Anders Blomberg

Physiology of osmotolerance in fungi.

Publisher Summary The response of a fungus to osmotic stress involves the integrated function of many components of cell metabolism. The dehydration stress is countered by an important mechanism that entails accumulation of polyols, primarily glycerol, to achieve an internal environment that is conducive for enzyme function and growth under water stress. The changes in the composition of the cytoplasm are controlled by systems for biosynthesis of polyols and for transport of inorganic ions. The intracellular retention of glycerol is controlled at the level of glycerol efflux and by systems for glycerol uptake. The osmoregulatory processes require energy to drive transport and carbon supply for polyol formation. The capacity for substrate uptake under osmotic stress and the efficiency operating the osmoregulatory processes are important in setting the limits for growth at low water potentials. The fungal response to changes in the external water potential must involve sensing as well as transduction of the received signal. Combined genetic and physiological analysis is required for a deeper understanding of fungus-water relations. Analysis at this level has revealed sequential induction of osmotically controlled genes in enteric bacteria and given exciting insights in signal transduction and regulation of the process.

Systematic exploration of essential yeast gene function with temperature-sensitive mutants

Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (∼45%) of the 1,101 essential yeast genes, with ∼30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)–based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes.

Trait Variation in Yeast Is Defined by Population History

A fundamental goal in biology is to achieve a mechanistic understanding of how and to what extent ecological variation imposes selection for distinct traits and favors the fixation of specific genetic variants. Key to such an understanding is the detailed mapping of the natural genomic and phenomic space and a bridging of the gap that separates these worlds. Here we chart a high-resolution map of natural trait variation in one of the most important genetic model organisms, the budding yeast Saccharomyces cerevisiae, and its closest wild relatives and trace the genetic basis and timing of major phenotype changing events in its recent history. We show that natural trait variation in S. cerevisiae exceeds that of its relatives, despite limited genetic variation, and follows the population history rather than the source environment. In particular, the West African population is phenotypically unique, with an extreme abundance of low-performance alleles, notably a premature translational termination signal in GAL3 that cause inability to utilize galactose. Our observations suggest that many S. cerevisiae traits may be the consequence of genetic drift rather than selection, in line with the assumption that natural yeast lineages are remnants of recent population bottlenecks. Disconcertingly, the universal type strain S288C was found to be highly atypical, highlighting the danger of extrapolating gene-trait connections obtained in mosaic, lab-domesticated lineages to the species as a whole. Overall, this study represents a step towards an in-depth understanding of the causal relationship between co-variation in ecology, selection pressure, natural traits, molecular mechanism, and alleles in a key model organism.

Automated screening in environmental arrays allows analysis of quantitative phenotypic profiles in Saccharomyces cerevisiae.

A methodology for large‐scale automated phenotypic profiling utilizing quantitative changes in yeast growth has been tested and applied to the analysis of some commonly used laboratory strains. This yeast‐adjusted methodology is based on microcultivation in 350 µl liquid medium, where growth is frequently optically recorded, followed by automated extraction of relevant variables from obtained growth curves. We report that cultivation at this micro‐scale displayed overall growth features and protein expression pattern highly similar to growth in well aerated medium‐scale (10 ml) culture. However, differences were also encountered, mainly relating to the respiratory potential and the production of stress‐induced proteins. Quantitative phenotypic profiles for the laboratory yeast strains W303, FY1679 and CEN‐PK.2 were screened for in environmental arrays, including 98 different conditions composed of low, medium and high concentrations of 33 growth inhibitors. We introduce the concepts phenotypic indexrate and phenotypic indexstationary, which relate to changes in rate of growth and the stationary phase optical density increment, respectively, in a particular environment relative a reference strain. The laboratory strains presented selective phenotypic profiles in both phenotypic indexes and the two features appeared in many cases to be independent characteristics. We propose the utilization of this methodology in large‐scale screening of the complete collection of yeast deletion mutants. Copyright

High-resolution yeast phenomics resolves different physiological features in the saline response

