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Inorganic sulfur fixation via a new homocysteine synthase allows yeast cells to cooperatively compensate for methionine auxotrophy [1]

['Jason S. L. Yu', 'Molecular Biology Of Metabolism Laboratory', 'The Francis Crick Institute', 'London', 'United Kingdom', 'Benjamin M. Heineike', 'Johannes Hartl', 'Department Of Biochemistry', 'Charité Universitätsmedizin', 'Berlin']

Date: 2022-12

The assimilation, incorporation, and metabolism of sulfur is a fundamental process across all domains of life, yet how cells deal with varying sulfur availability is not well understood. We studied an unresolved conundrum of sulfur fixation in yeast, in which organosulfur auxotrophy caused by deletion of the homocysteine synthase Met17p is overcome when cells are inoculated at high cell density. In combining the use of self-establishing metabolically cooperating (SeMeCo) communities with proteomic, genetic, and biochemical approaches, we discovered an uncharacterized gene product YLL058Wp, herein named Hydrogen Sulfide Utilizing-1 (HSU1). Hsu1p acts as a homocysteine synthase and allows the cells to substitute for Met17p by reassimilating hydrosulfide ions leaked from met17Δ cells into O-acetyl-homoserine and forming homocysteine. Our results show that cells can cooperate to achieve sulfur fixation, indicating that the collective properties of microbial communities facilitate their basic metabolic capacity to overcome sulfur limitation.

Funding: This work was supported by the Wellcome Trust IA grant (IA 200829/Z/16/Z to MR), the Bayerisches Staatsministerium für Bildung und Kultus, Wissenschaft und Kunst (Bavarian State Ministry of Education, Science and the Arts, BMBF) MSCoresys grant (031L0220 to MR), the European Commission CoBiotech project Sycolim (ID#33 to MR), European Commission Horizon 2020 research grant (ERC-SyG-202 951475 to MR), the Francis Crick Institute which receives its core funding from: Cancer Research UK (FC001134 to MR), UK Medical Research Council (FC001134 to MR) and the Wellcome Trust (FC001134 to MR) and the Swiss National Science Foundation Postdoc Mobility Fellowship 191052 to JH) The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Here, we studied the biochemical basis of the met17Δ phenotype and discovered a previously overlooked metabolic bypass that explains the growth of met17Δ in absence of organosulfur compounds. We found that an uncharacterized protein, which we herein have named Hydrogen Sulfide Utilizing-1 (Hsu1p, formerly YLL058Wp), encodes a metabolic enzyme that directly assimilates sulfur from hydrogen sulfide, thereby enabling cell growth by resolving methionine auxotrophy once critical concentrations of sulfide are leaked from met17Δ cells. We provide evidence that Hsu1p functions as a homocysteine synthase, performing the fixation of inorganic sulfide in place of Met17p, thereby generating the homocysteine required for methionine and cysteine biosynthesis.

Deletion of MET17 (met17Δ) renders cells auxotrophic for organosulfur compounds such as methionine, cysteine, homocysteine, or S-adenosyl methionine [ 15 – 18 ]. This led to the widespread use of the met17Δ allele as an auxotrophic marker for genetic experiments [ 19 ]. These alleles were crossed into many laboratory strains, including the W303 and S288C derivatives that gave rise to the yeast deletion collection [ 20 ]. However, met17Δ yeast exhibits an atypical growth phenotype, and cells overcome the autotrophy under specific conditions. When met17Δ cells are replicated as thick patches, they continue growth even in the absence of an organosulfur source. The phenomenon occurs in a temperature-sensitive manner, with growth of the auxotrophs being most evident at 22°C and decreasing linearly with temperature up to 37°C [ 21 ]. This cell density and temperature sensitive phenotype has also been described in distantly related yeasts like Candida albicans and Yarrowia lipolytica [ 22 , 23 ] and is hence not a species-specific phenomenon. Among the possible explanations for this paradoxical phenotype, Cost and Boeke [ 21 ] suggested that met17Δ could leak and share organosulfur metabolites between cells once they reached a critical cell density.

