Protection of Saccharomyces cerevisiae against Oxidative and Radiation-Caused Damage by Alkylhydroxybenzenes

The effects of C₇-alkylhydroxybenzene (₇-AHB) and p-hydroxyethylphenol (tyrosol), chemical analogs of microbial anabiosis autoregulators, on the viability of yeast cells under oxidative stress were investigated. The stress was caused by reactive oxygen species (ROS) produced under irradiation of cell suspensions using doses of 10–150 krad at an intensity of 194 rad/s or by singlet oxygen generated in cells photosensitized with chlorin e ₆ (10 g/l). C₇-AHB was found to exert a protective effect. The addition of 0.05–0.16 vol % of C₇-AHB to cell suspensions 30 min before irradiation protected yeast cells from radiation (50 krad). The protective effect of C₇-AHB manifested itself both in the preservation of cell viability during irradiation and in the recovery of their capacity to proliferate after irradiation. In our studies on photodynamic cell inactivation, the fact that the phenolic antioxidant C₇-AHB protects cells from intracellular singlet oxygen was revealed for the first time. The analysis of difference absorption spectra of oxidized derivatives of C₇-AHB demonstrated that the protective mechanism of ₇-AHB involves the scavenging of ROS resulting from oxidative stress. The fact that tyrosol failed to perform a photoprotective function suggests that the antioxidant properties of microbial ₇-AHB are not related to its chaperon functions. The results obtained make an important addition to the spectrum of known antioxidant and antistress effects of phenolic compounds.     

Properties of the phenotypic variants of Pseudomonas aurantiaca and P. fluorescens

Different capacity for phenotypic variation of Pseudomonas aurantiaca and P. fluorescens in populations of cyst-like resting cells (CRC) during their germination on solid media, was shown to be a characteristic trait of biodiversity for the dormant forms of these bacteria. This biodiversity manifests itself as qualitative and quantitative differences in the spectra and emergence frequency of phenotype variants, obtained by plating of CRC, and depends on the conditions of CRC formation and storage time. In P. aurantiaca, the variation was associated with transition of the wild-type S-colonial phenotype into the R-type or the more pigmented P-type. These transitions were most pronounced for the CRC obtained under nitrogen depletion (a twofold N limitation), as well as under the influence of a chemical analogue of microbial anabiosis autoinducers, C₁₂-AHB. In the latter case, the frequency of S?R and S?P transitions (up to 70% and 80%, respectively) depended on the C₁₂-AHB concentration (1.0 × 10^?4 M and 2.5 × 10^?4 M) and on the storage time of CRC suspensions (from 3 days to 1.3 months). In the CRC populations grown in nitrogen-deficient media, R-type appeared with a frequency of up to 45% after at least four months of storage. In the case of P. fluorescens, S?R transitions depended not only on the storage time of CRC and C₁₂-AHB concentrations, but also on the composition of the solid medium used for plating. Differences were shown between the R-, P-, and S-variants of P. aurantiaca in such morphological, physiological, and biochemical characteristics as the growth rate (μ_max) in a poor medium, biomass yield (Y _max), resistance to streptomycin and tetracycline (LD₅₀), and the productivity in extracellular proteases. The R-and S-variants of P. fluorescens differed in their growth characteristics, resistance to high salinity and oxidative stress, as well as in their sensitivity to exogenous introduction of chemical analogues of microbial autoregulators (C₁₂-AHB and C₇-AHB). Hence, both the formation of dormant forms of the various morphological types [1] and intrapopulation phenotypic variability observed during their germination are important for the survival strategy of pseudomonads under unfavorable environmental conditions.     

The role of alkylhydroxybenzenes in the adaptation of <Emphasis Type="Italic">Micrococcus luteus</Emphasis> to heat shock

