Molybdenum cofactor (Moco) is an essential prosthetic group that mediates the activity of 4 animal oxidases and is required for viability. Humans with mutations in the genes encoding Moco-biosynthetic enzymes suffer from Moco deficiency (MoCD), a neonatal lethal inborn error of metabolism. Caenorhabditis elegans has recently emerged as a useful and tractable genetic discovery engine for Moco biology. Here, we identify and characterize K10D2.7/moc-6, the C. elegans ortholog of human MOCS2A, a sulfur-carrier protein essential for Moco synthesis. Using CRISPR/Cas9 gene editing, we generate 3 null mutations in K10D2.7/moc-6 and with these alleles genetically demonstrate that K10D2.7/moc-6 is necessary for endogenous Moco synthesis in C. elegans.
","acknowledgements":"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.","authors":[{"affiliations":["Pediatrics and Rare Diseases Group, Sanford Research, Sioux Falls, SD 57104, USA"],"departments":[""],"credit":["investigation","methodology"],"email":"Jennifer.Snoozy@SanfordHealth.org","firstName":"Jennifer","lastName":"Snoozy","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":"","orcid":""},{"affiliations":["Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA"],"departments":[""],"credit":["investigation","methodology"],"email":"Peter_Breen@med.unc.edu","firstName":"Peter C","lastName":"Breen","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":null,"WBId":"","orcid":null},{"affiliations":["Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA"],"departments":[""],"credit":["conceptualization","fundingAcquisition","supervision","writing_reviewEditing"],"email":"ruvkun@molbio.mgh.harvard.edu","firstName":"Gary","lastName":"Ruvkun","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":null,"WBId":"","orcid":null},{"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"],"departments":["",""],"credit":["conceptualization","formalAnalysis","fundingAcquisition","investigation","methodology","supervision","visualization","writing_originalDraft","writing_reviewEditing"],"email":"kurt.warnhoff@sanfordhealth.org","firstName":"Kurt","lastName":"Warnhoff","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"0000-0002-9503-0557"}],"awards":null,"conflictsOfInterest":null,"dataTable":null,"extendedData":[],"funding":"This work was funded by an NIH grant to Gary Ruvkun (2R01GM044619-29), a Damon Runyon Fellowship to Kurt Warnhoff (DRG-2293-17), as well as startup funding for Kurt Warnhoff provided by Sanford Health.","image":{"url":"https://portal.micropublication.org/uploads/a1fc75c8da377ed23b9476acfcd48097.jpg"},"imageCaption":"(A) Amino acid alignment between human MOCS2A and C. elegans K10D2.7/MOC-6. Shaded amino acids (*) are identical. Strong (:) and weak (.) amino acid similarity are also displayed. (B) Nucleotide sequence for the wild type K10D2.7/moc-6 locus is displayed as are the sequences for newly generated K10D2.7/moc-6 deletions rae295, rae296, and rae297. Guide RNAs were designed targeting the 2 K10D2.7/moc-6 sequences displayed in red. The 2 corresponding PAM sites are displayed in blue. (C) Wild-type, rae295, rae296, and rae297 animals were cultured from synchronized L1 larvae for 72 hours on wild-type (Moco producing) and ΔmoaA mutant (Moco deficient) E. coli. Animal length was determined for each condition. (D) moc-6(rae296), cth-2(mg599); moc-6(rae296), and moc-6(rae296); cdo-1(mg622) mutant animals were cultured from synchronized L1 larvae for 72 hours on wild-type (Moco producing) and ΔmoaA mutant (Moco deficient) E. coli. Animal length was determined for each condition. For panels C and D, box plots display the median, upper, and lower quartiles, and whiskers indicate minimum and maximum data points. Sample size (n) is displayed for each experiment. (E) Hatching rates for wild-type and mutant C. elegans was determined. Moco+ diet indicates that mothers were cultured on wild-type E. coli while Moco- indicates that mothers were shifted to DmoaA mutant E. coli at the L4 stage of development, depriving animals of dietary Moco during reproductive adulthood. n is the number of embryos scored for hatching for each genotype and condition.
","imageTitle":"moc-6 is required for C. elegans Moco synthesis.
","methods":"Animal cultivation:
C. elegans strains were cultured using established protocols (Brenner 1974). Briefly, animals were cultured at 20°C on nematode growth media (NGM) seeded with wild-type E. coli (BW25113) unless otherwise noted. The wild-type C. elegans strain was Bristol N2. All C. elegans and E. coli strains used in this work are listed in the Reagents table.
Engineering deletions of moc-6/K10D2.7 using CRISPR/Cas9:
Genome engineering using CRISPR/Cas9 technology was performed using established techniques (Cho et al. 2013; Ghanta and Mello 2020). Briefly, 2 guide RNAs were designed and synthesized (IDT) that targeted the K10D2.7 locus (Fig. 1B). Cas9 (IDT) guide RNA ribonucleoprotein complexes were directly injected into the C. elegans germline (Choet al. 2013). Newly induced deletions were identified in the offspring of injected animals using a PCR-based screening approach. The DNA primers used to screen for new deletions were: 5’-tggtgtagcggcaagtacaa-3’ and 5’-tccctattttgcaccctttg-3’. We were able to isolate and homozygoze 3 independent deletions of K10D2.7; rae295, rae296, and rae297 (Fig. 1B).
C. elegans growth assays:
C. elegans were synchronized at the first stage of larval development (L1). L1 animals were cultured on NGM seeded with wild-type or ΔmoaA mutant E. coli. Animals were cultured for 72 hours at 20°C and live animals were 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. GraphPad Prism software was used for calculations of median, upper, and lower quartiles. No datapoints were excluded from this study based off statistical tests.
Quantification of embryo hatching:
To determine the hatching rate of wild-type and mutant C. elegans, we performed synchronized egg lays using young adult animals. Embryos were scored for hatching 12-24 hours after being laid. To test the effect of maternal dietary Moco on embryo hatching, mothers were initially grown on wild-type E. coli to the L4 stage of development and were shifted onto either wild-type or ΔmoaA mutant E. coli. Shifted L4 animals developed overnight into young adults on their respective E. coli diets and their progeny were assayed for their ability to hatch.
","reagents":"Organism | Strain | Genotype | Source |
C. elegans | N2 | Wild type | CGC |
C. elegans | USD954 | moc-6(rae295) III | This work |
C. elegans | USD955 | moc-6(rae296) III | This work |
C. elegans | USD956 | moc-6(rae297) III | This work |
C. elegans | GR2257 | cth-2(mg599) II | CGC |
C. elegans | GR2260 | cdo-1(mg622) X | CGC |
C. elegans | USD959 | moc-6(rae296) III; cdo-1(mg622) X | This work |
C. elegans | USD960 | cth-2(mg599) II; moc-6(rae296) III | This work |
E. coli | BW25113 | Wild type | NIG |
E. coli | JW0764-2 | ΔmoaA753::kan | NIG |
The nematode C. elegans has recently emerged as a tractable system for genetic discovery in Moco biology (Warnhoff and Ruvkun 2019; Warnhoff et al. 2021). In C. elegans, endogenous Moco biosynthesis is not required for growth, development, and reproduction. C. elegans mutants defective in Moco biosynthesis are viable because theyacquire and use Moco from their bacterial diet (Warnhoff and Ruvkun 2019; Warnhoff et al. 2021). Thus, C. elegans has 2 redundant pathways for maintaining cellular Moco: endogenous synthesis and dietary acquisition. C. elegans is the only known animal for which Moco biosynthesis is dispensable.
The human MOCS2A enzyme is required for Moco synthesis (Schwarz et al. 2009). MOCS2A is a small sulfur-carrier protein that is necessary for the conversion of cyclic pyranopterin monophosphate to molybdopterin. This reaction is the second step in the linear synthesis pathway of Moco from guanosine triphosphate (Schwarz et al.2009). Loss-of-function mutations in MOCS2A cause human Moco deficiency, a rare and lethal inborn error of metabolism (Johnson et al. 2001). However, the C. elegans orthologue of MOCS2A has not been molecularly identified or genetically studied. Using amino acid sequence analysis, we determined that the C. elegans K10D2.7 genomic locus encodes a protein with high amino acid similarity and identity to human MOCS2A (Fig. 1A) (Altschul et al.1990). To determine the role K10D2.7 plays in Moco biosynthesis, we used CRISPR/Cas9 to engineer 3 independent deletions of K10D2.7 (Fig. 1B) (Cho et al. 2013; Ghanta and Mello 2020). These K10D2.7 deletion alleles (rae295, rae296, and rae297) are likely null mutations because they eliminate >80% of the K10D2.7 coding sequence. As we have shown for other C. elegans mutations in Moco biosynthetic machinery, the K10D2.7 deletion alleles rae295, rae296, or rae297 cause no defects when animals are cultured under standard laboratory conditions feeding on wild-type E. coli which also synthesizes Moco. To determine if K10D2.7 is necessary for endogenous Moco synthesis, we cultured wild-type, rae295, rae296, and rae297 mutant animals on wild-type and ΔmoaA E. coli, a mutant bacterial strain that is unable to synthesize Moco. rae295, rae296, and rae297 mutant animals grew well on wild-type (Moco producing) E. coli but displayed a completely penetrant developmental arrest when cultured on ΔmoaA (Moco deficient) mutant E. coli (Fig. 1C). In contrast to K10D2.7 mutant animals, wild-type C. elegans grow well on wild-type or ΔmoaA mutant E. coli. By eliminating Moco from the diet, we revealed the defect in endogenous Moco synthesis caused by the rae295, rae296, and rae297 mutations. We conclude that K10D2.7 is required for C. elegans Moco synthesis and name this gene moc-6 (MOlybdenum Cofactor biosynthesis).
Moco deficiency in C. elegans also results in embryonic lethality when maternal animals are deprived of both endogenous and exogenous sources of Moco during reproductive adulthood (Warnhoff and Ruvkun 2019). moc-6 mutant C. elegans mothers were fed wild-type E. coli until the L4 stage of development, one stage prior to reproductive adulthood. These moc-6 mutant L4 mothers were then shifted onto wild-type or ΔmoaA mutant E. coli and the hatching rate of their progeny was determined. When moc-6 mutant animals were shifted to a Moco deficient diet, 0% of their embryos hatched (Fig. 1E). In contrast, 100% of moc-6 mutant embryos hatched when their mothers were fed wild-type E. coli. Hatching rates of wild-type embryos were not affected by the maternal diet (Fig. 1E). We conclude that maternal dietary Moco is sufficient to support the required Moco-dependent enzymes for embryonic development. Whether dietary Moco is acting maternally or embryonically to support embryonic development remains to be determined.
