[Bacterial Outer Membrane Nanovesicles: Structure, Biogenesis, Functions, and Application in Biotechnology and Medicine (Review)].

The review summarizes the comprehensive biochemical and physicochemical characteristics of extracellular membrane nanovesicles (EMN) derived from different kinds of bacteria. The EMN structure, composition, biogenesis, secretion mechanisms, formation conditions, functions, involvement in pathogenesis, and application in biotechnology and medicine are discussed.

[Excretion of extracellular membrane nanovesicles by aeromonas].

AIM: Study of extracellular membrane nanovesicles production by Aeromonas hydrophila and Aeromonas salmonicida bacteria on a subcellular level. MATERIALS AND METHODS: 4 strains of A. hydrophila: 342-1, E 8-8, H 336 and H 1-6-05 and 1 strain of A. salmonicida A-450 as well as intact Wistar line rats were used. Methods of transmission electron microscopy: ultrathin sectioning and negative contrasting were used. RESULTS. A. hydrophila and A. salmonicida bacteria produced in pure cultures excrete into the environment extracellular membrane nanovesicles. The size of these vesicles varies from 20 to 200 nm in diameter. The process of gemmation from bacterial cell and possibility of obtaining isolated membrane nanovesicles preparations is shown. These vesicles are detected in ultrathin sections of apical surface of intact rat intestine among accumulations ofparietal microorganisms that colonize mucous membrane. Extracellular membrane nanovesicles excreted by aeromonas are analogous by size and ultrastructure to vesicles of other species of gram-negative bacteria described in the literature. CONCLUSION: During production of A. hydrophila and A. salmonicida bacteria in vitro nanovesicles are formed from the outer membranes of the cells and excreted into the environment, the nanovesicles are similar to those detected in ultrathin sections of the surface of intestine of rats among accumulations of parietal microorganisms and in glycocalix between epitheliocyte microvilli.

Constitutive biosynthesis and localization of alcohol oxidase in the ethanol-insensitive catabolite repression mutant ecr1 of the yeast Pichia methanolica.

The activity and localization of alcohol oxidase (EC 1.1.3.13) have been studied in the Pichia methanolica mutant ecr1 defective in ethanol-induced catabolite repression of enzymes of methanol utilization. Ultrasctuctural, immunocytochemical, and biochemical analyses revealed the presence of peroxisomes containing active alcohol oxidase in the mutant grown in media with methanol, ethanol, and a mixture of both substrates. No alcohol oxidase was detected in the wild-type cells (ECR1) grown on ethanol-containing media. Mutant ecr1 growing in medium containing a mixture of different alcohols and the wild-type strain growing on methanol demonstrated similar buoyant density of peroxisomes (1.24-1.27 g/cm3)during isopicnic centrifugation of the organelles in sucrose density gradients. The integrated genetic, immunocytochemical, and biochemical data are in agreement with the model that synthesis, translocation into peroxisomes, and assembly of alcohol oxidase in P. methanolica may not require any regulatory signals induced by methanol.

[Physiologo-biochemical characteristics if Gluconobacter oxydans and prospects for its use in biotechnology and biosensor systems (review)]. / Fiziologo-biokhimicheskie osobennosti Gluconobacter oxydans i perspektivy ispol'zovaniia v biotekhnologii i biosensornykh sistemakh (obzor).

Gluconobacter oxydans possesses a unique organization of metabolic systems, which are characterized by reduction of major dissimilation pathways, surface localization of main oxidative enzymes responsible for partial oxidation of carbon substrates, high performance of electron-transport chains, and accumulation of partially oxidized metabolites in the medium. These features allow us to use the cells of these microorganisms in biotechnology for production of several food products and medicines. The use of G. oxydans in biosensors for estimation of concentrations of sugars, aldoses and polyalcohols is promising. Physiological and biochemical features of these microorganisms enabling their use in biotechnology and receptor elements of biosensors are reviewed.

[Formation of resting cells in microbial suspensions undergoing autolysis]. / Obrazovanie pokoiashchikhsia form v avtoliziruiushchikhsia suspenziiakh mikroorganizmov.

