A replication-time-controlling sequence element in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Eukaryotic replication origins are highly variable in their activity and replication timing. The nature and role of cis-acting regulatory sequences that control chromosomal replication timing is not well defined. In the fission yeast, Schizosaccharomyces pombe, a 200-bp late-replication-enforcing element (LRE), has been shown to enforce late replication of ARS elements in plasmids. Here, we show that a short (133-bp) fragment of the LRE (shLRE) is required for causing late replication of adjoining origins in its native as well as in an ectopic early-replicating chromosomal location. Active from both sides of an early-replicating origin, the shLRE is a bona fide cis-acting regulatory element that imposes late replication timing in the chromosome.     

Molecular phylogeny of pectinase genes <Emphasis Type="Italic">PGU</Emphasis> in the yeast genus <Emphasis Type="Italic">Saccharomyces</Emphasis>

Using yeast genome databases and literature data, phylogenetic analysis of pectinase PGU genes from 112 Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and the hybrid taxon S. pastorianus (syn. S. carlsbergensis) was carried out. A superfamily of divergent PGU genes was found. Natural interspecies transfer of the PGU gene both from S. cerevisiae to S. bayanus and from S. paradoxus to S. cerevisiae may, however, occur. Within the Saccharomyces species, identity of the PGU nucleotide sequences was 98.8–100% for S. cerevisiae, 86.1–95.7% for S. bayanus (var. uvarum), 94–98.3% for S. kudriavzevii, and 96.8–100% for S. paradoxus/S. cariocanus. For the first time, a family of polymeric PGU1b, PGU2b, PGU3b and PGU4b genes is documented for the yeast S. bayanus var. uvarum, a variety important for winemaking.     

The replication origin of <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis>

The putative replication origin of Azotobacter vinelandii was cloned as an autonomously replicating fragment after ligation to an antibiotic resistance cartridge. The resulting plasmids could be isolated and labelled by Southern hybridisation with the antibiotic resistance cartridge as probe and also visualised by electron microscopy. These plasmids integrated into the chromosome after a few generations, even in the recA mutant of A. vinelandii. The integrated copy of the plasmid was re-isolated from the chromosome and the DNA and its subfragments were cloned in the plasmid vector pBR322. A 200-bp DNA fragment was sufficient to allow the replication of pBR322 in an Escherichia coli polA strain. Electron microscopic analysis of this plasmid showed that replication initiated mostly within the A. vinelandii DNA fragment. The nucleotide sequence of the putative replication origin and its flanking regions was determined. In the sequence of the 200-bp fragment many of the distinctive features found in other replication origins are lacking. A greater variation from the consensus DnaA binding sequence was observed in A. vinelandii. Direct sequencing of the relevant genomic fragment was also carried after amplifying it from A. vinelandii chromosomal DNA by PCR. This confirmed that no rearrangements had taken place while the cloned fragment was resident in E. coli. It was shown by hybridisation that the 200-bp chromosomal origin fragment of A. vinelandii was present in three other field strains of Azotobacter spp.     

Identification of yeasts during alcoholic fermentation of <Emphasis Type="Italic">tchapalo</Emphasis>, a traditional sorghum beer from Côte d’Ivoire

This study investigated the diversity and dynamics of yeasts involved in alcoholic fermentation of a traditional sorghum beer from Côte d’Ivoire, tchapalo. A total of 240 yeast strains were isolated from fermenting sorghum wort inoculated with dry yeast from two geographic regions of Côte d’Ivoire (Abidjan and Bondoukou). Initial molecular identification to the species level was carried out using RFLP of PCR-amplified internal transcribed spacers of rDNA (ITS1-5.8S-ITS2). Ten different profiles were obtained from the restriction of PCR products with the three endonucleases HaeIII, CfoI and HinfI. Sequence analysis of the D1/D2 domain of the 26S rDNA and the ACT1 gene allowed us to assign these groups to six different species: Saccharomyces cerevisiae-like, Candida tropicalis, Pichia kudriavzevii, Pichia kluyveri, Kodamaea ohmeri and Meyerozyma caribbica. The most frequent species associated with tchapalo fermentation was S. cerevisiae-like (87.36%), followed by C. tropicalis (5.45%) and M. caribbica (2.71%). S. cerevisiae-like strains were diploid heterozygotes and exhibited three to four nucleotides divergence from the type strain in the D1/D2 domain and several indels in the more discriminant sequence of the intron of the ACT1 gene. During the process, the yeast species isolated and their frequencies varied according to the geographic origin of the dry yeast. The occurrence of some species was sporadic and only two non-Saccharomyces species were found in the final product.     