(A) The core and associated metabolic pathways that feed into and out of methionine and cysteine biosynthesis, including the gene names that encode for the respective enzymes in S. cerevisiae. Metabolites bearing sulfur are colored in light purple. (B) Liquid cultures of met17Δ strain in minimal media without methionine inoculated at 6 different cell densities (OD 600nm = 0.01 to 0.3) and cultured at 4 different temperatures (22, 26, 30, 37°C) for 48 h. Gray indicates prototrophic control strain cultured at 30°C. Lines indicate three replicates per condition. (C) Growth curves of gene deletion strains implicated in methionine metabolism, as compared with a prototrophic strain in minimal medium after 24 h. Adjacent images depict growth of indicated deletion strain on solid minimal media after 72 h. (D) Metabolomic analysis and measurement of HC, HS, and M in extracellular and intracellular fractions between exponentially growing prototrophic and SeMeCo cultures normalized to the final OD 600nm . Data are from 2 independent replicates containing 4 biologically independent samples (n = 8). Adjusted p-values from unpaired Wilcoxon rank sum test are as indicated; ns indicates no statistical significance. (E) Differential protein expression analysis of sulfur assimilation enzymes between sorted CFP+ (MET17) and CFP− (met17Δ), where n = 4 biologically independent replicates. (F) KEGG pathway analysis and hierarchical clustering of sorted cells as in (E) of all detected enzymes involved in cysteine and methionine metabolism characterized by associated metabolic pathways (colored). The data underlying this figure can be found in S1 Data , with the exception of (E) and (F), which can be found in the PRIDE with the dataset identifier PXD031160. HC, homocysteine; HS, homoserine; M, methionine; PRIDE, Proteomics Identification Database; SeMeCo, self-establishing metabolically cooperating.

In many eukaryotes, sulfur is primarily assimilated in the form of sulfate (SO 4 2− ), which becomes successively reduced to sulfide (S 2− ). A key enzyme in the eukaryotic sulfur assimilation process is homocysteine synthase, which, in S. cerevisiae, is encoded by the MET17 gene (also known as MET15 or MET25). Met17p catalyzes the fixation of inorganic sulfide with O-acetylhomoserine (OAHS) to form homocysteine ( Fig 1A ). Homocysteine is subsequently converted to methionine via the methionine synthase (Met6p) or into cysteine via cystathionine-β-synthase and cystathionine-ɣ-lyase (Cys4p and Cys3p, respectively) [ 11 – 13 ]. Homocysteine therefore provides the central organosulfur pool from which the key sulfur-bearing amino acids methionine and cysteine are synthesized [ 14 , 15 ].

Despite decades of effort, a large proportion of the coding sequences expressed from eukaryotic genomes lack complete functional annotation [ 1 – 4 ]. One contributing factor to this situation is that many laboratory experiments are conducted under a limited set of growth conditions that do not fully represent the range of conditions organisms are exposed to in nature [ 5 ]. The budding yeast Saccharomyces cerevisiae is a key model organism for the discovery and characterization of gene function in eukaryotes. Moreover, because of its importance in biotechnology, the response to varying carbon and nitrogen sources has been extensively studied in this organism [ 6 , 7 ]. However, although the utilization of sulfur is an equally fundamental process, other than the core genes that are involved in the assimilation and utilization of inorganic sulfur into metabolically useful organosulfur compounds, it remains largely undercharacterized [ 8 – 10 ].

