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Applied Biochemistry and Microbiology (v.48, #1)

Fungal spores: Dormancy, germination, chemical composition, and role in biotechnology (review) by E. P. Feofilova; A. A. Ivashechkin; A. I. Alekhin; Ya. E. Sergeeva (pp. 1-11).

This review is focused on one of the stages of ontogenesis distinctive by its particular tolerance to the action of unfavorable factors and ability to retain the genomic material for a long period of time, i.e., fungal spores. The major part is devoted to the characterization of the specific stage typical for spores, which is called dormancy. Data are presented characterizing the carbohydrate and lipid composition of spores, with special attention being paid to the role of carbohydrate protectors, in particular, trehalose and mannite, as well as to the role of rafts in the process of sporogenesis. The role of special compounds called autoinhibitors and autostimulators in the process of exit from dormancy is discussed. The final section deals with the role of spore seeding material in biotechnological processes. Data on the correlation between the chemical composition of spores, their ability to remain dormant, and the germination process are considered. Special biotechnological approaches are presented for the first; they allow for the preservation of the germinating ability of spores, intensification of sporogenesis, changes in the ratio of final fermentation products, and an increase in their yield.

Cyclic digene system as a control element of a bacterial biosensor by E. E. Stupak; I. V. Stupak (pp. 12-16).

The Escherichia coli JC158(pCIA12/pGFK5) strain carrying a cyclic digene system with a negative feedback on the pCIA12 plasmid reacting to the DNA damage by changing the synthesis level of reporter genes—GFP and β-galactosidase—was tested. The acquired phenotype was inherited by the next generations after the removal of the genotoxic action when the concentration of the DNA-damaging compounds was above the threshold level. A potential has been shown for the application of bacterial biosensors to monitor the presence of genotoxicants in the environment and to test the consequences of short-term exposures to toxic compounds.

Isolation and purification of Mn-peroxidase from Azospirillum brasilense SP245 by M. A. Kupryashina; N. Yu. Selivanov; V. E. Nikitina (pp. 17-20).

Homogenous Mn-peroxidase of a 26-fold purity grade was isolated from a culture of Azospirillum brasilense Sp245 cultivated on a medium containing 0.1 mM pyrocatechol. The molecular weight of the enzyme is 43 kD as revealed by electrophoresis in SDS-PAAG. It was shown that the use of pyrocatechol and 2,2′-azino-bis(3-ethylbenzotiazoline-6-sulfonate) at concentrations of 0.1 and 1 mM as inductors increased the Mn-peroxidase activity by a factor of 3.

Detoxification of high concentrations of trinitrotoluene by bacteria by I. P. Solyanikova; B. P. Baskunov; M. A. Baboshin; A. I. Saralov; L. A. Golovleva (pp. 21-27).

The ability of the strains-destructors of various aromatic compounds to utilize trinitrotoluene (TNT) up to concentration of 70 mg/l was shown. An increase in the TNT concentration from 100 to 150 mg/l did not inhibit its conversion rate by the Kocuria palustris RS32 strain. The Acinetobacter sp. VT11 strain utilized TNT as a sole substrate for growth; 3,5-dinitro-4-methyl anilide acetate and 2,6-dinitro-4-aminotoluene were identified as intermediates of TNT degradation by active strains of Pseudomonas sp. VT-7W and Kocuria rosea RS51. At the same time, 4-methyl-3,5-dinitroformamide was discovered for the first time upon the TNT destruction by the bacteria strains of Rhdococcus opacus 1G and Rhdococcus sp. VT-7. The active bacterial strains achieved an 82-90% destruction of TNT when they were introduced into the soil.

Biodegradation of polyhydroxyalkanoates by soil microbial communities of different structures and detection of PHA degrading microorganisms by A. N. Boyandin; S. V. Prudnikova; M. L. Filipenko; E. A. Khrapov; A. D. Vasil’ev; T. G. Volova (pp. 28-36).

