Microbial Cysteine Degradation Drives Cryptic Sulfide Redox Chemistry in the Gut

doi:10.21203/rs.3.rs-909548/v1

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Microbial Cysteine Degradation Drives Cryptic Sulfide Redox Chemistry in the Gut

https://doi.org/10.21203/rs.3.rs-909548/v1

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published 20 Oct, 2022

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Although microbial biochemistry shapes a dynamic environment in the gut, how bacterial metabolites such as hydrogen sulfide (H₂S) mechanistically alter the gut chemical landscape is poorly understood. Here we show for the first time that H₂S generated during cysteine metabolism drives the reduction of azo (R-N=N-R’) xenobiotics in bacterial cultures, human fecal microbial communities, and in vivo mouse models. Thus, chemical-chemical interactions, derived from microbial community metabolism, are a key missing feature shaping xenobiotic metabolism in the gut. Changing dietary levels of the H₂S xenobiotic redox partner Red 40 transiently decreases mouse fecal sulfide, confirming that a xenobiotic can attenuate sulfide concentration in vivo. Cryptic H₂S redox thus modulates sulfur homeostasis in the gut and the fate of xenobiotics to which humans are regularly exposed.

General Microbiology

Molecular Genetics

hydrogen sulfide

gut chemical landscape

cysteine metabolism

In microbial ecosystems, functional chemistry is dictated by microbial metabolism. Our gut microbiota produce millimolar quantities of hydrogen sulfide (H₂S/HS^-)¹, a highly redox active molecule. Sulfur is an essential element for all life and under physiological conditions in the human gut is found between the -2 and +6 redox states². Biogenic hydrogen sulfide is a Janus-faced molecule in the gut³; while it is toxic to colonocytes^1,4 and mediates antibiotic resistance in bacterial pathogens⁵, hydrogen sulfide also promotes mucosal integrity⁶ and can be neuroprotective⁷. Here we show for the first time that hydrogen sulfide abiotically transfers electrons to dietary and xenobiotic redox partners in the gut, alleviating accumulation of H₂S, modulating xenobiotic metabolism, and shaping the gut chemical landscape.

To investigate hydrogen sulfide redox chemistry with common xenobiotics in the gut, we used azo (R-N=N-R’) chemicals as model potential redox partners. Azo compounds are structurally diverse and common in Western foods and drugs⁸. Critically, physiological effects of many azo chemicals are dictated by reduction of the azo bond. For example, the prevalent azo food dye FD&C Red No. 40 (Red 40) is a potent inhibitor of the human intestinal transporter OATP2B1, while azoreduction of this dye by the gut microbiome rescues inhibition⁹. Azoreduction, however, does not always benefit the host. Cleavage of the azo bond in Red 40 was also recently found to induce colitis in mice overexpressing IL-23¹⁰.

Human luminal H₂S concentrations range from 0.5-3 mM¹¹. Consumption of animal protein correlates with higher luminal H₂S due to high levels of cysteine¹. The mammalian lumen also contains endogenous sulfur sources such as glycans (e.g. mucins) in the mucosa¹², which can be microbially degraded to H₂S. In the human gut, the mechanism of azoreduction is currently assumed to proceed enzymatically through the activity of microbial azoreductases¹³. However, outside of the host environment, in wastewater treatment of industrial azo dyes, azoreduction has also been shown to occur via abiotic chemical reduction mediated by the H₂S released as a byproduct of sulfate respiration by sulfate reducing bacteria. In abiotic azoreduction, two molecules of H₂S transfer four electrons to the azo bond directly or via a redox mediator, resulting in azo fission and oxidation of H₂S to zero valent sulfur (S⁰)¹⁴. Given the universal presence of H₂S in the human gut, we hypothesized that the same chemical fission of azo compounds via sulfide redox could occur.

