Cysteine Homeostasis Disturbance in Escherichia coli Caused by Exposure to Ciprofloxacin.

E. coli exposure to ciprofloxacin disturbs cysteine homeostasis; an increase in the intracellular concentration of cysteine is dangerous due to its ability to enhance ROS generation. Unlike wild-type bacteria, in which the cysteine content did not exceed the control level, cells of the gshA mutant lacking glutathione are characterized by increased concentration of intracellular cysteine in proportion to the concentrations of the antibiotic, despite the intensive export of cysteine into the medium. At low concentrations of ciprofloxacin, the mutant strain formed half as many colonies as the parent strain in the survival test. These findings attest to the important role of the incorporation of excess cysteine into glutathione as one of the mechanisms of cysteine homeostasis during the stress response to antibiotic.

[Changes in the Activity of Antioxidant Systems of Escherichia coli under Phosphate Starvation].

Changes in the activity of antioxidant systems in Escherichia coli during phosphate starvation have been studied. It is shown that starvation was accompanied by a decrease in the intensity of respiration, an increase in the rate of superoxide production, and a decrease in the level of ATP. Simultaneously, there was a decrease in H2O2 in the medium and a significant increase in the expression of the katG and katE genes which encode the HPI and HPII catalases, respectively. At the same time, there was no drop in the membrane potential, which may indicate the retention of normal membrane activity in starving cells. It has been shown for the first time that the transition of E. coli to phosphate starvation is accompanied by significant changes in the status of glutathione. The most important of these are associated with a decrease in the level of reduced glutathione in the medium (GSHout) and with a simultaneous increase in its content in the cytoplasm (GSHin), as well as a shift in the GSHin to oxidized glutathione form (GSSGin) ratio towards reductive values, and GSHout/GSSGout towards oxidative values. Among the mutants used in the work, the gor trxB double mutant, which is deficient in the synthesis of glutathione reductase and thioredoxin reductase, showed the most pronounced distinctive features. Compared to the parental strain, this mutant showed a multiple higher expression of katG::lacZ, the highest level of oxidized intra- and extracellular glutathione, and, accordingly, the lowest GSH/GSSG ratio in both compartments. In general, the data we obtained indicate that during phosphate starvation the interaction of the glutathione redox-system and regulons that control protection against reactive oxygen species creates conditions that allow maintaining the concentration of ROS below the toxic level. As a result, phosphate-starved E. coli cells can maintain high viability for a long period of time, which allows them to quickly resume growth after the addition of phosphate.

Effect of Changes in the Redox Status on Biofilm Formation in Escherichia coli.

Changes in the redox balance in the medium and in Escherichia coli cells significantly affect the ability of bacteria to form biofilms. An increase in the level of aeration in the culture of wild-type bacteria led to a 3-fold decrease in the mass of biofilms. Mutants lacking components of the glutathione and thioredoxin redox systems, as well as transporters involved in the transmembrane cycling of glutathione, demonstrated increased biofilm formation ability. The effect of exogenous glutathione on biofilm formation depended on the culturing conditions. The addition of 0.1-1 mM Trolox (a water-soluble analog of vitamin E) was accompanied by a 30-40% reduction in biofilm formation.

[The antioxidant characteristics of medicinal plant extracts from Western Siberia].

An antioxidant activity of the water-alcohol extracts of leaves of ten herbs from Western Siberia was studied. In vivo the capability of extracts to protect cells of Escherichia coli against the bacteriostatic action of H2O2 and the influence of the extracts on the expression of the antioxidant gene katG coding catalase-hydroperoxidase I were investigated. In vitro the radical-binding activity with DPhPG (1,1-diphenyl-2-picrylhydrazyl radical), the chelating capability with ferrozine, and total composition of flavonoids and tannins were determined. The extracts of Filipendula stepposa and Limonium gmelinii were characterized by the highest antioxidant activity. According to data, the test extracts could have an antioxidant effect on bacteria in different ways at once including the direct inhibition of ROS (reactive oxygen species), iron ion chelation and antioxidant gene induction.

[Role of Thiol Redox Systems in Escherichia coli Response to Thermal and Antibiotic Stresses].

