Cdc42p controls yeast-cell shape and virulence of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is characterized by a multiple budding phenotype and a polymorphic cell growth, leading to the formation of cells with extreme variations in shape and size. Since Cdc42 is a pivotal molecule in establishing and maintaining polarized growth for diverse cell types, as well as during pathogenesis of certain fungi, we evaluated its role during cell growth and virulence of the yeast-form of P. brasiliensis. We used antisense technology to knock-down PbCDC42's expression in P. brasiliensis yeast cells, promoting a decrease in cell size and more homogenous cell growth, altering the typical polymorphism of wild-type cells. Reduced expression levels also lead to increased phagocytosis and decreased virulence in a mouse model of infection. We provide genetic evidences underlying Pbcdc42p as an important protein during host-pathogen interaction and the relevance of the polymorphic nature and cell size in the pathogenesis of P. brasiliensis.

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and analysis of the CEN1-ADE1-CDC15 region.

To continue the systematic examination of the physical and genetic organization of an entire Saccharomyces cerevisiae chromosome, the DNA from the CEN1-ADE1-CDC15 region from chromosome I was isolated and characterized. Starting with the previously cloned ADE1 gene (J. C. Crowley and D. B. Kaback, J. Bacteriol. 159:413-417, 1984), a series of recombinant lambda bacteriophages containing 82 kilobases of contiguous DNA from chromosome I were obtained by overlap hybridization. The cloned sequences were mapped with restriction endonucleases and oriented with respect to the genetic map by determining the physical positions of the CDC15 gene and the centromeric DNA (CEN1). The CDC15 gene was located by isolating plasmids from a YCp50 S. cerevisiae genomic library that complemented the cdc15-1 mutation. S. cerevisiae sequences from these plasmids were found to be represented among those already obtained by overlap hybridization. The cdc15-1-complementing plasmids all shared only one intact transcribed region that was shown to contain the bona fide CDC15 gene by in vitro gene disruption and one-step replacement to delete the chromosomal copy of this gene. This deletion produced a recessive lethal phenotype that was also recessive to cdc15-1. CEN1 was located by finding a sequence from the appropriate part of the cloned region that stabilized the inheritance of autonomously replicating S. cerevisiae plasmid vectors. Finally, RNA blot hybridization and electron microscopy of R-loop-containing DNA were used to map transcribed regions in the 23 kilobases of DNA that went from CEN1 to CDC15. In addition to the transcribed regions corresponding to the ADE1 and ADC15 genes, this DNA contained five regions that gave rise to polyadenylated RNA, at least two regions complementary to 4S RNA species, and a Ty1 transposable element. Notably, a higher than average proportion of the DNA examined was transcribed into RNA.

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome.

Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae. A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector. Plasmids containing pyk1-complementing DNA were obtained from other investigators. Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes. These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector. A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined. Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3). Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome. The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.

Four Arabidopsis thaliana 14-3-3 protein isoforms can complement the lethal yeast bmh1 bmh2 double disruption.

The 14-3-3 proteins comprise a family of highly conserved proteins with multiple functions, most of which are related to signal transduction. Four isoforms from the plant Arabidopsis thaliana were able to complement the lethal disruption of the two Saccharomyces cerevisiae genes encoding 14-3-3 proteins; one complemented very poorly and one did not complement. However, the expression of the latter two isoforms was very low. These results show that at least four of the six A. thaliana isoforms are able to perform the same function(s) as the yeast 14-3-3 proteins.

Characterization of the yeast BMH1 gene encoding a putative protein homologous to mammalian protein kinase II activators and protein kinase C inhibitors.

We describe the identification and characterization of the BMH1 gene from the yeast Saccharomyces cerevisiae. The gene encodes a putative protein of 292 amino acids which is more than 50% identical with the bovine brain 14-3-3 protein and proteins isolated from sheep brain which are strong inhibitors of protein kinase C. Disruption mutants and strains with the BMH1 gene on multicopy plasmids have impaired growth on minimal medium with glucose as carbon source, i.e. a 30-50% increase in generation time. These observations suggest a regulatory function of the bmh1 protein. In contrast to strains with an intact or a disrupted BMH1 gene, strains with the BMH1 gene on multicopy plasmids hardly grew on media with acetate or glycerol as carbon source.

The Saccharomyces cerevisiae acetyl-coenzyme A synthetase encoded by the ACS1 gene, but not the ACS2-encoded enzyme, is subject to glucose catabolite inactivation.

