Adaptive response to wine selective pressures shapes the genome of a Saccharomyces interspecies hybrid

During industrial processes, yeasts are exposed to harsh conditions, which eventually lead to adaptation of the strains. In the laboratory, it is possible to use experimental evolution to link the evolutionary biology response to these adaptation pressures for the industrial improvement of a specific yeast strain. In this work, we aimed to study the adaptation of a wine industrial yeast in stress conditions of the high ethanol concentrations present in stopped fermentations and secondary fermentations in the processes of champagne production. We used a commercial Saccharomyces cerevisiae × S. uvarum hybrid and assessed its adaptation in a modified synthetic must (M-SM) containing high ethanol, which also contained metabisulfite, a preservative that is used during wine fermentation as it converts to sulfite. After the adaptation process under these selected stressful environmental conditions, the tolerance of the adapted strain (H14A7-etoh) to sulfite and ethanol was investigated, revealing that the adapted hybrid is more resistant to sulfite compared to the original H14A7 strain, whereas ethanol tolerance improvement was slight. However, a trade-off in the adapted hybrid was found, as it had a lower capacity to ferment glucose and fructose in comparison with H14A7. Hybrid genomes are almost always unstable, and different signals of adaptation on H14A7-etoh genome were detected. Each subgenome present in the adapted strain had adapted differently. Chromosome aneuploidies were present in S. cerevisiae chromosome III and in S. uvarum chromosome VII–XVI, which had been duplicated. Moreover, S. uvarum chromosome I was not present in H14A7-etoh and a loss of heterozygosity (LOH) event arose on S. cerevisiae chromosome I. RNA-sequencing analysis showed differential gene expression between H14A7-etoh and H14A7, which can be easily correlated with the signals of adaptation that were found on the H14A7-etoh genome. Finally, we report alterations in the lipid composition of the membrane, consistent with conserved tolerance mechanisms.

Long-term retainment of some chromosomal inversions in a local population of Belgica antarctica Jacobs (Diptera, Chironomidae)

Genome of antarctic endemic Belgica antarctica Jacobs has been sequenced. However, no set of inversion diagnostic markers has ever been assigned for the species. Using the classical method of polytene chromosome squash preparation, we found three heterozygous inversions located on the second (two heterozygous inversions) and third chromosomes (one heterozygous inversion) in the Belgica antarctica population of a cape of Wiencke Island, 500 m to SW from Port Lockroy. The chromosome set and chromosome variability did not differ from those described in the literature (Atchley and Davis 1979). Every salivary gland chromosome had its own markers by which it can be determined. However, we did not find a sex-linked inversion on chromosome III and heterozygous inversion on chromosome I, reported in earlier studies. For the first time, we observed a strong heterochromatin band in chromosome III at the telomere of one arm. Our data show not only the stability of the described inversions in the population but also the usefulness of the squash preparation technique in the studies of genetic variability of Belgica antarctica in present time.

The selfish yeast plasmid utilizes the condensin complex and condensed chromatin for faithful partitioning

Equipartitioning by chromosome association and copy number correction by DNA amplification are at the heart of the evolutionary success of the selfish yeast 2-micron plasmid. The present analysis reveals frequent plasmid presence near telomeres (TELs) and centromeres (CENs) in mitotic cells, with a preference towards the former. Inactivation of Cdc14 causes plasmid missegregation, which is correlated to the non-disjunction of TELs (and of rDNA) under this condition. Induced missegregation of chromosome XII, one of the largest yeast chromosomes which harbors the rDNA array and is highly dependent on the condensin complex for proper disjunction, increases 2-micron plasmid missegregation. This is not the case when chromosome III, one of the smallest chromosomes, is forced to missegregate. Plasmid stability decreases when the condensin subunit Brn1 is inactivated. Brn1 is recruited to the plasmid partitioning locus (STB) with the assistance of the plasmid-coded partitioning proteins Rep1 and Rep2. Furthermore, in a dihybrid assay, Brn1 interacts with Rep1-Rep2. Taken together, these findings support a role for condensin and/or condensed chromatin in 2-micron plasmid propagation. They suggest that condensed chromosome loci are among favored sites utilized by the plasmid for its chromosome-associated segregation. By homing to condensed/quiescent chromosome locales, and not over-perturbing genome homeostasis, the plasmid may minimize fitness conflicts with its host. Analogous persistence strategies may be utilized by other extrachromosomal selfish genomes, for example, episomes of mammalian viruses that hitchhike on host chromosomes for their stable maintenance.

