The regulatory effects of (p)ppGpp and indole on cAMP synthesis in Escherichia coli cells.

Bacterial stress adaptive response is formed due to changes in the cell gene expression profile in response to alterations in environmental conditions through the functioning of regulatory networks. The mutual influence of network signaling molecules represented by cells' natural metabolites, including indole and second messengers (p) ppGpp and cAMP, is hitherto not well understood, being the aim of this study. E. coli parent strain BW25141 ((p) ppGpp+) and deletion knockout BW25141ΔrelAΔspoT which is unable to synthesize (p)ppGpp ((p)ppGpp0) were cultivated in M9 medium supplemented with different glucose concentrations (5.6 and 22.2 mM) in the presence of tryptophan as a substrate for indole synthesis and in its absence. The glucose content was determined with the glucose oxidase method; the indole content, by means of HPLC; and the cAMP concentration, by ELISA. The onset of an increase in initially low intracellular cAMP content coincided with the depletion of glucose in the medium. Maximum cAMP accumulation in the cells was proportional to the concentration of initially added glucose. At the same time, the (p) ppGpp0 mutant showed a decrease in maximum cAMP levels compared to the (p)ppGpp+ parent, which was the most pronounced in the medium with 22.2 mM glucose. So, (p)ppGpp was able to positively regulate cAMP formation. The promoter of the tryptophanase operon responsible for indole biosynthesis is known to be under the positive control of catabolic repression. Therefore, in the cells of the (p)ppGpp+ strain grown in the tryptophan-free medium that were characterized by a low rate of spontaneous indole formation, its synthesis significantly increased in response to the rising cAMP level just after glucose depletion. However, this was not observed in the (p)ppGpp0 mutant cells with reduced cAMP accumulation. When tryptophan was added to the medium, both of these strains demonstrated high indole production, which was accompanied by a decrease in cAMP accumulation compared to the tryptophan-free control. Thus, under glucose depletion, (p)ppGpp can positively regulate the accumulation of both cAMP and indole, while the latter, in its turn, has a negative effect on cAMP formation.

Effects of polyamines and indole on the expression of ribosome hibernation factors in Escherichia coli at the translational level.

Polyamines and indole are small regulatory molecules that are involved in the adaptation to stress in bacteria, including the regulation of gene expression. Genes, the translation of which is under the regulatory effects of polyamines, form the polyamine modulon. Previously, we showed that polyamines upregulated the transcription of genes encoding the ribosome hibernation factors RMF, RaiA, SRA, EttA and RsfS in Escherichia coli. At the same time, indole affected the expression at the transcriptional level of only the raiA and rmf genes. Ribosome hibernation factors reversibly inhibit translation under stress conditions, including exposure to antibiotics, to avoid resource waste and to conserve ribosomes for a quick restoration of their functions when favorable conditions occur. In this work, we have studied the influence of indole on the expression of the raiA and rmf genes at the translational level and regulatory effects of the polyamines putrescine, cadaverine and spermidine on the translation of the rmf, raiA, sra, ettA and rsfS genes. We have analyzed the mRNA primary structures of the studied genes and the predicted mRNA secondary structures obtained by using the RNAfold program for the availability of polyamine modulon features. We have found that all of the studied genes contain specific features typical of the polyamine modulon. Furthermore, to investigate the influence of polyamines and indole on the translation of the studied genes, we have constructed the translational reporter lacZ-fusions by using the pRS552/λRS45 system. According to the results obtained, polyamines upregulated the expression of the rmf, raiA and sra genes, the highest expression of which was observed at the stationary phase, but did not affect the translation of the ettA and rsfS genes, the highest expression of which took place during the exponential phase. The stimulatory effects were polyamine-specific and observed at the stationary phase, when bacteria are under multiple stresses. In addition, the data obtained demonstrated that indole significantly inhibited translation of the raiA and rmf genes, despite the stimulatory effect on their transcrip- tion. This can suggest the activity of a posttranscriptional regulatory mechanism of indole on gene expression.

Effect of biogenic polyamines on sliding motility of mycobacteria in the presence of antibiotics.

