[The Factor Stabilizing the Bioluminescence of PVA-Immobilized Photobacteria].

Immobilization of photobacteria in the cryogel of polyvinyl alcohol (PVA) was carried out. Immobilization was found to result in increased intensity and stability of bioluminescence. The elements determining the stability of bioluminescence were investigated. Selection of the strain was found to be of the highest importance. Among immobilized cells, Photobacterium phosphoreum exhibited the most intense and prolonged light emission, while Vibrio harveyi showed the least one. The technological procedures for cryogenic immobilization of photobacteria were determined. The role of the environment of gel formation in the preservation of the bioluminescence activity was determined. In the gels formed in rich medium for submerged cultivation of photobacteria, almost 100% luminescence activity was preserved, while light emission was considerably prolonged. Bioluminescence intensity of the preparations was shown to depend significantly on pH of the incubation medium. The pH shift to acidic values during prolonged incubation of immobilized cells was shown to be one of the factors of bioluminescence quenching. The stress effects of cryogenic immobilization were found to have an insignificant effect on the temperature profile of bioluminescence. Decreased reduction rate of the luciferase flavin substrate was shown to be a possible reason for bioluminescence quenching.

Photobiosensors containing luminescent bacteria.

The scientific basis for producing luminescent biosensors containing free and immobilized luminescent bacteria is discussed. Modern technologies for engineering target objects, procedures used to immobilize bacteria in different carriers, as well as procedures for integral and specific biodetection of toxins are presented. Data regarding generation and application of biomonitoring for ecotoxicants derived from natural and genetically engineered photobacterial strains are analyzed. Special attention is given to immobilization of photobacteria in polyvinyl alcohol-containing cryogel. The main physicochemical, biochemical, and technological parameters for stabilizing luminescence in immobilized bacteria are described. Results of the application of immobilized photobacterial preparations both during discrete and continuous biomonitoring for different classes of ecotoxicants are presented.

[ATP pool and bioluminescence in psychrophilic bacteria Photobacterium phosphoreum].

Bioluminescence activity and ATP pool were investigated in the culture of psychrophilic bacteria Photobacterium phosphoreum collected-from the exponential and stationary growth phases, as well as immobilized in polyvinyl alcohol (PVA) cryogel. In liquid culture, ATP pool remained at an almost a constant level throughout the luminescence cycle (over 100 h). The ATP pool in the stationary-phase and PVA-immobilizedl cells remained constant throughout their incubation in the medium (over 200 h) and in 3% NaCl solution (over 100 h): Quantitative assessment of integral photon yield and ATP pool indicated that bioluminescence decay in growing or stationary cells was not caused by limitation by the energy substrates of the luciferase reaction. Kinetic and quantitative parameters of emission activity and ATP pool excluded the possibility of formation of the aldehyde substrate for luciferase via reduction of the relevant fatty acids in NADPH and ATP-dependent reductase reaction and its oxidation in the monooxygenase reaction. Our results indicate that the aliphatic aldehyde is not utilized in the process of light emission.

[Biosensors based on the luminous bacteria Photobaterium phosphoreum immobilized in polyvinyl alcohol cryogel for the monitoring of ecotoxicants].

Immobilization of Photobacterium phosphoreum bacteria in polyvinyl alcohol cryogel was performed in order to develop biosensors used for ecotoxicant biomonitoring. The immobilization procedure, storage, and application of the immobilized cells for biomonitoring were optimized. It was shown that the immobilized cells demonstrate significantly higher stability and a longer duration of light emission than free bacteria. A discrete analysis of heavy metals and chlorophenols was conducted using the obtained biosensor samples.

[The dependence of intracellular ATP level on the nutrition mode of the acidophilic bacteria Sulfobacillus thermotolerans and Alicyclobacillus tolerans].

The dynamics of the ATP pool in the aerobic spore-forming acidothermophilic mixotrophic bacteria Sulfobacillus thermotolerans Kr1T and Alicyclobacillus tolerans K1T were studied in the course of their chemolithoheterotrophic, chemoorganoheterotrophic, and chemolithoautotrophic growth. It was established that, during mixotrophic growth, the maximum ATP concentrations in the cells of S. thermotolerans Kr1 and A. tolerans K1 were 3.8 and 0.6 nmol/mg protein, respectively. The ATP concentrations in sulfobacilli and alicyclobacilli during organotrophic growth were 2.2 and 3.1 nmol/mg protein, respectively. In the cells of the obligately heterotrophic bacterium Alicyclobacillus cycloheptanicus 4006T, the maximum ATP concentration was several times higher and reached 12.3 nmol/mg protein. During lithotrophic growth, the maximum values of the ATP concentration in the cells of S. thermotolerans Kr1 and A. tolerans K1 were 0.3 and <0.1 nmol/mg protein, respectively; in the cells of the autotrophic bacterium Acidithiobacillus ferrooxidans TFBk, the ATP content was about 60-300 times higher (17.0 nmol/mg protein). It is concluded that low ATP content is among the possible causes of growth cessation of S. thermotolerans Kr1 and A. tolerans K1 under auto- and heterotrophic conditions after several culture transfers.

