Resistance acquisition of Thiobacillus thiooxidans upon cadmium and zinc ion addition and formation of cadmium ion-binding and zinc ion-binding proteins exhibiting metallothionein-like properties

Through subcultivations of Thiobacillus thiooxidans WU-79A in autotrophic media in which the concentrations of Cd²⁺ and Zn²⁺ were increased successively, Cd²⁺-resistant (CDR) and Zn²⁺-resistant strains (ZNR) were obtained. The growth of WU-79A was inhibited by the addition of 25 mM Cd²⁺ as well as Zn²⁺. However, CDR and ZNR could grow without any lag phase in media containing 200 mM Cd²⁺ and 250 mM Zn²⁺, respectively. CDR and ZNR were able to grow even in media containing up to 400 mM Cd²⁺ and 600 mM Zn²⁺, respectively, although they exhibited lag phases. CDR could grow in medium containing up to 250 mM Zn²⁺, as could ZNR in medium containing up to 200 mM Cd²⁺. Cd²⁺-binding and Zn²⁺-binding proteins were isolated from CDR and ZNR, respectively, by gel filtration and ion exchange chromatography. The molecular weights of both proteins were estimated to be approximately 13,000 by gel filtration. The fact that there was no strong absorption at 280 nm of the proteins suggested that they had few aromatic amino acids. Broad absorption bands which are typical of mercaptide (metal thiolate) complexes were detected. The properties of the proteins were spectrophotometrically similar to those of metallothionein.     

Cd<Superscript>2+</Superscript> extrusion by P-type Cd<Superscript>2+</Superscript>-ATPase of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> 17810R via energy-dependent Cd<Superscript>2+</Superscript>/H<Superscript>+</Superscript> exchange mechanism

Cd²⁺ is highly toxic to Staphylococcus aureus since it blocks dithiols in cytoplasmic 2-oxoglutarate dehydrogenase complex (ODHC) participating in energy conservation process. However, S. aureus 17810R is Cd²⁺-resistant due to possession of cadA-coded Cd²⁺ efflux system, recognized here as P-type Cd²⁺-ATPase. This Cd²⁺ pump utilizing cellular energy—ATP, ?μ _H ⁺ (electrochemical proton potential) and respiratory protons, extrudes Cd²⁺ from cytoplasm to protect dithiols in ODHC, but the mechanism of Cd²⁺ extrusion remains unknown. Here we propose that two Cd²⁺ taken up by strain 17810R via Mn²⁺ uniporter down membrane potential (?ψ) generated during glutamate oxidation in 100 mM phosphate buffer (high P_iB) are trapped probably by high affinity sites in cytoplasmic domain of Cd²⁺-ATPase, forming SCdS. This stops Cd²⁺ transport towards dithiols in ODHC, allowing undisturbed NADH production, its oxidation and energy conservation, while ATP could change orientation of SCdS towards facing transmembrane channel. Now, increased number of P_i-dependent protons pumped electrogenically via respiratory chain and countertransported through the channel down ?ψ, extrude two trapped cytoplasmic Cd²⁺, which move to low affinity sites, being then extruded into extracellular space via ?ψ-dependent Cd²⁺/H⁺ exchange. In 1 mM phosphate buffer (low P_iB), external Cd²⁺ competing with decreased number of P_i-dependent protons, binds to ψ_s of Cd²⁺-ATPase channel, enters cytoplasm through the channel down ?ψ via Cd²⁺/Cd²⁺ exchange and blocks dithiols in ODHC. However, Mg²⁺ pretreatment preventing external Cd²⁺ countertransport through the channel down ?ψ, allowed undisturbed NADH production, its oxidation and extrusion of two cytoplasmic Cd²⁺ via Cd²⁺/H⁺ exchange, despite low P_iB.     

Mobilization ofEscherichia coli R1 silver-resistance plasmid pJT1 by Tn5-Mob intoEscherichia coli C600

Summary Escherichia coli Rl is an Ag⁺-resistant strain that, as we have shown recently, harbours at least two large plasmids, pJT1 (83 kb) and pJT2 (77 kb). Tn5-Mob was introduced into theE. coli Rl host replicon via conjugation on membrane filters. The transfer functions of plasmid RP4-4 were provided in this process and Tn5-Mob clones mated withE. coli C600 yielded Ag⁺-resistant transconjugants. This mobilization procedure allowed transfer and expression of pJT1 Ag⁺ resistance inE. coli C600. Prior to use of Tn5-Mob mobilization, it was not possible to transfer Ag⁺-resistant determinant(s) intoE. coli by conjugation or transformation including high-voltage electroporation.E. coli C600 containing PJTI and PJT2 displayed decreased accumulation of Ag⁺ similar toE. coli R1.E. coli C600 could not tolerate 0.1 and 0.5 mM Ag⁺, rapidly accumulated Ag⁺ and became non-viable. Tn5-Mob mobilization may be useful in the study of metal resistance in bacteria, especially in strains not studied for resistance mechanisms.     