The response of the gram-positive bacterium micrococcus luteus to heat shock (4°C, 15 min) and the adaptogenic activity of alkylhydroxybenzenes (AHBs), which are extracellular growth-regulating substances of these bacteria, were studied. The perception of stress and the postshock behavior of M. luteus cells proved to depend on the growth phase and medium. The magnitude of the stress response was more pronounced in cultures grown on synthetic medium than in cultures grown on rich medium (nutrient broth). During exponential or linear growth, the cells were more sensitive to the temperature effect than during decelerated growth. In linearly growing m. luteus cultures, the amount of total intra- and extracellular alkylhydroxybenzenes, the anabiosis inducers, increased in response to heat shock. AHB redistribution between cells and culture liquid occurred in the course of stress and after stress. In micrococci exposed to heat shock, an increase in the AHB concentration both in cells and in culture liquid is likely a defense reaction of stress resistance. This conclusion was confirmed in experiments with the addition 30 min before the heat shock of a chemical analogue of the anabiosis inducer, C₇-AHB (12 mM), which protected M. luteus cells so that their intense growth was observed after shock without any lag. The protective effect of AHBs is a result of their ability to form complexes with enzyme macromolecules and stabilize them. The data obtained extend the knowledge of the stress-protective functions of low-molecular-weight autoregulators and of the role of intercellular communications in the stress response of bacterial cultures.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 26–33.Original Russian Text Copyright © 2005 by Stepanenko, Mulyukin, Kozlova, Nikolaev, El-Registan.     

Effect of alkylhydroxybenzenes,microbial anabiosis inducers,on the structural organization of <Emphasis Type="Italic">Pseudomonas aurantiaca</Emphasis> DNA and on the induction of phenotypic dissociation

We revealed a relationship between alkylhydroxybenzene (AHB)-induced changes in the structural organization of supramolecular complexes (SC) of the DNA of Pseudomonas aurantiaca and the phenotypic dissociation of this bacterium. The addition of 0.1–0.3 mM hexylresorcinol (C₆-AHB), a chemical analogue of microbial anabiosis autoinducers, caused the formation of cystlike refractile cells (CRC) in these gram-negative, nonsporulating bacteria. Inoculating pseudomonad CRC on solid nutrient media resulted in phenotypic dissociation of the microbial population that yielded several variants with different colony structure and morphology. This manifested itself in the conversion of the original S-colony-forming phenotype into the R form and in the formation of less pigmented colonies. These transitions were possibly linked to AHB-induced structural changes in the DNA. In vitro studies revealed that AHB could interact with DNA SC, resulting in their structural modification that manifested itself in changes in their viscoelasticity. DNA supramolecular complexes isolated from proliferating, stationary-phase, and anabiotic P. aurantiaca cells differed in their viscoelasticity and capacity to interact with AHB homologues with different hydrophobicity, such as hexylresorcinol and methylresorcinol (C₁-AHB). The DNA SC from actively proliferating cells were characterized by smaller viscoelasticity compared with those from stationary-phase and anabiotic cells, due to the difference in the DNA superspiralization degree and the physiological age of the bacteria involved. C₆-AHB produced a pronounced relaxing effect on the DNA SC from exponential-phase P. aurantiaca cells. The less hydrophobic C₁-AHB produced a similar relaxing effect on the DNA SC from stationary-phase cells. The curve of the dose-effect dependence of C₆-AHB had a breaking point within the submillimolar (10^–4 M) concentration range. These concentrations induce the formation of cystlike anabiotic pseudomonad cells that are characterized by an unstable phenotype and dissociate into distinct variants upon inoculation on solid media.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 157–165.Original Russian Text Copyright © 2005 by Mulyukin, Vakhrushev, Strazhevskaya, Shmyrina, Zhdanov, Suzina, Duda, Kozlova, El-Registan.     

Long-term preservation of DNA in aqueous solutions in the presence of the chemical analogues of microbial autoregulators

he fact of long-term preservation of the physicochemical properties of DNA molecules in aqueous solutions in complexes with methylresorcinol, hexylresorcinol, and tyrosol, the chemical analogues of microbial autoregulators (d₁ factors) from the group of alkylhydroxybenzenes (AOB), was established. Compared to the control variants of storage of aqueous DNA solutions, the AOB influence consisted in the sum of correlating effects: the prevention of DNA degradation (according to spectrophotometric parameters) and the preservation of its viscous characteristics and electrophoretic mobility. The initial DNA properties were preserved to the greatest degree in the presence of hexylresorcinol, the compound with the longest alkyl radical. Possible mechanisms of the protective action of alkylhydroxybenzenes in relation to DNA are discussed, namely, the prevention of its hydrolysis due to isolation from the aqueous environment and maintaining DNA stability in the dormant forms of microorganisms.     

