Nucleosomes affect local transformation efficiency

Genetic transformation is a natural process during which foreign DNA enters a cell and integrates into the genome. Apart from its relevance for horizontal gene transfer in nature, transformation is also the cornerstone of today's recombinant gene technology. Despite its importance, relatively little is known about the factors that determine transformation efficiency. We hypothesize that differences in DNA accessibility associated with nucleosome positioning may affect local transformation efficiency. We investigated the landscape of transformation efficiency at various positions in the Saccharomyces cerevisiae genome and correlated these measurements with nucleosome positioning. We find that transformation efficiency shows a highly significant inverse correlation with relative nucleosome density. This correlation was lost when the nucleosome pattern, but not the underlying sequence was changed. Together, our results demonstrate a novel role for nucleosomes and also allow researchers to predict transformation efficiency of a target region and select spots in the genome that are likely to yield higher transformation efficiency

Domestication and divergence of Saccharomyces cerevisiae beer yeasts

Whereas domestication of livestock, pets, and crops is well documented, it is still unclear to what extent microbes associated with the production of food have also undergone human selection and where the plethora of industrial strains originates from. Here, we present the genomes and phenomes of 157 industrial Saccharomyces cerevisiae yeasts. Our analyses reveal that today's industrial yeasts can be divided into five sublineages that are genetically and phenotypically separated from wild strains and originate from only a few ancestors through complex patterns of domestication and local divergence. Large-scale phenotyping and genome analysis further show strong industry-specific selection for stress tolerance, sugar utilization, and flavor production, while the sexual cycle and other phenotypes related to survival in nature show decay, particularly in beer yeasts. Together, these results shed light on the origins, evolutionary history, and phenotypic diversity of industrial yeasts and provide a resource for further selection of superior strains

Adaptation to high ethanol reveals complex evolutionary pathways

Tolerance to high levels of ethanol is an ecologically and industrially relevant phenotype of microbes, but the molecular mechanisms underlying this complex trait remain largely unknown. Here, we use long-term experimental evolution of isogenic yeast populations of different initial ploidy to study adaptation to increasing levels of ethanol. Whole-genome sequencing of more than 30 evolved populations and over 100 adapted clones isolated throughout this two-year evolution experiment revealed how a complex interplay of de novo single nucleotide mutations, copy number variation, ploidy changes, mutator phenotypes, and clonal interference led to a significant increase in ethanol tolerance. Although the specific mutations differ between different evolved lineages, application of a novel computational pipeline, PheNetic, revealed that many mutations target functional modules involved in stress response, cell cycle regulation, DNA repair and respiration. Measuring the fitness effects of selected mutations introduced in non-evolved ethanol-sensitive cells revealed several adaptive mutations that had previously not been implicated in ethanol tolerance, including mutations in PRT1, VPS70 and MEX67. Interestingly, variation in VPS70 was recently identified as a QTL for ethanol tolerance in an industrial bio-ethanol strain. Taken together, our results show how, in contrast to adaptation to some other stresses, adaptation to a continuous complex and severe stress involves interplay of different evolutionary mechanisms. In addition, our study reveals functional modules involved in ethanol resistance and identifies several mutations that could help to improve the ethanol tolerance of industrial yeasts

Ethanol exposure increases mutation rate through error-prone polymerases

International audienceEthanol is a ubiquitous environmental stressor that is toxic to all lifeforms. Here, we use the model eukaryote Saccharomyces cerevisiae to show that exposure to sublethal ethanol concentrations causes DNA replication stress and an increased mutation rate. Specifically, we find that ethanol slows down replication and affects localization of Mrc1, a conserved protein that helps stabilize the replisome. In addition, ethanol exposure also results in the recruitment of error-prone DNA polymerases to the replication fork. Interestingly, preventing this recruitment through mutagenesis of the PCNA/Pol30 polymerase clamp or deleting specific error-prone polymerases abolishes the mutagenic effect of ethanol. Taken together, this suggests that the mutagenic effect depends on a complex mechanism, where dysfunctional replication forks lead to recruitment of error-prone polymerases. Apart from providing a general mechanistic framework for the mutagenic effect of ethanol, our findings may also provide a route to better understand and prevent ethanol-associated carcinogenesis in higher eukaryotes

Involvement of the yeast PDK1 orthologs Pkh1-3 in nutrient-induced signaling.