Genetic Characterization of Mutations Related to Conidiophore Stalk Length Development in Aspergillus niger Laboratory Strain N402

Aspergillus niger is an important filamentous fungus in industrial biotechnology for the production of citric acid and enzymes. In the late 1980s, the A. niger N400/NRRL3 strain was selected for both fundamental and applied studies in relation to several processes including gluconic acid and protein production. To facilitate handling of A. niger, the N400 wild-type strain was UV mutagenized in two consecutive rounds to generate N401 and N402. N402 was used as a reference laboratory strain and exhibits the phenotypes with reduced conidiophore stalk length and reduced radial growth. The conidiophore stalk length and radial growth of A. niger strain N400 were determined and compared to N401 and N402. The length of N400 conidiophore stalks (2.52 ± 0.40 mm) was reduced in N401 and N402 to 0.66 ± 0.14 mm and 0.34 ± 0.06 mm, respectively. Whereas N400 reached a colony diameter of 6.7 ± 0.2 cm after 7 days, N401 and N402 displayed reduced radial growth phenotype (4.3 ± 0.1 and 4.1 ± 0.1, respectively). To identify the mutations (dubbed cspA and cspB) responsible for the phenotypes of N401 and N402, the genomes were sequenced and compared to the N400 genome sequence. A parasexual cross was performed between N400 and N402 derivatives to isolate segregants which allowed cosegregation analysis of single nucleotide polymorphisms and insertions and deletions among the segregants. The shorter conidiophore stalk and reduced radial growth in N401 (cspA) was found to be caused by a 9-kb deletion on chromosome III and was further narrowed down to a truncation of NRRL3_03857 which encodes a kinesin-like protein homologous to the A. nidulans UncA protein. The mutation responsible for the further shortening of conidiophore stalks in N402 (cspB) was found to be caused by a missense mutation on chromosome V in a hitherto unstudied C2H2 transcription factor encoded by the gene NRRL3_06646. The importance of these two genes in relation to conidiophore stalk length and radial growth was confirmed by single and double gene deletion studies. The mutations in the laboratory strain N402 should be taken into consideration when studying phenotypes in the N402 background.

Topoisomerase I Essentiality, DnaA-independent Chromosomal Replication, and Transcription-Replication Conflict in Escherichia coli

Topoisomerase I (Topo I) of <Escherichia coli, encoded by topA, acts to relax negative supercoils in DNA. Topo I deficiency results in hypernegative supercoiling, formation of transcription-associated RNA-DNA hybrids (R-loops), and DnaA- and oriC-independent constitutive stable DNA replication (cSDR), but some uncertainty persists as to whether topA is essential for viability in E. coli and related enterobacteria. Here we show that several topA alleles, including ΔtopA>, confer lethality in derivatives of wild-type E. coli strain MG1655. Viability in absence of Topo I was restored with two perturbations, neither of which reversed the hypernegative supercoiling phenotype: (i) in a reduced-genome strain MDS42, or (ii) by an RNA polymerase (RNAP) mutation rpoB*35 that has been reported to alleviate the deleterious consequences of RNAP backtracking and transcription-replication conflicts. Four phenotypes related to cSDR were identified for topA mutants: (i) One of the topA alleles rescued ΔdnaA lethality; (ii) in dnaA+ derivatives, Topo I deficiency generated a characteristic copy number peak in the terminus region of the chromosome; (iii) topA was synthetically lethal with rnhA (encoding RNase HI, whose deficiency also confers cSDR); and (iv) topA rnhA synthetic lethality was itself rescued by ΔdnaA. We propose that the terminal lethal consequence of hypernegative DNA supercoiling in E. colitopA mutants is RNAP backtracking during transcription elongation and associated R-loop formation, which in turn lead to transcription-replication conflicts and to cSDR.

