[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.

[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.

[The effect of Clp proteins on DnaK-dependent refolding of bacterial luciferases]. / Vliianie belkov semeistva Clp na DnaK-zavisimyi refolding bakterial'nykh liutsiferaz.

A study was made of the refolding of bacterial luciferases of Vibrio fischeri, V. harveyi, Photobacterium phosphoreum, and Photorhabdus luminescens. By reaction rate, luciferases were divided into two groups. The reaction rate constants of fast luciferases of V. fischeri and Ph. phosphoreum were about tenfold higher than those of slow luciferases of Ph. luminescens and V. harveyi. The order of increasing luciferase thermostability was Ph. phosphoreum, V. fischeri, V. harveyi, and Ph. luminescens. The refolding of thermoinactivated luciferases completely depended on the active DnaK-DnaJ-GrpE chaperone system. Thermolabile fast luciferases of V. fischeri and Ph. phosphoreum showed highly efficient rapid refolding. Slower and less efficient refolding was characteristic of thermostable slow luciferases of V. harveyi and Ph. luminescens. Chaperones of the Clp family were tested for effect on the efficiency of DnaK-dependent refolding of bacterial luciferases in Escherichia coli cells. The rate and extent of refolding were considerably lower in the clpB mutant than in wild-type cells. In E. coli cells with mutant clpA, clpP, of clpX showed a substantially lower luciferase refolding after heat shock.

Role of Hsp70 (DnaK-DnaJ-GrpE) and Hsp100 (ClpA and ClpB) chaperones in refolding and increased thermal stability of bacterial luciferases in Escherichia coli cells.

The role of chaperones Hsp70 (DnaK-DnaJ-GrpE) and Hsp100 (ClpA-ClpB-ClpX) in refolding of thermoinactivated luciferase from the marine bacterium Photobacterium fischeri and the terrestrial bacterium Photorhabdus luminescens has been studied. These luciferases are homologous, but differ greatly in the rate of thermal inactivation and the rate constant for the luminescence reaction. It was shown that refolding of thermoinactivated luciferases is completely determined by the DnaK-DnaJ-GrpE system. However these luciferases markedly differ in the rate and degree of refolding. The degree of refolding of thermolabile "quick" Ph. fischeri luciferase reaches 80% of the initial level over several minutes, whereas renaturation of thermostable "slow" Ph. luminescens luciferase proceeds substantially slower (the degree of renaturation reaches only ~7-8% of the initial level over tens of minutes). The measurement of the rate of thermal inactivation of luciferases in vivo in the cells of Escherichia coli wild strain and strains containing mutations in genes clpA, clpB, clpX showed that Ph. luminescens luciferase revealed reduced thermostability in mutant strain E. coli clpA-. It was shown that this effect was not connected with DnaK-dependent refolding. In the case of thermolabile Ph. fischeri luciferase, mutation in gene clpA has no effect on the shape of the curve of thermal inactivation. These data suggest that denatured Ph. luminescens luciferase has enhanced affinity with respect to chaperone ClpA in comparison with DnaK, whereas thermolabile Ph. fischeri luciferase is characterized by enhanced affinity with respect to chaperone DnaK. Denatured luciferase bound to ClpA does not aggregate and following refolding proceeds probably spontaneously and very quickly (over 1-2 min). It is evident that the process under discussion requires ATP, since the addition of uncoupler of oxidative phosphorylation carbonyl cyanide 3-chlorophenylhydrazone results in a sharp decrease in thermal stability of luciferase to the level typical of the enzyme in vitro. The enhanced thermosensitivity of luciferases was observed also in E. coli containing mutations in gene clpB. However, this effect, which takes place for Ph. fischeri luciferase as well as for Ph. luminescens luciferase, is determined by DnaK-dependent refolding and probably connected with the ability of chaperone ClpB to provide disaggregation of the proteins, resulting in their interaction with chaperones of the Hsp70 family (DnaK-DnaJ-GrpE).

[FMN-reductase from Escherichia coli and its effect on the activity of luciferase from marine bacterium Vibrio fischeri]. / FMN-reduktaza iz Escherichia coli i ee vliianie na aktivnost' liutsiferazy iz morskikh bakterii Vibrio fischeri.

Interactions of luciferases isolated from Vibrio fischeri 6 and Escherichia coli JM109(pF3) (bearing cloned V. fischeri luxAB genes) with FMN reductase isolated from E. coli JM109 were studied. FMN reductase formed a stable complex with luciferase, suggesting similar properties of the FMN reductases in the taxonomically close families Vibrionaceae and Enterobacteriaceae.

[Use of bacterial luciferase for determining monoamine oxidase activity]. / Ispol'zovanie bakterial'noi liutsiferazy dlia opredeleniia monoaminooksidaznoi aktivnosti.

A bioluminescence assay is proposed for measuring monoamine oxidase activity in different biological specimens (platelets, mitochondria). The assay is based on the bioluminescent reaction catalysed by bacterial luciferase and coupled to monoamine oxidase. Two modifications of the bioluminescence assay were used. In the first case, the bioluminescent system was added to monoamine oxidase preincubated with the substrates, while in the second case, all the components of the coupled enzymatic systems were directly mixed in a cell. The proposed bioluminescence assay is simple, highly sensitive and rapid, and could be especially useful for biomedical examinations.