[Inducible specific lux-biosensors for the detection of antibiotics: construction and main parameters].

Based on Escherichia coli, highly sensitive specific lux-biosensors for the detection of tetracycline and beta-lactam antibiotics, quinolones, and aminoglycosides have been obtained. To make biosensors, bacteria were used that contained fungal plasmids pTetA'::lux, pAmpC'::lux, pColD'::lux, and plbpA'::lux, in which transcription of the reporter Photorhabdus luminescens luxCDABE genes occurred from the inducible promoters of the tetA, ampC, cda, and ibpA genes, respectively. The main parameters (threshold sensitivity and response time) of lux-biosensors were measured. The high specificity of biosensors responding only to antibiotics of a certain type was demonstrated.

[Trigger factor dependent refolding of bacterial luciferases in Escherichia coli cells: kinetics, efficiency and effect of the bichaperone system, DnaKJE-ClpB].

Here were determined the basic parameters of the Tigger Factor (TF) -dependent refolding of thermal inactivated bacterial luciferases. The TF-dependent refolding is less efficient and requires more time than DnaKJE-dependent refolding. The increase in the intracellular concentration of TF leads to an apparent decrease in the level of the thermal inactivated bacterial luciferase refolding. For thermolabile luciferases, the level of TF-dependent refolding is significantly higher, than for thermostable luciferases: 30-40%--for the thermolabile Aliivibrio fischeri and Photobacterium leiognathi luciferases, and 10 and 0.5% for the thermostable Vibrio harveyi and Photorhabdus luminescens luciferases, respectively. The negative effect of the ClpB protein on the TF-dependent refolding was shown: in Escherichia coli clpB::kan TF-dependent refolding is more efficient than in the E. coli clpB+.

[SOS-repair--60 years].

This review integrates 60 years of research on SOS-repair and SOS-mutagenesis in procaryotes and eucaryotes, from Jean Weigle experiment in 1953 year (mutagenesis of lambda bacteriophage in UV-irradiated bacteria) to the latest achievements in studying SOS-mutagenesis on all living organisms--Eukarya, Archaea and Bacteria. A key role in establishing of a biochemical basis for SOS-mutagenesis belonges to the finding in 1998-1999 years that specific error-prone DNA polymerases (PolV and others) catalysed translesion synthesis on damaged DNA. This review focuses on recent studies addressing the new models for SOS-induced mutagenesis in Escherichia coli and Home sapiens cells.

[Comparative analysis of antirestriction activity of R64 ArdA and ArdB proteins].

Antirestriction proteins ArdA and ArdB are specific inhibitors of the type I restriction-modification enzymes. The transmissible plasmid R64 ardA and yfeB (ardB) genes were cloned in pUC18 and pZE21 vectors. It was shown that the R64 ArdA and ArdB proteins inhibit only restriction activity of the type I restriction-modification enzyme (EcoKI) in Escherichia coli K12 cells. The dependence of the effectiveness of the antirestriction activity of the ArdA and ArdB proteins on the intracellular concentration was determined. Antirestriction activity of ArdB is independent from the ClpXP protease. Transcription of yfeB (ardB) gene in R64 plasmid is realized from the yfeA promoter.

[Effects of the IbpAB AND ClpA chaperones on DnaKJE-dependent refolding of bacterial luciferases in Escherichia coli cells].

The rate and level of DnaKJE-dependent refolding of the thermoinactivated Aliivibrio fischeri luciferase are considerably lower in Escherichia coli ibpA and ibpB mutants than in wild type cells. The rate and level of refolding are lower in E. coli ibpB::kan than in ibpA::kan cells. The decline of refoldings level in E. coli clpA::kan makes progress only with the increase of thermoinactivation time of luciferase. Plasmids with the genes ibpAB don't compensate clpA mutation. It is supposed that small chaperones IbpAB and chaperone ClpA operate independently in a process of DnaKJE-dependent refolding of proteins at the different stages.

