Expression of a Neurospora crassa metallothionein and its variants in Escherichia coli.

The Neurospora crassa metallothionein (NC) synthesis gene was cloned and expressed in Escherichia coli in two different expression vectors (pING2 and pUA7), both under the regulation of the Salmonella typhimurium arabinose operon. Upon induction with arabinose, the pING2-NC vector expressed as inclusion body-localized AraB'::NC fusion protein of 21 kilodaltons. The pUA7-NC vector expressed a 5.3-kilodalton Lpp::NC fusion protein anchored to the outer membrane of the cell. Cells expressing the NC fusion proteins accumulated Cd2+ and Cu+ (between 2.3- and 11-fold) compared with nonexpressing cells. To generate novel forms of metal-binding peptides, a set of specific mutant genes for N. crassa NC was designed in which each cysteine residue was replaced with a subset of amino acids implicated in peptide-metal coordination (Asn, Asp, His, Lys, or Tyr residues). These mutant NC sequences were cloned into the two vectors and expressed in E. coli. One of the mutant proteins (containing His residues) showed accumulation of Cd2+ and Cu+ (threefold) from a mixture of 16 heavy metals species. None of the other heavy metals present in the culture was accumulated.     

Human metallothionein-II is synthesized as a stable membrane-localized fusion protein in Escherichia coli

A synthetic gene encoding human metallothionein-II (HMT) was cloned into the specially constructed high-copy-number expression vector, pUA7, and expressed in Escherichia coli. The plasmid construct includes the promoter/operator and regulatory sequences of the Salmonella typhimurium ara operon and part of the 5'-coding and all of the 3'-noncoding regions of the E. coli lpp. Upon induction with arabinose, the resulting Lpp::HMT fusion protein was produced 75,000-fold over uninduced cells, with a relatively stable mRNA (T1/2 of 8.3 min) and a completely stable protein. In addition, over 95% of the final fusion protein was localized in the outer membrane and was capable of binding heavy metals (especially cadmium) in vitro. Cells producing Lpp::HMT bioaccumulated heavy metals (e.g., cadmium) 66-fold over nonproducing cells.     

Transgenic Tobacco with αα Mutant Gene Has Higher Tolerance to Heavy Metals

Metallothionein (MT) has two domains, α and β domain. α domain preferred to bind Cd2+and Hg2+. Mouse metallothionein mutant αα has been constructed and expressed in E.coli, which has the same stability as the nature one but has stronger affinity to heavy metals. To testify the result in vivo, αα mutant gene was cloned into plant expression vector pE3 under the CaMV 35S promoter. A transgenic tobacco was obtained by using leaf discs of tobacco (Nicotiana tabacum L. cv. NC89) to Agrobacterium-mediated ααgene transfer. Southern blotting analysis indicated that the αα mutant gene was indeed integrated into the tobacco genome; Western blot indicated that the αα mutant gene was expressed in transgenic tobacco. It was also demonstrated that the transgenic tobacco with αα mutant gene have a little higher tolerance to heavy metals than that with natural MT gene. Moreover, the transgenic tobacco can accumulate more Cd2+ in its roots than natural, so that, it can decrease the concentration of Cd2+ in its leaves.     

Bioaccumulation of heavy metals in Escherichia coli expressing an inducible synthetic human metallothionein gene

A synthetic DNA coding for human hepatic metallothionein (HMT), fraction MT-2, has been cloned in the plasmid vector pING 1 and expressed in E. coli. Upon induction with arabinose, an araB′-HMT fusion protein is synthesized. The fusion protein is produced to a level of approximately 8% of the total protein in E. coli, with an apparent half-life of 50 min. Without arabinose, no fusion protein is expressed, demonstrating that the expression system is tightly regulated. In the presence of ¹⁰⁹Cd, the fusion protein binds the metal ion in vitro. Concurrently, a direct correlation is found between the expression in E. coli of the araB′-HMT fusion protein which binds Cd²⁺ in vitro and the bioaccumulation of Cd²⁺ in vivo. The bioaccumulation of Cu⁺ in E. coli, when the fusion protein is produced, has also been established.     

