Copper tolerance mediated by polyphosphate degradation and low-affinity inorganic phosphate transport system in Escherichia coli.

BACKGROUND: Metal tolerance in bacteria has been related to polyP in a model in which heavy metals stimulate the polymer hydrolysis, forming metal-phosphate complexes that are exported. As previously described in our laboratory, Escherichia coli cells grown in media containing a phosphate concentration >37 mM maintained an unusually high polyphosphate (polyP) level in stationary phase. The aim of the present work was to evaluate the influence of polyP levels as the involvement of low-affinity inorganic phosphate transport (Pit) system in E. coli copper tolerance. RESULTS: PolyP levels were modulated by the media phosphate concentration and/or using mutants in polyP metabolism. Stationary phase wild-type cells grown in high phosphate medium were significantly more tolerant to copper than those grown in sufficient phosphate medium. Copper addition to tolerant cells induced polyP degradation by PPX (an exopolyphosphatase), phosphate efflux and membrane polarization. ppk-ppx- (unable to synthesize/degrade polyP), ppx- (unable to degrade polyP) and Pit system mutants were highly sensitive to metal even in high phosphate media. In exponential phase, CopA and polyP-Pit system would act simultaneously to detoxify the metal or one could be sufficient to safeguard the absence of the other. CONCLUSIONS: Our results support a mechanism for copper detoxification in exponential and stationary phases of E. coli, involving Pit system and degradation of polyP. Data reflect the importance of the environmental phosphate concentration in the regulation of the microbial physiological state.

Inhibition of Penicillium expansum by an oxidative treatment.

Several oxidizing compounds such as sodium hypochlorite (NaClO) and hydrogen peroxide (H(2)O(2)) are used to control postharvest decay in fresh fruit due to their antimicrobial effects. Here, we applied these compounds in vitro, in the presence of CuSO(4), against Penicillium expansum, causal agent of apple blue mold. MICs were 50 mg L(-1) and 400 mmol L(-1) for NaClO and H(2)O(2), respectively, when these compounds were individually applied to conidia suspensions during 2 min. A combined oxidative treatment (OT) consisting on an incubation with 1 mg L(-1) NaClO and 200 mmol L(-1) H(2)O(2), in the presence of 6 mmol L(-1) CuSO(4), inhibited growth, conidial germination and fungal infectivity on apple. The fractional inhibitory concentration index for the interaction between NaClO and H(2)O(2) in the OT was 0.52 indicating a synergistic effect of the oxidizing compounds. These results suggest that the OT could be an interesting alternative for apple diseases postharvest control.

Cu(II)-reduction by Escherichia coli cells is dependent on respiratory chain components.

Copper is both an essential nutrient and a toxic element able to catalyze free radicals formation which damage lipids and proteins. Although the available copper redox species in aerobic environment is Cu(II), proteins that participate in metal homeostasis use Cu(I). With isolated Escherichia coli membranes, we have previously shown that electron flow through the respiratory chain promotes cupric ions reduction by NADH dehydrogenase-2 and quinones. Here, we determined Cu(II)-reductase activity by whole cells using strains deficient in these respiratory chain components. Measurements were done by the appearance of Cu(I) in the supernatants of cells exposed to sub-lethal Cu(II) concentrations. In the absence of quinones, the Cu(II)-reduction rate decreased ~70% in respect to the wild-type strain, while this diminution was about 85% in a strain lacking both NDH-2 and quinones. The decrease was ~10% in the absence of only NDH-2. In addition, we observed that quinone deficient strains failed to grow in media containing either excess or deficiency of copper, as we have described for NDH-2 deficient mutants. Thus, the Cu(II)-reduction by E. coli intact cells is mainly due to quinones and to a lesser extent to NDH-2, in a quinone-independent way. To our knowledge, this is the first in vivo demonstration of the involvement of E. coli respiratory components in the Cu(II)-reductase activity which contributes to the metal homeostasis.

Uptake of chromium by Salvinia minima: effect on plant growth, leaf respiration and carbohydrate metabolism.

Metabolic responses to chromium (Cr) exposure and metal uptake were investigated using Salvinia minima plants. Cr treatment reduced the dry weight of floating and submerged leaves, while photosynthetic pigments were not affected. Measurements of respiratory oxygen uptake with and without inhibitors (KCN and SHAM) demonstrated that total respiration, alternative oxidase capacity and residual respiration were higher in Cr-treated than in Cr-untreated leaves, but the highest values were observed in floating leaves. Cr affected the soluble sugar content. Sucrose concentration was, in general, higher in Cr-treated than in Cr-untreated leaves, while the glucose concentration showed an inverse pattern. Cr also affected soluble acid invertase activity, but affectation trend was different between both leaves. Highest values of invertase activity were observed in Cr-treated floating leaves. According to our data soluble acid invertase and sucrose seem to be related to alternative oxidase capacity and residual respiration in floating and submerged leaves exposed to Cr. Thereby, this study constitutes an important contribution to understand metabolic relationships between mitochondrial respiration, alternative respiratory pathway and soluble carbohydrates in plants exposed to heavy metals.