Results

The conditional nature of organosulfur auxotrophy in met17Δ yeast The ability of met17Δ yeast strains to overcome organosulfur auxotrophy at high cell density was first described by Cost and Boeke [19,21]. This phenotype is nonheritable, which ruled out adaptive, secondary mutations concurrent with met17Δ as the cause. We studied the phenotype in two haploid yeast strains in the BY4741 background [24]: (i) a prototrophic strain in which auxotrophies were repaired by genomic integration of the missing genes (prototroph or wild-type (WT): HIS3, LEU2, URA3, MET17); and (ii) an analogous strain in which the met17Δ allele was not repaired (met17Δ: HIS3, LEU2, URA3, met17Δ). As previously observed by Cost and Boeke [19,21], our met17Δ strain was also able to grow in the absence of methionine over the course of 8 days when replicated in thick patches, independently confirming the reproducibility of this phenomenon (S1A Fig). We started our investigations into the biochemical basis of this phenotype by ruling out amino acid contaminations in commercial yeast nitrogen broth (YNB), a common source of conflicting and unusual growth phenotypes. We validated the concentration of sulfur containing amino acids using liquid chromatography-selected reaction monitoring (LC-SRM) [25]. We detected levels of methionine, homocysteine, and glutathione at a concentration at least 100,000-fold lower than typical levels found in replete yeast media (S1B Fig). Therefore, the growth of met17Δ cells in the absence of methionine was not due to the presence of growth-relevant concentrations of sulfur-containing amino acids in the growth medium. To have a more experimentally tractable system to further our investigations, we next explored the conditions under which the growth phenotype was reproducible in liquid culture in a high-throughput, 96-well format. We inoculated the met17Δ strain into methionine-free medium at a range of initial densities (OD 600nm = 0.01 to 0.3) and cultured under four temperatures (22, 26, 30, and 37°C), recording the endpoint OD 600nm at 24 h (S1C Fig) and 48 h (Fig 1B) as a measure of growth. At typical yeast culture temperatures (22, 26, and 30°C), robust growth could be observed where the initial inoculation density was OD 600nm > 0.02, in agreement with previous observations [19]. Growth at the lowest inoculation density (OD 600nm = 0.01) was only observed when the culture temperature was at 26°C, confirming that culture temperatures alter the cell density threshold that permits growth. Similarly, growth was permissive at 37°C only when the initial cell density was high (OD 600nm ≥ 0.08). In contrast, the WT strains could robustly grow at 30°C irrespective of initial inoculation density (Fig 1B). Although we did not observe a strict correlation of temperature with growth as previously reported, growth was greatly enhanced at lower temperatures. Therefore, we established 25°C as the standard culture temperature for all subsequent experiments, with low-density (OD 600nm < 0.01) and high-density (OD 600nm ≥ 0.02) inoculums being nonpermissive and permissive densities for growth in the absence of organosulfur supplementation, respectively. These thresholds were independent of culture size (96-well versus 20 ml flasks; S1D Fig). Next, to probe whether the cell density limitation is specific to met17Δ or whether it is a more general phenomenon related to methionine or amino acid auxotrophy, we compared the growth of the met17Δ strain to other enzyme knockout strains of sulfur assimilation pathways (met1Δ, met3Δ, met5Δ, met7Δ, or met13Δ) or amino acid metabolism (argΔ, hisΔ, lysΔ, trpΔ, and aroΔ). All auxotrophic strains demonstrated growth deficiency, but only the met17Δ formed biomass close to that of the WT strain (Figs 1C and S1E). This result suggested that the ability to overcome the growth defect is not a common phenotype of methionine or amino acid auxotrophs but rather a specific phenotype that occurs upon the deletion of MET17. We speculated that either another enzyme replaces the function of Met17p in these conditions or else a metabolic shunt exists that allows yeast to circumvent the canonical sulfur utilization pathway to fuel growth.

The ability to overcome organosulfur auxotrophy does not require the exchange of homocysteine or methionine Cost and Boeke [21] speculated that met17Δ cells could overcome the growth defect through the sharing of organosulfur metabolites between cells to growth-relevant levels. Indeed, previous work from us and others has shown that communal yeast cells can effectively share metabolites to overcome auxotrophies [26,27]. In order to better understand the possible impact of metabolite exchange in the context of the ability of met17Δ cells to grow in the absence of methionine, we employed self-establishing metabolically cooperating (SeMeCo) communities, a synthetic yeast community designed to study metabolite exchange interactions between auxotrophs [27]. SeMeCo communities are composed of cells that express different combinations of auxotrophic markers and are established from a prototrophic founder cell through the stochastic loss of plasmids encoding one or more enzymes that compensate for metabolic deficiencies present in the genome. In the BY4741 background, these are met17Δ, his3Δ, leu2Δ, and ura3Δ, which induce methionine, histidine, leucine, and uracil auxotrophies, respectively. In SeMeCos, the degree of metabolite sharing influences the frequency of a respective auxotroph in the population. Examining data recently acquired by us revealed that met17Δ cells are the most dominant subpopulation within exponentially growing SeMeCo cultures (approximately 60%), and more frequent than cells harboring the three other metabolic deficiencies (S1F Fig) [28]. To understand the role that metabolite sharing plays in overcoming the loss of MET17 in the SeMeCo system, we recorded the metabolic profiles of SeMeCos by LC-SRM and compared these against the metabolic profiles of prototrophic communities. In SeMeCo communities dominated by met17Δ cells, we detected a relative increase in intracellular homocysteine and homoserine concentrations compared to prototrophic communities (Fig 1D, intracellular). However, extracellular concentrations of homocysteine and methionine were not statistically different, although homoserine levels were elevated upon the exclusion of an outlying measurement (Fig 1D, extracellular). This result suggested that indeed, the met17Δ metabolic deficiency might be overcome through the exchange of an upstream precursor rather than directly via methionine sharing.

