Description: Deep-sea isolate of the psychrotolerant yeast Cryptococcus sp. (NIOCC#PY13) obtained from polymetallic nodule-bearing sediments of the Central Indian Basin was examined for its capacity to grow in the presence of various concentrations of the heavy metal salts i.e. ZnSO 4 , CuSO 4 , Pb(CH 3 COO) 2 and CdCl 2 . It demonstrated considerable growth in the presence of 100 mg l -1 concentration of the above mentioned four heavy metal salts both at 30 as well as 15 °C. This strain tolerated comparatively higher levels of these four metal salts than other deep-sea and terrestrial yeast isolates belonging to Cryptococcus, Rhodotorula , Rhodosporidium and Sporidiobolus sp. Optimum pH for growth of this isolate ranged from 6 to 8 in the presence of heavy metal salts at these two temperatures. Scanning electron microscopic (SEM) studies exhibited altered cell surface morphology of the cells under the influence of heavy metals compared to that with control. The adsorption of heavy metals to the cells was demonstrated by FTIR and EDAX analysis. As evidenced by atomic absorption spectrophotometric (AAS) analysis, about 30-90% the heavy metals were removed from the culture supernatant after 4 days of growth at 30 °C. This deep-sea yeast isolate appears to be a potential candidate for bioremediation of metal contaminated sites. Moreover, its metal tolerance properties provide a significant insight on its ecological role and adaptations to grow in such extreme conditions. Copyright © 2013 John Wiley & Sons, Ltd.

Description: This unique study reports a new strain (BPU1) of Candida tropicalis isolated from the rumen of Malabari goat showing dual production of biosurfactant and polyhydroxybutyrate. C. tropicalis strain BPU1, a facultative anaerobe was tuned to become an aerobe in specially designed flask, the Benjamin flask. The puffy circular colonies were smooth, white-to-cream in color with pseudo-filaments. It fermented glucose, sucrose, maltose, dextrose, and not lactose and cellulose. It assimilated NH 4 SO 4 , peptone, glycine, arginine, and not NaNO 3 as nitrogen source. Interestingly, it utilized ground nut oil (up to 0.3%) in a specially designed basal mineral salt medium (BSM). Its ability for the dual production of a biosurfactant and polyhydroxybutyarates (PHB) was explored by various methods from the BSM-oil medium. The extracted biosurfactant from 6 days old culture was biochemically characterized as a complex of lipid and carbohydrate with an R f value of 0.88 by thin layer chromatography. Its PHB production was confirmed by specific staining methods with nile blue sulphate, Sudan Black B and Sudan 3. Briefly, this first ever report gives ample physical evidences for the dual production of a glycolipid (biosurfactant) and PHB by C. tropicalis strain BPU1 on a specially designed medium, which would open up elaborate research on this yeast. Copyright © 2013 John Wiley & Sons, Ltd.

Description: ABSTRACT Conventional isolation and detection methods for small RNAs from yeast cells have been designed for a limited number of samples. In order to be able to conduct a genome-wide assessment of how each gene product impacts upon small RNAs, we developed a rapid method for analyzing small RNAs from Saccharomyces cerevisiae wild-type (wt) and mutants cells in the deletion and temperature-sensitive (ts) collections. Our method implements three optimized techniques: a procedure for growing small yeast cultures in 96-deepwell plates, a fast procedure for small RNA isolation from the plates, and a sensitive nonradioactive Northern method for RNA detection. The RNA isolation procedure requires only 4 hours for processing 96 samples and is highly reproducible, and yields RNA of good quality and quantity. The nonradioactive Northern method employs digoxigenin (DIG)-labeled DNA probes and chemiluminescence. It detects femtomole-levels of small RNAs within 1-minute exposure time. We minimized the processing time for large-scale analysis and optimized the stripping and re-probing procedures for analyses of multiple RNAs from a single membrane. The method described is rapid, sensitive, safe, and cost-effective for genome-wide screens of novel genes involved in the biogenesis, subcellular trafficking, and stability of small RNAs. Moreover, it will be useful to educational laboratory class venues and to research institutions with limited access to radioisotopes or robots. Copyright © 2013 John Wiley & Sons, Ltd.