Previous genetic studies of the C. elegans Moco biosynthetic pathway demonstrated that Moco is required for the detoxification of sulfites produced by the sulfur amino acid metabolism pathway governed by cystathionase (cth-2) and cysteine dioxygenase (cdo-1). The activities of cth-2 and cdo-1 are required for the developmental arrest that occurs when C. elegans are defective in Moco synthesis and lack dietary Moco (Warnhoff and Ruvkun 2019). Loss-of-function/null mutations in cth-2 or cdo-1 suppress the developmental arrest displayed by moc mutant C. elegans grown on Moco-deficient E. coli (Warnhoff and Ruvkun 2019). To determine if the developmental arrest of moc-6 mutant C. elegans was also dependent upon cth-2 and cdo-1, we engineered cth-2; moc-6 and moc-6; cdo-1 double mutant animals and assessed their growth on wild-type and ΔmoaA mutant E. coli. In contrast to moc-6 single mutant animals, cth-2; moc-6 and moc-6; cdo-1 double mutant animals grew well on both wild-type and ΔmoaA mutant E. coli (Fig. 1D). cth-2 and cdo-1 single mutant animals grow well on both wild-type and ΔmoaA mutant E. coli (Warnhoff and Ruvkun 2019). Furthermore, mutations in cth-2 or cdo-1 suppressed the embryogenesis defects displayed by moc-6 mutant mothers fed on Moco-deficient E. coli (Fig. 1E). Together, these data demonstrate that cth-2 and cdo-1 inhibit normal development when Moco is deficient due to loss of both moc-6 and dietary Moco. CTH-2 and CDO-1 act in a catabolic pathway to produce sulfite from the sulfur amino acids methionine and cysteine. Sulfites are extremely toxic during Moco deficiency due to inactivity of the Moco-requiring sulfite oxidase enzyme, SUOX-1. Loss of cth-2 or cdo-1 prevents the metabolic production of sulfites, alleviates the cellular stress caused by inactive SUOX-1 (concomitant with Moco deficiency), and permits growth, development, and reproduction (Warnhoff and Ruvkun 2019). The mechanism by which sulfite promotes larval arrest and death remains enigmatic in both our C. elegans models and in human patients suffering from Moco deficiency.
This study demonstrates that moc-6 is required for endogenous C. elegans Moco biosynthesis and amino acid sequence analyses strongly suggest that moc-6 is orthologous to human MOCS2A.
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215: 403-10.","pubmedId":"2231712","doi":""},{"reference":"Brenner S. 1974. The genetics of Caenorhabditis elegans. Genetics 77: 71-94.","pubmedId":"4366476","doi":""},{"reference":"Cho SW, Lee J, Carroll D, Kim JS, Lee J. 2013. Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics 195: 1177-80.","pubmedId":"23979576","doi":""},{"reference":"Ghanta KS, Mello CC. 2020. Melting dsDNA Donor Molecules Greatly Improves Precision Genome Editing in Caenorhabditis elegans. Genetics 216: 643-650.","pubmedId":"32963112","doi":""},{"reference":"Johnson JL, Coyne KE, Rajagopalan KV, Van Hove JL, Mackay M, Pitt J, Boneh A. 2001. Molybdopterin synthase mutations in a mild case of molybdenum cofactor deficiency. Am J Med Genet 104: 169-73.","pubmedId":"11746050","doi":""},{"reference":"Schwarz G, Mendel RR, Ribbe MW. 2009. Molybdenum cofactors, enzymes and pathways. Nature 460: 839-47.","pubmedId":"19675644","doi":""},{"reference":"Warnhoff K, Hercher TW, Mendel RR, Ruvkun G. 2021. Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans. Genes Dev 35: 212-217.","pubmedId":"33446569","doi":""},{"reference":"Warnhoff K, Ruvkun G. 2019. Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification. Nat Chem Biol 15: 480-488.","pubmedId":"30911177","doi":""}],"title":"moc-6/MOCS2A is necessary for molybdenum cofactor synthesis in C. elegans
","reviews":[{"reviewer":{"displayName":"Stephan von Reuss"},"openAcknowledgement":true,"status":{"submitted":true}},{"reviewer":{"displayName":"Stephan von Reuss"},"openAcknowledgement":null,"status":{"submitted":false}}],"curatorReviews":[{"curator":{"displayName":"KJ Yook"},"openAcknowledgement":false,"submitted":null}]},{"id":"0931fcd1-d905-44fa-af7c-8f399eda2e44","decision":"accept","abstract":"Molybdenum cofactor (Moco) is an essential prosthetic group that mediates the activity of 4 animal oxidases and is required for viability. Humans with mutations in the genes encoding Moco-biosynthetic enzymes suffer from Moco deficiency, a neonatal lethal inborn error of metabolism. Caenorhabditis elegans has recently emerged as a useful and tractable genetic discovery engine for Moco biology. Here, we identify and characterize K10D2.7/moc-6, the C. elegans ortholog of human MOCS2A, a sulfur-carrier protein essential for Moco synthesis. Using CRISPR/Cas9 gene editing, we generate 3 null mutations in K10D2.7/moc-6 and with these alleles genetically demonstrate that K10D2.7/moc-6 is necessary for endogenous Moco synthesis in C. elegans.
","acknowledgements":"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.","authors":[{"affiliations":["Pediatrics and Rare Diseases Group, Sanford Research, Sioux Falls, SD 57104, USA"],"departments":[""],"credit":["investigation","methodology"],"email":"Jennifer.Snoozy@SanfordHealth.org","firstName":"Jennifer","lastName":"Snoozy","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":"","orcid":""},{"affiliations":["Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA"],"departments":[""],"credit":["investigation","methodology"],"email":"Peter_Breen@med.unc.edu","firstName":"Peter C","lastName":"Breen","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":null,"WBId":"","orcid":null},{"affiliations":["Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA"],"departments":[""],"credit":["conceptualization","fundingAcquisition","supervision","writing_reviewEditing"],"email":"ruvkun@molbio.mgh.harvard.edu","firstName":"Gary","lastName":"Ruvkun","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":null,"WBId":"","orcid":null},{"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"],"departments":["",""],"credit":["conceptualization","formalAnalysis","fundingAcquisition","investigation","methodology","supervision","visualization","writing_originalDraft","writing_reviewEditing"],"email":"kurt.warnhoff@sanfordhealth.org","firstName":"Kurt","lastName":"Warnhoff","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"0000-0002-9503-0557"}],"awards":null,"conflictsOfInterest":null,"dataTable":null,"extendedData":[],"funding":"This work was funded by an NIH grant to Gary Ruvkun (2R01GM044619-29), a Damon Runyon Fellowship to Kurt Warnhoff (DRG-2293-17), as well as an NIH grant to Kurt Warnhoff (5P20GM103620-09 7140).","image":{"url":"https://portal.micropublication.org/uploads/b81453f459fe3f5f59724123f2df230b.jpg"},"imageCaption":"(A) Amino acid alignment between human MOCS2A and C. elegans K10D2.7/MOC-6. Shaded amino acids (*) are identical. Strong (:) and weak (.) amino acid similarity are also displayed. (B) Nucleotide sequence for the wild type K10D2.7/moc-6 locus is displayed as are the sequences for newly generated K10D2.7/moc-6 deletions rae295, rae296, and rae297. Guide RNAs were designed targeting the 2 K10D2.7/moc-6 sequences displayed in red. The 2 corresponding PAM sites are displayed in blue. (C) Wild-type, rae295, rae296, and rae297 animals were cultured from synchronized L1 larvae for 72 hours on wild-type (Moco producing) and ΔmoaA mutant (Moco deficient) E. coli. Animal length was determined for each condition. (D) moc-6(rae296), cth-2(mg599); moc-6(rae296), and moc-6(rae296); cdo-1(mg622) mutant animals were cultured from synchronized L1 larvae for 72 hours on wild-type (Moco producing) and ΔmoaA mutant (Moco deficient) E. coli. Animal length was determined for each condition. For panels C and D, box plots display the median, upper, and lower quartiles, and whiskers indicate minimum and maximum data points. Sample size (n) is displayed for each experiment. *, indicates a statistically significant difference (p<0.01, unpaired t-test) in C. elegans growth on ΔmoaA E. coli when compared to corresponding growth on wild-type E. coli. (E) Hatching rates for wild-type and mutant C. elegans was determined. Moco+ diet indicates that mothers were cultured on wild-type E. coli while Moco- indicates that mothers were shifted to ΔmoaA mutant E. coli at the L4 stage of development, depriving animals of dietary Moco during reproductive adulthood. n is the number of embryos scored for hatching for each genotype and condition. * (red), indicates a statistically significant difference (p<.01, Chi-square test) in the hatching rates of the progeny of C. elegans mothers fed ΔmoaA E. coli when compared to the corresponding hatching rate when fed wild-type E. coli. * (black), indicates a statistically significant difference (p<.01, Chi-square test) in hatching rates of the progeny of cth-2(mg599); moc-6(rae296) and moc-6(rae296); cdo-1(mg622) double mutant C. elegans mothers fed ΔmoaA E. coli when compared to moc-6(rae296) single mutant mothers fed ΔmoaA E. coli.
","imageTitle":"moc-6 is required for C. elegans Moco synthesis.
","methods":"Animal cultivation:
C. elegans strains were cultured using established protocols (Brenner 1974). Briefly, animals were cultured at 20°C on nematode growth media (NGM) seeded with wild-type E. coli (BW25113) unless otherwise noted. The wild-type C. elegans strain was Bristol N2. All C. elegans and E. coli strains used in this work are listed in the Reagents table.
Engineering deletions of moc-6/K10D2.7 using CRISPR/Cas9:
Genome engineering using CRISPR/Cas9 technology was performed using established techniques (Cho et al. 2013; Ghanta and Mello 2020). Briefly, 2 guide RNAs were designed and synthesized (IDT) that targeted the K10D2.7 locus (Fig. 1B). Cas9 (IDT) guide RNA ribonucleoprotein complexes were directly injected into the C. elegans germline (Cho et al. 2013). Newly induced deletions were identified in the offspring of injected animals using a PCR-based screening approach. The DNA primers used to screen for new deletions were: 5’-tggtgtagcggcaagtacaa-3’ and 5’-tccctattttgcaccctttg-3’. We were able to isolate and homozygoze 3 independent deletions of K10D2.7; rae295, rae296, and rae297 (Fig. 1B).
C. elegans growth assays:
C. elegans were synchronized at the first stage of larval development (L1). L1 animals were cultured on NGM seeded with wild-type or ΔmoaA mutant E. coli. Animals were cultured for 72 hours at 20°C and live animals were 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. GraphPad Prism software was used to perform statistical tests as well as calculations of median, upper, and lower quartiles.
