[Effect of Pseudomonas aeruginosa exometabolites on planktonic and biofilm cultures of Escherichia coli].

AIM: Study the effect of P. aeruginosa exometabolites on planktonic and biofilm cultures of bioluminescent E. coli strain. MATERIALS AND METHODS: E. coli K12 TG1 (pF1 lux+ Ap(r)) recombinant bioluminescent strain, P. aeruginosa ATCC 27853 reference strain and 2 nosocomial isolates were used. Pyocyanin and pyoverdin content in supernatant of P. aeruginosa over-night cultures was evaluated according to E. Deziel et al. (2001). Planktonic and biofilm cultures of E. coli were obtained in 96-well plates (LB, statically, 37 degrees C), optical density of plankton, film biomass (OD600, OD580) and bioluminescence in plankton and biofilm were evaluated in microplate reader Infiniti M200 (Tecan, Austria). RESULTS: P. aeruginosa exometabolites increased the duration of lag-phase in E. coli, and short term exposition inhibited luminescence of planktonic cells. These effects are determined by bactericidal action ofpyocyanin and pyoverdin. Supernatants ofover-night cultures of P. aeruginosa inhibit formation of biofilm and disrupt the formed biofilm of E. coli. Effect of pyocyanin and pyoverdin on these processes is not established, other factors may have higher significance. CONCLUSION: Bioluminescence of E. coli K12 TGI that reflects the energetic status of the cell allows to evaluate and prognose the character of coexistence of P. aeruginosa in combined with E. coli planktonic and biofilm culture.

[Putrescine as a oxidative stress protecting factor in Escherichia coli]. / Putrestsin kak faktor zashchity Escherichia coli ot okislitel'nogo stressa.

The level of expression of oxyR, the gene that protects Escherichia coli against oxidative stress, was enhanced by physiological concentrations of the biogenic amine putrescine. This level was directly proportional to the degree of negative DNA supercoiling. 1,4-Diamino-2-butanone (DAB), a specific inhibitor of ornithine decarboxylase, the key enzyme of polyamine synthesis, produced an inhibitory effect on the level of oxyR expression under oxidative stress, which was relieved by the addition of putrescine. The direct relationship between putrescine concentration and the degree of negative DNA supercoiling suggests that the mechanism of action of putrescine as the modulator of oxyR transcription activity is based on both its direct influence on the gene expression level and its indirect effect mediated by topological DNA changes. Putrescine was shown to produce a protective effect if the DNA is damaged by reactive oxygen species.

[The role of putrescine in of oxidative stress defense genes expression regulation in Escherichia coli]. / Rol' putrestsina v reguliatsii ékspressii genov adaptatsii Escherichia coli k okislitel'nomu stressu.

The role of putrescine in the adaptive response of Escherichia coli grown aerobically in synthetic M9 medium with glucose to the H2O2-induced oxidative stress was studied. Under oxidative stress, the expression of the single-copy reporter gene fusions oxyR::lacZ and katG::lacZ was found to undergo biphasic changes, which were most pronounced in glucose-starved E. coli cells. The concentration-dependent activating effect of putrescine on the expression of the oxyR regulon genes was maximum when the oxyR gene was inhibited by high concentrations of hydrogen peroxide.

[Topological changes in DNA as a reflection of adaptation of Escherichia coli to oxidative stress under glucose starvation and transition to growth]. / Topologicheskie izmeneniia DNK kak otrazhenie adaptatsii Escherichia coli k okislitel'nomu stressu v usloviiakh golodaniia po gliukoze i pri perekhode k rostu.

Changes in the topological state of DNA occur in a starving Escherichia coli culture under oxidative stress caused by the addition of hydrogen peroxide. The addition of a carbon and energy source to this culture results in a second stress reaction. This supports previous data indicating that different mechanisms are responsible for the cell defense against oxidative stress in exponential and starving E. coli cultures. Polyamine synthesis is involved in the cell adaptation to the stress. Putrescine binding to DNA and its dissociation seem to modulate the DNA topological state, which regulates the expression of the adaptive genes. An increase in the activity of the polyamine-synthesizing system in response to oxidative stress leads to a putrescine flux across the cytoplasmic membrane, due to which the antioxidant activity of putrescine protects the membrane phospholipids and contributes to the restoration of the cell energy-generating function.

[The role of putrescine and the energy state of Escherichia coli in regulation of DNA topology during adaptation to oxidative stress]. / Rol' putresysina i énergeticheskogo sostoianiia Escherichia coli v re guliatsii topologii DNK pri adaptatsii k okislitel'nomu stressu.

An exponential-phase culture of E. coli responded to the addition of H2O2 by a decrease in DNA supercoiling induced by the lowering of the energy status of cells, potassium leakage, and breaking of polynucleotide chains. Extending the time of exposure of E. coli cells to hydrogen peroxide led to an increase in the intracellular pools of putrescine and potassium, promotion of cellular energy status, and the restoration of DNA supercoiling to values much in excess of the prestress level. The subsequent stabilization of the intracellular putrescine pool was accompanied by a release of this polyamine from the cell. Based on these results and those available in the literature, a mechanism of E. coli adaptation to oxidative stress is suggested that assigns roles to putrescine, potassium, and cellular energy status.

[Role of putrescine and potassium transport in regulating the topological state of DNA during adaptation of Escherichia coli to temperature stress]. / Rol' transporta putrestsina i kaliia v reguliatsii topologicheskogo sostoianiia aDNK v protsesse adaptatsii Escherichia coli k temperaturnomu stressu.

The effect of a temperature increase to 52 degrees C or the addition of ethanol (6%) to an exponential Escherichia coli culture on putrescine and potassium transport was studied. The first stage of heat shock was accompanied by a decrease in the extent of DNA supercoiling, due to the dissociation of the putrescine-DNA complex. The loss of potassium ions at this phase produced a synergistic effect. The second phase of the heat shock was characterized by a reversal in the direction of putrescine and potassium transport, which was accompanied by restoration of the prestress extent of DNA supercoiling. An increase in the ATP pool and cell energy charge resulting from the uncoupling of the energy metabolism and synthetic processes also played an important role in the restoration of the DNA initial topology at the second phase of the heat shock via the activation of the energy-dependent gyrase or the heat shock protein DnaK. A mechanism is suggested that explains the involvement of putrescine in the regulation of DNA topology, which is a universal regulator of gene expression under stress, heat shock in particular.