It was first determined whether H₂S can reduce azo food dyes and drugs⁸ in the absence of biological activity. In sterile anoxic buffer, H₂S transfers its electrons to the azo bond, resulting in bond cleavage and two aminated daughter compounds (Fig. 1a). Five structurally diverse azo compounds representing anti-inflammatory azo-prodrugs as well as food and industrial dyes, all relevant to human exposures, were added to sterile, anoxic buffer containing a physiological concentration (3 mM)^1,11 of hydrogen sulfide. Experiments were carried out in phosphate buffer, with or without flavin mononucleotide (FMN), because redox mediators like FMN and quinone compounds increase the rate of azoreduction by bacteria^15,16. The reaction proceeded for 6 hours and then was halted by precipitating sulfide with excess zinc acetate. Expected azoreduced aromatic amine products, confirmed by LCMS analysis, were generated at a rate reciprocal to the decrease in the azo parent compound (Fig. 1b, Supplementary Fig. 1). Furthermore, direct electron transfer from sulfide to the azo-prodrugs balsalazide (BSZ) and sulfasalazine (SSZ) was observed. Azoreduction of common food dyes Red 40 and Yellow 6, and the industrial dye Congo Red, were dependent upon FMN, indicating that FMN is an electron shuttle for reduction of these compounds.

To determine the relationship between the redox potential and propensity for H₂S reduction, cyclic voltammetry was used to obtain the redox potentials of the azo bonds in compounds from Fig. 1b. We observed a threshold of -0.55mV below which FMN was required for azoreduction (Fig. 1c). Characterizing abiotic electron transfer from H₂S to the azo bond in foods and drugs informs the mechanism of potential sulfide redox chemistry in the gut.

In the gut environment hydrogen sulfide is produced principally by bacterial degradation of thiols, particularly the amino acid cysteine¹¹. To directly interrogate the propensity for H₂S azoreduction in the background of expected enzymatic azoreduction, H₂S azoreduction from cysteine degradation was determined in a bacterium containing known azoreductases. E. coli K-12 W3100 was incubated under strict anoxic conditions with Red 40 at 0.25 mM, 0.5 mM, and 3.0 mM, with or without the addition of cysteine (Fig. 2a). Cysteine contains one thiol moiety and during dissimilatory metabolism releases stoichiometric H₂S¹⁷. Given the 2:1 ratio of H₂S to azo required for azoreduction, 2 mM cysteine theoretically supplies sufficient sulfur for the lower two Red 40 conditions, while the 3 mM Red 40 condition contains excess azo relative to supplemented sulfur. Controls included unamended LB media to determine the extent of enzymatic E. coli azoreduction and sulfide production in a standard medium under anoxic conditions, and serine supplemented media which provides an analogue amino acid missing the thiol moiety. Only cultures amended with cysteine eliminated Red 40 (Fig. 2a, top panel). Red 40 was completely azoreduced in 0.25 and 0.5 mM cultures within 7 and 9.5 hours, respectively. In the initial 5 hours of incubation, sulfide generation was equal among all cysteine conditions. During this time H₂S accumulated to ~ 2 mM, indicating near complete dissimilatory cysteine degradation. No Red 40 azo cleavage was observed by E. coli in anoxic LB. Cultures supplemented with serine behaved identically to LB media controls, indicating that any benefit from additional amino acid influenced neither azoreduction nor H₂S generation. The 3 mM cultures lost 0.82 mM Red 40 during the experimental window; this incomplete loss further indicates the lack of azoreductase activity under these conditions. To quantify the H₂S that donated its electrons to Red 40, H₂S in each Red 40 condition was compared to that in the Red 40-free control, which served as a reference for the maximum H₂S potential (Fig. 2b). After the initial 5 hours of incubation during which H₂S was equal among all cysteine conditions, Red 40 loss began and sulfide decreased in parallel with Red 40 loss until Red 40 was completely azoreduced. Only after complete Red 40 loss did sulfide concentrations again increase. Thus, the observed H₂S concentration as a function of time was not the absolute amount generated during cysteine degradation, but rather the net of H₂S generation and H₂S oxidation via Red 40. In the gut, where many compounds may serve as electron acceptors from H₂S, measured H₂S concentrations are a net value of these competing reactions.

