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The functions of nitrogen oxide (NO) in the regulation of the reversible processes of Fe-S cluster assembly in proteins and the formation of Escherichia coli biofilms have been investigated. S-nitrosoglutathione (GSNO) and crystalline nitrosyl complexes of iron with sulfur-containing aliphatic ligands cisaconite (CisA) and penaconite have been used as NO donors for the first time. Wild-type E. coli cells of the strain MC4100, mutants deltaiscA and deltasufA, and the double paralog mutant deltaiscA/sufA with deletions in the alternative pathways of Fe2+ supply for cluster assembly (all derived from the above-named strain) were used in this study. Plankton growth of bacterial cultures, the mass of mature biofilms, and the expression of the SoxRS[2Fe-2S] regulon have been investigated and shown to depend on strain genotype, the process of Fe-S cluster assembly in iron-sulfur proteins, NO donor structure, and the presence of Fe2+ chelator ferene in the incubation medium. The antibiotic ciprofloxacine (CF) was used as an inhibitor of E. coli biofilm formation in the positive control. NO donors regulating Fe-S cluster assembly in E. coli have been shown to control plankton growth of the cultures and the process of mature biofilm formation; toxic doses of NO caused a dramatic (3- to 4-fold) stimulation of cell entry into biofilms as a response to nitrosative stress; NO donors CisA and GSNO in physiological concentrations suppressed the formation of mature biofilms, and the activity of these compounds was comparable to that of CE Regulation of both Fe-S cluster assembly in iron-sulfur proteins and biofilm formation by NO is indicative of the connection between these processes in E. coli.

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Dinitrosyl iron complexes (DNICs) with thiol ligands--binuclear and mononuclear--inhibited aidB gene expression in E. coli cells. This process is due to the nitrosylation of the active center in iron-sulfur protein Fnr [4Fe-4S]2+ by low-molecular DNICs. The next step is transformation of the above DNICs into the DNICs with the thiol groups in the apo-form of Fnr protein. These nitrosylated proteins are characterized by the EPR signal with g perpendicular = 2.04 and g parallel 1 = 2,014. An addition of sulfur containing L-Cys or N-A-L-Cys as well as Na2S to the cells lead to the increasing in the aidB gene expression simultaneously with an appearance of the EPR signal with g perpendicular = 2.04 and g parallel = 2.02 as the characteristics of the DNICs with persulfide (R-S-S-) ligands. We suppose that the recovery of the aidB gene activity was due to the accumulation of inorganic sulfur in the cells and reconstruction of the active center in Fnr[4Fe-4S]2+. It appears that the above process is the function of L-cysteine-desulfurase protein which repaired the active center of Fnr[4Fe-4S]2+ protein using the sulfur from L-Cys or N-A-L-Cys after its deacetylation. On the other side the ions of inorganic sulfur being reacted with SH-groups led to the transformation of DNIC with thiol ligands into the persulfides. Na2S was the most potent activator of the aidB gene expression in our experiments.

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The SOS DNA repair pathway is induced in E. coli as a multifunctional cell response to a wide variety of signals: UV, X or gamma-irradiation, mitomycin C or nalidixic acid treatment, thymine starvation, etc. Triggering of the system can be used as a general and early sign of DNA damage. Additionally, the SOS-response is known to be an "error-prone" DNA repair pathway and one of the sources of genetic instability. Hypoxic conditions are established to be the major factor of genetic instability as well. In this paper we for the first time studied the SOS DNA repair response under hypoxic conditions induced by the well known aerobic SOS-inducers. The SOS DNA repair response was examined as a reaction of E. coli PQ37 [sfiA::lacZ] cells to UVC, NO-donating agents and 4NQO. Here we provide evidence that those agents were able to induce the SOS DNA repair response in E. coli at anaerobic growth conditions. The process does not depend on the transcriptional activity of the universal protein of E. col anaerobic growth Fnr [4Fe-4S]2+ or can not be referred to as an indicator of genetic instability in hypoxic conditions.

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In 2005 we have described in exponentially growing E. coli cells a new fundamental genetic phenomenon,--quasi-adaptive response to alkylating compounds (quasi-Ada). Phenotypic expression of quasi-Ada is similar to the true Ada response. However, in contrast to the letter, it develops in the course of pretreatment of the cells by a sublethal dose of nonalkylating agent, an NO-containing dinitrosyl iron complex with glutathione (DNICglu). To reveal the mechanisms of quasi-adaptation and its association with the function of the Ada regulatory protein, here we used a unique property of dual gene expression regulation of aidB1 gene, a part of the Ada-regulon, namely its relative independence from Ada protein in anaerobic conditions. Based on the results of aidB1 gene expression analysis an EPR spectra of E. coli MV2176 cells (aidB1::lacZ) in aerobic and anaerobic conditions after the corresponding treatments, we conclude that the function and the spatial structure of meAda and [(Cys-)2Fe+(NO+)2]Ada are identical and thus the nitrosylated protein represents a regulator of the Ada regulon gene expression during quasi-adaptation development.

