An improved gas distribution system for anaerobic screening of multiple microbial cultures.

A cultivation set-up for multiple cultures has been designed that can be used for anaerobic screening for quantitative changes in growth rate or other analyses, e.g. protein composition of different strains. The developed gas distribution system provides a reproducible level of anaerobicity in 30 cultivation flasks and resembles the open system of a high-performance bioreactor in that it ensures cultivation at atmospheric pressure and avoids supersaturation of carbon dioxide. The system is cheap and user-friendly and allows rapid screenings of many strains simultaneously.

Microaerobic glycerol formation in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae produces large amounts of glycerol as an osmoregulator during hyperosmotic stress and as a redox sink at low oxygen availability. NAD(+)-dependent glycerol-3-phosphate dehydrogenase in S. cerevisiae is present in two isoforms, coded for by two different genes, GPD1 and GPD2. Mutants for either one or both of these genes were investigated under carefully controlled static and dynamic conditions in continuous cultures at low oxygen transfer rates. Our results show that S. cerevisiae controls the production of glycerol in response to hypoxic conditions by regulating the expression of several genes. At high demand for NADH reoxidation, a strong induction was seen not only of the GPD2 gene, but also of GPP1, encoding one of the molecular forms of glycerol-3-phosphatase. Induction of the GPP1 gene appears to play a decisive role at elevated growth rates. At low demand for NADH reoxidation via glycerol formation, the GPD1, GPD2, GPP1, and GPP2 genes were all expressed at basal levels. The dynamics of the gene induction and the glycerol formation at low demand for NADH reoxidation point to an important role of the Gpd1p; deletion of the GPD1 gene strongly altered the expression patterns of the GPD2 and GPP1 genes under such conditions. Furthermore, our results indicate that GCY1 and DAK1, tentatively encoding glycerol dehydrogenase and dihydroxyacetone kinase, respectively, may be involved in the redox regulation of S. cerevisiae.

Fps1p controls the accumulation and release of the compatible solute glycerol in yeast osmoregulation.

The accumulation of compatible solutes, such as glycerol, in the yeast Saccharomyces cerevisiae, is a ubiquitous mechanism in cellular osmoregulation. Here, we demonstrate that yeast cells control glycerol accumulation in part via a regulated, Fps1p-mediated export of glycerol. Fps1p is a member of the MIP family of channel proteins most closely related to the bacterial glycerol facilitators. The protein is localized in the plasma membrane. The physiological role of Fps1p appears to be glycerol export rather than uptake. Fps1 delta mutants are sensitive to hypo-osmotic shock, demonstrating that osmolyte export is required for recovery from a sudden drop in external osmolarity. In wild-type cells, the glycerol transport rate is decreased by hyperosmotic shock and increased by hypo-osmotic shock on a subminute time scale. This regulation seems to be independent of the known yeast osmosensing HOG and PKC signalling pathways. Mutants lacking the unique hydrophilic N-terminal domain of Fps1p, or certain parts thereof, fail to reduce the glycerol transport rate after a hyperosmotic shock. Yeast cells carrying these constructs constitutively release glycerol and show a dominant hyperosmosensitivity, but compensate for glycerol loss after prolonged incubation by glycerol overproduction. Fps1p may be an example of a more widespread class of regulators of osmoadaptation, which control the cellular content and release of compatible solutes.

Improved ethanol production by glycerol-3-phosphate dehydrogenase mutants of Saccharomyces cerevisiae.

The anaerobic performance of gpd1 delta and gpd2 delta mutants of Saccharomyces cerevisiae was characterized and compared to that of a wild-type strain under well-controlled conditions by using a high-performance bioreactor. There was a 40% reduction in glycerol level in the gpd2 delta mutant compared to the wild-type. Also the gpd1 delta mutant showed a slight decrease in glycerol formation but to a much lesser degree. As a consequence, ethanol formation in the gpd2 delta mutant was elevated by 13%. In terms of growth, the gpd1 delta mutant and the wild-type were indistinguishable. The gpd2 delta mutant, on the other hand, displayed an extended lag phase as well as a reduced growth rate under the exponential phase. Even though glycerol-3-phosphate dehydrogenase 2 (GPD2) is the important enzyme under anaerobic conditions it can, at least in part, be substituted by GPD1. This was indicated by the higher expression level of GPD1 in the gpd2 delta mutant compared to the wild type. These results also show that the cells are able to cope and maintain redox balance under anaerobic conditions even if glycerol formation is substantially reduced, as observed in the gpd2 delta mutant. One obvious way of solving the redox problem would be to make a biomass containing less protein, since most of the excess NADH originates from amino acid biosynthesis. However, the gpd2 delta mutant did not show any decrease in the protein content of the biomass.

The DNA ligands influence the interactions between the herpes simplex virus 1 origin binding protein and the single strand DNA-binding protein, ICP-8.

The herpes simplex virus type 1 (HSV-1) origin binding protein, OBP, is a DNA helicase specifically stimulated by the viral single strand DNA-binding protein, ICP-8. The stimulation is dependent on direct protein-protein interactions between the C-terminal domain of OBP, delta OBP, and ICP 8 (Boehmer, P.E., Craigie, M.C., Stow, N.D., and Lehman, I.R. (1994) J. Biol. Chem. 269, 29329-29334). We have now observed that this interaction is dramatically influenced by the nature of the DNA ligand. Stable complexes between delta OBP, ICP 8, and double-stranded DNA, presented either as a specific duplex oligonucleotide or a restriction fragment containing the HSV-1 origin of replication, oriS, can be detected by gel chromatography and gel electrophoresis. In contrast, a single-stranded oligonucleotide, oligo(dT)65, will completely disrupt the complex between delta OBP and ICP 8. We therefore suggest that the interaction between delta OBP and ICP 8 serves to position the single strand DNA-binding protein with high precision onto single-stranded DNA at a replication fork or at an origin of DNA replication.