Effects of Environmental Factors and Metal Ions on Growth of the Red Alga Gracilaria Chorda Holmes (Gracilariales, Rhodophyta)

Gracilaria is a potentially valuable source of marine biopolymers such as proteins and polysaccharides. In order to select suitable culture conditions, growth and tolerance of Gracilaria chorda Holmes from Shikoku Island in southwest Japan were investigated under variations of temperature (5–30 ^∘C), photon irradiance (20–120 μmol photons m⁻² s⁻¹), and photoperiod (12:12 h, 14:10 h light:dark regime) in a unialgal culture. Gracilaria chorda showed wide tolerances for all factors investigated, which is characteristic of eurythermal species. Maximum growth was observed at 18–24 ^∘C. The optimum photon irradiance for the algal growth was 60–120 μmol photons m⁻²s⁻¹. Instead of using ordinary sea salt (NaCl) to prepare artificial seawater, ultra pure salt was adopted. Gracilaria chorda grew faster in artificial seawater made with ultra-pure salt than that made with ordinary sea salt, probably because the former medium was clear, while the latter was milky. Effects of some metal ions on the growth were tested with artificial seawater. Iron ions affected algal growth, but cobalt ions did not. This study enables us to determine suitable culture conditions for G. chorda. A scaled-up 30 l culture of G. chorda under such conditions was successful.     

Tolerance and phytoaccumulation of Chromium by three Azolla species

Azolla, an aquatic fern is ideal candidate for exploitation in constructed wetlands for treating metal-contaminated wastewaters. This study demonstrates the potential of Azolla spp. namely A. microphylla, A. pinnata and A.␣filiculoides to tolerate Cr ions in the growth environment and bioconcentrate them. These species could grow in presence of up to 10 μg ml⁻¹ Cr and showed biomass production 30–70% as compared to controls. Nitrogenase activity was not affected at 1–5 μg ml⁻¹ but at higher concentrations it diminished. There was no growth at higher concentrations of chromium. However, the necrosed biomass harvested from treatments containing higher concentrations of chromium, accumulated Cr to the levels 5000–15,000 μg g⁻¹. At increased levels of Cr, the metal was accumulated in higher amount in dry biomass. Bioconcentration Factor (BCF) ranged between 243 and 4617 for the three species. A. microphylla showed highest bioconcentration potential. Thus, these Azolla spp. can be exploited for treatment of tannery and other Cr contaminated wastewaters.     

A putative novel role for plant defensins: a defensin from the zinc hyper-accumulating plant, Arabidopsis halleri, confers zinc tolerance

The metal tolerance of metal hyper-accumulating plants is a poorly understood mechanism. In order to unravel the molecular basis of zinc (Zn) tolerance in the Zn hyper-accumulating plant Arabidopsis halleri ssp. halleri, we carried out a functional screening of an A. halleri cDNA library in the yeast Saccharomyces cerevisiae to search for genes conferring Zn tolerance to yeast cells. The screening revealed four A. halleri defensin genes (AhPDFs), which induced Zn but not cadmium (Cd) tolerance in yeast. The expression of AhPDF1.1 under the control of the 35S promoter in A. thaliana made the transgenic plants more tolerant to Zn than wild-type plants, but did not change the tolerance to Cd, copper (Cu), cobalt (Co), iron (Fe) or sodium (Na). Thus, AhPDF1.1 is able to confer Zn tolerance both to yeast and plants. In A. halleri, defensins are constitutively accumulated at a higher level in shoots than in A. thaliana. A. halleri defensin pools are Zn-responsive, both at the mRNA and protein levels. In A. thaliana, some but not all defensin genes are induced by ZnCl2 treatment, and these genes are not induced by NaCl treatment. Defensins, found in a very large number of organisms, are known to be involved in the innate immune system but have never been found to play any role in metal physiology. Our results support the proposition that defensins could be involved in Zn tolerance in A. halleri, and that a role for plant defensins in metal physiology should be considered.     

Sulphate uptake and metabolism in water hyacinth and salvinia during cadmium stress

Water hyacinth (Eichhornia crassipes (Mart.) Solms) and salvinia (Salvinia auriculata Aubl.) were exposed to toxic levels of Cd with the objective of evaluating its effect on sulphate uptake and metabolism. Plants were treated with 0 and 5 μmol L⁻¹ Cd for 3 days and, then sulphate uptake, ATP sulfurylase activity, soluble thiol content and Cd-binding complexes were determined. Water hyacinth showed a lower sulphate uptake, but its kinetic parameters were not affected by Cd. In salvinia, however, both V_max and affinity to sulphate (1/K_m) decreased with Cd treatment. The ATP sulfurylase activity increased in Cd-treated plant of both species, except in the roots of salvinia. In the presence of Cd water hyacinth always exhibited higher activity of this enzyme. The total soluble thiol content was always higher in water hyacinth. In Cd treated plants it increased in the leaves of water hyacinth, but decreased in salvinia. Cysteine content increased only in water hyacinth leaves, while γ-glutamylcysteine content increased in the two parts of the plants of both species after Cd treatment, especially in water hyacinth. Glutathione contents, on the contrary, after Cd treatment, reduced in both parts of the plants of water hyacinth but only in the leaves of salvinia. The unidentified thiol fraction content increased with Cd treatment in both species, especially in water hyacinth. Root and leaf extracts of both species showed peaks with maxima at A₂₆₅/A₂₈₀. In treated plants these peaks coincided with Cd content peaks indicating the formation of Cd-binding peptides. It was estimated that in the presence of Cd about 97% of Cd was associated with these complexes and water hyacinth had 28% more Cd-binding peptides than salvinia. Despite its lower sulphate uptake, water hyacinth showed higher rates of sulfur reduction and assimilation into soluble thiols. Possibly, glutathione is used in water hyacinth roots to synthesize hitherto unidentified Cd-binding peptides.     