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This work is a part of a directional search for new crystal donors of nitric oxide (NO), which are promising for complex chemotherapy. The relationships between the physico-chemical properties of NO donors, their genotoxic and mutagenic activities, and the dependence on intracellular iron were studied. New crystal NO donors (di- and trinitrosyl iron complexes with synthetic ligands) were examined for the first time and compared with known NO donors containing natural ligands. All but one compound induced expression of the Escherichia coli sfiA gene belonging to the SOS regulon and exerted a mutagenic effect on Salmonella typhimurium TA1535. These effects were fully or significantly inhibited by the iron(II)-chelating agent o-phenanthrolin, depending on the mono- or binuclear structure of the ligands. The rate of donating free NO in solution did not positively correlate with the genotoxic activity of the crystal NO donors. The genetic activity of all NO donors proved to depend on intracellular iron.

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In vitro experiments showed that O6-benzylguanine (O6-benzG, 0.2 microM) fully inhibited the repair activity of human O6-alkylguanine-DNA alkyltransferase (MGMT) due to the formation of S-benzylcytosine in the protein acceptor site. O6-benzG at concentrations increased many times (up to 800 microM) failed to inhibit the repair activity of the Escherichia coli Ada protein, the structural and functional analog of MGMT. It has been shown for the first time that O6-benzG stimulates the regulatory activity of the Ada protein. In experiments with N-nitroso-N-methylurea (NMU), the pretreatment of Escherichia coli cells with O6-benzG at a sublethal concentration of 10 microM led to a twofold enhancement of transcription at the Ada-dependent promoter of the alkA gene in control cells and ensured transcription enhanced 1.6-1.7 times at alkA and alkB promoters in cells with the induced "classical" Ada response. Apparently, an increase in the regulatory activity of the Ada protein was associated with the formation of the stable protein molecule having the strong affinity for alkA and alkB promoters after transfer of the benzyl group from O6-benzG to the acceptor site Cys-69 in the N-terminal domain of Ada protein. O6-benzG did not affect the regulative activity of Ada in alternative quasi-adaptive responses to NMU.

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An original hypothesis of a quasi-adaptive response to nitrosomethylurea (NMU) in Escherichia coli cells was verified experimentally. In contrast to the true Ada response, which is induced in cells pretreated with a sublethal dose of NMU, a quasi-adaptive response was induced using NO-containing dinitrosyl iron complex with glutathione (DNICglu). Quasi-adaptation increased expression of the Ada regulon and cell resistance to the cytotoxic and mutagenic effects of NMU. The levels of alkA, alkB, and aidB gene expression in quasi-adaptation were higher than in the true Ada response. Thus, experimental evidence was obtained for the alternative mechanism regulating the function of the Ada sensory protein in controlling expression of the Ada regulon during the adaptive response. The free iron--chelating agent o-phenanthroline (OP) facilitated degradation of DNICglu (by electron paramagnetic resonance (EPR) spectra) and considerably or completely inhibited gene expression in the quasi-adaptive response. The new phenomenon extends the functional range of NO compounds to include a role in genetic signal transduction within the Ada response system in addition to similar roles in the SoxRS, SOS, and OxyR systems in E. coli.

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A convenient preparative synthesis of 2'-amino-2'-deoxyuridine was developed. Starting from 2'-amino-2'-deoxyuridine and 2'-amino-2'-deoxycytidine, monomers for the phosphoamidite oligonucleotide synthesis were obtained that carry a linker with methoxyoxalamide groups in position 2'. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.

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A number of monomers for the standard phosphoamidite oligodeoxynucleotide synthesis that carry reactive methoxyoxalamide groups attached to the thymidine, 2'-deoxycytidine, and 2'-deoxyadenosine heterocyclic bases were prepared. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.

