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Obtaining the necessary molybdenum cofactor for sulfite oxidase activity in the nematode C. elegans surprisingly involves a dietary source

  • Author Footnotes
    + These authors contributed equally to this work.
    Kevin D. Oliphant
    Footnotes
    + These authors contributed equally to this work.
    Affiliations
    Department of Plant Biology, Braunschweig University of Technology, 38106 Braunschweig, Germany
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  • Author Footnotes
    + These authors contributed equally to this work.
    Robin R. Fettig
    Footnotes
    + These authors contributed equally to this work.
    Affiliations
    Pediatrics and Rare Diseases Group, Sanford Research, Sioux Falls, SD 57104, USA

    Department of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, SD 57069, USA
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  • Author Footnotes
    + These authors contributed equally to this work.
    Jennifer Snoozy
    Footnotes
    + These authors contributed equally to this work.
    Affiliations
    Pediatrics and Rare Diseases Group, Sanford Research, Sioux Falls, SD 57104, USA
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  • Ralf R. Mendel
    Affiliations
    Department of Plant Biology, Braunschweig University of Technology, 38106 Braunschweig, Germany
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  • Kurt Warnhoff
    Correspondence
    Corresponding author: Kurt Warnhoff,
    Affiliations
    Pediatrics and Rare Diseases Group, Sanford Research, Sioux Falls, SD 57104, USA

    Department of Pediatrics, Sanford School of Medicine, University of South Dakota, Sioux Falls, SD 57105, USA
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  • Author Footnotes
    + These authors contributed equally to this work.
Open AccessPublished:November 21, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102736

      Abstract

      Molybdenum cofactor (Moco) is a prosthetic group necessary for the activity of four unique enzymes, including the essential sulfite oxidase (SUOX-1). Moco is required for life; humans with inactivating mutations in the genes encoding Moco-biosynthetic enzymes display Moco deficiency, a rare and lethal inborn error of metabolism. Despite its importance to human health, little is known about how Moco moves among and between cells, tissues, and organisms. The prevailing view is that cells that require Moco must synthesize Moco de novo. Although, the nematode Caenorhabditis elegans appears to be an exception to this rule and has emerged as a valuable system for understanding fundamental Moco biology. C. elegans has the seemingly unique capacity to both synthesize its own Moco as well as acquire Moco from its microbial diet. However, the relative contribution of Moco from the diet or endogenous synthesis has not been rigorously evaluated or quantified biochemically. We genetically removed dietary or endogenous Moco sources in C. elegans and biochemically determined their impact on animal Moco content and SUOX-1 activity. We demonstrate that dietary Moco deficiency dramatically reduces both animal Moco content and SUOX-1 activity. Furthermore, these biochemical deficiencies have physiological consequences; we show that dietary Moco deficiency alone causes sensitivity to sulfite, the toxic substrate of SUOX-1. Altogether, this work establishes the biochemical consequences of depleting dietary Moco or endogenous Moco synthesis in C. elegans and quantifies the surprising contribution of the diet to maintaining Moco homeostasis in C. elegans.