Isogenic knockout mutants of Escherichia coli deficient in components of the glutathione and thioredoxin redox systems and growing at various temperatures (20-46°C) exhibited considerable differences in growth rate and survival, as well as in expression of the antioxidant genes. In the parent strain E. coli BW25113 (wt) treated with ciprofloxacin, ampicillin, or streptomycin, dependence of survival from growth temperature was a V-shaped curve with the maximum sensitivity within the range corresponding to high growth rates (40-44°C). Significant inverse correlation was observed between log CFU at different temperatures and specific growth rate prior to antibiotic addition. This applied to most of the mutants, which exhibited higher resistance to the three antibiotics tested at nonoptimal temperatures (20 and 46°C) than at 37 and 40°C. No correlation was found between resistance to stress and antibiotics and expression of the antioxidant genes. The role of global regulators ppGpp and σ(s) in E. coli resistance to antibiotics and nonoptimal temperatures was shown.

Effects of menadione and hydrogen peroxide on glutathione status in growing Escherichia coli.

Menadione (MD) and H2O2 caused distinct effects on glutathione status in growing Escherichia coli. Treatment of E. coli AB1157 with 1-25 mM H2O2 did not result in an appreciable decrease in intracellular total glutathione (reduced glutathione [GSH] + oxidized glutathione [GSSG]). Only when cells were treated with 25 mM H2O2 an increase in GSSG and a decrease in the GSH:GSSG ratio were observed. In cells deficient in catalase HPI, such effect was observed even at 10 mM H2O2. The exposure of E. coli AB1157 to MD caused a dose-dependent decrease in intracellular total glutathione, an increase in GSSG, and a decrease in the ratio of GSH:GSSG. In E. coli deficient in cytosolic superoxide dismutase activity, a decrease in total glutathione after incubation with 0.2 mM MD was not accompanied by an increase in GSSGin, and the ratio of GSHin:GSSGin was three times higher than in the wild-type cells. The changes in the redox status of extracellular glutathione under the action of both oxidants were similar. Although the catalase activity increased several times after exposure to both oxidants, there were little or no changes in the activity of enzymes related to glutathione metabolism. A possible role of changes in redox status of glutathione under oxidative stress is discussed.

The role of antioxidant enzymes in response of Escherichia coli to osmotic upshift.

Aerobic growth of Escherichia coli sodAsodB and katE mutants lacking cytosolic superoxide dismutases and catalase hydroperoxidase II was inhibited by osmotic upshift to a greater extent than of their wild-type parent strains. The fur mutation leading to an intracellular overload of iron also increased sensitivity of growing E. coli cells to osmotic upshift. Using lacZ fusions, it was shown that expression of antioxidant genes soxS and katE was stimulated by an increase in osmolarity. These data suggest that in aerobically growing E. coli cells, moderate osmotic upshift causes activation of certain antioxidant systems.

Induction of the alkylation-inducible aidB gene of Escherichia coli by cytoplasmic acidification and N-ethylmaleimide.

We have studied the effects of changes in intracellular pH and the influence of thiol reagents on the induction of the DNA damage-inducible genes of Escherichia coli, aidB, alkA, and alkB. Under aerobic conditions in the absence of alkylating agents aidB, but not alkA or alkB, was induced by an acidification of cytoplasm or by treatment with the sulfhydryl reagent N-ethylmaleimide. Alkaline shift and thiosalicyclic acid did not affect the induction of aidB and alkB. The induction of alkA increased under the alkaline shift but not in the case of treatment with reducing agents. Compared with the aidB gene, a component of the SOS system, the sulA (sfiA) gene, responded to changes in cytoplasmic pH and in the level of intracellular thiols in an opposite way. SulA induction was observed under alkaline shift and after treatment with thiosalicylic acid.

Acidification of Escherichia coli and Salmonella typhimurium cytoplasm reduces the mutagenic effect of N-methyl-N'-nitro-N-nitrosoguanidine.

Preliminary acidification of the cytoplasm of E. coli cells growing at pH 6.9 by adding to the medium 50 mM of sodium acetate or propionate reduced the mutagenic effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) to almost the spontaneous level. In experiments with S. typhimurium the protective effects of cytoplasm acidification against the mutagenic effect of MNNG was observed at pH 5.5 and was absent at a medium pH of 6.9. Alkalinization of the cytoplasm by adding 80 mM of methylamine to the growth medium at pH 8.1 did not modify the effect of MNNG on the cells of E. coli and S. typhimurium. Alkalinization of the cytoplasm of E. coli B/r and K12 was followed by a reduction of the intracellular non-protein SH group level by 25 and 50%, respectively. It is supposed that the protective effect of acidification may be due to a decline in the productivity of mutagenically more active intermediates of MNNG when the pH is reduced and the associated fall of the level of intracellular non-protein thiols occurs. The above situation may serve as a model for studying the effects of MNNG and other alkylating agents on cells differing in physiological status.