Biodegradation of microbial linear polymers of hydroxyalkanoic acids (polyhydroxyalkanoates, PHAs) by soil microbial communities of different structures has been studied during two field seasons in different weather conditions. This process was shown to be influenced by the polymer chemical composition, temperature, humidity, and the microbial soil component. The PHA degradation was accompanied by a decrease in the polymer molecular weight and an increase in the degree of crystallinity, indicating the preferential destruction of the amorphous phase compared to the crystalline one. The quantity of the true PHA destructors developing at the surface of the polymer samples was lower than the quantity of accompanying bacteria. The dominant PHA degrading microorganisms under the test conditions were identified as bacteria of the genera Variovorax, Stenotrophomonas, Acinetobacter, Pseudomonas, Bacillus, and Xanthomonas and as micromycetes from Penicillium, Paecilomyces, Acremonium, Verticillium, and Zygosporium.

Degradation of polyisoprene rubber by newly isolated bacillus sp. AF-666 from soil by A. A. Shah; F. Hasan; Z. Shah; Mutiullah; A. Hameed (pp. 37-42).

Various microorganisms were screened for their ability to degrade polyisoprene rubber (natural rubber latex gloves). Strain AF-666, newly isolated from a soil sample, was selected as the best strain having the ability to grow on polyisoprene containing plates. The strain identified as Bacillus sp. AF-666, was found to degrade polyisoprene rubber, both on basal agar plates (latex overlay) as well as in liquid medium. Qualitative analysis of degradation was done through scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. SEM showed changes in surface morphology, like appearance of pits and cracks, and marked difference in transmittance spectra of test and control due to changes in the functional groups, was detected through FTIR. CO₂ evolution as a result of rubber degradation, was calculated gravimetrically by Sturm Test. About 4.43 g/1 of CO₂ was produced in case of test, whereas, 1.57 g/1 in case of control. The viable number of cells (CFU/ml) was also higher in test than in control. Present study may provide an opportunity for further studies on the applications of biotechnological processes as a tool for rubber waste management.

Isolation, identification and characterization of fluoride resistant bacteria: Possible role in bioremediation by S. Chouhan; U. Tuteja; S. J. S. Flora (pp. 43-50).

Microorganisms found in industrial effluents and near the sites of the contamination can be used to indicate pollution and detoxify the contaminated water resources. Emergence of xenobiotic resistant bacteria among them might be potential application in bioremediation. The objective of this study was to isolate and characterize fluoride resistant bacteria from soil and water samples of different regions of India. Five isolates were recovered from different samples which were found to be fluoride resistant. Two of them effectively reduced the fluoride from their media. Through the current study it can be predicted that fluoride pollution results in selective pressure that leads to the development of fluoride resistant among bacterial populations, probably through the mechanism which involved high affinity anion binding compounds called ionophores. Resistant microbes may play a bioremediative role by transforming and concentrating these anions so that they are less available and less dangerous.

Characterization of the fungicidal activity of Calothrix elenkinii using chemical methods and microscopy by C. Natarajan; R. Prasanna; V. Gupta; P. Dureja; L. Nain (pp. 51-57).

An investigation was directed towards biochemical characterization of cyanobacterium Calothrix elenkinii and analysis of the chemical nature and mode of action of its fungicidal metabolite(s) against oomycete Pythium debaryanum. Biochemical characterization of the culture in terms of carbohydrate utilization revealed the facultative nature of C. elenkinii. Unique antibiotic markers were also found for this strain. 16S rDNA sequencing of the strain revealed 98% similarity with Calothrix sp. PCC7101. The fungicidal activity was tested by disc diffusion assay of different fractions of the culture filtrate. A minimum inhibitory concentration of 10 μl was recorded for ethyl acetate fraction of the 7-weeks old culture filtrates. HPLC, followed by NMR spectral analysis demonstrated the presence of a substituted benzoic acid in the ethyl acetate fraction. Microscopic examination revealed distinct granulation, followed by disintegration of the hyphae of Pythium sp., indicating the presence of an active metabolite in the culture filtrates of Calothrix sp. The fungicidal activity of C. elenkinii can be attributed to the presence of 3-acetyl-2-hydroxy-6-methoxy-4-methyl benzoic acid. This is the first report of a benzoic acid derivative having fungicidal activity in cyanobacteria.