Description: Pgt1p encodes a glutathione transporter in S . pombe , orthologous to the S . cerevisiae glutathione transporter, Hgt1p. Despite high similarity to Hgt1p, Pgt1p failed to display functionality during heterologous expression in S . cerevisiae . In the present study we employed a genetic strategy to investigate the reason behind the non functionality of pgt1 + in S . cerevisiae . Functional mutants were isolated after in vitro mutagenesis. Several mutants were obtained and 4 mutants analyzed. Among these, 3 yielded different point mutations in the N-terminal region (301 bp to 350 bp) of the transporter before the first transmembrane domain, while one mutant contained a deletion of 42 nucleotide within the same region. The mutant pgt1 + proteins not only expressed and localized correctly, but displayed high affinity glutathione transport capabilities in S . cerevisae . Comparison of the wild-type pgt1 + with the functional mutants revealed that a loss in protein expression was responsible for lack of functionality of wild type pgt1 + in S . cerevisiae . The mRNA levels in wild-type and mutants were comparable, suggesting that the block was in translation. The formation of a strong stem-loop structure appeared to be responsible for inefficient translation in pgt1 + and disruption of these structures in the mutants was probabily permitting translation. This was confirmed by making silent mutations in this region of WT pgt1 + which led to their functionality in S . cerevisiae . This genetic strategy to relieve functional blocks in expression should greatly facilitate the study of these and other transporters from more intractable genetic organisms in a heterologous expression system. Copyright © 2012 John Wiley & Sons, Ltd.

Description: ABSTRACT Kl Pdr1p is a single Kluyveromyces lactis homologue of Saccharomyces cerevisiae Sc Pdr1p/ Sc Pdr3p, the main transcriptional regulators of genes involved in S. cerevisiae multidrug resistance. KlPDR1 deletion leads to a sharp increase in K. lactis drug susceptibility. The presence of putative PDRE and YRE regulatory elements in the KlPDR1 gene promoter suggests an autoregulation of its transcription as well as its control by Kl Yap1p, the transcription factor involved in oxidative stress response. In this work, one plasmid-borne Klpdr1-1 allele that led to amino acid substitution (L273P) in the Kl Pdr1p was isolated. Overexpression of the Klpdr1-1 allele from a multicopy plasmid in the K. lactis wild-type and Klpdr1Δ mutant strain increased the tolerance of transformants to oligomycin. The plasmid-borne Klpdr1-1 allele increased the activation of the ScPDR5 promoter and complemented the drug hypersensitivity of the Saccharomyces cerevisiae pdr1Δ pdr3Δ mutant strain. The results indicate that L273P amino acid substitution is the result of a gain-of-function mutation in the KlPDR1 gene that confers the Kl Pdr1p hyperactivity as revealed by a high expression of the ABC transporter gene KlPDR5 leading to multidrug resistance and rhodamine 6 G efflux out of the cells. Copyright © 2013 John Wiley & Sons, Ltd.

Description: A previous study showed that the use of nitrate by Dekkera bruxellensis might be an advantageous trait when ammonium is limited in sugarcane substrate for ethanol fermentation. The aim of the present work was to evaluate the influence of nitrate on the yeast physiology during cell growth under oxygen limitation in different carbon sources. If nitrate was the sole source of nitrogen, D . bruxellensis cells presented slower growth, diminished sugar consumption and growth-associated ethanol production, when compared to ammonia. These results were corroborated by the increased expression of genes involved in pentose-phosphate pathway, tricarboxylic acid cycle and ATP synthesis. The presence of ammonium in the mixed medium restored most parameters to the standard conditions. This work may open up a line of investigations to establish the connection between nitrate assimilation and the energetic metabolism in D . bruxellensis and their influence on its fermentative capacity in oxygen-limited or oxygen-depleted conditions. Copyright © 2013 John Wiley & Sons, Ltd.