Influence of chemical analogues of microbial autoregulators on the sensitivity of DNA to UV radiation

We established that chemical analogues of alkylhydroxybenzenes (AHB), belonging to alkylresorcinols and functioning as microbial autoregulatory d₁ factors, enhance the UV resistance of various DNA molecules of different origin and conformation. These include the linear DNA of the λ phage, bovine spleen DNA, and the DNA of the pUC19 plasmid that is composed of a number of annular (supercoiled and relaxed) and linearized molecules. Irradiating DNA with UV light (λ = 254 nm) in the presence of methylresorcinol (MR) or hexylresorcinol (HR) results in comparatively insignificant DNA destruction as evidenced by our data on the electrophoretic mobility pattern in agarose gel. Using the linear HindIII restricts of the λ phage DNA, we revealed that the protective effect of AHB varies depending on their chemical structure (it is more manifest with HR than MR) and the concentration. Importantly, the effect of HR on bovine spleen DNA was based on its protective activity and manifested itself after a long incubation period. Studies using the pUC19 plasmid demonstrated that AHB, apart from increasing the resistance of linearized DNA molecules to UV irradiation, prevented both the supercoiled annular-supercoiled relaxed and the supercoiled relaxed-linearized transitions. The possible mechanisms of the UV-protective effect of AHB on DNA and their contributions to the resistance of dormant microbial forms to environmental factors are discussed.     

Mechanisms of Interaction between DNA and Chemical Analogues of Microbial Anabiosis Autoinducers

The alkylhydroxybenzene (AHB) autoregulatory factors d₁ (fd₁) of microorganisms have been found to directly interact with highly polymeric DNA. This circumstance results in changes, related to alterations in the topology of this macromolecule, in DNA physicochemical properties. The physicochemical properties of DNA in the presence of chemical analogues of microbial AHBs (methylresorcinol; hexylresorcinol; and 2-(4-hydroxyphenyl)ethane-1-ol, also known as tyrosol) were investigated using adsorption spectrophotometry, fluorometry, heat denaturation, viscosimetry, and electrophoresis in agarose gel. A number of concordant effects pointing to DNA-AHB interactions were revealed that manifest themselves in the hypochromic properties of the resulting complexes, an increase in their melting temperature and viscosity, a decrease in their electrophoretic mobility, and a change in the fluorescent properties of AHBs upon complexation with DNA. Such alterations were particularly significant in the presence of hexylresorcinol, which possessed the maximum alkyl radical length among the fd₁ analogues tested. Using atomic force microscopy, we visualized the micellelike DNA structures forming in the presence of AHBs. The results obtained provided the basis for developing a hypothetical model of the interaction between the biopolymer macromolecule and low-molecular-weight AHBs that takes into account the differences in the hydrophobicity of individual AHB homologues functioning as ligands. In terms of our model, we discuss AHB involvement in the stabilization of DNA and alteration of its topology, i.e., in the process related to intragenomic rearrangements, which account for the intrapopulational variability of bacteria, including dissociation processes.     

Regulation of the functional activity of lysozyme by alkylhydroxybenzenes

In our study, we investigated the capacity of alkylhydroxybenzenes (AHB), which are microbial anabiosis autoinducers, for alteration of the enzymatic activity of the hen egg-white lysozyme, as well as the efficiency of hydrolysis of specific (peptidoglycan) and nonspecific (chitin) substrates catalyzed by lysozyme. AHB homologues (C7-AHB and C12-AHB), which differ in their hydrophobicity and effects in their interaction with lysozyme, were used as modifying agents. C7-AHB stimulated enzymatic activity within the whole range of concentrations used (10^?7?10^?3 M). More hydrophobic C12-AHB exhibited this ability only at low concentrations and inhibited fermentative activity at high concentrations, acting as a mixed-type inhibitor. Both AHB homologues caused changes in the hydrophobicity of lysozyme molecules. An increase in the affinity level between the C7-AHB-modified enzyme and the nonspecific substrate (colloidal chitin or cell wall polymers of Saccharomyces sp.) was observed, which manifested itself in the enhancement of the hydrolysis rate by 200–500% (as compared to the native enzyme). A significant effect on the efficiency of the lysozyme-catalyzed modifications of the substrate (peptidoglycan, colloidal chitin) structure as a result of its complexation with AHB was demonstrated. A stabilizing effect of C7-AHB and C12-AHB was revealed, which ensured a high level of activity of the AHB-modified enzyme (as compared to the control) after heat treatment (functional stability), as well as at nonoptimal temperatures of catalysis (operational stability). The biological significance of lysozyme modification with AHB and the practical aspects of its application are discussed.     

