RESUMO
The discrete steps of transcriptional rewiring have been proposed to occur neutrally to ensure steady gene expression under stabilizing selection. A conflict-free switch of a regulon between regulators may require an immediate compensatory evolution to minimize deleterious effects. Here, we perform an evolutionary repair experiment on the Lachancea kluyveri yeast sef1Δ mutant using a suppressor development strategy. Complete loss of SEF1 forces cells to initiate a compensatory process for the pleiotropic defects arising from misexpression of TCA cycle genes. Using different selective conditions, we identify two adaptive loss-of-function mutations of IRA1 and AZF1. Subsequent analyses show that Azf1 is a weak transcriptional activator regulated by the Ras1-PKA pathway. Azf1 loss-of-function triggers extensive gene expression changes responsible for compensatory, beneficial, and trade-off phenotypes. The trade-offs can be alleviated by higher cell density. Our results not only indicate that secondary transcriptional perturbation provides rapid and adaptive mechanisms potentially stabilizing the initial stage of transcriptional rewiring but also suggest how genetic polymorphisms of pleiotropic mutations could be maintained in the population.
Assuntos
Redes Reguladoras de Genes , Fatores de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Mutação , FenótipoRESUMO
Prior and extensive plastic rewiring of a transcriptional network, followed by a functional switch of the conserved transcriptional regulator, can shape the evolution of a new network with diverged functions. The presence of three distinct iron regulatory systems in fungi that use orthologous transcriptional regulators suggests that these systems evolved in that manner. Orthologs of the transcriptional activator Sef1 are believed to be central to how iron regulatory systems developed in fungi, involving gene gain, plastic network rewiring, and switches in regulatory function. We show that, in the protoploid yeast Lachancea kluyveri, plastic rewiring of the L. kluyveri Sef1 (Lk-Sef1) network, together with a functional switch, enabled Lk-Sef1 to regulate TCA cycle genes, unlike Candida albicans Sef1 that mainly regulates iron-uptake genes. Moreover, we observed pervasive nonfunctional binding of Sef1 to its target genes. Enhancing Lk-Sef1 activity resuscitated the corresponding transcriptional network, providing immediate adaptive benefits in changing environments. Our study not only sheds light on the evolution of Sef1-centered transcriptional networks but also shows the adaptive potential of nonfunctional transcription factor binding for evolving phenotypic novelty and diversity.
Assuntos
Redes Reguladoras de Genes , Plásticos , Candida albicans/genética , Plásticos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Leveduras/genéticaRESUMO
The first committed step in the leucine biosynthetic pathway is catalyzed by α-isopropylmalate synthase (α-IPMS, EC 2.3.3.13), which in the Saccaromycotina subphylum of Ascomycete yeasts is frequently encoded by duplicated genes. Following a gene duplication event, the two copies may be preserved presumably because the encoded proteins diverge in either functional properties and/or cellular localization. The genome of the petite-negative budding yeast Lachancea kluyveri includes two SAKL0E10472 (LkLEU4) and SAKL0F05170 g (LkLEU4BIS) paralogous genes, which are homologous to other yeast α-IPMS sequences. Here, we investigate whether these paralogous genes encode functional α-IPMS isozymes and whether their functions have diverged. Molecular phylogeny suggested that the LkLeu4 isozyme is located in the mitochondria and LkLeu4BIS in the cytosol. Comparison of growth rates, leucine intracellular pools and mRNA levels, indicate that the LkLeu4 isozyme is the predominant α-IPMS enzyme during growth on glucose as carbon source. Determination of the kinetic parameters indicates that the isozymes have similar affinities for the substrates and for the feedback inhibitor leucine. Thus, the diversification of the physiological roles of the genes LkLEU4 and LkLEU4BIS involves preferential transcription of the LkLEU4 gene during growth on glucose and different subcellular localization, although ligand interactions have not diverged.