Natural variation in the sequestosome-related gene, sqst-5, underlies zinc homeostasis in Caenorhabditis elegans

Zinc is an essential trace element that acts as a co-factor for many enzymes and transcription factors required for cellular growth and development. Altering intracellular zinc levels can produce dramatic effects ranging from cell proliferation to cell death. To avoid such fates, cells have evolved mechanisms to handle both an excess and a deficiency of zinc. Zinc homeostasis is largely maintained via zinc transporters, permeable channels, and other zinc-binding proteins. Variation in these proteins might affect their ability to interact with zinc, leading to either increased sensitivity or resistance to natural zinc fluctuations in the environment. We can leverage the power of the roundworm nematode Caenorhabditis elegans as a tractable metazoan model for quantitative genetics to identify genes that could underlie variation in responses to zinc. We found that the laboratory-adapted strain (N2) is resistant and a natural isolate from Hawaii (CB4856) is sensitive to micromolar amounts of exogenous zinc supplementation. Using a panel of recombinant inbred lines, we identified two large-effect quantitative trait loci (QTL) on the left arm of chromosome III and the center of chromosome V that are associated with zinc responses. We validated and refined both QTL using near-isogenic lines (NILs) and identified a naturally occurring deletion in sqst-5, a sequestosome-related gene, that is associated with resistance to high exogenous zinc. We found that this deletion is relatively common across strains within the species and that variation in sqst-5 is associated with zinc resistance. Our results offer a possible mechanism for how organisms can respond to naturally high levels of zinc in the environment and how zinc homeostasis varies among individuals.

Detection of the DNA primary structure modifications induced by the base analog 6-n-hydroxylaminopurine in the alpha-test in yeast saccharomyces cerevisiae

Background. The alpha-test allows to detect inherited genetic changes of different types, as well as phenotypic expression of primary DNA lesions before the lesions are fixed by repair. Here we investigate ability of the alpha-test to detect base modifications induced by 6-N-hydroxylaminopurine (HAP) and determine frequency of inherited and non-inherited genetic changes in yeast strains treated with HAP. Materials and methods. The alpha-test is based on mating type regulation and detects cell type switch from to a in heterothallic yeast Saccharomyces cerevisiae. The frequency of mating type switching reflects level of both spontaneous and induced by a mutagen DNA instability. The alpha-test may be performed in two variants: illegitimate hybridization and cytoduction. Conducting both complementary tests and analysis of phenotypes of the illegitimate hybrids and cytoductants allows to detect the full spectrum of genetic events that lead to mating type switching, such as chromosome III loss and chromosome III arm loss, mutations and temporary lesions, recombination and conversion. Results. HAP increases the frequency of illegitimate hybridization by 5-fold, and illegitimate cytoduction by 10-fold. A large proportion of the primary lesions induced by HAP causes temporary mating type switch and the remainder parts are converted into inherited point mutations. Conclusion. The alpha-test can detect HAP-induced base modifications and may be used to investigate the ratio between correct and error-prone processing of such primary DNA lesions. Like other genetic toxicology tests the alpha-test has limitations, which are discussed.

