Tarakdas Basu

Mechanism of antibacterial activity of copper nanoparticles

In a previous communication, we reported a new method of synthesis of stable metallic copper nanoparticles (Cu-NPs), which had high potency for bacterial cell filamentation and cell killing. The present study deals with the mechanism of filament formation and antibacterial roles of Cu-NPs in E. coli cells. Our results demonstrate that NP-mediated dissipation of cell membrane potential was the probable reason for the formation of cell filaments. On the other hand, Cu-NPs were found to cause multiple toxic effects such as generation of reactive oxygen species, lipid peroxidation, protein oxidation and DNA degradation in E. coli cells. In vitro interaction between plasmid pUC19 DNA and Cu-NPs showed that the degradation of DNA was highly inhibited in the presence of the divalent metal ion chelator EDTA, which indicated a positive role of Cu(2+) ions in the degradation process. Moreover, the fast destabilization, i.e. the reduction in size, of NPs in the presence of EDTA led us to propose that the nascent Cu ions liberated from the NP surface were responsible for higher reactivity of the Cu-NPs than the equivalent amount of its precursor CuCl2; the nascent ions were generated from the oxidation of metallic NPs when they were in the vicinity of agents, namely cells, biomolecules or medium components, to be reduced simultaneously.

How does plasmid DNA penetrate cell membranes in artificial transformation process of Escherichia coli

Artificial transformation of Escherichia coli with plasmid DNA in presence of CaCl2 is a widely used technique in recombinant DNA technology. However, exact mechanism of DNA transfer across cell membranes is largely obscure. In this study, measurements of both steady state and time-resolved anisotropies of fluorescent dye trimethyl ammonium diphenyl hexatriene (TMA-DPH), bound to cellular outer membrane, indicated heat-pulse (0°C→42°C) step of the standard transformation procedure had lowered considerably outer membrane fluidity of cells. The decrease in fluidity was caused by release of lipids from cell surface to extra-cellular medium. A subsequent cold-shock (42°C→0°C) to the cells raised the fluidity further to its original value and this was caused by release of membrane proteins to extra-cellular medium. When the cycle of heat-pulse and cold-shock steps was repeated, more release of lipids and proteins respectively had taken place, which ultimately enhanced transformation efficiency gradually up to third cycle. Study of competent cell surface by atomic force microscope showed release of lipids had formed pores on cell surface. Moreover, the heat-pulse step almost depolarized cellular inner membrane. In this communication, we propose heat-pulse step had two important roles on DNA entry: (a) Release of lipids and consequent formation of pores on cell surface, which helped DNA to cross outer membrane barrier, and (b) lowering of membrane potential, which facilitated DNA to cross inner membrane of E. coli.

Plasmid DNA binds to the core oligosaccharide domain of LPS molecules of E. coli cell surface in the CaCl2-mediated transformation process.

In the standard procedure for artificial transformation of E. coli by plasmid DNA, cellular competence for DNA uptake is developed by suspending the cells in ice-cold CaCl2 (50-100 mM). It is believed that CaCl2 helps DNA adsorption to the lipopolysaccharide (LPS) molecules on E. coli cell surface; however, the binding mechanism is mostly obscure. In this report, we present our findings of an in-depth study on in vitro interaction between plasmid DNA and E. coli LPS, using different techniques like absorption and circular dichroism spectroscopy, isothermal titration calorimetry, electron and atomic force microscopy, and so on. The results suggest that the Ca(II) ions, forming coordination complexes with the phosphates of DNA and LPS, facilitate the binding between them. The binding interaction appears to be cooperative, reversible, exothermic, and enthalpy-driven in nature. Binding of LPS causes a partial transition of DNA from B- to A-form. Finer study with the hydrolyzed products of LPS shows that only the core oligosaccharide domain of LPS is responsible for the interaction with DNA. Moreover, the biological significance of this interaction becomes evident from the observation that E. coli cells, from which the LPS have been leached out considerably, show higher efficiency of transformation, when transformed with plasmid-LPS complex rather than plasmid DNA alone.