Production of enzyme preparations on the basis of Penicillum canescens recombinant strains with a high ability for the hydrolysis of plant materials by P. V. Volkov; A. M. Rozhkova; A. G. Pravilnikov; R. M. Andrianov; G. S. Dotsenko; A. O. Bekkarevich; A. V. Koshelev; O. N. Okunev; I. N. Zorov; A. P. Sinitsin (pp. 58-64).

An enzyme preparation has been produced on the basis of Penicillium canescens strains with the activity of cellibiohydrolase I, II; endo-1,4-β-gluconase of Penicillium verruculosum; and β-glucosidase of Aspergillus niger. It was shown that for the most effective hydrolysis of aspen wood pulp the optimal ratio of cellobiohydrolase and endo-1,4-β-gluconase in enzyme preparations was 8: 2 (by protein). It was also established that the homologous xylanase secreted by the Penicillium canescens fungus is a required component for the enzyme complex for hydrolysis of the hemicellulose matrix of aspen wood.

Branched glucan from the fruiting bodies of Piptoporus betulinus (Bull.:Fr) Karst. by D. N. Olennikov; S. V. Agafonova; A. V. Rokhin; T. A. Penzina; G. B. Borovskii (pp. 65-70).

A new glucan, namely, piptoporane I, with a molecular mass of 270 kDa was isolated from fruiting bodies of Piptoporus betulinis (Bull.:Fr.) Karst. (Fomitopsidacaeae). Using a combination of physicochemical methods, it was established that piptoporane I was a branched glucan with a backbone consisting of α-(1 → 3)-glucopyranose residues substituted at the C-6 position by single residues of β-D-glucopyranose by 17.3%. A polysaccharide with such a structure was isolated for the first time from the fungus genus Piptoporus.

Enzyme immunoassay of the secondary metabolites of micromycetes as components of lichen substances by G. P. Kononenko; A. A. Burkin; T. Yu. Tolpysheva (pp. 71-76).

The composition of low-molecular biologically active metabolites typical of microscopic fungi has been studied in blastemas of fruticose lichens of the genera Cladonia, Cetraria, Evernia, Bryoria, and Usnes. The enzyme immunoassay method showed the presence of sterigmatocystin, emodin, mycophenolic acid, citrinin, alternariol, and diacetoxyscirpenol, which occurred regularly and, in most cases, at a frequency of 55 to 100%. The highest levels of accumulation were 0.001–0.003% for emodin, 0.0002% for alternariol and citrinin, 0.0001% for sterigmatocystin and mycophenolic acid, and 0.00005% of the weight of air-dry material for diacetoxyscirpenol. Other metabolites (cyclopiazonic acid, ergot alkaloids, ochratoxin A, PR toxin, deoxynivalenol, zearalenone, and fumonisins) were detected in these lichens less frequently (sometimes only upon the expansion of the territory of sampling), and their content was no more than 0.00005%. The peculiarities of the component composition and the levels of accumulation of fungal metabolites in lichens of different taxonomic affiliation were discussed.

Lipoxygenase from the leaves of wheat grown under different water supply conditions by M. D. Permyakova; A. V. Permyakov; S. V. Osipova; T. A. Pshenichnikova (pp. 77-82).

Lipoxygenase (LOG) in protein fractions isolated from the leaves of substituted wheat lines was investigated. Three molecular forms of the enzyme were detected. A water deficiency caused the induction of a membrane-bound form (mLOG) and resulted in a decrease in the activity of “soluble” enzymes (s1LOG) and (s2LOG) in most genotypes. A correlation analysis demonstrated the dependence between the level of enzymatic activity and indices of resistance to drought. A genetic control of the s1LOG and s2LOG activity at an optimal water supply level was associated with chromosomes 1A, 1D, 3A, 5A, 5B, and 5D, while under the conditions of the modeled soil drought, it was associated with chromosomes 1B and 1D.

Influence of environmental factors on the generation of nitric oxide in the roots of etiolated pea seedlings by A. K. Glyan’ko; N. B. Mitanova; A. V. Stepanov (pp. 83-89).

