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New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
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Applied Biochemistry and Microbiology (v.40, #4)
Extracellular Factors of Bacterial Adaptation to Unfavorable Environmental Conditions by Yu. A. Nikolaev (pp. 327-336).
Data on extracellular compounds of bacteria involved in their adaptation to unfavorable environmental conditions are reviewed, including high or low temperatures, growth-inhibiting or bactericidal concentrations of toxic substances (oxidants, phenols, and heavy metals) and antibiotics, deviation of pH values from optimum levels, and salinity of the medium. Chemically, the compounds identified belong to diverse types (proteins, hydrocarbons, organic acids, nucleotides, amino acids, lipopeptides, volatile substances, etc.). Most of them remain unidentified, and their properties are studied using biological testing. It has been proposed to view extracellular adaptation factors (EAFs) as a new group of biologically active substances. EAFs may be divided into several subgroups by the mechanism of action. These subgroups include protectors (stabilizers), signaling molecules inducing defense responses, regulators (e.g., adhesion regulators) not acting as inducers, and antidotes (neutralizers). The fields of EAF study include screening (search for new compounds using biological tests), identification, and research into the mechanisms of action. EAFs may find use in biotechnology, medicine, agriculture, and environmental protection.
Inhibition of Urease by Cyclic β-Triketones and Fluoride Ions by E. I. Tarun; D. B. Rubinov; D. I. Metelitza (pp. 337-344).
Competitive inhibition of soybean urease by 11 cyclic β-triketones was studied in aqueous solutions at pH 7.4 and 36°C. This process was characterized quantitatively by the inhibition constant (K i), which showed a strong dependence on the structure of the organic chelating agents (nickel atoms in urease) and varied from 58.4 to 847 μM. Under similar conditions, the substrate analogue (hydroxyurea) acted as a weak urease inhibitor (K i = 6.47 mM). At 20°C, competitive inhibition of urease with the ligand of nickel atoms (fluoride anion) was pH-dependent. At pH 3.85–6.45, the value of K i for the process ranged from 36.5 to 4060 μM. Three nontoxic cyclic β-triketones with K i values of 58.4, 71.4, and 88.0 μM (36°C) were the most potent inhibitors of urease. Their efficacy was determined by the presence of three >C=O– groups in the molecule and minimum steric hindrances to binding with metal sites in soybean urease.
A Study of the Adhesive Glycoprotein-Inactivating Protein from Mammalian Blood Serum by V. P. Yamskova; E. Yu. Rybakova; A. A. Vinogradov; V. V. Vecherkin; I. A. Yamskov (pp. 345-350).
A protein with a molecular weight of 70 kDa was isolated from bovine blood serum and purified to a homogenous state. This protein reversibly inhibited the adhesive serum glycoprotein with a molecular weight of 12 kDa, which displayed biological activity at ultralow doses. Amino acid analysis showed that the protein inactivator belongs to the group of prealbumins from vertebrate blood serum. The secondary structure of its molecule was characterized by a considerable number of α-helices. The conditions for inactivation of serum glycoprotein were studied. The interaction between the serum glycoprotein and the protein inactivator occurred over a long period of time (1 day). It should be emphasized that the presence of calcium ions was a necessary condition for the inactivation of the serum glycoprotein. The data suggest that inactivation of serum glycoprotein results from the formation of a molecular complex consisting of the protein inactivator and the glycoprotein, which is related to the carbon–protein interaction.
Bromoacyl Analogues of Phosphatidylcholine with Intramolecular Fluorescence Quenching and Their Use as Substrates for Continuous Monitoring of Phospholipase A2 Activity by S. V. Babitskaya; M. A. Kisel; P. A. Kisselev (pp. 351-356).
Two new derivatives of phosphatidylcholine with intramolecular fluorescence quenching were obtained by substituting residues of pyrene butyric and bromine-containing fatty acids for acyl chains. The two compounds can be used for quantitative evaluation of the catalytic activity of pancreatic phospholipase A2 in kinetic mode.
Formation of DNA Complexes with Actinomycins in Aqueous Solutions and Films by I. V. Savintsev; N. L. Vekshin (pp. 357-364).
The mechanisms of DNA interaction with actinomycin D (AMD), 7-amino-actinomycin D (7-AAMD), and ethidium bromide (EtBr) were studied in aqueous solutions and in the condensed state (films coating plates). The use of the methods of absorption (UV, IR, and visible spectral ranges) and fluorescence (steady-state, polarization, and phase-modulation) spectroscopy revealed that (1) the formation of DNA complexes with 7-AAMD in solution was not accompanied by energy transfer from photoexcited nucleotides to phenoxazone chromophore and (2) the mechanism of ligand incorporation was distinct from stacking. In the film of the DNA–7-AAMD complex, which simulated the native state in a biological cell, the energy transfer efficiency was high. This indicates that a stacking-type mechanism underlies actinomycin intercalation into DNA. In the presence of high concentrations of 7-AAMD in the film, DNA denatured and its double-helical structure degraded. In the DNA–AMD complex, the native B-form of DNA molecule was conserved both in films and in solution.