Quantification of embryo hatching:
To determine the hatching rate of wild-type and mutant C. elegans, we performed synchronized egg lays using young adult animals. Embryos were scored for hatching 12-24 hours after being laid. To test the effect of maternal dietary Moco on embryo hatching, mothers were initially grown on wild-type E. coli to the L4 stage of development and were shifted onto either wild-type or ΔmoaA mutant E. coli. Shifted L4 animals developed overnight into young adults on their respective E. coli diets and their progeny were assayed for their ability to hatch.
","reagents":"Organism | Strain | Genotype | Source |
C. elegans | N2 | Wild type | CGC |
C. elegans | USD954 | moc-6(rae295) III | This work |
C. elegans | USD955 | moc-6(rae296) III | This work |
C. elegans | USD956 | moc-6(rae297) III | This work |
C. elegans | GR2257 | cth-2(mg599) II | CGC |
C. elegans | GR2260 | cdo-1(mg622) X | CGC |
C. elegans | USD959 | moc-6(rae296) III; cdo-1(mg622) X | This work |
C. elegans | USD960 | cth-2(mg599) II; moc-6(rae296) III | This work |
E. coli | BW25113 | Wild type | NIG |
E. coli | JW0764-2 | ΔmoaA753::kan | NIG |
The nematode C. elegans has recently emerged as a tractable system for genetic discovery in Moco biology (Warnhoff and Ruvkun 2019; Warnhoff et al. 2021). In C. elegans, endogenous Moco biosynthesis is not required for growth, development, and reproduction. C. elegans mutants defective in Moco biosynthesis are viable because they acquire and use Moco from their bacterial diet (Warnhoff and Ruvkun 2019; Warnhoff et al. 2021). Thus, C. elegans has 2 redundant pathways for maintaining cellular Moco: endogenous synthesis and dietary acquisition. C. elegans is the only known animal for which Moco biosynthesis is dispensable.
The human MOCS2A enzyme is required for Moco synthesis (Schwarz et al. 2009). MOCS2A is a small sulfur-carrier protein that is necessary for the conversion of cyclic pyranopterin monophosphate to molybdopterin. This reaction is the second step in the linear synthesis pathway of Moco from guanosine triphosphate (Schwarz et al. 2009). Loss-of-function mutations in MOCS2A cause human Moco deficiency, a rare and lethal inborn error of metabolism (Johnson et al. 2001). However, the C. elegans orthologue of MOCS2A has not been molecularly identified or genetically studied. Using amino acid sequence analysis, we determined that the C. elegans K10D2.7 genomic locus encodes a protein with high amino acid similarity and identity to human MOCS2A (Fig. 1A) (Altschul et al.1990). To determine the role K10D2.7 plays in Moco biosynthesis, we used CRISPR/Cas9 to engineer 3 independent deletions of K10D2.7 (Fig. 1B) (Cho et al. 2013; Ghanta and Mello 2020). These K10D2.7 deletion alleles (rae295, rae296, and rae297) are likely null mutations because they eliminate >80% of the K10D2.7 coding sequence. As we have shown for other C. elegans mutations in Moco biosynthetic machinery, the K10D2.7 deletion alleles rae295, rae296, or rae297 cause no defects when animals are cultured under standard laboratory conditions feeding on wild-type E. coli which also synthesizes Moco. To determine if K10D2.7 is necessary for endogenous Moco synthesis, we cultured wild-type, rae295, rae296, and rae297 mutant animals on wild-type and ΔmoaA E. coli, a mutant bacterial strain that is unable to synthesize Moco. rae295, rae296, and rae297 mutant animals grew well on wild-type (Moco producing) E. coli but displayed a completely penetrant developmental arrest when cultured on ΔmoaA (Moco deficient) mutant E. coli (Fig. 1C). In contrast to K10D2.7 mutant animals, wild-type C. elegans grow well on wild-type or ΔmoaA mutant E. coli, although we observed a subtle, but statistically significant, reduction in the growth of wild-type animals on ΔmoaA mutant E. coli (Fig. 1C). By eliminating Moco from the diet, we revealed the defect in endogenous Moco synthesis caused by the rae295, rae296, and rae297 mutations. We conclude that K10D2.7 is required for C. elegans Moco synthesis and name this gene moc-6 (MOlybdenum Cofactor biosynthesis).
Moco deficiency in C. elegans also results in embryonic lethality when maternal animals are deprived of both endogenous and exogenous sources of Moco during reproductive adulthood (Warnhoff and Ruvkun 2019). moc-6 mutant C. elegans mothers were fed wild-type E. coli until the L4 stage of development, one stage prior to reproductive adulthood. These moc-6 mutant L4 mothers were then shifted onto wild-type or ΔmoaA mutant E. coli and the hatching rate of their progeny was determined. When moc-6 mutant animals were shifted to a Moco deficient diet, 0% of their embryos hatched (Fig. 1E). In contrast, 100% of moc-6 mutant embryos hatched when their mothers were fed wild-type E. coli. Hatching rates of wild-type embryos were not affected by the maternal diet (Fig. 1E). We conclude that maternal dietary Moco is sufficient to support the required Moco-dependent enzymes for embryonic development. Whether dietary Moco is acting maternally or embryonically to support embryonic development remains to be determined.
Previous genetic studies of the C. elegans Moco biosynthetic pathway demonstrated that Moco is required for the detoxification of sulfites produced by the sulfur amino acid metabolism pathway governed by cystathionase (cth-2) and cysteine dioxygenase (cdo-1). The activities of cth-2 and cdo-1 are required for the developmental arrest that occurs when C. elegans are defective in Moco synthesis and lack dietary Moco (Warnhoff and Ruvkun 2019). Loss-of-function/null mutations in cth-2 or cdo-1 suppress the developmental arrest displayed by moc mutant C. elegans grown on Moco-deficient E. coli (Warnhoff and Ruvkun 2019). To determine if the developmental arrest of moc-6 mutant C. elegans was also dependent upon cth-2 and cdo-1, we engineered cth-2; moc-6 and moc-6; cdo-1 double mutant animals and assessed their growth on wild-type and ΔmoaA mutant E. coli. In contrast to moc-6 single mutant animals, cth-2; moc-6 and moc-6; cdo-1 double mutant animals grew well on both wild-type and ΔmoaA mutant E. coli (Fig. 1D). cth-2 and cdo-1 single mutant animals grow well on both wild-type and ΔmoaA mutant E. coli (Warnhoff and Ruvkun 2019). Furthermore, mutations in cth-2 or cdo-1 suppressed the embryogenesis defects displayed by moc-6 mutant mothers fed on Moco-deficient E. coli (Fig. 1E). Although, we find that moc-6; cdo-1 double mutant embryos derived from mothers fed ΔmoaA mutant E. coli do not hatch at the same rate as wild type. Further studies are required to account for this observation. Together, these data demonstrate that cth-2 and cdo-1 inhibit normal development when Moco is deficient due to loss of both moc-6 and dietary Moco. CTH-2 and CDO-1 act in a catabolic pathway to produce sulfite from the sulfur amino acids methionine and cysteine. Sulfites are extremely toxic during Moco deficiency due to inactivity of the Moco-requiring sulfite oxidase enzyme, SUOX-1. Loss of cth-2 or cdo-1 prevents the metabolic production of sulfites, alleviates the cellular stress caused by inactive SUOX-1 (concomitant with Moco deficiency), and permits growth, development, and reproduction (Warnhoff and Ruvkun 2019). The mechanism by which sulfite promotes larval arrest and death remains enigmatic in both our C. elegans models and in human patients suffering from Moco deficiency.
This study demonstrates that moc-6 is required for endogenous C. elegans Moco biosynthesis and amino acid sequence analyses strongly suggest that moc-6 is orthologous to human MOCS2A.
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215: 403-10.","pubmedId":"2231712","doi":""},{"reference":"Brenner S. 1974. The genetics of Caenorhabditis elegans. Genetics 77: 71-94.","pubmedId":"4366476","doi":""},{"reference":"Cho SW, Lee J, Carroll D, Kim JS, Lee J. 2013. Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics 195: 1177-80.","pubmedId":"23979576","doi":""},{"reference":"Ghanta KS, Mello CC. 2020. Melting dsDNA Donor Molecules Greatly Improves Precision Genome Editing in Caenorhabditis elegans. Genetics 216: 643-650.","pubmedId":"32963112","doi":""},{"reference":"Johnson JL, Coyne KE, Rajagopalan KV, Van Hove JL, Mackay M, Pitt J, Boneh A. 2001. Molybdopterin synthase mutations in a mild case of molybdenum cofactor deficiency. Am J Med Genet 104: 169-73.","pubmedId":"11746050","doi":""},{"reference":"Schwarz G, Mendel RR, Ribbe MW. 2009. Molybdenum cofactors, enzymes and pathways. Nature 460: 839-47.","pubmedId":"19675644","doi":""},{"reference":"Warnhoff K, Hercher TW, Mendel RR, Ruvkun G. 2021. Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans. Genes Dev 35: 212-217.","pubmedId":"33446569","doi":""},{"reference":"Warnhoff K, Ruvkun G. 2019. Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification. Nat Chem Biol 15: 480-488.","pubmedId":"30911177","doi":""}],"title":"moc-6/MOCS2A is necessary for molybdenum cofactor synthesis in C. elegans
","reviews":[],"curatorReviews":[{"curator":{"displayName":"KJ Yook"},"openAcknowledgement":false,"submitted":"1645122281624"}]},{"id":"9db6bcea-0904-438f-96d1-a469c5e7027e","decision":"publish","abstract":"Molybdenum cofactor (Moco) is an essential prosthetic group that mediates the activity of 4 animal oxidases and is required for viability. Humans with mutations in the genes encoding Moco-biosynthetic enzymes suffer from Moco deficiency, a neonatal lethal inborn error of metabolism. Caenorhabditis elegans has recently emerged as a useful and tractable genetic discovery engine for Moco biology. Here, we identify and characterize K10D2.7/moc-6, the C. elegans ortholog of human MOCS2A, a sulfur-carrier protein essential for Moco synthesis. Using CRISPR/Cas9 gene editing, we generate 3 null mutations in K10D2.7/moc-6 and with these alleles genetically demonstrate that K10D2.7/moc-6 is necessary for endogenous Moco synthesis in C. elegans.