Synergistic antifungal activity of sodium hypochlorite, hydrogen peroxide, and cupric sulfate against Penicillium digitatum.

Oxidizing compounds such as sodium hypochlorite (NaCIO) and hydrogen peroxide (H2O2) are widely used in food sanitization because of their antimicrobial effects. We applied these compounds and metals to analyze their antifungal activity against Penicillium digitatum, the causal agent of citrus green mold. The MICs were 300 ppm for NaClO and 300 mM for H2O2 when these compounds were individually applied for 2 min to conidia suspensions. To minimize the concentration of these compounds, we developed and standardized a sequential treatment for conidia that resulted in loss of viability on growth plates and loss of infectivity on lemons. The in vitro treatment consists of preincubation with 10 ppm of NaClO followed by incubation with 100 mM H2O2 and 6 mM CuSO4 (cupric sulfate). The combination of NaClO and H2O2 in the presence of CuSO4 produces a synergistic effect (fractional inhibitory concentration index of 0.36). The sequential treatment applied in situ on lemon peel 24 h after the fruit was inoculated with conidia produced a significant delay in the fungal infection. The in vitro treatment was effective on both imazalil-sensitive and imazalil-resistant strains of P. digitatum and Geotrichum candidum, the causal agent of citrus sour rot. However, this treatment inhibited 90% of mycelial growth for Penicillium italicum (citrus blue mold). These results indicate that sequential treatment may be useful for postharvest control of citrus fruit diseases.

Protection against oxidative stress in Escherichia coli stationary phase by a phosphate concentration-dependent genes expression.

Escherichia coli gradually decline the capacity to resist oxidative stress during stationary phase. Besides the aerobic electron transport chain components are down-regulated in response to growth arrest. However, we have previously reported that E. coli cells grown in media containing at least 37mM phosphate maintained ndh expression in stationary phase, having high viability and low NADH/NAD(+) ratio. Here we demonstrated that, in the former condition, other aerobic respiratory genes (nuoAB, sdhC, cydA, and ubiC) expression was maintained. In addition, reactive oxygen species production was minimal and consequently the levels of thiobarbituric acid-reactive substances and protein carbonylation were lower than the expected for stationary cells. Interestingly, defense genes (katG and ahpC) expression was also maintained during this phase. Our results indicate that cells grown in high phosphate media exhibit advantages to resist endogenous and exogenous oxidative stress in stationary phase.

Solar and supplemental UV-B radiation effects in lemon peel UV-B-absorbing compound content-seasonal variations.

Effects of solar and supplemental UV-B radiation on UV-B-absorbing compounds and malondialdehyde (MDA) accumulations in the peel of lemons collected in summer and winter were analyzed. UV-B-absorbing compounds were higher in flavedo than in albedo tissue in both seasons; however, the highest values were observed in summer. These compounds were also higher in outer than in inner flavedo surface. Lemons were categorized as sun-, semisun- and shaded-lemon according to localization inside the tree canopy. Depending on-tree localization UV-B-absorbing compounds were higher in flavedo of sun-lemon than in semisun- and shaded-lemon. Supplementary UV-B radiation (22 kJ m(-2) day(-1) UV-BBE) induced UV-B-absorbing compound synthesis in on-tree and postharvest lemons. Two minutes of supplemental UV-B irradiation in summer lemons produced a strong increment (300%) of UV-B-absorbing compound content, whereas in winter lemons a slight increase (30%) was observed only after 3 min of irradiation. By contrast, UV-B-absorbing compound accumulation was not observed in albedo. MDA accumulation showed approximately a similar trend of UV-B-absorbing compounds. According to our results, solar UV-B was not required for UV-B-absorbing compound accumulation in lemon peel. Relationships between UV-B-absorbing compounds, MDA, reactive oxygen species and pathogen protection are also discussed.

A critical phosphate concentration in the stationary phase maintains ndh gene expression and aerobic respiratory chain activity in Escherichia coli.

Escherichia coli NADH dehydrogenase-2 (NDH-2) is a primary dehydrogenase in aerobic respiration that shows cupric-reductase activity. The enzyme is encoded by ndh, which is highly regulated by global transcription factors. It was described that the gene is expressed in the exponential growth phase and repressed in late stationary phase. We report the maintenance of NDH-2 activity and ndh expression in the stationary phase when cells were grown in media containing at least 37 mM phosphate. Gene regulation was independent of RpoS and other transcription factors described to interact with the ndh promoter. At this critical phosphate concentration, cell viability, oxygen consumption rate, and NADH/NAD+ ratio were maintained in the stationary phase. These physiological parameters gradually changed, but NDH-2 activity remained high for up to 94 h. Phosphate seems to trigger an internal signal in the stationary phase mediated by systems not yet described.

