Effect of ferric and cupric ions on the inactivation rate of dextransucrase

When ferric ion was added to solutions of the enzyme dextransucrase, first-order followed by second-order inactivation behavior was observed. The initial rapid activity loss was attributed to a ferric ion interacting with the thiol group of the native monomer to form a less active enzyme-ion complex; the second inactivation stage involved enzyme-ion complex aggregation and disulfide cross-link formation. In contrast, Cu²⁺ ion inactivation demonstrated simple first-order kinetics. As with Fe³⁺, Cu²⁺ ions can form complexes with enzyme thiol groups. However, unlike ferric ions, cupric ions can also strongly interact with the imidazole ring of histidine. Since the dextransucrase active site contains two key histidines, imidazole-cupric-ion interactions could potentially inhibit enzymatic activity. Thus, it was hypothesized that first-order Cu²⁺ inactivation kinetics involved the adsorption of this ion to the enzyme's activity site. The addition of a reducing agent such as dithiothreitol can inhibit the second enzyme aggregation stage by breaking disulfide cross-links but cannot restrict the initial formation of metal-enzyme complexes.     

Genome-wide analysis of nucleotide-level variation in commonly used Saccharomyces cerevisiae strains

Ten years have passed since the genome of Saccharomyces cerevisiae-more precisely, the S288c strain-was completely sequenced. However, experimental work in yeast is commonly performed using strains that are of unknown genetic relationship to S288c. Here, we characterized the nucleotide-level similarity between S288c and seven commonly used lab strains (A364A, W303, FL100, CEN.PK, summation 1278b, SK1 and BY4716) using 25mer oligonucleotide microarrays that provide complete and redundant coverage of the approximately 12 Mb Saccharomyces cerevisiae genome. Using these data, we assessed the frequency and distribution of nucleotide variation in comparison to the sequenced reference genome. These data allow us to infer the relationships between experimentally important strains of yeast and provide insight for experimental designs that are sensitive to sequence variation. We propose a rational approach for near complete sequencing of strains related to the reference using these data and directed re-sequencing. These data and new visualization tools are accessible online in a new resource: the Yeast SNPs Browser (YSB; http://gbrowse.princeton.edu/cgi-bin/gbrowse/yeast_strains_snps) that is available to all researchers.     

Effect of some heavy metal ions on copper-induced metallothionein synthesis in the yeast Saccharomyces cerevisiae

Copper-induced metallothionein (MT) synthesis in Saccharomyces cerevisiae was investigated in order to associate this exclusively with Cu²⁺ in vivo, when cultured in nutrient medium containing other heavy metal ions. Expression of the CUP1 promoter/lacZ fusion gene was inhibited by all heavy metal ions tested, especially Cd²⁺ and Mn²⁺. By adding Cd²⁺ and Mn²⁺ at 10 M concentration, the -galactosidase activity decreased by about 80% and 50% of the maximum induction observed with 1 mM CuSO₄, respectively. Furthermore, cell growth was markedly inhibited by combinations of 1 mM-Cu²⁺ and 1 M-Cd²⁺. Therefore, the yeast S. cerevisiae could not rely on MT synthesis as one of the copper-resistance mechanisms, when grown in a Cd²⁺ environment. In contrast, the presence of Mn²⁺ in the nutrient medium showed alleviation rather than growth inhibition by high concentrations of Cu²⁺. The recovery from growth inhibition by Mn²⁺ was due to decreased Cu²⁺ accumulation. Inhibitory concentrations of Co²⁺, Ni²⁺ and Zn²⁺ on expression of the CUP1p/lacZ fusion gene were at least one order of magnitude higher than that of Cd²⁺ and Mn²⁺. These results are discussed in relation to Cu²⁺ transport and Cu-induced MT synthesis in the copper-resistance mechanism of the yeast S. cerevisiae.     

FTIR spectroscopic discrimination of Saccharomyces cerevisiae and Saccharomyces bayanus strains

In this study, we tested the potential of Fourier-transform infrared absorption spectroscopy to screen, on the one hand, Saccharomyces cerevisiae and non-S. cerevisiae strains and, on the other hand, to discriminate between S. cerevisiae and Saccharomyces bayanus strains. Principal components analysis (PCA), used to compare 20 S. cerevisiae and 21 non-Saccharomyces strains, showed only 2 misclassifications. The PCA model was then used to classify spectra from 14 Samos strains. All 14 Samos strains clustered together with the S. cerevisiae group. This result was confirmed by a routinely used electrophoretic pattern obtained by pulsed-field gel electrophoresis. The method was then tested to compare S. cerevisiae and S. bayanus strains. Our results indicate that identification at the strain level is possible. This first result shows that yeast classification and S. bayanus identification can be feasible in a single measurement.     