Genetic and biochemical evidences reveal novel insights into the mechanism underlying <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Sae2-mediated abrogation of DNA replication stress

In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) protein complex plays pivotal roles in double-strand break (DSB) repair, replication stress and telomere length maintenance. Another protein linked to DSB repair is Sae2, which regulates MRX persistence at DSBs. However, very little is known about its role in DNA replication stress and repair. Here, we reveal a crucial role for Sae2 in DNA replication stress. We show that different mutant alleles of SAE2 cause hypersensitivity to genotoxic agents, and when combined with Δmre11 or nuclease-defective mre11 mutant alleles, the double mutants are considerably more sensitive suggesting that the sae2 mutations synergize with mre11 mutations. Biochemical studies demonstrate that Sae2 exists as a dimer in solution, associates preferentially with single-stranded and branched DNA structures, exhibits structure-specific endonuclease activity and cleaves these substrates from the 5′ end. Furthermore, we show that the nuclease activity is indeed intrinsic to Sae2. Interestingly, sae2^G270D protein possesses DNA-binding activity, but lacks detectable nuclease activity. Altogether, our data suggest a direct role for Sae2 nuclease activity in processing of the DNA structures that arise during replication and DNA damage and provide insights into the mechanism underlying Mre11-Sae2-mediated abrogation of replication stress-related defects in S. cerevisiae.     

Comparison of L-lactate dehydrogenases of different origins produced in the cells of yeast <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

The expression of L-lactate dehydrogenase genes ldh1 (Bos taurus), ldhA (Homo sapiens), ldhA (Rhizopus oryzae), ldh1 (Lactobacillus plantarum), and ldh1 (Lactobacillus pentosus) in the cells of yeast Schizosaccharomyces pombe VKPM U-3106 has been investigated. The catalytic characteristics of the enzymes encoded by these genes have been compared, and the intensity of lactic acid synthesis by the recombinant strains obtained has been evaluated. The enzymatic activity of L-lactate dehydrogenases from L. plantarum and L. pentosus was the highest (approximately 2 to 2.5 times higher than that of the mammalian enzymes), and these enzymes therefore appear to have the highest potential for the development of lactic-acid producing strains of yeast S. pombe.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains associated with the production of cachaça: identification and characterization by traditional and molecular methods (PCR,PFGE and mtDNA-RFLP)

A study of the genetic diversity of populations of Saccharomyces cerevisiae was conducted in ten different cachaça producers (alambiques) in the southern state of Minas Gerais, Brazil. A total of 106 isolates were identified by PCR using the primer SCREC114, specific to S. cerevisiae, by pulsed-field gel electrophoresis (PFGE) and by restriction fragment polymorphism of mitochondrial DNA analysis (RFLP-mtDNA). PCR showed a product of amplification to 61 isolates, enabling a rapid identification of S. cerevisiae in different alambiques. Nine different profiles were found by PFGE; all the yeasts identified as S. cerevisiae by PCR had profiles similar to that of the marker S. cerevisiae, highlighting the specificity of primer SCREC114. RFLP-mtDNA, using four different enzymes, enabled the grouping of strains of S. cerevisiae, with 80%–100% similarity. Some alambiques that had a higher frequency of S. cerevisiae characterized by PCR and PFGE, had a lower level of genetic diversity determined by RFLP-mtDNA, indicating the ability of these strains to lead the fermentative process.     