["Quorum sensing" regulation of lux gene expression and the structure of lux operon in marine bacteria Alivibrio logei].

A group of luminescent strains of marine bacteria Alivibrio logei has been isolated (basins of the Okhotsk, White and Bering Seas). Strains A. logei were shown to be psycrophiic bacteria with an optimal growth temperature of approximately 15 degrees C. Biolumiscent characteristics of strains were studied, and the expression of lux genes was shown to be regulated by the "quorum sensing" system. The A. logei lux operon was cloned in Escherichia coli cells and the structure of this operon and its nucleotide sequence were determined. The structure of A. logei lux operon differs markedly from that in the closely related species of luminescent marine bacteria A. fischeri. In the structure of the A. logei lux operon, the the luxI gene is absent in front of luxC, and a fragment containing luxR2-luxI genes is located immediately after luxG gene. Luminescent psycrophiic marine bacteria of A. logei are assumed to be widely distributed in cold waters of northern seas.

[Proteolytic control of expression of Vibrio fischeri lux-operon genes in Escherichia coli cells].

The key elements of the regulatory system activating expression of the lux-operon genes in the sea bacteria Vibrio fischeri are the LuxR protein (an activator oftranscription) and N-(3-oxohexanoyl) L-homoserine lactone (an autoinducer, AI). It is shown that the ATP-dependent proteases ClpXP and Lon take part in the negative control of expression of the lux-operon genes and that AI protects the LuxR protein from proteolysis.

[The C-terminal domain of the Vibrio fischeri transcription activator LuxR is not essential for degradation by Lon protease].

The Vibrio fischer luxICDABEG genes are activated by autoinducer N-(3-oxohexanoyl)homoserine lactone and the LuxR protein. The LuxR contains 250 aa and consists of two domains. The C-domain, that extends from around residue 162 to the C-terminus, is thought to bind lux regulatory DNA and activate transcription of the luxICDABEG genes. The N-terminal domain, which binds the autoinducer, consists of about 70% residues of LuxR. In E. coli C-terminal domain can activate the lux genes in the absence of autoinducer. Previously it was shown that the ATP-dependent Lon protease of E. coli takes part in the negative regulation of the transcription of the V. fischeri lux operon and that LuxR is a target of Lon protease. Comparative analysis of effects of Lon protease on the V. fischeri luxICDABEG genes expression was made. Special constructed hybrid plasmids which permit the regulation of luxR, luxR 5'-deletion mutation were used and luICDABEG genes were activated independently and quantitatively. We show that the full length LuxR, but not C-terminal domain is a target protein for Lon protease. The transcription activity by full length LuxR protein isobserved when its intracellular concentration is about two order lower than that of its C-terminal domain.

[Antirestriction proteins ardA and Ocr as effective inhibitors of the type I restriction-modification enzymes].

Genes encoding antirestriction proteins (antirestrictases, inasmuch as the antirestriction proteins inhibit the activity of restriction-modification systems, but have no proper enzyme activity, the name antirestrictase is only tentative) are included in the composition of conjugative plasmids (genes ardABC) and some bacteriophages (genes ocr and darA). Antirestriction proteins inhibit of the type I restriction-modification enzymes and thus protect unmodified DNA of plasmids and bacteriophages from degradation. Antirestriction proteins belong to the "protein mimicry of DNA" family: the spatial structure is like the B-form of DNA, and therefore the antirestriction proteins operated on the principle of concurrent inhibition replacing DNA in the complex with the restriction-modification enzyme. Based on the prepared in vitro mutant forms of ArdA and Ocr, and also on natural proteins ArdA selectively inhibiting restriction activity of the type I enzymes, but not affecting their methylase activity, we have developed a model of complex formation between the antirestriction proteins and the restriction-modification enzymes R2M2S. Antirestriction proteins are capable of competing displacement of the DNA strand from two sites which are situated as follows: 1) in S-subunit (enzyme contact with the specific DNA site) and 2) in R-subunit (through this unit translocation of the DNA strand occurs followed by its degradation). Analysis of estriction and antimodification activities of proteins ArdA and Ocr depending on the expression level of genes ardA and ocr was performed (the cloning of the genes was done under strictly regulated promoter).