Duplication processes in Saccharomyces cerevisiae haploid strains

Duplication is thought to be one of the main processes providing a substrate on which the effects of evolution are visible. The mechanisms underlying this chromosomal rearrangement were investigated here in the yeast Saccharomyces cerevisiae. Spontaneous revertants containing a duplication event were selected and analyzed. In addition to the single gene duplication described in a previous study, we demonstrated here that direct tandem duplicated regions ranging from 5 to 90 kb in size can also occur spontaneously. To further investigate the mechanisms in the duplication events, we examined whether homologous recombination contributes to these processes. The results obtained show that the mechanisms involved in segmental duplication are RAD52-independent, contrary to those involved in single gene duplication. Moreover, this study shows that the duplication of a given gene can occur in S.cerevisiae haploid strains via at least two ways: single gene or segmental duplication.     

Synergistic anion-directed coordination of ferric and cupric ions to bovine serum transferrin--an inorganic perspective

A series of new iron(III) and copper(II) complexes of bovine serum transferrin (BTf), with carbonate and/or oxalate as the synergistic anion, are presented. The complexes [Fe(2)(CO(3))(2)BTf], [Fe(2)(C(2)O(4))(2)BTf], [Cu(2)(CO(3))(2)BTf] and [Cu(C(2)O(4))BTf] were prepared by standard titrimetric techniques. The oxalate derivatives were also obtained from the corresponding carbonate complexes by anion-displacement. The site-preference of the transition metal-oxalate synergism has facilitated the preparation and isolation of the mononuclear complex [Cu(C(2)O(4))BTf], the mixed-anion complexes [Cu(2)(CO(3))(C(2)O(4))BTf] and [Fe(2)(CO(3))(C(2)O(4))BTf] and the mixed-metal complex [FeCu(C(2)O(4))(2)BTf]. The sensitivity of electron paramagnetic resonance (EPR) spectroscopy to the nature of the synergistic anions at the specific-binding sites of the transferrins has made this physical technique particularly indispensable to this study. None of the other members of the transferrin family of proteins has ever been demonstrated to bind the ferric and cupric ions one after the other, each occupying a separate specific-binding site of the same transferrin molecule, as a response to the coordination restrictions imposed by the oxalate ion. The bathochromic shift of the visible p(pi)-d(pi*) CT band for iron(III)-BTf and the hypsochromic shift of the p(pi)-d(sigma*) CT band for copper(II)-BTf, on replacing carbonate by oxalate as the associated anion, are consistent with the relative positions of these anionic ligands in the spectrochemical series and the nature of the d-type acceptor orbitals involved in the CT transitions. The binding and spectroscopic properties of bovine serum transferrin--a serum transferrin--very nearly mirror those of human serum transferrin, but differ significantly from those of human lactoferrin.     

Urea transport-defective strains of Saccharomyces cerevisiae.

Experiments characterizing the urea active transport system in Saccharomyces cerevisiae indicate that (i) formamide and acetamide are strong competitive inhibitors of urea accumulation, (ii) uptake is maximal at pH 3.3 and is 80% inhibited at pH 6.0, and (iii) adenosine 5'-triphosphate generated by glycolysis in conjunction with formation of an ion gradient is likely the driving force behind urea transport. Mutant strains were isolated that are unable to accumulate urea at external concentrations of 0.25 mM. These strains also exhibit a depressed growth rate on 10 mM urea, indicating existence of a relationship between the active transport and facilitated diffusion modes of urea uptake.     

Lipid synthesis in inositol-starved Saccharomyces cerevisiae

Lipid synthesis was analyzed in an inositol-requiring mutant of Saccharomyces cerevisiae (MC13). Both rates and cellular amounts of [U-14C]acetate incorporation into phospholipids, triacylglycerols, free sterols and steryl esters were elevated in an inositol-starved culture compared to the supplemented control at a time when the deprived culture was losing viability (inositol-less death). The rates at a later time were greatly reduced. During the period when de novo lipid synthesis was high in the starved culture, phospholipid turnover and presumed conversion to triacylglycerols was also accelerated; no differences were apparent in the turnover of the sterol fractions between the two cultures. No change in the fractional percent of ergosterol or of the sterol precursors could be attributed to inositol starvation. The synthesis and maintenance of membrane lipids (phospholipids and free sterols) and their coupling in cellular metabolism are discussed in light of these results.     