Biodegradation of Gossypol by Mixed Fungal Cultures in Minimal Medium

The fungal cultures, namely – Pleurotus sajor-caju MTCC 1806, Saccharomyces cerevisiae MTCC 6933 and Candida tropicalis MTCC 1406 and their combinations, C. tropicalis + S. cerevisiae, P. sajor-caju + S. cerevisiae and C. tropicalis + P. sajor-caju were grown in minimal medium containing 100 ppm of gossypol as the sole carbon and energy source. The culture supernatants of C. tropicalis + S. cerevisiae and P. sajor-caju + S. cerevisiae had low residual gossypol levels of 29 and 25 ppm, respectively. In the present study, we attempted to isolate gossypol-degrading enzyme and biodegraded gossypol from the culture supernatants of C. tropicalis + S. cerevisiae and P. sajor-caju + S. cerevisiae. The specific activity of laccase in the purified enzyme extracts of the C. tropicalis + S. cerevisiae and P. sajor-caju + S. cerevisiae treated samples was 425 and 224 U/mg, respectively. In SDS-PAGE, the gossypol-degrading enzyme was revealed as 3 bands of molecular weights ranging from 45 to 66 kDa. The characterization of biodegraded gossypol by FTIR analysis showed a reduction in aldehydes (C-H) stretches in samples treated with fungi. Mass spectrometry analysis revealed that the monoisotopic mass of the biodegraded gossypol was 474 g/mol.     

A novel killer protein from <Emphasis Type="Italic">Pichia kluyveri</Emphasis> isolated from an Algerian soil: purification and characterization of its in vitro activity against food and beverage spoilage yeasts

A novel killer protein (Pkkp) secreted by a Pichia kluyveri strain isolated from an Algerian soil was active against food and beverage spoilage yeasts of the genera Dekkera, Kluyveromyces, Pichia, Saccharomyces, Torulaspora, Wickerhamomyces and Zygosaccharomyces. After purification by gel filtration chromatography Pkkp revealed an apparent molecular mass of 54 kDa with SDS-PAGE. Minimum inhibitory concentrations (MICs) of purified Pkkp exhibited a high in vitro activity against Dekkera bruxellensis (MICs from 64,000- to 256,000-fold lower than that exhibited by potassium metabisulphite) and Saccharomyces cerevisiae (MICs from 32,000- to 64,000- fold lower than potassium sorbate). No in vitro synergistic interactions (calculated by FIC index ? Σ FIC) were observed when Pkkp was used in combination with potassium metabisulphite, potassium sorbate, or ethanol. Pkkp exhibited a dose–response effect against D. bruxellensis and S. cerevisiae in a low-alcoholic drink and fruit juice, respectively. The results of the present study suggest that Pkkp could be proposed as a novel food-grade compound useful for the control of food and beverage spoilage yeasts.     

In vitro investigation of <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis> strains for potential probiotic properties

In this study, 23 Debaryomyces hansenii strains, isolated from cheese and fish gut, were investigated in vitro for potential probiotic properties i.e. (1) survival under in vitro GI (gastrointestinal) conditions with different oxygen levels, (2) adhesion to Caco-2 intestinal epithelial cells and mucin, and (3) modulation of pro- and anti-inflammatory cytokine secretion by human monocyte-derived dendritic cells. As references two commercially available probiotic Saccharomyces cerevisiae var. boulardii (S. boulardii) strains were included in the study. Our results demonstrate that the different D. hansenii yeast strains had very diverse properties which could potentially lead to different probiotic effects. One strain of D. hansenii (DI 09) was capable of surviving GI stress conditions, although not to the same degree as the S. boulardii strains. This DI 09 strain, however, adhered more strongly to Caco-2 cells and mucin than the S. boulardii strains. Additionally, two D. hansenii strains (DI 10 and DI 15) elicited a higher IL-10/IL-12 ratio than the S. boulardii strains, indicating a higher anti-inflammatory effects on human dendritic cells. Finally, one strain of D. hansenii (DI 02) was evaluated as the best probiotic candidate because of its outstanding ability to survive the GI stresses, to adhere to Caco-2 cells and mucin and to induce a high IL-10/IL-12 ratio. In conclusion, this study shows that strains of D. hansenii may offer promising probiotic traits relevant for further study.     