The absence of Red 40 azoreduction in control E. coli cultures indicated that enzymatic activity is not responsible for Red 40 loss under these conditions. To confirm this, enzymatic activity was eliminated while preserving all metabolites of cysteine, including volatile H₂S, by heat inactivation of overnight cultures in sealed vials (121°C, 25 minutes). H₂S accumulated to ~2 mM, indicating complete cysteine degradation. After a 1-hour re-equilibration at 37°C, Red 40 was spiked in at the three experimental concentrations. The results following heat inactivation mirrored the live E. coli incubations – Red 40 loss was observed only in the spent medium of E. coli grown with supplemented cysteine (Fig. 2c). No azoreduction was detected in control cultures. Thus, a metabolite of cysteine degradation was responsible for abiotic azoreduction in the absence of enzymatic and other biological activity.

Sulfide was measured in parallel with Red 40 to elucidate the mechanism of sulfide attenuation observed in E. coli exposed to Red 40. In the absence of Red 40, sulfide concentrations were constant (Fig. 2c). The addition of Red 40 stimulated loss of sulfide proportional to the concentration of Red 40. Sulfide concentrations decreased in an approximate 2:1 stoichiometric ratio to the concentration of reduced Red 40 (Fig. 2d). In live biological systems sulfide can be simultaneously generated and oxidized, obscuring direct observation of the redox reaction. The stoichiometric, concomitant decrease of sulfide and Red40 seen in the absence of microbial activity illustrates the cryptic nature of sulfide redox chemistry.

The prevalence of azo-compounds in Western diets as food dyes is of particular concern because the compounds themselves as well as their metabolites can have physiological effects⁹. Given cysteine’s abundance in dietary protein, we investigated the relative change in sulfide accumulation and azoreduction induced by nutritional and endogenous sulfur sources. A healthy human microbiome was recapitulated from a human fecal sample cultured in anoxic defined media, with subcultures supplemented with different physiological sulfur (i.e. thiol) sources. These included the amino acid cysteine, bovine serum albumin (BSA) as an analog of dietary animal protein, and the highly thiolated endogenous glycoprotein mucin. Thiol-supplemented anaerobic cultures produced more sulfide (Fig. 3a) and reduced Red 40 at a higher rate (Fig. 3b) than thiol-free counterparts. The rate of azoreduction was inversely proportional to the maximum amount of H₂S generated in each condition, with significant differences in the rates of Red 40 reduction among groups (one-way ANOVA (F(6, 14) = 8.828, p< 0.001). Notably, H₂S did not begin to accumulate above background in any of the cultures until the azo substrate was exhausted at approximately 4 hours (Fig. 3a), similar to the pattern observed in live E. coli cultures (Fig. 2a). Thus, we observe cryptic sulfide redox chemistry dictating the concentration of sulfide in real time, providing direct evidence that sulfide azoreduction in the human gut is distinct from enzymatic azoreduction and is a fundamental contributor to total azoreduction by human gut microbiota.

