Description: The Fps1p is an aquaglyceroporin important for turgor regulation of Saccharomyces cerevisiae . Previously we reported the involvement of Fps1p in the yeast killing action of killer toxin HM-1. The fps1 cells showed high HM-1-resistant phenotype in hypotonic medium, and HM-1-susceptible phenotype in hypertonic medium. This osmotic dependency in HM-1-susceptibility was similar to those observed in Congo Red, but different from those observed in other cell wall disturbing agents. These results indicate that HM-1 exerts the fungicidal activity mainly by binding and inserting into yeast cell wall structure, rather than inhibiting 1,3- beta -glucan synthase. Next we determined that HM-1-susceptibility and diphospho-MAP kinase inductions in S. cerevisiae . In wild-type cell, expressions of diphospho-Hog1p and -Slt2p, and mRNA transcription of CWP1 and HOR2 were induced within 1 h after an addition of HM-1. The ssk1 and pbs2 cells, but not sho1 and hkr1 cells, showed HM-1-sensitive phenotypes and lacked in inductions of phospho-Hog1p in response to HM-1. The mid2, rom2 and bck1 cells showed HM-1-sensitive phenotypes, and decreased inductions of phospho-Slt2p in response to HM-1. From these results, we postulated that Sln1-Ypd1-Ssk1-branch of high osmolality glycerol (HOG) -pathway and plasma membrane sensors of cell wall integrity (CWI) -pathway detect cell wall stresses caused by HM-1. And we suggested that activations of both HOG- and CWI-pathways have an important role in the adaptive response to HM-1 toxicity. Copyright © 2012 John Wiley & Sons, Ltd.

Description: Ideal reporter genes for temporal transcription programs have short half-lives that restrict their detection to the window in which their transcripts are present and translated. In an effort to meet this criterion for reporters of transcription in individual living cells, we adapted the ubiquitin fusion strategy for programmable N-end rule degradation to generate an N-degron version of green fluorescent protein (GFP) with a half-life of ~7 min. The GFP variant we used here (designated GFP*) has excellent fluorescence brightness and maturation properties, which make the destabilized reporter well suited for tracking the induction and attenuation kinetics of gene expression in living cells. These attributes are illustrated by its ability to track galactose and pheromone induced transcription in S. cerevisiae . We further show that the fluorescence measurements using the short-lived N-degron GFP* reporter gene accurately predict the transient mRNA profile of the prototypical pheromone induced FUS1 gene. Copyright © 2012 John Wiley & Sons, Ltd.

Description: ABSTRACT Methanol-inducible gene promoters in methanol-utilizing yeasts are used in high level heterologous gene expression systems. Generally, expression of methanol-inducible genes is completely repressed by the presence of glucose. In this study, we identified the MIG1 gene in Candida boidinii , which encodes a homolog of the glucose repressor Mig1p of Saccharomyces cerevisiae . Disruption of the CbMIG1 gene had no growth effect on various carbon sources. Activation of the methanol-inducible AOD1 gene, which encodes alcohol oxidase, was increased in the early stage of methanol induction when cells of the CbMIG1 -disrupted strain were transferred from glucose medium to methanol medium. Furthermore, CbMig1p tagged with yellow fluorescent protein was primarily localized in the nucleus of glucose-grown cells, but was diffuse in the cytosol of methanol-grown cells. This cytosolic diffusion in methanol-grown cells occurred in a CbMsn5p-dependent manner. These results suggest that CbMig1p is a negative regulator involved in regulation of methanol-inducible gene expression in C. boidinii . Copyright © 2012 John Wiley & Sons, Ltd.

Description: ABSTRACT Although fragmentation of DNA has been observed in cells undergoing freezing procedures, a mutagenic effect of subzero temperature treatment has not been proved by induction and isolation of mutants in nuclear DNA (nDNA). We supply in this communication evidence for mutagenicity of freezing on nDNA of Saccharomyces cerevisiae cells. In absence of cryoprotectors, cooling for 2 h at +4 °C and freezing for 1 h at −10 °C and 16 h at −20 °C with a cooling rate of 3 °C/min resulted in induction of frame-shift and reverse mutations in microsatellite and coding regions of nDNA. The subzero temperature exposure has also a strong recombinogenic effect evidenced by induction of gene–conversion and crossingover events. Freezing induces mutations and enhances recombination with a frequency equal or higher than that of methylmethanesulfonate at comparable survival rates. The signals for the appearance of nDNA lesions induced by freezing are detected and transduced by the DNA damage pathway. Extracellular cryoprotectors did not prevent the mutagenic effect of freezing while accumulation of trehalose inside cells reduced nDNA cryodamages. Freezing of cells is accompanied with generation of high ROS levels and the oxidative stress raised during the freeze/thaw process is the most likely reason for the DNA damaging effect. Experiments with mitochondrial rho - mutants or scavengers of ROS indicated that mutagenic and recombinogenic effects of subzero temperatures can be decreased but not eliminated by reduction of ROS level. The complete protection against cryodamages in nDNA required simultaneous usage of intracellular cryoprotector and ROS scavenger during the freeze/thaw process. Copyright © 2012 John Wiley & Sons, Ltd.

