Simon V. Avery

Microbial cell individuality and the underlying sources of heterogeneity

Single cells in genetically homogeneous microbial cultures exhibit marked phenotypic individuality, a biological phenomenon that is considered to bolster the fitness of populations. Major phenotypes that are characterized by heterogeneity span the breadth of microbiology, in fields ranging from pathogenicity to ecology. The cell cycle, cell ageing and epigenetic regulation are proven drivers of heterogeneity in several of the best-known phenotypic examples. However, the full contribution of factors such as stochastic gene expression is yet to be realized.

Molecular targets of oxidative stress

Aerobic life requires organisms to resist the damaging effects of ROS (reactive oxygen species), particularly during stress. Extensive research has established a detailed picture of how cells respond to oxidative stress. Attention is now focusing on identifying the key molecular targets of ROS, which cause killing when resistance is overwhelmed. Experimental criteria used to establish such targets have differing merits. Depending on the nature of the stress, ROS cause loss of essential cellular functions or gain of toxic functions. Essential targets on which life pivots during ROS stress include membrane lipid integrity and activity of ROS-susceptible proteins, including proteins required for faithful translation of mRNA. Protein oxidation also triggers accumulation of toxic protein aggregates or induction of apoptotic cell death. This burgeoning understanding of the principal ROS targets will offer new possibilities for therapy of ROS related diseases.

Metal toxicity in yeasts and the role of oxidative stress.

Publisher Summary Metal toxicity continues to be a major problem at several levels, and there is a pressing need for the deleterious effects of metals at the cellular and molecular level. This chapter discusses a major role of free radical generation in the toxicities of many metals. A number of mechanisms have been proposed to underlie metal induced Reactive Oxygen Species (ROS) generation, and these mechanisms tend to differ between metals according principally to whether they are redox active or inactive. Several yeast groups are currently focusing on metal homeostasis, which may have steered some attention away from exploiting yeasts for direct studies of metal toxicity. The extent of the contribution to whole-cell metal toxicity made by damage to each potential target has not been determined, and this is not helped by the fact that almost all studies have tended to focus on one type of target and/or whole cells exclusively. The evidence to date indicates that each of the major cellular macromolecules (lipids, proteins, and nucleic acid) can be a target for metal toxicity. The relative importance of membrane damage in determining the metal sensitivities of different yeasts may depend on the organisms lipid compositions, specifically their polyunsaturated fatty acid contents.

Destabilized green fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry.

Green fluorescent protein (GFP) has many advantages as a reporter molecule, but its stability makes it unsuitable for monitoring dynamic changes in gene expression, among other applications. Destabilized GFPs have been developed for bacterial and mammalian systems to counter this problem. Here, we extend such advances to the yeast model. We fused the PEST‐rich 178 carboxyl‐terminal residues of the G1 cyclin Cln2 to the C terminus of yEGFP3 (a yeast‐ and FACS‐optimized GFP variant), creating yEGFP3‐Cln2PEST. We tested the hybrid protein after integrating modules harbouring the yEGFP3 or yEGFP3–CLN2PEST ORFs into the Saccharomyces cerevisiae genome. yEGFP3– Cln2PEST had a markedly shorter half‐life (t½) than yEGFP3; inhibition of protein synthesis with cycloheximide lead to a rapid decline in GFP content and fluorescence (t½ ∼30 min) in cells expressing yEGFP3–Cln2PEST, whereas these parameters were quite stable in yEGFP3‐expressing cells (t½ ∼7 h). We placed yEGFP3–CLN2PEST under the control of the CUP1 promoter, which is induced only transiently by copper. This transience was readily discernible with yEGFP3–Cln2PEST, whereas yEGFP3 reported only on CUP1 switch‐on, albeit more slowly than yEGFP3–Cln2PEST. Cell cycle‐regulated transcriptional activation/inactivation of the CLN2 promoter was also discernible with yEGFP3– Cln2PEST, using cultures that were previously synchronized with nocodazole. In comparison to CLN2, expression from the ACT1 promoter was stable after release from nocodazole. We also applied a novel flow‐cytometric technique for cell cycle analysis with asynchronous cultures. The marked periodicities of CLN2 and CLB2 (mitotic cyclin) transcription were readily evident from cellular yEGFP3–Cln2PEST levels with this non‐perturbing approach. The results represent the first reported successful destabilization of a yeast–GFP. This new construct expands the range of GFP applications open to yeast workers. Copyright

