Antistress cross-protection of UV-irradiated yeast cells with participation of extracellular peptide factors

Antistress effect of extracellular peptides on UV-irradiated yeast of different phylogenetic groups was studied. Yeast from different ecotopes and taxonomic groups exposed to UV radiation of a lethal intensity showed a protective effect and reactivating effect with participation of extracellular peptides. The highest protective activity was found in peptide reactivation factors (RFs) of bakery yeast—Saccharomyces cerevisiae, Kluyveromyces fragilis, and Candida utilis; the highest reactivating activity was exhibited by factors of the above-mentioned cultures and Debariomyces hansenii. Cross-protective and reactivating effects of RFs of yeast belonging to different taxonomic groups were demonstrated. Cross-protection increased two to three times after preexposure of reactivation factors to UV light (activation) in contrast to their reactivating effect.     

Characterization of extracellular yeast peptide factors and their stress-protective effect on probiotic lactic acid bacteria

Protective effect of the extracellular peptide fraction (reactivating factors, RF) produced by yeasts of various taxonomic groups (Saccharomyces cerevisiae, Kluyveromyces lactis, Candida utilis, and Yarrowia lipolytica) on probiotic lactic acid bacteria (LAB) Lactobacillus casei, L. acidophilus, and L. reuteri under bile salt (BS)-induced stress was shown. RF of all yeasts were shown to be of peptide nature; the active component of the S. cerevisiae RF was identified as a combination of low-molecular polypeptides with molecular masses of 0.6 to 1.5 kDa. The protective and reactivating effects of the yeast factors were not species-specific and were similar to those of the Luteococcus japonicus subsp. casei RF. In BS-treated cells of the tester bacteria, a protective effect was observed after 10-min preincubation of the LAB cell suspension with yeast RF: the number of surviving cells (CFU) was 2 to 4.5 times higher than in the control. The reactivating effect was observed when RF was added to LAB cell suspensions not later than 15 min after stress treatment. It was less pronounced than the protector effect, with the CFU number 1 to 3 times that of the control. Both the protector and the reactivating effects were most pronounced in the S. cerevisiae and decreased in the row C. utilis > K. lactis > Y. lipolytica. The efficiency of protective action of yeast RF was found to depend on the properties of recipient LAB cells, with the L. casei strain being most sensitive to BS treatment. In both variants, the highest protective effect of RF (increase in the CFU number) was observed for L. acidophilus, while the least pronounced one was observed for L. casei. The reasons for application of the LAB strains combining high stress resistance and high response to stress-protecting metabolites, including RF factors, as probiotics, is discussed.     

Reactivating effect of Escherichia coli exometabolites on UV-irradiated cells

Wild-type and mutant (AB 1157 and K-12) strains of Escherichia coli were shown to synthesize the logarithmic growth phase, exometabolites reactivating UV-irradiated cells of producer strains. The exometabolites of the strain K-12 were of protein nature and had a molecular weight of no more than 10 kDa. The reactivating activity of these exometabolites was inversely related to bacterial survival and slightly increased under the influence of stress factors. The reactivating factor of Luteococcus casei had a cross-reactivating and protective effect on UV-irradiated cells of E. coli strain K-12. Due to activation of the reactivating factor after UV irradiation and heating, the cross-protective effect increased more than threefold. The reactivating effect remained unchanged under these conditions. The protein exometabolites of E. coli did not induce cross-stress response in L. casei.     

