recordings showed no clear effect of the S1 domain on α7-AChR-mediated currents. We conclude that the binding of the SARS-CoV-2 spike protein to the human α7-AChR's orthosteric sites-and thus, its competition with ACh, choline, or nicotine-is unlikely to be a relevant aspect of this complex disease.

ACBP/DBI protein neutralization confers autophagy-dependent organ protection through inhibition of cell loss, inflammation, and fibrosis.

Acyl-coenzyme A (CoA)-binding protein (ACBP), also known as diazepam-binding inhibitor (DBI), is an extracellular feedback regulator of autophagy. Here, we report that injection of a monoclonal antibody neutralizing ACBP/DBI (α-DBI) protects the murine liver against ischemia/reperfusion damage, intoxication by acetaminophen and concanavalin A, and nonalcoholic steatohepatitis caused by methionine/choline-deficient diet as well as against liver fibrosis induced by bile duct ligation or carbon tetrachloride. α-DBI downregulated proinflammatory and profibrotic genes and upregulated antioxidant defenses and fatty acid oxidation in the liver. The hepatoprotective effects of α-DBI were mimicked by the induction of ACBP/DBI-specific autoantibodies, an inducible Acbp/Dbi knockout or a constitutive Gabrg2F77I mutation that abolishes ACBP/DBI binding to the GABAA receptor. Liver-protective α-DBI effects were lost when autophagy was pharmacologically blocked or genetically inhibited by knockout of Atg4b. Of note, α-DBI also reduced myocardium infarction and lung fibrosis, supporting the contention that it mediates broad organ-protective effects against multiple insults.

Choline degradation in Paracoccus denitrificans: identification of sources of formaldehyde.

Paracoccus denitrificans is a facultative methylotroph that can grow on methanol and methylamine as sole sources of carbon and energy. Both are oxidized to formaldehyde and then to formate, so growth on C1 substrates induces the expression of genes encoding enzymes required for the oxidation of formaldehyde and formate. This induction involves a histidine kinase response regulator pair (FlhSR) that is likely triggered by formaldehyde. Catabolism of some complex organic substrates (e.g., choline and L-proline betaine) also generates formaldehyde. Thus, flhS and flhR mutants that fail to induce expression of the formaldehyde catabolic enzymes cannot grow on methanol, methylamine, and choline. Choline is oxidized to glycine via glycine betaine, dimethylglycine, and sarcosine. By exploring flhSR growth phenotypes and the activities of a promoter and enzyme known to be upregulated by formaldehyde, we identify the oxidative demethylations of glycine betaine, dimethylglycine, and sarcosine as sources of formaldehyde. Growth on glycine betaine, dimethylglycine, and sarcosine is accompanied by the production of up to three, two, and one equivalents of formaldehyde, respectively. Genetic evidence implicates two orthologous monooxygenases in the oxidation of glycine betaine. Interestingly, one of these appears to be a bifunctional enzyme that also oxidizes L-proline betaine (stachydrine). We present preliminary evidence to suggest that growth on L-proline betaine induces expression of a formaldehyde dehydrogenase distinct from the enzyme induced during growth on other formaldehyde-generating substrates.IMPORTANCEThe bacterial degradation of one-carbon compounds (methanol and methylamine) and some complex multi-carbon compounds (e.g., choline) generates formaldehyde. Formaldehyde is toxic and must be removed, which can be done by oxidation to formate and then to carbon dioxide. These oxidations provide a source of energy; in some species, the CO2 thus generated can be assimilated into biomass. Using the Gram-negative bacterium Paracoccus denitrificans as the experimental model, we infer that oxidation of choline to glycine generates up to three equivalents of formaldehyde, and we identify the three steps in the catabolic pathway that are responsible. Our work sheds further light on metabolic pathways that are likely important in a variety of environmental contexts.