Isolation of quiescent and nonquiescent cells from yeast stationary-phase cultures

Quiescence is the most common and, arguably, most poorly understood cell cycle state. This is in part because pure populations of quiescent cells are typically difficult to isolate. We report the isolation and characterization of quiescent and nonquiescent cells from stationary-phase (SP) yeast cultures by density-gradient centrifugation. Quiescent cells are dense, unbudded daughter cells formed after glucose exhaustion. They synchronously reenter the mitotic cell cycle, suggesting that they are in a G(0) state. Nonquiescent cells are less dense, heterogeneous, and composed of replicatively older, asynchronous cells that rapidly lose the ability to reproduce. Microscopic and flow cytometric analysis revealed that nonquiescent cells accumulate more reactive oxygen species than quiescent cells, and over 21 d, about half exhibit signs of apoptosis and necrosis. The ability to isolate both quiescent and nonquiescent yeast cells from SP cultures provides a novel, tractable experimental system for studies of quiescence, chronological and replicative aging, apoptosis, and the cell cycle.     

Diarylacylhydrazones: Clostridium-selective antibacterials with activity against stationary-phase cells

Current antibiotics for treating Clostridium difficile infections (CDI), that is, metronidazole, vancomycin and more recently fidaxomicin, are mostly effective but treatment failure and disease relapse remain as significant clinical problems. The shortcomings of these agents are attributed to their low selectivity for C. difficile over normal gut microflora and their ineffectiveness against C. difficile spores. This Letter reports that certain diarylacylhydrazones identified during a high-throughput screening/counter-screening campaign show selective activity against two Clostridium species (C. difficile and Clostridium perfringens) over common gut commensals. Representative examples are shown to possess activity similar to vancomycin against clinical C. difficile strains and to kill stationary-phase C. difficile cells, which are responsible for spore production. Structure–activity relationships with additional synthesised analogues suggested a protonophoric mechanism may play a role in the observed activity/selectivity and this was supported by the well-known protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) showing selective anti-Clostridium effects and activity similar to diarylacylhydrazones against stationary-phase C. difficile cells. Two diarylacylhydrazones were shown to be non-toxic towards human FaDu and Hep G2 cells indicating that further studies with the class are warranted towards new drugs for CDI.     

Consequences of cytochrome c oxidase assembly defects for the yeast stationary phase

The assembly of cytochrome c oxidase (COX) is essential for a functional mitochondrial respiratory chain, although the consequences of a loss of assembled COX at yeast stationary phase, an excellent model for terminally differentiated cells in humans, remain largely unexamined. In this study, we show that a wild-type respiratory competent yeast strain at stationary phase is characterized by a decreased oxidative capacity, as seen by a reduction in the amount of assembled COX and by a decrease in protein levels of several COX assembly factors. In contrast, loss of assembled COX results in the decreased abundance of many mitochondrial proteins at stationary phase, which is likely due to decreased membrane potential and changes in mitophagy. In addition to an altered mitochondrial proteome, COX assembly mutants display unexpected changes in markers of cellular oxidative stress at stationary phase. Our results suggest that mitochondria may not be a major source of reactive oxygen species at stationary phase in cells lacking an intact respiratory chain.     

mRNA translation in yeast during entry into stationary phase

The expression of some Saccharomyces cerevisiae genes is induced as cells enter stationary phase. Their mRNAs are translated during a period in the growth cycle when the translational apparatus is relatively inert, thereby raising the possibility that these mRNAs compete effectively for a limiting pool of translation factors. To test this idea, the translation of mRNAs carrying different 5′-leaders was compared during exponential growth and after entry into stationary phase upon glucose starvation. Closely related sets of lacZ mRNAs, carrying 5′-leaders from the PYK1, PGK1, RpL3, Rp29, HSP12, HSP26 or THI4 mRNAs, were studied. These mRNAs displayed differing translational efficiencies during exponential growth, but their relative translatabilities were not significantly affected by entry into stationary phase, indicating that they compete just as effectively under these conditions. Polysome analysis revealed that the wild-type PYK1, ACT1 and HSP26 mRNAs are all translated efficiently during stationary phase, when the translational apparatus is relatively inert. Also, significant levels of the translation initiation factors eIF-2α, eIF-4E and eIF-4A were maintained during the growth cycle. These data are consistent with the idea that, while translational activity decreases dramatically during entry into stationary phase, yeast cells maintain excess translational capacity under these conditions. Received: 31 March 1998 / Accepted: 4 May 1998     

Lon protease promotes survival of <Emphasis Type="Italic">Escherichia coli</Emphasis> during anaerobic glucose starvation

In Escherichia coli, Lon is an ATP-dependent protease which degrades misfolded proteins and certain rapidly-degraded regulatory proteins. Given that oxidatively damaged proteins are generally degraded rather than repaired, we anticipated that Lon deficient cells would exhibit decreased viability during aerobic, but not anaerobic, carbon starvation. We found that the opposite actually occurs. Wild-type and Lon deficient cells survived equally well under aerobic conditions, but Lon deficient cells died more rapidly than the wild-type under anaerobiosis. Aerobic induction of the Clp family of ATP-dependent proteases could explain these results, but direct quantitation of Clp protein established that its level was not affected by Lon deficiency and overexpression of Clp did not rescue the cells under anaerobic conditions. We conclude that the Lon protease supports survival during anaerobic carbon starvation by a mechanism which does not depend on Clp. Shen Luo and Megan McNeill contributed equally to this research.     