We present a methodology for gene functional prediction based on extraction of physiologically relevant growth variables from all viable haploid yeast knockout mutants. This quantitative phenomics approach, here applied to saline cultivation, identified marginal but functionally important phenotypes and allowed the precise determination of time to adapt to an environmental challenge, rate of growth, and efficiency of growth. We identified ≈500 salt-sensitive gene deletions, the majority of which were previously uncharacterized and displayed salt sensitivity for only one of the three physiological features. We also report a high correlation to protein–protein interaction data; in particular, several salt-sensitive subcellular networks indicating functional modules were revealed. In contrast, no correlation was found between gene dispensability and gene expression. It is proposed that high-resolution phenomics will be instrumental in systemwide descriptions of intragenomic functional networks.

Purification and Characterization of Two Isoenzymes of DL-Glycerol-3-phosphatase from Saccharomyces cerevisiae IDENTIFICATION OF THE CORRESPONDING GPP1 AND GPP2 GENES AND EVIDENCE FOR OSMOTIC REGULATION OF Gpp2p EXPRESSION BY THE OSMOSENSING MITOGEN-ACTIVATED PROTEIN KINASE SIGNAL TRANSDUCTION PATHWAY

The existence of specific DL-glycerol-3-phosphatase (EC) activity in extracts of Saccharomyces cerevisiae was confirmed by examining strains lacking nonspecific acid and alkaline phosphatase activities. During purification of the glycerol-3-phosphatase, two isozymes having very similar molecular weights were isolated by gel filtration and anion exchange chromatography. By microsequencing of trypsin-generated peptides the corresponding genes were identified as previously sequenced open reading frames of unknown function. The two genes, GPP1 (YIL053W) and GPP2 (YER062C) encode proteins that show 95% amino acid identity and have molecular masses of 30.4 and 27.8 kDa, respectively. The intracellular concentration of Gpp2p increases in cells subjected to osmotic stress, while the production of Gpp1p is unaffected by changes of external osmolarity. Both isoforms have a high specificity for DL-glycerol-3-phosphate, pH optima at 6.5, and KG3Pm in the range of 3-4 mM. The osmotic induction of Gpp2p is blocked in cells that are defective in the HOG-mitogen-activated protein kinase pathway, indicating that GPP2 is a target gene for this osmosensing signal transduction pathway. Together with DOG1 and DOG2, encoding two highly homologous enzymes that dephosphorylate 2-deoxyglucose-6-phosphate, GPP1 and GPP2 constitute a new family of genes for low molecular weight phosphatases.

Metabolic and regulatory changes associated with growth of Saccharomyces cerevisiae in 1.4 M NaCl. Evidence for osmotic induction of glycerol dissimilation via the dihydroxyacetone pathway

The salt-instigated protein expression of Saccharomyces cerevisiae during growth in either 0.7 or 1.4 M NaCl was studied by two-dimensional polyacrylamide gel electrophoresis. The 73 protein spots that were identified as more than 3-fold responsive in 1.4 M NaCl were further grouped by response class (halometric, low-salt, and high-salt regulation). Roughly 40% of these responsive proteins were found to decrease in expression, while at higher magnitudes of change (>8-fold) only induction was recorded. Enolase 1 (Eno1p) was the most increasing protein by absolute numbers per cell, but not by -fold change, and the enzymes involved in glycerol synthesis, Gpd1p and Gpp2p, were also induced to a similar degree as Eno1p. We furthermore present evidence for salt induction of glycerol dissimilation via dihydroxyacetone and also identify genes putatively encoding the two enzymes involved; dihydroxyacetone kinase (DAK1 and DAK2) and glycerol dehydrogenase (YPR1 and GCY1). The GPD1, GPP2, GCY1, DAK1, and ENO1 genes all displayed a halometric increase in the amount of transcript. This increase was closely linked to the salt-induced rate of protein synthesis of the corresponding proteins, indicating mainly transcriptional regulation of expression for these genes. A consensus element with homology to the URS sequence of the ENO1 promoter was found in the promoters of the GPD1, GPP2, GCY1, and DAK1 genes.