[Use of the zwitterion detergent Sulfobetain-12 for preparation of virosomes and investigation of their immunogenic and protective properties]. / Ispol'zovanie tsvitterionnogo detergenta sul'fobetaina-12 dlia polucheniia virosom i izuchenie ikh immunogennykh i protektivnykh svoistv.

Virosomes were prepared by using the zwitterion detergent sulfobetaine-12. The virosomes included the surface antigens and virus-specific lipids of influenza virus, strain A/PR/8/34. Immunogenic and protective properties of the surface antigens in the micellar form and as a complex with the virosomes were studied. The surface antigens of this complex, like the intact virus, were found to possess the high immunogenic and protective activity in relation to the following infection with the homologous pathogenic virus.

[Effect of pH on cea gene expression in cells irradiated with UV-light and unirradiated Escherichia coli cells]. / Vliianie pH na ékspressiiu gena cea u obluchennykh UF-svetom i neobluchennykh kletok Escherichia coli.

The effect of pH on the expression of the cea gene encoding colicin EI in Escherichia coli was investigated by measuring the beta-galactosidase activity in the UV-irradiated growing cells carrying the cea-lacZ fusion. Maximum activity was observed at pH 7, and inhibition of expression was observed at pH 6 and pH 8. Treatment of the irradiated cells with 50-mM acetate increased inhibition at pH 6.0-7.5. No correlation between cea expression and the rate of cell growth was observed at different pH levels. Preliminary treatment with acetate at pH 7 reduced the expression of the recA gene, which participates in the regulation of the cea gene to 33% in irradiated cells and to 25% in nonirradiated cells.

[Dynamics of the redox potential in a fed-batch culture of E. coli]. / Dinamika redoks-potentsiala v kul'ture E. coli v rezhime periodicheskogo kul'tivirovaniia.

Dynamics of Eh, pH, pO2 and optical density in E. coli cultures under glucose and ammonium exhaustion were studied. It has been shown that changes in the redox potential accompanying the exhaustion of these substances in aerobic cultures are the leaps by their character and reflect the physiological state of cells and changes in the structure of cell surface. A relationship between the changes in the redox potential and in the electrochemical potential of H ions (delta mu H) is suggested.

[Changes in redox potentials during transitional processes in pure and mixed cultures of Escherichia coli and Serratia marcescens]. / Izmenenie redoks-potentsiala pri perekhodnykh protsessakh v chistykh i smeshannykh kul'turakh Escherichia coli i Serratia marcescens.

There were studied transitional processes accompanying the beginning of growth under glucose addition and stopping of growth under glucose exhaustion in pure and mixed aerobic cultures of Escherichia coli and Serratia marcescens. Continued record of Eh, pH, and CO2 showed that these processes sharply differ from each other in their character in pure and mixed cultures, it is particularly related to the changes of the redox potential. There is no characteristic change in the redox potential in pure culture of E. coli at growth termination in the case when S. marcescens cells are present in the culture.

[Protection from virulent Salmonella groups B and D after the peroral immunization of chicks with a hybrid of Salmonella typhimurium and Salmonella dublin]. / Zashchita ot virulentnykh sal'monell grupp B i D posle peroral'noi immunizatsii tsypliat gibridom Salmonella typhimurium i Salmonella dublin.

Chickens over 10 days old, infected orally with virulent salmonellae, were found to remain alive. Histologic investigation showed the development of mild enteritis and more pronounced, lasting for more than two weeks, inflammation of the cecum, dissemination and focal lesions in the liver (granulomas, necrosis). In experiments on the oral immunization of 3-day old chickens the bivalent hybrid of S. typhimurium vaccine strain 274 and S. dublin induced only pronounced blast transformation in lymphatic follicles of the cecum, hyperplasia of activated macrophages and formation of granulomas from these macrophages and lymphocytes. After oral challenge of the immunized chickens with virulent salmonellae of group B (S. typhimurium) and group D (S. enteritidis, S. gallinarum-pullorum) the chickens exhibited sharply pronounced protection against adhesion, colonization and invasion, and a few penetrating bacteria were rapidly destroyed by immune macrophages. Hybrid strain 274/O9 proved to be suitable for use as oral bivalent vaccine against salmonellosis in chickens.

[A morphological evaluation of the protective properties of noninvasive recombinant strains of Shigella]. / Morfologicheskaia otsenka protektivnosti neinvazivnykh rekombinantnykh shtammov shigell.