Under experimentally selected conditions favoring spontaneous or induced autolysis of cell suspensions, the asporogenous bacteria Escherichia coli and Methylococcus capsulatus, the bacilli Bacillus cereus (under conditions of suppressed sporulation), and the yeast Saccharomyces cerevisiae were shown to be capable of forming cystlike resting cells. Their number was influenced by (1) cell density in the suspensions; (2) the presence of Ca2+ ions in nutrient-limited medium; (3) pH of medium; and (4) autolysis rate, dependent on the concentration of oleic acid (a chemical analogue of the autolysis-inducing d2 factor) introduced into the cell suspensions.

[Formation of a resting form of Bacillus cereus and Micrococcus luteus]. / Obrazovanie pokoiashchikhsia form Bacillus cereus i Micrococcus luteus.

Under certain cultivation conditions, the bacteria Bacillus cereus and Micrococcus luteus form cystlike refractive cells (up to 60% of the total number) that retain viability over a long time, are metabolically inactive and thermotolerant and possess specific ultrastructure. These properties allow them to be attributed to a new type of resting forms of microorganisms.

Molecular forms of lipases and their localization in the fungus Rhizopus microsporus by immuno-electron microscopy.

Several lipases differing in their molecular masses (24, 32, 43, 66 and 98 kDa and 28, 40, 45 and 69 kDa) were found in Rhizopus microsporus UzLT-4B and UzLT-5C, respectively. The lipases in each strain were immunologically related. Strain UzLT-5C grown on a medium with lipid substrate secreted lipases of 32, 66 and 98 kDa whereas strain UzLT-4B produced lipases of 45 and 69 kDa on the same medium. Immuno-electron microscopy indicated that the intracellular lipases were in peripheral zones of the hyphae, primarily in the periplasm and adjacent vesicles. Immobilization in Ca-alginate gel revealed unusual structures in the cell wall. These structures accumulated lipases and, apparently, exported the enzymes out of the cell.

[Degradation of alkylsulfates by a Pseudomonas aeruginosa culture immobilized on a polyvinyl alcohol fiber]. / Razrushenie alkilsul'fatov kul'turoi Pseudomonas aeruginosa, immobilizovannoi na polivinilspirtovom volokne.

Pseudomonas aeruginosa cells capable of destroying alkyl sulfates, anionic surfactants, were immobilised on activated polyvinyl alcohol fibres. The immobilised cells could decompose SDS. When the immobilised cells were used repeatedly, their biomass increased but the activity hardly changed.

[Immobilization of E. coli cells in polyacrylamide-based microporous cryogels]. / Immobilizatsiia kletok E. coli v maloporistye kriogeli na osnove poliakrilamida.

E. coli cells were immobilized in polyacrylamide cryogel by three ways: (1) introduction of cells in the reaction mixture followed by cryopolymerization; (2) the filling of the cryogel pores followed by cell fixation with diluted glutaric dialdehyde (GDA), and (3) the filling of the macropores of the polymeric matrix with modified surface. The ultrastructure of the gels and immobilized cells as well as distribution of attachment of the cells immobilized by different techniques were studied. The first type of immobilization was characterized by the highest quantity of the biomass in the gel (by protein) and by a sharp decrease of the cell viability. The second failed to retain the cells in the pores, and the GDA treatment significantly decreased the viability index. The latter technique was the mildest and completely maintained the viability of the population. However, the biomass content was lower as compared to the first type of immobilization, but could be considerably increased by the GDA treatment.

[Selection of Escherichia coli and Pseudomonas putida cultures with an enhanced resistance to immobilization on polyacrylamide gel]. / Selektivnyi otbor kul'tur Escherichia coli i Pseudomonas putida s povyshennoi ustoichivosti k protsessu immobilizatsii v poliakrilamidnom gele.