met17Δ cells up-regulate enzymes involved in sulfur assimilation, cysteine and methionine metabolism We have previously recorded proteomes from auxotrophs and prototrophs isolated from SeMeCo cultures using fluorescence activated cell sorting, and a CFP marker expressed from the same plasmid as the auxotrophic marker ([28]; PRIDE project: PXD031160). A differential expression analysis comparing specifically the MET17 and met17Δ subpopulations revealed a clear up-regulation of sulfur assimilation enzymes (Met3p, Met14p, Met5p, and Met10p) and enzymes of the methionine salvage pathway (Meu1p, Spe3p, Spe4p, Bat1p, and Bat2p) in the MET17 strain (Fig 1F). This result is consistent with a typical feedback response to the lack of methionine. Counterintuitively, however, several of the downstream enzymes, including Met6p and Gsh1p, that should carry no flux in the absence of Met17p, had increased protein levels (Fig 1A and 1F). In addition, the abundance of several core enzymes of the methionine cycle (Met6p, Sam1p, Sam2p), serine (Ser1p, Ser33p), and cysteine (Cys3p, Cys4p) biosynthetic pathways were also increased, although this was not observed with all enzymes (Sah1p, Ser3p). This data argued that homocysteine could be further metabolized despite the absence of Met17p, which would suggest the existence of a metabolic bypass to the homocysteine synthase activity of Met17p.

Hydrogen sulfide fixation drives growth in the absence of methionine We speculated that the intracellular accumulation of homocysteine in met17Δ cells (Fig 1D) could indicate the presence of an enzyme-catalyzed reaction that forms homocysteine independent of Met17p. As sulfide overflow is a defining characteristic of met17Δ cells [19,21], one possible explanation would be the presence of an enzyme that can utilize sulfide or its protonated forms (hydrosulfide (HS−) and hydrogen sulfide (H 2 S)) to incorporate sulfur and form homocysteine. To test this hypothesis, we exploited the cell density dependency of met17Δ cells in liquid culture that leads to the resolution of organosulfur auxotrophy as a readout (Figs 1B and S1C and S1D). When conditioned medium from met17Δ cells cultured either at high-density (OD 600nm > 0.02, permissive) or low-density (OD 600nm < 0.01, nonpermissive) was filtered and inoculated with fresh cells at low density, only the filtered medium from the high-density culture could support cell growth. Moreover, this property was abolished upon precipitation of H 2 S with lead acetate (Fig 2A). To further substantiate whether H 2 S could be responsible for the growth, we supplemented two low-density cultures with sodium hydrosulfide (NaHS) that dissociates to form hydrosulfide ions and, subsequently, both aqueous and gaseous forms of H 2 S (Fig 2B, reactions (ii-iv)). Taking advantage of the phase equilibria of H 2 S, one culture was left unsealed to drive the equilibria towards the formation and dissipation of gaseous H 2 S from the system (Fig 2B, reaction (iv)). Growth was only observed in the sealed culture, consistent with the assumption that a critical concentration of H 2 S is required to overcome the met17Δ auxotrophy (Fig 2C). In parallel, we manipulated the phase equilibria in the opposite direction, allowing the accumulation of aqueous H 2 S in low-density cultures via outgassing from either a high-density culture or a concentrated solution of NaHS by connecting the vessels with a small piece of rubber tubing to create a closed system. In both situations, growth was observed in the low-density culture (Fig 2D). To further substantiate that it is H 2 S, which resolves the auxotrophy, we evaluated its presence and uptake across different cultures and growth conditions, exploiting the chemical properties of 7-azido-4-methylcoumarin (AzMC). In the presence of H 2 S, AzMC undergoes selective reduction of the azido moiety to form 7-amino-4-methylcoumarin (AMC), which fluoresces when exposed to ultraviolet light [29]. When minimal media was compared with cultures inoculated at low density, no significant difference in AMC fluorescence was observed. Conversely, the fluorescence in NaHS-supplemented cultures decreased in the presence of cells, suggesting utilization of H 2 S and its concomitant depletion from the medium (Fig 2E). This fluorescence assay also indicated that high-density cultures had higher levels of aqueous H 2 S, relative to low-density cultures, orthogonally confirmed by the precipitation of lead sulfide in high-density cultures (Fig 2A). PPT PowerPoint slide