[Inhibitory effects of phenolic ecotoxicants on photobacteria at various pH values].

Kinetic characteristics of light emission by intact cells of the photobacteria Photobacterium phosphoreum and Vibrio harveyi at pH 5.5, 7.0, and 8.0 were studied as well as specific features of inhibitory effects of 2,4-di- and 2,4,5-triphenoxyacetic acids (2,4-D and 2,4,5-T), pentachlorophenol (PCP), and 2,6-dimethylphenol (2,6-DMP) at the same pH values. Nonstationarity of emission kinetics was observed at all the pH values studied. Exponential luminescence decay in a 60-sec range was observed at pH 5.5; a 5-min luminescence activation, at pH 7.0 and 8.0. The cell respiratory activity drops by over one order of magnitude at pH 5.5 compared with the activities at pH 7.0 and 8.0. The inhibitory effects of 2,4-D, 2,4,5-T, and PCP differ by one-two orders of magnitude depending on pH. The maximal cell sensitivity to these compounds appears at pH 5.5; the minimal, at pH 8.0. The effect of 2,6-DMP is independent of pH. As is demonstrated, it is hydrophobicity of the molecule and pK values of the toxicants that determine the inhibitory effect. Characteristic of the substrate-starved photobacterial cells are higher sensitivity to chlorophenolic compounds compared with the cells provided with high energy supply at all the pH values.

[Effect of Na+ and K+ ions on the luminescence of intact Vibrio harveyi cells at different pH values]. / Vliianie ionov Na+, K+ na liuminestsentnuiu aktivnost' intaktnykh kletok Vibrio harveyi pri razlichnykh pH.

The bioluminescent activity of intact Vibrio harveyi cells loaded with different concentrations of NaCl and KCl at different pH values was studied. In the pH range of 6.5-8.5, the effect of Na+ was significantly higher than that of K+ at all concentrations studied. Maximum luminescent activity was observed in cells loaded with 0.68 M NaCl. When Na+ was nonuniformly distributed on the plasma membrane, the cell luminescence kinetics was nonstationary in the 20-min range: during incubation, the luminescence intensity increased at pH 6.5 and decreased at pH 8.5. The activation and damping rate constants depended on the Na+ gradient value. The maximum of luminescent activity shifted during incubation from pH 8.5 to 6.5-7.0. The luminescence kinetics in the systems with KCl was stationary; the maximum level of luminescence was observed in the pH range of 7.0-7.5. Under Na(+)-controlled conditions, the cell respiration and luminescence changed in synchronism. The protonophore CCP at a concentration of 20 microM completely inhibited luminescence at pH 6.5 and was ineffective at pH 8.5.

[Inhibition of bacterial bioluminescence by chlorophenols]. / Ingibirovanie bakterial'noi bioliuminestsentsii khlorfenolami.

Photobacteria were used as a test object for rapid monitoring of ecotoxicants. Specific inhibitory effects of phenol and its chlorinated derivatives (2-chlorophenol, 2,3-dichlorophenol, pentachlorophenol, 2,4-dichlorophenoxyacetic acid, and 2,4,5-trichlorophenoxyacetic acid) on bioluminescence and respiration of intact cells, as well as on the emission activity of the bioluminescence system and luciferase itself, were studied. The toxic effect on the photobacterial cells was found to increase as the number of chlorine atoms in the chlorophenol molecule increases. However, this trend was not observed in cell-free systems (purified luciferase or the protein fraction of a cell-free extract treated with (NH)4SO4 at 40-75% saturation). Bacterial cells have a higher threshold sensitivity to chlorophenols in comparison to the sensitivity of the bioluminescence enzyme system or luciferase. Neutral phenols inhibit luciferase by competing with decanal, whereas a mixed mechanism of inhibition with this substrate is typical of phenoxyacetic acids. With respect to FMNH2, all chlorophenols tested in this work were uncompetitive inhibitors. Oxygen uptake by photobacteria was shown to be insensitive to chlorophenols, at least within the concentration range that was effective in bioluminescence inhibition. The results of this study suggest that bacterial bioluminescence system is not the primary target of the chlorophenol-induced effect on photobacteria.

Bioluminescence decay kinetics in the reaction of bacterial luciferase with different aldehydes.