Hydrolysis of Heparin by the Immobilized Enzymatic Complex from Streptomyces kurssanovii by G. E. Bannikova; P. P. Stolbushkina; N. N. Drozd; G. A. Vikhoreva; V. P. Varlamov; V. A. Makarov; A. V. Panov (pp. 365-369).
The possibility of obtaining low-molecular-weight heparins using a chitinolytic enzymatic complex immobilized on Silochrom has been demonstrated. The optimal conditions of this process (sodium acetate buffer, pH 7.0–7.5; temperature, 40–45°C; and duration of hydrolysis, 3 h) were determined. Depending on the ratio between heparin and the immobilized enzymatic complex, samples with molecular weights varying from 1.7 to 4.7 kDa, were obtained. These complexes inhibited factor Xa 2.0–3.7 times more effectively than the original heparin.
Hydrolysis of Peptides by Immobilized Bacterial Peptide Hydrolases by A. D. Neklyudov; E. K. Denyakina (pp. 370-375).
The feasibility of hydrolysis of a mixture of peptides with an enzyme from the bacterium Xanthomonas rubrilineans, displaying a peptidase activity and immobilized on aluminum oxide, was studied. Kinetic schemes and equations allowing one to approach quantitative descriptions of peptide hydrolysis in complex mixtures containing free amino acids and peptides were obtained. It was demonstrated that as a result of hydrolysis, the content of free amino acids in hydrolysates decreased 2.5- to 3-fold and the molecular weight of the constituent peptides, 2-fold.
Effect of Iron Hydroxide on Phosphate Removal during Anaerobic Digestion of Activated Sludge by V. P. Stabnikov; S. T.-L. Tay; D.-Kh. Tay; V. N. Ivanov (pp. 376-380).
The addition of iron (III) hydroxide during methanogenic digestion of activated sludge by anaerobic sludge displaying an iron-reducing activity resulted in a microbial reduction of iron (III) with the formation of iron (II), capable of precipitating phosphates. The feasibility of eliminating 66.6 to 99.6% of the dissolved phosphate at initial concentrations of 1000 to 3500 mg PO3- 4/l by adding 6420 mg/l iron (III) hydroxide into a reactor for anaerobic fermentation of activated sludge was analyzed. The optimal ratio of iron (III) added to dissolved phosphate removed (mg) providing a 95% removal amounted to 2 : 1. These results may be used in new technology for anaerobic wastewater treatment with phosphate removal.
Effect of Cultivation Conditions on the Growth and Activities of Sulfur Metabolism Enzymes and Carboxylases of Sulfobacillus thermosulfidooxidans subsp. asporogenes Strain 41 by M. A. Egorova; I. A. Tsaplina; L. M. Zakharchuk; T. I. Bogdanova; E. N. Krasil'nikova (pp. 381-387).
The moderately thermophilic acidophilic bacterium Sulfobacillus thermosulfidooxidans subsp. asporogenes strain 41 is capable of utilizing sulfides of gold–arsenic concentrate and elemental sulfur as a source of energy. Growth in the presence of S0 under auto- or mixotrophic conditions was less stable than in media containing iron monoxide. The enzymes involved in the oxidation of sulfur inorganic compounds—thiosulfate-oxidizing enzyme, tetrathionate hydrolase, rhodanase, adenylyl phosphosulfate reductase, sulfite oxidase, and sulfur oxygenase—were determined in the cells of the sulfobacilli grown in mineral medium containing 0.02% yeast extract and either sulfur or iron monoxide and thiosulfate. Cell-free extracts of the cultures grown in the medium with sulfur under auto- or mixotrophic conditions displayed activity of the key enzyme of the Calvin cycle—ribulose bisphosphate carboxylase—and several other enzymes involved in the heterotrophic fixation of carbon dioxide. Activities of carboxylases depended on the composition of the cultivation media.
Microorganisms of Lake Baikal and Lake Nyasa as Indicators of Anthropogenic Influence: Prospects for Use in Biotechnology by V. A. Verkhozina; E. V. Verkhozina; D. A. Gonchar; V. S. Dedkov; S. Kh. Degtyarev; Yu. S. Kusner (pp. 388-391).
Restriction endonucleases (RENs) were detected in 650 microbial strains isolated from water columns and bottom sediments of deep rift lakes, Baikal (Russia) and Nyasa (Southeastern Africa). They enzymes included unique (Fan I, Aca I, and Sse 91) and very rare (Bsi I, and Cci N I) species not typical of aquatic ecosystems. Water columns, deep cores, and bottom sediments of pure areas of the lakes contained no microorganisms with new RENs. Thus, the inshore areas of Lake Baikal, having been exposed to anthropogenic influences, may contain mutant bacterial strains expressing RENs that have not been described previously.
Carotenoids and Fatty Acids in Red Yeasts Sporobolomyces roseus and Rhodotorula glutinis by P. Davoli; V. Mierau; R. W. S. Weber (pp. 392-397).