Protective and reactivating effect of the protein exometabolite on yeast cells inactivated by the ultraviolet irradiation

It has been shown that Saccharomyces cerevisiae, Kluyveromyces lactis, and Candida utilis strains produce the protein exometabolites, which has a protective and reactivating effect on the ultraviolet irradiated yeast cells. The protective effect of the preliminary ultraviolet irradiated (activated) protein exometabolite of all strains increased 2–3 times, though its reactivating activity did not change. Yarrowia lipolytica yeast cells, isolated from the areas with the high daily irradiation, and Endomyces magnusii, the obligate fungi parasites, were characterized by the highest ultraviolet tolerance in comparison with the other strains. However, they did not produce the exometabolites with the antistress effect. Luteococcus casei reactivating factor demonstrated protective and reactivating cross-action in relation to the ultraviolet irradiated S. cerevisiae, K. lactis, and C. utilis cells and were inactive in relation to Y. lipolytica and E. magnusii. Using killer and nonkiller S. cerevisiae strain, it has been shown that the peptide exometabolite accumulation was not associated with toxin production.     

Reactivating effect of Escherichia coli exometabolites on UV-irradiated cells

Wild-type and mutant (AB 1157 and K-12) strains of Escherichia coli were shown to synthesize the logarithmic growth phase, exometabolites reactivating UV-irradiated cells of producer strains. The exometabolites of the strain K-12 were of protein nature and had a molecular weight of no more than 10 kDa. The reactivating activity of these exometabolites was inversely related to bacterial survival and slightly increased under the influence of stress factors. The reactivating factor of Luteococcus casei had a cross-reactivating and protective effect on UV-irradiated cells of E. coli strain K-12. Due to activation of the reactivating factor after UV irradiation and heating, the cross-protective effect increased more than threefold. The reactivating effect remained unchanged under these conditions. The protein exometabolites of E. coli did not induce cross-stress response in L. casei.     

Biological effect of extracellular peptide factor from Luteococcus japonicus subsp. casei on probiotic bacteria

The biological effect of the extracellular peptide reactivating factor (RF) from Luteococcus casei on cells of probiotic cultures was studied. The RF showed the protective and reactivating effects on the Bifidobacterium bifidum cells under the action of bile salts and an acidic stress. Also, it acted as a cryoprotector during lyophilisation and long-term culture storage. The RF and the L. casei culture liquid (CL) were shown to have bifidogenic properties. The degree of protection and reactivation of lactic-acid bacteria under the action of bile salts depended on the particular strain properties. The maximum degree of protection (more than thirteen-fold) and reactivation (close to three-fold) was found in Lactobacillus casei, while the minimum values were characteristic of Lactobacillus reuterii. The resistance of lactobacilli to bile was increased in the row of L. acidophilus, L. casei, L. plantarum, L. rhamnosus, and L. reuterii correlating with the RF protection degree.     

Effect of the reactivating factor of Luteococcus japonicus subsp. casei on the expression of SOS response genes

The effect of the extracellular peptide reactivating factor (RF) synthesized by Luteococcus casei on stress response of Escherichia coli cells subjected to UV irradiation was studied. For these studies, we constructed a test strain carrying the umuD-lacZ operon. The expression rate of this operon reflects the rate of SOS response. Protective effect of RF, defined as the number of cells retaining the colony-forming activity (CFU) after UV irradiation (49–1166 J/m²), was dose-dependent, species-nonspecific, and increasing with increase of the stress load. RF was demonstrated to possess the properties of a direct adaptogen: 15 min of preincubation with RF caused a 1.5–6-fold decrease in expression of the umuD SOS response gene in UV-treated cells, concurrently with a 1.2–7.5 times increase in the number of viable cells (those having retained their colony-forming activity). The probable mechanisms of the protective effect of RF are being discussed.     

THE INFLUENCE OF PROTEINS ON THE REACTIVATION OF YEAST INVERTASE

1. Acid-inactivated yeast invertase could not be regenerated in the presence of the proteolytic enzymes trypsin, pepsin, and chymotrypsin. 2. Certain foreign proteins of non-enzymatic nature partially inhibited the reactivation of acid-inactivated invertase. 3. Certain proteins as gelatin, lacto-globulin, and carbohydrate-free horse crystalbumin did not prevent the reactivation of invertase at all. 4. Highly purified reactivated invertase was shown to exhibit an effect typical of original native invertase; that is, acceleration of its activity in presence of foreign protein at pH 3.0. 5. Native invertase was not digested by trypsin and chymotrypsin. 6. The addition of trypsin and chymotrypsin to reactivating invertase did not affect the invertase which had already reverted to the active form, but prevented further reactivation of inactive invertase.     