Description: ABSTRACT Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent deep mycosis in Latin America. The production of eicosanoids during fungal infection has been associated with the biology of these microorganisms and modulation of host immune response. The aim of our study was to evaluate whether P. brasiliensis strains with high or low virulence produce leukotriene B4 (LTB 4 ), using endogenous and/or exogenous sources of arachidonic acid (AA). Moreover, we assessed whether this fungus might use the same metabolic pathway, described for mamalian cells, that involves the lipoxygenase (LOX) enzyme. The association between the production of this eicosanoid and fungus survival and growth was also evaluated. Our results showed that P. brasiliensis , irrespective of its virulence, produces high levels of LTB 4 using endogenous AA. In addition, in cultures treated with exogenous AA, LTB 4 levels were significantly higher, showing that this fungus also uses exogenous sources of fatty acids. Treatment with MK886, which blocks the activity of lipoxygenase, by inhibiting FLAP (five-lipoxygenase-activating protein) or with NDGA (Nordihydroguaiaretic acid), a non-selective lipoxygenase inhibitor, resulted in a significant reduction in LTB 4 levels, indicating that the fungus produces this eicosanoid by using the LOX pathway or an enzyme with biochemically similar function. The significant reduction in viability detected in cultures treated with these inhibitors is, however, restored by adding exogenous LTB 4 , confirming the role of this eicosanoid in fungus survival. Moreover, the addition of LTB 4 to cultures capable of producing LTs induces fungal growth. These results provide a foundation for additional studies on the contributions of LTB 4 in P. brasiliensis virulence. Copyright © 2012 John Wiley & Sons, Ltd.

Description: ABSTRACT This study was undertaken to evaluate the apparent viscosity within the vacuoles of the single Saccharomyces cerevisiae cells by steady-state fluorescence anisotropy measurements of quinacrine using wide-field fluorescence polarization microscopy combined with computer image analysis. Quinacrine was shown to be rather specifically accumulated within the vacuoles of the cells. This accumulation was effectively reversed by the ATP depletion of the cells with no detectable binding of the dye within the vacuoles. Quinacrine fluorescence anisotropy in the sucrose solutions of various viscosities obeyed the Perrin equation. The fluorescence anisotropy of quinacrine was measured in the vacuoles of 39 cells. From cell to cell, this parameter changed in a range from 0.032 up to 0.086. Using the Perrin plot as a calibration curve, apparent viscosity values of the vacuolar milieu were calculated for each cell. The population of the cells studied was heterogeneous with regard to the vacuolar viscosity, which was in the range from 3.5 ± 0.4 cP to 14.06 ± 0,64 cP. There was a characteristic distribution of the frequencies of the cells with the apparent viscosities within certain limits, and the cells with the viscosity values in the range from 5 to 6 cP were the most frequent. No relation between the size of the vacuoles and their apparent viscosity was found. Copyright © 2012 John Wiley & Sons, Ltd.

Description: ABSTRACT Nep1 methylates the hypermodified ψ1191 base of 18 S rRNA and has an additional essential function during ribosome biogenesis. It is strongly conserved in eukaryotes and a point mutation causes the human Bowen-Conradi syndrome. To identify ∆ nep1 specific genetic interactions viable deletions were screened genome wide (SGA). Due to its essential function we used for the first time, query strain (Δ nep1 ) with two additive suppressor conditions (mc RPS19B, nop6-1 ). Nep1 interacting genes correspond to ribosome biogenesis ( RPS18A , RPS18B, RRP8, EFG1, UTP30 ), to ribosome quality control ( UBP3 , BRE5 , UBP6 ) and to ribosome functional control ( DOM34 , no-go decay). Deletions in ribosome quality and functional control genes were synthetically sick with Δ nep1 . They cope with malfunctions and the respective deletions strengthen the Δ nep1 growth deficiency. Except for Δ rps18b , deletions in the identified ribosome biogenesis genes were synthetically lethal with Δ nep1 . While the synthetic lethalities of ∆ rrp8 and ∆ efg1 may result from additive defects, the ∆ utp30 deletion seems to be in close functional relationship. The ∆ utp30 deletion itself has no phenotype but it enforced all nep1-1 ts mutant phenotypes. Furthermore, its overexpression partially restored the nep1-1 ts growth deficiency. Our genetic and biochemical data suggest that Utp30 and Nep1 act together during pre-ribosomal complex formation and along with Rps18 provide the surface for the Rps19 assembly to the 90 S pre-ribosome. Copyright © 2012 John Wiley & Sons, Ltd.

