Unravelling metabolic cross-feeding in a yeast-bacteria community using 13 C-based proteomics.

Cross-feeding is fundamental to the diversity and function of microbial communities. However, identification of cross-fed metabolites is often challenging due to the universality of metabolic and biosynthetic intermediates. Here, we use 13 C isotope tracing in peptides to elucidate cross-fed metabolites in co-cultures of Saccharomyces cerevisiae and Lactococcus lactis. The community was grown on lactose as the main carbon source with either glucose or galactose fraction of the molecule labelled with 13 C. Data analysis allowing for the possible mass-shifts yielded hundreds of peptides for which we could assign both species identity and labelling degree. The labelling pattern showed that the yeast utilized galactose and, to a lesser extent, lactic acid shared by L. lactis as carbon sources. While the yeast provided essential amino acids to the bacterium as expected, the data also uncovered a complex pattern of amino acid exchange. The identity of the cross-fed metabolites was further supported by metabolite labelling in the co-culture supernatant, and by diminished fitness of a galactose-negative yeast mutant in the community. Together, our results demonstrate the utility of 13 C-based proteomics for uncovering microbial interactions.

Bioactivity assessment of natural compounds using machine learning models trained on target similarity between drugs.

Natural compounds constitute a rich resource of potential small molecule therapeutics. While experimental access to this resource is limited due to its vast diversity and difficulties in systematic purification, computational assessment of structural similarity with known therapeutic molecules offers a scalable approach. Here, we assessed functional similarity between natural compounds and approved drugs by combining multiple chemical similarity metrics and physicochemical properties using a machine-learning approach. We computed pairwise similarities between 1410 drugs for training classification models and used the drugs shared protein targets as class labels. The best performing models were random forest which gave an average area under the ROC of 0.9, Matthews correlation coefficient of 0.35, and F1 score of 0.33, suggesting that it captured the structure-activity relation well. The models were then used to predict protein targets of circa 11k natural compounds by comparing them with the drugs. This revealed therapeutic potential of several natural compounds, including those with support from previously published sources as well as those hitherto unexplored. We experimentally validated one of the predicted pair's activities, viz., Cox-1 inhibition by 5-methoxysalicylic acid, a molecule commonly found in tea, herbs and spices. In contrast, another natural compound, 4-isopropylbenzoic acid, with the highest similarity score when considering most weighted similarity metric but not picked by our models, did not inhibit Cox-1. Our results demonstrate the utility of a machine-learning approach combining multiple chemical features for uncovering protein binding potential of natural compounds.

The HIV-1 Nucleocapsid Regulates Its Own Condensation by Phase-Separated Activity-Enhancing Sequestration of the Viral Protease during Maturation.

A growing number of studies indicate that mRNAs and long ncRNAs can affect protein populations by assembling dynamic ribonucleoprotein (RNP) granules. These phase-separated molecular 'sponges', stabilized by quinary (transient and weak) interactions, control proteins involved in numerous biological functions. Retroviruses such as HIV-1 form by self-assembly when their genomic RNA (gRNA) traps Gag and GagPol polyprotein precursors. Infectivity requires extracellular budding of the particle followed by maturation, an ordered processing of ∼2400 Gag and ∼120 GagPol by the viral protease (PR). This leads to a condensed gRNA-NCp7 nucleocapsid and a CAp24-self-assembled capsid surrounding the RNP. The choreography by which all of these components dynamically interact during virus maturation is one of the missing milestones to fully depict the HIV life cycle. Here, we describe how HIV-1 has evolved a dynamic RNP granule with successive weak-strong-moderate quinary NC-gRNA networks during the sequential processing of the GagNC domain. We also reveal two palindromic RNA-binding triads on NC, KxxFxxQ and QxxFxxK, that provide quinary NC-gRNA interactions. Consequently, the nucleocapsid complex appears properly aggregated for capsid reassembly and reverse transcription, mandatory processes for viral infectivity. We show that PR is sequestered within this RNP and drives its maturation/condensation within minutes, this process being most effective at the end of budding. We anticipate such findings will stimulate further investigations of quinary interactions and emergent mechanisms in crowded environments throughout the wide and growing array of RNP granules.

High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding.

Genetic screens using high-throughput fluorescent microscopes have generated large datasets, contributing many cell biological insights. Such approaches cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) reveals detailed cellular ultrastructure but requires time-consuming sample preparation, limiting throughput. Here we describe a robust method for screening by high-throughput EM. Our approach uses combinations of fluorophores as barcodes to uniquely mark each cell type in mixed populations and correlative light and EM (CLEM) to read the barcode of each cell before it is imaged by EM. Coupled with an easy-to-use software workflow for correlation, segmentation, and computer image analysis, our method, called "MultiCLEM," allows us to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate several uses for MultiCLEM with 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher throughput, and can be used in multiple ways to enable EM to become a powerful screening technique.