Activation of the alternative oxidase of Yarrowia lipolytica by adenosine 5'-monophosphate

The study of the effect of nucleoside phosphates on the activity of cyanide-resistant oxidase in the mitochondria and the submitochondrial particles of Yarrowia lipolytica showed that adenosine monophosphate (5'-AMP, AMP) did not stimulate the respiration of the intact mitochondria. The incubation of the mitochondria at room temperature (25 degrees C) for 3-5 h or their treatment with ultrasound, phospholipase A, and detergent Triton X-100 at a low temperature inactivated the cyanide-resistant alternative oxidase. The inactivated alternative oxidase could be reactivated by AMP. The reactivating effect of AMP was enhanced by azolectin. Some other nucleoside phosphates also showed reactivating ability in the following descending order. AMP = GMP > GDP > GTP > XMP > IMP. The apparent reaction rate constant Km for AMP upon the reactivation of the alternative oxidase of mitochondria treated with Triton X-100 or incubated at 25 degrees C was 12.5 and 20 microM, respectively. The Km for AMP upon the reactivation of the alternative oxidase of submitochondrial particles was 15 microM. During the incubation of yeast cells under conditions promoting the development of alternative oxidase, the content of adenine nucleotides (AMP, ADP, and ATP) in the cells and their respiration tended to decrease. The subsequent addition of cyanide to the cells activated their respiration, diminished the intracellular content of ATP three times, and augmented the content of AMP five times. These data suggest that the stimulation of cell respiration by cyanide may be due to the activation of alternative oxidase by AMP.     

Stress-protective and cross action of the extracellular reactivating factor of the microorganisms of the domains Bacteria, Archaea, and Eukaryota

Cross protection of members of the domains Bacteria, Archaea, and lower Eukaryota from stress factors due to the action of extracellular low-molecular metabolites with adaptogenic functions was shown. The adaptogen produced by Luteococcus japonicus subsp. casei and described previously as a reactivating factor (RF) was shown to protect the yeasts Saccharomyces cerevisiae, archaea Haloarcula marismorti, and the cells of higher eukaryotes (HeLa) against weak stressor impacts. Production of an archaeal extracellular metabolite with a weak adaptogenic effect of the producer cells and capable of a threefold increase in survival of heat-inactivated yeast cells was discovered. Our results confirm the similarity of the compensatory adaptive reactions in prokaryotes (bacteria and archaea) and eukaryotes.     

Molecular basis for specificities of reactivating factors for adenosylcobalamin-dependent diol and glycerol dehydratases

Adenosylcobalamin-dependent diol and glycerol dehydratases are isofunctional enzymes and undergo mechanism-based inactivation by a physiological substrate glycerol during catalysis. Inactivated holoenzymes are reactivated by their own reactivating factors that mediate the ATP-dependent exchange of an enzyme-bound, damaged cofactor for free adenosylcobalamin through intermediary formation of apoenzyme. The reactivation takes place in two steps: (a) ADP-dependent cobalamin release and (b) ATP-dependent dissociation of the resulting apoenzyme-reactivating factor complexes. The in vitro experiments with purified proteins indicated that diol dehydratase-reactivating factor (DDR) cross-reactivates the inactivated glycerol dehydratase, whereas glycerol dehydratase-reactivating factor (GDR) did not cross-reactivate the inactivated diol dehydratase. We investigated the molecular basis of their specificities in vitro by using purified preparations of cognate and noncognate enzymes and reactivating factors. DDR mediated the exchange of glycerol dehydratase-bound cyanocobalamin for free adeninylpentylcobalamin, whereas GDR cannot mediate the exchange of diol dehydratase-bound cyanocobalamin for free adeninylpentylcobalamin. As judged by denaturing PAGE, the glycerol dehydratase-DDR complex was cross-formed, although the diol dehydratase-GDR complex was not formed. There were no specificities of reactivating factors in the ATP-dependent dissociation of enzyme-reactivating factor complexes. Thus, it is very likely that the specificities of reactivating factors are determined by the capability of reactivating factors to form complexes with apoenzymes. A modeling study based on the crystal structures of enzymes and reactivating factors also suggested why DDR cross-forms a complex with glycerol dehydratase, and why GDR does not cross-form a complex with diol dehydratase.     