Some characteristics of membrane Cd<Superscript>2+</Superscript> transport in rat thymocytes: an analysis using Fluo-3

Although cadmium-induced apoptosis of lymphocytes is one of common features in the immunotoxicity of cadmium, the membrane pathway for intracellular cadmium accumulation is not fully elucidated. To characterize membrane Cd²⁺ transport of rat thymocytes, the change in intracellular Cd²⁺ concentration under various conditions was examined by the use of Fluo-3, a fluorescent probe for monitoring the change in intracellular concentration of divalent metal cations. The membrane Cd²⁺ transport was estimated by the augmentation of Fluo-3 fluorescence induced by bath application of CdCl₂. Lowering temperature strongly suppressed the augmentation of Fluo-3 fluorescence by CdCl₂, suggesting that the metabolic process can be involved in membrane Cd²⁺ transport. External acidification (decreasing pH) and membrane depolarization by adding KCl attenuated the augmentation, indicating the requirement of electrochemical driving force for membrane Cd²⁺ transport into the cells. Bath application of CaCl₂ and ZnCl₂ equally decreased the augmentation, suggesting their competition with Cd²⁺ at the membrane transport. The augmentation by CdCl₂ was lesser in the cells treated with N-ethylmaleimide inducing chemical depletion of cellular thiols. The result suggests the contribution of sulfhydryl groups to membrane Cd²⁺ transport. Taken together, it is suggested that the cells possess a temperature-sensitive membrane Cd²⁺ pathway, driven by electrochemical gradient of Cd²⁺ and transmembrane potential, with competitive binding site. Based on the characteristics described above, it is unlikely that the membrane Cd²⁺ transport in rat thymocytes is attributed to a single transport system although it has characteristics that are similar to those of divalent cation transporter 1.     

A metallothionein-like protein of rice (rgMT) functions in E. coli and its gene expression is induced by abiotic stresses

A metallothionein-like (rgMT) gene was isolated from a rice (Oryza sativa L.) root cDNA library that was prepared from plants grown under NaHCO₃ stress. The rgMT gene expression was induced in rice leaves and roots under several abiotic stresses from salts (NaCl and NaHCO₃), drought (PEG) and metals (CuCl_2, ZnCl₂, CdCl₂). The results suggested that the rgMT gene was expressed in response to environmental stresses. The rgMT gene was expressed in Escherichia coli, and the final yield of the purified rgMT protein was 4.8 mg g⁻¹ dry cells. Tolerance of E. coli expressing GST-rgMT fusion protein to Cu²⁺, Zn²⁺ and Cd²⁺ was enhanced, and cells dry weight increased 0.04 mg, 0.17 mg and 0.07 mg in 1 ml culture treated with either CuCl₂, ZnCl₂ or CdCl₂, respectively, compared with control after 6 h culture.     

Selective inhibition of L-glutamate and gammaaminobutyrate transport in nerve ending particles by aluminium,manganese, and cadmium chloride

AlCl₃, MnCl₂, and CdCl₂ inhibited the rates of accumulation of ¹⁴C] L-glutamate and ³H] gammaaminobutyrate (GABA) in purified rat forebrain nerve-ending particles in a dose-dependent fashion. The concentrations that would give 50% inhibition (IC₅₀) of GABA transport were 316 μM, 7.4 mM, and 1.4 mM, respectively. Ca²⁺ (1 mM) enhanced the inhibitory effect of Al³⁺ (IC₅₀ decreased to 149 μM) but antagonized that of Mn²⁺ (IC₅₀ = 10 mM) and Cd²⁺ (IC₅₀ = 2.1 mM). For glutamate transport 1 mM Ca²⁺ changed the IC₅₀ values from 299 to 224 μm for Al³⁺, 7.1 to 10 mM for Mn²⁺, and 2 to 3 mM for Cd²⁺. In contrast, the rates of accumulation of ¹⁴C] 2-deoxy-glucose and ³H] L-phenylalanine were mostly unaffected by these metal ions. The results indicate that Al³⁺, Mn²⁺, and Cd²⁺ exerted selective and differential effects on the transport systems of neurotransmitter substances in the synaptosomal membrane.     

