Nickel and cobalt resistance engineered in Escherichia coli by overexpression of serine acetyltransferase from the nickel hyperaccumulator plant Thlaspi goesingense.
ABSTRACT: The overexpression of serine acetyltransferase from the Ni-hyperaccumulating plant Thlaspi goesingense causes enhanced nickel and cobalt resistance in Escherichia coli. Furthermore, overexpression of T. goesingense serine acetyltransferase results in enhanced sensitivity to cadmium and has no significant effect on resistance to zinc. Enhanced nickel resistance is directly related to the constitutive overactivation of sulfur assimilation and glutathione biosynthesis, driven by the overproduction of O-acetyl-L-serine, the product of serine acetyltransferase and a positive regulator of the cysteine regulon. Nickel in the serine acetyltransferase-overexpressing strains is not detoxified by coordination or precipitation with sulfur, suggesting that glutathione is involved in reducing the oxidative damage imposed by nickel.
Project description:When growing in its native habitat, Thlaspi goesingense can hyperaccumulate 1.2% of its shoot dry weight as nickel. We reported previously that both constitutively elevated activity of serine acetyltransferase (SAT) and concentration of glutathione (GSH) are involved in the ability of T. goesingense to tolerate nickel. A feature of SAT is its feedback inhibition by L-cysteine. To understand the role of this regulation of SAT by Cys on GSH-mediated nickel tolerance in T. goesingense, we characterized the enzymatic properties of SATs from T. goesingense. We demonstrate that all three isoforms of SAT in T. goesingense are insensitive to inhibition by Cys. Further, two amino acids (proline and alanine) in the C-terminal region of the cytosolic SAT (SAT-c) from T. goesingense are responsible for converting the enzyme from a Cys-sensitive to a Cys-insensitive form. Furthermore, the Cys-insensitive isoform of SAT-c confers elevated resistance to nickel when expressed in Escherichia coli and Arabidopsis thaliana, supporting a role for altered regulation of SAT by Cys in nickel tolerance in T. goesingense.
Project description:Metal resistance determinants have traditionally been found in cultivated bacteria. To search for genes involved in nickel resistance, we analyzed the bacterial community of the rhizosphere of Erica andevalensis, an endemic heather which grows at the banks of the Tinto River, a naturally metal-enriched and extremely acidic environment in southwestern Spain. 16S rRNA gene sequence analysis of rhizosphere DNA revealed the presence of members of five phylogenetic groups of Bacteria and the two main groups of Archaea mostly associated with sites impacted by acid mine drainage (AMD). The diversity observed and the presence of heavy metals in the rhizosphere led us to construct and screen five different metagenomic libraries hosted in Escherichia coli for searching novel nickel resistance determinants. A total of 13 positive clones were detected and analyzed. Insights about their possible mechanisms of resistance were obtained from cellular nickel content and sequence similarities. Two clones encoded putative ABC transporter components, and a novel mechanism of metal efflux is suggested. In addition, a nickel hyperaccumulation mechanism is proposed for a clone encoding a serine O-acetyltransferase. Five clones encoded proteins similar to well-characterized proteins but not previously reported to be related to nickel resistance, and the remaining six clones encoded hypothetical or conserved hypothetical proteins of uncertain functions. This is the first report documenting nickel resistance genes recovered from the metagenome of an AMD environment.
Project description:The ability of Thlaspi goesingense to hyperaccumulate Ni seems to be governed in part by enhanced accumulation of Ni within leaf vacuoles. We have characterized genes from T. goesingense encoding putative vacuolar metal ion transport proteins, termed metal tolerance proteins (TgMTPs). These proteins contain all of the features of cation-efflux family members, and evidence indicates they are derived from a single genomic sequence (TgMTP1) that gives rise to an unspliced (TgMTP1t1) and a spliced (TgMTP1t2) transcript. Heterologous expression of these transcripts in yeast lacking the TgMTP1 orthologues COT1 and ZRC1 complements the metal sensitivity of these yeast strains, suggesting that TgMTP1s are able to transport metal ions into the yeast vacuole in a manner similar to COT1 and ZRC1. The unspliced and spliced TgMTP1 variants differ within a histidine-rich putative metal-binding domain, and these sequence differences are reflected as alterations in the metal specificities of these metal ion transporters. When expressed in yeast, TgMTP1t1 confers the highest level of tolerance to Cd, Co, and Zn, whereas TgMTP1t2 confers the highest tolerance to Ni. TgMTP1 transcripts are highly expressed in T. goesingense compared with orthologues in the nonaccumulators Arabidopsis thaliana, Thlaspi arvense, and Brassica juncea. We propose that the high-level expression of TgMTP1 in T. goesingense accounts for the enhanced ability of this hyperaccumulator to accumulate metal ions within shoot vacuoles.