Nutriënten zijn belangrijke regulatoren van groei. Wanneer een essentiee l nutriënt, zoals een koolstof- , stikstof- of fosfaatbron niet meer aan wezig is in hun omgeving, zullen gistcellen stoppen met groeien en overg aan in een soort van slapende toestand, stationaire fase genoemd. In dez e toestand kunnen cellen periodes zonder voedsel overleven, onder andere door hun verhoogde stresstolerantie. Toevoegen van het ontbrekende nutr iënt, in de aanwezigheid van een fermenteerbare koolstofbron, activeert de Fermenteerbaar Groei Medium (FGM) signaalweg. Dit zorgt ervoor dat de cellen de stationaire fase eigenschappen verliezen en opnieuw beginnen te groeien. Verschillende eiwitten in de plasma membraan van gist werden geïdentific eerd die niet alleen de toegevoegde nutriënten in de cel transporteren, maar tegelijkertijd ook dienst doen als nutriëntsensor en zo de FGM sign aalweg activeren. Helaas zijn er momenteel weinig andere componenten van deze signaalweg gekend. Behalve proteïne kinase < U&gt;A (PKA) is het kinase Sch9 (het gist homoloog van de zoogdier eiwitten PKB en S6K) het enige andere eiwit waarvan we weten da t het nodig is voor activatie van de FGM signaalweg door een stikstofbro n. In deze studie hebben we onderzocht wat het belang is van de gist eiwitt en Pkh1-3 (kinasen die sterk gelijken op het zoogdier eiwit PDK1) voor d e juiste respons op het toevoegen van nutriënten. Zowel de katalytische subeenheden van PKA (de eiwitten Tpk1-3) als het Sch9 kinase hebben een mogelijke PDK1 fosforylatiesite, wat erop duidt dat deze eiwitten mogeli jk substraten zijn van Pkh1-3. Om te achterhalen of Pkh1-3 ook effectief een rol spelen in FGM signalering, bestudeerden we eerst hun belang in de activatie van het enzyme trehalase, één van de doelwitten van de FGM signaalweg. In cellen zonder Pkh1-3 is de trehalase activatie door zowel glucose als door een stikstofbron sterk verlaagd. De katalytische subeenheden van PKA zijn nodig voor zowel stik stof- als glucose-geïnduceerde activatie van de FGM signaalweg. Pkh1 fos foryleert Tpk1 inderdaad in vitro, maar deze fosforylatie is in vivo&amp;nbs p;niet afhankelijk van Pkh1-3. Bovendien blijkt dat factoren waarvan gew eten is dat ze PKA activiteit verhogen, zoals bvb. toevoegen van glucose aan glucose-gedepriveerde cellen, niet zorgen voor een toename in PDK1 site fosforylatie van Tpk1. Hoewel we voorlopig niet weten welk kinase d eze site in Tpk1 fosforyleert, tonen onze resultaten wel dat deze fosfor ylatie zeer belangrijk is voor de activiteit van PKA. Bovendien is fosfo rylatie ook nodig voor binding van de regulatorische subeenheid. Dit lig t in de lijn van wat gekend is over het belang van deze site in zoogdier PKA. We tonen hier voor de eerste keer dat Pkh1-3 nodig zijn voor stikstofsig nalering in gist, en dat zij Sch9 fosforyleren. Bovendien verdwijnt deze fosforylatie als er geen stikstof aanwezig is, en keert ze terug wannee r er terug stikstof (en alle andere componenten die nodig zijn voor groe i) aanwezig is. Op deze manier lijkt de fosforylatie van Sch9 sterk same n te hangen met de groei van de cel. Onze gegevens wijzen dus op een nie uwe functie van de gist Pkh eiwitten, naast hun reeds gekende rol in cel integriteitssignalering. Andere onderzoekers toonden recent aan dat ook het TORC1 eiwitcomplex Sc h9 fosforyleert op een stikstofafhankelijke manier. Samen met onze resul taten, duidt dit op een centrale rol voor Sch9 in stikstofsignalering in gist.status: publishe

How do regulatory networks evolve and expand throughout evolution?

Throughout evolution, regulatory networks need to expand and adapt to accommodate novel genes and gene functions. However, the molecular details explaining how gene networks evolve remain largely unknown. Recent studies demonstrate that changes in transcription factors contribute to the evolution of regulatory networks. In particular, duplication of transcription factors followed by specific mutations in their DNA-binding or interaction domains propels the divergence and emergence of new networks. The innate promiscuity and modularity of regulatory networks contributes to their evolvability: duplicated promiscuous regulators and their target promoters can acquire mutations that lead to gradual increases in specificity, allowing neofunctionalization or subfunctionalization.publisher: Elsevier articletitle: How do regulatory networks evolve and expand throughout evolution? journaltitle: Current Opinion in Biotechnology articlelink: http://dx.doi.org/10.1016/j.copbio.2015.02.001 content_type: article copyright: Copyright © 2015 The Authors. Published by Elsevier Ltd.status: publishe

How do yeast cells become tolerant to high ethanol concentrations?

The brewer's yeast Saccharomyces cerevisiae displays a much higher ethanol tolerance compared to most other organisms, and it is therefore commonly used for the industrial production of bioethanol and alcoholic beverages. However, the genetic determinants underlying this yeast's exceptional ethanol tolerance have proven difficult to elucidate. In this perspective, we discuss how different types of experiments have contributed to our understanding of the toxic effects of ethanol and the mechanisms and complex genetics underlying ethanol tolerance. In a second part, we summarize the different routes and challenges involved in obtaining superior industrial yeasts with improved ethanol tolerance.status: publishe

Experimental evolution yields insight into dynamics of adaptation to ethanol

status: publishe

Experimental evolution yields insight into dynamics of adaptation to ethanol

status: publishe