      Keywords

      Introduction

      Molybdenum cofactor (Moco) is a prosthetic group that was present in the last universal common ancestor and continues to be synthesized in all domains of life by a conserved biosynthetic pathway (Fig. 1) (
      • Weiss M.C.
      • Sousa F.L.
      • Mrnjavac N.
      • Neukirchen S.
      • Roettger M.
      • Nelson-Sathi S.
      • Martin W.F.
      The physiology and habitat of the last universal common ancestor.
      ,
      • Zhang Y.
      • Gladyshev V.N.
      Molybdoproteomes and evolution of molybdenum utilization.
      ). Moco supports the activity of 4 animal enzymes: sulfite oxidase, xanthine dehydrogenase, aldehyde oxidase, and mitochondrial amidoxime reducing component (
      • Schwarz G.
      • Mendel R.R.
      • Ribbe M.W.
      Molybdenum cofactors, enzymes and pathways.
      ,
      • Mendel R.R.
      The molybdenum cofactor.
      ). Thus, Moco is essential to support core metabolic pathways such as sulfur amino acid and purine metabolism.
      Figure thumbnail gr1
      Figure 1Intersection of Moco biosynthesis and sulfur amino acid metabolism in C. elegans. The C. elegans Moco biosynthetic and sulfur amino acid metabolism (simplified) pathways are displayed (enzymes, red). Structures of the following Moco-biosynthetic intermediates are displayed: guanosine triphosphate (GTP), cyclic pyranopterin monophosphate (cPMP), molybdopterin (MPT), molybdopterin adenosine monophosphate (MPT-AMP), and molybdenum cofactor (Moco).
      Moco was initially revealed in the 1960s through elegant genetic studies in the fungus Aspergillus nidulans (
      • Cove D.J.
      • Pateman J.A.
      Independently segregating genetic loci concerned with nitrate reductase activity in Aspergillus nidulans.
      ,
      • Pateman J.A.
      • Cove D.J.
      • Rever B.M.
      • Roberts D.B.
      A COMMON CO-FACTOR FOR NITRATE REDUCTASE AND XANTHINE DEHYDROGENASE WHICH ALSO REGULATES THE SYNTHESIS OF NITRATE REDUCTASE.
      ). Chemical mutagenesis screens were performed, leading to the isolation of mutants defective in both nitrate reductase and xanthine dehydrogenase activity. These mutants affected multiple genetic loci outlining the genes necessary for synthesizing CNX, a cofactor common to nitrate reductase and xanthine dehydrogenase (
      • Pateman J.A.
      • Cove D.J.
      • Rever B.M.
      • Roberts D.B.
      A COMMON CO-FACTOR FOR NITRATE REDUCTASE AND XANTHINE DEHYDROGENASE WHICH ALSO REGULATES THE SYNTHESIS OF NITRATE REDUCTASE.
      ). CNX is now known as Moco.
      Paralleling the genetic understanding of Moco deficiency in A. nidulans, human Moco deficiency was first documented in 1978 as a combined deficiency of xanthine dehydrogenase and sulfite oxidase, two Moco-requiring enzymes (
      • Duran M.
      • Beemer F.A.
      • van de Heiden C.
      • Korteland J.
      • de Bree P.K.
      • Brink M.
      • Wadman S.K.
      • Lombeck I.
      Combined deficiency of xanthine oxidase and sulphite oxidase: a defect of molybdenum metabolism or transport?.
      ). Causative genetic lesions in human patients affected the orthologous biosynthetic pathway identified initially in A. nidulans. Patients presented with a severe range of symptoms as neonates, including difficulty feeding, neurological dysfunction, and eye lens dislocation. Symptoms typically appear within the first week of life, and patients generally die within three years of birth (
      • Spiegel R.
      • Schwahn B.C.
      • Squires L.
      • Confer N.
      Molybdenum cofactor deficiency: A natural history.
      ). Excitingly, a therapy has been developed to treat a subset of patients suffering from Moco deficiency (
      • Veldman A.
      • Santamaria-Araujo J.A.
      • Sollazzo S.
      • Pitt J.
      • Gianello R.
      • Yaplito-Lee J.
      • Wong F.
      • Ramsden C.A.
      • Reiss J.
      • Cook I.
      • Fairweather J.
      • Schwarz G.
      Successful treatment of molybdenum cofactor deficiency type A with cPMP.
      ). Patients with defects in cPMP synthesis can receive supplemental cPMP, which is converted to Moco by the healthy downstream Moco biosynthetic machinery. Unfortunately, cPMP therapy is only effective for patients with mutations in the first step in Moco synthesis. Supplementation with mature Moco seems a logical therapy to treat all forms of Moco deficiency. However, the instability and sensitivity of Moco to oxidation have proven experimentally limiting and precluded it from therapeutic consideration. Moco is a unique pterin and its chemical nature was ultimately resolved by studying its stable degradation products, one of which is known as “FormA” (
      • Johnson J.L.
      • Hainline B.E.
      • Rajagopalan K.V.
      • Arison B.H.
      The pterin component of the molybdenum cofactor. Structural characterization of two fluorescent derivatives.
      ,
      • Kramer S.P.
      • Johnson J.L.
      • Ribeiro A.A.
      • Millington D.S.
      • Rajagopalan K.V.
      The structure of the molybdenum cofactor. Characterization of di-(carboxamidomethyl)molybdopterin from sulfite oxidase and xanthine oxidase.
      ).
      Given its unstable nature, very little is known about how Moco moves within and between cells. Most Moco-requiring cells are believed to synthesize Moco de novo. However, recent advances in the nematode Caenorhabditis elegans have demonstrated that Moco can move not only between cells and tissues but between disparate organisms (
      • Snoozy J.
      • Breen P.C.
      • Ruvkun G.
      • Warnhoff K.
      moc-6/MOCS2A is necessary for molybdenum cofactor synthesis in C. elegans.
      ,
      • Warnhoff K.
      • Hercher T.W.
      • Mendel R.R.
      • Ruvkun G.
      Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans.
      ,
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ). Unlike other animals studied, C. elegans with null mutations in genes encoding the Moco-biosynthetic enzymes are viable and fertile. These genes are termed moc in C. elegans for molybdenum cofactor biosynthesis. moc mutant animals are viable because C. elegans stably acquires mature Moco from its bacterial diet. This dietary Moco is stabilized by Moco-binding/using proteins and is transported by unknown mechanisms throughout the C. elegans animal to support the activity of its Moco-requiring enzymes (
      • Warnhoff K.
      • Hercher T.W.
      • Mendel R.R.
      • Ruvkun G.
      Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans.
      ). Unsurprisingly, Moco is essential for C. elegans viability: animals lacking both endogenous Moco synthesis and dietary Moco arrest during larval development and die. This lethality is caused by the inactivity of sulfite oxidase (SUOX-1), a Moco-requiring enzyme that oxidizes the lethal toxin sulfite to sulfate (
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ). Sulfite oxidase is also essential in humans. Patients with inactivating mutations in sulfite oxidase develop clinical and biochemical features that mirror Moco deficiency, highlighting that SUOX-1 is likely the key Moco-requiring enzyme supporting animal life (
      • Duran M.
      • Beemer F.A.
      • van de Heiden C.
      • Korteland J.
      • de Bree P.K.
      • Brink M.
      • Wadman S.K.
      • Lombeck I.
      Combined deficiency of xanthine oxidase and sulphite oxidase: a defect of molybdenum metabolism or transport?.
      ,
      • Mudd S.H.
      • Irreverre F.
      • Laster L.
      Sulfite oxidase deficiency in man: demonstration of the enzymatic defect.
      ).
      We reason that dietary Moco must enter the C. elegans animal through the intestine. However, the primary tissue responsible for Moco synthesis and use seems to be the hypodermis, a distal tissue. Tissue-specific expression of the moc-1 gene, encoding a Moco biosynthesis enzyme (Fig. 1), strongly rescues moc-1(-) null mutant phenotypes when transcribed from a hypodermal-specific promoter. moc-1 expression in muscle cells also rescues moc-1(-) mutant phenotypes, albeit to a lesser extent (
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ). Furthermore, only hypodermal expression of suox-1 was sufficient to rescue the lethality of a suox-1(-) null mutant animal (
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ). These results suggest Moco is both synthesized and acting primarily in the C. elegans hypodermis. Considering these findings, the functionality of dietary Moco is even more impressive: dietary Moco must travel from the environment, through the intestine, and be distributed to the hypodermis where it ultimately supports SUOX-1 activity. This journey likely involves the stable crossing of multiple cell membranes via an unknown mechanism.
      Here, we adapt biochemical protocols to quantify the contribution of dietary Moco vs. endogenously synthesized Moco. We use genetic strategies to remove either source of Moco and evaluate the impact on i) the activity of the Moco-requiring SUOX-1 enzyme and ii) C. elegans Moco content found in crude C. elegans extracts. Furthermore, we evaluate the physiological impact of removing dietary Moco vs. endogenously synthesized Moco by examining growth and development under pharmacological and genetic conditions where sulfite concentrations are toxic to animal growth. We demonstrate that dietary Moco and endogenously synthesized Moco are non-redundantly promoting Moco accumulation and SUOX-1 activity. Furthermore, removing either source of Moco causes sulfite sensitivity, indicating that both dietary Moco and endogenous Moco synthesis are required to promote optimal C. elegans fitness.

      Results

      Biochemical quantification of C. elegans SUOX-1 activity and Moco content:

      To biochemically evaluate C. elegans Moco homeostasis, we generated crude extracts from large cultures of young-adult animals. Animals were cultured on solid agar media seeded with a monoculture of E. coli, the food source of C. elegans cultivated in the laboratory. Young-adult animals were collected, extensively washed to remove E. coli cells, and then snap-frozen in liquid nitrogen. Samples were subsequently lysed using a bead beater, and protein concentrations were determined via Bradford protein assay to allow for downstream normalization.
      These crude extracts were then used to determine SUOX-1 activity and Moco content. We first analyzed samples that we anticipated would have maximal and minimal SUOX-1 activity and Moco content to validate these approaches. We reasoned that wild-type C. elegans fed wild-type (Moco+) E. coli would have high levels of SUOX-1 activity and Moco as both endogenous Moco synthesis and dietary Moco uptake are functional in this context. In contrast, we used the moc-4; cdo-1 double mutant C. elegans fed ΔmoaA mutant (Moco-) E. coli as the sample where we anticipated little to no SUOX-1 activity or Moco content (
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ). To validate that wild-type E. coli are Moco replete and ΔmoaA mutant E. coli are Moco deficient, we performed high-pressure liquid chromatography (HPLC) analyses and observed Moco content as detected by the fluorescent derivative dephospho-FormA (dpFormA) (
      • Johnson J.L.
      • Hainline B.E.
      • Rajagopalan K.V.
      • Arison B.H.
      The pterin component of the molybdenum cofactor. Structural characterization of two fluorescent derivatives.
      ). Indeed, we detected a strong dpFormA peak when analyzing wild-type E. coli and did not detect a peak in ΔmoaA mutant E. coli (Fig. S1). moc-4; cdo-1 double mutant C. elegans cannot synthesize their own Moco due to the null mutation in the moc-4 gene that encodes molybdopterin synthase (Fig. 1). moc-4 mutant animals typically display 100% penetrant larval arrest and death when cultured on Moco- E. coli. However, this lethality can be suppressed by mutations in cdo-1, which encodes cysteine dioxygenase (
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ). CDO-1 is a critical enzyme in the catabolism of sulfur amino acids and promotes the production of sulfites, the critical toxin when Moco is absent (Fig. 1) (
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ,
      • Gunnison A.F.
      Sulphite toxicity: a critical review of in vitro and in vivo data.
      ,
      • Bailey J.L.
      • Cole R.D.
      Studies on the reaction of sulfite with proteins.
      ,
      • Würfel M.
      • Häberlein I.
      • Follmann H.
      Inactivation of thioredoxin by sulfite ions.
      ). cdo-1 inactivation prevents the accumulation of sulfites and allows C. elegans animals to grow in the absence of endogenous and dietary Moco sources. Thus, we speculated that moc-4; cdo-1 double mutant animals cultured on Moco- E. coli would be completely Moco deficient and lack SUOX-1 activity.
      To determine SUOX-1 activity of these samples, we modified an assay based on a photometrically quantifiable reduction of cytochrome c (Fig. 2A) (
      • Cohen H.J.
      • Betcher-Lange S.
      • Kessler D.L.
      • Rajagopalan K.V.
      Hepatic sulfite oxidase. Congruency in mitochondria of prosthetic groups and activity.
      ). In this reaction, sulfite is converted to sulfate, and cytochrome c acts as an electron donor. The resulting reduced form of cytochrome c has an increased absorption at OD550, which we detect and quantify. Aligning with our expectations, we readily detected SUOX-1 activity from extracts of wild-type C. elegans fed Moco+ E. coli (0.33 U/mg), while we were unable to detect SUOX-1 activity from moc-4; cdo-1 double mutant animals fed Moco- E. coli (Fig. 2B,C). moc-6 encodes another component of the Moco-biosynthetic machinery and is necessary for C. elegans Moco synthesis (Fig. 1) (
      • Snoozy J.
      • Breen P.C.
      • Ruvkun G.
      • Warnhoff K.
      moc-6/MOCS2A is necessary for molybdenum cofactor synthesis in C. elegans.
      ). Supporting our findings with moc-4; cdo-1 animals, we were unable to detect SUOX-1 activity in crude extracts from moc-6; cdo-1 double mutant animals fed Moco- E. coli (Fig. 2C). These results align well with prior genetic data and validate this assay to quantify SUOX-1 activity from crude C. elegans extracts (
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ).
      Figure thumbnail gr2
      Figure 2Quantifying SUOX-1 activity in crude extracts from C. elegans. A) Simplified reaction mechanism for SUOX-1 by which sulfite (SO32-) is oxidized to sulfate (SO42-). Sulfite-dependent SUOX-1 activity is detected via the concomitant reduction of cytochrome c. B) SUOX-1 activity was detected in crude extracts from wild-type C. elegans fed wild-type (Moco+) E. coli (black) or moc-4(ok2571); cdo-1(mg622) mutant C. elegans fed ΔmoaA (Moco-) E. coli (red). Units of SUOX-1 activity per mg of protein are displayed. C) SUOX-1 activity is displayed for wild-type and mutant C. elegans fed either wild-type (Moco+) or ΔmoaA (Moco-) E. coli. All biological replicates, the sample size, mean, and standard deviation are displayed for each condition. N/D indicates no SUOX-1 activity was detected in any sample. ****, p<0.0001, ordinary one-way ANOVA with Dunnet’s post-hoc analysis. Note, the wild-type data in C are also displayed in A, A, and A, to allow for appropriate graphical comparisons of data.
      To determine Moco content of these same samples, crude extracts were oxidized and subsequently treated with alkaline phosphatase. Iodine-dependent oxidation and dephosphorylation convert highly-unstable Moco and MPT to the stable and fluorescent derivative, dephospho-FormA (dpFormA) (Fig. 3A) (
      • Johnson J.L.
      • Hainline B.E.
      • Rajagopalan K.V.
      • Arison B.H.
      The pterin component of the molybdenum cofactor. Structural characterization of two fluorescent derivatives.
      ). Oxidized metabolites were then analyzed via HPLC (
      • Hercher T.W.
      • Krausze J.
      • Hoffmeister S.
      • Zwerschke D.
      • Lindel T.
      • Blankenfeldt W.
      • Mendel R.R.
      • Kruse T.
      Insights into the Cnx1E catalyzed MPT-AMP hydrolysis.
      ). dpFormA was detected with an average elution time of 5:40 minutes and quantified using established methods and standards (
      • Klewe A.
      • Kruse T.
      • Lindel T.
      Aminopyrazine Pathway to the Moco Metabolite Dephospho Form A.
      ). Supporting our genetic hypotheses, we readily detected dpFormA in crude extracts of wild-type C. elegans fed Moco+ E. coli (1.3 pmol/mg) and were not able to detect Moco in moc-4; cdo-1 mutant animals fed Moco- E. coli (Fig. 3B,C). We also analyzed crude extracts from moc-6; cdo-1 double mutant animals fed Moco- E. coli and were unable to detect Moco-derived dpFormA (Fig. 3C). To further validate that the peak highlighted in Fig. 3B is dpFormA, the same samples were prepped and analyzed without alkaline phosphatase treatment. To be detected on a reversed-phase column, FormA must be dephosphorylated, or it will not interact with the solid phase and elute in the void volume. Without alkaline phosphatase treatment, we did not see the highlighted peak in any of our samples, supporting our claim that this peak is dpFormA (Fig. S2). These results align well with expectations and validate this assay for quantifying Moco content from crude C. elegans extracts.
      Figure thumbnail gr3
      Figure 3Quantifying Moco content in crude extracts from C. elegans. A) A schematic of the conversion of Moco to dephospho-FormA (dpFormA) in the presence of an acidic iodine environment and treatment with alkaline phosphatase. B) HPLC measurements of Moco-derived dpFormA from crude extracts of wild-type C. elegans fed wild-type (Moco+) E. coli (black) or moc-4(ok2571); cdo-1(mg622) mutant C. elegans fed ΔmoaA (Moco-) E. coli (blue). The dpFormA peak is indicated (black arrow). C) dpFormA content is displayed for wild-type, and mutant C. elegans fed either wild-type (Moco+) or ΔmoaA (Moco-) E. coli. All biological replicates, the sample size, mean, and standard deviation are displayed for each condition. N/D indicates no dpFormA was detected in any sample. ****, p<0.0001, ordinary one-way ANOVA with Dunnet’s post-hoc analysis. Note, the wild-type data in C are also displayed in B and B to allow for appropriate graphical comparisons of data.

      Dietary Moco is necessary to promote SUOX-1 activity and Moco content in C. elegans:

      Having established new methodology for quantifying SUOX-1 activity and Moco content in C. elegans, we sought to address some fundamental questions regarding Moco homeostasis. Principally, how much of the total Moco content of C. elegans is derived from the diet as compared to endogenous synthesis? To address this question, we generated C. elegans samples where we deprived the animals of either i) dietary Moco (wild-type C. elegans fed Moco- E. coli) or ii) endogenous Moco synthesis (moc-4 mutant C. elegans fed Moco+ E. coli). Extracts were generated from these samples, and SUOX-1 activity and Moco content were determined.
      Wild-type C. elegans fed Moco- E. coli displayed only 11% SUOX-1 activity compared to wild-type C. elegans fed a Moco+ diet (Fig. 4A). This surprising result demonstrates that dietary Moco is necessary to promote SUOX-1 activity in wild-type C. elegans. Similar results were observed when we analyzed the same samples for Moco content. Wild-type C. elegans fed Moco- E. coli displayed only 18% Moco content when compared to the same animals fed a Moco+ diet (Fig. 4B). These data demonstrate that dietary Moco is necessary to promote Moco accumulation in wild-type C. elegans.
      Figure thumbnail gr4
      Figure 4Dietary Moco is necessary to promote SUOX-1 activity and Moco accumulation. A) SUOX-1 activity and B) dpFormA content are displayed for wild-type C. elegans fed either wild-type (Moco+) or ΔmoaA (Moco-) E. coli. All biological replicates, the sample size, mean, and standard deviation are displayed for each condition. ****, p<0.0001, Welch’s t test.