[Antirestriction and antimodification activities of the T7 Ocr protein: effect of mutations in interface].

Antirestriction protein Ocr (bacteriophage T7) is specific inhibitor of the type I restriction-modification enzymes. The bacteriophage T7 0.3 (ocr) gene is cloned in pUC18 vector. It was shown that T7 Ocr protein inhibits both restriction and modification activities of the type I restriction-modification enzyme (EcoKI) in Escherichia coli K12 cells. The mutation form of Ocr-Ocr F53D A57E, which inhibits only the restriction activity of EcoKI-enzyme, was constructed. The T7 0.3 (ocr) and the Photorhabdus luminescens luxCDABE genes were cloned in pZ-series vectors with the P(ltet0-1) promoter which is tightly repressible by the TetR repressor. Controlling the expression of the lux-genes encoding bacterial luciferase demonstrates that the P(ltet0-1) promoter can be regulated over and up to 5000 fold range by supplying anhydrotetracycline (aTc) to the E. coli MG1655Z1 tetR+ cells. It was determined the dependence of the effectiveness of the antirestriction activity of the Ocr and Ocr F53D A57E proteins on the intracellular concentration. It was shown that the values of the dissociation constants K(d) for Ocr and Ocr F53D A57E proteins with EcoKI enzyme differ in 1000 times: Kd (Ocr) = 10(-10) M, K(d) (Ocr F53D A57E) = 10(-7) M.

[Induction of oxidative stress and SOS response in Escherichia coli by plant extracts: the role of hydroperoxides and the synergistic effect of simultaneous treatment with cisplatinum].

Plasmids containing a transcription fusion of Escherichia coli katG, soxS, and resA promoters to the Photorhabdus luminescens lux operon (luxCDABE) were constructed. The bioluminescence method of assessing oxidative stress and SOS response in E. coli cells was applied to test the genotoxicity of cisplatinum and vegetable extracts. Strains MG1655 (pKatG-lux) and MG1655 (pSoxS-lux) were used in the oxidative stress procedure. Strain MG1655(pRecA-lux) was used to test the genotoxicity of the chemicals. All vegetable extracts induced oxidative stress and SOS response. A marked synergistic response was observed when MG1655 (pRecA-lux) cells were exposed to both cisplatinum and vegetable extracts; the level of luminescence measured in the presence of both inducers was much higher than the sum of the levels of luminescence observed with vegetable extracts or cisplatinum alone. The hydroperoxide content in vegetable extracts and in X63-Ag8.6.5.3 myeloma cells was determined. Vegetable extracts were shown to inhibit the HeLa cell growth.

[Influence of mutations at genes of global transcriptional regulators on production of autoinducer AI-2 Quorum Sensing in the system of Escherichia coli].

The control of gene expression in response to an increase in the bacterial population density (Quorum Sensing) involves low-molecular-weight signal molecules (autoinducers, AI). AI-2 and synthase LuxS mediating its synthesis are widely distributed in Gram-negative and Gram-positive bacteria. In this work, the data were obtained on the role of global regulators of gene expression in AI-2 synthesis in Escherichia coli cells. The mutation inactivating gene rpoS (encodes sigma S subunit of RNA polymerase) was shown to drastically decrease an amount of active AI-2 in the culture medium. Mutations at gene rpoN that encodes sigma N subunit of RNA polymerase and also at gene lon, which encodes Lon proteinase, on the contrary, increase an amount of active AI-2 in supernatants of cultures. Mutant strains lacking histone-like proteins H-NS and StpA accumulate a slightly higher amount of AI-2 than the isogenic wild-type strain: however, an amount of AI-2 decreased in the culture medium of the double mutant devoid of both these proteins.