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TIFF original image Download: Fig 2. Hydrogen sulfide is essential for the rescue of organosulfur auxotrophy of met17Δ. (A) met17Δ cultures inoculated at low starting density (OD 600nm < 0.01) and high densities (OD 600nm ≥ 0.02) (upper panel, left and middle). One flask of high-density inoculation was supplemented with Pb(II) acetate to precipitate H 2 S, forming lead sulfide (upper panel, right). Conditioned media from these three cultures was subsequently filtered, replenished with YNB and glucose, and reinoculated at low density (lower panel). Data are representative from two biologically independent experiments. (B) Reaction schemes. Hydrolysis of sulfide is in equilibrium with hydrosulfide and hydrogen sulfide (i, iii, iv). NaHS can serve as a hydrosulfide donor (ii) in solution. Hydrogen sulfide preferentially undergoes phase transition to a gaseous state (iv) in unsealed vessels or establishes a dynamic equilibrium with its aqueous counterpart in sealed vessels. (C) Growth of met17Δ cultures supplemented with NaHS and inoculated at low density, sealed vs. unsealed. (D) Cultures inoculated with the met17Δ strain where the vessels are connected via rubber tubing to facilitate gaseous exchange, either between a high-density culture or a high-concentration solution of NaHS (20 mM). Schematic indicates the reactions occurring at each stage to facilitate the growth of low-density cultures. (E) Quantification of hydrogen sulfide production and uptake via conversion of AzMC to AMC [21] between unsupplemented and NaHS supplemented cultures inoculated at low density in the presence (purple) or absence (gray) of met17Δ cells. Blue and red bars indicate a separate experiment where hydrogen sulfide production was quantified met17Δ cultures inoculated at either low (blue) or high (red) density, in a similar experiment as shown in (A). P values calculated via two-tailed Student t test, where n = 3 independent replicates. (F) Isotopic tracing experiment with pathway map indicating the biosynthetic transfer of isotopic S34 from ammonium sulfate to methionine (left group) and glutathione (right group) via methionine and cysteine biosynthetic pathways, respectively. Pie charts indicate the percentage of S34 for cultures supplemented with S34-labeled ammonium sulfate. The data underlying this figure can be found in S1 Data. AMC, 7-amino-4-methylcoumarin; NaHS, sodium hydrosulfide; YNB, yeast nitrogen broth. https://doi.org/10.1371/journal.pbio.3001912.g002 Finally, to test if sulfur does indeed transition from sulfate through to the biosynthesis of cysteine and methionine in the absence of MET17, we performed isotopic tracing with S34-labeled ammonium sulfate (Figs 2F and S2A). Uptake of this isotope label by sulfur assimilation via the up-regulation of the associated enzymes should lead to S34 dissemination into all subsequent sulfur bearing derivatives via sulfide overflow (Figs 1A and S2B). We quantified the ratios of S32 and S34 sulfur in methionine and reduced glutathione, the downstream products derived from homocysteine and cysteine biosynthesis, respectively, using liquid chromatography mass spectrometry (LC–MS). When native (S32) ammonium sulfate was supplied, the assay detected S34 at its expected environmental isotopic abundance (S2B Fig; 4% to 6%). Conversely, when we supplied S34-labeled ammonium sulfate, S34-methionine (91.4%) and S34-reduced glutathione (67.9%) accumulated to levels well above natural abundances, indicative of sulfur transfer from inorganic sulfate to organic sulfur-bearing metabolites. Thus, met17Δ assimilated inorganic sulfur from ammonium sulfate, and yeast cells are competent in the use of H 2 S as a source for methionine and cysteine biosynthesis.