Exogenous Abscisic Acid Alleviates Cadmium Toxicity by Restricting Cd2+ Influx in Populus euphratica Cells

Abscisic acid (ABA), a widely known phytohormone involved in the plant response to abiotic stress, plays a vital role in mitigating Cd²⁺ toxicity in herbaceous species. However, the role of ABA in ameliorating Cd²⁺ toxicity in woody species is largely unknown. In the present study, we investigated ABA restriction on Cd²⁺ uptake and the relevance to Cd²⁺ stress alleviation in Cd²⁺-hypersensitive Populus euphratica. ABA (5 μM) markedly improved cell viability and growth but reduced membrane permeability in CdCl₂ (100 μM)-stressed P. euphratica cells. Moreover, ABA significantly increased the activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX), contributing to the scavenging of Cd²⁺-elicited H₂O₂ within P. euphratica cells during the period of CdCl₂ exposure (100 μM, 24–72 h). ABA alleviation of Cd²⁺ toxicity was mainly the result of ABA restriction of Cd²⁺ uptake under Cd²⁺ stress. Steady-state and transient flux recordings showed that ABA inhibited Cd²⁺ entry into Cd²⁺-shocked (100 μM, 30 min) and short-term-stressed P. euphratica cells (100 μM, 24–72 h). Non-invasive micro-test technique data showed that H₂O₂ (3 mM) stimulated the Cd²⁺-elicited Cd²⁺ influx but that the plasma membrane (PM) Ca²⁺ channel inhibitor LaCl₃ blocked it, suggesting that the Cd²⁺ influx was through PM Ca²⁺-permeable channels. These results suggested that ABA up-regulated antioxidant enzyme activity in Cd²⁺-stressed P. euphratica and that these enzymes scavenged the Cd²⁺-elicited H₂O₂ within cells. The entry of Cd²⁺ through the H₂O₂-mediated Ca²⁺-permeable channels was subsequently restricted; thus, Cd²⁺ buildup and toxicity were reduced in the Cd²⁺-hypersensitive species, P. euphratica.

     

Actin assembly by cadmium ions

Cadmium is a highly toxic metal entering cells by a variety of mechanisms. Its toxic action is far from being completely understood, although specific interaction with the cellular calcium metabolism has been indicated. Metal ions that influence intracellular Ca²⁺ concentrations or compete with Ca²⁺ for protein binding sites may exert an effect on actin filaments, whose assembly and disassembly are both regulated by a number of calcium-dependent factors. Cadmium is such a metal. Much evidence demonstrates that cadmium interferes with the dynamics of actin filaments in various types of cells. Here we show that, at high (0.8–1.0 mM) concentrations, CdCl₂ causes actin denaturation. At such Cd²⁺ concentrations, actin precipitates (really actin, as shown by SDS-PAGE, see Fig. 1B) in the form of irregular, disordered clots, clearly appreciable by electron microscopy. Denaturation seems to be reversible since, after Cd²⁺ removal by dialysis, the polymerizability of sedimented actin is restored almost completely. On the other hand, at concentrations ranging from 0.25 to 0.6 mM, CdCl₂ is more effective as an actin polymerizing agent than both MgCl₂ and CaCl₂. The Cd-related increase in the actin assembly rate is ascribable to an enhanced nucleation rather than to an increased monomer addition to filament growing ends. The latter, in contrast, appears quite slow. Critical concentration measurements revealed that the extent of polymerization of both Mg- and Cd-assembled actin are very close (C_c ranges from 0.25 to 0.5 μM), while Ca-polymerized actin shows a polymerization extent markedly lower (C_c=4.0 μM). By both the fluorescent Ca²⁺ chelator Quin-2 assay and limited proteolysis of actin by trypsin and α-chymotrypsin, the real substitution of G-actin-bound Ca²⁺ by Cd²⁺ has been appreciated. The increase in Quin-2 fluorescence after addition of excess CdCl₂ indicates that, in our experimental conditions, Ca²⁺ tightly-bound to actin is partially (60–70%) replaced by Cd²⁺, forming Cd-actin. Electrophoretic patterns after limited proteolysis reveal that the trypsin cleavage sites in the segment 61–69 of the actin polypeptide chain are less accessible in Cd-actin than in Ca-actin, although the cation-dependent effect is less pronounced in Cd-actin than in Mg-actin. Our results are consistent with some of the consequences on microfilament organization observed in Cd²⁺-treated cells; however, considering the positive effect of Cd²⁺ on actin polymerization in solution we have noticed that this was never observed in vivo. A different indirect effect of Cd²⁺ on some cellular event(s) influencing cytoplasmic actin polymerization appears to be reasonable. © 1997 Elsevier Science B.V. All rights reserved.     