Oxygen responses of the free-living anaerobic amoeboflagellate Psalteriomonas lanterna

Abstract The effects of O₂ on growth of the anaerobic amoeboflagellate Psalteriomonas lanterna were studied. The organism tolerates low oxygen tensions (about 1% O₂ atm. sat.) and under these conditions growth was stimulated in mixed populations. Catalase could not be found in the cells, whereas superoxide dismutase was present. Addition of O₂ resulted in loss of the methanogenic endosymbionts and favoured the transformation to amoeba cells. Symbiont-free cells did not grow under anaerobic conditions probably due to the accumulation of H₂.     

Horizontal gene transfer promoted evolution of the ability to propagate under anaerobic conditions in yeasts

The ability to propagate under anaerobic conditions is an essential and unique trait of brewers or bakers yeast (Saccharomyces cervisiae). To understand the evolution of facultative anaerobiosis we studied the dependence of de novo pyrimidine biosynthesis, more precisely the fourth enzymic activity catalysed by dihydroorotate dehydrogenase (DHODase), on the enzymes of the respiratory chain in several yeast species. While the majority of yeasts possess a mitochondrial DHODase, Saccharomyces cerevisiae has a cytoplasmatic enzyme, whose activity is independent of the presence of oxygen. From the phylogenetic point of view, this enzyme is closely related to a bacterial DHODase from Lactococcus lactis. Here we show that S. kluyveri, which separated from the S. cerevisiae lineage more than 100 million years ago, represents an evolutionary intermediate, having both cytoplasmic and mitochondrial DHODases. We show that these two S. kluyveri enzymes, and their coding genes, differ in their dependence on the presence of oxygen. Only the cytoplasmic DHODase promotes growth in the absence of oxygen. Apparently a Saccharomyces yeast progenitor which had a eukaryotic-like mitochondrial DHODase acquired a bacterial gene for DHODase, which subsequently allowed cell growth gradually to become independent of oxygen.Communicated by C. P. Hollenberg     

Isolation and characterization of polyphosphates from the yeast cell surface

When cells of Saccharomyces fragilis are subjected to osmotic shock, they release a limited amount of inorganic polyphosphate into the medium, which represents about 10% of the total cellular content. The osmotic shock procedure causes no substantial membrane damage, as judged from the unimpaired cell viability, limited K⁺ leakage and low percentage of stained cells. It is therefore suggested that this polyphosphate fraction is localized outside the plasma membrane. The released polyphosphate fraction differs from the remaining cellular polyphosphates in two respects: the mean chain length of the shock-sensitive fraction is significantly higher than that of the total cellular polyphosphates and its metabolic turnover rate, subsequent to pulsing with [³²P]orthophosphate is much lower compared to the rest of the cellular polyphosphate. Incubation of intact cells with the anion exchange resin Dowex AG 1-X4 results in the release of high molecular weight polyphosphates. These results suggest that the osmotic shock-sensitive polyphosphate fraction has specific characteristics in both its cellular localization and metabolism.     

Isolation and characterization of ethanol tolerant yeast strains

Yeast strains are commonly associated with sugar rich environments. Various fruit samples were selected as source for isolating yeast cells. The isolated cultures were identified at Genus level by colony morphology, biochemical characteristics and cell morphological characters. An attempt has been made to check the viability of yeast cells under different concentrations of ethanol. Ethanol tolerance of each strain was studied by allowing the yeast to grow in liquid YEPD (Yeast Extract Peptone Dextrose) medium having different concentrations of ethanol. A total of fifteen yeast strains isolated from different samples were used for the study. Seven strains of Saccharomyces cerevisiae obtained from different fruit sources were screened for ethanol tolerance. The results obtained in this study show a range of tolerance levels between 7%-12% in all the stains. Further, the cluster analysis based on 22 RAPD (Random Amplified polymorphic DNA) bands revealed polymorphisms in these seven Saccharomyces strains.     