      C. elegans Moco biosynthesis is necessary to promote SUOX-1 activity and Moco content:

      Given the critical role of dietary Moco in supporting SUOX-1 activity and animal Moco content, we wondered about the relative importance of endogenous Moco synthesis. To evaluate this, we cultured moc-4 mutant C. elegans on Moco+ E. coli, restricting the animal’s Moco source to the diet. We generated crude extracts from these samples and determined SUOX-1 activity and Moco content. moc-4 mutant animals had 28% of the SUOX-1 activity displayed by their wild-type counterparts and 64% of the Moco content (Fig. 5). We saw similar results when evaluating moc-6 mutant animals fed Moco+ E. coli which displayed 39% of wild-type SUOX-1 activity and 65% of Moco content (Fig. 5). These data demonstrate that endogenous Moco synthesis is necessary to promote SUOX-1 activity and Moco accumulation in C. elegans.
      Figure thumbnail gr5
      Figure 5Endogenous Moco synthesis is necessary to promote SUOX-1 activity and Moco accumulation. A) SUOX-1 activity and B) dpFormA content are displayed for wild-type, moc-4, and moc-6 mutant C. elegans fed wild-type (Moco+) E. coli. All biological replicates, the sample size, mean, and standard deviation are displayed for each condition. *, p<0.05 and ****, p<0.0001, ordinary one-way ANOVA with Dunnet’s post-hoc analysis.

      The C. elegans suox-1(gk738847) mutation causes reduced SUOX-1 activity and sulfite sensitivity:

      SUOX-1 is an essential enzyme in both C. elegans and humans (
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ,
      • Mudd S.H.
      • Irreverre F.
      • Laster L.
      Sulfite oxidase deficiency in man: demonstration of the enzymatic defect.
      ). To evaluate the role of suox-1 in C. elegans biology, we use the hypomorphic suox-1(gk738847) allele as a genetic tool as it reduces suox-1 function but does not cause overt developmental phenotypes (
      • Warnhoff K.
      • Hercher T.W.
      • Mendel R.R.
      • Ruvkun G.
      Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans.
      ,
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ,
      • Thompson O.
      • Edgley M.
      • Strasbourger P.
      • Flibotte S.
      • Ewing B.
      • Adair R.
      • Au V.
      • Chaudhry I.
      • Fernando L.
      • Hutter H.
      • Kieffer A.
      • Lau J.
      • Lee N.
      • Miller A.
      • Raymant G.
      • Shen B.
      • Shendure J.
      • Taylor J.
      • Turner E.H.
      • Hillier L.W.
      • Moerman D.G.
      • Waterston R.H.
      The million mutation project: a new approach to genetics in Caenorhabditis elegans.
      ). gk738847 is a missense mutation resulting in the amino acid substitution D391N. Aspartic acid 391 is highly conserved from C. elegans to humans (
      • Altschul S.F.
      • Gish W.
      • Miller W.
      • Myers E.W.
      • Lipman D.J.
      Basic local alignment search tool.
      ). To characterize the impact of the D391N substitution to SUOX-1 activity, we prepared crude extracts from young adult wild-type and suox-1(gk738847) animals cultured on Moco+ E. coli. SUOX-1 activity of these extracts was then evaluated. Our data demonstrate that suox-1(gk738847) mutant animals displayed 4% SUOX-1 activity compared to their wild-type counterparts (Fig. 6A). Despite this dramatic reduction in SUOX-1 activity, suox-1(gk738847) mutant animals appear superficially wild type when cultured under standard laboratory conditions. To probe for fitness defects caused by reduced SUOX-1 activity, we cultured wild-type and suox-1(gk738847) animals on Moco+ bacteria exposed to various concentrations of supplemental sulfite. Wild-type C. elegans fed Moco+ bacteria tolerate high sulfite well, displaying a half-maximal inhibitory concentration (IC50) of 0.0077M supplemental sulfite. In contrast, suox-1(gk738847) mutant animals are sensitive to sulfite displaying an IC50 of 0.0024M supplemental sulfite (Fig. 6B,D). These results align well with previous studies (
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ). Taken together, these data biochemically demonstrate the effect of the suox-1(gk738847) D391N mutation on the activity of SUOX-1 in crude C. elegans extracts. Furthermore, we show that suox-1 is essential for tolerating high supplemental sulfite and establish a sulfite-sensitivity assay for detecting physiological outcomes of reduced SUOX-1 activity.
      Figure thumbnail gr6
      Figure 6Dietary Moco and endogenous Moco synthesis are non-redundantly required for sulfite tolerance. A) SUOX-1 activity is displayed for wild-type and suox-1(gk738847) mutant C. elegans fed wild-type (Moco+) E. coli. All biological replicates, the sample size, mean, and standard deviation are displayed for each condition. ****, p<0.0001, Welch’s t test. B,C) Wild-type and mutant C. elegans were synchronized at L1 stage and cultured on wild-type (Moco+) or ΔmoaA mutant (Moco-) E. coli supplemented with various concentrations of sulfite (0, 0.0001, 0.001, 0.0025, 0.005, 0.01, and 0.02M). Animal length was measured after 72 hours of growth at 20°C. Data points represent the average of 3 (suox-1, Moco+ (purple), and moc-4, Moco+ (blue)) or 4 (wild-type, Moco+ (black), and wild type, Moco- (red)) biological replicates. For each biological replicate, 15 or more individual C. elegans animals were imaged and measured at each sulfite concentration. Data points indicate mean and error bars display standard deviation. IC50 was calculated by non-linear regression analyses and shading indicates the 95% confidence interval. IC50 and R-squared values are displayed. Note that the data displayed for wild-type C. elegans fed Moco+ E. coli in B are identical to the same control displayed in C. D) Representative individuals are displayed at the critical 0.005M supplemental sulfite concentration. Scale bar is 250μm. Yellow arrowhead indicates embryos in the uterus of the gravid adult animal.

