The effect of simulated copper stress on genes associated with the metabolism of polyphosphates and polyhydroxyalkanoates in Bacillus thuringiensis Y1

Aims@#Bacillus thuringiensis Y1 is a copper-tolerant bacterium that can serve as a model for the elucidation of the mechanism of energy metabolism under simulated copper stress. This study aimed to elucidate the effects of simulated copper stress on the genes associated with the biosynthesis of polyhydroxyalkanoates (PAH) and the metabolism of polyphosphates (PP).@*Methodology and results @#The gene expression study involved the growth of the bacterium in nutrient broth supplemented with two concentrations of copper sulphate (0.4 mM and 0.8 mM), followed by RNA extraction and quantification of four and 11 genes associated with the metabolism of polyphosphate (PP) and polyhydroxyalkanoates (PAHs) respectively, using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Three genes associated with polyphosphate metabolism, which are polyphosphate kinase (ppk), exopolyphosphatase (ppx) and NAD kinase (ppnk), were all shown to be upregulated by both 0.4 mM and 0.8 mM copper, except for the 5’-nucleotidase (surE) gene that was downregulated under the second treatment. Among the 11 genes associated with the metabolism of polyhydroxyalkanoates, only the 3-ketoacyl-CoA-thiolase (phaA) gene was upregulated in both treatments and highly expressed in the second treatment; the majority were downregulated and repressed.@*Conclusion, significance and impact of study @#The study demonstrated that copper induces the metabolism of polyphosphates in B. thuringiensis Y1 that serve as an alternative source of energy under copper stress. This model can be extended to the study of other species of Bacillus under environmental stress.

Effects of copper stress on accumulation of three medicinal compositions and expression of two key enzyme genes in biosynthesis of Atractylodes lancea / 中草药

Objective: To explore the effect on the accumulation of medicinal compositions β-eudesmol, atractylon, atractylodin and key enzyme genes 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and farnesyl pyrophosphate synthase (FPPS) expression in biosynthesis of Atractylodes lancea under copper stress. Methods: Under copper stress, the expression of key enzyme genes HMGR and FPPS in A. lancea was determined by real-time fluorescence quantitative PCR; the content of three medicinal components in A. lancea were determined by HPLC; The correlation analysis was performed with SPSS, and DPS software for grey correlation analysis. Results: When the copper stress concentration was within 100 mg/kg, the expression of FPPS and the content of atractylon in the rhizomes of A. lancea increased slightly. However, when the copper concentration continued to increase, the expression levels of HMGR and FPPS and three medicinal components content of A. lancea showed a different degrees of downward trend. The expression levels of HMGR and FPPS were positively correlated with the content of β-eudesmol, atractylon, and atractylodin (P < 0.05) under copper stress. Grey relational analysis showed that the content of β-eudesmol and atractylon in the rhizomes was significantly correlated with the expression of HMGR and FPPS of A. lancea under copper stress. The expression of FPPS gene had the larger contribution on the composition of β-eudesmol and atractylon. However, the correlation between the content of atractylodin and the expression of these two key enzyme genes was relatively small. Conclusion: This study clarified the change regulation of two key enzyme gene expression and the content of three medicinal compositions, and revealed the relationship between β-eudesmol, atractylon and HMGR and FPPS, the key enzymes in terpene biosynthesis of A. lancea under copper stress. It contributed to the further study of the molecular regulation mechanism of the synthesis of medicinal constituents under copper stress and provided a theoretical basis for improving the quality of A. lancea.

Endophytic fungus Purpureocillium sp. A5 protect mangrove plant Kandelia candel under copper stress

Abstract Mangrove is an important ecosystem in the world. Mangrove ecosystems have a large capacity in retaining heavy metals, and now they are usually considered as sinks for heavy metals. However, the mechanism of why the soil of mangrove ecosystems can retain heavy metal is not certain. In this research, endophytic fungus Purpureocillium sp. A5 was isolated and identified from the roots of Kandelia candel. When this fungus was added, it protected the growth of K. candel under Cu stress. This can be illustrated by analyzing chlorophyll A and B, RWC and WSD to leaves of K. candel. Purpureocillium sp. A5 reduces uptake of Cu in K. candel and changes the pH characterization of soil. Furthermore, A5 increase the concentration of Cu complexes in soil, and it enhanced the concentration of carbonate-bound Cu, Mn-Fe complexes Cu and organic-bound Cu in soil. Nevertheless, a significant reduction of the Cu ion was noted among A5-treated plants. This study is significant and illustrates a promising potential use for environmental remediation of endophytes, and also may partially explain the large capacity of mangrove ecosystems in retaining heavy metals.