The Yeast Glutaredoxins Are Active as Glutathione Peroxidases

The yeast Saccharomyces cerevisiaecontains two glutaredoxins, encoded by GRX1 andGRX2, which are active as glutathione-dependent oxidoreductases. Our studies show that changes in the levels of glutaredoxins affect the resistance of yeast cells to oxidative stress induced by hydroperoxides. Elevating the gene dosage ofGRX1 or GRX2 increases resistance to hydroperoxides including hydrogen peroxide, tert-butyl hydroperoxide and cumene hydroperoxide. The glutaredoxin-mediated resistance to hydroperoxides is dependent on the presence of an intact glutathione system, but does not require the activity of phospholipid hydroperoxide glutathione peroxidases (GPX1–3). Rather, the mechanism appears to be mediated via glutathione conjugation and removal from the cell because it is absent in strains lacking glutathione-S-transferases (GTT1,GTT2) or the GS-X pump (YCF1). We show that the yeast glutaredoxins can directly reduce hydroperoxides in a catalytic manner, using reducing power provided by NADPH, GSH, and glutathione reductase. With cumene hydroperoxide, high pressure liquid chromatography analysis confirmed the formation of the corresponding cumyl alcohol. We propose a model in which the glutathione peroxidase activity of glutaredoxins converts hydroperoxides to their corresponding alcohols; these can then be conjugated to GSH by glutathione-S-transferases and transported into the vacuole by Ycf1.

Phenotypic heterogeneity: differential stress resistance among individual cells of the yeast Saccharomyces cerevisiae.

Phenotypic heterogeneity describes non-genetic variation that exists between individual cells within isogenic populations. Such heterogeneity is readily evident in the differential sensitivity to stress of genetically identical cells and can be fundamental to the fitness and persistence of an organism. Consequently, phenotypic heterogeneity is currently receiving increased attention from the scientific community. Here, we present the first review of this important subject, with an account that focuses on the factors that contribute to cell-to-cell diversity in the model eukaryotic micro-organism Saccharomyces cerevisiae. Some of the key parameters that drive non-genetic heterogeneity include cell cycle progression, cell ageing, mitochondrial activity, epigenetic regulation and potentially also stochastic variation. The relative significance of these and other parameters in generating heterogeneity is discussed. We refer in particular to differential stress sensitivity in S. cerevisiae, although other relevant phenomena such as phenotypic switching in Candida spp. are also addressed.

Copper-induced oxidative stress in Saccharomyces cerevisiae targets enzymes of the glycolytic pathway

Increased cellular levels of reactive oxygen species are known to arise during exposure of organisms to elevated metal concentrations, but the consequences for cells in the context of metal toxicity are poorly characterized. Using two‐dimensional gel electrophoresis, combined with immunodetection of protein carbonyls, we report here that exposure of the yeast Saccharomyces cerevisiae to copper causes a marked increase in cellular protein carbonyl levels, indicative of oxidative protein damage. The response was time dependent, with total‐protein oxidation peaking approximately 15 min after the onset of copper treatment. Moreover, this oxidative damage was not evenly distributed among the expressed proteins of the cell. Rather, in a similar manner to peroxide‐induced oxidative stress, copper‐dependent protein carbonylation appeared to target glycolytic pathway and related enzymes, as well as heat shock proteins. Oxidative targeting of these and other enzymes was isoform‐specific and, in most cases, was also associated with a decline in the proteins’ relative abundance. Our results are consistent with a model in which copper‐induced oxidative stress disables the flow of carbon through the preferred glycolytic pathway, and promotes the production of glucose‐equivalents within the pentose phosphate pathway. Such re‐routing of the metabolic flux may serve as a rapid‐response mechanism to help cells counter the damaging effects of copper‐induced oxidative stress.

Caesium accumulation and interactions with other monovalent cations in the cyanobacterium Synechocystis PCC 6803

