Periodic Reporting for period 1 - Yeast H2S Signalling (Quantitatively deciphering a novel metabolic pathway triggered by sulfide gas)
Okres sprawozdawczy: 2021-10-01 do 2023-09-30
Excreted chemicals form a major component of the language through which living cells communicate with each other. When these chemicals are gaseous, they can mediate rapid interactions between seemingly unconnected cell populations. Gaseous mediators are widespread in biology. For instance, plants use an elaborate vocabulary of volatile compounds to attract pollinators or warn conspecifics of predators. Some gases such as hydrogen sulfide are produced by most cell types and can impact tissue in diverse ways: while high doses of sulfide can be toxic, in controlled doses, the gas can regulate blood vessel diameter, enhance biofilm formation in the gut microbiome, and affect longevity in a range of organisms such as yeast, flies and rodents. Interestingly, many other volatile sulfur compounds are produced in microbial cells, but their functions are not well understood. Better knowledge of the metabolic pathways that produce and consume volatile compounds thus holds potential for novel therapeutics.
The overall aim of this project was to identify a hidden pathway in the budding yeast, Saccharomyces cerevisiae, which can assimilate sulfide into organic sulfur compounds—essential nutrients for all cell types. Our preliminary work had revealed gaps in the current understanding of yeast sulfur metabolism; even when a key gene involved in sulfide assimilation (MET17) was deleted, yeast could assimilate sulfide through an unknown alternative mechanism, however only in cultures exceeding a threshold cell density. Thus, to understand how these yeast populations overcome their metabolic defect in a density-dependent manner, we aimed to 1) develop quantitative tools to understand their sulfide-response, 2) identify gene(s) in the alternative sulfide metabolism, and 3) reveal the function of these genes when the primary route of sulfide assimilation was not perturbed.
Synergistically combining mathematical modelling and quantitative experiments, we elucidated the mechanism by which MET17-lacking yeast overcome their metabolic defect. We found that the uncharacterized locus YLL058W carries out sulfide assimilation in the absence of MET17, albeit at a low efficiency, making the growth outcome sensitive to factors that affect sulfide accumulation: cell density and gas escape. Thus, our research resolved misconceptions and revealed novel aspects of sulfur metabolism in budding yeast. Furthermore, we developed generalisable quantitative tools for studying diverse chemical-mediated interactions in microbial communities.
The overall aim of this project was to identify a hidden pathway in the budding yeast, Saccharomyces cerevisiae, which can assimilate sulfide into organic sulfur compounds—essential nutrients for all cell types. Our preliminary work had revealed gaps in the current understanding of yeast sulfur metabolism; even when a key gene involved in sulfide assimilation (MET17) was deleted, yeast could assimilate sulfide through an unknown alternative mechanism, however only in cultures exceeding a threshold cell density. Thus, to understand how these yeast populations overcome their metabolic defect in a density-dependent manner, we aimed to 1) develop quantitative tools to understand their sulfide-response, 2) identify gene(s) in the alternative sulfide metabolism, and 3) reveal the function of these genes when the primary route of sulfide assimilation was not perturbed.
Synergistically combining mathematical modelling and quantitative experiments, we elucidated the mechanism by which MET17-lacking yeast overcome their metabolic defect. We found that the uncharacterized locus YLL058W carries out sulfide assimilation in the absence of MET17, albeit at a low efficiency, making the growth outcome sensitive to factors that affect sulfide accumulation: cell density and gas escape. Thus, our research resolved misconceptions and revealed novel aspects of sulfur metabolism in budding yeast. Furthermore, we developed generalisable quantitative tools for studying diverse chemical-mediated interactions in microbial communities.
It is well established that budding yeast can synthesize essential organic sulfur forms (organosulfurs) from inorganic sulfate. Sulfate is first reduced to sulfide, and the Met17 enzyme then catalyzes the addition of sulfide to an organic backbone. According to this conventional knowledge, MET17 should be required for the growth of yeast on inorganic sulfate, and yeast lacking MET17 (henceforth referred to as “met17-”) should require organosulfur supplements to grow. Surprisingly, we found that met17- were able to grow on sulfate, albeit only at sufficiently high cell densities. In the absence of MET17, yeast cultures accumulated the sulfide produced from sulfate reductio. Once sulfide levels became sufficiently high, an alternative pathway enabled the population growth of met17-. Indeed, the propensity of met17- to grow on sulfate could be promoted either by increasing cell numbers (thus increasing sulfide production) or reducing gas escape from the culturing set-up (thus decreasing sulfide loss). Through quantitative modelling, we showed that this sulfide-response likely results from the inefficiency of the alternative pathway at metabolizing sulfide; the pathway only produces sufficient organosulfurs to fuel cell division at high sulfide levels.