In Saccharomyces cerevisiae, the structural genes ACS1 and ACS2 each encode an isoenzyme of acetyl-CoA synthetase (ACS; EC 6.2.1.1). Involvement of glucose catabolite repression in regulation of the two isoenzymes was investigated by following ACS activity after glucose pulses (100 mM) to ethanol-limited chemostat cultures. In wild-type S. cerevisiae and in an isogenic strain in which ACS2 had been disrupted, ACS activity decreased after a glucose pulse. No such inactivation was observed in a strain in which ACS1 was disrupted. Western blots demonstrated that the ACS1 product, but not the ACS2 product, was degraded after a glucose pulse. Inactivation kinetics of the ACS1 product resembled those of isocitrate lyase.

Overproduction of acetyl-coenzyme A synthetase isoenzymes in respiring Saccharomyces cerevisiae cells does not reduce acetate production after exposure to glucose excess.

To investigate whether the production of acetate which occurs after exposure of respiring Saccharomyces cerevisiae cells to excess glucose can be reduced by overproduction of acetyl-CoA synthetase (ACS, EC 6.2.1.1), the ACS1 and ACS2 genes were introduced on multi-copy plasmids. For each isoenzyme, the level in glucose-limited chemostat cultures was increased by 3-6-fold, relative to an isogenic reference strain. However, ACS overproduction did not result in a reduced production of acetate after a glucose pulse (100 mmol l-1) to these cultures. This indicates that a limited capacity of ACS is not the sole cause of acetate accumulation in S. cerevisiae.

Attachment of MAL32-encoded maltase on the outside of yeast cells improves maltotriose utilization.

The fermentation of maltotriose, the second most abundant fermentable sugar in wort, is often incomplete during high-gravity brewing. Poor maltotriose consumption is due to environmental stress conditions during high-gravity fermentation and especially to a low uptake of this sugar by some industrial strains. In this study we investigated whether the use of strains with an alpha-glucosidase attached to the outside of the cell might be a possible way to reduce residual maltotriose. To this end, the N-terminal leader sequence of Kre1 and the carboxy-terminal anchoring domain of either Cwp2 or Flo1 were used to target maltase encoded by MAL32 to the cell surface. We showed that Mal32 displayed on the cell surface of Saccharomyces cerevisiae laboratory strains was capable of hydrolysis of alpha-1,4-linkages, and that it increased the ability of a strain lacking a functional maltose permease to grow on maltotriose. Moreover, the enzyme was also expressed and found to be active in an industrial strain. These data show that expressing a suitable maltase on the cell surface might provide a means of modifying yeast for more complete maltotriose utilization in brewing and other fermentation applications.

Post-transcriptional control of the Saccharomyces cerevisiae proteome by 14-3-3 proteins.

UNLABELLED: 14-3-3 proteins form a family of conserved eukaryotic proteins binding to over 200 different proteins involved in nearly all cellular processes. The yeast Saccharomyces cerevisiae has two genes encoding 14-3-3 proteins, BMH1 and BMH2. As 14-3-3 proteins are essential in most S. cerevisiae strains, we constructed a bmh mutant with suboptimal 14-3-3 protein activity. Here, we report the effect of these bmh mutations on the proteome as determined by two-dimensional gel electrophoresis and mass spectrometry. We identified 26 proteins of which the levels increased by more than 2.0-fold and 51 proteins of which the levels decreased by more than 2.0-fold in the bmh mutant compared with those of the wild-type strain. For only 9 of these proteins, a more than 2.0-fold alteration was found at the transcriptional level. The levels of many proteins involved in gluconeogenesis, including Fba1, Eno1, Eno2, Tpi1, Pck1, Mdh2, Tdh2, Tdh3, and Gpm1, increased in the mutant, whereas the levels of several proteins involved in amino acid biosynthesis and translation and heat shock proteins were lower. Our studies indicate that 14-3-3 proteins control the S. cerevisiae proteome at the post-transcriptional level, in agreement with the binding of 14-3-3 proteins to proteins involved in protein synthesis and degradation. In addition, our studies suggest a key role in the regulation of carbohydrate metabolism by 14-3-3 proteins. KEYWORDS: 14-3-3 proteins * Saccharomyces cerevisiae * proteome * gluconeogenesis * BMH1 * BMH2.

Maltotriose utilization in lager yeast strains: MTT1 encodes a maltotriose transporter.