Effect of a chemical analogue of autoinducers of microbial anabiosis on the Ca<Superscript>2+</Superscript> response of mycelial fungi

The microbial alkylhydroxybenzenes (AHB), which are anabiosis autoinducers also termed d₁ factors, participate in the stress response of mycelial fungi, as determined from changes in intracellular Ca²⁺ concentration. By using the genetically modified strain Aspergillus awamori 66A, which produces the recombinant Ca²⁺-dependent protein aequorin, the dynamics of Ca²⁺ was studied in the cytosol of cells exposed to mechanical shock in the presence of protective doses (0.001–0.01% w/vol) of a chemical AHB analogue, 4-n-hexylresorcinol. As under stressful conditions, Ca²⁺ concentration increases in the cell cytosol in response to an enhanced AHB level in a growing fungal culture; thus, AHB is perceived by cells as a stress signal. The level of cell response, which was determined from the amplitude of luminescence dependent on the Ca²⁺ concentration in the cytosol, was related to the physiological age of the cells and the AHB concentration. Micromycete preincubation with AHB was found to protect cells from subsequent stress; this was reflected in the Ca²⁺ response. The protective AHB effect was manifested as (1) a significant decrease in the amplitude of luminescence and, thus, in Ca²⁺ accumulation in the cytosol during subsequent mechanical stress (as compared to the control—mechanical stress only); (2) development of a secondary Ca²⁺ response, which was not observed in the control; and (3) a high level of Ca²⁺ retained in the cytosol for a long time in the presence of AHB (as compared to the control without preincubation with AHB). The mechanisms underlying the AHB effect on Ca²⁺ transport systems are discussed.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 741–750.Original Russian Text Copyright © 2004 by Kozlova, Kupriyanova-Ashina, Egorov, El-Registan.     

Non-species-specific effects of unacylated homoserine lactone and hexylresorcinol, low molecular weight autoregulators, on the growth and development of bacteria

We conducted a comparative study of the effects of α-amino-γ-butyrolactone, the common structural element of extracellular microbial regulators of the homoserine lactone (HSL) group, and of 4-n-hexylresorcinol, an autoregulator of the alkylhydroxybenzene (AHB) group, on the growth and development of grampositive and gram-negative bacteria. We revealed non-species-specific effects of HSL and AHB and characterized their concentration dependencies. The addition of 10^?5?10^?3 M HSL or 10^?5?10^?4 M AHB during the exponential growth phase of the cultures grown on balanced media resulted in cell division arrest and accelerated the transition to the stationary phase that culminated in endospore formation in Bacillus cereus, Alicyclobacillus tolerans, and Sulfobacillus thermosulfidooxidans. When bacilli grew under the cultivation conditions that resulted in a low-zero spore percentage, 10^?4?10^?3 M HSL cancelled the inhibition of spore formation. In the gram-negative bacteria Pseudomonas aurantiaca and Azotobacter vinelandii, AHB at concentrations of 10^?4 to (1.5?2.5)×10^?4 M induced the formation of dormant cells. Studies with the actinobacterium Streptomyces avermitilis revealed that the HSL effect varied depending on the age of the test cultures. The addition of 10^?4 M HSL during the lag phase of a submerged streptomycete culture accelerated its transition to the stationary phase and induced the formation of endospores, the dormant cells that are regarded as alternatives to exospores (conidia). If HSL (3.64 and 4.55 mg per 1 cm² disc) was locally added to a surface S. avermitilis culture, the growing mycelium formed rings that differed in their density, in the extent of the development of aerial mycelium, and in the presence/absence of exospores. Ring-shaped growth of streptomycete mycelia was also induced by 0.075–0.75 mg of AHB; however, unlike HSL, AHB repressed exospore formation. The data on non-species-specific effects of HSL and AHB suggest that they may perform regulatory functions at the microbial community level.