Yeast Creates a Niche for Symbiotic Lactic Acid Bacteria through Nitrogen Overflow.

Many microorganisms live in communities and depend on metabolites secreted by fellow community members for survival. Yet our knowledge of interspecies metabolic dependencies is limited to few communities with small number of exchanged metabolites, and even less is known about cellular regulation facilitating metabolic exchange. Here we show how yeast enables growth of lactic acid bacteria through endogenous, multi-component, cross-feeding in a readily established community. In nitrogen-rich environments, Saccharomyces cerevisiae adjusts its metabolism by secreting a pool of metabolites, especially amino acids, and thereby enables survival of Lactobacillus plantarum and Lactococcus lactis. Quantity of the available nitrogen sources and the status of nitrogen catabolite repression pathways jointly modulate this niche creation. We demonstrate how nitrogen overflow by yeast benefits L. plantarum in grape juice, and contributes to emergence of mutualism with L. lactis in a medium with lactose. Our results illustrate how metabolic decisions of an individual species can benefit others.

A cascade of iron-containing proteins governs the genetic iron starvation response to promote iron uptake and inhibit iron storage in fission yeast.

Iron is an essential cofactor, but it is also toxic at high levels. In Schizosaccharomyces pombe, the sensor glutaredoxin Grx4 guides the activity of the repressors Php4 and Fep1 to mediate a complex transcriptional response to iron deprivation: activation of Php4 and inactivation of Fep1 leads to inhibition of iron usage/storage, and to promotion of iron import, respectively. However, the molecular events ruling the activity of this double-branched pathway remained elusive. We show here that Grx4 incorporates a glutathione-containing iron-sulfur cluster, alone or forming a heterodimer with the BolA-like protein Fra2. Our genetic study demonstrates that Grx4-Fra2, but not Fep1 nor Php4, participates not only in iron starvation signaling but also in iron-related aerobic metabolism. Iron-containing Grx4 binds and inactivates the Php4 repressor; upon iron deprivation, the cluster in Grx4 is probably disassembled, the proteins dissociate, and Php4 accumulates at the nucleus and represses iron consumption genes. Fep1 is also an iron-containing protein, and the tightly bound iron is required for transcriptional repression. Our data suggest that the cluster-containing Grx4-Fra2 heterodimer constitutively binds to Fep1, and upon iron deprivation the disassembly of the iron cluster between Grx4 and Fra2 promotes reverse metal transfer from Fep1 to Grx4-Fra2, and de-repression of iron-import genes. Our genetic and biochemical study demonstrates that the glutaredoxin Grx4 independently governs the Php4 and Fep1 repressors through metal transfer. Whereas iron loss from Grx4 seems to be sufficient to release Php4 and allow its nuclear accumulation, total or partial disassembly of the Grx4-Fra2 cluster actively participates in iron-containing Fep1 activation by sequestering its iron and decreasing its interaction with promoters.

Cells lacking pfh1, a fission yeast homolog of mammalian frataxin protein, display constitutive activation of the iron starvation response.

Friedreich ataxia is a genetic disease caused by deficiencies in frataxin. This protein has homologs not only in higher eukaryotes but also in bacteria, fungi, and plants. The function of this protein is still controversial. We have identified a frataxin homolog in fission yeast, and we have analyzed whether its depletion leads to any of the phenotypes observed in other organisms. Cells deleted in pfh1 are sensitive to growth under aerobic conditions, display increased levels of total iron, hallmarks of oxidative stress such as protein carbonylation, decreased aconitase activity, and lower levels of oxygen consumption compared with wild-type cells. This mitochondrial protein seems to be important for iron and/or reactive oxygen species homeostasis. We have analyzed the proteome of cells devoid of Pfh1, and we determined that gene products up- and down-regulated upon iron depletion in wild-type cells are constitutively misregulated in this mutant. Because of the particular signaling pathway components governing the iron starvation response in fission yeast, our experiments suggest that cells lacking Pfh1 display a decrease of cytosolic available iron that triggers activation of Grx4, the common regulator of the iron starvation gene expression program. Our Schizosaccharomyces pombe Δpfh1 strain constitutes a new and useful model system to study Friedreich ataxia.

Lifespan extension by calorie restriction relies on the Sty1 MAP kinase stress pathway.