      Dietary Moco and endogenous Moco synthesis are non-redundantly required for sulfite tolerance:

      Our biochemical analyses demonstrate that wild-type C. elegans fed a diet of Moco- E. coli show decreased SUOX-1 activity (Fig. 4A). Like suox-1(gk738847) mutant animals, wild-type C. elegans provided Moco- E. coli appear superficially healthy under standard culture conditions (
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ). We hypothesized that the reductions in SUOX-1 activity caused by a Moco- diet would cause sulfite sensitivity. To test this, we exposed wild-type C. elegans cultured on Moco+ or Moco- E. coli to various concentrations of supplemental sulfite and analyzed growth and development. Wild-type C. elegans fed Moco+ E. coli tolerate sulfite well and display an IC50 of 0.0077M supplemental sulfite. By contrast, wild-type animals fed Moco- E. coli were sensitive to supplemental sulfite, displaying an IC50 of 0.0024M supplemental sulfite (Fig. 6C,D). These data demonstrate that dietary Moco is necessary for sulfite tolerance in C. elegans.
      Given the requirement of dietary Moco to support sulfite tolerance, we wondered if endogenous Moco synthesis would also be essential for this process. To test the role of endogenously synthesized Moco in sulfite tolerance, we analyzed moc-4 null mutant C. elegans that cannot synthesize Moco (
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ). We exposed moc-4 mutant C. elegans cultured on Moco+ E. coli to various concentrations of supplemental sulfite and analyzed their growth and development. When compared to wild-type animals fed Moco+ bacteria, we found that moc-4 mutant C. elegans grown on Moco+ E. coli were sensitive to supplemental sulfite, displaying an IC50 of 0.0044M supplemental sulfite (Fig. 6C,D). Taken together, these data demonstrate that dietary Moco and endogenous Moco synthesis are non-redundantly required for C. elegans to tolerate high environmental sulfite.

      Dietary Moco is essential for the development of suox-1(gk738847) mutant C. elegans:

      Our biochemical analyses of Moco content and SUOX-1 activity paired with our pharmacological experiments with supplemental sulfite suggest a model for Moco homeostasis whereby endogenous synthesis and dietary acquisition of Moco are acting non-redundantly to promote healthy Moco content in C. elegans, thus promoting SUOX-1 activity. To further test this model, we used the suox-1(gk738847) allele as a sensitized genetic background. We observed the development of these mutant animals when we altered i) dietary Moco availability and ii) endogenous Moco synthesis.
      To test the role dietary Moco plays in supporting the development of suox-1(gk738847) mutant C. elegans, we cultured suox-1(gk738847) mutant animals on Moco+, Moco-, and mixtures of Moco+/Moco- E. coli. suox-1(gk738847) mutant animals grew well on Moco+ E. coli and 50:50 mixtures of Moco+ and Moco- E. coli, reaching fertile adulthood within 72 hours of development post first larval (L1) stage (Fig. 7). However, we observe dose-dependent developmental delay in suox-1(gk738847) mutant animals at reduced fractions of Moco+ E. coli (IC50 of 0.06 Moco+ E. coli in Moco- E. coli, Fig. S3A). Furthermore, suox-1(gk738847) animals displayed the most substantial reduction in growth and development when cultured on completely Moco- E. coli, failing to progress through larval development within the 72-hour assay (Fig. 7, Fig. S3A). Importantly, wild-type C. elegans reached fertile adulthood in our developmental assays on all mixtures of Moco+ and Moco- E. coli (Fig. S3B). These data align well with our previous studies and demonstrate that dietary Moco is required for the growth and development of suox-1(gk738847) mutant animals (
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ).
      Figure thumbnail gr7
      Figure 7Dietary Moco is essential for C. elegans development when suox-1 activity is compromised. A) suox-1(gk738847) mutant C. elegans were synchronized at the L1 stage and cultured on mixtures of wild-type (Moco+) and ΔmoaA mutant (Moco-) E. coli. Animal length was measured after 72 hours of growth at 20°C. Box plots display the median, upper, and lower quartiles, while whiskers indicate minimum and maximum data points. Sample size is 15 individuals per experiment. ****, p<0.0001, ordinary one-way ANOVA with Tukey’s post-hoc analysis. B) Representative individuals from A are displayed. Scale bar is 250μm. Yellow arrowheads indicate embryos in the uterus of gravid adult animals.

      Endogenous Moco synthesis is essential for the development of suox-1(gk738847) mutant C. elegans:

      We then sought to test the role of endogenous Moco synthesis in supporting the growth and development of suox-1(gk738847) mutant C. elegans. To test this, we attempted to engineer double mutant strains of C. elegans by combining the suox-1(gk738847) allele with various mutations in C. elegans Moco-biosynthetic enzymes (moc-1(ok366), moc-4(ok2571), moc-5(mg589), and moc-6(rae296)) (
      • Snoozy J.
      • Breen P.C.
      • Ruvkun G.
      • Warnhoff K.
      moc-6/MOCS2A is necessary for molybdenum cofactor synthesis in C. elegans.
      ,
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ). In our attempts to construct these double mutants, we were unable to isolate viable double mutant strains. Furthermore, dead larvae were observed in all stages of mutant construction where we would have expected the double mutant to emerge. Thus, we hypothesized a synthetic lethal interaction between mutations in moc genes and the suox-1(gk738847) hypomorphic allele. To test this hypothesis, we constructed a strain where suox-1(gk738847) was homozygous, and the moc-4(ok2571) allele was balanced by the tmC18 balancer (
      • Dejima K.
      • Hori S.
      • Iwata S.
      • Suehiro Y.
      • Yoshina S.
      • Motohashi T.
      • Mitani S.
      An Aneuploidy-Free and Structurally Defined Balancer Chromosome Toolkit for Caenorhabditis elegans.
      ). Given this strain, we could synchronize animals at the L1 stage and unequivocally evaluate the growth and development of moc-4(-); suox-1(gk738847), and moc-4(+)/moc-4(-); suox-1(gk738847) animals and compare their growth to suox-1(gk738847) single mutant animals. These mutant C. elegans were fed Moco+ E. coli throughout the experiment. Consistent with our earlier failed efforts to generate moc; suox-1(gk738847) double mutant animals, moc-4(-); suox-1(gk738847) double mutant C. elegans derived from the balancer strain grew extremely slowly, did not reach fertile adulthood, and displayed larval lethality (Fig. 8). By contrast, moc-4(+)/moc-4(-); suox-1(gk738847) and suox-1(gk738847) mutant animals grew well, reaching fertile adulthood within 72 hours of development post L1 stage (Fig. 8). These data demonstrate that when suox-1 activity is reduced via the gk738847 mutation, endogenous Moco synthesis is essential. These results support a model whereby dietary Moco and endogenous Moco synthesis are non-redundantly promoting Moco accumulation in C. elegans. Increased Moco content is likely directly promoting SUOX-1 activity, which promotes sulfite tolerance (Fig. 9).
      Figure thumbnail gr8
      Figure 8C. elegans Moco synthesis is essential when suox-1 activity is compromised. A) suox-1(gk738847), moc-4(+)/moc-4(ok2571); suox-1(gk738847), and moc-4(ok2571); suox-1(gk738847) mutant C. elegans were synchronized at the L1 stage and cultured on wild-type (Moco+) E. coli. Animal length was measured after 72 hours of growth at 20°C. Box plots display the median, upper, and lower quartiles, while whiskers indicate minimum and maximum data points. Sample size is 15 individuals per experiment. ****, p<0.0001, ordinary one-way ANOVA, with Tukey’s post-hoc analysis. Note, because moc-4; suox-1 double mutant animals are not viable, they (along with moc-4(+)/moc-4(-); suox-1 animals) were derived from the balanced strain USD1011 (see Experimental procedures) (
      • Dejima K.
      • Hori S.
      • Iwata S.
      • Suehiro Y.
      • Yoshina S.
      • Motohashi T.
      • Mitani S.
      An Aneuploidy-Free and Structurally Defined Balancer Chromosome Toolkit for Caenorhabditis elegans.
      ). B) Representative individuals from A are displayed. Scale bar is 250μm. Yellow arrowheads indicate embryos in the uterus of gravid adult animals.
      Figure thumbnail gr9
      Figure 9Moco homeostasis in C. elegans. In healthy control animals, wild-type C. elegans grown on wild-type E. coli (Moco+), SUOX-1 function and Moco content are high, leading to normal development and sulfite tolerance. SUOX-1 activity and Moco content are dramatically reduced when animals are fed a Moco- diet or when the endogenous synthesis of Moco is disrupted. These deficiencies result in sensitivity to sulfite (SO32-).