Genome sequence of <Emphasis Type="Italic">Candida versatilis</Emphasis> and comparative analysis with other yeast

The genome of Candida versatilis was sequenced to understand its characteristics in soy sauce fermentation. The genome size of C. versatilis was 9.7 Mb, the content of G + C was 39.74 %, scaffolds of N50 were 1,229,640 bp in length, containing 4711 gene. There were predicted 269 tRNA genes and 2201 proteins with clear function. Moreover, the genome information of C. versatilis was compared with another salt-tolerant yeast Zygosaccharomyces rouxii and the model organism Saccharomyces cerevisiae. C. versatilis and Z. rouxii genome size was close and both smaller than 12.1 for the Mb of S. cerevisiae. Using the OrthoMCL protein, three genomes were divided into 4663 groups. There were about 3326 homologous proteins in C. versatilis, Z. rouxii and S. cerevisiae.     

Gene expression profiles of the thermotolerant yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain KKU-VN8 during high-temperature ethanol fermentation using sweet sorghum juice

Objectives

To investigate gene expression profiles of the thermotolerant yeast Saccharomyces cerevisiae strain KKU-VN8, a potential high-ethanol producer, in response to various stresses during high-temperature ethanol fermentation using sweet sorghum juice (SSJ) under optimal conditions.

Results

The maximal ethanol concentration obtained by S. cerevisiae KKU-VN8 using SSJ at 40 °C was 66.6 g/l, with a productivity of 1.39 g/l/h and a theoretical ethanol yield of 81%. Quantitative RT-PCR assays were performed to investigate the gene expression profiles of S. cerevisiae KKU-VN8. Differential expression of genes encoding heat-shock proteins (HSP82, HSP104, SSA4), genes involved in trehalose metabolism (TPS1, TPS2, NTH1) and genes involved the glycolytic pathway (ADH1, ADH2, CDC19) at various time points during fermentation was observed. The expression levels of HSP82, HSP104, SSA4, ADH1 and CDC19 were significantly higher than those of the controls (10.2-, 4-, 8-, 8.9- and 5.9-fold higher, respectively). In contrast, the expression levels of TPS1, TPS2, NTH1 and ADH2 were approx. 2-fold less than those of the controls.

Conclusions

The highly expressed genes encoding heat-shock proteins, HSP82 and SSA4, potentially play an important role in helping S. cerevisiae KKU-VN8 cope with various stresses that occur during high-temperature fermentation, leading to higher ethanol production efficiency.

     

Defective DNA Synthesis in Permeabilized Yeast Mutants

THE simple eukaryote, Saccharomyces cerevisiae, is suitable for combined genetic and biochemical analysis of the cell division cycle. More than forty temperature-sensitive mutants of S. cerevisiae defective in fifteen genes that control various steps of the yeast cell cycle have been detected by screening a collection of mutants with time-lapse photomicroscopy¹. Mutations in two genes, cdc4 and cdc8, result in defective DNA synthesis at the restrictive temperature². The product of cdc8 is apparently required throughout the period of DNA synthesis, because if a strain defective in this gene is shifted to 36° C within the S period, DNA replication ceases. In contrast, the product of cdc4 is apparently required only at the initiation of DNA synthesis because when a strain carrying a defect in this gene is shifted to 36° C, DNA replication already in progress is not impaired. Cells defective in cdc4, however, fail to initiate new rounds of DNA synthesis at the restrictive temperature. Based on these observations the DNA mutants have been tentatively classified as defective in DNA replication (cdc8) and in the initiation of DNA synthesis (cdc4).     

Identification and polymorphism of pectinase genes <Emphasis Type="Italic">PGU</Emphasis> in the <Emphasis Type="Italic">Saccharomyces bayanus</Emphasis> complex

Pectinase (endo-polygalacturonase) is the key enzyme splitting plant pectin. The corresponding single gene PGU1 is documented for the yeast S. cerevisiae. On the basis of phylogenetic analysis of the PGU nucleotide sequence available in the GenBank, a family of divergent PGU genes is found in the species complex S. bayanus: S. bayanus var. uvarum, S. eubayanus, and hybrid taxon S. pastorianus. The PGU genes have different chromosome localization.     