","acknowledgements":"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.","authors":[{"affiliations":["Pediatrics and Rare Diseases Group, Sanford Research, Sioux Falls, SD 57104, USA"],"departments":[""],"credit":["investigation","methodology"],"email":"Jennifer.Snoozy@SanfordHealth.org","firstName":"Jennifer","lastName":"Snoozy","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":"","orcid":""},{"affiliations":["Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA"],"departments":[""],"credit":["investigation","methodology"],"email":"Peter_Breen@med.unc.edu","firstName":"Peter C","lastName":"Breen","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":null,"WBId":"","orcid":null},{"affiliations":["Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA"],"departments":[""],"credit":["conceptualization","fundingAcquisition","supervision","writing_reviewEditing"],"email":"ruvkun@molbio.mgh.harvard.edu","firstName":"Gary","lastName":"Ruvkun","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":null,"WBId":"","orcid":null},{"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"],"departments":["",""],"credit":["conceptualization","formalAnalysis","fundingAcquisition","investigation","methodology","supervision","visualization","writing_originalDraft","writing_reviewEditing"],"email":"kurt.warnhoff@sanfordhealth.org","firstName":"Kurt","lastName":"Warnhoff","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"0000-0002-9503-0557"}],"awards":null,"conflictsOfInterest":null,"dataTable":null,"extendedData":[],"funding":"This work was funded by an NIH grant to Gary Ruvkun (2R01GM044619-29), a Damon Runyon Fellowship to Kurt Warnhoff (DRG-2293-17), as well as an NIH grant to Kurt Warnhoff (5P20GM103620-09 7140).","image":{"url":"https://portal.micropublication.org/uploads/49a5db63c1afe40157fabc215e9210f5.jpg"},"imageCaption":"(A) Amino acid alignment between human MOCS2A and C. elegans K10D2.7/MOC-6. Shaded amino acids (*) are identical. Strong (:) and weak (.) amino acid similarity are also displayed. (B) Nucleotide sequence for the wild type K10D2.7/moc-6 locus is displayed as are the sequences for newly generated K10D2.7/moc-6 deletions rae295, rae296, and rae297. Guide RNAs were designed targeting the 2 K10D2.7/moc-6 sequences displayed in red. The 2 corresponding PAM sites are displayed in blue. (C) Wild-type, rae295, rae296, and rae297 animals were cultured from synchronized L1 larvae for 72 hours on wild-type (Moco producing) and ΔmoaA mutant (Moco deficient) E. coli. Animal length was determined for each condition. (D) moc-6(rae296), cth-2(mg599); moc-6(rae296), and moc-6(rae296); cdo-1(mg622) mutant animals were cultured from synchronized L1 larvae for 72 hours on wild-type (Moco producing) and ΔmoaA mutant (Moco deficient) E. coli. Animal length was determined for each condition. For panels C and D, box plots display the median, upper, and lower quartiles, and whiskers indicate minimum and maximum data points. Sample size (n) is displayed for each experiment. *, indicates a statistically significant difference (p<0.01, unpaired t-test) in C. elegans growth on ΔmoaA E. coli when compared to corresponding growth on wild-type E. coli. (E) Hatching rates for wild-type and mutant C. elegans was determined. Moco+ diet indicates that mothers were cultured on wild-type E. coli while Moco- indicates that mothers were shifted to ΔmoaA mutant E. coli at the L4 stage of development, depriving animals of dietary Moco during reproductive adulthood. n is the number of embryos scored for hatching for each genotype and condition. * (red), indicates a statistically significant difference (p<0.01, Chi-square test) in the hatching rates of the progeny of C. elegans mothers fed ΔmoaA E. coli when compared to the corresponding hatching rate when fed wild-type E. coli. * (black), indicates a statistically significant difference (p<0.01, Chi-square test) in hatching rates of the progeny of cth-2(mg599); moc-6(rae296) and moc-6(rae296); cdo-1(mg622) double mutant C. elegans mothers fed ΔmoaA E. coli when compared to moc-6(rae296) single mutant mothers fed ΔmoaA E. coli.
","imageTitle":"moc-6 is required for C. elegans Moco synthesis.
","methods":"Animal cultivation:
C. elegans strains were cultured using established protocols (Brenner 1974). Briefly, animals were cultured at 20°C on nematode growth media (NGM) seeded with wild-type E. coli (BW25113) unless otherwise noted. The wild-type C. elegans strain was Bristol N2. All C. elegans and E. coli strains used in this work are listed in the Reagents table.
Engineering deletions of moc-6/K10D2.7 using CRISPR/Cas9:
Genome engineering using CRISPR/Cas9 technology was performed using established techniques (Cho et al. 2013; Ghanta and Mello 2020). Briefly, 2 guide RNAs were designed and synthesized (IDT) that targeted the K10D2.7 locus (Fig. 1B). Cas9 (IDT) guide RNA ribonucleoprotein complexes were directly injected into the C. elegans germline (Cho et al. 2013). Newly induced deletions were identified in the offspring of injected animals using a PCR-based screening approach. The DNA primers used to screen for new deletions were: 5’-tggtgtagcggcaagtacaa-3’ and 5’-tccctattttgcaccctttg-3’. We were able to isolate and homozygoze 3 independent deletions of K10D2.7; rae295, rae296, and rae297 (Fig. 1B).
C. elegans growth assays:
C. elegans were synchronized at the first stage of larval development (L1). L1 animals were cultured on NGM seeded with wild-type or ΔmoaA mutant E. coli. Animals were cultured for 72 hours at 20°C and live animals were 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. GraphPad Prism software was used to perform statistical tests as well as calculations of median, upper, and lower quartiles.
Quantification of embryo hatching:
To determine the hatching rate of wild-type and mutant C. elegans, we performed synchronized egg lays using young adult animals. Embryos were scored for hatching 12-24 hours after being laid. To test the effect of maternal dietary Moco on embryo hatching, mothers were initially grown on wild-type E. coli to the L4 stage of development and were shifted onto either wild-type or ΔmoaA mutant E. coli. Shifted L4 animals developed overnight into young adults on their respective E. coli diets and their progeny were assayed for their ability to hatch.
","reagents":"Organism | Strain | Genotype | Source |
C. elegans | N2 | Wild type | CGC |
C. elegans | USD954 | moc-6(rae295) III | This work |
C. elegans | USD955 | moc-6(rae296) III | This work |
C. elegans | USD956 | moc-6(rae297) III | This work |
C. elegans | GR2257 | cth-2(mg599) II | CGC |
C. elegans | GR2260 | cdo-1(mg622) X | CGC |
C. elegans | USD959 | moc-6(rae296) III; cdo-1(mg622) X | This work |
C. elegans | USD960 | cth-2(mg599) II; moc-6(rae296) III | This work |
E. coli | BW25113 | Wild type | NIG |
E. coli | JW0764-2 | ΔmoaA753::kan | NIG |
The nematode C. elegans has recently emerged as a tractable system for genetic discovery in Moco biology (Warnhoff and Ruvkun 2019; Warnhoff et al. 2021). In C. elegans, endogenous Moco biosynthesis is not required for growth, development, and reproduction. C. elegans mutants defective in Moco biosynthesis are viable because they acquire and use Moco from their bacterial diet (Warnhoff and Ruvkun 2019; Warnhoff et al. 2021). Thus, C. elegans has 2 redundant pathways for maintaining cellular Moco: endogenous synthesis and dietary acquisition. C. elegans is the only known animal for which Moco biosynthesis is dispensable.
The human MOCS2A enzyme is required for Moco synthesis (Schwarz et al. 2009). MOCS2A is a small sulfur-carrier protein that is necessary for the conversion of cyclic pyranopterin monophosphate to molybdopterin. This reaction is the second step in the linear synthesis pathway of Moco from guanosine triphosphate (Schwarz et al. 2009). Loss-of-function mutations in MOCS2A cause human Moco deficiency, a rare and lethal inborn error of metabolism (Johnson et al. 2001). However, the C. elegans orthologue of MOCS2A has not been molecularly identified or genetically studied. Using amino acid sequence analysis, we determined that the C. elegans K10D2.7 genomic locus encodes a protein with high amino acid similarity and identity to human MOCS2A (Fig. 1A) (Altschul et al.1990). To determine the role K10D2.7 plays in Moco biosynthesis, we used CRISPR/Cas9 to engineer 3 independent deletions of K10D2.7 (Fig. 1B) (Cho et al. 2013; Ghanta and Mello 2020). These K10D2.7 deletion alleles (rae295, rae296, and rae297) are likely null mutations because they eliminate >80% of the K10D2.7 coding sequence. As we have shown for other C. elegans mutations in Moco biosynthetic machinery, the K10D2.7 deletion alleles rae295, rae296, or rae297 cause no defects when animals are cultured under standard laboratory conditions feeding on wild-type E. coli which also synthesizes Moco. To determine if K10D2.7 is necessary for endogenous Moco synthesis, we cultured wild-type, rae295, rae296, and rae297 mutant animals on wild-type and ΔmoaA E. coli, a mutant bacterial strain that is unable to synthesize Moco. rae295, rae296, and rae297 mutant animals grew well on wild-type (Moco producing) E. coli but displayed a completely penetrant developmental arrest when cultured on ΔmoaA (Moco deficient) mutant E. coli (Fig. 1C). In contrast to K10D2.7 mutant animals, wild-type C. elegans grow well on wild-type or ΔmoaA mutant E. coli, although we observed a subtle, but statistically significant, reduction in the growth of wild-type animals on ΔmoaA mutant E. coli (Fig. 1C). By eliminating Moco from the diet, we revealed the defect in endogenous Moco synthesis caused by the rae295, rae296, and rae297 mutations. We conclude that K10D2.7 is required for C. elegans Moco synthesis and name this gene moc-6 (MOlybdenum Cofactor biosynthesis).
Moco deficiency in C. elegans also results in embryonic lethality when maternal animals are deprived of both endogenous and exogenous sources of Moco during reproductive adulthood (Warnhoff and Ruvkun 2019). moc-6 mutant C. elegans mothers were fed wild-type E. coli until the L4 stage of development, one stage prior to reproductive adulthood. These moc-6 mutant L4 mothers were then shifted onto wild-type or ΔmoaA mutant E. coli and the hatching rate of their progeny was determined. When moc-6 mutant animals were shifted to a Moco deficient diet, 0% of their embryos hatched (Fig. 1E). In contrast, 100% of moc-6 mutant embryos hatched when their mothers were fed wild-type E. coli. Hatching rates of wild-type embryos were not affected by the maternal diet (Fig. 1E). We conclude that maternal dietary Moco is sufficient to support the required Moco-dependent enzymes for embryonic development. Whether dietary Moco is acting maternally or embryonically to support embryonic development remains to be determined.
