K. Sigler

Oxidative stress in microorganisms--I. Microbial vs. higher cells--damage and defenses in relation to cell aging and death.

Oxidative stress in microbial cells shares many similarities with other cell types but it has its specific features which may differe in prokaryotic and eukaryotic cells. We survey here the properties and actions of primary sources of oxidative stress, the role of transition metals in oxidative stress and cell protective machinery of microbial cells, and compare them with analogous features of other cell types. Other features to be compared are the action of reactive oxygen species (ROS) on cell constituents, secondary lipid-or protein-based radicals and other stress products. Repair of oxidative injury by microorganisms and proteolytic removal of irreparable cell constituents are briefly described. Oxidative damage of aerobically growing microbial cells by endogenously formed ROS mostly does not induce changes similar to the aging of multiplying mammalian cells. Rapid growth of bacteria and yeast prevents accumulation of impaired macromolecules which are repaired, diluted or eliminated. During growth some simple fungi, such as yeast orPodospora spp., exhibit aging whose primary cause seems to be fragmentation of the nucleolus or impairment of mitochondrial DNA integrity. Yeast cell aging seems to be accelerated by endogenous oxidative stress. Unlike most growing microbial cells, stationaryphase cells gradually lose their viability because of a continuous oxidative stress, in spite of an increased synthesis of antioxidant enzymes. Unlike in most microorganisms, in plant and animal cells a severe oxidative stress induces a specific programmed death pathway-apoptosis. The scant data on the microbial death mechanisms induced by oxidative stress indicate that in bacteria cell death can result from activation of autolytic enzymes (similarly to the programmed mother-cell death at the end of bacillar sporulation). Yeast and other simple eukaryotes contain components of a proapoptotic pathway which are silent under normal conditions but can be activated by oxidative stress or by manifestation of mammalian death genes, such asbak orbax. Other aspects, such as regulation of oxidative-stress response, role of defense enzymes and their control, acquisition of stress tolerance, stress signaling and its role in stress response, as well as cross-talk between different stress factors, will be the subject of a subsequent review.

How microorganisms use hydrophobicity and what does this mean for human needs

Cell surface hydrophobicity (CSH) plays a crucial role in the attachment to, or detachment from the surfaces. The influence of CSH on adhesion of microorganisms to biotic and abiotic surfaces in medicine as well as in bioremediation and fermentation industry has both negative and positive aspects. Hydrophobic microorganisms cause the damage of surfaces by biofilm formation; on the other hand, they can readily accumulate on organic pollutants and decompose them. Hydrophilic microorganisms also play a considerable role in removing organic wastes from the environment because of their high resistance to hydrophobic chemicals. Despite the many studies on the environmental and metabolic factors affecting CSH, the knowledge of this subject is still scanty and is in most cases limited to observing the impact of hydrophobicity on adhesion, aggregation or flocculation. The future of research seems to lie in finding a way to managing the microbial adhesion process, perhaps by steering cell hydrophobicity.

Trachydiscus minutus, a new biotechnological source of eicosapentaenoic acid

The yellow-green alga Trachydiscus minutus (class Xanthophyta) was cultivated in a standard medium and in media without sulfur and nitrogen. Its yield after a 16-d cultivation reached 13 g dry mass per 1 L medium. The content of oligoenoic (‘polyenoic’) fatty acid (PUFA), i.e. eicosapentaenoic (EPA), was in excess of 35 % of total fatty acids; the productivity was thus 88 mg/L per d. This result makes the alga a very prospective organism that may serve as a new biotechnological source of single cell oil.

Monitoring of membrane potential changes inSaccharomyces cerevisiae by diS-C3(3) fluorescence