Maltotriose is the second most abundant fermentable sugar in wort and, due to incomplete fermentation, residual maltotriose in beer causes both quality and economic problems in the brewing industry. To identify genes that might improve utilization of maltotriose, we developed a library containing genomic DNA from four lager strains and a laboratory Saccharomyces cerevisiae strain and isolated transformants that could grow on YP/2% maltotriose in the presence of 3 mg/l of the respiratory inhibitor antimycin A. In this way we found a gene which shared 74% similarity with MPH2 and MPH3, 62% similarity with AGT1 and 91% similarity with MAL61 and MAL31, all encoding known maltose transporters. Moreover, the gene shared an even higher similarity (98%) with the uncharacterized Saccharomyces pastorianus mty1 gene (M. Salema-Oom, unpublished; NCBI Accession No. AJ491328). Therefore, we named the gene MTT1 (mty1-like transporter). We showed that the gene was present in four different lager strains but was absent from the laboratory strain CEN.PK113-7D. The ORF in the plasmid isolated from the library lacks 66 base pairs from the 3'-end of MTT1 but instead contains 54 bp of the vector. We named this ORF MTT1alt (NCBI Accession No. DQ010174). 14C-Maltose and repurified 14C-maltotriose were used to show that MTT1 and, especially, MTT1alt, encode maltose transporters for which the ratio between activities to maltotriose and maltose is higher than for most known maltose transporters. Introduction of MTT1 or MTT1alt into lager strain A15 raised maltotriose uptake by about 17% or 105%, respectively.

An evaluation of the rate constants for the adsorption of the defective phage PBS Z to Bacillus subtilis.

The adsorption of the defective phage PBS Z of Bacillus subtilis has previously been assumed to proceed in two steps, a reversible adsorption of extended phages followed by contraction of the adsorbed particles (Steensma, 1981a). This model, also used for other phages, explained the biphasic character of the adsorption curve, but a discrepancy was found between the calculated and observed concentrations of adsorbed, extended phages. Computer simulations indicated that this might be caused by inhomogeneity of the phage preparations with respect to their adsorption properties and that in that case other models would also fit the experimental data. Discrimination between the models was not possible on the basis of the available information on PBS Z and it was therefore concluded that the values reported previously for the rate constants (Steensma, 1981a) should be used with caution.

Adsorption of the defective phage PBS Z1 to Bacillus subtilis 168 Wt.

Three aspects of the adsorption of the defective phage PBS Z1 to Bacillus subtilis 168 Wt have been investigated. These are the kinetics, the number of receptors on the cell wall and the character of these receptors. The reaction constants for the binding of phages onto receptors, for the dissociation of the phage-receptor complex and for the transition from reversible to irreversible binding of the phages were calculated from adsorption curves obtained by an enzyme-linked immunosorbent assay (ELISA). They were 1.8 x 10(-13), 6.7 x 10(-2) and 9.0 x 10(-3) respectively. The maximum number of phages adsorbed per cell was 2700, a number limited by the surface area of the cells. Apart from the receptors on the cell wall, receptors on the cell membrane were found. This was concluded from additional adsorption experiments with stable L-forms and contracted phages. Based on these results, together with data from the literature on bacteriocins, phage ghosts and yeast killer factors, a hypothesis concerning the first stage of killing by defective phages has been formulated.

Effect of defective phages on the cell membrane of Bacillus subtilis and partial characterization of the phage protein involved in killing.

In order to investigate whether defective phages of Bacillus subtilis killed sensitive bacteria by a lysis from without mechanism, the minimal number of phages required for killing was determined. This figure was found to vary with the m.o.i., giving a value of 1 on extrapolation to an m.o.i. of 0. This excluded lysis from without as the only killing mechanism, although it might play a role at high m.o.i.s. This was confirmed by experiments on leakage of ATP and u.v.-absorbing material, the uptake of oxygen and the effect of the phages on the membrane potential. Apart from a short, initial leakage of ATP, the cell membrane was not affected at low m.o.i.s. These results lead to the conclusion that at low m.o.i.s. the phages acted on a cytoplasmic component. Treatment of defective phages for 10 min at pH 2.5 resulted in breakdown of the phages without complete abolition of the killing activity. The active component, which was shown not to be DNA, could not be isolated from the mixture, but SDS gel electrophoresis of PBS X and a non-killing mutant of this phage suggested that a protein with a mol. wt. of 85000 was involved in killing.

Review: the dominant flocculation genes of Saccharomyces cerevisiae constitute a new subtelomeric gene family.

The quality of brewing strains is, in large part, determined by their flocculation properties. By classical genetics, several dominant, semidominant and recessive flocculation genes have been recognized. Recent results of experiments to localize the flocculation genes FLO5 and FLO8, combined with the in silicio analysis of the available sequence data of the yeast genome, have revealed that the flocculation genes belong to a family which comprises at least four genes and three pseudogenes. All members of this gene family are located near the end of chromosomes, just like the SUC, MEL and MAL genes, which are also important for good quality baking or brewing strains. Transcription of the flocculation genes is repressed by several regulatory genes. In addition, a number of genes have been found which cause cell aggregation upon disruption or overexpression in an as yet unknown manner. In total, 33 genes have been reported that are involved in flocculation or cell aggregation.

Effect of calcium ions on the infection of Bacillus subtilis by bacteriophage SF 6.