Previous genetic studies of the C. elegans Moco biosynthetic pathway demonstrated that Moco is required for the detoxification of sulfites produced by the sulfur amino acid metabolism pathway governed by cystathionase (cth-2) and cysteine dioxygenase (cdo-1). The activities of cth-2 and cdo-1 are required for the developmental arrest that occurs when C. elegans are defective in Moco synthesis and lack dietary Moco (Warnhoff and Ruvkun 2019). Loss-of-function/null mutations in cth-2 or cdo-1 suppress the developmental arrest displayed by moc mutant C. elegans grown on Moco-deficient E. coli (Warnhoff and Ruvkun 2019). To determine if the developmental arrest of moc-6 mutant C. elegans was also dependent upon cth-2 and cdo-1, we engineered cth-2; moc-6 and moc-6; cdo-1 double mutant animals and assessed their growth on wild-type and ΔmoaA mutant E. coli. In contrast to moc-6 single mutant animals, cth-2; moc-6 and moc-6; cdo-1 double mutant animals grew well on both wild-type and ΔmoaA mutant E. coli (Fig. 1D). cth-2 and cdo-1 single mutant animals grow well on both wild-type and ΔmoaA mutant E. coli (Warnhoff and Ruvkun 2019). Furthermore, mutations in cth-2 or cdo-1 suppressed the embryogenesis defects displayed by moc-6 mutant mothers fed on Moco-deficient E. coli (Fig. 1E). Although, we find that moc-6; cdo-1 double mutant embryos derived from mothers fed ΔmoaA mutant E. coli do not hatch at the same rate as wild type. Further studies are required to account for this observation. Together, these data demonstrate that cth-2 and cdo-1 inhibit normal development when Moco is deficient due to loss of both moc-6 and dietary Moco. CTH-2 and CDO-1 act in a catabolic pathway to produce sulfite from the sulfur amino acids methionine and cysteine. Sulfites are extremely toxic during Moco deficiency due to inactivity of the Moco-requiring sulfite oxidase enzyme, SUOX-1. Loss of cth-2 or cdo-1 prevents the metabolic production of sulfites, alleviates the cellular stress caused by inactive SUOX-1 (concomitant with Moco deficiency), and permits growth, development, and reproduction (Warnhoff and Ruvkun 2019). The mechanism by which sulfite promotes larval arrest and death remains enigmatic in both our C. elegans models and in human patients suffering from Moco deficiency.
This study demonstrates that moc-6 is required for endogenous C. elegans Moco biosynthesis and amino acid sequence analyses strongly suggest that moc-6 is orthologous to human MOCS2A.
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215: 403-10.","pubmedId":"2231712","doi":""},{"reference":"Brenner S. 1974. The genetics of Caenorhabditis elegans. Genetics 77: 71-94.","pubmedId":"4366476","doi":""},{"reference":"Cho SW, Lee J, Carroll D, Kim JS, Lee J. 2013. Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics 195: 1177-80.","pubmedId":"23979576","doi":""},{"reference":"Ghanta KS, Mello CC. 2020. Melting dsDNA Donor Molecules Greatly Improves Precision Genome Editing in Caenorhabditis elegans. Genetics 216: 643-650.","pubmedId":"32963112","doi":""},{"reference":"Johnson JL, Coyne KE, Rajagopalan KV, Van Hove JL, Mackay M, Pitt J, Boneh A. 2001. Molybdopterin synthase mutations in a mild case of molybdenum cofactor deficiency. Am J Med Genet 104: 169-73.","pubmedId":"11746050","doi":""},{"reference":"Schwarz G, Mendel RR, Ribbe MW. 2009. Molybdenum cofactors, enzymes and pathways. Nature 460: 839-47.","pubmedId":"19675644","doi":""},{"reference":"Warnhoff K, Hercher TW, Mendel RR, Ruvkun G. 2021. Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans. Genes Dev 35: 212-217.","pubmedId":"33446569","doi":""},{"reference":"Warnhoff K, Ruvkun G. 2019. Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification. Nat Chem Biol 15: 480-488.","pubmedId":"30911177","doi":""}],"title":"moc-6/MOCS2A is necessary for molybdenum cofactor synthesis in C. elegans
","reviews":[],"curatorReviews":[{"curator":{"displayName":"KJ Yook"},"openAcknowledgement":false,"submitted":null}]},{"id":"84f3ff46-426a-40aa-95ea-3f464c2724e4","decision":"publish","abstract":"Molybdenum cofactor (Moco) is an essential prosthetic group that mediates the activity of 4 animal oxidases and is required for viability. Humans with mutations in the genes encoding Moco-biosynthetic enzymes suffer from Moco deficiency, a neonatal lethal inborn error of metabolism. Caenorhabditis elegans has recently emerged as a useful and tractable genetic discovery engine for Moco biology. Here, we identify and characterize K10D2.7/moc-6, the C. elegans ortholog of human MOCS2A, a sulfur-carrier protein essential for Moco synthesis. Using CRISPR/Cas9 gene editing, we generate 3 null mutations in K10D2.7/moc-6 and with these alleles genetically demonstrate that K10D2.7/moc-6 is necessary for endogenous Moco synthesis in C. elegans.
\n","acknowledgements":"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.\n","authors":[{"affiliations":["Pediatrics and Rare Diseases Group, Sanford Research, Sioux Falls, SD 57104, USA"],"departments":[""],"credit":["Investigation","Methodology"],"email":"Jennifer.Snoozy@SanfordHealth.org","firstName":"Jennifer","lastName":"Snoozy","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA"],"departments":[""],"credit":["Investigation","Methodology"],"email":"Peter_Breen@med.unc.edu","firstName":"Peter C","lastName":"Breen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA"],"departments":[""],"credit":["Conceptualization","Funding acquisition","Supervision","Writing - review and editing"],"email":"ruvkun@molbio.mgh.harvard.edu","firstName":"Gary","lastName":"Ruvkun","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"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"],"departments":["",""],"credit":["Conceptualization","Formal analysis","Funding acquisition","Investigation","Methodology","Supervision","Visualization","Writing - original draft","Writing - review and editing"],"email":"kurt.warnhoff@sanfordhealth.org","firstName":"Kurt","lastName":"Warnhoff","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":null}],"awards":null,"conflictsOfInterest":null,"dataTable":null,"extendedData":[],"funding":"This work was funded by an NIH grant to Gary Ruvkun (2R01GM044619-29), a Damon Runyon Fellowship to Kurt Warnhoff (DRG-2293-17), as well as an NIH grant to Kurt Warnhoff (5P20GM103620-09 7140).","image":{"url":"https://portal.micropublication.org/uploads/micropub.biology.000531.jpeg"},"imageCaption":"(A) Amino acid alignment between human MOCS2A and C. elegans K10D2.7/MOC-6. Shaded amino acids (*) are identical. Strong (:) and weak (.) amino acid similarity are also displayed. (B) Nucleotide sequence for the wild type K10D2.7/moc-6 locus is displayed as are the sequences for newly generated K10D2.7/moc-6 deletions rae295, rae296, and rae297. Guide RNAs were designed targeting the 2 K10D2.7/moc-6 sequences displayed in red. The 2 corresponding PAM sites are displayed in blue. (C) Wild-type, rae295, rae296, and rae297 animals were cultured from synchronized L1 larvae for 72 hours on wild-type (Moco producing) and ΔmoaA mutant (Moco deficient) E. coli. Animal length was determined for each condition. (D) moc-6(rae296), cth-2(mg599); moc-6(rae296), and moc-6(rae296); cdo-1(mg622) mutant animals were cultured from synchronized L1 larvae for 72 hours on wild-type (Moco producing) and ΔmoaA mutant (Moco deficient) E. coli. Animal length was determined for each condition. For panels C and D, box plots display the median, upper, and lower quartiles, and whiskers indicate minimum and maximum data points. Sample size (n) is displayed for each experiment. *, indicates a statistically significant difference (p<0.01, unpaired t-test) in C. elegans growth on ΔmoaA E. coli when compared to corresponding growth on wild-type E. coli. (E) Hatching rates for wild-type and mutant C. elegans was determined. Moco+ diet indicates that mothers were cultured on wild-type E. coli while Moco- indicates that mothers were shifted to ΔmoaA mutant E. coli at the L4 stage of development, depriving animals of dietary Moco during reproductive adulthood. n is the number of embryos scored for hatching for each genotype and condition. * (red), indicates a statistically significant difference (p<0.01, Chi-square test) in the hatching rates of the progeny of C. elegans mothers fed ΔmoaA E. coli when compared to the corresponding hatching rate when fed wild-type E. coli. * (black), indicates a statistically significant difference (p<0.01, Chi-square test) in hatching rates of the progeny of cth-2(mg599); moc-6(rae296) and moc-6(rae296); cdo-1(mg622) double mutant C. elegans mothers fed ΔmoaA E. coli when compared to moc-6(rae296) single mutant mothers fed ΔmoaA E. coli.
\n","imageTitle":"Figure 1. moc-6 is required for C. elegans Moco synthesis","methods":"Animal cultivation:
\nC. elegans strains were cultured using established protocols (Brenner 1974). Briefly, animals were cultured at 20°C on nematode growth media (NGM) seeded with wild-type E. coli (BW25113) unless otherwise noted. The wild-type C. elegans strain was Bristol N2. All C. elegans and E. coli strains used in this work are listed in the Reagents table.
\nEngineering deletions of moc-6/K10D2.7 using CRISPR/Cas9:
\nGenome engineering using CRISPR/Cas9 technology was performed using established techniques (Cho et al. 2013; Ghanta and Mello 2020). Briefly, 2 guide RNAs were designed and synthesized (IDT) that targeted the K10D2.7 locus (Fig. 1B). Cas9 (IDT) guide RNA ribonucleoprotein complexes were directly injected into the C. elegans germline (Cho et al. 2013). Newly induced deletions were identified in the offspring of injected animals using a PCR-based screening approach. The DNA primers used to screen for new deletions were: 5’-tggtgtagcggcaagtacaa-3’ and 5’-tccctattttgcaccctttg-3’. We were able to isolate and homozygoze 3 independent deletions of K10D2.7; rae295, rae296, and rae297 (Fig. 1B).
\nC. elegans growth assays:
\nC. elegans were synchronized at the first stage of larval development (L1). L1 animals were cultured on NGM seeded with wild-type or ΔmoaA mutant E. coli. Animals were cultured for 72 hours at 20°C and live animals were 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. GraphPad Prism software was used to perform statistical tests as well as calculations of median, upper, and lower quartiles.