Bioinformatic analysis of sulfide generating enzymes in the gut microbiota was consistent with this direct assessment of hydrogen sulfide production by the human microbiome and highlights the importance of abiotic H₂S redox in human gut physiology. Sequences of nine genes encoding enzymes with sulfidogenic cysteine degradation activity^5,17–19 were searched against the Metaquery database²⁰. The proteins CBS (K01697), CSE (K01758), CysK (K01738), CysM (K12339), CyuA (COG3681), MalY (K14155), MetC (K01760), SseA (K01011) and TnaA (K01667) were analyzed for mean copy number per cell in 2,271 annotated metagenomic samples. CysK and MalY are both present at >1 copy per cell (Fig. 3c). The lower abundances of the other seven proteins suggest that subsets of the human gut microbiome make different contributions to sulfide production. We therefore sought to identify these enzymes in common human gut microbes with completely sequenced genomes to assess the distribution of sulfidogenic cysteine degradation in the human gut (Fig 3d, Supplementary Table 1). We found that multiple sulfidogenic cysteine degrading enzymes are present in the genomes of diverse, abundant, gut microbes. Of note are 7 cysteine degrading enzymes in both Bacteroides ovatus and Enterococcus faecalis, microbes recently implicated in inducing colitis via Red 40 azoreduction¹⁰. In conjunction with the E. coli and fecal microcosm data, cysteine degradation to H₂S lends mechanistic support by which B. ovatus and E. faecalis can azoreduce Red 40 and Yellow 6 to the colitis-inducing product 5-amino-6-hydroxynaphthalene-2-sulfonic acid. Together, these results support the conclusion that H₂S biogenesis through cysteine metabolism is a “core” function of the human gut microbiome, regardless of individual community composition (Fig. 3c, d). The pervasiveness of the 9 sulfidogenic cysteine degrading enzymes among diverse human microbiomes shifts focus away from individual microbes as reducers of Red 40 and towards Red 40 reduction as a community-level function.

These results indicate that many common gut bacteria can release H₂S from cysteine and thus modulate redox chemistry in the gut environment. We therefore directly tested the potential for diet to influence H₂S biogenesis and H₂S-azo redox. Healthy mice with intact, uncontrolled microbiomes were fed purified diets differing in cysteine and Red 40 concentrations. The diets contained a standard concentration of cysteine (4 g/kg)²¹, double that level (8 g/kg), or with cysteine eliminated from the diet (0 g/kg) (Supplementary Table 2) (3 separate cohorts, N=30, 10/diet). Methionine, an essential sulfur amino acid, was present at 1.6 g/kg in all three diets²². After two weeks, mice with uncontrolled microbiomes fed 0 g/kg cysteine produced significantly less fecal sulfide than those consuming a standard cysteine diet (Fig. 4a; Wilcoxon rank-sum test). Mice consuming 8 g/kg, did not differ in fecal sulfide compared to the standard diet (Supplementary Fig. 2). Tuning hydrogen sulfide via cysteine changes was therefore achieved through decreasing cysteine, but not increasing cysteine. We next addressed if hydrogen sulfide in mouse guts was decreased by administration of azo compounds as we observed in E. coli and human fecal microcosms. A controlled diet with standard cysteine levels (4 g/kg) was fed to two cohorts of mice, and after two weeks Red 40 was added to the diet of one cohort (N=20, 10 each ± Red 40). There was a transient decrease in hydrogen sulfide post-Red 40 administration (Fig. 4b), confirming that an azo-containing xenobiotic can attenuate sulfide concentration in animal guts. Hydrogen sulfide electron transfer to the azo redox partner Red 40 therefore alters fecal H₂S concentrations in vivo. Cryptic H₂S redox is thus responsible for gut chemistry previously thought to be solely enzymatic. Sulfide concentrations returned to pre-Red 40 levels after 4 days, suggesting the existence of complex sulfur electron transfer occurring between the gut chemical landscape, microbiota, and host on longer time scales.

Here, we establish gut sulfur redox transformation in vivo, initiating from the thiol group of cysteine that is liberated as H₂S and then oxidized to zero valent sulfur (Fig. 4c). Altering concentrations of the reactants in diet, i. e. cysteine as a sulfide source or an azo compound as an oxidizing agent, alters metabolite concentrations in the gut environment. These data establish a microbiome-mediated role in the abiotic redox pairing of sulfide with dietary and pharmaceutical compounds and may inform human physiological effects of gut redox state and sulfur homeostasis^3,23,24.