Yeast CUP1 protects HeLa cells against copper-induced stress

As an essential trace element, copper can be toxic in mammalian cells when present in excess. Metallothioneins (MTs) are small, cysteine-rich proteins that avidly bind copper and thus play an important role in detoxification. Yeast CUP1 is a member of the MT gene family. The aim of this study was to determine whether yeast CUP1 could bind copper effectively and protect cells against copper stress. In this study, CUP1 expression was determined by quantitative real-time PCR, and copper content was detected by inductively coupled plasma mass spectrometry. Production of intracellular reactive oxygen species (ROS) was evaluated using the 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay. Cellular viability was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the cell cycle distribution of CUP1 was analyzed by fluorescence-activated cell sorting. The data indicated that overexpression of yeast CUP1 in HeLa cells played a protective role against copper-induced stress, leading to increased cellular viability (P<0.05) and decreased ROS production (P<0.05). It was also observed that overexpression of yeast CUP1 reduced the percentage of G1 cells and increased the percentage of S cells, which suggested that it contributed to cell viability. We found that overexpression of yeast CUP1 protected HeLa cells against copper stress. These results offer useful data to elucidate the mechanism of the MT gene on copper metabolism in mammalian cells.

Studies on antioxidant enzymes in Canna indica plant under copper stress.

Bright red-flowered Canna indica L. plants were subjected to grow in nutrient solution supplemented with five different concentrations (0, 5, 10, 30 and 50 µM) of CuCl2 to study antioxidant defense responses of the plant. Accumulation of Cu was dose-dependent and much higher in the roots (108- 191µg g-1 d. wt.) than in the leaves (23.36 - 40.43 µg g-1 d.wt.). Total ascorbate content did not changed in both tissues, but ascorbate redox state decreased (0.570-0.640) in Cu-treated Canna roots. In contrast, both total and reduced glutathione contents increased (387-591.9 nmol g-1 f. wt.) considerably in roots, accompanied with enhanced activities of dehydroascorbate reductase (153.3-160 nmol mg-1 protein) and glutathione reductase (67-87.5 nmol mg-1 protein). No significant change, however, was observed for monodehydroascorbate reductase activity in both tissues of the treated plant. The efficient scavenging of hydrogen peroxide was performed by normal (control level) activities of both ascorbate peroxidase and catalase in leaf and increased activity of only catalase in root, preventing its accumulation at toxic concentrations (despite high superoxide dismutase activity) and subsequent damage of membrane lipids by peroxidation. Together, these ensured normal dry weight of leaves and roots, indicating tolerance of Canna indica plant to Cu-induced oxidative stress.

Copper induced oxidative stress in tea (Camellia sinensis) leaves.

Tea [Camellia sinensis L. (O.) Kuntze] is an economically important plantation crop of India but is prone to attack by several fungal pathogens. Copper based fungicides are being used for decades to control fungal diseases in tea which may lead to accumulation of copper in the soil. The biochemical responses to increasing concentrations of copper (50 to 700 µM) were investigated in the leaves of two cultivars of tea commonly grown in the Darjeeling hills. Exposure to excess Cu resulted in increased lipid peroxidation (level of TBARS increased from 3.5 µmol g-1 f.wt. in control to 12 µmol g-1 f.wt. in TS-520 plants exposed to 700 mM of Cu), reduced chlorophyll content (from 83.7 µg g-1 f.wt. in control to 22.5 µg g-1 f.wt. in TS-520 plants exposed to 700 µM of Cu), higher levels of phenolic compounds(total phenol content increased from 4.54 mg g-1 f.wt. in control to 5.79 mg g-1 f.wt. in TS-520 plants exposed to 400 µM of Cu) and an increase in peroxidase enzyme levels. Two new peroxidase isozymes (POD1 and POD2) were detected in plants exposed to Cu. In addition, biochemical responses in two tested cultivars, TS-462 and TS-520 differed significantly. TS-520 was found to be more sensitive to increasing concentrations of Cu. Superoxide dismutase activity increased progressively from 2.55 U mg-1 protein in control to 5.59 U mg-1 protein in TS-462 but declined from 4.75 U mg-1 protein in control to 3.33 U mg-1 protein in TS-520 when exposed to Cu concentrations higher than 400 µM. A sharp increase in the activity of ascorbate peroxidase (from 0.53 units in control to 2.37 units in plants exposed to 400 µM of Cu) was noticed at the 10th day of exposure in the more tolerant cultivar. On the other hand, catalase levels increased only marginally (from 8.4 to 10.1 units in TS 520 and 8.7 to 10.9 units in TS 462) in both the cultivars. From this study, it appears that Cu exposure led to the production of reactive oxygen species in the leaves resulting in significant lipid peroxidation. Tea plants try to mitigate this oxidative damage through accumulation of phenolic compounds and induction of antioxidant enzymes.