A targeted genetic screen identifies the bypass enzyme responsible for H 2 S-mediated growth in the absence of methionine Having established homocysteine formation via H 2 S fixation as the process by which methionine and cysteine auxotrophy could be overcome, we next used a targeted genetic approach to identify the enzyme responsible. We started by identifying strains in the yeast deletion collection that are associated with sulfur metabolism [20]. We selected 15 strains (S1 Table) that carried a deletion in the sulfur metabolism-associated gene in addition to deletions of four auxotrophic markers in their genome that include MET17 (his3Δleu2Δura3Δmet17Δ) based on two criteria: (i) the deleted gene product was a part of either the sulfur assimilation or organosulfur biosynthetic pathways (S2B Fig); or (ii) the deleted gene product was identified as an ortholog to Met17p according to the eggNOG database [30–32] (Fig 1A and S2 Table). These selected deletion strains were cultured in synthetic drop-in media and tested for met17Δ growth in the presence of NaHS. In a background of MET17 deletion, sul1/2Δ, met3Δ, met14Δ, met5Δ, and met10Δ strains demonstrated robust growth only in the presence of NaHS, while the met6Δ strain lacking the methionine synthase was unable to grow. These growth phenotypes indicated that H 2 S utilization and the resolution of organosulfur auxotrophy in the presence of NaHS was independent of sulfur assimilation, but dependent on sulfur utilization (Figs 3A and S2B and S2C). Critically, these experiments also revealed the dependency of the H 2 S utilization pathway on homoserine-O-acetyltransferase that is responsible for the conversion of homoserine to OAHS as the met2Δ strain was also unable to grow (Fig 3B). Since OAHS is a substrate shared by both Met17p and Str2p, this implies a similar catalytic mechanism (Fig 1A) [17]. Of the strains deficient in Met17p orthologs, the met17Δcys3Δ strain also could be rescued by NaHS supplementation, albeit with an extended lag phase, which suggests the existence of a direct route towards cysteine formation via H 2 S, possibly via cysteine synthase activity (S2C and S3B Figs, gray pathway; [14,33,34]). Notably, the str2Δ and str3Δ strains also had a lengthened lag phase upon NaHS supplementation. This suggested that the degree of transsulfuration from cysteine to homocysteine may act to regulate the reaction efficiency, although neither Str2p nor Str3p appears to directly catalyze the bypass reaction itself. Intriguingly, we observed that YLL058WΔ led to the loss of the ability to utilize H 2 S for growth (Fig 3A). This defect was not observed in strains deficient in the other Met17p orthologs (irc7Δ, YML082WΔ, and YHR112CΔ), indicating that YLL058Wp could potentially function as the bypass enzyme. We next asked if the gene products of STR2 and YLL058W could operate as a homocysteine synthase in place of Met17p to rescue organosulfur auxotrophy. Str2p has been shown to be a cystathionine-γ-synthase, catalyzing the conversion of cysteine to cystathionine using OAHS as a substrate (Fig 1A). When operating in tandem with Str3p, this permits the transsulfuration of cysteine to methionine [14,35]. Since Str2p shares the same substrate as Met17p, there was a possibility it could incorporate sulfur via a secondary or promiscuous reaction. The catalytic properties of our other candidate YLL058Wp have not previously been experimentally characterized, although based on its close phylogenetic relationship to Str2p, we hypothesized that it could possess cystathionine-γ-synthase activity or at least interact with similar substrates (S2E Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 3. Identification and characterization of YLL058Wp/Hsu1p as the homocysteine synthase responsible for hydrogen sulfide fixation. (A) A targeted genetic screen of knockout strains lacking in addition to met17Δ, the genes encoding the homoserine (Met2p), methionine (Met6p) biosynthesis pathways, and selected orthologs of Met17p: Str3p, Str2p, and YLL058Wp. Cultures were inoculated at low density either in the presence or absence of NaHS, which rescues growth in met17Δ strains. Growth curves were recorded over 84 h with 3 biologically independent replicates for each strain (n = 3) as shown. A mutant strain carrying met17Δ alone was used as control. Complete or partial loss of NaHS rescue phenotype in these