Clones of Escherichia coli (A4, A70, G60) and Pseudomonas putida (A70, G30) with an elevated resistance to the process of immobilization in polyacrylamide gel and to the action of monomeric acrylamide were selected from the parent E. coli IBPM B115 and P. putida. The isolated cultures remained resistant to the above actions for a long time. The frequency at which cells with the elevated resistance appeared was comparable with the frequency of bacterial mutations. The plasmid analysis did not reveal the presence of plasmid DNA in the cells of the isolated cultures. The decrease in the viability index of bacterial populations caused by their immobilization in polyacrylamide gel and by the action of monomeric acrylamide did depend on the growth phase. The cells were more resistant to these actions in the stationary phase. The isolated cultures were more resistant as compared to the parent cultures irrespective of the growth phase.

[Protective action of antioxidants on Escherichia coli cells immobilized in polyacrylamide gel]. / Zashchitnoe deistvie antioksidantov na Escherichia coli pri immobilizatsii kletok v poliakrilamidnom gele.

The object of this work was to find out whether antioxidants could be used for weakening the effect of free radicals on Escherichia coli cells immobilized in polyacrylamide gel. Some of the antioxidants soluble in lipids and water (ionol, Epigid, glutathione) protected the cells against the action of free radicals produced in the process of acrylamide polymerization, and increased the viability of the immobilized bacteria.

[Prediction of microorganism resistance to the immobilization process in polyacrylamide gel]. / O prognozirovanii ustoichivosti mikroorganizmov k protsessu immobiliztasii v poliakrilamidnom gele.

It is shown that the immobilization of bacterial cells in polyacrylamide gel or their exposure to monomer acrylamide results in a quantitatively similar decrease of their viability. It is indicated that acrylamide treatment may be used as a test for measuring the resistance of microbial populations to polyacrylamide gel immobilization and predicting the survival rate of microorganisms incorporated.

[Action of acrylamide on Escherichia coli cells]. / Deistvie akrilamida na kletki Escherichia coli.

The action of acrylamide on Escherichia coli B was studied: short-term action at high concentrations, long-term action at low doses under the normal conditions of growth and in the process of cell immobilization in polyacrylamide gel. Such a treatment was found to cause considerable structural changes in the cells. Division of the cells was inhibited and they reached giant sizes when grown in media containing 1-2% of acrylamide. The phenomenon might be used for differentiation of living and dead E. coli cells.

[Influence of various factors in polyacrylamide gel immobilization on the viability of Escherichia coli B cells]. / Vliianie nekotorykh faktorov immobilizatsii v poliakrilamidnom gele na zhiznesposobnost' populiatsii kletok Escherichia coli B.

The entrapment of an E. coli cell population into polyacrylamide gel (PAAG) is associated with a drastic decline of its viability. The effect of immobilization factors (the reagents used for PAAG preparation, their mixtures and the elevated temperature) upon the viability of the E. coli cell population was investigated by the method of microcultural analysis. It was found that all the components of the polymerization mixture, except for acrylamide, as well as the washed-off granules of polymerized del did not appreciably influence that viability of the cell population. Acrylamide at a concentration of 10% appeared very toxic. The decrease of its concentration to 5% and of the initial temperature of the polymerization mixture markedly lowered the toxic effect and, consequently, increased the viability of the population of immobilized E. coli cells.

Physiological and biochemical properties and morphology of Saccharomyces cerevisiae VKMu-488 cells incorporated into polyacrylamide gel.

The enzymatic activity, viability, respiratory activity, and ultrastructural changes in saccharomyces cerevisiae VKMu-488 cells, which carry out the stereospecific 17 beta-reduction of methyl esters, was studied. The 17 beta-hydroxysteroid dehydrogenase activity of yeasts in gel is four times lower than that of free cells and is unstable. The decrease in the viability and respiratory activity immediately after immobilization, the disturbance in the ultrastructure of the cells in gel along with the progressive lysis of the cells in the course of the transformation indicate that polymerization has a stressful effect on this culture. It was found that the immobilized yeasts can grow on the surface of the gel in the presence and absence of nutrient medium. A single incubation of granules containing cells in nutrient medium greatly stabilizes the original activity of the immobilized cells. The activation and stabilization of the activity are probably due to the participation of a heterogeneous population in the transformation: the original population incorporated into the gel and the new population which grows in the gel after immobilization as well as to the stability of the ultrastructural organization of this mixed population in the course of repeated transformations of secoketone.