The development ofChlorella vulgaris cells exposed to cadmium at successive stages of their life cycle

The effect of CdCl₂ in a concentration range 0.01-10.0 g m^-3 on the growth ofChlorella vulgaris under synchronous cultivation conditions was determined. The general biological activity, the growth multiplication factor, the cell size and shape and intracellular arrangement showed disturbances of synchronization that depended on Cd²⁺ concentration. The highest inhibition of all mentioned parameters was observed when Cd²⁺ was administered after the second hour of synchronous cultivation, whereas the administration after 6 or 8 h did not induce any significant effect.     

Cadmium uptake in Elodea canadensis leaves and its interference with extra‐ and intra‐cellular pH

This study investigated cadmium (Cd) uptake in Elodea canadensis shoots under different photosynthetic conditions, and its effects on internal (cytosolic) and external pH. The plants were grown under photosynthetic (light) or non‐photosynthetic (dark or in the presence of a photosynthetic inhibitor) conditions in the presence or absence of CdCl₂ (0.5 μm ) in a medium with a starting pH of 5.0. The pH‐sensitive dye BCECF‐AM was used to monitor cytosolic pH changes in the leaves. Cadmium uptake in protoplasts and leaves was detected with a Cd‐specific fluorescent dye, Leadmium Green AM, and with atomic absorption spectrophotometry. During cultivation for 3 days without Cd, shoots of E. canadensis increased the pH of the surrounding water, irrespective of the photosynthetic conditions. This medium alkalisation was higher in the presence of CdCl₂. Moreover, the presence of Cd also increased the cation exchange capacity of the shoots. The total Cd uptake by E. canadensis shoots was independent of photosynthetic conditions. Protoplasts from plants exposed to 0.5 μm CdCl₂ for 3 days did not exhibit significant change in cytosolic [Cd²⁺] or pH. However, exposure to CdCl₂ for 7 days resulted in increased cytosolic [Cd²⁺] as well as pH. The results suggest that E. canadensis subjected to a low CdCl₂ concentration initially sequesters Cd into the apoplasm, but under prolonged exposure, Cd is transported into the cytosol and subsequently alters cytosolic pH. In contrast, addition of 10–50 μm CdCl₂ directly to protoplasts resulted in immediate uptake of Cd into the cytosol.     

Modulation of adrenal cell functions by cadmium salts. 4. Ca2+-dependent sites affected by CdCl2 during basal and ACTH-stimulated steroid synthesis

