Viviana A. Rapisarda

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.

Quenching of bathocuproine disulfonate fluorescence by Cu(I) as a basis for copper quantification.

In this paper we report the up to now ignored fluorescence properties of the specific Cu(I)-chelator bathocuproine disulfonate and their application in assays of total copper and Cu(I). The method is based on the linear quenching of the bathocuproine disulfonate emission at 770 nm (lambda(ex)580 nm) by increasing concentrations of Cu(I), at pH 7.5. Copper concentrations as low as 0.1 microM can be determined. Other metal ions (iron, manganese, zinc, cadmium, cobalt, nickel) do not interfere. The procedure for total copper determination in proteins includes HCl treatment to release the copper, neutralization to pH 7.5 in the presence of citrate to stabilize the copper, and reduction of the copper to Cu(I) by ascorbate in the presence of the chelator. This assay gave results coincident with the analysis by atomic absorption spectroscopy in two selected proteins. In addition, conditions are described (omitting HCl treatment and reduction by ascorbate) for direct measurement of Cu(I) in native proteins, as illustrated for the Escherichia coli NADH dehydrogenase-2. Data show that the fluorometric assays described in this paper are simple and convenient procedures for total copper and direct Cu(I) quantification in determined biological samples.

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.

Cellular damage induced by a sequential oxidative treatment on Penicillium digitatum

Aim:  To investigate the cellular damage on Penicillium digitatum produced by a sequential oxidative treatment (SOT), previously standardized in our laboratory, to prevent the conidia growth. Lethal SOT consists of 2‐min preincubation with 10 ppm NaClO followed by 2‐min incubation with 6 mmol l−1 CuSO4 and 100 mmol l−1 H2O2 at 25°C.

Polyphosphate Degradation in Stationary Phase Triggers Biofilm Formation via LuxS Quorum Sensing System in Escherichia coli

In most natural environments, association with a surface in a structure known as biofilm is the prevailing microbial life-style of bacteria. Polyphosphate (polyP), an ubiquitous linear polymer of hundreds of orthophosphate residues, has a crucial role in stress responses, stationary-phase survival, and it was associated to bacterial biofilm formation and production of virulence factors. In previous work, we have shown that Escherichia coli cells grown in media containing a critical phosphate concentration >37 mM maintained an unusual high polyP level in stationary phase. The aim of the present work was to analyze if fluctuations in polyP levels in stationary phase affect biofilm formation capacity in E. coli. Polymer levels were modulated by the media phosphate concentration or using mutant strains in polyP metabolism. Cells grown in media containing phosphate concentrations higher than 25 mM were defective in biofilm formation. Besides, there was a disassembly of 24 h preformed biofilm by the addition of high phosphate concentration to the medium. These phenotypes were related to the maintenance or re-synthesis of polyP in stationary phase in static conditions. No biofilm formation was observed in ppk−ppx− or ppk−ppx−/ppk+ strains, deficient in polyP synthesis and hydrolysis, respectively. luxS and lsrK mutants, impaired in autoinducer-2 quorum sensing signal metabolism, were unable to form biofilm unless conditioned media from stationary phase wild type cells grown in low phosphate were used. We conclude that polyP degradation is required for biofilm formation in sufficient phosphate media, activating or triggering the production of autoinducer-2. According to our results, phosphate concentration of the culture media should be carefully considered in bacterial adhesion and virulence studies.

Phosphate-Enhanced Stationary-Phase Fitness of Escherichia coli Is Related to Inorganic Polyphosphate Level

We found that Escherichia coli grown in media with >37 mM phosphate maintained a high polyphosphate level in late stationary phase, which could account for changes in gene expression and enzyme activities that enhance stationary-phase fitness.

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.

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.

Amphipathic C-terminal region of Escherichia coli NADH dehydrogenase-2 mediates membrane localization

Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a membrane-bound flavoprotein. Bioinformatics approaches suggested the involvement of NDH-2 C-terminal region in membrane anchorage. Here, we demonstrated that NDH-2 is a peripheral membrane protein and that its predicted C-terminal amphipathic Arg390-Ala406 helix is sufficient to bind the protein to lipid membranes. Additionally, a cytosolic NDH-2 protein (Trun-3), lacking the last 43 aminoacids, was purified and characterized. FAD cofactor was absent in purified Trun-3. Upon the addition of FAD, Trun-3 maximum velocity was similar to native NDH-2 rate with ferricyanide and MTT acceptors. However, Trun-3 activity was around 5-fold lower with quinones. No significant difference in K(m) values was observed for both enzymes. For the first time, an active and water soluble NDH-2 was obtained, representing a major improvement for structural/functional characterizations.

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.