      Discussion

      Dietary E. coli are a major source of Moco for C. elegans:

      The free-living nematode C. elegans acquires Moco from its bacterial diet or by synthesizing it de novo from GTP. Under standard laboratory conditions, either source of Moco is sufficient to promote growth, development, and reproduction. Thus, when growth conditions are ideal, these pathways appear to operate redundantly to support life. However, this conclusion relies exclusively on measuring the rate of development and fertility (
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ).
      To expand our understanding of Moco homeostasis, we produced C. elegans extracts from animals lacking either endogenous Moco synthesis or dietary Moco. These extracts were used to quantify Moco content and SUOX-1 activity, providing biochemical clarity to our genetic system (
      • Snoozy J.
      • Breen P.C.
      • Ruvkun G.
      • Warnhoff K.
      moc-6/MOCS2A is necessary for molybdenum cofactor synthesis in C. elegans.
      ,
      • Warnhoff K.
      • Hercher T.W.
      • Mendel R.R.
      • Ruvkun G.
      Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans.
      ,
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ). We found that C. elegans mutants lacking endogenous Moco biosynthesis displayed reduced Moco content and SUOX-1 activity. moc-4 and moc-6 mutant C. elegans fed Moco+ E. coli showed 64% and 65% of Moco content and 28% and 39% SUOX-1 activity, respectively, compared to wild-type controls. These decreases were expected as Moco synthesis has long been understood to be an essential source of Moco for the cell.
      Surprisingly, we found even more severe defects when evaluating the biochemical impacts of a Moco-deficient diet. Wild-type C. elegans fed a Moco- diet displayed 18% of the Moco content and 11% of SUOX-1 activity compared to wild-type animals fed a Moco+ diet. This demonstrates that the C. elegans diet substantially contributes to Moco homeostasis in C. elegans animals. Thus, dietary Moco and endogenous Moco synthesis function non-redundantly to promote Moco content and SUOX-1 activity.
      Having demonstrated the biochemical impacts of either dietary or endogenous Moco loss, we wondered whether these deficiencies in Moco content or SUOX-1 activity would cause a fitness defect. To evaluate this, we employed sulfite as pharmacological intervention. Sulfite is a useful tool as it is a toxin and the primary substrate of SUOX-1. Sulfite is typically produced as a byproduct of sulfur amino acid metabolism (
      • Stipanuk M.H.
      Metabolism of Sulfur-Containing Amino Acids: How the Body Copes with Excess Methionine, Cysteine, and Sulfide.
      ). Thus, maintaining SUOX-1 activity is critical for sulfite detoxification and C. elegans survival. We observed that C. elegans lacking either source of Moco displayed sulfite sensitivity, demonstrating the non-redundant roles of dietary and endogenous Moco in promoting sulfite tolerance. Supporting this result, we found that both sources of Moco were also required for life in a suox-1(gk738847) hypomorphic mutant background.
      Given these results, we propose the model that when food is abundant, C. elegans animals rely on both endogenous Moco synthesis and dietary Moco to support Moco homeostasis (Fig. 9). While endogenous Moco synthesis has long been established to be critical for supporting Moco homeostasis, our work demonstrates the equal importance of dietary Moco in the life and survival of C. elegans.

      Are two sources of Moco better than one?

      Why would the C. elegans genome retain Moco biosynthetic machinery if the animals can acquire the cofactor so efficiently from the diet? Why is Moco not a vitamin for C. elegans, as is the case for other essential coenzymes (
      • Bito T.
      • Matsunaga Y.
      • Yabuta Y.
      • Kawano T.
      • Watanabe F.
      Vitamin B12 deficiency in Caenorhabditis elegans results in loss of fertility, extended life cycle, and reduced lifespan.
      ,
      • Watson E.
      • MacNeil L.T.
      • Ritter A.D.
      • Yilmaz L.S.
      • Rosebrock A.P.
      • Caudy A.A.
      • Walhout A.J.
      Interspecies systems biology uncovers metabolites affecting C. elegans gene expression and life history traits.
      ,
      • Rao A.U.
      • Carta L.K.
      • Lesuisse E.
      • Hamza I.
      Lack of heme synthesis in a free-living eukaryote.
      )? Logic dictates that there must be a fitness advantage to maintaining both strategies of increasing cellular Moco. We speculate that acquiring Moco from dietary microbes (when they are abundant) is energetically favorable compared to de novo Moco synthesis from GTP. However, endogenous Moco synthesis is likely a more reliable Moco source during the life of a wild C. elegans individual. This is because wild C. elegans have a “boom-and-bust” life cycle (
      • Frézal L.
      • Félix M.A.
      C. elegans outside the Petri dish.
      ). When conditions are ideal, newly hatched C. elegans pass through four larval stages and reach fertile adulthood in about three days. However, under stressful conditions (low food, high population density, high temperature), C. elegans enter an alternative non-feeding diapause state known as dauer (

      Hu, P. J. (2007) Dauer. WormBook, 1-19

      ). Dauer larvae are stress-resistant and can survive months as they disperse and seek new food. Wild C. elegans live the majority of their lives as non-feeding dauer larvae that are likely reliant on endogenous Moco synthesis (
      • Félix M.A.
      • Braendle C.
      The natural history of Caenorhabditis elegans.
      ). As dauer larvae locate food (usually microbes found on rotting fruit or plant stems) and return to favorable conditions, they resume development and reach fertile adulthood (

      Hu, P. J. (2007) Dauer. WormBook, 1-19

      ). During this “boom” time, dietary Moco would be abundant as ∼70% of bacterial genomes encode Moco biosynthetic enzymes (
      • Zhang Y.
      • Gladyshev V.N.
      Molybdoproteomes and evolution of molybdenum utilization.
      ). Yet, the food source will eventually be consumed, dauer larvae will emerge in the population, and the cycle will repeat. Considering this natural history, redundant sources of Moco seem logical. When no food is available for weeks/months, the animals must rely on endogenous Moco synthesis to survive. However, once a microbial food source is identified, C. elegans additionally harvest Moco from their diet, promoting Moco accumulation and enzymatic function within the animals.