At 22 degrees C the bioluminescence decay kinetics in the in vitro reaction catalysed by Vibrio harveyi luciferase in the presence of different aldehydes--nonanal, decanal, tridecanal and tetradecanal did not follow the simple exponential pattern and could be fitted to a two-exponential process. One more principal distinction from the first-order kinetics is the dependence of the parameters on aldehyde concentration. The complex bioluminescence decay kinetics are interpreted in terms of a scheme, where bacterial luciferase is able to perform multiple turnovers using different flavin species to produce light. The initial phase of the bioluminescent reaction appears to proceed mainly with fully reduced flavin as the substrate while the final one results from the involvement of flavin semiquinone in the catalytic cycle.

The inhibition of bacterial bioluminescence by xenobiotics.

The effect of various xenobiotic substrates of microsomal cytochrome P-450, including dimethylaniline, ethylmorphine, hexabarbital and aminopyrine, on the bioluminescence of the bacteria Vibrio fischeri and the bacterial luciferase mixed-function oxidase system is described. These compounds are effective inhibitors of the luminescence reaction. The inhibition provided evidence for the competitive nature of the interactions between xenobiotics and an aliphatic aldehyde, which is a substrate of bacterial luciferase, at the binding site for cytochrome P-450. The bioluminescence method is suitable for the analysis of metabolism and detoxication of various xenobiotics.

[Lactate dehydrogenase activity and its isoenzyme in a system coupled with bacterial luciferase]. / Issledovanie aktivnosti laktatdegidrogenazy i ee izofermentov v sopriazhennoi sisteme s bakterial'noi liutsiferazoi.

Properties of a coupled system "LDH-bacterial luciferase" were studied. The light-generating enzymatic system from luminescent bacteria Photobacterium fisheri was used as an indicator of the dehydrogenase activity. Kinetic parameters of the coupled system were studied using commercially available preparation of LDH and the enzyme from human blood plasma. The luminescent activity of bacterial preparation was shown to correlate with the activity of LDH and its isoenzymes within wide range of blood plasma concentrations.

[Inhibitory analysis of the luminescent electron transport chain of Photobacterium fischeri]. / Ingibitornyi analiz liuminestsentnoí elektrontransportnoi tsepi Photobacterium fischeri.

The quenching of luminescence of bacterial luciferase from Photobacterium fischeri by non-specific electron acceptors and inhibitors of dehydrogenases was studied. The inhibition of the luminescent reaction obeys the non-competitive mechanism with NADH, FMN and aliphatic aldehyde. The inhibitors compete with cytochrome c for NADH -- cytochrome c oxido-reductase. It is concluded that lumiredoxin, a FeS-containing protein, is the most sensitive component of the luminescent electron transport chain.

[Cytochromes of the luminescent bacterium, Photobacterium fischeri, their solubilization and relationship to luminescence]. / Tsitokhromy liuminestsentnykh bakterii Photobacterium fischeri, ikh soliubilizatsiia i sviaz' so svecheniem.

The hemoprotein composition of the luminescent bacterium Photobacterium fischeri was studied, in particular, the distribution of cytochromes among the bacterial fractions, viz. cell-free extract, supernatant, "particles", protein preparation. The hemochromogenic analysis has shown that the principal hemoproteins of Photobacterium fischeri are cytochromes, with hemes of the b and c type. The activity of luciferase is distributed with hemoproteins. The purified preparation of luciferase contains cytochrome of the b type that has been identified as mixed functions oxidase--P-450.

[Electron transport systems of Photobacterium fischeri]. / Elektrontransportnye sistemy Photobacterium fischeri.

The composition of cytochromes was studied in Photobacterium fischeri 6 at different growth phases and under various conditions of cultivation. The electron transport chains of the bacterium are characterized by the presence of cytochromes b and c types. The terminal oxidases are cytochromes o, a2+a1 and P-450. The hemoprotein P-450 functions as a mixed function oxidase. The qualitative composition of cytochromes does not depend on the growth phase of the bacterium but does on the conditions of cultivation. The bacterium synthesizes cytochromes a2+a1 in the conditions of oxygen deficiency. The synthesis of cytochrome o depends on the composition of the medium.

[Ion currents in mitochondrial and liposome membranes induced by acid action]. / Ionnye potoki v membranakh mitokhoncrii i liposom, indutsirovannye deistviem kislot.

It is shown that potassium ion yield from mitochondria observed under low pH of the incubation medium and at the addition of n-(N,N-di-2-Chloridethyl)aminophenylacidic acid and low concentrations of 2,4-dinitrophenol results from the formation of non-electrogenic (nigericyn-like) carrier. The data are obtained which show that under experimental conditions a weak electrogenic transport of ions is observed along with non-electrogenic ion transport in the membranes.