The two inducible responses, SOS and heat-shock, in Escherichia coli act synergistically during Weigle reactivation of the bacteriophage ϕX174

Purpose: The objective of this study was to investigate how Escherichia coli cells responded at the level of DNA repair, when the cells were subjected to UV (ultraviolet) radiation and heat-stress to induce a DNA repair system (SOS) and heat-shock response, respectively. Materials and methods: The experiments were performed to study the Weigle reactivation of the bacteriophage ϕX174 in its host E. coli C/1 cells. Two distinct techniques, top layer agar plating and Western blotting, were employed to measure the plaque count of viable phages and to demonstrate the heat-shock response respectively. Results: Repair of UV-inactivated bacteriophages in UV-irradiated E. coli cells is known as Weigle reactivation. In the case of the single-stranded DNA containing bacteriophage ϕX174, Weigle reactivation occurs only through the inducible SOS repair response. Here we report that when UV-irradiated E. coli cells were transferred to higher temperature, the consequent heat-shock enhanced the reactivation of UV-inactivated ϕX174 over normal Weigle reactivation; the enhancement being maximum when the cells were shifted from 30 – 47°C and incubated there for 30 min. The extent of increase of reactivation was less, when the cells were first subjected to heat-shock and then irradiated by UV. Besides heat, ethanol (5 – 10% volume/volume [v/v]), an established heat-shock inducer, also caused enhancement of phage reactivation and the maximum enhancement occurred at 8% v/v ethanol. Conclusion: We suggest that the SOS and heat-shock responses in E. coli act synergistically in the reactivation of UV-damaged bacteriophage ϕX174.

Green synthesized cerium oxide nanoparticle: A prospective drug against oxidative harm

Cerium oxide nanoparticle (CeONP) of size 2-3nm was synthesized by a new, simple and green method at ambient temperature, using cerium nitrate as prime precursor and Aloe vera leaf extract as stabilizing agent. Of the two oxidation states (+3) and (+4) of cerium, it was dominantly present in (+3) state in CeONP and cyclic conversion of Ce(III)O→Ce(IV)O→Ce(III)O by reaction with H2O2 implied uninterrupted antioxidant property of CeONP. Moreover, the higher oxygen defect in the crystal lattice produced particles with higher antioxidant activity. CeONP was found to neutralize the deleterious effects of H2O2 viz., cell death, generation of intracellular reactive oxygen species and loss of connectivity in mouse neural cells. Therefore, CeONP might have potential use in future as an anti-oxidant drug.

Mechanism of protonophores-mediated induction of heat-shock response in Escherichia coli

BackgroundProtonophores are the agents that dissipate the proton-motive-force (PMF) across E. coli plasma membrane. As the PMF is known to be an energy source for the translocation of membrane and periplasmic proteins after their initial syntheses in cell cytoplasm, protonophores therefore inhibit the translocation phenomenon. In addition, protonophores also induce heat-shock-like stress response in E. coli cell. In this study, our motivation was to investigate that how the protonophores-mediated phenomena like inhibition of protein translocation and induction of heat-shock proteins in E. coli were correlated.ResultsInduction of heat-shock-like response in E. coli attained the maximum level after about 20 minutes of cell growth in the presence of a protonophore like carbonyl cyanide m-chloro phenylhydrazone (CCCP) or 2, 4-dinitrophenol (DNP). With induction, cellular level of the heat-shock regulator protein sigma-32 also increased. The increase in sigma-32 level was resulted solely from its stabilization, not from its increased synthesis. On the other hand, the protonophores inhibited the translocation of the periplasmic protein alkaline phosphatase (AP), resulting its accumulation in cell cytosol partly in aggregated and partly in dispersed form. On further cell growth, after withdrawal of the protonophores, the previously accumulated AP could not be translocated out; instead the AP-aggregate had been degraded perhaps by an induced heat-shock protease ClpP. Moreover, the non-translocated AP formed binary complex with the induced heat-shock chaperone DnaK and the excess cellular concentration of DnaK disallowed the induction of heat-shock response by the protonophores.ConclusionOur experimental results suggested that the protonophores-mediated accumulation and aggregation of membrane proteins (like AP) in cell cytosol had signaled the induction of heat-shock proteins in E. coli and the non-translocated protein aggregates were possibly degraded by an induced heat-shock protease ClpP. Moreover, the induction of heat-shock response occurred by the stabilization of sigma-32. As, normally the DnaK-bound sigma-32 was known to be degraded by the heat-shock protease FtsH, our experimental results further suggested that the engagement of DnaK with the non-translocated proteins (like AP) had made the sigma-32 free and stable.