In previous studies, nonlethal CdCl₂ concentrations apparently inhibited basal Y-1 mouse adrenal tumor cell endogenous mitochondrial cholesterol conversion to pregnenolone. In addition, CdCl₂ inhibited all agents stimulating both plasma membrane-dependent cAMP synthesis and 20-hydroxy-4-pregnen-3-one (20DHP) secretion. Bypassing the plasma membrane using dibutyryl-cAMP (dbcAMP) stimulated cytoplasmic cholesterol metabolism and 20DHP secretion in the presence of CdCl₂. Since CdCl₂ competed at metabolic steps requiring Ca²⁺ in other tissues, experiments were designed to examine Cd²⁺ competition with Ca²⁺ during steroidogenesis. Sets of cells incubated with either medium or adrenocorticotropin (ACTH) with or without CdCl₂ were also treated with 0, 1.0, 5.0 or 10.0 mmol/L CaCl₂ in the presence or absence of EGTA, a relatively specific Ca²⁺, but not Cd²⁺, chelating agent. Another experimental cell set incubated with either medium or ACTH, with or without CdCl₂, was treated with or without 1 mmol/L A23187, an ionophore specifically facilitating extracellular Ca²⁺ transfer across plasma membranes. Besides determining Ca²⁺ involvement in steroidogenesis using steroid secretion as an endpoint, we directly measured Ca²⁺ concentrations using intracellular fura-2 fluorescence. Following loading with 2 mol/L fura-2, cells remained untreated or medium was infused with CdCl₂, ACTH, ACTH/CdCl₂ or ACTH followed after 50 s by CdCl₂. Using Ca²⁺-supplemented media, we observed that Cd²⁺ inhibition of ACTH-stimulated 20DHP secretion was completely reversed. Standard Ca²⁺-containing medium supplemented with Ca²⁺ also enhanced maximally stimulated 20DHP secretion by ACTH. 20DHP secretion by ACTH-treated and ACTH/Cd²⁺-treated cells was only reduced by EGTA, when Ca²⁺ was not supplemented. The ionophore A23187 increased basal and ACTH-stimulated 20DHP secretion by Cd²⁺-treated cells, suggesting that extracellular Ca²⁺ resources may compete against Cd²⁺ effects on plasma membrane cAMP synthesis and on basal cholesterol metabolism by mitochondria. No time-dependent change in Ca²⁺ concentrations occurred within untreated cell suspensions. ACTH stimulation caused a 25 s burst in Ca²⁺ concentrations before returning to basal, steady-state levels. Cd²⁺ also stimulated intracellular fura-2 fluorescence. Untreated cell suspensions infused with Cd²⁺ exhibited a continuous rise in intracellular fluorescence. ACTH/CdCl₂-treated cells exhibited a hyperbolic rise in intracellular fluorescence over the 300 s study period. Cells treated with Cd²⁺ 50 s after ACTH treatment initially exhibited the 25 s fluorescence burst followed by a Cd²⁺-induced hyperbolic rise in intracellular Cd²⁺. These fluorescence measurements suggested that cytoplasmic Ca²⁺ changes do not appear to be necessary for basal 20DHP synthesis and secretion; only a 25 s burst in intracellular Ca²⁺ is necessary to a slightly higher plateau level for stimulated 20DHP synthesis and secretion. Cd²⁺ freely enters the cell under basal conditions and Cd²⁺ entry is accelerated by ACTH stimulation. Data were consistent with Ca²⁺ being required for optimal stimulated steroid production and Cd²⁺ probably competing with Ca²⁺ during basal mitochondrial cholesterol metabolism and plasma membrane ACTH-stimulated cAMP generation.     

Expression,purification, and characterization of recombinant mangrove glutamine synthetase

To expand our knowledge about the relationship of nitrogen use efficiency and glutamine synthetase (GS) activity in the mangrove plant, a cytosolic GS gene from Avicennia marina has been heterologously expressed in and purified from Escherichia coli. Synthesis of the mangrove GS enzyme in E. coli was demonstrated by functional genetic complementation of a GS deficient mutant. The subunit molecular mass of GSI was ~40 kDa. Optimal conditions for biosynthetic activity were found to be 35 °C at pH 7.5. The Mg²⁺-dependent biosynthetic activity was strongly inhibited by Ni²⁺, Zn²⁺, and Al³⁺, whereas was enhanced by Co²⁺. The apparent K _m values of AmGLN1 for the substrates in the biosynthetic assay were 3.15 mM for glutamate, and 2.54 mM for ATP, 2.80 mM for NH₄ ⁺ respectively. The low affinity kinetics of AmGLN1 apparently participates in glutamine synthesis under the ammonium excess conditions.     

Cd2+ Effect on Photosynthetic Apparatus in Synechococcus elongatus and Spinach (Spinacia oleracea L.)

Thylakoid membranes (TM) of the cyanobacterium Synechococcus elongatus were exposed for 30 min to the influence of 0, 10, 100, and 1 000 mM CdCl₂ (= Cd₀, Cd₁₀, Cd₁₀₀, and Cd₁₀₀₀). Cd₁₀ and Cd₁₀₀ caused some increase in activity of photosystem 2, PS2 (H₂O DCPIP), while distinct inhibition was observed with Cd₁₀₀₀. We also observed a similar effect when measuring oxygen evolution (H₂O PBQ + FeCy). Chloroplasts of spinach (Spinacia oleracea L.) were incubated for 30 min with 0, 15, 30, and 60 mM CdCl₂ (= Cd₀, Cd₁₅, Cd₃₀, and Cd₆₀). All concentrations studied inhibited the PS2 activity, the effect being stronger with increasing concentration of Cd²⁺. The photosynthetic oxygen evolution activity was also influenced most distinctly by the highest concentration employed, i.e. Cd₆₀. Electrophoretic analysis of the protein composition of cyanobacterium TM showed chief changes in the molecular mass regions of M_r 29 000 and 116 000, while with spinach chloroplasts the most distinct differences were observed in the regions of M_r 15 000 and 50 000. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) activity in cyanobacterial spheroplasts still remained on the 40 % level in the case of Cd₁₀₀₀, but it decreased down to approx. 2.5 % in the Cd₆₀ sample of spinach chloroplasts.     