Expression of human metallothionein III and its metalloabsorption capability in Escherichia coli

Human metallothionein III (MT III) gene was synthesized with Escherichia coli preference codon usage and expressed in E. coli in glutathione-S-transferase (GST) fusion form. The recombinant MT III was released by proteinase Factor Xa digestion and purified with the yield of 2 mg/L culture, and its specific Cd2+ binding capability was confirmed. E. coli strain BL21(DE3), expressing MT III, showed metal tolerance between 0.1 and 0.5 mM Cd2+ and bacterial growth was inhibited at 1 mM Cd2+. MT III expressing E. coli strain showed binding discrimination between different metal ions in combination use, with the preference order of Cd2+ > Cu2+ > Zn2+. It absorbed different metal ions with relatively constant ratio and showed a cumulative absorption capability for mixed heavy metals.     

Expression of BjMT2, a metallothionein 2 from Brassica juncea, increases copper and cadmium tolerance in Escherichia coli and Arabidopsis thaliana, but inhibits root elongation in Arabidopsis thaliana seedlings

The protective function of a plant type-2 metallothionein was analysed after expression in Escherichia coli and in Arabidopsis thaliana seedlings. BjMT2 from Brassica juncea was expressed in E. coli as a TrxABjMT2 fusion protein. After affinity chromatography and cleavage from the TrxA domain, pure BjMT2 protein was obtained which strongly reacted with the thiol reagent monobromobimane. Escherichia coli cells expressing the TrxABjMT2 fusion were more tolerant to Cu2+ and Cd2+ exposure than control strains. Likewise, when BjMT2 cDNA was expressed in A. thaliana under the regulation of the 35S promoter, seedlings exhibited an increased tolerance against Cu2+ and Cd2+ based on shoot growth and chlorophyll content. Analysis of transiently transformed cells of A. thaliana and tobacco leaves by confocal laser scanning microscopy (CLSM) revealed exclusive cytosolic localization of a BjMT2::EGFP (enhanced green fluorescent protein) fusion protein in control and heavy metal-exposed plant cells. Remarkably, ectopic expression of BjMT2 reduced root growth in the absence of heavy metal exposure, whereas in the presence of 50 or 100 microM Cu2+ root growth in control and transgenic lines was identical. The results indicate that in A. thaliana, root and shoot development are differentially affected by ectopic expression of BjMT2.     

Functional expression of AtHMA4, a P1B-type ATPase of the Zn/Co/Cd/Pb subclass

Mechanisms are required by all organisms to maintain the concentration of essential heavy metals (e.g. Zn and Cu) within physiological limits and to minimise the detrimental effects of non-essential heavy metals (e.g. Cd). Heavy-metal P-type ATPases (HMAs) are a subgroup of the P-type ATPase superfamily that may contribute to metal homeostasis in plants. We cloned and characterised a member of this family, AtHMA4, from Arabidopsis thaliana that clusters with the Zn/Co/Cd/Pb subclass of HMAs on phylogenetic analysis. Sequencing of the AtHMA4 cDNA showed that it contained the conserved motifs found in all P-type ATPases and also motifs that are characteristic of heavy-metal ATPases. Escherichia coli mutants defective in the HMAs, CopA and ZntA, were used in functional complementation studies. AtHMA4 was able to restore growth at high [Zn] in the zntA mutant but not at high [Cu] in the copA mutant, suggesting a role in zinc transport. Heterologous expression of AtHMA4 in Saccharomyces cerevisiae made the yeast more resistant to Cd but did not affect sensitivity to other metals compared with vector-transformed controls. The organ specificity of AtHMA4 was analysed in Arabidopsis and showed that AtHMA4 was expressed in a range of tissues with highest expression in roots. AtHMA4 was upregulated in roots exposed to elevated levels of Zn and Mn but downregulated by Cd. Possible physiological roles of this transporter in Arabidopsis are discussed.     

Characterization of the PIB-Type ATPases present in Thermus thermophilus

P(IB)-type ATPases transport heavy metals (Cu(2+), Cu(+), Ag(+), Zn(2+), Cd(2+), Co(2+)) across biomembranes, playing a key role in homeostasis and in the mechanisms of biotolerance of these metals. Three genes coding for putative P(IB)-type ATPases are present in the genome of Thermus thermophilus (HB8 and HB27): the TTC1358, TTC1371, and TTC0354 genes; these genes are annotated, respectively, as two copper transporter (CopA and CopB) genes and a zinc-cadmium transporter (Zn(2+)/Cd(2+)-ATPase) gene. We cloned and expressed the three proteins with 8His tags using a T. thermophilus expression system. After purification, each of the proteins was shown to have phosphodiesterase activity at 65°C with ATP and p-nitrophenyl phosphate (pNPP) as substrates. CopA was found to have greater activity in the presence of Cu(+), while CopB was found to have greater activity in the presence of Cu(2+). The putative Zn(2+)/Cd(2+)-ATPase was truncated at the N terminus and was, surprisingly, activated in vitro by copper but not by zinc or cadmium. When expressed in Escherichia coli, however, the putative Zn(2+)/Cd(2+)-ATPase could be isolated as a full-length protein and the ATPase activity was increased by the addition of Zn(2+) and Cd(2+) as well as by Cu(+). Mutant strains in which each of the three P-type ATPases was deleted singly were constructed. In each case, the deletion increased the sensitivity of the strain to growth in the presence of copper in the medium, indicating that each of the three can pump copper out of the cells and play a role in copper detoxification.     