Project description:Generally in the nickel converter slag, metals are mainly in the form of sulfides, which are difficult to separate from slag. Although metal oxides in the slag, such as NiO, CoO, and Cu2O, are easily reduced into metal using carbon, the presence of sulfur inhibits the reduction reaction. In this study, the addition of Fe2O3 to nickel converter slag produced desulfurized slag, which enhanced the carbothermal reduction process. Increasing the desulfurization rate promoted the conversion of sulfides into oxides in slag, which significantly increased the activity of NiO, Cu2O, and Fe2O3. However, the residual sulfur content had no significant effect on the activity of FeO and CoO, due to the high initial FeO content and cobalt existing mainly in the form of oxides. The optimum addition of Fe2O3 was 15.0 g per 100 g nickel slag, while the desulfurization ratio was 36.84% and the rates of nikel, cobalt and copper recovery were 95.33%, 77.73%, and 73.83%, respectively.
Project description:An integrated molecular and physiological investigation of the fundamental mechanisms of heavy metal accumulation was conducted in Thlaspi caerulescens, a Zn/Cd-hyperaccumulating plant species. A heavy metal transporter cDNA, ZNT1, was cloned from T. caerulescens through functional complementation in yeast and was shown to mediate high-affinity Zn(2+) uptake as well as low-affinity Cd(2+) uptake. It was found that this transporter is expressed at very high levels in roots and shoots of the hyperaccumulator. A study of ZNT1 expression and high-affinity Zn(2+) uptake in roots of T. caerulescens and in a related nonaccumulator, Thlaspi arvense, showed that alteration in the regulation of ZNT1 gene expression by plant Zn status results in the overexpression of this transporter and in increased Zn influx in roots of the hyperaccumulating Thlaspi species. These findings yield insights into the molecular regulation and control of plant heavy metal and micronutrient accumulation and homeostasis, as well as provide information that will contribute to the advancement of phytoremediation by the future engineering of plants with improved heavy metal uptake and tolerance.
Project description:Here, we describe the isolation of two nickel-induced genes in Paramecium caudatum, NCI16 and PcGST1, by subtractive hybridization. NCI16 encoded a predicted four-transmembrane domain protein (?16 kDa) of unknown function, and PcGST1 encoded glutathione S-transferase (GST; ?25 kDa) with GST and glutathione peroxidase (GPx) activities. Exposing cells to cobalt chloride also caused the moderate upregulation of NCI16 and PcGST1 mRNAs. Both nickel sulfate and cobalt chloride dose dependently induced NCI16 and PcGST1 mRNAs, but with different profiles. Nickel treatment caused a continuous increase in PcGST1 and NCI16 mRNA levels for up to 3 and 6 days, respectively, and a notable increase in H?O? concentrations in P. caudatum. NCI16 expression was significantly enhanced by incubating cells with H?O?, implying that NCI16 induction in the presence of nickel ions is caused by reactive oxygen species (ROS). On the other hand, PcGST1 was highly induced by the antioxidant tert-butylhydroquinone (tBHQ) but not by H2O2, suggesting that different mechanisms mediate the induction of NCI16 and PcGST1. We introduced a luciferase reporter vector with an ?0.42-kb putative PcGST1 promoter into cells and then exposed the transformants to nickel sulfate. This resulted in significant luciferase upregulation, indicating that the putative PcGST1 promoter contains a nickel-responsive element. Our nickel-inducible system also may be applicable to the efficient expression of proteins that are toxic to host cells or require temporal control.