Either calorie restriction, loss-of-function of the nutrient-dependent PKA or TOR/SCH9 pathways, or activation of stress defences improves longevity in different eukaryotes. However, the molecular links between glucose depletion, nutrient-dependent pathways and stress responses are unknown. Here, we show that either calorie restriction or inactivation of nutrient-dependent pathways induces lifespan extension in fission yeast, and that such effect is dependent on the activation of the stress-dependent Sty1 mitogen-activated protein (MAP) kinase. During transition to stationary phase in glucose-limiting conditions, Sty1 becomes activated and triggers a transcriptional stress programme, whereas such activation does not occur under glucose-rich conditions. Deletion of the genes coding for the SCH9-homologue, Sck2 or the Pka1 kinases, or mutations leading to constitutive activation of the Sty1 stress pathway increase lifespan under glucose-rich conditions, and importantly such beneficial effects depend ultimately on Sty1. Furthermore, cells lacking Pka1 display enhanced oxygen consumption and Sty1 activation under glucose-rich conditions. We conclude that calorie restriction favours oxidative metabolism, reactive oxygen species production and Sty1 MAP kinase activation, and this stress pathway favours lifespan extension.

Genome-wide screen of genes required for caffeine tolerance in fission yeast.

BACKGROUND: An excess of caffeine is cytotoxic to all eukaryotic cell types. We aim to study how cells become tolerant to a toxic dose of this drug, and the relationship between caffeine and oxidative stress pathways. METHODOLOGY/PRINCIPAL FINDINGS: We searched for Schizosaccharomyces pombe mutants with inhibited growth on caffeine-containing plates. We screened a collection of 2,700 haploid mutant cells, of which 98 were sensitive to caffeine. The genes mutated in these sensitive clones were involved in a number of cellular roles including the H(2)O(2)-induced Pap1 and Sty1 stress pathways, the integrity and calcineurin pathways, cell morphology and chromatin remodeling. We have investigated the role of the oxidative stress pathways in sensing and promoting survival to caffeine. The Pap1 and the Sty1 pathways are both required for normal tolerance to caffeine, but only the Sty1 pathway is activated by the drug. Cells lacking Pap1 are sensitive to caffeine due to the decreased expression of the efflux pump Hba2. Indeed, ?hba2 cells are sensitive to caffeine, and constitutive activation of the Pap1 pathway enhances resistance to caffeine in an Hba2-dependent manner. CONCLUSIONS/SIGNIFICANCE: With our caffeine-sensitive, genome-wide screen of an S. pombe deletion collection, we have demonstrated the importance of some oxidative stress pathway components on wild-type tolerance to the drug.

Promoter-driven splicing regulation in fission yeast.

The meiotic cell cycle is modified from the mitotic cell cycle by having a pre-meiotic S phase that leads to high levels of recombination, two rounds of nuclear division with no intervening DNA synthesis and a reductional pattern of chromosome segregation. Rem1 is a cyclin that is only expressed during meiosis in the fission yeast Schizosaccharomyces pombe. Cells in which rem1 has been deleted show decreased intragenic meiotic recombination and a delay at the onset of meiosis I (ref. 1). When ectopically expressed in mitotically growing cells, Rem1 induces a G1 arrest followed by severe mitotic catastrophes. Here we show that rem1 expression is regulated at the level of both transcription and splicing, encoding two proteins with different functions depending on the intron retention. We have determined that the regulation of rem1 splicing is not dependent on any transcribed region of the gene. Furthermore, when the rem1 promoter is fused to other intron-containing genes, the chimaeras show a meiotic-specific regulation of splicing, exactly the same as endogenous rem1. This regulation is dependent on two transcription factors of the forkhead family, Mei4 (ref. 2) and Fkh2 (ref. 3). Whereas Mei4 induces both transcription and splicing of rem1, Fkh2 is responsible for the intron retention of the transcript during vegetative growth and the pre-meiotic S phase.

Mitochondrial dysfunction increases oxidative stress and decreases chronological life span in fission yeast.

BACKGROUND: Oxidative stress is a probable cause of aging and associated diseases. Reactive oxygen species (ROS) originate mainly from endogenous sources, namely the mitochondria. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the effect of aerobic metabolism on oxidative damage in Schizosaccharomyces pombe by global mapping of those genes that are required for growth on both respiratory-proficient media and hydrogen-peroxide-containing fermentable media. Out of a collection of approximately 2700 haploid yeast deletion mutants, 51 were sensitive to both conditions and 19 of these were related to mitochondrial function. Twelve deletion mutants lacked components of the electron transport chain. The growth defects of these mutants can be alleviated by the addition of antioxidants, which points to intrinsic oxidative stress as the origin of the phenotypes observed. These respiration-deficient mutants display elevated steady-state levels of ROS, probably due to enhanced electron leakage from their defective transport chains, which compromises the viability of chronologically-aged cells. CONCLUSION/SIGNIFICANCE: Individual mitochondrial dysfunctions have often been described as the cause of diseases or aging, and our global characterization emphasizes the primacy of oxidative stress in the etiology of such processes.