Bacterial sorption of heavy metals

Four bacteria, Bacillus cereus, B. subtilis, Escherichia coli, and Pseudomonas aeruginosa, were examined for the ability to remove Ag+, Cd2+, Cu2+, and La3+ from solution by batch equilibration methods. Cd and Cu sorption over the concentration range 0.001 to 1 mM was described by Freundlich isotherms. At 1 mM concentrations of both Cd2+ and Cu2+, P. aeruginosa and B. cereus were the most and least efficient at metal removal, respectively. Freundlich K constants indicated that E. coli was most efficient at Cd2+ removal and B. subtilis removed the most Cu2+. Removal of Ag+ from solution by bacteria was very efficient; an average of 89% of the total Ag+ was removed from the 1 mM solution, while only 12, 29, and 27% of the total Cd2+, Cu2+, and La3+, respectively, were sorbed from 1 mM solutions. Electron microscopy indicated that La3+ accumulated at the cell surface as needlelike, crystalline precipitates. Silver precipitated as discrete colloidal aggregates at the cell surface and occasionally in the cytoplasm. Neither Cd2+ nor Cu2+ provided enough electron scattering to identify the location of sorption. The affinity series for bacterial removal of these metals decreased in the order Ag greater than La greater than Cu greater than Cd. The results indicate that bacterial cells are capable of binding large quantities of different metals. Adsorption equations may be useful for describing bacterium-metal interactions with metals such as Cd and Cu; however, this approach may not be adequate when precipitation of metals occurs.     

Bacterial sorption of heavy metals.

Four bacteria, Bacillus cereus, B. subtilis, Escherichia coli, and Pseudomonas aeruginosa, were examined for the ability to remove Ag+, Cd2+, Cu2+, and La3+ from solution by batch equilibration methods. Cd and Cu sorption over the concentration range 0.001 to 1 mM was described by Freundlich isotherms. At 1 mM concentrations of both Cd2+ and Cu2+, P. aeruginosa and B. cereus were the most and least efficient at metal removal, respectively. Freundlich K constants indicated that E. coli was most efficient at Cd2+ removal and B. subtilis removed the most Cu2+. Removal of Ag+ from solution by bacteria was very efficient; an average of 89% of the total Ag+ was removed from the 1 mM solution, while only 12, 29, and 27% of the total Cd2+, Cu2+, and La3+, respectively, were sorbed from 1 mM solutions. Electron microscopy indicated that La3+ accumulated at the cell surface as needlelike, crystalline precipitates. Silver precipitated as discrete colloidal aggregates at the cell surface and occasionally in the cytoplasm. Neither Cd2+ nor Cu2+ provided enough electron scattering to identify the location of sorption. The affinity series for bacterial removal of these metals decreased in the order Ag greater than La greater than Cu greater than Cd. The results indicate that bacterial cells are capable of binding large quantities of different metals. Adsorption equations may be useful for describing bacterium-metal interactions with metals such as Cd and Cu; however, this approach may not be adequate when precipitation of metals occurs.     

Bacterium-based heavy metal biosorbents: enhanced uptake of cadmium by E. coli expressing a metallothionein fused to beta-galactosidase

We investigated the potential utility of a recombinant E. coli that expresses the human metallothionein II gene as a fusion protein with beta-galactosidase as a heavy metal biosorbent. E. coli cells expressing the metallothionein fusion demonstrated enhanced binding of Cd2+ compared to cells that lack the metallothionein. It was shown that the metallothionein fusion was capable of efficiently removing Cd2+ from solutions. Approximately 40% of the Cd2+ accumulated by the recombinant cells free in suspension was associated with the outer cell membrane, and 60% of that was present in the cytoplasm.     