Solvent treatment for β-d-galactosidase release from yeast cells

A method is presented for the release of β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) from yeast cells. Enzyme release is attained by mixing yeast cells with concentrated solvents (20 to 95%) and subsequently suspending and agitating the cells in buffer. Many solvents, including isopropanol, ethanol and methanol, were found to be effective. Enzyme release into buffer was relatively slow: 10–20 h was required for maximum yields. The release of protease and β-d-galactosidase was monitored. β-d-Galactosidase solubilization was achieved in high yield: 90% of the intracellular enzyme was released into the buffer. Because this method exhibits resistance to yield loss due to microbial degradation and is not sensitive to small changes in solvent in buffer concentration or treatment time, it is particularly suited to industrial-scale enzyme recoveries.     

Dielectrophoretic properties of yeast cells dividing by budding and by transversal fission

The dielectrophoretic behaviour of yeast cells dividing by budding or by transversal fission was analyzed. The results obtained show that the dielectrophoretic yield is a linear function of alternating voltage, cell concentration and the square root of the time of collection in all the species assayed. Dependence of the rate of collection on the frequency of the voltage applied (between 0.2 and 5 MHz) was also found. This behaviour is similar in the three microorganisms studied. The scale factor correlating the frequency spectrum for Saccharomyces cerevisiae and Saccharomycopsis lipolytica is proportional to cell size. However, these results can not be extended to Schizosaccharomyces pombe. A relationship between the dielectrophoretic yield and the age of the culture and the consumption of glucose has been established for the three yeast strains. Dielectrophoresis also permits the differentiation between viable and non-viable cells.     

The interaction of nitrite with photosynthetic electron transport under anaerobic conditions

Chlorella cells were examined in a modulated oxygen polarograph under aerobic and anaerobic conditions. At light intensities below about 600 ergs · cm^?2 · s^?1 of 650 nm light, the oxygen yield and phase lag are lower under anaerobic conditions. Addition of 25 mM sodium nitrite increases both these parameters to values close to those found in the presence of oxygen. It is proposed that nitrite is reduced by Photosystem I thus diverting electrons from the cyclic electron transport pathway. The intersystem electron transport chain becomes more oxidized and this suppresses a backflow of electrons to the oxidizing side of Photosystem II, hence increasing the oxygen yield and the phase lag. The removal of oxygen from the bathing medium also alters the response of dark adapted _Chlorella to a series of saturating light flashes. In terms of the Kok model of Photosystem II (Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475) there is a large increase in the parameter α. Addition of nitrite reverses this change and virtually restores the response seen in the presence of oxygen. The deactivation of the S₂ state is greatly speeded up in the absence of oxygen but the addition of nitrite again reverses this.     

Anaerobiosis induces complex changes in sterol esterification pattern in the yeast Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae is auxotrophic for ergosterol in the absence of oxygen. We showed that complex changes in esterification of exogenously supplied sterols were also induced by anaerobiosis. Utilization of oleic acid for sterol esterification was significantly impaired in anaerobic cells. Furthermore, anaerobic cells fed different sterols exhibited striking variation in esterification efficiency (high levels of sterol esters for cholesterol and sitosterol, low levels for ergosterol, lanosterol or stigmasterol). Relative activities of two yeast acylCoA:sterol acyltransferases (Are1p and Are2p) changed in response to anaerobiosis: while Are2p was dominant under aerobic conditions, Are1p provided the major activity in the absence of oxygen. Our results indicate that sterol esters may fulfil different roles in aerobic and anaerobic cells.     

Non-homologous end joining dependency of gamma-irradiation-induced adaptive frameshift mutation formation in cell cycle-arrested yeast cells

There is a strong selective pressure favoring adaptive mutations which relieve proliferation-limiting adverse living conditions. Due to their importance for evolution and pathogenesis, we are interested in the mechanisms responsible for the formation of such adaptive, gain-of-fitness mutations in stationary-phase cells. During previous studies on the occurrence of spontaneous reversions of an auxotrophy-causing frameshift allele in the yeast Saccharomyces cerevisiae, we noticed that about 50% of the adaptive reversions depended on a functional non-homologous end joining (NHEJ) pathway of DNA double-strand break (DSB) repair. Here, we show that the occasional NHEJ component Pol4, which is the yeast ortholog of mammalian DNA polymerase lambda, is not required for adaptive mutagenesis. An artificially imposed excess of DSBs by gamma-irradiation resulted in a dramatic increase in the incidence of adaptive, cell cycle arrest-releasing frameshift reversions. By the use of DNA ligase IV-deficient strains we detected that the majority of the gamma-induced adaptive mutations were also dependent on a functional NHEJ pathway. This suggests that the same mutagenic NHEJ mechanism acts on spontaneously arising as well as on ionizing radiation-induced DSBs. Inaccuracy of the NHEJ repair pathway may extensively contribute to the incidence of frameshift mutations in resting (non-dividing) eukaryotic cells, and thus act as a driving force in tumor development.