Hydrogen sulfide is a driver of redox chemistry, both altered by, and in turn altering, dietary and pharmaceutical compounds. Therefore, our characterization of microbially produced hydrogen sulfide as a modifier of gut redox is a novel mechanism by which gut microbiota shape their environment, revealing dynamic chemical-chemical interactions, derived from community microbiota metabolism, as essential in structuring the human gastrointestinal ecosystem. Hydrogen sulfide plays a role in cardiac homeostasis^25,26, neuroprotection in the brain⁷, inflammatory bowel disease and colorectal cancer⁶and microbial defense against pathogens²⁴, highlighting the potential far-reaching significance of microbial sulfide production and cryptic sulfur redox in the human body.

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Data Availability Statement

The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files.

Acknowledgments

The authors thank Ali Ryan (Northumbria University) for sharing his knowledge of azoreductase enzymes, and the laboratories of Steven Almo and Tyler Grove (Albert Einstein College of Medicine) for experimental and intellectual support in related work. Libusha Kelly was supported by a United States Department of Defense Cancer Research Program Career Development Award (CA171019). Reese Hitchings was supported by the Albert Einstein College of Medicine PhD in Clinical Investigation training grant TL1 TR001072. Leonard Augenlicht was supported by the National Cancer Institute Department of Cancer Prevention Nutrition grants 1R01CA214625 and 1R01CA229216. Support was also received by the Albert Einstein Cancer Center core support grant P30CA013330.

Author Contributions

SW, RH, LK, and LA drafted the manuscript. SW performed E. coli experiments, metagenomic analysis, and mouse fecal sulfide determination. RH performed abiotic and fecal slurry experiments, as well as mass spectrometry. KP performed all mouse experimentation and maintained the mouse colony. LA guided mouse experiments and diet formulation. TY performed cyclic voltammetry of azo compounds. EG guided electrochemistry experiments. MM and ZC assisted with fecal slurry experiments. SK assisted with metagenomic analysis. All authors edited manuscript and contributed intellectually.

Competing Interest Declaration

The authors declare no competing interests.

Abiotic Azoreduction Assay

The capacity for H₂S to abiotically reduce azo compounds common in foods and drugs was investigated in a sterile, anoxic phosphate buffer medium. Serum bottles were prepared by sparging with ultrapure (99.9%) N₂flowed over a heated copper catalyst to remove impurities²⁷. Bottles were stoppered and capped under positive N₂pressure and autoclaved 3 times on successive days. The medium contained final concentrations of 3 mM sulfide delivered as Na₂S, 50 mM NaPO₄ buffer (pH 6.3), 25 μM FMN, and 500 μM azo-compound. Bottles were incubated at 37°C with shaking and sampled using sterile anoxic technique. To halt the reaction, 5 mM ZnAc was pre-dispensed into tubes so the sample was immediately submerged in ZnAc solution. Samples were vortexed for 1min and stored at -80°C. For mass spectrometry analysis, samples were centrifuged at 14,000xg for 5 min, and the supernatants were analyzed. Redox potentials of the experimental azo compounds were calculating using a multipotentiostat (CHI 1040C A2728)²⁸. Three cycles of cyclic voltammetry scans were collected using a rod-shaped glassy carbon working electrode, carbon counter electrodes and silver/silver-chloride (Ag/AgCl) paste reference electrode connected to the multipotentiostat at various scan rate settings to observe the reduction potentials of test molecules.

LC-MS of Azo-compounds and Azoreduction Products

Liquid Chromatography

Chromatography was performed on an Agilent 1200-series HPLC coupled to a 6130 Single-Quadrupole Mass Spectrometer (Agilent Technologies, Santa Clara, CA). Liquid chromatography was performed using an Agilent Poroshell 120 EC-C18, 21x50mm, 2.7nm. The mobile phases used were 2mM NH₄Ac in H₂O +0.1% formic acid (mobile phase A), and 2mM NH₄Ac in MeOH +0.1% formic acid (mobile phase B). The column was maintained at 25°C, while the autosampler was maintained at 4°C. Elution schemes for each drug and metabolite are reported in Supplementary Table 3.