Nowadays, sliding is the least investigated mode of bacterial motility. Sliding is a process of passive movement on the surface of semi-liquid mediums which was originally described for mycobacteria and other bacterial species deprived of the organelles specialized for movement. Some mycobacteria are able to colonize surfaces, including tissues of macro-organisms, using glycopeptidolipids localized in the cell envelope for this aim. This is a serious problem for effective therapy of mycobacteriosis caused by nontuberculosis mycobacteria. Furthermore, animal tissues contain biogenic polyamines, which can increase tolerance of microorganisms to stresses, including antibiotics, and modulate cell motility. Therefore, studying mutual effects of biogenic polyamines and antibiotics on the expansion of mycobacteria is important for medicine. Mycobacterial strains, including the parent Mycolicibacterium smegmatis mc2 155 and strains containing single (ΔrelMsm) or double (ΔrelMsmΔrelZ) deletions, were used as the objects of this study. The content of glycopeptidolipids was determined using thin layer chromatography. Sliding motility was assessed by measuring the area of the sliding colony. The effectiveness of antibiotics was measured by comparison of the areas of sliding colonies in the presence of comparable concentrations of antibiotics. The polyamines spermidine and spermine had different effects on the sliding of mycobacteria through an increase or decrease in the colony areas. At the same time, polyamines had neither bactericidal nor bacteriostatic effects. The polyamines contained in the medium decreased the bactericidal effects of the antibiotics streptomycin or isoniazid, but enhanced the effects of DMNP, a synthetic analogue of the natural antibiotic erogorgiaene. Rifampicin was the most effective of all antibiotics investigated here. Moreover, we found that glycopeptidolipids are, apparently, not the only regulators of mycobacterial sliding.

[Adaptive functions of Escherichia coli polyamines in response to sublethal concentrations of antibiotics].

Escherichia coli exposure to sublethal antibiotic concentrations induced an increase in cell polyamine contents. Maximum accumulation of putrescine and spermidine in response to antibiotics-induced oxidative stress preceded the increment of cadaverine, the content of which was dependent on the rpoS expression level and reached the maximum in response to fluoroquinolones. The polyamine positive modulating effects on rpoS expression increased in the following order: cadaverine-putrescine-spermidine. The reason for cadaverine accumulation was the increase in activities of lysine decarboxylases CadA and Ldc. High cadaverine accumulation in the cells exposed to fluoroquinolones and cephalosporins resulted in the reduction of porin permeability; so it was considered as a response aimed at cell protection against antibiotic penetration into the cell. Netilmycin, unlike other antibiotics, did not substantially affect the lysine decarboxylase activity and cellular polyamine pools.

Dependence of protective functions of Escherichia coli polyamines on strength of stress caused by superoxide radicals.

Mechanisms of antioxidant effect of polyamines were studied in dependence on the strength of superoxide stress. Under conditions of weak stress, polyamines from Escherichia coli cultures were shown to function mainly as a scavenger of free superoxide radicals, whereas under conditions of strong stress they mainly acted as positive modulators of antioxidant genes. Spectrofluorimetry was used to show that both polyamine-dependent mutants and wild type cells treated with inhibitors of polyamine synthesis contained an elevated amount of free oxygen radicals, which could be decreased to the normal level by addition of exogenous polyamines. Under conditions of strong stress, polyamines positively influenced expression of the soxRS regulon genes of antioxidant defense, which was accompanied by an increase in the quantity (activity) of their gene products, such as glucose-6-phosphate dehydrogenase (Zwf) and fumarase (FumC). These effects led to an increase in the number of live cells in the cultures subjected to superoxide stress.

Role of polyamines in formation of multiple antibiotic resistance of Escherichia coli under stress conditions.

Under stress conditions, polyamines decreased the permeability of the outer membrane of Escherichia coli. This effect is caused by at least three mechanisms providing for an increase in the resistance to antibiotics transported through porin channels (fluoroquinolones, beta-lactams): a positive modulation of the gene micF transcription (its product antisense RNA inhibits the synthesis of porin proteins on the translational level); a positive effect on the cell content of the multiple stress resistance factor sigma(S) (it is accompanied by a decrease in the porin transport because of suppression of ompF transcription and induction of cadaverine synthesis); a direct inhibition of the transport activity of porin channels. The production of cadaverine in E. coli cells significantly increased in response to various antibiotics, and this was likely to be a manifestation of oxidative stress.

Putrescine as a modulator of the level of RNA polymerase sigma S subunit in Escherichia coli cells under acid stress.

Metabolites accumulated in the culture medium of Escherichia coli cells induce expression of the rpoS gene encoding the alternative sigmaS subunit of RNA polymerase, which controls adaptation of E. coli to acid stress during growth in glucose-mineral medium. The effect of acetate and succinate as end products of E. coli metabolism has been investigated on the levels of transcription, translation, and sigmaS protein stability. These end products mainly influenced the stability of the RNA polymerase sigmaS subunit. Under conditions of acid stress caused by acetate addition, the content of polyamines in the cells and medium decreased, whereas artificial rpoS gene switch-off by antisense RNA was accompanied by increase in polyamine level. Addition of polyamine to E. coli cells treated with acetate and especially with succinate caused a significant concentration-dependent stimulatory effect on rpoS expression. Thus, induction of the rpoS regulon depends on the combined action of the investigated metabolites determining adequate control of gene expression under conditions of acid stress. A scheme for metabolic pathways describing the role of putrescine in the maintenance of intracellular pH and polyamine pool homeostasis during E. coli adaptation to acid stress is proposed.