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Development of the adaptive response (AR) to the SoxRS-inducers-menadione (O2(-.)-donor), dinitrosyl-iron complex (NO donor) and their simultaneous action was studied in E. coli. Two AR parameters were used: an increasing in viability and decreasing in the soxS gene (SoxRS-regulon) expression in adapted cells. It was shown that namely peroxynitrite (ONOO-), being formed inside the cells from O2-. and NO, was the most cytotoxic agent among the drugs tested. On the one side, an increase in resistance to menadione treatment was selectively demonstrated in adapted E. coli delta oxyR mutant cells, defective in OxyR-regulon activity. On the other side, a decrease in soxS gene expression was marked in the experiments with menadione, as well So, an AR to O2-. superoxide anion was selectively regulated by the SoxRS DNA-repair pathway. OxyR-regulon that is selectively activated by the most redox-cycling agents and controls AR to these agents doesn't provide development of the AR to O2-..

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A new photoreactive oligonucleotide derivative was synthesized with a perfluoroarylazido group attached to the 2'-position of the ribose fragment of the 5'-terminal nucleotide. Using this conjugate, photoreactive DNA duplexes were produced which contained single-stranded regions of different length, single-stranded breaks (nicks), and also ds duplex with a photoreactive group inside one of the chains. These structures imitate DNA intermediates generated at different stages of DNA replication and repair. The interaction of replication protein A (RPA) with the resulting DNA structures was studied using photoaffinity modification and gel retardation assay. Independently of the DNA structure, only the large subunit of RPA (p70) was crosslinked to photoreactive DNAs, and the intensity of its labeling increased with decrease in the size of the single-stranded region and was maximal in the case of the nick-containing DNA structure. By gel retardation, the most effective binding of RPA to this structure was shown, whereas the complexing of RPA with DNA containing the unmodified nick and also with the full duplex containing the photoreactive group inside the chain was significantly less effective. The data suggest that RPA should be sensitive to such damages in the double-stranded DNA structure.

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Having one electron with unpaired spin, nitric oxide (NO) shows high reactivity and activates or inhibits free radical chain reactions. NO toxic and genotoxic effects appear to be the result of intracellular formation of peroxinitrite that can induce some cellular damages, including DNA strand breaks, DNA base oxidation, destruction of the key enzymes, etc. Taking into account the character of DNA damages being formed under NO activity, we proposed a formation of the SOS signal and induction the SOS DNA repair response in E. coli cells treated with NO physiological donors--DNIC and GSNO. The ability of NO donor compounds to induce the SOS DNA response in E. coli PQ37 with sfiA::lacZ operon fusion is reported here at the first time. So, the SOS DNA repair response induction is one of the function of nitric oxide.

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Oxidative stress formed in Escherichia coli cells is known to bring about a complex induction of alternative DNA repair processes, including SOS, SoxRS, and heat-shock response (HSR). The modification by heat shock of the expression of sfiA and soxS genes induced by oxidative agents H2O2, menadione and 4-nitroquinoline-1-oxide (4NQO) was studied for the first time. Quantitative parameters of gene expression were examined in E. coli strains with fused genes (promoters) sfiA::lacZ and soxS::lacZ. The expression of these genes induced by cell treatment with H2O2, but not menadione or 4NQO, was shown to decrease selectively after exposure to heat shock. Since genetic activity of menadione and 4NQO depends mainly on the formation of superoxide anion O2-, it is assumed that the effect of selective inhibition by heat-shock of sfiA and soxS gene expression in experiments with H2O2 is connected with activity of DnaK heat shock protein, which, unlike other heat-shock proteins, cannot be induced by superoxide anion O2-.

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Activation of the Escherichia coli SoxRS-regulon by nitric oxide (NO) and its physiological donors (S-nitrosothiol (GS-NO) and dinitrosyl iron complexes with glutathione (DNIC(glu)) and cysteine (DNIC(cys)) ligands) has been studied. To elucidate the molecular mechanisms of signal transduction via nitrosylation of Fe-S-centers in SoxR, the ability of pure NO and NO-producing agents to activate the SoxRS-regulon in E. coli cells bearing a soxS::lacZ operon (promoter) fusion has been compared. EPR spectroscopy of whole cells has been used to monitor the formation of inducible protein-DNIC complexes. DNIC(cys), GS-NO, and pure NO appeared to be potent inducers of soxS expression, whereas DNIC(glu) was considerably less efficient. Thus, lower in vitro stability of DNIC(cys) was in contrast with its higher biological activity. Pretreatment of the cells with o-phenanthroline, a chelating agent for iron, prevented soxS expression by GS-NO. Treatment of intact E. coli cells with DNIC, GS-NO, and NO at equimolar concentration 150 microM resulted in formation of a single EPR-detectable DNIC-type signal with g = 2.03. The initial stage in the SoxR transcription activity is supposed to include two steps: first, DNIC primers are formed from exogenous NO and free iron, and then these DNIC disintegrate SoxR [2Fe-2S] clusters and thus activate SoxRS-regulon transcription.

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