cultures indicate involvement of the deleted enzyme in the metabolic bypass (purple). Images are representative of the endpoint following growth curve capture. (B) Simplified pathway that summarizes the deletion mutants with a no growth (red) or delayed growth (orange) phenotype during NaHS-mediated growth rescue. Metabolites bearing sulfur are marked in purple. Gray pathway indicates the possible conversion from OAS to cysteine that we observe in vitro with YLL058Wp analogous to Met17p. (C) Methionine limitation screen comparing met17Δstr2Δ and met17ΔYLL058WΔ. Low amounts of methionine were provided to fuel the first phase of growth, allowing cells to reach a critical density at which sufficient hydrogen sulfide leaked from cells to allow a second phase of growth. In this setup, NaHS supplementation enables the second phase of growth to proceed only if the bypass is functional by allowing cells to freely use H 2 S as a sulfur source regardless of limited concentrations of methionine. (D) Schematic indicating the setup of in vitro enzymatic reactions and growth assays used to assess the ability of OAHS reaction supernatants to rescue the indicated deletion strains (met17Δ, met17ΔYLL058WΔ, met17Δstr2Δ). Enzyme was sourced both from GFP immunoprecipitation (Hsu1-GFP) and recombinant (His-Hsu1, His-Str2, His-Met17) sources. (E) Quantification of homocysteine via Ellman’s reagent and spectrometry using postreaction supernatants where immunoprecipitated Hsu1p-GFP was incubated with OAHS as the substrate. RCT_0, 25, and 100 indicate bead volumes of enzyme–resin slurry used in the reaction. RCT(-PLP or -EDTA) indicates reactions whereby pyridoxal 5′-phosphate (PLP) or EDTA has been omitted from the reaction buffer. Data are mean thiol concentrations ± SD where n = 3 biologically independent replicates. Student t test was used to test for significance between indicated samples (***, p < 0.0001, ns = no significance). (F) Enzyme assay as in (E) utilizing recombinant enzymes purified by Ni-NTA affinity chromatography. Data are mean thiol concentrations ± SD where n = 3 biologically independent replicates. Student t test was used to test for significance between indicated samples (*, p < 0.05, ns = no significance). (G) Growth assays whereby each strain was inoculated at low density and supplemented with 50 μl of the indicated in vitro reaction product (n = 3). Final OD 600nm measured following 5 days of outgrowth. For OAHS reaction supplements, data are mean OD 600nm ±SD where n = 3 biologically independent replicates. OD 600nm reached in minimal media and supplementation with homocysteine or methionine (n = 1) are shown for comparison. (H) Growth assay as in (G) using postreaction supernatant generated from recombinant Hsu1 (rHsu1). Growth curves were captured for met17Δ and met17Δstr2Δ over 3 days, and for met17Δhsu1Δ over 5 days with 3 biologically independent replicates for each strain (n = 3). Schemes for Fig 3A, 3C, and 3D were created through BioRender.com. The data underlying this figure can be found in S1 Data. EDTA, ethylenediaminetetraacetic acid; NaHS, sodium hydrosulfide; Ni-NTA, nickel-NTA; OAHS, O-acetylhomoserine; OAS, O-acetylserine. https://doi.org/10.1371/journal.pbio.3001912.g003 To characterize how YLL058Wp and Str2p contributed to the bypass reaction, we devised an experiment in which we supplemented only a limited concentration of methionine into minimal media that was rapidly depleted (Fig 3C). In essence, the assay separates growth into two phases; the first phase is driven by methionine until the cells have reached the critical cell density that is required by the cells to overcome the MET17 deficiency using an unknown bypass enzyme. The met17Δstr2Δ strain maintained robust growth, both in the presence and absence of supplemented NaHS, indicating that the bypass reaction does not require Str2p. In contrast, growth of the met17ΔYLL058WΔ strain slowed after the initial biomass accumulation phase that is driven by the presence of methionine and did not achieve robust growth even in the presence of ample NaHS (Fig 3C). Thus, YLL058Wp is necessary for the cell to assimilate sulfide and sufficient to catalyze homocysteine biosynthesis from OAHS and sulfide, while Str2p appears to indirectly affect the bypass reaction. Considering the H 2 S dependency of YLL058Wp, we henceforth refer to it as Hydrogen Sulfide Utilizing-1 (Hsu1p).

