Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification

Abstract

The kingdoms of life share many small molecule cofactors and coenzymes. Molybdenum cofactor (Moco) is synthesized by many archaea, bacteria, and eukaryotes, and is essential for human development. The genome of Caenorhabditis elegans contains all of the Moco biosynthesis genes, and surprisingly these genes are not essential if the animals are fed a bacterial diet that synthesizes Moco. C. elegans lacking both endogenous Moco synthesis and dietary Moco from bacteria arrest development, demonstrating interkingdom Moco transfer. Our screen of Escherichia coli mutants identifies genes necessary for synthesis of bacterial Moco or transfer to C. elegans. Developmental arrest of Moco-deficient C. elegans is caused by loss of sulfite oxidase, a Moco-requiring enzyme, and is suppressed by mutations in either C. elegans cystathionine gamma-lyase or cysteine dioxygenase, blocking toxic sulfite production from cystathionine. Thus, we define the genetic pathways for an interkingdom dialogue focused on sulfur homeostasis.

Figure 1:. Moco acquisition and biosynthesis are redundantly required for life in C. elegans.

(A) By protein sequence homology to human and bacterial Moco biosynthetic enzymes, the inferred C. elegans Moco biosynthesis pathway is displayed. (B) The growth of wild-type and moc-1(ok366) animals on either wild-type or ΔMoaA E. coli (lab microbial diet) or 6 bacterial strains native to the Caenorhabditis ecosystem (natural microbial diet) was quantified after 72 hours of growth from the first larval stage. (C,D) The growth of moc-1(ok366) animals on either wild-type or mutant E. coli was quantified after 72 hours of growth from the first larval stage. Before being fed to C. elegans, the wild-type and mutant E. coli strains were initially cultured without (C, Control) or with 100μm sodium molybdate (D, +100μm molybdate) in LB. The mutant E. coli strains used here are nonpolar deletions of the indicated genes, except for the MobA mutant strain which still has the Kanamycin resistance cassette inserted into the MobA open reading frame. Box plots display the median, upper, and lower quartiles while whiskers indicate minimum and maximum data points. Sample size (n) is displayed for each experiment.

Figure 2:. C. elegans acquire cPMP and Moco from dietary E. coli.

(A) Wild-type, (B) moc-5(mg589), (C) moc-4(ok2571), (D) moc-3(ku300), (E) moc-2(mg595), and (F) moc-1(ok366) animals were cultured from synchronized L1 larvae for 72 hours on wild-type and mutant E. coli disrupted at distinct steps in Moco biosynthesis. E. coli mutations used here are nonpolar and should not disrupt other genes in their operons. Animal lengths were determined for each condition. The moc-1(ok366) mutation is a 1.4kb deletion that is predicted to be a null allele. The moc-4(ok2571) mutation is a 1.8kb deletion that is predicted to be a null allele. moc-2(mg595) encodes a (Gly72Stop) followed by a frameshift that is likely a null allele. moc-3(ku300) is an established genetic reagents that represents a likely loss-of-function or null allele. moc-5(mg589) is a missense mutation (G303R) substitution loss-of-function or null allele. Box plots display the median, upper, and lower quartiles while whiskers indicate minimum and maximum data points. Sample size (n) is displayed for each experiment.

Figure 3:. cth-2 and cdo-1 are necessary for the growth arrest and death caused by Moco deficiency.

(A) moc-1(ok366), cth-2(mg599);moc-1(ok366), and cdo-1(mg622) moc-1(ok366) mutant animals were synchronized at the L1 stage and cultured on wild-type and ΔMoaA E. coli for approximately 48 hours. Animal lengths were determined for each condition. Box plots display the median, upper, and lower quartiles while whiskers indicate minimum and maximum data points. Sample size (n) is the number of individuals assayed and is displayed for each experiment. (B) Cartoons of CTH-2 (upper) and CDO-1 (lower) proteins. Colored vertical lines indicate amino acid substitutions identified in our screen as suppressors of (green) moc-2(mg595), (red) moc-3(ku300), or (blue) moc-4(ok2571) mutant C. elegans when cultured on ΔMoaA E. coli. *, reference alleles for each gene.

Figure 4:. Endogenously produced sulfites inhibit growth and development during Moco deficiency.

(A) Simplified cartoon of sulfur amino acid catabolism beginning with methionine. Here we highlight the roles of CTH/cth-2, CDO/cdo-1, and the Moco requiring enzyme SUOX/suox-1. CBS is cystathionine beta synthase and AAT is aspartate aminotransferase. (B-F) Wild-type, cth-2(mg599), cdo-1(mg622), cth-2(mg599);moc-1(ok366), cdo-1(mg622) moc-1(ok366) and cth-2(mg599); cdo-1(mg622) moc-1(ok366) animals were cultured from the first larval stage on ΔMoaA E. coli supplemented with various concentrations of (B-E) cysteine or (F) 5mM sulfite. Animals were cultured for 48 hours and animal lengths were quantified. (B-D) Average values and standard deviation are displayed for each concentration of supplemental cysteine. (E,F) Growth of C. elegans mutants was scored on (E) 3mM supplemental cysteine or (F) 5mM supplemental sulfite. Box plots display the median, upper, and lower quartiles while whiskers indicate minimum and maximum data points. Sample size (n) is the number of individuals assayed and is displayed for each experiment. The data in Figure 4E summarizes the data in Figures 4B–D, highlighting the critical 3mM concentration of supplemental cysteine.

Figure 5:. suox-1 hypomorphic allele phenocopies Moco deficiency in C. elegans.

Analyses of the suox-1(gk738847) hypomorphic allele: cth-2(mg599), cdo-1(mg622), suox-1(gk738847), cth-2(mg599);suox-1(gk738847), and cdo-1(mg622) suox-1(gk738847) animals were cultured from the first larval stage on (A) wild-type or (B) ΔMoaA E. coli. (C-F) Wild-type or suox-1(gk738847) mutant animals were cultured from the first larval stage on (C) wild-type E. coli, or wild-type E. coli supplemented with (D) 3mM cysteine, (E) 5mM sulfite, or (F) 5mM sulfate. Animals were cultured for 72 hours and animal lengths were quantified. Box plots display the median, upper, and lower quartiles while whiskers indicate minimum and maximum data points. Sample size (n) is displayed for each experiment. Please note, the wild-type data points displayed in Figure 5C–F are also displayed in Supplementary Fig.5, as these assays were performed in parallel and utilize the same wild-type controls.

Figure 6:. Tissue specific rescue of suox-1, moc-1, and cth-2.

(A) Nontransgenic moc-1(ok366) and moc-1(ok366) transgenic animals expressing moc-1 under the control of the moc-1 (native, gray), sng-1 (neurons, green), vha-6 (intestine, purple), myo-3 (muscle, orange), or col-10 (hypodermis, blue) promoters were cultured for 72 hours from the first larval stage on MoaA mutant E. coli and animal lengths were quantified. Each rescue construct was tested with 3 independently isolated transgenes. (B) Nontransgenic cth-2(mg599); moc-1(ok366) and cth-2(mg599); moc-1(ok366) transgenic animals expressing cth-2 under the control of the cth-2 (native, gray), sng-1 (neurons, green), vha-6 (intestine, purple), myo-3 (muscle, orange), or col-10 (hypodermis, blue) promoters were cultured for 72 hours from the first larval stage on MoaA mutant E. coli and animal lengths were quantified. Each rescue construct was tested with 2–3 independently isolated transgenes. Box plots display the median, upper, and lower quartiles while whiskers indicate minimum and maximum data points. Sample size (n) is the number of individuals assayed and is displayed for each experiment.