Adsorption Behavior of Tween 80 on Soil in the Presence of CdCl2 and DDT

Batch experiments were conducted to investigate the adsorption behavior of Tween 80 in the systems composed of Tween 80, CdCl₂, and/or DDT. The results show that Cd²⁺ from CdCl₂ is the functional fraction influencing the adsorption of Tween 80 to soil, rather than Cl^?. Moreover, DDT can induce the increase of the critical micelle concentration (CMC) of Tween 80, which further impacts the Tween 80 adsorption behavior. The Tween 80 adsorption to soil in the Cd²⁺-DDT coexisted system follows the Langmuir isotherm, as in the Tween 80-Cd²⁺ or -DDT systems. Cd²⁺ and/or DDT decrease(s) the adsorption capacity of Tween 80 to soil, and the magnitude of decrease is dependent on the concentration of coexisting pollutants. Although DDT has a stronger inhibitory effect on Tween 80 adsorption than Cd²⁺ under the same DDT/Cd²⁺ concentrations, the coexistence of Cd²⁺ and DDT has an antagonistic effect on the adsorption of Tween 80. This effect is impacted by the concentrations of the coexisting pollutants, and is a result of the complex interaction among the three pollutants.     

Molecular characterization of Alr1105 a novel arsenate reductase of the diazotrophic cyanobacterium Anabaena sp. PCC7120 and decoding its role in abiotic stress management in Escherichia coli

This paper constitutes the first report on the Alr1105 of Anabaena sp. PCC7120 which functions as arsenate reductase and phosphatase and offers tolerance against oxidative and other abiotic stresses in the alr1105 transformed Escherichia coli. The bonafide of 40.8 kDa recombinant GST+Alr1105 fusion protein was confirmed by immunoblotting. The purified Alr1105 protein (mw 14.8 kDa) possessed strong arsenate reductase (Km 16.0 ± 1.2 mM and Vmax 5.6 ± 0.31 μmol min^?1 mg protein^?1) and phosphatase activity (Km 27.38 ± 3.1 mM and Vmax 0.077 ± 0.005 μmol min^?1 mg protein^?1) at an optimum temperature 37 °C and 6.5 pH. Native Alr1105 was found as a monomeric protein in contrast to its homologous Synechocystis ArsC protein. Expression of Alr1105 enhanced the arsenic tolerance in the arsenate reductase mutant E. coli WC3110 (?arsC) and rendered better growth than the wild type W3110 up to 40 mM As (V). Notwithstanding above, the recombinant E. coli strain when exposed to CdCl₂, ZnSO₄, NiCl₂, CoCl₂, CuCl₂, heat, UV-B and carbofuron showed increase in growth over the wild type and mutant E. coli transformed with the empty vector. Furthermore, an enhanced growth of the recombinant E. coli in the presence of oxidative stress producing chemicals (MV, PMS and H₂O₂), suggested its protective role against these stresses. Appreciable expression of alr1105 gene as measured by qRT-PCR at different time points under selected stresses reconfirmed its role in stress tolerance. Thus the Alr1105 of Anabaena sp. PCC7120 functions as an arsenate reductase and possess novel properties different from the arsenate reductases known so far.     

Influence of activator and inhibitors of Ca2+ channels on proliferative activity in Tetrahymena pyriformis infusoria

It was determined that change in DNA content in macronuclei occurs in the T. pyriformis infusoria under the influence of an activator (caffeine) and inhibitors of Ca²⁺ channels (verapamil), NiCl₂, and CdCl₂. Caffeine (10 mM) stimulates DNA synthesis. Verapamil (5 ??M), CdCl₂ (125 ??M), and NiCl₂ (100 ??M) decrease DNA content in macronuclei by 30 min after proliferative stimulation. By 4 h of incubation, there is, on average, 10% less DNA in macronuclei of Tetrahymena preprocessed with verapamil than in the control cells. The cells preprocessed with CdCl₂ and NiCl₂ differ from the control cells by lower DNA content almost at all studied periods, but they restore the level of nuclear DNA by 4 h. It is assumed that trans-mission of proliferative signals in the T. pyriformis has a Ca²⁺-dependent character.     