\nQuantification of embryo hatching:
\nTo determine the hatching rate of wild-type and mutant C. elegans, we performed synchronized egg lays using young adult animals. Embryos were scored for hatching 12-24 hours after being laid. To test the effect of maternal dietary Moco on embryo hatching, mothers were initially grown on wild-type E. coli to the L4 stage of development and were shifted onto either wild-type or ΔmoaA mutant E. coli. Shifted L4 animals developed overnight into young adults on their respective E. coli diets and their progeny were assayed for their ability to hatch.
\n","reagents":"| Organism | \nStrain | \nGenotype | \nSource | \n
| C. elegans | \nN2 | \nWild type | \nCGC | \n
| C. elegans | \nUSD954 | \nmoc-6(rae295) III | \nThis work | \n
| C. elegans | \nUSD955 | \nmoc-6(rae296) III | \nThis work | \n
| C. elegans | \nUSD956 | \nmoc-6(rae297) III | \nThis work | \n
| C. elegans | \nGR2257 | \ncth-2(mg599) II | \nCGC | \n
| C. elegans | \nGR2260 | \ncdo-1(mg622) X | \nCGC | \n
| C. elegans | \nUSD959 | \nmoc-6(rae296) III; cdo-1(mg622) X | \nThis work | \n
| C. elegans | \nUSD960 | \ncth-2(mg599) II; moc-6(rae296) III | \nThis work | \n
| E. coli | \nBW25113 | \nWild type | \nNIG | \n
| E. coli | \nJW0764-2 | \nΔmoaA753::kan | \nNIG | \n
The nematode C. elegans has recently emerged as a tractable system for genetic discovery in Moco biology (Warnhoff and Ruvkun 2019; Warnhoff et al. 2021). In C. elegans, endogenous Moco biosynthesis is not required for growth, development, and reproduction. C. elegans mutants defective in Moco biosynthesis are viable because they acquire and use Moco from their bacterial diet (Warnhoff and Ruvkun 2019; Warnhoff et al. 2021). Thus, C. elegans has 2 redundant pathways for maintaining cellular Moco: endogenous synthesis and dietary acquisition. C. elegans is the only known animal for which Moco biosynthesis is dispensable.
\nThe human MOCS2A enzyme is required for Moco synthesis (Schwarz et al. 2009). MOCS2A is a small sulfur-carrier protein that is necessary for the conversion of cyclic pyranopterin monophosphate to molybdopterin. This reaction is the second step in the linear synthesis pathway of Moco from guanosine triphosphate (Schwarz et al. 2009). Loss-of-function mutations in MOCS2A cause human Moco deficiency, a rare and lethal inborn error of metabolism (Johnson et al. 2001). However, the C. elegans orthologue of MOCS2A has not been molecularly identified or genetically studied. Using amino acid sequence analysis, we determined that the C. elegans K10D2.7 genomic locus encodes a protein with high amino acid similarity and identity to human MOCS2A (Fig. 1A) (Altschul et al. 1990). To determine the role K10D2.7 plays in Moco biosynthesis, we used CRISPR/Cas9 to engineer 3 independent deletions of K10D2.7 (Fig. 1B) (Cho et al. 2013; Ghanta and Mello 2020). These K10D2.7 deletion alleles (rae295, rae296, and rae297) are likely null mutations because they eliminate >80% of the K10D2.7 coding sequence. As we have shown for other C. elegans mutations in Moco biosynthetic machinery, the K10D2.7 deletion alleles rae295, rae296, or rae297 cause no defects when animals are cultured under standard laboratory conditions feeding on wild-type E. coli which also synthesizes Moco. To determine if K10D2.7 is necessary for endogenous Moco synthesis, we cultured wild-type, rae295, rae296, and rae297 mutant animals on wild-type and ΔmoaA E. coli, a mutant bacterial strain that is unable to synthesize Moco. rae295, rae296, and rae297 mutant animals grew well on wild-type (Moco producing) E. coli but displayed a completely penetrant developmental arrest when cultured on ΔmoaA (Moco deficient) mutant E. coli (Fig. 1C). In contrast to K10D2.7 mutant animals, wild-type C. elegans grow well on wild-type or ΔmoaA mutant E. coli, although we observed a subtle, but statistically significant, reduction in the growth of wild-type animals on ΔmoaA mutant E. coli (Fig. 1C). By eliminating Moco from the diet, we revealed the defect in endogenous Moco synthesis caused by the rae295, rae296, and rae297 mutations. We conclude that K10D2.7 is required for C. elegans Moco synthesis and name this gene moc-6 (MOlybdenum Cofactor biosynthesis).
\nMoco deficiency in C. elegans also results in embryonic lethality when maternal animals are deprived of both endogenous and exogenous sources of Moco during reproductive adulthood (Warnhoff and Ruvkun 2019). moc-6 mutant C. elegans mothers were fed wild-type E. coli until the L4 stage of development, one stage prior to reproductive adulthood. These moc-6 mutant L4 mothers were then shifted onto wild-type or ΔmoaA mutant E. coli and the hatching rate of their progeny was determined. When moc-6 mutant animals were shifted to a Moco deficient diet, 0% of their embryos hatched (Fig. 1E). In contrast, 100% of moc-6 mutant embryos hatched when their mothers were fed wild-type E. coli. Hatching rates of wild-type embryos were not affected by the maternal diet (Fig. 1E). We conclude that maternal dietary Moco is sufficient to support the required Moco-dependent enzymes for embryonic development. Whether dietary Moco is acting maternally or embryonically to support embryonic development remains to be determined.
\nPrevious genetic studies of the C. elegans Moco biosynthetic pathway demonstrated that Moco is required for the detoxification of sulfites produced by the sulfur amino acid metabolism pathway governed by cystathionase (cth-2) and cysteine dioxygenase (cdo-1). The activities of cth-2 and cdo-1 are required for the developmental arrest that occurs when C. elegans are defective in Moco synthesis and lack dietary Moco (Warnhoff and Ruvkun 2019). Loss-of-function/null mutations in cth-2 or cdo-1 suppress the developmental arrest displayed by moc mutant C. elegans grown on Moco-deficient E. coli (Warnhoff and Ruvkun 2019). To determine if the developmental arrest of moc-6 mutant C. elegans was also dependent upon cth-2 and cdo-1, we engineered cth-2; moc-6 and moc-6; cdo-1 double mutant animals and assessed their growth on wild-type and ΔmoaA mutant E. coli. In contrast to moc-6 single mutant animals, cth-2; moc-6 and moc-6; cdo-1 double mutant animals grew well on both wild-type and ΔmoaA mutant E. coli (Fig. 1D). cth-2 and cdo-1 single mutant animals grow well on both wild-type and ΔmoaA mutant E. coli (Warnhoff and Ruvkun 2019). Furthermore, mutations in cth-2 or cdo-1 suppressed the embryogenesis defects displayed by moc-6 mutant mothers fed on Moco-deficient E. coli (Fig. 1E). Although, we find that moc-6; cdo-1 double mutant embryos derived from mothers fed ΔmoaA mutant E. coli do not hatch at the same rate as wild type. Further studies are required to account for this observation. Together, these data demonstrate that cth-2 and cdo-1 inhibit normal development when Moco is deficient due to loss of both moc-6 and dietary Moco. CTH-2 and CDO-1 act in a catabolic pathway to produce sulfite from the sulfur amino acids methionine and cysteine. Sulfites are extremely toxic during Moco deficiency due to inactivity of the Moco-requiring sulfite oxidase enzyme, SUOX-1. Loss of cth-2 or cdo-1 prevents the metabolic production of sulfites, alleviates the cellular stress caused by inactive SUOX-1 (concomitant with Moco deficiency), and permits growth, development, and reproduction (Warnhoff and Ruvkun 2019). The mechanism by which sulfite promotes larval arrest and death remains enigmatic in both our C. elegans models and in human patients suffering from Moco deficiency.
\nThis study demonstrates that moc-6 is required for endogenous C. elegans Moco biosynthesis and amino acid sequence analyses strongly suggest that moc-6 is orthologous to human MOCS2A.