[Mutation clpA::kan in gene encoding the chaperone of Hsp100-family decreases DnaK-dependent refolding efficiency of proteins in Escherichia coli cells].

The rate and level of DnaK-dependent refolding of the thermoinactivated Vibrio fischeri luciferase were considerably lower in clpA mutant (clpA::kan) then in wild type cells. The decline of level of refolding makes progress with the increase of thermoinactivation time of enzyme. The mutation in clpP gene has no influence on kinetic and level of luciferase refolding. It was shown that the approximately equal amounts of the DnaKJE chaperone are synthesized under "heat shock" induction in E. coli clpA+ and E. coli clpA::kan cells. We suppose that the chaperone ClpA (like homological chaperone ClpB) is involved in the disaggregation process of denaturized proteins and that results to the increase of refolding efficacy. This in vivo phenomenon occurs only under long time incubation of cells at a high temperature, i.e. when protein aggregates of large size poorly refoldable by the DnaKJE system are formed.

[Host factors in the regulation of the Vibrio fischeri lux operon in Escherichia coli cells].

It has been shown that the chaperonin GroEL, together with GroES co-chaperonin and Lon ATP-dependent protease are involved in the regulation of expression of the Vibrio fischeri lux operon in Escherichia coli cells. The cells of E. coli groE (pF1)- bearing a plasmid with the complete V. fischeri lux regulon were weakly luminescent. The cells of E. coli lonA (pF1) displayed intense bioluminescence. The same effects also occurred in mutant E. coli strains bearing a hybrid plasmid pVFR1, where the luxR gene and the regulatory region of the V. fischeri lux operon were inserted before the Photorhabdus luminescens luxCDABE cassette. The V. fischeri luxR gene was cloned in the pGEX-KG vector with the formation of a hybrid gene gst-luxR. It was shown that affinity chromatography of the product of expression, the chimeric protein GST-LuxR, on a column with glutathione-agarose resulted in its copurification with the proteins GroEL and Lon. Consequently, LuxR, the transcription activator of the lux operon, forms complexes with these proteins. It is supposed that GroEL/GroES is responsible for the folding of the LuxR protein, and Lon protease degrades the LuxR protein either before its folding into an active globule or at denaturing.

[Antirestriction and antimodification activities of the ArdA protein encoded by the IncI1 transmissive plasmids R-64 and ColIb-P9].

Proteins of the Ard family are specific inhibitors of type I restriction-modification enzymes. The ArdA of R64 is highly homologous to ColIb-P9 ArdA, differing only by four amino acid residues of the overall 166. However, unlike ColIb-P9 ArdA, which inhibits both the endonuclease and the methylase activities of EcoKI, the R64 ArdA protein inhibits only the endonuclease activity of this enzyme. The mutant forms of R64 ArdA--A29T, S43A, and Y75W, capable of partially reversing the protein to ColIb-P9 ArdA form--were produced by directed mutagenesis. It was demonstrated that only Y75W mutation of these three variants essentially influenced the functional activity of ArdA: the antimodification activity was restored to approximately 90-99%. It is assumed that R64 ArdA inhibits formation of the complex between unmodified DNA and the R subunit of the type I restriction-modification enzyme EcoKI (R2M2S), which translocates and cleaves DNA. ColIb-P9 ArdA protein is capable of forming the DNA complex not only with the R subunit, but also with the S subunit, which contacts sK site (containing modified adenine residues) in DNA. ArdA bound to the specific sK site inhibits concurrently the endonuclease and methylase activities of EcoKI (R2M2S), while ArdA bound to the nonspecific site in the R subunit blocks only its endonuclease activity.

[Antirestriction activity of the IncI1 transmissive plasmid R64 ArdA protein]. / Antirestriktsionnaia aktivnost' belka ArdA, kodiruemogo transmissivnoi plazmidoi R64.