      Moco bioavailability in C. elegans and beyond:

      Moco is an ancient and essential prosthetic group; Moco synthesizing and requiring enzymes were present in the last universal common ancestor and persist in all domains of life today (
      • Weiss M.C.
      • Sousa F.L.
      • Mrnjavac N.
      • Neukirchen S.
      • Roettger M.
      • Nelson-Sathi S.
      • Martin W.F.
      The physiology and habitat of the last universal common ancestor.
      ,
      • Zhang Y.
      • Gladyshev V.N.
      Molybdoproteomes and evolution of molybdenum utilization.
      ). Thus, it seems likely that Moco biology uncovered in the nematode C. elegans will be found throughout the tree of life. Our discovery that dietary Moco promotes Moco content, SUOX-1 activity, and rescues C. elegans Moco deficiency lays additional intellectual groundwork for developing Moco supplementation as a therapy to treat human Moco deficiency (
      • Warnhoff K.
      • Hercher T.W.
      • Mendel R.R.
      • Ruvkun G.
      Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans.
      ,
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ). However, uptake of exogenous Moco has yet to be examined or observed in another organism. It is crucial to determine if human cells are competent to acquire exogenous Moco as this is a critical next step in developing therapeutic Moco.
      In addition to therapeutic considerations, insights into Moco biology from the nematode C. elegans reveal many new fundamental questions about Moco biology: how is Moco moving from bacteria to C. elegans? How is Moco stably harvested from the exogenous donor proteins? Once harvested, how is Moco traversing cell membranes? Are there animal Moco chaperones facilitating these processes? Given the ancient and conserved nature of Moco, we expect pathways and phenomena uncovered in C. elegans to be at play across the diversity of life.

      Experimental procedures

      Animal cultivation

      C. elegans strains were cultured using established protocols (
      • Brenner S.
      The genetics of Caenorhabditis elegans.
      ). Briefly, animals were cultured at 20°C on nematode growth media (NGM) seeded with wild-type E. coli (OP50) unless otherwise noted. The wild-type C. elegans strain was Bristol N2. All mutant alleles and transgenes used in this study have been previously published.
      Additional C. elegans strains used in this work and their origins are described below:
      GR2253 [moc-4(ok2571) I] (
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ),
      FX30167 [tmC18 [dpy-5(tmIs1200)] I] (
      • Dejima K.
      • Hori S.
      • Iwata S.
      • Suehiro Y.
      • Yoshina S.
      • Motohashi T.
      • Mitani S.
      An Aneuploidy-Free and Structurally Defined Balancer Chromosome Toolkit for Caenorhabditis elegans.
      ),
      USD989 [moc-4(ok2571) I; cdo-1(mg622) X] was engineered for this study,
      USD1011 [moc-4(ok2571)/tmC18 [tmIs1236] I; suox-1(gk738847) X] was engineered for this study
      USD955 [moc-6(rae296) III] (
      • Snoozy J.
      • Breen P.C.
      • Ruvkun G.
      • Warnhoff K.
      moc-6/MOCS2A is necessary for molybdenum cofactor synthesis in C. elegans.
      ),
      USD959 [moc-6(rae296) III; cdo-1(mg622) X] (
      • Snoozy J.
      • Breen P.C.
      • Ruvkun G.
      • Warnhoff K.
      moc-6/MOCS2A is necessary for molybdenum cofactor synthesis in C. elegans.
      ),
      GR2256 [moc-5(mg589) X] (
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ),
      GR2254 [moc-1(ok366) X] (
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ),
      GR2260 [cdo-1(mg622) X] (
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ), and
      GR2269 [suox-1(gk738847) X] (
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ).
      To generate strains USD989 and USD1011, animals were mated using standard C. elegans husbandry and critical genetic loci were monitored as described here. The moc-4(ok2571) deletion was monitored by polymerase chain reaction using the following primers: 5’-gcagttatgaggcgaaggag-3’, 5’-tcgagcccactctttctctg-3’, and 5’-cgtcacacgagaatgcaaga-3’ (
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ). The cdo-1(mg622) single nucleotide polymorphism was monitored by PCR amplification and Sanger sequencing using the following primers: 5’-gcaaatgagtggcgaagatt-3’ and 5’-cgccgattgactaacctcat-3’ (
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ). The tmC18 [dpy-5(tmIs1200)] balancer was monitored by the expression of the dominant Pmyo-2::Venus fluorescent transgene in combination with a recessive dumpy (Dpy) phenotype caused by dpy-5(tmIs1200) (
      • Dejima K.
      • Hori S.
      • Iwata S.
      • Suehiro Y.
      • Yoshina S.
      • Motohashi T.
      • Mitani S.
      An Aneuploidy-Free and Structurally Defined Balancer Chromosome Toolkit for Caenorhabditis elegans.
      ).
      Additional E. coli strains used in this work were:
      BW25113 (wild type, Moco+) and
      JW0764-2 (ΔmoaA753::kan, Moco-).

      Protein and metabolite extraction from C. elegans:

      Synchronized populations of 10,000 to 20,000 C. elegans animals were cultured at 20°C and harvested at the young adult stage of development. To remove dietary bacterial cells from the sample, animals were washed in excess M9 buffer three times and then incubated in M9 buffer for 1 hour at 20°C with gentle rocking. Animals were subsequently washed one additional time with excess M9 buffer, pelleted, and flash frozen in liquid nitrogen. All samples were cultured on either BW25113 (wild type, Moco+) or JW0764-2 (ΔmoaA753::kan, Moco-) E. coli for the entire experiment. However, if moc-mutant C. elegans are cultured on ΔmoaA (Moco-) E. coli from early larval stages, they undergo larval arrest and death (
      • Snoozy J.
      • Breen P.C.
      • Ruvkun G.
      • Warnhoff K.
      moc-6/MOCS2A is necessary for molybdenum cofactor synthesis in C. elegans.
      ,
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ). To allow for biochemical analyses of moc-4 and moc-6 single mutant animals cultured on ΔmoaA (Moco-) E. coli, these mutant strains were initially cultured on Moco+ E. coli until the L4 stage of development (48 hours post L1 synchronization). These L4 larvae were then collected, extensively washed as described above, and re-introduced onto new culture dishes containing NGM seeded with Moco- E. coli. Thus, dietary Moco was depleted at a later stage in development, as previously described (Table S1) (
      • Snoozy J.
      • Breen P.C.
      • Ruvkun G.
      • Warnhoff K.
      moc-6/MOCS2A is necessary for molybdenum cofactor synthesis in C. elegans.
      ,
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ).
      For total protein and metabolite extraction, flash-frozen C. elegans samples were lysed in 400 μl of lysis buffer (20 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, pH 7.5) using a FastPrep-24 (M.P. Biomedicals Irvine, USA) four times for 30 seconds at 6.5 m/s with 5-minute breaks and then incubated for 30 minutes on ice. Protein concentrations were determined using a Bradford protein assay (Carl Roth Karlsruhe, Germany). Absorption was measured using a Multiskan GO microplate spectrophotometer (ThermoFisher Scientific Waltham, USA).

      Detection of sulfite oxidase activity:

      Sulfite oxidase activity measurements were based on a sulfite-dependent enzymatic reduction of cytochrome c (
      • Cohen H.J.
      • Betcher-Lange S.
      • Kessler D.L.
      • Rajagopalan K.V.
      Hepatic sulfite oxidase. Congruency in mitochondria of prosthetic groups and activity.
      ). 10 – 50 μg protein of total crude extract from C. elegans were used for quantification. 50 μl of each dilution were loaded into a 96-well plate. 180 μl of the SUOX-1 activity buffer (100 mM Tris-HCl, 0.1 mM EDTA, 0.04 mM cytochrome cox, pH 8.5) were added to the well. Subsequently, samples were incubated for 5 minutes on ice. The reaction was initiated by adding 20 μl of a 5 mM sodium sulfite solution. As a control, 20 μl ddH2O was added instead. Immediately after adding sulfite, an absorption change at 550 nm was measured in a Multiskan GO microplate spectrophotometer (ThermoFisher Scientific Waltham, USA). The detection was set over a time course of 60 min with 720 measurements. For visualization, the ddH2O control was subtracted from the sample with sulfite, which was used to plot a time-dependent OD change. The SUOX-1 activity was determined from the slope of the linear range of the curve. 1U is defined as the amount of protein required to reach an absorption increase of 1.0 per minute.