Attempt was made to measure the membrane potential in yeast cells by the electrochromic probe di-4-ANEPPS (dibutylaminonaphthylethylene pyridinium propyl sulfonate) which has previously been used for measuring action potentials in neurons [1, 2]. This probe is believed to provide fluorescent response to changes in transmembrane electric field in nanoseconds by changing its fluorescence intensity due to an underlying wavelength shift of emission maximum. The requirements for successful measurement are (1) defined dependence of the fluorescence response on change in membrane potential, (2) low probe toxicity at the concentrations used, (3) reproducible incorporation of the probe solely into the outer layer of the membrane lipid bilayer (incorporation into the inner layer would give rise to two probe pools whose respective responses to membrane potential changes would be mutually opposite, hampering the measurement), (4) absence of any penetration of the probe into the cell. The fluorescence of the electrochromic probe was measured in suspensions of intact cells, protoplasts and phosphatidylserine/phosphatidylcholine (20/80) liposomes. Tentative adjustment of membrane potential was done by incubating the samples in 3.5-150 mmol/L KC1, the overall molarity being adjusted in each case to 150 mmol/L by choline chloride. The effect of nonuniform staining of individual cells on the excitation spectrum of the probe was eliminated by measuring the ratio of fluorescence intensities at excitation wavelengths of 450 and 530 nm [3, 4]. The measurements showed that (1) the probe responds to membrane potential change by an electrochromic shift; (2) the cell wall hampers the penetration of the probe to the plasma membrane of yeast cells; (3) the actual equilibration of the probe in cell suspension should take 10-15 min but in fact the staining intensity keeps on rising even at longer intervals; (4) this is due to the fact that the probe is not incorporated solely into the plasma membrane but spreads gradually into the cells and liposomes, which causes persistent variations in fluorescence response to membrane potential change. This penetration brings about a fluorescence change mimicking a decrease in membrane potential, i.e. membrane depolarization. The probe is therefore suitable for monitoring membrane potential in yeast only over short periods of time (up to 30 min). Longer monitoring will require either a modified staining protocol or derivatization of the probe molecule. As found by using the dioctyl derivative di-8-ANEPPS, extending the aliphatic chains of the di-4-ANEPPS molecule does not prevent the dye from penetrating into the cell or liposome interior and, in addition, impairs staining.

Monitoring the kinetics and performance of yeast membrane ABC transporters by diS-C3(3) fluorescence.

Kinetic features (initial start-up phase, drug pumping velocity and efficiency as dependent on drug concentration and growth phase) of yeast plasma membrane multidrug resistance ABC pumps were studied by monitoring the uptake of the fluorescent potentiometric dye diS-C3(3), which has been found to be expelled from the cells by these pumps. The monitoring was done with Saccharomyces cerevisiae mutants AD1-8 and AD1-3 deleted in different ABC pumps, and in their pump-competent parent strain US50-18C overexpressing transcriptional activators Pdr1p and Pdr3p. On addition to the cells, diS-C3(3) is expelled by the Pdr5p, Yor1p and Snq2p pumps with overlapping substrate specificity. The pump action can be assessed as a difference between the dye uptake curve for pump-competent and pump-deleted cells. The pump-mediated dye efflux, which shows an initial lag of various lengths, maintains a certain residual intracellular dye level. In the absence of external glucose the dye efflux ability of the pumps depends on the growth phase; late exponential and stationary cells can maintain the export for tens of minutes, whereas exponential cells keep up the pump action for limited time periods. This may reflect an insufficient number of pump molecules in the membrane or an effect of insufficient pump energization from endogenous sources. This effect is not mediated by changes in membrane potential because lowered membrane potential caused by inhibition of the plasma membrane H+-ATPase does not affect the pump action.

Factors underlying membrane potential-dependent and -independent fluorescence responses of potentiometric dyes in stressed cells: diS-C3(3) in yeast

The redistribution fluorescent dye diS-C(3)(3) responds to yeast plasma membrane depolarisation or hyperpolarisation by Delta psi-dependent outflow from or uptake into the cells, reflected in changes in the fluorescence maximum lambda(max) and fluorescence intensity. Upon membrane permeabilisation the dye redistributes between the cell and the medium in a purely concentration-dependent manner, which gives rise to Delta psi-independent fluorescence responses that may mimic Delta psi-dependent blue or red shift in lambda(max). These lambda(max) shifts after cell permeabilisation depend on probe and ion concentrations inside and outside the cells at the moment of permeabilisation and reflect (a) permeabilisation-induced Delta psi collapse, (b) changing probe binding capacity of cell constituents (inverse to the ambient ionic strength) and (c) hampering of probe equilibration by the poorly permeable cell wall. At low external ion concentrations, cell permeabilisation causes ion outflow and probe influx (hyperpolarisation-like red shift in lambda(max)) caused by an increase in the probe-binding capacity of the cell interior and, in the case of heat shock, protein denaturation unmasking additional probe-binding sites. At high external ion levels minimising net ion efflux and at high intracellular probe concentrations at the moment of permeabilisation, the Delta psi collapse causes a blue lambda(max) shift mimicking an apparent depolarisation.