\n","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215: 403-10.","pubmedId":"2231712","doi":""},{"reference":"Brenner S. 1974. The genetics of Caenorhabditis elegans. Genetics 77: 71-94.","pubmedId":"4366476","doi":""},{"reference":"Cho SW, Lee J, Carroll D, Kim JS, Lee J. 2013. Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics 195: 1177-80.","pubmedId":"23979576","doi":""},{"reference":"Ghanta KS, Mello CC. 2020. Melting dsDNA Donor Molecules Greatly Improves Precision Genome Editing in Caenorhabditis elegans. Genetics 216: 643-650.","pubmedId":"32963112","doi":""},{"reference":"Johnson JL, Coyne KE, Rajagopalan KV, Van Hove JL, Mackay M, Pitt J, Boneh A. 2001. Molybdopterin synthase mutations in a mild case of molybdenum cofactor deficiency. Am J Med Genet 104: 169-73.","pubmedId":"11746050","doi":""},{"reference":"Schwarz G, Mendel RR, Ribbe MW. 2009. Molybdenum cofactors, enzymes and pathways. Nature 460: 839-47.","pubmedId":"19675644","doi":""},{"reference":"Warnhoff K, Hercher TW, Mendel RR, Ruvkun G. 2021. Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans. Genes Dev 35: 212-217.","pubmedId":"33446569","doi":""},{"reference":"Warnhoff K, Ruvkun G. 2019. Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification. Nat Chem Biol 15: 480-488.","pubmedId":"30911177","doi":""}],"title":"moc-6/MOCS2A is necessary for molybdenum cofactor synthesis in C. elegans","reviews":[{"reviewer":{"displayName":"Stephan von Reuss"},"openAcknowledgement":true,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"KJ Yook"},"openAcknowledgement":false,"submitted":null}]}]}},"species":{"species":[{"value":"acer saccharum","label":"Acer saccharum","imageSrc":"","imageAlt":"","mod":"TreeGenes","modLink":"https://treegenesdb.org","linkVariable":""},{"value":"achillea millefolium","label":"Achillea millefolium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"acinetobacter baylyi","label":"Acinetobacter baylyi","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"actinobacteria bacterium","label":"Actinobacteria bacterium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adelges tsugae","label":"Adelges tsugae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adenocaulon chilense","label":"Adenocaulon chilense","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aedes japonicus","label":"Aedes japonicus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aegorhinus vitulus","label":"Aegorhinus vitulus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alaimidae","label":"Alaimidae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"allobates femoralis","label":"Allobates femoralis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alnus glutinosa","label":"Alnus glutinosa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alosa aestivalis","label":"Alosa aestivalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alosa pseudoharengus","label":"Alosa pseudoharengus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alternaria alternata","label":"Alternaria alternata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"amynthas agrestis","label":"Amynthas Agrestis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ancylostoma caninum","label":"Ancylostoma caninum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ancylostoma ceylanicum","label":"Ancylostoma ceylanicum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anemone multifida","label":"Anemone multifida","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anguilla rostrata","label":"Anguilla rostrata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anisakis simplex","label":"Anisakis simplex","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anomala albopilosa","label":"Anomala albopilosa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anthomyiidae sp","label":"Anthomyiidae sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anthomyiidae sp","label":"Anthomyiidae sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"arabidopsis","label":"Arabidopsis","imageSrc":"arabidopsis.png","imageAlt":"Arabidopsis graphic by Zoe Zorn CC BY 4.0","mod":"TAIR","modLink":"https://arabidopsis.org","linkVariable":""},{"value":"architeuthis dux","label":"Architeuthis dux","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"arion vulgaris","label":"Arion vulgaris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"armeria","label":"Armeria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"artemia","label":"Artemia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"arthrobacter sp.","label":"Arthrobacter sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ascaridia","label":"Ascaridia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ascaridia galli","label":"Ascaridia galli","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"asparagopsis taxiformis","label":"Asparagopsis taxiformis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"astatotilapia burtoni","label":"Astatotilapia burtoni","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"avena sativa","label":"Avena sativa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aves","label":"Aves","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus","label":"Bacillus (firmicutes)","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus cereus","label":"Bacillus cereus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus mycoides","label":"Bacillus mycoides","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus subtilis","label":"Bacillus subtilis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus thuringiensis","label":"Bacillus thuringiensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus toyonensis","label":"Bacillus toyonensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus wiedmannii","label":"Bacillus wiedmannii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacteria","label":"Bacteria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacteriophage","label":"Bacteriophage","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bactrocera","label":"Bactrocera sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"batrachospermum gelatinosum","label":"Batrachospermum gelatinosum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"betula lenta","label":"Betula lenta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"betula nigra","label":"Betula nigra","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bombus dahlbohmii","label":"Bombus dahlbohmii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bombus terrestris","label":"Bombus terrestris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bombyx mori","label":"Bombyx mori","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bos taurus","label":"Bos Taurus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"brachygobius doriae","label":"Brachygobius doriae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"brassica oleracea","label":"Brassica oleracea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"brassica rapa","label":"Brassica rapa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"brugia malayi","label":"Brugia malayi","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"burkholderia thailandensis","label":"Burkholderia thailandensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"buttiauxella","label":"Buttiauxella","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caenorhabditis brenneri","label":"Caenorhabditis brenneri","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"caenorhabditis briggsae","label":"Caenorhabditis briggsae","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"c. elegans","label":"Caenorhabditis elegans","imageSrc":"c-elegans.jpg","imageAlt":"C. elegans graphic by Zoe Zorn CC BY 4.0","mod":"WormBase","modLink":"https://wormbase.org","linkVariable":""},{"value":"caenorhabditis inopinata","label":"Caenorhabditis inopinata","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"caenorhabditis japonica","label":"Caenorhabditis japonica","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"caenorhabditis nigoni","label":"Caenorhabditis nigoni","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caenorhabditis remanei","label":"Caenorhabditis remanei","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"caenorhabditis tropicalis","label":"Caenorhabditis tropicalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"calidifontibacillus","label":"Calidifontibacillus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"calidifontibacillus erzuremensis","label":"Calidifontibacillus erzuremensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"calliphora sp","label":"Calliphora sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caltha sagittata","label":"Caltha sagittata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cambarus latimanus","label":"Cambarus latimanus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"candida albicans","label":"Candida albicans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"canis familiaris","label":"Canis familiaris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cannabis sativa","label":"Cannabis sativa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caretta caretta","label":"Caretta caretta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cassiopea xamachana","label":"Cassiopea xamachana","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caulobacter vibrioides","label":"Caulobacter vibrioides","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cephalopods","label":"Cephalopoda","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cerastium arvense","label":"Cerastium arvense","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ceriodaphnia","label":"Ceriodaphnia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ceroglossus suturalis","label":"Ceroglossus suturalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chaetoceros","label":"Chaetoceros","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chamaecrista fasciculata","label":"Chamaecrista fasciculata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chilicola chalcidiformis","label":"Chilicola chalcidiformis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chitinimonas","label":"Chitinimonas","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chlamydomonas reinhardtii","label":"Chlamydomonas reinhardtii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chromobacterium","label":"Chromobacterium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chrysemys picta","label":"Chrysemys picta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chrysoperla rufilabris","label":"Chrysoperla rufilabris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"citrus","label":"Citrus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"clavibacter sp.","label":"Clavibacter sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"colinus virginianus","label":"Colinus virginianus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"crassostrea virginica","label":"Crassostrea virginica","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"crithidia fasciculata","label":"Crithidia fasciculata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cutibacterium acnes","label":"Cutibacterium acnes","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cyanobacteria","label":"Cyanobacteria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"daphnia","label":"Daphnia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"daphnia pulex","label":"Daphnia pulex","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"diabrotica virgifera","label":"Diabrotica virgifera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"diabrotica virgifera virgifera virus 1","label":"Diabrotica virgifera virgifera virus 1","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"d. discoideum","label":"Dictyostelium discoideum","imageSrc":"dicty.png","imageAlt":"D. discoideum","mod":"dictyBase","modLink":"http://dictybase.org","linkVariable":""},{"value":"diptera","label":"Diptera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dotocryptus bellicosus","label":"Dotocryptus bellicosus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"drechmeria coniospora","label":"Drechmeria coniospora","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"drosophila","label":"Drosophila","imageSrc":"drosophila.png","imageAlt":"Drosophila graphic by Zoe Zorn CC BY 4.0","mod":"FlyBase","modLink":"https://flybase.org/doi/","linkVariable":"doi"},{"value":"dryopteris campyloptera","label":"Dryopteris campyloptera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dryopteris expansa","label":"Dryopteris expansa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dryopteris intermedia","label":"Dryopteris intermedia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dugesia dorotocephala","label":"Dugesia dorotocephala","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"elasmobranchii","label":"Elasmobranchii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"embryophyta","label":"Embryophyta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"enoploteuthis chunii","label":"Enoploteuthis chunii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"enterobacter aerogenes","label":"Enterobacter aerogenes","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"enterococcus raffinosus","label":"Enterococcus raffinosus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"epichloë coenophiala","label":"Epichloë coenophiala","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"equus caballus","label":"Equus caballus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"erigeron sp","label":"Erigeron sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"eristalis","label":"Eristalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"eruca vesicaria","label":"Eruca vesicaria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"erwinia carotovora","label":"Erwinia carotovora","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"erythronium americanum","label":"Erythronium americanum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"escherichia coli","label":"Escherichia coli","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"eukaryota","label":"Eukaryotes","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"felis catus","label":"Felis catus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"francisella novicida","label":"Francisella novicida","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"francisella tularensis","label":"Francisella tularensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"fraxinus americana","label":"Fraxinus americana","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"fucus distichus","label":"Fucus distichus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"fungi","label":"Fungi","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"gasteropelecus sp.","label":"Gasteropelecus sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"geranium sp","label":"Geranium sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"girardia","label":"Girardia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"glaucomys volans","label":"Glaucomys volans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"glycine max","label":"Glycine max","imageSrc":"","imageAlt":"","mod":"Soybase","modLink":"https://soybase.org","linkVariable":""},{"value":"glyptemys insculpta","label":"Glyptemys insculpta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"gossypium hirsutum","label":"Gossypium hirsutum","imageSrc":"","imageAlt":"","mod":"CottonGen","modLink":"https://www.cottongen.org/","linkVariable":""},{"value":"gromphadorhina portentosa","label":"Gromphadorhina portentosa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"gryllodes sigillatus","label":"Gryllodes sigillatus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"haliotis rufescens","label":"Haliotis rufescens","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hepacivirus hominis","label":"Hepatitis C Virus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"herpes simplex virus type 1","label":"Herpes simplex virus type 1","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"human","label":"Human","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"human coronavirus oc43","label":"Human coronavirus OC43","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hydra vulgaris","label":"Hydra vulgaris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hydropsyche sp","label":"Hydropsyche sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hymenoptera","label":"Hymenoptera","imageSrc":"","imageAlt":"","mod":"Hymenoptera Genome Database","modLink":"https://hymenoptera.elsiklab.missouri.edu/","linkVariable":""},{"value":"hypochaeris radicata","label":"Hypochaeris radicata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hypodynerus vespiformis","label":"Hypodynerus vespiformis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"iflaviridae","label":"Iflaviridae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"iflavuris","label":"Iflavirus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ipomoea hederacea","label":"Ipomoea