Infection of Bacillus subtilis 168Wt by SF 6 resulted in a rapid reduction in the number of phages. This could be counteracted by the addition of calcium, barium or strontium ions. At the optimum concentration of 7.5 x 10(-2) M, the number of p.f.u. remained constant until lysis began. Although cultures of another host. B. subtilis 31 try- his-, at the end of the logarithmic growth phase produced a substance which inactivated free phages, this was not the major cause of the reduction in the numbers of p.f.u. during infection experiments at low Ca2+ concentrations. The diminution of the number of p.f.u. was therefore attributed to the fact that at least one of the steps of the lytic cycle was calcium dependent. Adsorption of SF 6 was equally effective in media containing high or low concentrations of calcium ions. Infection experiments with phages whose DNA had been labelled radioactively revealed that, at high concentrations of calcium ions, the label remained associated with the host cells until lysis commenced. At low concentrations, however, a dissociation between phage DNA and the host was found, although adsorption took place at a normal rate. From these experiments we concluded that a high concentration of calcium ions was required for the penetration of phage DNA. Similar experiments with phages whose protein coat had been labelled showed the same results, indicating that desorption of the inactivated phages occurred. Both electron microscopy and column chromatogarphy with hydroxyapatite showed that a considerable fraction of the inactivated phages had ejected their DNA into the medium. A hypothesis explaining these results is presented.

An enzyme-linked immunosorbent assay (ELISA) for PBS Z1, a defective phage of Bacillus subtilis.

The sensitivity, reproducibility and specificity of an enzyme-linked immunosorbent assay (ELISA) for the defective phage PBS Z1 of Bacillus subtilis have been investigated. It was shown that phages in concentrations between 10(8) and 2.5 X 10(10) particles/ml could be assayed with this method. The coefficient of variation for concentrations between 5 X 10(8) and 5 X 10(9) particles/ml was approx. 10%. From some other Bacillus phages tested, only the defective phages resembling PBS Z1 in morphology were detected efficiently with the ELISA for PBS Z1. A comparison is made between ELISA and other assays for PBS Z1.

A counting method for determining the burst size of defective phages from Bacillus subtilis.

The defective phages of Bacillus subtilis cannot be counted by plating as they do not form plaques. In addition, counting under the electron microscope with latex spheres as an internal standard is not possible. The reliability of a method using Escherichia coli phage T4 as a substitute for the latex spheres has been tested and the results compared with those of other methods. Using this method, we determined the burst sizes of the defective phages PBS X, PBS Y and PBS Z under various conditions.

The acetyl co-enzyme A synthetase genes of Kluyveromyces lactis.

Two Kluyveromyces lactis genes encoding acetyl co-enzyme A synthetase isoenzymes were isolated. One we named KlACS1, as it has high similarity to the ACS1 gene of Saccharomyces cerevisiae. The other gene, KlACS2, showed more similarity to S. cerevisiae ACS2 than to KlACS1 or ScACS1. This suggests that divergence of the two isogenes occurred before the evolutionary separation of the species and that the different functions have been conserved. In line with this idea is the regulation of transcription of the genes. The mode of regulation appeared to be maintained between ScACS1 and KlACS1 and between ScACS2 and KlACS2. The KlACS1 transcript was absent in glucose-grown cells, whereas transcription levels in ethanol- and acetate-grown cells were high. Disruption of the KlACS1 gene did not result in growth defects on glucose or ethanol. The growth rate on acetate, however, was reduced by a factor of two. KlACS2 was expressed at similar levels during growth on glucose and acetate, whereas expression on ethanol was slightly higher. A null mutant in this gene showed a reduced growth rate on all three carbon sources. Taken together, these data suggest that KlACS2 is used during growth on glucose and that KlACS1 is most dominant during growth on acetate. Strains in which both ACS genes are deleted could only be retrieved when a plasmid containing the ACS2 gene was present, suggesting that the double mutant is lethal. Tetrad analysis confirmed that non-viable spores with a deduced Klacs1Klacs2 genotype germinated but could not divide further. It therefore appears that, as in S. cerevisiae, the pyruvate dehydrogenase bypass formed by the enzymes pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl co-enzyme A synthetase is essential for growth. These results are in apparent contradiction with the growth on glucose of a strain with a disruption in the only structural pyruvate decarboxylase gene of K. lactis. Residual enzyme activity might, however, account for this discrepancy, or Acs fulfils an additional as yet unknown function, separate from its enzymatic activity.

Sequence of the CDC10 region at chromosome III of Saccharomyces cerevisiae.

A 4.74 kb DNA fragment from the right arm of chromosome III of Saccharomyces cerevisiae, adjacent to the centromere region was sequenced. Four open reading frames with an ATG initiation codon and larger than 200 bp were found in this fragment. The largest open reading frame of 966 bp was identified as the CDC10 gene.