The article studies the nitric oxide (NO) levels in the roots of etiolated seedlings of sowing peas (Pisum sativum L.) using the DAF-2DA fluorescent probe and fluorescent microscopy. Cross sections of roots of 100–150 µm (the site of a root which is 10–15 mm from the apex) are analyzed. It is shown that the level of NO in the roots after 24 h increased by more than a factor of 2 in the versions with NaNO₂ and sodium nitroprusside. At feeding the seedlings with KNO₃, a peak in the accumulation of NO in the roots (twofold increase) was observed after 30 min. Fertilizing seedlings with L-arginine (2 mM) increased the intensity of the fluorescence of the root sections by more than a factor of 2. The inoculation of seedlings of rhizobia (Rhizobium leguminosarum bv. viceae) contributed to the reduction of NO on the background of the control (H2O) and sodium nitroprusside and nitrogen compounds. Scavengers of NO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), hemoglobin) and inhibitors of nitrate reductase and animal NO synthase (sodium tungstate and aminoguanidine hydrochloride) reduced the level of NO in the roots. The results are discussed in relation to the role of NO in plants under the influence of biotic and abiotic factors.

Involvement of salicylic acid and nitric oxide in protective reactions of wheat under the influence of heavy metals by I. R. Gil’vanova; A. R. Enikeev; S. Yu. Stepanov; Z. F. Rakhmankulova (pp. 90-94).

This article studies the effect of salicylic acid (SA) and nitric oxide (NO) on Triticum aestivum L. wheat plants exposed to the influence of high concentrations of copper and zinc compounds. It is shown that heavy metals (HMs) caused a decrease in the growth parameters in the overground and underground plant parts and contributed to a sharp deterioration in the energy balance and the situation regarding oxidative stress. SA and NO exerted a protective effect, which was expressed in the increased ability to accumulate shoot and root mass, stabilize the energy balance, and reduced lipid peroxidation. Resemblance of protective reactions of wheat plants affected by SA and NO appeared as the alteration of energy (Rtot/Pnet and alternative respiration) and antioxidant (MDA) balance of studied plants was identified. The resemblance possibly evidences of common signaling pathway for SA and NO caused by effect of toxic concentrations of heavy metals.

Elicitor activity of chitosan and arachidonic acid: Their similarity and distinction by N. I. Vasyukova; N. G. Gerasimova; G. I. Chalenko; O. L. Ozeretskovskaya (pp. 95-101).

Two elicitors—chitosan and arachidonic acid—induced the same defense responses in potatoes, stimulating the processes of wound reparation and inducing the formation of phytoalexins, inhibitors of proteinase, and active forms of oxygen. However, chitosan induced the defense potential of plant tissues at concentrations higher than those of arachidonic acid. The protective action of chitosan was defined by two parameters, i.e., the ability to induce the immune responses in plant tissues and to exhibit a toxic effect on the pathogen development, causing late blight and seedling blight, whereas the elicitor effect of arachidonic acid depended on its ability to induce the defense potential of plant tissues only.

Influence of the composition of essential oils on their antioxidant and antiradical properties by T. A. Misharina; E. S. Alinkina; L. D. Fatkulina; A. K. Vorobyova; I. B. Medvedeva; E. B. Burlakova (pp. 102-107).

The antiradical and antioxidant properties of essential oil mixtures (EOMs) with various compositions in model systems of hexanal autooxidation, thermal oxidation of methyl linoleate and β-carotene, and in a reaction with the stable diphenylpicrylhydrazyl radical were studied and compared. It was found that all studied EOMs exhibited antiradical activity. The highest antiradical activity was observed for an EOM containing monoterpene hydrocarbons as the main components (the phenol content was low). The antioxidant activity of all EOMs was from 60 to 98% and depended on the model system composition and the method of assay. The mixture with the maximum phenol content exhibits the highest antioxidant activity level in the hexanal autooxidation system. EOMs with a high content of phenols and α- and γ-terpinenes were the most efficient antioxidants in the β-carotene model system. The study confirmed the possibility to vary the antioxidant and antiradical properties of essential oils by preparing their mixtures with a specific composition.

Erratum to: “Synthesis of 3_Hydroxybutyrate_CO_4_Hydroxybutyrate Copolymers by Hydrogen_Oxidizing Bacteria” by T. G. Volova; N. O. Zhila; G. S. Kalacheva; V. A. Sokolenko; E. J. Sinski (pp. 108-108).

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Applied Biochemistry and Microbiology (v.48, #1)