In vitro interaction between calf thymus DNA and Escherichia coli LPS in the presence of divalent cation Ca2

With increasing addition of Escherichia coli LPS to calf thymus DNA, both dissolved in CaCl2, absorption maxima of DNA at 260 nm decreased gradually with the appearance of isosbastic points at both ends of spectra, which implied some binding between DNA and LPS. Hill plot of absorbance data showed that the binding interaction was positive cooperative in nature. For any fixed concentration of DNA and LPS, extent of interaction increased as concentration of CaCl2 was raised from 1.0 to 100 mM, signifying the electrostatic nature of the interaction, mediated through Ca2+ ion. Stepwise addition of EDTA, a chelating agent for divalent cations, to DNA-LPS bound complex gradually reversed the spectral shift with increase in absorbance at 260 nm, which implied opening up of the complex, that is, reversible nature of the interaction. Circular dichroism spectral changes of DNA by the addition of LPS indicated partial transition of DNA from B to A form. Isothermal titration calorimetric (ITC) study showed that the DNA-LPS binding was an exothermic and enthalpy-driven phenomenon. Moreover, in the presence of 100 mM CaCl2, binding constant of the interaction was found to be 2.6 x 10(4) M(-1) and 3.1 x 10(4) M(-1) from the analysis of Hill plot and ITC result, respectively. DNA-melting study showed that the LPS binding had increased the melting temperature of DNA, indicating more stabilization of DNA double helix. The binding of LPS to DNA made the complex resistant to digestion with endonucleases EcoRI and DNase I.

GroEL to DnaK chaperone network behind the stability modulation of σ32 at physiological temperature in Escherichia coli

The stability of heat‐shock transcription factor σ32 in Escherichia coli has long been known to be modulated only by its own transcribed chaperone DnaK. Very few reports suggest a role for another heat‐shock chaperone, GroEL, for maintenance of cellular σ32 level. The present study demonstrates in vivo physical association between GroEL and σ32 in E. coli at physiological temperature. This study further reveals that neither DnaK nor GroEL singly can modulate σ32 stability in vivo; there is an ordered network between them, where GroEL acts upstream of DnaK.

Over expression of inducible proteins in Escherichia coli by treatment with ethanol

It is reported that ethanol enhances DNA synthesis in E. coli cells [Basu, T and Poddar, R K (1994), Folia. Microbiol., 39, 3‐6]. This communication reports that during growth of E. coli in the presence of 5% v/v ethanol, the derepressed expression of the cytoplasmic enzymes β‐galactosidase and D‐serine deaminase per cell increased approximately three fold, while that of the periplasmic enzyme alkaline phosphatase decreased approximately 40% compared to control cell levels. However, in cells transformed with the plasmid pSM 456, bearing phoA‐lacZ fusion, the level of induced synthesis of the hybrid protein PhoA‐LacZ, controlled by the phoA promoter, was elevated by 25% in the presence of 5% v/v ethanol. This result suggests that the induction of the alkaline phosphatase precursor has also been enhanced by the ethanol treatment; but the inhibition in the export of the precursor across the cytoplasmic membrane, by the influence of ethanol, may represent the reason for the deficient expression of active alkaline phosphatase. It is proposed that there is an ethanol‐mediated increase in DNA synthesis, resulting in gene amplification, which may enhance the synthesis of inducible proteins in ethanol‐treated cells.

Calcium phosphate-quercetin nanocomposite (CPQN): A multi-functional nanoparticle having pH indicating, highly fluorescent and anti-oxidant properties

Calcium phosphate quercetin nanocomposite (CPQN) i.e., quercetin entrapped in calcium phosphate nanoparticle was synthesized by a precipitation method at 80°C, taking ammonium hydrogen phosphate, calcium nitrate and quercetin as precursors and sodium citrate as stabilizer. The nanocomposite suspension had different color at different pH values, a property that could render the nanoparticle a pH indicator. Besides color, the particles also had different size, shape, stability and quercetin content with change of pH. In addition, the CPQN was highly fluorescent having two sharp emission peaks at 460 and 497nm, when excited at 370nm; by this property it behaved as an effective fluorophore to label biological cell. Moreover, the nanocomposite had potential anti-oxidant property, for which mortality of mouse neuroblastoma cell N2A, by H2O2-induced oxidative stress, was found to be lowered by the pre-treatment of the cells with CPQN.