Lennart Adler

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.

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.

A gene encoding sn‐glycerol 3‐phosphate dehydrogenase (NAD+) complements an osmosensitive mutant of Saccharomyces cerevisiae

Osmoregulatory mutants of Saccharomyces cerevisiae with a defect in their capacity to readjust the cell volume/buoyant density after osmotically induced dehydration were enriched by density gradient centrifugation. Colonies derived from cells that remained dense after dehydration were screened for sensitivity to high concentrations of NaCl and defects in their osmotically induced production and intracellular accumulation of glycerol. The isolated osg (osmosensitive gtycerol defective) mutants were recessive in heterozygous diploids and fell into four complementation groups (osg1‐osg4). The osg1‐1 mutant, described in this work, is unable to grow at low water potential and shows a decreased capacity for glycerol production and a strongly reduced activity of NAD+‐dependent sn‐glycerol 3‐phosphate dehydrogenase (GPD), an enzyme in the glycerol‐producing pathway. Complementation of the osg1‐1 salt sensitivity defect with a low copy yeast genomic library led to the cloning of GPD1, encoding an S. cerevisiae GPD consisting of 391 amino acids and sharing 47‐50% identity with GPD from other sources. Micro‐sequencing of the N‐terminus of purified S. cerevisiae GPD revealed a 20‐amino‐acid sequence that was identical to a nucleotide‐deduced amino acid sequence in GPD1, but indicated that the enzyme is produced with an N‐terminal extension that is removed from the functional enzyme. Subcellular fractionation does not indicate, however, that the putative pre‐sequence targets GPD to any organelle; the enzyme appears to be located in the cytoplasm. Chromoblot and tetrad analysis were used to position the GPD1 gene to chromosome IV, with a distance of about 18 cM from trp1.

The importance of the glycerol 3-phosphate shuttle during aerobic growth of Saccharomyces cerevisiae.

Maintenance of a cytoplasmic redox balance is a necessity for sustained cellular metabolism. Glycerol formation is the only way by which Saccharomyces cerevisiae can maintain this balance under anaerobic conditions. Aerobically, on the other hand, several different redox adjustment mechanisms exist, one of these being the glycerol 3‐phosphate (G3P) shuttle. We have studied the importance of this shuttle under aerobic conditions by comparing growth properties and glycerol formation of a wild‐type strain with that of gut2Δ mutants, lacking the FAD‐dependent glycerol 3‐phosphate dehydrogenase, assuming that the consequent blocking of G3P oxidation is forcing the cells to produce glycerol from G3P. To impose different demands on the redox adjustment capability we used various carbon sources having different degrees of reduction.

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.

Osmoregulation in Saccharomyces cerevisiae Studies on the osmotic induction of glycerol production and glycerol 3-phosphate dehydrogenase (NAD+)

Production of glycerol and a key enzyme in glycerol production, glycerol 3‐phosphate dehydrogenase (NAD+) (GPD), was studied in Saccharomyces cerevisiae cultured in basal media or media of high salinity, with glucose, raffinose or ethanol as the sole carbon source. At high salinity, glycerol production was stimulated with all carbon sources and glycerol was accumulated to high intracellular concentration in cells grown on glucose and raffinose. Cells grown on ethanol accumulated glycerol to a lower level but showed an increased content of trehalose at high salinity. However, the trehalose concentration corresponded only to about 20% of the glycerol level, and did not compensate for the shortfall in intracellular osmolyte content. Immunoblot analysis demonstrated an increased production of GPD at high salinity. This increase was osmotically mediated but was lower when glycerol was substituted for NaCl or sorbitol as the stress‐solute. The enzyme also appeared to be subject to glucose repression; the specific activity of GPD was significantly lower in cells grown on glucose, than on raffinose or ethanol.