Role of putrescine in regulation of the sigmaS subunit of RNA polymerase in Escherichia coli cells on transition to stationary phase.

Expression of the rpoS gene encoding the sigmaS subunit of E. coli RNA polymerase was studied on transition to the stationary phase and under conditions of starvation. Polyamines, in particular putrescine, are involved in regulation of the rpoS expression by concentration-dependent stimulation at the levels of translation and stability of the protein sigmaS. The stimulatory effect of exogenous putrescine inversely depended on its content in the cell and was the most pronounced in the beginning of the exponential growth. Possible mechanisms of the polyamine effect on the rpoS expression on post-transcriptional and post-translational levels are discussed.

Mechanisms of protective functions of Escherichia coli polyamines against toxic effect of paraquat, which causes superoxide stress.

The toxic effect of paraquat, mainly caused by production of superoxide radicals, results in the induction of polyamine synthesizing enzymes and their products in cells of exponentially growing E. coli cultures. The activity of ornithine decarboxylase increases approximately twofold and the activity of lysine decarboxylase increases 1.4-fold. Unlike cadaverine, putrescine and spermidine stimulate expression of the soxRS regulon genes, and this depends on the polyamine concentration and is specific for different genes of the regulon. Of six genes studied, the maximum (to 130%), minimum (about 40%), and average (60-80%) stimulation was observed for the stress induction of nfo (endonuclease IV), sodA (superoxide dismutase), and the soxS gene of the transcriptional regulator, respectively. Addition of paraquat to exponentially growing E. coli culture results in oscillations of the topological state of DNA. Putrescine prevents the drop in the extent of DNA supercoiling caused by the damaging effect of free radicals during the first minutes of stress and increases it during the restoration (the peak of the transcriptional activity of the soxRS regulon genes). These effects are due to properties of putrescine as a DNA protector and modulator of its topological state. The ability of putrescine to decrease the mutation rate under conditions of superoxide stress induced by addition of paraquat is shown by the example of rifampicin resistance.

Polyamines as modulators of gene expression under oxidative stress in Escherichia coli.

Activity of enzymes of polyamine synthesis and contents of their products increased in E. coli cells in response to oxidative stress caused by addition of hydrogen peroxide to an exponentially growing culture. Putrescine and spermidine added to the culture medium in physiological concentrations significantly increased expression of genes oxyR and katG responsible for defense against oxidative stress, whereas cadaverine had no effect. The role of polyamines as modulators of the gene expression was confirmed by experiments with an inhibitor of polyamine synthesis, 1,3-diaminopropane, which decreased the level of cell polyamines and thus abolished the ability of the cell to induce oxyR expression under oxidative stress. A genetic method gave similar results: under oxidative stress mutants with disorders in polyamine synthesis displayed a significantly decreased level of induction of the oxyR and katG genes, and this level was recovered on addition of putrescine. In the presence of inhibitors of DNA-gyrase, nalidixic acid and novobiocin, the oxyR expression depended on the extent of DNA supercoiling. Putrescine decreased the inhibitory effects of nalidixic acid and novobiocin, and this confirmed its properties of a stimulator of DNA supercoiling. Resistance to rifampicin was studied to exemplify the mutation rate under oxidative stress. Putrescine decreased twofold the level of mutations and increased the number of viable cells in the culture exposed to oxidative stress.

[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.

Interaction of alcohol dehydrogenase with tert-butylhydroperoxide: stimulation of the horse liver and inhibition of the yeast enzymes.

Preincubation of horse liver alcohol dehydrogenase (HLADH) with the oxidative agent, tert-butyl hydroperoxide (tBOOH) results in a twofold stimulation of the ethanol dehydrogenase activity of this enzyme. This stimulation was dependent on tBOOH concentration up to 100 mM; above this concentration tBOOH did not further stimulate ethanol oxidation by HLADH. Active-site-directed reagents and classical ADH binary complexes were used to probe the possible mechanism of this activating effect. The rate and extent of stimulation by tBOOH is strongly reduced by binary complexes with NAD(+) or NADH, whose pyrophosphate groups bind to Arg-47 and Arg-369. In contrast stimulation by tBOOH was not prevented by AMP or the sulfhydryl reagents dithiothreitol and glutathione, suggesting, respectively, a lack of role for Lys-228 and sulfhydryl group oxidation in the stimulation by tBOOH. In contrast to the liver enzyme, treatment of yeast ADH (YADH) with tBOOH irreversibly inhibited its ethanol dehydrogenase activity. Inhibition of YADH by tBOOH approximated first-order rate kinetics with respect to enzyme at fixed concentrations of tBOOH between 0.5 to 300 mM. Four -SH groups per molecule of YADH were modified by tBOOH, whereas only two -SH groups were modified in HLADH. The stimulation of HLADH by tBOOH is suggested to be due to destabilization of the catalytic Zn-coordination sphere and amino acids associated with coenzyme binding in the active site, while inactivation of YADH appears to be associated with -SH group oxidation by the 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.