SUMMARY: Growth of Synechocystis PCC 6803 in BG-11 medium supplemented with 1 mM-CsCl resulted in intracellular accumulation of Cs+ to a final level of approximately 510 nmol (109 cells)-1 after incubation for 10 d. The doubling time was increased by 64% and the final cell yield was decreased by 70% during growth in the presence of Cs+ as compared to growth in control BG-11 medium. When the total monovalent cation concentration of the medium was doubled by adding either K+ or Na+, levels of accumulated Cs+ were decreased by approximately 50% to 220 and 270 nmol (109 cells)-1, respectively, after 28 d with little inhibition of growth being apparent. Short-term experiments revealed that extracellular K+ and Na+ inhibited Cs+ accumulation to a similar extent, with 90% inhibition of Cs+ accumulation occurring at the highest concentrations used (50 mM-K+ or Na+; 1 mM-Cs+). In all experiments, Cs+ accumulation resulted in a reduction in intracellular K+, except when cells were grown in K+-depleted medium, although a stoichiometric relationship was not apparent, the amount of Cs+ accumulated generally being greater than the amount of K+ released. Cs+ accumulation had no discernible effect on intracellular Na+. When K+, Na+, Rb+, Li+ or Tl+ were supplied at equimolar (1 mM) concentrations to Cs+, only Tl+ significantly reduced Cs+ accumulation. However, an approximately 50% inhibition of Cs+ accumulation resulted when concentrations of K+, Na+, Rb+ or Li+ were increased to 10 mM, which suggests that Cs+ may have a higher affinity for the monovalent cation transport system than K+, Rb+ and TI+ also caused a decrease in intracellular K+, whereas Na+ and Li+ stimulated K+ uptake. Cs+ accumulation was dependent on the external Cs+ concentration and showed a linear relationship to external Cs+ concentrations≤2 mM over 12 h incubation. However, prolonged incubation in external Cs+ concentrations≥ 0·8 mM resulted in Cs+ release from the cells and after 48 h, similar amounts of Cs+ and K+ were present in cells incubated at these higher concentrations. Cs+ accumulation was energy- and pH-dependent. Incubation in the light at 4 °C, or in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), or at 22 °C in the dark resulted in decreased Cs+ accumulation and decreased K+ release from the cells. Increased amounts of Cs+ were accumulated as the pH of the external medium was increased, with maximal accumulation [approximately 1330 nmol Cs+ (109 cells)-1 after 24 h incubation] occurring at pH 10, the highest pH value used. It is suggested that an important mechanism of Cs+ toxicity in Synechocystis PCC 6803 arises through replacement of cellular K+ by Cs+. The possible role of primary producers such as cyanobacteria in the mobilization of this radionuclide in aquatic habitats is discussed.

Glutathione and Gts1p drive beneficial variability in the cadmium resistances of individual yeast cells

Phenotypic heterogeneity among individual cells within isogenic populations is widely documented, but its consequences are not well understood. Here, cell‐to‐cell variation in the stress resistance of Saccharomyces cerevisiae, particularly to cadmium, was revealed to depend on the antioxidant glutathione. Heterogeneity was decreased strikingly in gsh1 mutants. Furthermore, cells sorted according to differing reduced‐glutathione (GSH) contents exhibited differing stress resistances. The vacuolar GSH‐conjugate pathway of detoxification was implicated in heterogeneous Cd resistance. Metabolic oscillations (ultradian rhythms) in yeast are known to modulate single‐cell redox and GSH status. Gts1p stabilizes these oscillations and was found to be required for heterogeneous Cd and hydrogen‐peroxide resistance, through the same pathway as Gsh1p. Expression of GTS1 from a constitutive tet‐regulated promoter suppressed oscillations and heterogeneity in GSH content, and resulted in decreased variation in stress resistance. This enabled manipulation of the degree of gene expression noise in cultures. It was shown that cells expressing Gts1p heterogeneously had a competitive advantage over more‐homogeneous cell populations (with the same mean Gts1p expression), under continuous and fluctuating stress conditions. The results establish a novel molecular mechanism for single‐cell heterogeneity, and demonstrate experimentally fitness advantages that depend on deterministic variation in gene expression within cell populations.

Modulation of Chaperone Gene Expression in Mutagenized Saccharomyces cerevisiae Strains Developed for Recombinant Human Albumin Production Results in Increased Production of Multiple Heterologous Proteins

ABSTRACT The yeast Saccharomyces cerevisiae has been successfully established as a commercially viable system for the production of recombinant proteins. Manipulation of chaperone gene expression has been utilized extensively to increase recombinant protein production from S. cerevisiae, focusing predominantly on the products of the protein disulfide isomerase gene PDI1 and the hsp70 gene KAR2. Here we show that the expression of the genes SIL1, LHS1, JEM1, and SCJ1, all of which are involved in regulating the ATPase cycle of Kar2p, is increased in a proprietary yeast strain, developed by several rounds of random mutagenesis and screening for increased production of recombinant human albumin (rHA). To establish whether this expression contributes to the enhanced-production phenotype, these genes were overexpressed both individually and in combination. The resultant strains showed significantly increased shake-flask production levels of rHA, granulocyte-macrophage colony-stimulating factor, and recombinant human transferrin.