Assuntos
2-Isopropilmalato Sintase , Saccharomycetales , 2-Isopropilmalato Sintase/química , 2-Isopropilmalato Sintase/genética , 2-Isopropilmalato Sintase/metabolismo , Glucose/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Leucina/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismoRESUMO
Since more than a decade ago, Saccharomyces cerevisiae has been used as a model to dissect complex traits, revealing the genetic basis of a large number of traits in fine detail. However, to have a more global view of the genetic architecture of traits across species, the examination of the molecular basis of phenotypes within non-conventional species would undoubtedly be valuable. In this respect, the Saccharomycotina yeasts represent ideal and potential non-model organisms. Here we sought to assess the feasibility of genetic mapping by bulk segregant analysis in the protoploid Lachancea kluyveri (formerly S. kluyveri) yeast species, a distantly related species to S. cerevisiae For this purpose, we designed a fluorescent mating-type marker, compatible with any mating-competent strains representative of this species, to rapidly create a large population of haploid segregants (>10(5) cells). Quantitative trait loci can be mapped by selecting and sequencing an enriched pool of progeny with extreme phenotypic values. As a test bed, we applied this strategy and mapped the causal loci underlying halotolerance phenotypes in L. kluyveri Overall, this study demonstrates that bulk segregant mapping is a powerful way for investigating the genetic basis of natural variations in non-model yeast organisms and more precisely in L. kluyveri.
Assuntos
Mapeamento Cromossômico/métodos , Locos de Características Quantitativas , Saccharomycetales/genética , Genes Reporter , Coloração e RotulagemRESUMO
Branched chain amino acid aminotransferases (BCATs) catalyze the last step of the biosynthesis and the first step of the catabolism of branched chain amino acids. In Saccharomyces cerevisiae, BCATs are encoded by the ScBAT1 and ScBAT2 paralogous genes. Analysis of Lachancea kluyveri genome sequence, allowed the identification of the LkBAT1 locus, which could presumably encode a BCAT. A second unlinked locus (LkBAT1bis), exhibiting sequence similarity to LkBAT1 was also identified. To determine the function of these putative BCATs, L. kluyveri mutant strains lacking LkBAT1, LkBAT1bis or both genes were generated and tested for VIL metabolism. LkBat1 displayed branched chain aminotransferase activity and is required for VIL biosynthesis and catabolism. However, Lkbat1Δ mutant is a valine and isoleucine auxotroph and a leucine bradytroph indicating that L. kluyveri harbors an alternative enzyme(s) involved in leucine biosynthesis. Additionally, heterologous reciprocal gene complementation between S. cerevisiae and L. kluyveri orthologous LkBAT1, ScBAT1 and ScBAT2 genes, confirmed that the mitochondrial LkBat1 functions as BCAT in S. cerevisiae, restoring wild type phenotype to the ScBAT1 null mutant. Conversely, LkBAT1bis did not display a role in BCAAs metabolism. However, when ethanol was used as carbon source, deletion of LkBAT1bis in an Lkbat1Δ null strain resulted in an extended 'lag' growth phase, pointing to a potential function of LkBAT1 and LkBAT1bis in the aerobic metabolism of L. kluyveri. These results confirm the BCAT function of LkBAT1 in L. kluyveri, and further support the proposition that the BCAT function in ancestral-type yeasts has been distributed in the two paralogous genes present in S. cerevisiae.
Assuntos
Saccharomycetales/enzimologia , Transaminases/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Isoleucina/genética , Isoleucina/metabolismo , Leucina/genética , Leucina/metabolismo , Mitocôndrias/metabolismo , Saccharomycetales/genética , Transaminases/genética , Valina/genética , Valina/metabolismoRESUMO
A healthy gut microbiota generally improves the performance of its insect host. Although the effects can be specific to the species composition of the microbial community, the role of gut microbiota in determining water balance has not been well explored. We used axenic and gnotobiotic (reared with a known microbiota) Drosophila melanogaster to test three hypotheses about the effects of gut yeasts on the water balance of adult flies: 1) that gut yeasts would improve desiccation survival in adult flies; 2) that larval yeasts would improve adult desiccation survival; 3) that the effects would be species-specific, such that yeasts closely associated with D. melanogaster in nature are more likely to be beneficial than those rarely found in association with D. melanogaster. We used Saccharomyces cerevisiae (often used in Drosophila cultures, but rarely associated with D. melanogaster in nature), Lachancea kluyveri (associated with some species of Drosophila, but not D. melanogaster), and Pichia kluyveri (associated with D. melanogaster in nature). Adult inoculation with yeasts had no effect on survival of desiccating conditions. Inoculation with P. kluyveri as larvae did not change desiccation survival in adults; however, rearing with L. kluyveri or S. cerevisiae reduced adult desiccation survival. We conclude that adult inoculation with gut yeasts has no impact on desiccation survival, but that rearing with yeasts can have either no or detrimental effect. The effects appear to be species-specific: P. kluyveri did not have a negative impact on desiccation tolerance, suggesting some level of co-adaptation with D. melanogaster. We note that S. cerevisiae may not be an appropriate species for studying the effects of gut yeasts on D. melanogaster.