Arousing sleeping genes: shifts in secondary metabolism of metal tolerant actinobacteria under conditions of heavy metal stress

Numerous microbial habitats are strongly influenced by elevated levels of heavy metals. This type of habitat has developed either due to ore mining and metal processing or by pedogenesis above metal-rich base rocks. Most actinobacteria are soil-borne microbes with a remarkable capability for the synthesis of a broad variety of biologically active secondary metabolites. One major obstacle in identifying secondary metabolites, however, is the known phenomenon of sleeping gene clusters which are present, but silent under standard screening conditions. Here, we proceed to show that sleeping gene clusters can be awakened by the induction in heavy metal stress. Both, a chemical and a biological screening with extracts of supernatant and biomass of 10 strains derived from metal contaminated and non-contaminated environments was carried out to assay the influence of heavy metals on secondary metabolite patterns of metal tolerant actinobacteria. Metabolite patterns of cultures grown in complex and minimal media were compared to nickel (or cadmium) spiked parallels. Extracts of some strains grown in the presence of a metal salt displayed intense antibiosis against Escherichia coli, Mycobacterium smegmatis, Staphylococcus aureus and Candida albicans. Contrarily to the widely held opinion of metals as hindrance in secondary metabolism, metals thus can induce or enhance synthesis of possibly potent and medically relevant metabolites in metal tolerant strains. Hence, re-screening of existing strain libraries as well as identification of new strains from contaminated areas are valid strategies for the detection of new antibiotics in the future.     

Reduction of molybdate by sulfate-reducing bacteria

Molybdate is an essential trace element required by biological systems including the anaerobic sulfate-reducing bacteria (SRB); however, detrimental consequences may occur if molybdate is present in high concentrations in the environment. While molybdate is a structural analog of sulfate and inhibits sulfate respiration of SRB, little information is available concerning the effect of molybdate on pure cultures. We followed the growth of Desulfovibrio gigas ATCC 19364, Desulfovibrio vulgaris Hildenborough, Desulfovibrio desulfuricans DSM 642, and D. desulfuricans DSM 27774 in media containing sub-lethal levels of molybdate and observed a red-brown color in the culture fluid. Spectral analysis of the culture fluid revealed absorption peaks at 467, 395 and 314 nm and this color is proposed to be a molybdate–sulfide complex. Reduction of molybdate with the formation of molybdate disulfide occurs in the periplasm D. gigas and D. desulfuricans DSM 642. From these results we suggest that the occurrence of poorly crystalline Mo-sulfides in black shale may be a result from SRB reduction and selective enrichment of Mo in paleo-seawater.     

Abortiporus biennis tolerance to insoluble metal oxides: oxalate secretion, oxalate oxidase activity, and mycelial morphology

The ability of Abortiporus biennis to tolerate and solubilize toxic metal oxides (Cu₂O, Al₂O₃, ZnO, CuFe₂O₄Zn, CdO, and MnO₂) incorporated into agar media was investigated and the growth rate, oxalic acid secretion, and mycelial morphology were monitored. Among the tested metal oxides, formation of clear zones underneath the mycelium growing on Cu₂O- and ZnO-amended plates was observed. ZnO, CdO and Cu₂O caused the highest rate of fungal growth inhibition. An increased level of oxalic acid concentration was detected as a response of A. biennis to the presence of Cu₂O, MnO₂, ZnO and CuFe₂O₄Zn in growth medium. The oxalate oxidase (OXO) was found to be responsible for oxalic acid degradation in A. biennis cultivated in metal-amended media. An increased level of OXO was observed in media amended with Cu₂O, ZnO and MnO₂. Confocal microscopy used in this study revealed changes in mycelial morphology which appeared as increased hyphal branching, increased septation and increased spore number.     

Zinc transport by respiratory epithelial cells and interaction with iron homeostasis

Despite recurrent exposure to zinc through inhalation of ambient air pollution particles, relatively little information is known about the homeostasis of this metal in respiratory epithelial cells. We describe zinc uptake and release by respiratory epithelial cells and test the postulate that Zn²⁺ transport interacts with iron homeostasis in these same cells. Zn²⁺ uptake after 4 and 8 h of exposure to zinc sulfate was concentration- and time-dependent. A majority of Zn²⁺ release occurred in the 4 h immediately following cell exposure to ZnSO₄. Regarding metal importers, mRNA for Zip1 and Zip2 showed no change after respiratory epithelial cell exposure to zinc while mRNA for divalent metal transporter (DMT)1 increased. Western blot assay for DMT1 protein supported an elevated expression of this transport protein following zinc exposure. RT-PCR confirmed mRNA for the metal exporters ZnT1 and ZnT4 with the former increasing after ZnSO₄. Cell concentrations of ferritin increased with zinc exposure while oxidative stress, measured as lipid peroxides, was decreased supporting an anti-oxidant function for Zn²⁺. Increased DMT1 expression, following pre-incubations of respiratory epithelial cells with TNF-α, IFN-γ, and endotoxin, was associated with significantly decreased intracellular zinc transport. Finally, incubations of respiratory epithelial cells with both zinc sulfate and ferric ammonium citrate resulted in elevated intracellular concentrations of both metals. We conclude that exposure to zinc increases iron uptake by respiratory epithelial cells. Elevations in cell iron can possibly affect an increased expression of DMT1 and ferritin which function to diminish oxidative stress. Comparable to other metal exposures, changes in iron homeostasis may contribute to the biological effects of zinc in specific cells and tissues.