The Extracellular Protein of Luteococcus japonicus subsp. casei Reactivates Cells Inactivated by UV Irradiation or Heat Shock

The culture liquid of Luteococcus japonicus subsp. casei was found to be able to reactivate cells of this bacterium inactivated by UV irradiation or heat shock. The antistress activity of the culture liquid was due to the presence of an extracellular exometabolite of a protein nature with a molecular mass of more than 10 kDa. When the bacterium was grown in a nutrient broth or glucose-containing mineral medium, the antistress protein was secreted by cells in the logarithmic growth phase. The reactivating effect of the antistress protein was inversely proportional to the survival rate of stressed cells.     

Antioxidative and antimutagenic activity of yeast cell wall mannans in vitro

Antioxidative and antimutagenic effect of yeast cell wall mannans, in particular, extracellular glucomannan (EC-GM) and glucomannan (GM-C.u.) both from Candida utilis, mannan from Saccharomyces cerevisiae (M-S.c.) and mannan from Candida albicans (M-C.a.) was evaluated. Luminol-dependent photochemical method using trolox as a standard showed that EC-GM, GM-C.u., M-S.c. and M-C.a. have relatively good antioxidative properties. EC-GM exhibited the highest antioxidative activity, followed by GM-C.u. and M-S.c. M-C.a. showed the least antioxidative activity. These mannans were experimentally confirmed to exhibit different, statistically significant antimutagenic activity in reducing damage of chloroplast DNA of the flagellate Euglena gracilis induced by ofloxacin and acridine orange (AO). We suggest that the antimutagenic effect of EC-GM, GM-C.u., M-S.c. and M-C.a. against ofloxacin is based on their ability to scavenge reactive oxygen radicals. With AO, the reduction of the chloroplast DNA lession could be a result of the absorptive capacity of the mannans. The important characteristics of mannans isolated from the yeast cell walls, such as good water solubility, relatively small molecular weight (15-30kDa), and antimutagenic effect exerted through different mode of action, appear to be a promising features for their prospective use as a natural protective (antimutagenic) agents.     

Coexistence of microbial populations and autostabilization of regulating factors in continuous culture: theory and experiments

The paper summarizes the author’s theoretical and experimental researches aimed at studying the rule of stable coexistence of interacting microbial populations within same trophic level. Populations of yeast and algae interact in open continuous cultures through regulating factors (RFs), which come together by the ability to be released or taken up by a microbial population and affect the growth of this and other population. Theoretical and experimental studies show that in steady state, the number of coexisting species is not greater than the number of RFs. Two-dimensional regions with different resultant species compositions of experimental equilibrium communities are plotted in the coordinates of “input levels of RFs”. This is perhaps the first study showing that the background steady-state levels of RFs in the system are not related to their input levels. This effect has been termed autostabilization of RFs, and its theoretical basis has been developed. A new criterion of intra- and inter-population microbial interactions has been introduced for RFs—growth acceleration response to a change in population density. Based on the proposed new criterion, experimental and theoretical estimates of the intensity and the sign of interactions between populations are given, allowing the quantification of their complex relationships, which was earlier unattainable. An integrated approach to detection of RFs has been proposed based on this criterion and the autostabilization effect.     

Chaperone activity of DsbC.