Identification and specificities of N‐terminal acetyltransferases from Saccharomyces cerevisiae

N‐terminal acetylation can occur cotranslationally on the initiator methionine residue or on the penultimate residue if the methionine is cleaved. We investigated the three N‐terminal acetyltransferases (NATs), Ard1p/Nat1p, Nat3p and Mak3p. Ard1p and Mak3p are significantly related to each other by amino acid sequence, as is Nat3p, which was uncovered in this study using programming alignment procedures. Mutants deleted in any one of these NAT genes were viable, but some exhibited diminished mating efficiency and reduced growth at 37°C, and on glycerol and NaCl‐containing media. The three NATs had the following substrate specificities as determined in vivo by examining acetylation of 14 altered forms of iso‐1‐cytochrome c and 55 abundant normal proteins in each of the deleted strains: Ard1p/Nat1p, subclasses with Ser‐, Ala‐, Gly‐ and Thr‐termini; Nat3p, Met‐Glu‐ and Met‐Asp‐ and a subclass of Met‐Asn‐termini; and Mak3p subclasses with Met‐Ile‐ and Met‐Leu‐termini. In addition, a special subclass of substrates with Ser‐Glu‐ Phe‐, Ala‐Glu‐Phe‐ and Gly‐Glu‐Phe‐termini required all three NATs for acetylation.

Cloning and characterization of GPD2, a second gene encoding sn‐glycerol 3‐phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae, and its comparison with GPD1

We have cloned and characterized a homologue of the previously isolated GPD1 gene, encoding sn‐glycerol 3‐phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae. This second gene, called GPD2, encodes a protein of 384 amino acids that shares 69% sequence identity with GPD1. Like GPD1 it has an amino‐terminal extension of unknown function. GPD2 is located on chromosome VII and cross‐hybridizes with GPD1 at chromosome IV as well as with an unknown homologue at chromosome XV. Disruption of the GPD2 gene did not reveal any observable phenotypic effects, whereas overexpression resulted in a slight, but significant, increase of GPD enzyme activity in wild‐type cells. Analysis of gene transcription by a CAT‐reporter gene fused to the GPD promoters revealed decreased transcriptional activity of the GPD2 promoter in cells grown on non‐fermentable as opposed to fermentable carbon sources, and no induction in cells exposed to high osmolarity or heat shock. Similar analysis of GPD1 demonstrated an 8–17‐fold higher basal level of transcription compared to GPD2. Furthermore, such analysis revealed that the GPD1 promoter was it induced by increased osmolarity essentially independent of the type of stress solute used, the level of GPD1 transcription being increased about sevenfold in cells growing at 1.4M NaCl.

Assessing the complex architecture of polygenic traits in diverged yeast populations

Phenotypic variation arising from populations adapting to different niches has a complex underlying genetic architecture. A major challenge in modern biology is to identify the causative variants driving phenotypic variation. Recently, the baker’s yeast, Saccharomyces cerevisiae has emerged as a powerful model for dissecting complex traits. However, past studies using a laboratory strain were unable to reveal the complete architecture of polygenic traits. Here, we present a linkage study using 576 recombinant strains obtained from crosses of isolates representative of the major lineages. The meiotic recombinational landscape appears largely conserved between populations; however, strain‐specific hotspots were also detected. Quantitative measurements of growth in 23 distinct ecologically relevant environments show that our recombinant population recapitulates most of the standing phenotypic variation described in the species. Linkage analysis detected an average of 6.3 distinct QTLs for each condition tested in all crosses, explaining on average 39% of the phenotypic variation. The QTLs detected are not constrained to a small number of loci, and the majority are specific to a single cross‐combination and to a specific environment. Moreover, crosses between strains of similar phenotypes generate greater variation in the offspring, suggesting the presence of many antagonistic alleles and epistatic interactions. We found that subtelomeric regions play a key role in defining individual quantitative variation, emphasizing the importance of the adaptive nature of these regions in natural populations. This set of recombinant strains is a powerful tool for investigating the complex architecture of polygenic traits.