Mass Spectrometry

Ions were analyzed using single ion monitoring in either positive or negative mode, using m/z for each metabolite reported in Supplementary Table 3. Drying gas was set at 12 L/min, nebulizer pressure at 35 psig, drying gas at 350°C, fragmentor at 70V, and capillary voltage at 3000V. Identity of all compounds was verified by retention time and m/z of authentic, commercially available standards, except for the azoreduction products of Yellow 6 and Congo red, which were unavailable commercially. For these compounds, we identified species with predicted m/z values that increased in intensity at the same relative rate at which the parent compound decreased in our abiotic experiment.

Statistical analysis

Statistical analysis was performed in R (version 3.6.3,²⁹) and plotted with the package ggplot2 (ggplot2 3.3.3,³⁰) or in Graphpad Prism 8.

Hydrogen sulfide analysis

Hydrogen sulfidewas quantified using a modified methylene blue detection assay^31,32. For liquid microbial culture, 30 µL sample was collected using strict anoxic technique and immediately dispensed into 157 µL ZnAc (28 g/L). This was diluted with 1.26 mL H₂O, followed by the addition of 113 µL Cline’s Reagent (N,N-dimethyl-p-phenylenediamine and FeCl₃·6H₂O in 50% HCl). The reaction was incubated at room temperature for 20 minutes to allow complete color development. Samples were centrifuged to remove particulates and 100 µL supernatant was transferred into Corning Costar half area 96-well flat bottom plates (Corning Inc., Corning, NY). Hydrogen sulfide concentrations were quantified using UV-vis detection at a wavelength of 670 nm on a BioTek Synergy H4 plate reader (BioTek Instruments, Winooski, VT) and quantified using Gen5 version 1.11 software.

E. coli pure cultures

E. coli K-12 W3100 (Carolina Biological, Burlington, NC) was grown in anoxic LB supplemented with 25 mM FMN and 30 mM succinate for all experiments. To minimize headspace and encourage hydrogen sulfide partitioning to the liquid fraction, 8 mL cultures were grown in 10 mL serum bottles under an N₂ atmosphere, sealed with butyl rubber stoppers and aluminum crimps, and incubated at 37°C. All conditions were established in triplicate. Samples were extracted from sealed bottles by syringe using sterile anaerobic technique.

Live E. coli

Sulfide azoreduction and enzymatic azoreductase activity were compared in growth phase E. coli cultures. Cysteine served as a readily metabolized thiol source. The scheme of culture conditions can be seen in Supplemental Table 4. Experimental conditions received 2 mM L-cysteine and Red 40 amended at 3.0 mM, 0.5 mM, or 0.25 mM. To quantify the maximum potential sulfide generated from cysteine, cultures were spiked with 2 mM cysteine but no Red 40 (“No Red 40”). Three control conditions were established. The first, “Enzymatic Control”, monitored E. coli azoreduction and hydrogen sulfide production in bottles receiving 0.25 mM Red 40 but no supplemental amino acid. The second set of controls received 2 mM L-serine as a nonsulfur cysteine analog, to account for any growth advantage effect of excess amino acid (“AA Control”). These control cultures served to quantify baseline enzymatic azoreductase activity, while the cysteine amended cultures would in theory azoreduce Red 40 via both enzymatic azoreductase activity and sulfide azoreduction. A sterile, uninoculated media control containing media and 0.25 mM Red 40 was also monitored for abiotic activity in the media itself (“Background”). Each condition was monitored for Red 40 loss and hydrogen sulfide generation.

Heat-inactivated E. coli

The E. coli experiment was replicated using heat-killed E. coli to eliminate all enzymatic activity, including azoreductases. Cultures of E. coli were incubated with the addition of either 2 mM cysteine or serine for 18 hours and then heat-inactivated by autoclave. Autoclaving the sealed bottles allowed for cell death and enzyme denaturing without breaching the headspace, maintaining both dissolved and gas phase H₂S. Following 1 hour of cooling and equilibration, H₂S concentration was measured and bottles then immediately amended with 3.0, 0.5, or 0.25 mM Red 40. An additional 25 mM FMN was supplemented to replace any portion lost to heat-inactivation. H₂S and Red 40 concentrations were monitored for 6 hours.