A genetic screen revealed that the uncharacterized genetic locus YLL058W (also known as HSU1) is responsible for sulfide assimilation in the absence of MET17. To reveal the function of HSU1 in wildtype yeast (i.e. when MET17 carries out sulfide assimilation), we conducted various functional analyses. The Hsu1 protein was expressed when yeast were either sulfur-starved or exposed to detrimentally high sulfide levels. However, HSU1 did not confer any detectable fitness advantage to yeast under either sulfur starvation or sulfide exposure. Interestingly, HSU1 provided an advantage during the assimilation of the sulfur-containing amino acid methionine, but a mild disadvantage in assimilating other sulfur sources such as the plant-derived organosulfur S-methylmethionine. Thus, HSU1 has a complex contribution to sulfur assimilation in budding yeast.
The results of this projected are publicly available on the preprint server bioRxiv and will shortly be available as a peer-reviewed publication in highly accredited open-access journal PLOS Biology. Additionally, the research has been presented in various international and national conferences with audiences of diverse scientific expertise covering microbiology, metabolism, evolution, ecology and mathematical biology. Furthermore, the quantitative tools developed herein are available for free use through the public repository Zenodo.
A genetic screen revealed that the uncharacterized genetic locus YLL058W (also known as HSU1) is responsible for sulfide assimilation in the absence of MET17. To reveal the function of HSU1 in wildtype yeast (i.e. when MET17 carries out sulfide assimilation), we conducted various functional analyses. The Hsu1 protein was expressed when yeast were either sulfur-starved or exposed to detrimentally high sulfide levels. However, HSU1 did not confer any detectable fitness advantage to yeast under either sulfur starvation or sulfide exposure. Interestingly, HSU1 provided an advantage during the assimilation of the sulfur-containing amino acid methionine, but a mild disadvantage in assimilating other sulfur sources such as the plant-derived organosulfur S-methylmethionine. Thus, HSU1 has a complex contribution to sulfur assimilation in budding yeast.
The results of this projected are publicly available on the preprint server bioRxiv and will shortly be available as a peer-reviewed publication in highly accredited open-access journal PLOS Biology. Additionally, the research has been presented in various international and national conferences with audiences of diverse scientific expertise covering microbiology, metabolism, evolution, ecology and mathematical biology. Furthermore, the quantitative tools developed herein are available for free use through the public repository Zenodo.
Budding yeast has been a popular model organism; much of our understanding of metabolism in higher organisms originated from pathways revealed in this eukaryotic microorganism. The fact that even after half a century of studies, we could reveal novel aspects of sulfur metabolism in yeast highlights that there remains considerable potential for discovery in metabolism. Revealing new metabolites and pathways could hold the key to outstanding healthcare concerns. Sulfide, with its beneficial physiological effects on various tissues, highlights the hidden therapeutic potential of sulfur metabolism.
The insights gained during this project have opened various avenues of enquiry regarding the function(s) of HSU1. Our work and that from other labs suggest that HSU1 is a multicatalytic enzyme involved in the synthesis of various volatile sulfur compounds. Similar enzymes found in plants and bacteria have been studied as anticancer therapeutics, though their application is impeded by the high immunogenicity of these enzymes. Finding new eukaryotic sources for such enzymes could thus provide new candidates for therapeutic application. Furthermore, volatile sulfur compounds have a significant contribution to wine aroma, but their metabolism remains poorly understood. Revealing the pathways underlying the synthesis of these compounds can enable the wine and fermentation industries to optimize their products. Thus, the ongoing research arising from this project holds broad societal implications.
Finally, our work has also contributed useful methodology to the scientific community. Gas-mediated interactions are common in biology but remain understudied since volatiles can be challenging to quantify. Our work suggests that if chemical transitions occur at a considerably faster timescale than the biological processes, the liquid-gas partitioning of the chemical mediator may not need to be modelled dynamically, thus considerably simplifying the quantitative modelling of gas-mediated interactions. In sum, our work has not only raised awareness towards the often-overlooked gaseous metabolites in microbial cultures, but has also provided tools to study gas-mediated interactions in other, more complex microbial communities.
The insights gained during this project have opened various avenues of enquiry regarding the function(s) of HSU1. Our work and that from other labs suggest that HSU1 is a multicatalytic enzyme involved in the synthesis of various volatile sulfur compounds. Similar enzymes found in plants and bacteria have been studied as anticancer therapeutics, though their application is impeded by the high immunogenicity of these enzymes. Finding new eukaryotic sources for such enzymes could thus provide new candidates for therapeutic application. Furthermore, volatile sulfur compounds have a significant contribution to wine aroma, but their metabolism remains poorly understood. Revealing the pathways underlying the synthesis of these compounds can enable the wine and fermentation industries to optimize their products. Thus, the ongoing research arising from this project holds broad societal implications.
Finally, our work has also contributed useful methodology to the scientific community. Gas-mediated interactions are common in biology but remain understudied since volatiles can be challenging to quantify. Our work suggests that if chemical transitions occur at a considerably faster timescale than the biological processes, the liquid-gas partitioning of the chemical mediator may not need to be modelled dynamically, thus considerably simplifying the quantitative modelling of gas-mediated interactions. In sum, our work has not only raised awareness towards the often-overlooked gaseous metabolites in microbial cultures, but has also provided tools to study gas-mediated interactions in other, more complex microbial communities.