Project description:Metal hyperaccumulator plants have previously been characterized by transcriptomics, but reports on other profiling techniques are scarce. Protein profiles of Thlaspi caerulescens accessions La Calamine (LC) and Lellingen (LE) and lines derived from an LCxLE cross were examined here to determine the co-segregation of protein expression with the level of zinc (Zn) hyperaccumulation. Although hydrophobic proteins such as membrane transporters are not disclosed, this approach has the potential to reveal other proteins important for the Zn hyperaccumulation trait. Plants were exposed to metals. Proteins were separated using two-dimensional electrophoresis and those showing differences among accessions, lines or metal exposures were subjected to mass-spectrometric analysis for identification. Crossing decreased the number of different proteins in the lines compared with the parents, more so in the shoots than in the roots, but the frequencies of Zn-responsive proteins were about the same in the accessions and the selection lines. This supports the finding that the Zn accumulation traits are mainly determined by the root and that Zn accumulation itself is not the reason for the co-segregation. This study demonstrates that crossing accessions with contrasting Zn accumulation traits is a potent tool to investigate the mechanisms behind metal hyperaccumulation. Four tentatively identified root proteins showed co-segregation with high or low Zn accumulation: manganese superoxide dismutase, glutathione S-transferase, S-formyl glutathione hydrolase, and translation elongation factor 5A-2. However, these proteins may not be the direct determinants of Zn accumulation. The role of these and other tentatively identified proteins in Zn accumulation and tolerance is discussed.
Project description:Nickel and its compounds, which are well-documented carcinogens, induce the Warburg effect in normal cells by stabilizing hypoxia-inducible factor 1? (HIF-1?). Melatonin has shown diverse anticancer properties for its reactive oxygen species- (ROS-) scavenging ability. Our aim was to explore how melatonin antagonized a nickel-induced increment in aerobic glycolysis. In the current work, a normal human bronchial epithelium cell line (BEAS-2B) was exposed to a series of nonlethal doses of NiCl2, with or without 1?mM melatonin. Melatonin attenuated nickel-enhanced aerobic glycolysis. The inhibition effects on aerobic glycolysis were attributed to the capability of melatonin to suppress the regulatory axis comprising HIF-1?, microRNA210 (miR210), and iron-sulfur cluster assembly scaffold protein (ISCU1/2). N-Acetylcysteine (NAC) manifested similar effects as melatonin in scavenging ROS, maintaining prolyl-hydroxylase activity, and mitigating HIF-1? transcriptional activity in nickel-exposed cells. Our results indicated that ROS generation contributed to nickel-caused HIF-1? stabilization and downstream signal activation. Melatonin could antagonize HIF-1?-controlled aerobic glycolysis through ROS scavenging.
Project description:White birch (Betula papyrifera) is a dominant tree species of the Boreal Forest. Recent studies have shown that it is fairly resistant to heavy metal contamination, specifically to nickel. Knowledge of regulation of genes associated with metal resistance in higher plants is very sketchy. Availability and annotation of the dwarf birch (B. nana) enables the use of high throughout sequencing approaches to understanding responses to environmental challenges in other Betula species such as B. papyrifera. The main objectives of this study are to 1) develop and characterize the B. papyrifera transcriptome, 2) assess gene expression dynamics of B. papyrifera in response to nickel stress, and 3) describe gene function based on ontology. Nickel resistant and susceptible genotypes were selected and used for transcriptome analysis. A total of 208,058 trinity genes were identified and were assembled to 275,545 total trinity transcripts. The transcripts were mapped to protein sequences and based on best match; we annotated the B. papyrifera genes and assigned gene ontology. In total, 215,700 transcripts were annotated and were compared to the published B. nana genome. Overall, a genomic match for 61% transcripts with the reference genome was found. Expression profiles were generated and 62,587 genes were found to be significantly differentially expressed among the nickel resistant, susceptible, and untreated libraries. The main nickel resistance mechanism in B. papyrifera is a downregulation of genes associated with translation (in ribosome), binding, and transporter activities. Five candidate genes associated to nickel resistance were identified. They include Glutathione S-transferase, thioredoxin family protein, putative transmembrane protein and two Nramp transporters. These genes could be useful for genetic engineering of birch trees.
Project description:Agrobacterium sp. strain 33MFTa1.1 was isolated for functional host-microbe interaction studies from the Thlaspi arvense root-associated microbiome. The complete genome is comprised of a circular chromosome of 2,771,937?bp, a linear chromosome of 2,068,443?bp, and a plasmid of 496,948?bp, with G+C contents of 59%, 59%, and 58%, respectively.