Biological Culture Red 40 Analysis

Red 40 concentrations were quantified using UV-vis detection at a wavelength of 489 nm in Corning Costar half area 96-well flat bottom plates (Corning Inc., Corning, NY) on a BioTek Synergy H4 plate reader (BioTek Instruments, Winooski, VT) and quantified using Gen5 version 1.11 software. Timepoints were diluted 1:1 with water and quantification was performed by linear regression of known standards.

Fecal Microcosm Azoreduction

The ability of a complex fecal microbiome community to azoreduce Red 40 both with and without dietary and endogenous sulfur sources was determined in fecal microcosms. Microcosms were established as 1% feces (w/v) in defined chemical medium as described in detail below. A freshly voided fecal sample was collected from a volunteer with no known health conditions who had not used antibiotics within 6 months. This study was approved by the Einstein Institutional Review Board (IRB#2013-2895). Defined chemical medium was prepared under anaerobic conditions by boiling and sparging with ultrapure (99.9%) N₂ scrubbed as before, with the following final concentrations: 9 mM Na₂HPO₄, 31 mM NaH₂PO₄, 24 mM NaHCO₃, 0.3 mM (NH₄)₂SO₄, 14.4 mM NaCl, 25 µM riboflavin, 20.5 µM CaCl₂ꞏ 2H₂O, 50.5 µM MnCl₂ ꞏ 4H₂O, 98.4 µM MgCl₂ ꞏ 6H₂O, 4.2 µM CoCl₂ ꞏ 6H₂O, Widdel trace elements andvitamins⁴¹, 0.77 µM (0.5 µg/mL) hemin, 100 µM DTT and 20 mM glucose. 8 mL cultures were grown in 10 mL serum bottles under an N₂ atmosphere, sealed with butyl rubber stoppers and aluminum crimps, and incubated at 37°C. All conditions were established in triplicate. Base media before addition of feces was added to serum bottles as sterile controls. Fecal sample was homogenized in 10mL media with sterile glass beads by vigorous shaking and added anoxically to the base media to a final concentration of 1% feces (w/v). This unamended fecal slurry was placed in serum bottles to serve as heat-killed controls and immediately autoclaved before incubating at 37°C for 12 hours. This process was repeated twice before supplementation with Red 40. For the Mucin, BSA, and Formaldehyde controls, fecal slurry was supplemented with 0.4% (w/v) type III porcine gastric mucin, 1% Bovine Serum Albumin, and 4% paraformaldehyde, respectively, before aliquoting into serum bottles. Cysteine and Serine conditions were supplemented with 10 mM of the appropriate amino acid; Background was not supplemented to show baseline hydrogen sulfide production. Once all culture bottles had been assembled, they were supplemented with Red 40 to a final concentration of 500 µM (Supplementary Table 5).

Cysteine Degrading Gene Analysis

Nine genes encoding enzymes with characterized cysteine-degrading activity^17–19,24 were evaluated in human gut metagenomic samples and in commensal gut microbes: CBS (K01697), CSE (K01758), CysK (K01738), CysM (K12339), CyuA (COG3681), MalY (K14155), MetC (K01760), SseA (K01011) and TnaA (K01667).

Metaquery Analysis for Mean Copy Number Per Cell in Human Gut Microbiomes

The KEGG or COG orthologue of each gene was searched within the 2,522 annotated metagenomic samples in the Metaquery²⁰ database. For each gene, the copy number per cell in each metagenomic sample was averaged and reported in Figure 3c.