[Phasic changes in reproductive potential, intracellular polyamines and antilysozyme activity in Escherichia coli in periodic culture]. / Fazovye izmeneniia reproduktivnogo potentsiala, vnutrikletochnykh poliaminov i antilizotsimnoi aktivnosti u Escherichia coli v periodicheskoi kul'ture.

Growth-phase associated changes in and relationships between the specific growth rate (mu) characterizing the reproductive capacity of the cells, the contents of intracellular biogenic polyamines (BPA), such as putrescine (P), cadaverine (C), and spermidine (S), and antilysozyme activity (ALA) were studied in 37 strains of Escherichia coli grown in batch culture on solid medium. A decrease in mu upon the transition of the culture to the stationary growth phase was accompanied by a decrease in the pool of free BPA, mainly P and C, and by the appearance of ALA. The interrelations between the parameters studied were described as a complex of direct and negative correlations; the combination of low initial P and C contents, reduced P/S and C/S ratios, and a high level of ALA was designated as a factor of slight inhibition of E. coli reproduction. It is argued that BPA and ALA are integrated in a system controlling both the metabolism and stability of peptidoglycan in E. coli.

[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.

[Exchange of putrescine and potassium between cells and media as a factor in the adaptation of Escherichia coli to hyperosmotic shock]. / Obmen putrestsina i kaliia mezhdu kletkoi i sredoi kak faktor adaptatsii Escherichia coli k giperosmoticheskomu shoku.

Putrescine/potassium exchange in response to hyperosmotic stress was studied. The addition of 0.3 M NaCl or 0.44 M sucrose to an exponentially growing E. coli culture induced potassium uptake and putrescine release from the cell. Potassium added to an osmotically stressed potassium-deficient culture was readily absorbed by cells; this was accompanied by the loss of intracellular putrescine, both free and bound. Since DNA is the main binding site of putrescine, the loss of bound putrescine caused a relaxation of DNA supercoiling. The increase in the intracellular content of potassium not only restored but also enhanced DNA supercoiling as compared to the initial level. In vitro experiments showed the degree of plasmid DNA supercoiling to rise drastically at potassium concentrations of 300-500 mM, while different putrescine concentrations affected this parameter differently. Thus, the physiological concentrations of putrescine (below 1 mM) greatly augmented DNA supercoiling, whereas higher concentrations (5-10 mM) exerted a relaxing effect. A change in DNA supercoiling in vivo in response to osmotic stress is the result of competition between biogenic and abiogenic cations for the sites of binding to polyanionic DNA structures. A change in DNA topology serves as the regulatory factor controlling the expression of genes responsible for cell adaptation to osmotic stress.

[Role of putrescine and potassium transport in the adaptation of Escherichia coli to ammonium starvation]. / Rol' transporta putrestsina i kaliia v adaptatsii Escherichia coli k golodaniiu po ammoniiu.

Ammonium depletion in the nutrient medium induced an active transport of putrescine into the cell, which was not associated with its utilization as a nitrogen source. The uptake of putrescine was accompanied by a proportional release of potassium from the cells. Quantitative analysis of free as well as weakly and tightly bound pools of putrescine showed that this diamine was bound mainly to DNA. Topological studies by the plasmid method indicated an increase in the DNA supercoiling in response to the putrescine transport. In vitro experiments made it possible to establish an ambiguous dependence of DNA topology on putrescine content--its physiological concentrations (0.5-1.0 mM) enhanced DNA supercoiling, while higher concentrations (2-10 mM) caused gradual relaxation of DNA. A possible physiological significance of these effects in adaptive response of cells to ammonium deficiency is discussed.

[Distribution of polyamines in Escherichia coli and their role in potassium exchange between the cell and the environment during aerobiosis-anaerobiosis transitions]. / Raspredelenie poliaminov u Escherichia coli i ikh rol' v obmene kaliia mezhdu kletkoi i sredoi v protsesse aérobno-anaérobnykh perekhodov.

The transition of E. coli cells to anaerobiosis is accompanied by the onset of two cellular cation flows in different directions: potassium is released into the environment, and putrescine enters the cells. If aerobic conditions are reestablished, the cation flows change directions. Under anaerobiosis, the cell components bind putrescine. Investigation of the chemical interactions resulting in putrescine binding and metabolic conversion under anaerobic conditions revealed DNA to be the main target in this process. The driving forces and mechanisms of cation transfer are discussed, and the involvement of putrescine in topological changes in the DNA and the development of adaptive cell responses to anaerobiosis is considered.