Retromobilization of heavy metal resistance genes in unpolluted and heavy metal polluted soil

Abstract: Retromobilization of the nonconjugative (Tra⁻Mob⁺) IncQ vector, pMOL155, and the non-mobilizable (Tra⁻Mob⁻) vector, pMOL149, by means of the IncP plasmids RP4 and pULB113 (RP4::Mu3A), was studied in plate matings and in soil microcosms, and compared with direct and triparental mobilization. Both vectors harbour the czc genes, originating from Alcaligenes eutrophus , which code for resistance to Co, Zn, and Cd. The donor of the czc genes was Escherichia coli which did not express these genes. The recipient, Alcaligenes eutrophus , expressed the czc genes very well. Retromobilization, direct and triparental mobilization of pMOL155 was observed in sterile soil. Both the addition of nutrients and heavy metals significantly enhanced the number of (retro)transconjugants. Retromobilization was also detected in nutrient amended nonsterile soil, but the presence of the autochthonous soil biota strongly reduced the number of retrotransconjugants and also prevented their increase upon application of heavy metals to the soil. Retromobilization of the czc genes, cloned in pMOL149, by using pULB113 was also observed, yet only in sterile, nutrient amended, heavy metal polluted soil.     

Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins

A novel strategy using synthetic phytochelatins is described for the purpose of developing microbial agents for enhanced bioaccumulation of toxic metals. Synthetic genes encoding for several metal-chelating phytochelatin analogs (Glu-Cys)(n)Gly (EC8 (n = 8), EC11 (n = 11), and EC20 (n = 20)) were synthesized, linked to a lpp-ompA fusion gene, and displayed on the surface of E. coli. For comparison, EC20 was also expressed periplasmically as a fusion with the maltose-binding protein (MBP-EC20). Purified MBP-EC20 was shown to accumulate more Cd(2+) per peptide than typical mammalian metallothioneins with a stoichiometry of 10 Cd(2+)/peptide. Cells displaying synthetic phytochelatins exhibited chain-length dependent increase in metal accumulation. For example, 18 nmoles of Cd(2+)/mg dry cells were accumulated by cells displaying EC8, whereas cells exhibiting EC20 accumulated a maximum of 60 nmoles of Cd(2+)/mg dry cells. Moreover, cells with surface-expressed EC20 accumulated twice the amount of Cd(2+) as cells expressing EC20 periplasmically. The ability to genetically engineer ECs with precisely defined chain length could provide an attractive strategy for developing high-affinity bioadsorbents suitable for heavy metal removal.     

Influence of heavy metal ions on the electrophysical properties of Anacystis nidulans and Escherichia coli cells]

The influence of heavy metal ions (Ag+, Cu2+, Cd2+, Pb2+, Mn2+, Zn2+, Gd3+, 1 microM-1 mM) on Anacystis nidulans and Escherichia coli cells has been studied by means of electrophoresis and electro-orientation spectroscopy methods. It has been shown that changes of cell electrophoretic mobility (EM) and low-frequency (20 Hz) electro-orientation effect (EOE) observed with the increase of metal cation concentration characterize the adsorption of these ions on surface layers of cell envelopes. The degree and the character of these changes depend on cation valency and the initial value of cell EM. At the same time different changes of EM and EOE as a result of the multivalent cation adsorption allows to conclude that in that case the anisotropy of the cell surface increases. Cell damages were determined by changes in high-frequency EOE of cells which indicated the disturbance of barrier properties of their cytoplasmic membrane. Toxic effects of Ag+, Cu2+, Cd2+ ions on cells of both species and of Pb2+ on E.coli cells were observed. By toxic effects on the cytoplasmic membrane these ions could be placed in the order: for A.nidulans cells--Ag+ greater than Cu2+ greater than Cd2+; for E.coli cells Ag+ greater than Cu2+ greater than Cd2+ greater than Pb2+. Higher toxicity of heavy metals on E.coli cells seems to be connected with the more negative charge of deep layers of the cell surface.     

融和蛋白GST-SUMO-MT在大肠杆菌中的表达、纯化及其活性研究

将编码融合蛋白GST-SUMO-MT的DNA片段连接到大肠杆菌表达载体pET-28a中,构建重组表达质粒pET-GS-MT并转化到大肠杆菌Origami（DE3）中。20℃,1mM的IPTG诱导20h后,获得分子量约为43Kd的融合蛋白,表达量占菌体上清总蛋白的38.4％。利用谷胱甘肽交联琼脂糖（Glutathione Sepharose 4B）凝胶柱和Sephardex G-25分子筛联用可以得到纯度为95%以上的融合蛋白,得率约为70mg/L。该融合蛋白可与GST抗体产生阳性反应。融合蛋白GST-SUMO-MT可以显著提高宿主对Cd2+、Zn2+和Cu2+离子聚积的能力,其耐受能力比对照组分别提高4.2倍、4倍及1.6倍。此外,原子吸收光谱法测定,每分子GST-SUMO-MT可以结合2-3个Cd2+离子。     