Engineering of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> to utilize xylan as a sole carbohydrate source by co-expression of an endoxylanase,xylosidase and a bacterial xylose isomerase

Xylan represents a major component of lignocellulosic biomass, and its utilization by Saccharomyces cerevisiae is crucial for the cost effective production of ethanol from plant biomass. A recombinant xylan-degrading and xylose-assimilating Saccharomyces cerevisiae strain was engineered by co-expression of the xylanase (xyn2) of Trichoderma reesei, the xylosidase (xlnD) of Aspergillus niger, the Scheffersomyces stipitis xylulose kinase (xyl3) together with the codon-optimized xylose isomerase (xylA) from Bacteroides thetaiotaomicron. Under aerobic conditions, the recombinant strain displayed a complete respiratory mode, resulting in higher yeast biomass production and consequently higher enzyme production during growth on xylose as carbohydrate source. Under oxygen limitation, the strain produced ethanol from xylose at a maximum theoretical yield of ~90 %. This study is one of only a few that demonstrates the construction of a S. cerevisiae strain capable of growth on xylan as sole carbohydrate source by means of recombinant enzymes.     

Cytogenetic evidences on the evolutionary relationships between the tetraploids of the section <Emphasis Type="Italic">Rhizomatosae</Emphasis> and related diploid species (<Emphasis Type="Italic">Arachis</Emphasis>, Leguminosae)

Rhizomatosae is a taxonomic section of the South American genus Arachis, whose diagnostic character is the presence of rhizomes in all its species. This section is of particular evolutionary interest because it has three polyploid (A. pseudovillosa, A. nitida and A. glabrata, 2n?=?4x?=?40) and only one diploid (A. burkartii, 2n?=?2x?=?20) species. The phylogenetic relationships of these species as well as the polyploidy nature and the origin of the tetraploids are still controversial. The present study provides an exhaustive analysis of the karyotypes of all rhizomatous species and six closely related diploid species of the sections Erectoides and Procumbentes by cytogenetic mapping of DAPI/CMA heterochromatin bands and 5S and 18–26S rDNA loci. Chromosome banding showed variation in the DAPI heterochromatin distribution pattern, which, together with the number and distribution of rDNA loci, allowed the characterization of all species studied here. The bulk of chromosomal markers suggest that the three rhizomatous tetraploid species constitute a natural group and may have at least one common diploid ancestor. The cytogenetic data of the diploid species analyzed evidenced that the only rhizomatous diploid species—A. burkartii—has a karyotype pattern different from those of the rhizomatous tetraploids, showing that it is not likely the genome donor of the tetraploids and the non-monophyletic nature of the section Rhizomatosae. Thus, the tetraploid species should be excluded from the R genome, which should remain exclusively for A. burkartii. Instead, the karyotype features of these tetraploids are compatible with those of different species of the sections Erectoides and Procumbentes (E genome species), suggesting the hypothesis of multiple origins of these tetraploids. In addition, the polyploid nature and the group of diploid species closer to the tetraploids are discussed.     

Genome-wide identification and characterization of replication origins by deep sequencing

Background

DNA replication initiates at distinct origins in eukaryotic genomes, but the genomic features that define these sites are not well understood.

Results

We have taken a combined experimental and bioinformatic approach to identify and characterize origins of replication in three distantly related fission yeasts: Schizosaccharomyces pombe, Schizosaccharomyces octosporus and Schizosaccharomyces japonicus. Using single-molecule deep sequencing to construct amplification-free high-resolution replication profiles, we located origins and identified sequence motifs that predict origin function. We then mapped nucleosome occupancy by deep sequencing of mononucleosomal DNA from the corresponding species, finding that origins tend to occupy nucleosome-depleted regions.