Microbioluminescent study of the general toxicity and mutagenicity of pollutants

Bacterial bioluminescence was applied to detection of general toxicity (MIT test) and genotoxicity (SOS-lux test) of some chemicals, seawater, and fresh water. The SOS-induced luminescence of E. coli WP2s (cda::luxCDABE) cells was higher than in E. coli C 600 (cda::luxCDABE) at 37°C and pH 6.5. The mutagenic effect of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide determined from the induction of E. coli WP2s cell luminescence was detected at lower concentrations than in the assessment of reversion frequencies. General toxicity was demonstrated by using luminescence inhibition for hydrogen peroxide, Zn²⁺, and Cd²⁺ at low concentrations. Regions of the Krasnodar Krai where sea and fresh waters exerted toxic action on luminescence were determined by the microbioluminescent method.     

Significantly improved Escherichia coli tolerance and accumulation of Cd2+, Zn2+ and Cu2+ expressing Streptococcus thermophilus StGCS-GS with high glutathione content

Streptococcus thermophilus γ-glutamylcysteine synthetase-glutathione synthetase (StGCS-GS) which synthesized glutathione (GSH) without limit feedback inhibition was over-expressed as a fusion protein of TrxA-StGCS-GS to analyze its possibly functional role in heavy metal tolerance of Escherichia coli (BL21). For comparative analyses, Arabidopsis γ-glutamylcysteine synthetase (AtGCS) and glutathione synthetase (AtGS) were introduced into Escherichia coli (E. coli) in the same manner, respectively. The results showed that the growth and survivability of E. coli over-expressing TrxA-StGCS-GS were slightly influenced by 1 mM Cd²⁺, Zn²⁺ and Cu²⁺ toxicity, and it could withstand duration of these heavy metal stresses competently. In contrast, the two strains over-expressing TrxA-AtGCS and TrxA-AtGS were impacted apparently; the BL21 empty strain was even almost suppressed. Meanwhile, a much higher bioaccumulation of Cd²⁺, Zn²⁺, Cu²⁺ ions and glutathione content were observed in the strain over-expressing TrxA-StGCS-GS than in the other comparison strains. It could be concluded that over-expression of StGCS-GS offered a more significant enhancement of heavy metal tolerance to E. coli with superior GSH content to accumulate considerable heavy metal.     

Cloning expression and analysis of phytochelatin synthase (pcs) gene from Anabaena sp. PCC 7120 offering multiple stress tolerance in Escherichia coli

Phytochelatin synthase (PCS) is involved in the synthesis of phytochelatins (PCs), plays role in heavy metal detoxification. The present study describes for first time the functional expression and characterization of pcs gene of Anabaena sp. PCC 7120 in Escherichia coli in terms of offering protection against heat, salt, carbofuron (pesticide), cadmium, copper, and UV-B stress. The involvement of pcs gene in tolerance to above abiotic stresses was investigated by cloning of pcs gene in expression vector pGEX-5X-2 and its transformation in E. coli BL21 (DE3). The E. coli cells transformed with pGEX-5X-pcs showed better growth than control cells (pGEX-5X-2) under temperature (47 °C), NaCl (6% w/v), carbofuron (0.025 mg ml⁻¹), CdCl₂ (4 mM), CuCl₂ (1 mM), and UV-B (10 min) exposure. The enhanced expression of pcs gene revealed by RT-PCR analysis under above stresses at different time intervals further advocates its role in tolerance against above abiotic stresses.     

Cadmium modulates NADPH oxidase activity and expression in sunflower leaves

The production of reactive oxygen species (ROS) and the ways by which ROS are generated are very important facts related to heavy metal toxicity in plants. In this work, superoxide anion (O₂ ^·−) generation diminished in cadmium treated sunflower (Helianthus annuus L.) leaf discs, and this reduction was time and Cd-concentration dependent. In line with these findings, we observed that NADPH-dependent oxidase activity was significantly inhibited by 0.1 and 0.5 mM Cd²⁺ treatments and the expression of the NADPH oxidase putative gene related to O₂ ^·− synthesis in sunflower leaves was 83 % inhibited by 0.1 mM CdCl₂ and almost completely depleted by 0.5 mM CdCl₂.