Description: In this study, a previously developed mini-bioreactor, the Biocurve, was used to identify an informative stimulus–response experiment. The identified stimulus–response experiment was a modest 50% shift-up in glucose uptake rate ( q GLC) that unexpectedly resulted in a disproportionate transient metabolic response. The 50% shift-up in q GLC in the Biocurve resulted in a near tripling of the online measured oxygen uptake ( q O 2 ) and carbon dioxide production ( q CO 2 ) rates, suggesting a considerable mobilization of glycogen and trehalose. The 50% shift-up in q GLC was subsequently studied in detail in a conventional bioreactor (4 l working volume), which confirmed the results obtained with the Biocurve. Especially relevant is the observation that the 50% increase in glucose uptake rate led to a three-fold increase in glycolytic flux, due to mobilization of storage materials. This explains the unexpected ethanol and acetate secretion after the shift-up, in spite of the fact that after the shift-up the q GLC was far less than the critical value. Moreover, these results show that the correct in vivo fluxes in glucose pulse experiments cannot be obtained from the uptake and secretion rates only. Instead, the storage fluxes must also be accurately quantified. Finally, we speculate on the possible role that the transient increase in dissolved CO 2 immediately after the 50% shift-up in q GLC could have played a part in triggering glycogen and trehalose mobilization. Copyright © 2012 John Wiley & Sons, Ltd.

Description: This paper developed a novel strategy to improve the fluorescence in situ hybridization–flow cytometry (FISH–FCM) enumeration performance in filamentous yeast species in activated sludge by snailase partial digestion to fully disaggregate filamentous yeast chains into single cells. A 2 h 2% snailase partial digestion liberated more rod-shaped yeast single cells from intertwined filamentous yeast samples than did sonication disaggregation, based on an optical microscopic observation and the forward-light-scatter frequency histogram of FCM analysis. However, adding snailase resulted in a fluorescence-quenching phenomenon of the hybridized filamentous yeast cells, which was minimized by lowering the snailase concentration. An approximately 3 h 0.5% snailase partial digestion conducted between sonication and hybridization significantly improved the FISH–FCM enumeration performance for filamentous yeast species by 37%. The results presented here will facilitate the rapid detection, identification and exact enumeration of specific filamentous fungal species in environmental samples. Copyright © 2012 John Wiley & Sons, Ltd.

Description: The bottom-fermenting lager yeast Saccharomyces pastorianus has been proposed to be allotetraploid containing two S. cerevisiae (Sc)-type and two S. bayanus (Sb)-type chromosomes. This chromosomal constitution likely explains why recessive mutants of S. pastorianus have not been previously reported. Here, we describe construction of a ura3 deletion strain derived from the lager strain Weihenstephan34/70 by targeted transformation and subsequent loss of heterozygosity (LOH). Initially, deletion constructs of the Sc and Sb types of URA3 were constructed in laboratory yeast strains in which a TDH3p-hygro allele conferring hygromycin B resistance replaced Sc URA3 and a KanMX cassette conferring G-418 resistance replaced Sb URA3 . The lager strain was then transformed with these constructs to yield a heterozygous URA3 disruptant, (Sc URA3 + /Sc ura3Δ::TDH3p-hygro , Sb URA3 + /Sb ura3Δ::KanMX ) which was plated on 5-fluoroorotic acid (5-FOA) plates to generate the desired Ura - homozygous disruptant, (Sc ura3Δ::TDH3p-hygro /Sc ura3Δ::TDH3p-hygro Sb ura3Δ::KanMX /Sb ura3Δ::KanMX ) through LOH. This ura3 deletion strain was then used to construct a bottom-fermenting yeast transformant overexpressing ATF1 that encodes an enzyme that produces acetate esters. The ATF1 -overexpressing transformant produced significantly more acetate esters than the parent strain. The constructed ura3∆ lager strain will be a useful host for constructing strains of relevance to brewing. Copyright © 2012 John Wiley & Sons, Ltd.