Polyhydric alcohol production and intracellular amino acid pool in relation to halotolerance of the yeast Debaryomyces hansenii

Changes in polyol production and the intracellular amino acid pool were followed during the growth cycle of Debaryomyces hansenii in 4 mM and 2.7 M NaCl media. The intracellular levels of polyols were markedly enhanced by high salinity, the dominant solutes being glycerol in log phase cells and arabinitol in stationary phase cells. At low salinity arabinitol was the most prominent intracellular solute throughout the growth cycle. There were no major changes in the composition of the total amino acid pool with changes in cultural salinity. The amount of total free amino acids related to cell dry weight was 15–50% lower in cells cultured in 2.7 M NaCl as compared to 4 mM NaCl media.After subtraction of contributions from intracellular polyols the calculated cellular C/N ratio was found to be unaffected by cultural age and salinity during the late log and early stationary phase. On prolonged incubation of stationary phase cells, this ratio decreased, particularly at high salinity. The sensitivity of cells towards exposure to high salinity was measured in terms of the length of the lag phase after transference to 2.7 M NaCl media. This lag phase decreased with increasing intracellular polyol concentrations. At a given polyol content, stationary phase cells were considerably less sensitive than were log phase cells.When cultured at high salinity the mutant strain, 26-2b, grew more slowly and retained less of the total polyol produced during the early growth stages than did the wildtype. Exogenously supplied mannitol, arabinitol, and glycerol stimulated the growth of the mutant in saline media. Erythritol was without effect.

Strategies for enhancing fermentative production of glycerol - A review

The present paper reviews the metabolic basis of different methods for fermentative glycerol production. The most important microbial production organism is the yeast Saccharomyces cerevisiae but other yeast species, as well as molds, algae, and bacteria are of potential interest for glycerol production. A large variety of methods have been applied to increase the fermentative glycerol yield. The first methods were based on physiological control, e.g. chemically induced overproduction of glycerol through NADH entrapment by the addition of chemical steering agents (such as bisulfite). More recently, genetic engineering of the glycolytic pathway has been used to improve production, involving modulated function of e.g. triose phosphate isomerase, phosphoglycerate mutase, PDC or alcohol dehydrogenase. Direct intervention in the glycerol pathway, such as overexpression of G3P dehydrogenase, has also been tried. The applied strategies can be divided into three principal groups; (a) deactivation or down-regulation of NADH oxidation sites alternative to G3P dehydrogenase, (b) increase of NADH generation or, (c) direct changes in the carbon flux to glycerol.

The role of glycerol in osmotolerance of the yeast Debaryomyces hansenii

SUMMARY: Transfer of growing cells of the salt-tolerant yeast Debaryomyces hansenii to media of higher salinity resulted in an increased production and intracellular accumulation of glycerol, which was proportional to the magnitude of the shift in salinity. Stress solutes other than NaCl, when added in iso-osmolar concentrations, promoted the accumulation of similar amounts of glycerol. Cells grown at high salinity rapidly lost glycerol when returned to media of lower salinity and the loss was greater when the cells were transferred to more dilute media. A mutant strain of D. hansenii showed poor glycerol production and was inhibited by NaCl at concentrations about half the maximum tolerated by the wild-type. Growth of this mutant occurred at otherwise inhibitory NaCl concentrations if the medium was supplemented with a low concentration of glycerol. The added glycerol was intracellularly accumulated to levels that increased with salinity and were only slightly lower than the corresponding wild-type levels. Glycerol additions above the growth promoting level had little effect on growth rate but caused substantial shortening of the lag phase. Osmoprotectants other than glycerol did not permit growth to occur. The mutant was isolated as a glycerol non-utilizer but displayed growth in glycerol media at increased NaCl concentrations.

NADH-reductive stress in Saccharomyces cerevisiae induces the expression of the minor isoform of glyceraldehyde-3-phosphate dehydrogenase ( TDH1 )

Abstract A strain of Saccharomyces cerevisiae lacking the GPD2 gene, encoding one of the glycerol-3-phosphate dehydrogenases, grows slowly under anaerobic conditions, due to reductive stress caused by the accumulation of cytoplasmic NADH. We used 2D-PAGE to study the effect on global protein expression of reductive stress in the anaerobically grown gpd2Δ strain. The most striking response was a strongly elevated expression of Tdh1p, the minor isoform of glyceraldehyde-3-phosphate dehydrogenase. This increased expression could be reversed by the addition of acetoin, a NADH-specific redox sink, which furthermore largely restored anaerobic growth of the gpd2Δ strain. Additional deletion of the TDH1 gene (but not of TDH2 or TDH3) improved anaerobic growth of the gpd2Δ strain. We therefore propose that TDH1 has properties not displayed by the other TDH isogenes and that its expression is regulated by reductive stress caused by an excess of cytoplasmic NADH.