Rhodotorula glutinis and Sporobolomyces roseus, grown under different aeration regimes, showed differential responses in their carotenoid content. At higher aeration, the concentration of total carotenoids increased relative to the biomass and total fatty acids in R. glutinis, but the composition of carotenoids (torulene > β-carotene > γ-carotene > torularhodin) remained unaltered. In contrast, S. roseus responded to enhanced aeration by a shift from the predominant β-carotene to torulene and torularhodin, indicating a biosynthetic switch at the γ-carotene branch point of carotenoid biosynthesis. The overall levels of total carotenoids in highly aerated flasks were 0.55 mol-percent and 0.50 mol-percent relative to the total fatty acids in R. glutinis and S. roseus (respectively), and 206 and 412 μg g–1 dry weight (respectively).
Effect of 5-Azacytidine on the Light-Sensitive Formation of Sexual and Asexual Reproductive Structures in wc-1 and wc-2 Mutants of Neurospora crassa by S. Yu. Filippovich; G. P. Bachurina; M. S. Kritsky (pp. 398-403).
Under nitrogen starvation conditions, illumination by blue light of wc-1 and wc-2 mutants of the ascomycete Neurospora crassa failed to stimulate the formation of protoperithecia and inhibit condition (contrary to what was observed in the mycelium of the wild-type fungus). The data obtained indicate that wc-1 and wc-2 genes of N. crassa are involved in the light-dependent formation of protoperithecia and conidia. The effects of 5-azacytidine (an inhibitor of DNA methylation) under the same experimental conditions suggest that the balance between the formation of sexual and asexual reproductive structures, maintained in N. crassa, depends on genome methylation processes sensitive to the action of light, which is mediated by the photoreceptor complex of WC proteins.
Comamonas testosteroni Strain TI as a Potential Base for a Microbial Sensor Detecting Surfactants by L. A. Taranova; A. P. Fesay; G. V. Ivashchenko; A. N. Reshetilov; M. Winther-Nielsen; J. Emneus (pp. 404-408).
Strain Comamonas testosteroni TI, capable of degrading the nonionic surfactant (NIS) nonylphenolethoxylate (OP-10), was used for constructing a pilot cellular biosensor. The lower NIS detection limit for the biosensor was 0.25 mg/l. We studied the substrate specificity of the biosensor with respect to a wide range of organic compounds: surfactants, polyaromatic compounds (PAC), carbohydrates, alcohols, etc. It was shown that the biosensor based on Comamonas testosteroni TI did not respond to glucose, which was an advantage over the formerly described biosensor based on Pseudomonas rathonis T. The amplitude of the sensor response remained stable for 10 days.
Determination of Tetanus Toxin and Toxoid by ELISA Using Monoclonal Antibodies by M. A. Burkin; V. V. Sviridov; O. V. Perelygina (pp. 409-414).
The procedure for obtaining monoclonal antibodies TT-1, TT-2, and TT-3 against tetanus toxin/toxoid is described. It is shown that the commercial DTP vaccine and tetanus toxoid conjugated with a low-molecular-weight hapten can both be used as immunogens. Monoclonal antibodies TT-1 and TT-2 neutralized tetanus toxin in vivo. The monoclonal antibodies obtained were used to design and compare several schemes of quantitative determination of tetanus toxoid and toxin by ELISA. A more sensitive competitive ELISA allowed the detection of as much as 0.01 EC/ml toxoid and 50 LD50/ml toxin.
Interaction of Perch Fucolectin with Lewis Antigens by V. E. Piskarev; T. L. Busheva; I. A. Yamskov (pp. 415-417).
The interaction of fucolectin of perch Perca fluviatilis (PFL) with a set of Lewis antigens was studied by monitoring changes in its tryptophan fluorescence. PFL bound Lec (H type 1)-pentasaccharide (K a = 6.6 × 103 М–1) and H type 6-trisaccharide (K a = 2.5 × 103 М–1); bound, although less strongly, with Leb-hexasaccharide (K a = 4.0 × 102 М–1); and failed to interact with Lea-, Lex-, and Led-containing oligosaccharides. PFL belongs to a new type of the fucolectins recognizing H-disaccharide Fucα1-2Gal within various antigens, including H type 1/2 and Leb.
Effect of Ambiol on the Ultrastructure of Mitochondria in the Apical Cells of Tubers of Original and Transgenic Potato Plants by T. A. Platonova; A. S. Evsyunina; N. P. Korableva (pp. 418-425).
The ultrastructure of the mitochondrial apparatus of apical tuber cells of original and transgenic (defensin gene-transfected) potatoes have been compared in normal and ambiol-treated plants, using morphometric approaches. No qualitative or quantitative differences were found between the mitochondria of original and transgenic plants under normal conditions (control). Treatment with ambiol produced only quantitative differences (in the number of mitochondria and their volume) between the cells of original and transgenic plants. This observation has been attributed to (1) changes in the physiology and biochemistry of transgenic plants, induced by the expression of the gene of defensin (hormonal balance, functional activity of the plasmalemmata, etc.), and (2) direct effects of ambiol.