In 2-3 weeks after the oral immunization of rabbits, made in one or two administrations, with attenuated two-marker S. dysenteriae 1 strain VS-12 and recombinant S. dysenteriae VS-12/S. sonnei NR-18 and S. flexneri y433/S. sonnei NR-18 pronounced immunological reaction developed in the mucous membrane of the small intestine: blast transformation follicles of Peyer's patches, an increase in the number of lymphoblasts and plasmocytes in the cupolae of follicles and in intestinal villi, and an increase in the number of lymphocytes and macrophages in the intestinal epithelium with their release into the lumen of the intestine after challenge with virulent shigellae. The protective potency of these recombinants after challenge with massive doses of virulent shigellae was found to be high, which was shown by quantitative evaluation of the decrease of adhesion, invasiveness and cytotoxicity, suppression of epithelial lesions and development of inflammation in the intestinal mucosa.

[The use of a live Salmonella bivalent vaccine for decreasing the circulation of salmonellae in a poultry plant]. / Primenenie zhivoi sal'monelleznoi bivalentnoi vaktsiny dlia snizheniia tsirkuliatsii sal'monell na ptitsefabrike.

S. typhimurium (expressing antigen 09 of S.dublin) recombinant vaccine strain was tested at a large poultry-breeding farm under the conditions of permanent circulation of salmonellae. The oral immunization of laying hens in three administrations with doses of 5 x 10(8), 5 x 10(9) and 5 x 10(9) microbial cells induced an increase in titers of antibodies only to group D salmonellae, while in immunized chickens (each receiving a dose of 3 x 10(8) microbial cells) antibodies to both group B and group D salmonellae were detected. The vaccination of laying hens and chickens produced a protective effect due to the development of immunity and the decrease of Salmonella contamination of eggs laid by immunized hens, thus reducing the risk of infection at incubator stations. Total mortality during the first 3 months of life was essentially lower (p < 0.001) among immunized chickens (4.41 +/- 0.09%) than in the control group of nonimmunized chickens (5.91 +/- 0.10%).

[A morphological evaluation of the harmlessness of noninvasive recombinant Shigella strains]. / Morfologicheskaia otsenka bezvrednosti neinvazivnykh rekombinantnykh shtammov shigell.

The interaction of Shigella recombinant strains (with attenuating chromosomal mutations, with transposon-neutralized plasmid gene mutations, the hybrids of both strains), incapable of inducing keratoconjunctivitis in guinea pigs, with cells Hep-2 and enterocytes in the ligated loop of the small intestine of rabbits was studied. These strains retained, to varying extent, pronounced adhesiveness, but practically lost their invasiveness (though in Peyer's patches the translocation of bacteria by M-cells was observed) and cytotoxicity, as well as their capacity to multiply in epithelial cells and to cause destructive inflammation in the intestine. According to these criteria of evaluation, morphological investigations confirm the safety of the recombinant shigellae under study.

[The effect of calcium gluconate on the acute and chronic toxicity of doxorubicin in mice]. / Vliianie gliukonata kal'tsiia na ostruiu i khronicheskuiu toksichnost' doksorubitsina u myshei.

The effect of calcium gluconate on the toxicity of the anthracycline antibiotic doxorubicin (DOX) in mice was studied. Calcium gluconate showed a significant protective action with respect to the DOX acute toxicity. When DOX was used in the lethal doses, up to the LD50s calcium gluconate protected all the mice from death. At the DOX LD100 or higher the antitoxic effect of calcium gluconate manifested itself in a lower death rate and/or in a higher lifespan of the animals (at least 2-fold). When DOX was used for the treatment course its chronic toxicity in the presence of calcium gluconate was 2 or more times lower by all the quantitative indices: the lifespan of the animals that died, the maximum and minimum total lethal doses of DOX, the latent period before the first mouse death, the overall duration of the DOX treatment course. The antitoxic effect of calcium gluconate also manifested itself in a lower DOX acute and chronic toxicity with respect to the gastrointestinal tract. Thus, calcium gluconate proved to be an effective DOX antitoxic modificator which provided the use of about 2 times higher single and courses doses of DOX in mice.

[Genotoxic effects of the complex interaction of industrial factors]. / Genotoksicheskie éffekty kompleksnogo vozdeistviia proizvodstvennykh faktorov.

Results of genoepidemiological study of reproduction function of workers in plastic goods production are presented. Incidence of spontaneous abortions (SA) in workers' families was analyzed. Frequency of early and late SA in women working in hazard production and wives of men working in harmful production is discussed. Differences in SA incidence in hazardous risk groups and controls were discovered.