      Quantification of Moco/MPT using FormA:

      Quantifying the Moco and molybdopterin (MPT) content of crude C. elegans extracts was performed by oxidizing Moco/MPT in a well-defined environment to FormA, a stable and fluorescent Moco/MPT oxidation product (
      • Johnson J.L.
      • Hainline B.E.
      • Rajagopalan K.V.
      • Arison B.H.
      The pterin component of the molybdenum cofactor. Structural characterization of two fluorescent derivatives.
      ). For oxidation of Moco, 500 μg of crude protein extract in 400 μl of 100 mM Tris-HCL pH 7.2 were used. Samples were oxidized with 50 μl oxidation solution (1% I2, 2% KI in 1 M HCl) for 16 hours in the dark at room temperature. After oxidation, the samples were centrifuged at 10,000 x g for 10 min, and the supernatant was transferred to a new reaction tube. 55 μl 1% ascorbic acid solution were added to stop the oxidation reaction. Subsequently, 200μl 1 M Tris, 13 μl MgCl2, and 2 μl alkaline phosphatase (Roche Basel, Switzerland) were added and incubated overnight in the dark at room temperature. The HPLC detection of dpFormA was performed at room temperature using a reversed-phase C-18 column (250 mm × 4.6 mm, 5 μm, ReproSil-Pur Basic C-18 HD) with an Agilent 1100 HPLC system containing a fluorescence detector (
      • Hercher T.W.
      • Krausze J.
      • Hoffmeister S.
      • Zwerschke D.
      • Lindel T.
      • Blankenfeldt W.
      • Mendel R.R.
      • Kruse T.
      Insights into the Cnx1E catalyzed MPT-AMP hydrolysis.
      ). The specific parameters were set with a flow rate of 1 ml min−1 using an isocratic run with 5 mM ammonium acetate and 15% (v/v) methanol as the mobile phase. For the fluorescence detection λ ex = 302 nm, λ em = 451 nm were set. For data analysis, OpenLab CDS Version 2.2.0.600 was utilized. Calibration was carried out using synthetic dpFormA (
      • Klewe A.
      • Kruse T.
      • Lindel T.
      Aminopyrazine Pathway to the Moco Metabolite Dephospho Form A.
      ).
      To monitor dpFormA content in E. coli, bacterial strains were cultivated by inoculation from glycerol stocks in 50 ml LB medium and grown overnight at 37°C in a shaking incubator. Bacteria were centrifuged at 10,000 x g for 10 min at 4°C. Subsequently, the supernatant was discarded, and the pellet was resuspended with 25 ml chilled 100 mM Tris-HCI (pH 7.2). After this washing step, the cells were centrifuged again at 10,000 x g for 10 min at 4°C, and the supernatant was discarded. The cell pellets were resuspended with 1.5 ml 100mM Tris-HCI (pH 7.2). Cell lysis and FormA oxidation were performed as described for C. elegans, except we used 0.1 mm glass beads to disrupt the bacteria.

      C. elegans growth assays:

      Growth assays were performed as previously described with minor modifications (
      • Warnhoff K.
      • Hercher T.W.
      • Mendel R.R.
      • Ruvkun G.
      Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans.
      ,
      • Warnhoff K.
      • Ruvkun G.
      Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification.
      ). Briefly, wild-type and mutant C. elegans were synchronized at the L1 stage. To synchronize animals, embryos were harvested from gravid adult mothers via treatment with a bleach and sodium hydroxide solution (

      Stiernagle, T. (2006) Maintenance of C. elegans. WormBook, 1-11

      ). Embryos developed overnight in M9 solution causing them to hatch and arrest development at the L1 stage. Subsequently, animals were cultured on NGM seeded with wild-type or mutant E. coli for 72 hours at 20°C. Live animals were then imaged using an SMZ25 stereomicroscope (Nikon) equipped with an ORCA-Flash4.0 camera (Hamamatsu). Images were captured using NIS-Elements software (Nikon) and processed using ImageJ. Animal length was measured from the tip of the head to the end of the tail.
      For sulfite sensitivity experiments, the dietary E. coli were pelleted and re-suspended with various concentrations of sodium sulfite (Millipore Sigma) in water. The E. coli (5X concentrated)-sulfite slurries were then seeded onto the NGM media as a food source for C. elegans. The concentration of sulfite displayed assumes the sulfite diffuses evenly throughout the 10ml of NGM media. A limitation to this method is that sulfite oxidizes to sulfate in water, and thus the presented concentrations likely overestimate the sulfite concentration experienced by C. elegans in these assays (
      • Gómez-Otero E.
      • Costas M.
      • Lavilla I.
      • Bendicho C.
      Ultrasensitive, simple and solvent-free micro-assay for determining sulphite preservatives (E220-228) in foods by HS-SDME and UV-vis micro-spectrophotometry.
      ,
      • Lindgren M.
      • Cedergren A.
      • Lindberg J.
      Conditions for sulfite stabilization and determination by ion chromatography.
      ). To mitigate sulfite oxidation, we worked quickly to limit the time between establishment of the culture dishes (NGM, E. coli, sulfite) and the addition of the appropriate C. elegans animals. Growth assays were then performed as described above.
      For experiments where Moco+ and Moco- bacterial diets were mixed, BW25113 and JW0764-2 E. coli were first cultured overnight in LB at 37°C in a shaking incubator. Overnight cultures were then concentrated ten times. These 10X concentrated stocks were then mixed yielding various fractions of Moco+ (wild type) E. coli in Moco- (ΔmoaA) mutant E. coli. These E. coli mixtures were then seeded onto NGM supplemented with the antibiotic Streptomycin, preventing additional E. coli growth. Growth assays were performed as described above.

      Statistics

      Welch’s unpaired t-test or Ordinary One-Way analysis of variance (ANOVA) were used to determine significant differences between two or more than two groups. Dunnet’s post-hoc analysis was employed to determine significant differences between groups when compared to a reference group. A Tukey’s post-hoc test was used to determine significance between all comparisons. The analyses used are indicated in the appropriate figure legends. A p-value < 0.05 was considered statistically significant for all analyses. To characterize dose-response curves, nonlinear regression with IC50 analyses were utilized and incorporated experimentally-derived minimum and maximum animal length constraints. 95% confidence intervals are displayed in shading along each regression curve presented in the figures. R-squared values for each curve are presented in the appropriate figure panels. All analyses were completed using GraphPad Prism software.

      Data availability

      All data are presented within the manuscript.

      Supporting information

      This article contains supporting information.

      Funding information

      Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award numbers P20GM103620 (7140) and R35GM146871. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This research was also funded by a grant of the Deutsche Forschungsgemeinschaft [GRK 2223/1].

      Conflict of interest

      The authors declare no conflict of interest.

      Acknowledgments

      We thank the Caenorhabditis Genetics Center for providing C. elegans strains. We thank the National Institute of Genetics, National BioResource Project (NIG, Japan) for providing E. coli strains.

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