DsbC, a periplasmic disulfide isomerase of Gram-negative bacteria, displays about 30% of the activities of eukaryotic protein disulfide isomerase (PDI) as isomerase and as thiol-protein oxidoreductase. However, DsbC shows more pronounced chaperone activity than does PDI in promoting the in vitro reactivation and suppressing aggregation of denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during refolding. Carboxymethylation of DsbC at Cys98 decreases its intrinsic fluorescence, deprives of its enzyme activities, but lowers only partly its chaperone activity in assisting GAPDH reactivation. Simultaneous presence of DsbC and PDI in the refolding buffer shows an additive effect on the reactivation of GAPDH. The assisted reactivation of GAPDH and the protein disulfide oxidoreductase activity of DsbC can both be inhibited by scrambled and S-carboxymethylated RNases, but not by shorter peptides, including synthetic 10- and 14-mer peptides and S-carboxymethylated insulin A chain. In contrast, all the three peptides and the two nonnative RNases inhibit PDI-assisted GAPDH reactivation and the reductase activity of PDI. DsbC assists refolding of denatured and reduced lysozyme to a higher level than does PDI in phosphate buffer and does not show anti-chaperone activity in HEPES buffer. Like PDI, DsbC is also a disulfide isomerase with chaperone activity but may recognize different folding intermediates as does PDI.     

Photoreactivation and dark repair in UV-treated microorganisms: effect of temperature

Because of the lack of readily available information about the influence of temperature on microorganism reactivation processes subsequent to inactivation with UV radiation, a series of batch reactivation studies were performed at 5, 10, 15, 20, 25, and 30 degrees C. A special effort was made to model the reactivation process to enable the effect of the temperature variable to be quantified. Because an earlier-proposed kinetic model (K. Kashimada, N. Kamiko, K. Yamamoto, and S. Ohgaki, Water Sci. Technol. 33:261-269, 1996), a first-order saturation type, does not adequately fit the data obtained in experiments of reactivation in conditions of light and darkness, a modification of that model is proposed. The new model, which actually coincides with the classical logistic equation, incorporates two kinetic parameters: the maximum survival ratio (Sm) and the second-order reactivation rate constant (k2). In order to interpret correctly the reactivation occurring in conditions of darkness, a new term for the decay is added to the logistic equation. The new model accurately fits the data obtained in reactivation experiments, permitting the interpretation of the kinetic parameters Sm, k2, and M (for only repair in darkness), where M is mortality, a zero-order decay rate constant, and their relationship with various environmental conditions, such as microbial type, light, and temperature. The parameters Sm and k2 (and M for reactivation in conditions of darkness) show exponential dependence on the reactivating temperature, and it is possible to predict their values and hence the reactivation curve from the equations proposed in this work.     

Production of wild-type and peptide fusion cutinases by recombinant Saccharomyces cerevisiae MM01 strains

This study focused on the growth of Saccha-romyces cerevisiae MM01 recombinant strains and the respective production of three extracellular heterologous cutinases: a wild-type cutinase and two cutinases in which the primary structure was fused with the peptides (WP)(2) and (WP)(4), respectively. Different cultivation and strategies were tested in a 2-L shake flask and a 5-L bioreactor, and the respective cell growth and cutinase production were analyzed and compared for the three yeast strains. The highest cutinase productions and productivities were obtained in the fed-batch culture, where wild-type cutinase was secreted up to a level of cutinase activity per dry cell weight (specific cell activity) of 4.1 Umg(-1) with activity per protein broth (specific activity) of 266 Umg(-1), whereas cutinase-(WP)(2) was secreted with a specific cell activity of 2.1 Umg(-1) with a specific activity of 200 Umg(-1), and cutinase-(WP)(4) with a specific cell activity of 0.7 Umg(-1) with a specific activity of 15 Umg(-1). The results indicate that the fusion of hydrophobic peptides to cutinase that changes the physical properties of the fused protein limits cutinase secretion and subsequently leads to a lower plasmid stability and lower yeast cell growth. These effects were observed under different cultivation conditions (shake flask and bioreactor) and cultivation strategies (batch culture versus fed-batch culture).     