Conclusions

The sequences that specify origins are evolutionarily plastic, with low complexity nucleosome-excluding sequences functioning in S. pombe and S. octosporus, and binding sites for trans-acting nucleosome-excluding proteins functioning in S. japonicus. Furthermore, chromosome-scale variation in replication timing is conserved independently of origin location and via a mechanism distinct from known heterochromatic effects on origin function. These results are consistent with a model in which origins are simply the nucleosome-depleted regions of the genome with the highest affinity for the origin recognition complex. This approach provides a general strategy for understanding the mechanisms that define DNA replication origins in eukaryotes.     

Effects of Probiotics on Survival,Growth and Digestive Enzymes Activities in Freshwater Prawn <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis> (De Man 1879)

A study was conducted to examine the effects of three probiotics, Lactobacillus sporogenes, Bacillus subtilis and Saccharomyces cerevisiae on the survival, growth and digestive enzymes activities of the freshwater prawn Macrobrachium rosenbergii post larvae (PL). The probiotics, L. sporogenes (4 %), B. subtilis (3 %) and S. cerevisiae (4 %) were taken and mixed with basal diet. Diet without probiotics served as control. These probiotics diets were fed to M. rosenbergii PL for a period of 60 days. After the feeding trail, the growth parameters such as survival, weight gain, specific growth rate and protein efficiency rate were found to be significantly (P < 0.05) higher in 4 % S. cerevisiae incorporated diet fed PL when compared with control. In the case of feed conversion rate just the reverse was seen (P < 0.05) at this concentration. This indicates its superior quality among different concentrations of probiotics tested. Activities of digestive enzymes, such as protease, amylase and lipase were significantly (P < 0.05) higher at this concentration (4 % S. cerevisiae). Some of essential and non-essential amino acids also significantly elevated in probiotics supplemented diet fed prawns. This study indicated that probiotics, S. cerevisiae incorporated diets were beneficial for M. rosenbergii in terms of increasing growth, enzyme and amino acid production.     

Expression of the catalytic subunits of pol α and pol δ from fission yeast<Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

This paper reports on expression and posttranslational modifications of the catalytic subunits of pol α and pol δ from fission yeastSchizosaccharomyces pombe. Okadaic acid treatment ofS. pombe spheroplasts in amounts known to inhibit phosphatases, 1 and 2A resulted in decreased proteolysis of both pol α and pol δ. Computer analysis of pol α and pol δ sequences confirmed the presence of consensus motifs for protein phosphorylation. Indirect immunofluorescence microscopy ofS. pombe cells showed nuclear location of both proteins in wild type cells. However, whereas cells transformed with a vector expressing pol α produced a clear increase of the nuclear signal no increase was detectable in cells transformed with pol δ. This observation suggests the existence of a mechanism limiting thecell concentration of pol δ in the cell. Constitutive expression ofS. pombe pol δ inE. coli was possible only with vectors containing truncated forms of its gene, indicating a toxic effect of pol δ onE. coli growth.     

Cell Surface Display of MerR on <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for Biosorption of Mercury

The metalloregulatory protein MerR which plays important roles in mer operon system exhibits high affinity and selectivity toward mercury (II) (Hg²⁺). In order to improve the adsorption ability of Saccharomyces cerevisiae for Hg²⁺, MerR was displayed on the surface of S. cerevisiae for the first time with an α-agglutinin-based display system in this study. The merR gene was synthesized after being optimized and added restriction endonuclease sites EcoR I and Mlu I. The display of MerR was indirectly confirmed by the enhanced adsorption ability of S. cerevisiae for Hg²⁺ and colony PCR. The hydride generation atomic absorption spectrometry was applied to measure the Hg²⁺ content in water. The engineered yeast strain not only showed higher tolerance to Hg, but also their adsorption ability was much higher than that of origin and control strains. The engineered yeast could adsorb Hg²⁺ under a wide range of pH levels, and it could also adsorb Hg²⁺ effectively with Cd²⁺ and Cu²⁺ coexistence. Furthermore, the engineered yeast strain could adsorb ultra-trace Hg²⁺ effectively. The results above showed that the surface-engineered yeast strain could adsorb Hg²⁺ under complex environmental conditions and could be used for the biosorption and bioremediation of environmental Hg contaminants.