The transmissive plasmid IncI1 R64 contains the ardA gene encoding the ArdA antirestriction protein. The R64 ardA gene locating in the leading region of plasmid R64 has been cloned and their sequence has been determined. Antirestriction proteins belonging to the Ard family are specific inhibitors of type I restriction-modification enzymes. The IncI1 ColIb-P9 and R64 are closely related plasmids, and the latter specifies an ArdA homologue that is predicted to be 97.6% (162 residues from 166) identical at the amino acid sequence level with the ColIb = P9 equivalent. However, the R64 ArdA selectively inhibits the restriction activity of EcoKi enzyme leaving significant levels of modification activity under conditions in which restriction was almost completely prevented. The ColIb-P9 ArdA inhibits restriction endonuclease and methyltransferase activities simultaneously. It is hypothesized that the ArdA protein forms two complexes with the type I restriction-modification enzyme (R2M2S): (1) with a specific region in the S subunit involved in contact with the sK site in DNA; and (2) with nonspecific region in the R subunit involved in DNA translocation and degradation by restriction endonuclease. The association of the ColIb-P9 ArdA with the specific region inhibits restriction endonuclease and methyltransferase activities simultaneously, whereas the association of the R64 ArdA with a nonspecific region inhibits only restriction endonuclease activity of the R2M2S enzyme.

[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.

[The effects of the regulatory proteins RcsA and RcsB on the expression of the Vibrio fischeri lux operon in Escherichia coli]. / Vliianie belkov-reguliatorov RcsA i RcsB na ékspressiiu lux-operona Vibrio fischeri v kletkakh Escherichia coli.

A study was made of the effect of RcsA and RcsB on the Vibrio fischeri lux expression in Escherichia coli. RcsA suppressed the LuxR activity and thereby inhibited expression of the lux genes coding for luciferase and reductase. In osmotic shock, RcsA-RcsB activated lux expression and, consequently, bioluminescence of E. coli cells in the early log phase.

[Osmotic shock induces expression of Vibrio fischeri lux genes in Escherichia coli cells]. / Osmoticheskii shok indutsiruet ékspressiiu lux-genov Vibrio fischeri v kletkakh Escherichia coli.

The effect of osmotic shock on the expression of genes in the lux regulon of marine bacteria Vibrio fischeri was studied in cells of Escherichia coli. Bioluminescence of cells was shown to drastically increase, when cells were exposed to osmotic shock at the early logarithmic growth phase. The expression of lux genes induced by osmotic shock is determined by the two-component regulatory system RcsC-RcsB. A nucleotide sequence in the regulatory region of the luxR gene homologous to the RcsB-box consensus of E. coli is assumed to be a primary site for this system.

[Role of "anti-restriction" motif in functional activity of anti-restriction protein ArdA pKM101 (incN)]. / Rol' "antirestriktsionnogo" motiva v funktsional'noi aktivnosti antirestriktazy ArdA pKM101 (incN).

A number of mutant forms of the antirestriction protein ArdA encoded by the ardA gene located in a transmissive IncN plasmid pKM101 have been constructed. Proteins belonging to the Ard family are specific inhibitors of type I restriction--modification enzymes. Single mutational substitutions of negatively charged amino acid residues located in the "antirestriction motif" with hydrophobic alanine, E134A, E137A, D144A, or a double substitution E134A, E137A do not affect the antirestriction activity (Ard) of ArdA but almost completely abolish the antimodification activity (Amd). Mutational substitutions F107D and A110D in the assumed interface ArdA, which determines contact between monomers in the active dimer (Ard)2, cause an approximately 100-fold decrease in the antirestriction protein activity. It is hypothesized that the ArdA protein forms two complexes with the type I restriction--modification enzyme (R2M2S): (1) with a specific region in the S subunit involved in contact with the sK site in DNA; and (2) with a nonspecific region in the R subunit involved in DNA translocation and degradation by restriction endonucleases. The association of ArdA with the specific region inhibits restriction endonuclease and methyltransferase activities simultaneously, whereas the association of ArdA with a nonspecific region inhibits only restriction endonuclease activity of the R2M2S enzyme.