Yeast ATM and ATR kinases use different mechanisms to spread histone H2A phosphorylation around a DNA double-strand break

One of the hallmarks of DNA damage is the rapid spreading of phosphorylated histone H2A (γ-H2AX) around a DNA double-strand break (DSB). In the budding yeastSaccharomyces cerevisiae, nearly all H2A isoforms can be phosphorylated, either by Mec1ATRor Tel1ATMcheckpoint kinases. We induced a site-specific DSB with HO endonuclease at theMATlocus on chromosome III and monitored the formation of γ-H2AX by chromatin immunoprecipitation (ChIP)-qPCR in order to uncover the mechanisms by which Mec1ATRand Tel1ATMpropagate histone modifications across chromatin. With either kinase, γ-H2AX spreads as far as ∼50 kb on both sides of the lesion within 1 h; but the kinetics and distribution of modification around the DSB are significantly different. The total accumulation of phosphorylation is reduced by about half when either of the two H2A genes is mutated to the nonphosphorylatable S129A allele. Mec1 activity is limited by the abundance of its ATRIP partner, Ddc2. Moreover, Mec1 is more efficient than Tel1 at phosphorylating chromatin intrans—at distant undamaged sites that are brought into physical proximity to the DSB. We compared experimental data to mathematical models of spreading mechanisms to determine whether the kinases search for target nucleosomes by primarily moving in three dimensions through the nucleoplasm or in one dimension along the chromatin. Bayesian model selection indicates that Mec1 primarily uses a three-dimensional diffusive mechanism, whereas Tel1 undergoes directed motion along the chromatin.

Genome-wide Cas9 binding specificity in Saccharomyces cerevisiae

The CRISPR system has become heavily utilized in biomedical research as a tool for genomic editing as well as for site-specific chromosomal localization of specific proteins. For example, we developed a CRISPR-based methodology for enriching a specific genomic locus of interest for proteomic analysis in Saccharomyces cerevisiae, which utilized a guide RNA-targeted, catalytically dead Cas9 (dCas9) as an affinity reagent. To more comprehensively evaluate the genomic specificity of using dCas9 as a site-specific tool for chromosomal studies, we performed dCas9-mediated locus enrichment followed by next-generation sequencing on a genome-wide scale. As a test locus, we used the ARS305 origin of replication on chromosome III in S. cerevisiae. We found that enrichment of this site is highly specific, with virtually no off-target enrichment of unique genomic sequences. The high specificity of genomic localization and enrichment suggests that dCas9-mediated technologies have promising potential for site-specific chromosomal studies in organisms with relatively small genomes such as yeasts.

The Synaptonemal Complex Central Region Modulates Crossover Pathways and Feedback Control of Meiotic Double-strand Break Formation

SummaryThe synaptonemal complex (SC) is a proteinaceous structure that mediates homolog engagement and genetic recombination during meiosis. Zip-Mer-Msh (ZMM) proteins promote crossover (CO) formation and initiate SC formation. In SC elongation, the SUMOylated SC component Ecm11 and its interacting protein Gmc2 facilitate the polymerization of Zip1, a SC-central region component in budding yeast. Through physical recombination, cytological, and genetic analyses, we here demonstrate that ecm11 and gmc2 mutants exhibit chromosome-specific defects in meiotic recombination. CO frequencies were reduced on a short chromosome (chromosome III), whereas CO and non-crossover (NCO) frequencies were increased on a long chromosome (chromosome VII). Further, persistent double-strand breaks (DSBs) occurred in unsynapsed chromosome regions during the late prophase, suggesting the presence of a negative regulation of DSB formation. The Ecm11-Gmc2 (EG) complex could participate in joint molecule (JM) processing and/or double-Holliday junction resolution for CO-designated recombination of the ZMM-dependent pathway. However, absence of the EG complex ameliorated the JM-processing defect in zmm mutants, suggesting a role of these proteins in suppression of ZMM-independent recombination. Therefore, the EG complex fosters ZMM-dependent processing and resolution of JMs while suppressing ZMM-independent JM processing and late DSB formation. Hence, EG-mediated SC central regions, which display properties similar to those of liquid crystals, may function as a compartment for sequestering recombination proteins in and out of the process to ensure meiosis specificity during recombination.