Factors affecting the outcome of the acidification power test of yeast quality: Critical reappraisal

Brewery bottom yeast strain 95 from thePilsner Urquell propagation unit was used to reappraise the efficiency of the acidification power (AP) test consisting in determining the spontaneous (oxygen-induced) and glucose-induced medium acidification caused by yeast and lactic acid bacteria under standard conditions, and used widely for assessing and predicting the vitality of industrial strains. AP was evaluated in yeast stored for different periods of time (0–28 d) at 4 °C, at different temperatures before and during the test (0–55 °C), and at different concentrations of cells and glucose and different cells-to-glucose ratios. All these factors had a strong effect on acidification kinetics and the AP value. By contrast, the duration of the lag period between yeast collection and the test (0–6 h) had no perceptible effect on the AP value. The best results were achieved at saturation concentrations of cells (>10 g pressed yeast or ≈14 g yeast slurry per 100 mL) and glucose (≈3 %) and at 25 °C. Since an exact evaluation of acidification characteristics depends strongly on the kinetics of the process, the AP test should include monitoring the time course of the acidification.

Viability and formation of conjugated dienes in plasma membrane lipids of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Rhodotorula glutinis and Candida albicans exposed to hydrophilic, amphiphilic and hydrophobic pro-oxidants.

Effects of four lipid peroxidation-inducing pro-oxidants-amphiphilictert-butyl hydroperoxide (TBHP), hydrophobic 1,1′-azobis(4-cyclohexanecarbonitrile) (ACHN), hydrophilic Fe11 and 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH)-on cell growth and on generation of peroxidation products in isolated plasma membrane lipids were determined in four yeast species (S. cerevisiae, S. pombe, R. glutinis andC. albicans) differing in their plasma membrane lipid composition. TBHP and ACHN inhibited cell growth most strongly, Fe11 and AAPH exerted inhibitory action for about 2 h, with subsequent cell growth resumption.S. cerevisiae strain SP4 was doped during growth with unsaturated linoleic (18∶2) and linolenic (18∶3) acids to change its resistance to lipid peroxidation. Its plasma membranes then contained some 30% of these acids as compared with some 1.3% of 18∶2 acid found in undopedS. cerevisiae, while the content of (16∶1) and (18∶1) acids was lower than in undopedS. cerevisiae. The presence of linoleic and linolenic acids inS. cerevisiae cells lowered cell survival and increased the sensitivity to pro-oxidants. Peroxidationgenerated conjugated dienes (CD) were measured in pure TBHP- and ACHN-exposed fatty acids used as standards. The CD level depended on the extent of unsaturation and the pro-oxidant used. The TBHP-induced CD production in a mixture of oleic acid and its ester was somewhat lower than in free acid and ester alone. In lipids isolated from the yeast plasma membranes, the CD production was time-dependent and decreased after a 5–15-min pro-oxidant exposure. ACHN was less active than TBHP. The most oxidizable were lipids fromS. cerevisiae plasma membranes doped with linoleic and linolenic acids and fromC. albicans with indigenous linolenic acid.

Effect of Antioxidants on Saccharomyces cerevisiae Mutants Deficient in Superoxide Dismutases

S. cerevisiae strain Δsod1 lacking Cu,Zn-superoxide dismutase and Δsod1Δsod2 mutant lacking both Cu,Zn-SOD and Mn-superoxide dismutase displayed strongly reduced aerobic growth on glucose, glycerol and lactate; Δsod2 deletion had no effect on aerobic growth on glucose and largely precluded growth on glycerol and lactate. The oxygen-induced growth defects and their alleviation by antioxidants depended on growth conditions, in particular on oxygen supply to cells. Under strong aeration, vitamins A and E had a low effect, 100 µmol/L quercetin alleviated the growth defects of all three mutants while β-carotene had no growth-restoring effect. The superoxide producer paraquat inhibited the aerobic growth of all three mutants in a concentration-dependent manner. Low concentrations of antioxidants had no effect on paraquat toxicity while higher concentrations supported the toxic effect of the agent.

Monitoring the growth and stress responses of yeast cells by two-dimensional fluorescence spectroscopy: First results

S. cerevisiae growth and responses to different treatments were monitored by two-dimensional fluorescence spectroscopy, which simultaneously detects the fluorescence of a number of cells’ own fluorophores. Growth curves of cultures of free cells were measured by means of tryptophan fluorescence in nonfluorescent culture medium and a flow-through system at a suitable excitation/emission beam geometry. Fast responses of the cells to anaerobic-aerobic transition or addition of glucose, methanol or cyanide, which could not be measured in this system because of the time delay inherent in transporting the cells from the culture flask to the cuvette, were monitored with cells immobilized in alginate. The major fluorescence changes caused by these treatments belonged to NAD(P)H which is a good indicator of the redox state of the cells.