Hsu1p is a homocysteine and cysteine synthase that fixes sulfur from hydrogen sulfide Having identified an enzyme required for the bypass reaction, we next sought to further characterize the levels and activity of Hsu1p, using a yeast strain that expresses a Hsu1-GFP fusion protein in an auxotrophic background lacking Met17p (leu2Δura3Δmet17Δ) [36]. We first verified by PCR the identity of the strain isolated from this library (S3A Fig). Monitoring GFP expression via flow cytometry, we observed that the GFP fluorescence increased 1.5-fold in minimal media supplemented with NaHS, indicating that HSU1 expression is responsive to sulfide levels (S3B Fig). We next immunoprecipitated Hsu1-GFP from yeast cells grown under these conditions. Then, we tested in vitro if the immunopurified protein was capable of transferring the sulfur from H 2 S onto OAHS to form homocysteine (Figs 3D and S3C). We also tested the ability of the enzyme to function as a cysteine synthase using O-acetylserine (OAS) as an alternative substrate, since this could potentially explain the growth of the met17Δcys3Δ strain in the presence of NaHS (Figs 3B and S3D). We quantified the formation of homocysteine or cysteine from OAHS or OAS, respectively, upon covalent linkage of inorganic sulfide to organic backbones using Ellman’s reagent and spectrophotometry [37]. While the detection of homocysteine was hampered by the presence of a high background, the reaction of OAS to cysteine could be followed without strong interference. Cysteine synthase activity was observed at high enzyme concentrations (RCT_100; S3D Fig). To further validate homocysteine synthase activity in Hsu1p and to overcome the issue of high background, we generated recombinant versions of Hsu1p and Met17p by expressing the proteins in E. coli and purifying them via affinity chromatography (nickel-NTA (Ni-NTA)) and molecular weight cutoff (MWCO) filtration. We again tested for homocysteine synthase activity, this time using the purified recombinant enzymes in the presence or absence of OAHS substrate and reduced levels of PLP and ethylenediaminetetraacetic acid (EDTA) (Figs 3F and S3F). We observed an increase in homocysteine in the presence of OAHS for both Met17p and Hsu1p, again indicating that Hsu1p possesses homocysteine synthase activity. However, high enzyme levels were also required, indicating that this might be a secondary catalytic activity. We therefore tested whether the increase in homocysteine levels that we observed was relevant to our phenotype and linked the enzyme assay to a growth assay. Specifically, we asked whether supplementation with postreaction supernatant could rescue the growth of strains that previously had a growth defect in the absence of methionine or NaHS supplementation (met17Δ, met17Δhsu1Δ, met17Δstr2Δ, and met17Δcys3Δ). Supernatants obtained from the in vitro OAHS and OAS reactions (RCT_0, RCT_25, and RCT_100) were supplemented into minimal media without methionine and inoculated with the above strains. At 5 days postinoculation, the endpoint OD 600nm of the OAHS supplemented cultures were compared against cultures directly supplemented with 0.15 mM methionine and homocysteine. Critically, we observed that supplementation with postreaction supernatant generated in the presence of immunoprecipitated Hsu1-GFP restores growth in the mutant backgrounds, akin to direct supplementation with an organosulfur compound (Fig 3G). Conversely, the supernatants taken from both OAHS and OAS reactions did not support growth of any tested strain in the absence of the immunoprecipitated enzyme (RCT_0; Figs 3G and S3E). Similarly, growth profiles captured from cultures supplemented with the OAHS postreaction supernatant generated in the presence of recombinant Hsu1p could also rescue the growth of the deletion strains, converse to supernatants that did not contain OAHS (Fig 3H). Hence, the reaction products formed in the presence of Hsu1p were sufficient to rescue the growth defects of the organosulfur auxotrophs.

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