人NDRG1的融合表达、纯化及抗体制备

NDRG1是在N myc缺陷的小鼠胚胎组织中发现的一异常高表达的新基因 .在研究高同型半胱氨酸血症引起动脉粥样硬化的机制时 ,在培养的人脐静脉内皮细胞 (HUVEC)中发现了人NDRG1 .为了研究人NDRG1的功能以及结构与功能之间的关系 ,用RT PCR技术 ,从人脑总RNA中克隆NDRG1cDNA ,进行序列测定后 ,将NDRG1插入pPROEXHTb表达载体中 ,转化E .coliDH5α感受态细胞 ,并在LB培养基中获得高效可溶表达 .表达的 6His NDRG1融合蛋白经Ni2 + NTA偶联的琼脂糖珠纯化 ,通过圆二色性分析其二级结构 ,结果为 :α螺旋 :2 3 6 ,β片层 :1 8 6 ,转角 :2 5 7,无规卷曲 :32 0 .用此融合表达的蛋白免疫家兔 ,制备得到高效价的抗体 ,利用固定于硝酸纤维素膜上的NDRG1抗原亲和吸附纯化抗血清提高了抗体的特异性 ,为进一步研究NDRG1的功能打下了良好的基础     

The effect of the trematode invasion and accumulation of heavy metals onto the pond snail (Mollusca: Gastropoda: Lymnaeidae)

The cumulating of heavy metals (Cu, Cd, Pb, Zn) by the pond snail Lymnaea stagnalis from river and pond populations in a norm and under the trematode infection was examined. The copper and cadmium were accumulated by all specimens, mainly in the mantle and leg, in less content--in the hepatopancreas, and in the least content--in the shell and haemolymph. The lead was accumulated most intensively by the river specimens in the shell and mantle, while in the pond specimens it was accumulated in the mantle and leg. The greatest content of zink was found in the mantle, leg, and hepatopancreas. According to the ratio of cumulation the heavy metals by the river and pond populations the following series of ions were obtained respectively: Pb > Zn > Cu > Cd and Zn > Pb > Cu > Cd. The difference between uninfected specimens and one infected with trematodes was recorded in the ratio of cumulated heavy metals, in the ratio of benthic biological accumulation, and also based on bending force between metals compared in pairs. An intensity of differences was directly proportional to the intensity of the trematode infection.     

胁迫时间与非毒性离子对重金属抑制拟南芥种子发芽及幼苗生长的影响

测定了Hg²⁺、Cd²⁺、Cu²⁺、Pb²⁺单一重金属胁迫对拟南芥种子发芽和幼苗生长的影响.结果表明,重金属对幼苗生长的毒性大于对种子发芽的毒性,以抑制种子发芽的IC₅₀为指标,4种重金属的毒性顺序为Hg²⁺＞Cd²⁺＞Pb²⁺/Cu²⁺,以幼苗生长为指标,则毒性顺序为:Cu²⁺＞Hg²⁺＞Cd²⁺/Pb²⁺,并随着胁迫时间延长,种子萌发率下降.此外,不同重金属在不同发芽时段对种子的毒性也不尽相同,Cd²⁺的毒性在种子吸水后的0～12 h大于12～24 h,而Hg²⁺毒性在12～24 h大于0～12 h,其中,种皮对减轻重金属毒性起着十分重要的作用.通过非毒性离子(Ca²⁺、Mg²⁺、K⁺、Na⁺)与重金属离子(Hg²⁺、Cd²⁺、Cu²⁺、Pb²⁺)交互作用对拟南芥种子发芽及幼苗生长效应的研究发现, mmol·L^-1的Ca²⁺、Mg²⁺、K⁺、Na⁺可以增强Hg²⁺对种子发芽的毒性,但对Cd²⁺的毒性却没有影响.对于幼苗来说,Ca²⁺、Mg²⁺、K⁺、Na⁺可以显著增强Hg²⁺的毒性,Ca²⁺可以缓解Cd²⁺的毒性,但却增加Cu²⁺的毒性,K⁺可以缓解Pb²⁺对幼苗的毒害作用.最后,本文对重金属的毒害机理进行了探讨.