Gut Commensal Genomic Analysis of Cysteine Degrading Genes

To identify the presence or absence of the nine cysteine degrading genes, databases of homologous sequences for each gene were constructed and searched against 24,758 NCBI bacterial genomes (Genbank, April 2021³³).

The reference protein database for each gene was derived from the sequence from the organism in which it was originally characterized¹⁷. A KEGG web server³⁴ BLASTP search of these genes, limited to bacteria, provided 500 closely related orthologues (≤ 1e^-75) from other species.

CyuA (formerly YhaM¹⁹) is not catalogued in the KEGG database. Reference sequences were instead retrieved from COG3681 in the EggNOG database (EggNOG 4.5.1³⁵). To reduce the number of sequences to approximately 500 to facilitate alignment, sequences were clustered at 77% identity with usearch cluster_fast (Usearch 8,³⁶) and centroids were used in the reference database.

Sequences in each gene’s reference protein database were aligned using MUSCLE with default parameters (MUSCLE 3.8.1551,³⁷) and alignments were used to construct Hidden Markov Models using hmmbuild with default parameters (hmmer 3.3.2,³⁸). The 9 hmms were searched against the 24,758 bacterial genomes using hmmsearch with a stringent cutoff of 1e^-10. One hit in a genome indicated presence of the gene in a particular genome; multiple hits were ignored (Supplementary Table 6). We then narrowed the output to a subset of bacteria commonly found in the human gut microbiome. To identify relevant gut microbes we procured 8,548 metagenomic samples of patients from 51 studies using the R package curatedMetagenomicData³⁹. For each of these samples, we downloaded a list of relative abundances of 6,832 taxa annotated using the MetaPhlAn2⁴⁰ software for each of these studies. We computed the mean relative abundance across studies for each taxa and selected organisms with ≥0.05% mean relative abundance and manual curation. Finally, different strains within the same species with the same pattern of cysteine degrading genes were clustered. By doing so we were able to observe intra-species diversity of cysteine degrading genes while eliminating duplicates.

A phylogenetic tree of the stool organisms of interest was constructed using 16S rRNA nucleotide sequences associated with each genome assembly in NCBI. 16S sequences were aligned using MUSCLE with default parameters via the EMBL-EBI search and analysis tool ⁴¹. A maximum likelihood tree was constructed using the IQ-TREE web server with 1000 ultrafast bootstrap analysis, an automatically selected substitution model and otherwise default parameters⁴² and visualized in iTOL⁴³.

Mouse Housing and Diet

To assess the influence of varying dietary cysteine concentrations on fecal H₂S, three separate cohorts of 10 3-week old male weanling C57BL/6J mice were randomly assigned to each of three diets varying in cysteine (Control, 0 Cys, and High Cys; Supplementary Table 2) (N = 30, 10/diet). 2-5 mice per cage were co-housed with ad-libitum access to their assigned diet and water. We then followed this experiment with a crossover experiment adding Red 40 to the control diet as a potential redox partner for H₂S. For the crossover experiment, two cohorts of 6 3-week old male weanling C57BL/6J mice were assigned to either the Control diet or Red 40 diet (Supplementary Table 2) (N=12, 6/diet). Each cohort of 6 mice was split into two cages, 3 mice per cage.

Mouse Fecal Sulfide Sampling

Fecal sulfide was measured twice per week in all mice consuming Control, 0 Cys, or High Cys diets. Mice co-housed in each condition were placed in a clean collection tray and allowed to freely defecate for one hour, at which point 7-9 random fecal pellets were collected and separately placed in pre-weighed tubes containing 157 µL of 28 g/L Zinc Acetate. These samples were vortexed and stored at 4°C until analysis by Cline assay. During the crossover experiment fecal sulfide was measured more frequently, 2-5 times per week. Each mouse was placed in a separate collection tray to ensure one pellet per mouse, and, following defecation, pellets were collected and stored as above.

There is NO Competing Interest.

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Microbial Cysteine Degradation Drives Cryptic Sulfide Redox Chemistry in the Gut

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