The Cu(II)-reductase NADH dehydrogenase-2 of Escherichia coli improves the bacterial growth in extreme copper concentrations and increases the resistance to the damage caused by copper and hydroperoxide.

NADH dehydrogenase-2 (NDH-2) from Escherichia coli respiratory chain is a membrane-bound cupric-reductase encoded by ndh gene. Here, we report that the respiratory system of a ndh deficient strain suffered a faster inactivation than that of the parental strain in the presence of tert-butyl hydroperoxide due to endogenous copper. The inactivation was similar for both strains when copper concentration increased in the culture media. Furthermore, several ndh deficient mutants grew less well than the corresponding parental strains in media containing either high or low copper concentrations. A mutant strain complemented with ndh gene almost recovered the parental phenotype for growing in copper limitation or excess. Then, NDH-2 gives the bacteria advantages to diminish the susceptibility of the respiratory chain to damaging effects produced by copper and hydroperoxides and to survive in extreme copper conditions. These results suggest that NDH-2 contributes in the bacterial oxidative protection and in the copper homeostasis.

Evidence for Cu(I)-thiolate ligation and prediction of a putative copper-binding site in the Escherichia coli NADH dehydrogenase-2.

NADH dehydrogenase-2 (NDH-2) from Escherichia coli is a membrane-bound flavoprotein linked to the respiratory chain. We have previously shown that this enzyme has cupric reductase activity that is involved in hydroperoxide-induced oxidative stress. In this paper we present spectroscopic evidence that NDH-2 contains thiolate-bound Cu(I) with luminescence properties. Purified NDH-2 exhibits an emission band at 670nm with excitation wavelengths of 280 and 580nm. This emission is quenched by the specific Cu(I) chelator bathocuproine disulfonate, but not by EDTA. The luminescence intensity is sensitive to the enzyme substrates and, thus, the Cu(I)-thiolate chromophore reflects the redox and/or conformational states of the protein. There is one copper atom per polypeptide chain of the purified NDH-2, as determined by atomic absorption spectroscopy. Bioinformatics allowed us to recognize a putative copper-binding site and to predict four structural/functional domains in NDH-2: (I) the FAD-binding domain, (II) the NAD(H)-binding domain, (III) the copper-binding domain, and (IV) the domain of anchorage to the membrane containing two transmembrane helices, at the C-terminus. A NDH-2 topology model, based on the secondary structure prediction, is proposed. This is the first description of a copper-containing NADH dehydrogenase. Comparative sequence analysis allowed us to identify a branch of homologous dehydrogenases that bear a similar metal-binding motif.

Un nuevo método químico específico para la determinación de la heparina y otros glicosaminoglicanos naturales / New chemical method for determination of heparin and other glycosaminoglycans

El objetivo de la investigación fue la obtención de un método que permitiera la determinación de la heparina y otros glicosaminoglicanos (GAG) naturales en las cantidades pequeñas en que se los encuentra en los líquidos biológicos. El método debía ser sencillo y de la mayor especificidad posible. Se ha logrado poner a punto la reacción de los hidratos de carbono con el indol en medio ácido, de manera tal que, dependiendo de las condiciones de una hidrólisis previa también en medio ácido, sea específica, bien para la heparina y el dermatán sulfato, o bien para el condroitín 4-sulfato o el condroitín 6-sulfato. La albúmina humana y las sales inorgánicas no interfieren con la reacción, lo que valoriza el método para su aplicación a material biológica. La sencillez de la técnica, pues no requiere ninguna precipitación previa, acrecienta en mayor medida su valor potencial para la Bioquímica analítica (AU)

Un nuevo método químico específico para la determinación de la heparina y otros glicosaminoglicanos naturales / New chemical method for determination of heparin and other glycosaminoglycans

El objetivo de la investigación fue la obtención de un método que permitiera la determinación de la heparina y otros glicosaminoglicanos (GAG) naturales en las cantidades pequeñas en que se los encuentra en los líquidos biológicos. El método debía ser sencillo y de la mayor especificidad posible. Se ha logrado poner a punto la reacción de los hidratos de carbono con el indol en medio ácido, de manera tal que, dependiendo de las condiciones de una hidrólisis previa también en medio ácido, sea específica, bien para la heparina y el dermatán sulfato, o bien para el condroitín 4-sulfato o el condroitín 6-sulfato. La albúmina humana y las sales inorgánicas no interfieren con la reacción, lo que valoriza el método para su aplicación a material biológica. La sencillez de la técnica, pues no requiere ninguna precipitación previa, acrecienta en mayor medida su valor potencial para la Bioquímica analítica