Cross-effects of extracellular factors of adaptation to stress in Luteococcus casei and Saccharomyces cerevisiae

Saccharomyces cerevisiae yeasts (lower eukaryotes) were shown to produce a protein exometabolite with reactivation activity. We demonstrated cross-effects of extracellular protein factors of adaptation to stress (heat and UV irradiation) in yeasts and Luteococcus casei bacteria. The possibility for isolation and partial purification of protein exometabolites from the culture liquid of yeasts and bacteria by similar methods, as well as the similarity of elution profiles for the active proteins in high-performance liquid chromatography, suggests that the proteins (or fragments thereot) of the organisms studied are homologous.     

Translational compensation of a frameshift mutation affecting herpes simplex virus thymidine kinase is sufficient to permit reactivation from latency

Herpes simplex virus thymidine kinase is important for reactivation of virus from its latent state and is a target for the antiviral drug acyclovir. Most acyclovir-resistant isolates have mutations in the thymidine kinase gene; however, how these mutations confer clinically relevant resistance is unclear. Reactivation from explanted mouse ganglia was previously observed with a patient-derived drug-resistant isolate carrying a single guanine insertion within a run of guanines in the thymidine kinase gene. Despite this mutation, low levels of active enzyme were synthesized following an unusual ribosomal frameshift. Here we report that a virus, generated from a pretherapy isolate from the same patient, engineered to lack thymidine kinase activity, was competent for reactivation. This suggested that the clinical isolate contains alleles of other genes that permit reactivation in the absence of thymidine kinase. Therefore, to establish whether thymidine kinase synthesized via a ribosomal frameshift was sufficient for reactivation under conditions where reactivation requires this enzyme, we introduced the mutation into the well-characterized strain KOS. This mutant virus reactivated from latency, albeit less efficiently than KOS. Plaque autoradiography revealed three phenotypes of reactivating viruses: uniformly low thymidine kinase activity, mixed high and low activity, and uniformly high activity. We generated a recombinant thymidine kinase-null virus from a reactivating virus expressing uniformly low activity. This virus did not reactivate, confirming that mutations in other genes that would influence reactivation had not arisen. Therefore, in strains that require thymidine kinase for reactivation from latency, low levels of enzyme synthesized via a ribosomal frameshift can suffice.     

Reactivation of dormant and nonculturable bacterial forms from paleosoils and subsoil permafrost

Methods of reactivating the dormant forms (DFs) and nonculturable cells (NCs) of the bacterial communities of buried paleosoils and subsoil permafrost stored for long periods of time (thousands to millions of years), including completely sterile samples (CFU = 0), were developed. They were based on washing the DFs and NCs to remove anabiosis autoinducers (spore germination autoinhibitors) and introducing low molecular weight extracellular growth regulators of microbial or plant origin, such as alkylhydroxybenzenes of the alkylresorcinol subtype, indoleacetic acid, and wheat germ agglutinin. It was revealed that the dormant communities of permafrost and buried soils differed in their sensitivity to reactivating factors, probably due to different natural storage conditions of the tested soil substrates and the heterogeneity of dormant populations. The latter was confirmed by FISH (fluorescent in situ hybridization): applying the reactivation methods to the cells of the dormant permafrost community resulted in an increase in the number of metabolically active cells from 5 to 77% of their total number. In contrast, the addition of microbial anabiosis autoinducers (C₁₂-AHB) to background surface soil and permafrost samples caused the transition of bacterial cells to the dormant or the nonculturable state, depending on the C₁₂-AHB concentration and the sensitivity of the cells from the control soil or permafrost’ to it. The data obtained contribute to our knowledge concerning the role of intercellular communication factors and the survival of microorganisms under extreme environmental conditions.