Description: Objectives The study aims to determine the distribution of candidal species in the oral cavity and differentiate the candidal species based on PCR amplification including Hinf I and Msp I digestion in order to assess the effectiveness of using the rDNA region for species identification. Methods Samples from saliva as well as the palate, tongue and cheek mucosa surfaces were collected from 45 individuals consisting of three groups: periodontal disease patients, denture wearers, and the control group. The samples were serially diluted, spread on BHI and YPD agar plates and scored for Colony-Forming Units (CFUs). Fifteen random candidal colonies were isolated and subjected to genomic DNA extraction based on glass beads disruption. Four primers were used to amplify regions in the rDNA, and the ITSI-5.8S-ITSII PCR product was digested by Hinf I and Msp I restriction enzymes. Results The microbial loads on all the sites of denture wearers was found to be significantly higher than the control, while only the microbial loads on the tongue of the periodontal disease group was significantly higher than the control. Meanwhile, there was no significant difference at other sites. The restriction fragment lengths of the clinical samples were compared to that of seven control species, allowing the differentiation of all seven species and the identification of fourteen species from the clinical samples. The Msp I restriction digest was not able to distinguish between C. albicans and C. dubliniensis , whereas the Hinf I digest could not distinguish between C. tropicalis and C. parapsilosis . Conclusion PCR-RFLP of the candidal rDNA region has potential for species identification. Copyright © 2012 John Wiley & Sons, Ltd.

Description: ABSTRACT The phospholipid metabolism of Saccharomyces cerevisiae plays a central role in its adaptation to low temperatures. In order to detect the key genes in this adaptation, various phospholipid mutants from the EUROSCARF collection of Saccharomyces cerevisiae BY4742 were tested to ascertain whether the suppression of some genes could improve the fermentation vitality of the cells at low temperature. The cell vitality and phospholipid composition of these mutants were analysed. Some knockouts improved ( hmn1∆ ) or impaired ( cho2∆ and psd1∆ ) their vitality at low temperature (13 °C), but were not affected at optimum temperature (25 °C). A common trait of the mutants that had some defect in vitality was a lower concentration of phosphatidylcholine and/or phosphatidylethanolamine. The supplementation with choline allowed them to recover viability, probably by synthesis through Kennedy's pathway. Hmn1∆ showed a lower concentration of phosphatidylcholine, which explains the dominant role of the de novo pathway in cellular phosphatidylethanolamine and phosphatidylcholine vs . the Kennedy's pathway. The absence of such genes as CRD1 or OPI3 produced important changes in phospholipid composition. Cardiolipin was not detected in crd1∆ but phosphatidylglycerol circumvents most of the functions assigned to CL. The considerable reduction in PC diminished the cell vitality of opi3∆ at both temperatures although the decrease at 13 °C was more marked. Copyright © 2012 John Wiley & Sons, Ltd.

Description: ABSTRACT The yeast Yarrowia lipolytica presents specific physiological, metabolic and genomic characteristics, which differentiate it from the model yeast Saccharomyces cerevisiae . Those properties led several research groups to use this yeast as a model for basic knowledge. Thanks to the development of advanced genetic tools and of -omic approaches, significant progress was achieved in the understanding of specific biological processes. This review, after a short presentation of this model yeast, will briefly highlight the different use of Y. lipolytica for basic knowledge and the advantages gained by exploiting this non conventional yeast. Future perspectives in employing this yeast for basic knowledge in the field of RNA splicing and genome evolution, and for the study of lipid metabolism will also be discussed. Copyright © 2012 John Wiley & Sons, Ltd.

Description: Three RNA polymerases coexist in the ribosomal DNA of S. cerevisiae . RNAP-I transcribes the 35S rRNA , RNAP-III transcribes the 5S rRNA , and RNAP-II is found in both intergenic non-coding regions. Previously, we demonstrated that RNAP-II molecules bound to the intergenic non-coding regions (IGS) of the ribosomal locus are mainly found in a stalled conformation, and the stalled polymerase mediates chromatin interactions, which isolate RNAP-I from the RNAP-III transcriptional domain. Besides, RNAP-II transcribes both IGS regions at low levels using different cryptic promoters. This report demonstrates that RNAP-II also transcribes two sequences located in the 5′- and 3′-ends of the 35S rRNA gene that overlap with the sequences of the 35S rRNA precursor transcribed by RNAP-I. The sequence located at the promoter region of RNAP-I, called p-RNA transcript, binds to the transcription termination-related protein, Reb1p. While the T-RNA sequence, located in the termination sites of RNAP-I gene, contains the stem-loop recognised by Rtn1p, which is necessary for proper termination of RNAP-I. Because of their location, these small RNAs may play a key role in the initiation and termination of RNAP-I transcription. To correctly synthesise proteins, eukaryotic cells may retain a mechanism that connects the three main polymerases. This report suggests that cryptic transcription by RNAP-II may be required for normal transcription by RNAP-I in the ribosomal locus of S. cerevisiae . Copyright © 2012 John Wiley & Sons, Ltd.