Accumulation and intracellular compartmentation of lithium ions in Saccharomyces cerevisiae

Abstract Accumulation of Li⁺ in Saccharomyces cerevisiae X2180-1B occured via an apparent stoichiometric relationship of 1: 1 (K⁺/Li⁺) when S. cerevisiae was incubated in the presence of 5 and 10 mM LiCl for 3 h. Other cellular cations (Mg²⁺, Ca²⁺ and Na⁺) did not vary on Li⁺ accumulation, although lithium chemistry dictates a degree of similarity to Group I and II metal cations. Compartmentation of Li⁺ was mainly in the vacuole which accounted for 85% of the Li⁺ accumulated after a 6-h incubation period. The remainder was located in the cytosol with negligible amounts being bound to cell fragments including the cell wall. Transmission electron microscopy of Li⁺-loaded cells revealed enlarged vacuoles compared with control cells. This asymmetric cellular distribution may therefore enhance tolerance of S. cerevisiae to Li⁺ and ensure that essential metabolic processes in the cytosol are not disrupted.     

Study on agglutinating factors from flocculent Saccharomyces cerevisiae strains

The lectin-like theory suggest that yeast flocculation is mediated by an aggregating lectinic factor. In this study we isolated an agglutinating factor, which corresponds to lectin, from whole cells by treating the flocculent wild-type Saccharomyces cerevisiae NCYC 625 strain and its weakly flocculent mutant [rho degrees ] with EDTA and two non-ionic surfactants (Hecameg and HTAC). The dialysed crude extracts obtained in this way agglutinated erythrocytes and this hemagglutination was specifically inhibited by mannose and mannose derivatives. However, SDS-PAGE profiles showed that the three reagents had different effects on the yeast cells. The non-ionic surfactants appeared to be the most efficient, as their extracts possessed the highest specific agglutinating activity. The products released by the wild-type strain presented a higher specific agglutinating activity than those released by the [rho degrees ] mutant. Purification of the agglutinating factor from extracts of both strains by affinity chromatography revealed two active bands of relative mass of 26 and 47 kDa on SDS-PAGE. Mass spectrometry analysis by MALDI-TOF, identified a 26 kDa band as the triose phosphate isomerase (TPI) whereas a 47 kDa band was identical to enolase. Edman degradation showed that the N-terminal sequences of these proteins were similar to TPI and enolase, respectively. The difference in the flocculation behaviour of the two strains is due to changes in the protein composition of the cell wall and in the protein structure involved in cell-cell recognition.     

Design and construction of acetyl-CoA overproducing Saccharomyces cerevisiae strains

Saccharomyces cerevisiae has increasingly been engineered as a cell factory for efficient and economic production of fuels and chemicals from renewable resources. Notably, a wide variety of industrially important products are derived from the same precursor metabolite, acetyl-CoA. However, the limited supply of acetyl-CoA in the cytosol, where biosynthesis generally happens, often leads to low titer and yield of the desired products in yeast. In the present work, combined strategies of disrupting competing pathways and introducing heterologous biosynthetic pathways were carried out to increase acetyl-CoA levels by using the CoA-dependent n-butanol production as a reporter. By inactivating ADH1 and ADH4 for ethanol formation and GPD1 and GPD2 for glycerol production, the glycolytic flux was redirected towards acetyl-CoA, resulting in 4-fold improvement in n-butanol production. Subsequent introduction of heterologous acetyl-CoA biosynthetic pathways, including pyruvate dehydrogenase (PDH), ATP-dependent citrate lyase (ACL), and PDH-bypass, further increased n-butanol production. Recombinant PDHs localized in the cytosol (cytoPDHs) were found to be the most efficient, which increased n-butanol production by additional 3 fold. In total, n-butanol titer and acetyl-CoA concentration were increased more than 12 fold and 3 fold, respectively. By combining the most effective and complementary acetyl-CoA pathways, more than 100 mg/L n-butanol could be produced using high cell density fermentation, which represents the highest titer ever reported in yeast using the clostridial CoA-dependent pathway.