hederacea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ischnomera","label":"Ischnomera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ischnomera ruficollis","label":"Ischnomera ruficollis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"julidochromis marlieri","label":"Julidochromis marlieri","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"juniperus virginiana","label":"Juniperus virginiana","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"kluyveromyces marxianus","label":"Kluyveromyces marxianus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"l. casei","label":"L. casei","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lacticaseibacillus casei","label":"Lacticaseibacillus casei","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"larentiinae sp","label":"Larentiinae sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"laurus nobilis","label":"Laurus nobilis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lepidoptera","label":"Lepidoptera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"leucanthemum vulgare","label":"Leucanthemum vulgare","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"linepithema humile","label":"Linepithema humile","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"liometopum occidentale","label":"Liometopum occidentale","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lolium arundinaceum","label":"Lolium arundinaceum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lumbriculus variegatus","label":"Lumbriculus variegatus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lumbricus terrestris","label":"Lumbricus terrestris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lupinus polyphyllus","label":"Lupinus polyphyllus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lycorma delicatula","label":"Lycorma delicatula","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lynx rufus","label":"Lynx rufus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"magnaporthe oryzae","label":"Magnaporthe oryzae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"mammalia","label":"Mammalia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"manihot esculenta","label":"Manihot esculenta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"medicago lupulina","label":"Medicago lupulina","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"meloidogyne","label":"Meloidogyne","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"mimus polyglottos","label":"Mimus polyglottos","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bryophyta","label":"Mosses","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"mouse","label":"Mouse","imageSrc":"","imageAlt":"","mod":"MGI","modLink":"https://informatics.jax.org","linkVariable":""},{"value":"m. minutoides","label":"Mus minutoides","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"mycobacterium smegmatis","label":"Mycobacterium smegmatis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"nakaseomyces glabratus","label":"Nakaseomyces glabratus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"nauphoeta cinerea","label":"Nauphoeta cinerea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"neurospora","label":"Neurospora","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"n. benthamiana","label":"Nicotiana benthamiana","imageSrc":"","imageAlt":"","mod":"Solgenomics Network","modLink":"https://solgenomics.net/organism/Nicotiana_benthamiana/genome","linkVariable":""},{"value":"nicotiana tabacum","label":"Nicotiana tabacum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"noctuidae","label":"Noctuidae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"noctuidae sp","label":"Noctuidae sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"nothobranchius furzeri","label":"Nothobranchius furzeri","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"onchocerca volvulus","label":"Onchocerca volvulus","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"orconectes virilis","label":"Orconectes virilis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ormia ochracea","label":"Ormia ochracea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"o. sativa","label":"Oryza sativa","imageSrc":"","imageAlt":"","mod":"Gramene","modLink":"https://www.gramene.org/","linkVariable":""},{"value":"other","label":"Other","imageSrc":"","imageAlt":"","mod":null,"modLink":null,"linkVariable":null},{"value":"oxalis enneaphylla","label":"Oxalis enneaphylla","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"paenarthrobacter nicotinovorans","label":"Paenarthrobacter nicotinovorans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"paenarthrobacter nicotinovorans","label":"Paenarthrobacter nicotinovorans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pantoea","label":"Pantoea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pantoea agglomerans","label":"Pantoea agglomerans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"papaver sp","label":"Papaver sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"paramecium bursaria","label":"Paramecium bursaria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"partitiviridae","label":"Partitiviridae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pelodiscus sinensis","label":"Pelodiscus sinensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"perezia recurvata","label":"Perezia recurvata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"petromyzon marinus","label":"Petromyzon marinus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"photinus pyralis","label":"Photinus pyralis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"photinus pyralis associated partiti-like virus","label":"Photinus pyralis associated partiti-like virus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"photinus pyralis iflavirus 1","label":"Photinus pyralis iflavirus 1","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"physcomitrium patens","label":"Physcomitrium patens","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pinus strobus","label":"Pinus strobus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pinus taeda","label":"Pinus taeda","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"platycheirus","label":"Platycheirus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"plectus sambesii","label":"Plectus sambesii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pogonomyrmex occidentalis","label":"Pogonomyrmex occidentalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"poncirus trifoliata","label":"Poncirus trifoliata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"populus deltoides","label":"Populus deltoides","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"potato virus y","label":"Potato virus Y","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"primula magellanica","label":"Primula magellanica","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pristionchus pacificus","label":"Pristionchus pacificus","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"prunus persica","label":"Prunus persica","imageSrc":"","imageAlt":"","mod":"Genome Database for Rosaceae","modLink":"https://www.rosaceae.org/","linkVariable":""},{"value":"psalmopoeus iriminia","label":"Psalmopoeus iriminia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudanabaena sp.","label":"Pseudanabaena sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas","label":"Pseudomonas","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas aeruginosa","label":"Pseudomonas aeruginosa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas glycinae","label":"Pseudomonas glycinae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas putida","label":"Pseudomonas putida","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas syringae","label":"Pseudomonas syringae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pterophyllum scalare","label":"Pterophyllum scalare","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"python regius","label":"Python regius","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"quercus macrocarpa","label":"Quercus macrocarpa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ralstonia solanacearum","label":"Ralstonia solanacearum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ranitomeya imitator","label":"Ranitomeya imitator","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ranunculus peduncularis","label":"Ranunculus peduncularis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"rat","label":"Rat","imageSrc":"","imageAlt":"","mod":"RGD","modLink":"https://rgd.mcw.edu","linkVariable":""},{"value":"rheinheimera","label":"Rheinheimera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ribes rubrum","label":"Ribes rubrum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"sars-cov-2","label":"SARS-CoV-2","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"s. cerevisiae","label":"Saccharomyces cerevisiae","imageSrc":"yeast.png","imageAlt":"Yeast graphic by Zoe Zorn CC BY 4.0","mod":"SGD","modLink":"https://yeastgenome.org","linkVariable":""},{"value":"saccharomyces paradoxus","label":"Saccharomyces paradoxus ","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"s. uvarum","label":"Saccharomyces uvarum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"schistosoma","label":"Schistosoma","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"schizosaccharomyces japonicus","label":"Schizosaccharomyces japonicus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"s. pombe","label":"Schizosaccharomyces pombe","imageSrc":"pombe.png","imageAlt":"Pombe graphic by Zoe Zorn © Caltech","mod":"PomBase","modLink":"https://www.pombase.org/reference/PMID:","linkVariable":"pmId"},{"value":"schmidtea mediterranea","label":"Schmidtea mediterranea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"senecio sp","label":"Senecio sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"simocephalus","label":"Simocephalus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"siraitia grosvenorii","label":"Siraitia grosvenorii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"solanum lycopersicum","label":"Solanum lycopersicum","imageSrc":"","imageAlt":"","mod":"Solgenomics Network","modLink":"https://solgenomics.net/organism/1/view/","linkVariable":""},{"value":"sorghum","label":"Sorghum","imageSrc":"","imageAlt":"","mod":"SorghumBase","modLink":"https://www.sorghumbase.org","linkVariable":""},{"value":"spiroplasma eriocheiris","label":"Spiroplasma eriocheiris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"staphylococcus aureus","label":"Staphylococcus aureus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"staphylococcus epidermidis","label":"Staphylococcus epidermidis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"steinernema carpocapsae","label":"Steinernema carpocapsae","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"https://wormbase.org","linkVariable":""},{"value":"steinernema hermaphroditum","label":"Steinernema hermaphroditum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"stenotrophomonas geniculata","label":"Stenotrophomonas geniculata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"streptococcus gordonii ","label":"Streptococcus gordonii ","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"streptococcus mutans","label":"Streptococcus mutans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":" streptococcus pneumoniae","label":"Streptococcus pneumoniae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"s. purpuratus","label":"Strongylocentrotus purpuratus","imageSrc":"","imageAlt":"","mod":"Echinobase","modLink":"https://www.echinobase.org","linkVariable":""},{"value":"strongyloides ratti","label":"Strongyloides ratti","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"sulfolobus","label":"Sulfolobus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"symphoricarpos albus","label":"Symphoricarpos albus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"syncirsodes","label":"Syncirsodes","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"synechococcus elongatus","label":"Synechococcus elongatus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"syrphidae","label":"Syrphidae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tarantobelus jeffdanielsi","label":"Tarantobelus jeffdanielsi","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"taraxacum officinale","label":"Taraxacum officinale","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tatochila theodice","label":"Tatochila theodice","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tetrahymena","label":"Tetrahymena","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tetramorium immigrans","label":"Tetramorium immigrans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tomato brown rugose fruit virus","label":"ToBRFV","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"trachemys scripta","label":"Trachemys scripta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tribolium castaneum","label":"Tribolium castaneum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"trichoptera","label":"Trichoptera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"trichuris muris","label":"Trichuris muris","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"trifolium repens","label":"Trifolium repens","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"trypoxylus dichotomus","label":"Trypoxylus dichotomus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tsuga canadensis","label":"Tsuga canadensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ulva expansa","label":"Ulva expansa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"universal","label":"Universal","imageSrc":"","imageAlt":"","mod":null,"modLink":null,"linkVariable":null},{"value":"vargula hilgendorfii","label":"Vargula hilgendorfii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"vespula vulgaris","label":"Vespula vulgaris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"virus","label":"Virus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"watasenia scintillans","label":"Watasenia scintillans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"wolbachia pipientis","label":"Wolbachia pipientis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"xenopus","label":"Xenopus","imageSrc":"xenopus.png","imageAlt":"Xenopus graphic by Zoe Zorn CC BY 4.0","mod":"XenBase","modLink":"https://xenbase.org","linkVariable":""},{"value":"xenorhabdus griffiniae","label":"Xenorhabdus griffiniae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"yramea cytheris","label":"Yramea cytheris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"zaprionus indianus","label":"Zaprionus indianus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"zea mays","label":"Zea mays","imageSrc":"","imageAlt":"","mod":"MaizeGDB","modLink":"https://www.maizegdb.org","linkVariable":""},{"value":"zebrafish","label":"Zebrafish","imageSrc":"zebrafish.png","imageAlt":"Zebrafish graphic by Zoe Zorn CC BY 4.0","mod":"ZFIN","modLink":"https://zfin.org","linkVariable":""}]}},"pageContext":{"id":"18f8df10-93fd-436e-9c33-8df4746ede12","citedBy":[{"title":"Hypoxia-inducible factor promotes cysteine homeostasis inCaenorhabditis elegans","url":"http://dx.doi.org/10.1101/2023.05.04.538701","datePublished":"May 2023","firstAuthor":"Warnhoff, K"},{"title":"Hypoxia-inducible factor induces cysteine dioxygenase and promotes cysteine homeostasis in Caenorhabditis elegans","url":"http://dx.doi.org/10.7554/elife.89173","journal":"eLife","datePublished":"February 2024","firstAuthor":"Warnhoff, K"},{"title":"Hypoxia-inducible factor induces cysteine dioxygenase and promotes cysteine homeostasis in Caenorhabditis elegans","url":"http://dx.doi.org/10.7554/elife.89173.3","journal":"eLife","datePublished":"February 2024","firstAuthor":"Warnhoff, K"},{"title":"Hypoxia-inducible factor induces cysteine dioxygenase and promotes cysteine homeostasis in Caenorhabditis elegans","url":"http://dx.doi.org/10.7554/elife.89173","journal":"eLife","datePublished":"February 2024","firstAuthor":"Warnhoff, K"},{"title":"Robustness and variability inCaenorhabditis elegansdauer gene expression","url":"http://dx.doi.org/10.1101/2024.08.15.608164","datePublished":"August 2024","firstAuthor":"Corchado, JC"},{"title":"The parasitic nematode Haemonchus contortus lacks molybdenum cofactor synthesis, leading to sulphite sensitivity and lethality in vitro","url":"https://doi.org/10.1016/j.ijpara.2024.11.004","journal":"International Journal for Parasitology","datePublished":"November 2024","firstAuthor":"Brinzer, RA"},{"title":"XDH-1 inactivation causes xanthine stone formation inC. eleganswhich is inhibited by SULP-4-mediated anion exchange in the excretory cell","url":"https://doi.org/10.1101/2025.01.24.634556","datePublished":"January 2025","firstAuthor":"Snoozy, J"},{"title":"XDH-1 inactivation causes xanthine stone formation in Caenorhabditis elegans which is inhibited by SULP-4-mediated anion exchange in the excretory cell","url":"https://doi.org/10.1371/journal.pbio.3003410","journal":"PLOS Biology","datePublished":"September 2025","firstAuthor":"Snoozy, J"}],"parsedCsv":{"csvHeader":[],"csvData":[]}}}, "staticQueryHashes": ["2114697108"]}