No abstract

This work describes the effects of the cell surface display of a synthetic phytochelatin in the highly metal tolerant bacterium Cupriavidus metallidurans CH34. The EC20sp synthetic phytochelatin gene was fused between the coding sequences of the signal peptide (SS) and of the autotransporter β-domain of the Neisseria gonorrhoeae IgA protease precursor (IgAβ), which successfully targeted the hybrid protein toward the C. metallidurans outer membrane. The expression of the SS-EC20sp-IgAβ gene fusion was driven by a modified version of the Bacillus subtilis mrgA promoter showing high level basal gene expression that is further enhanced by metal presence in C. metallidurans. The recombinant strain showed increased ability to immobilize Pb 2+ , Zn 2+ , Cu 2+ , Cd 2+ , Mn 2+ , and Ni 2+ ions from the external medium when compared to the control strain. To ensure plasmid stability and biological containment, the MOB region of the plasmid was replaced by the E. coli hok/sok coding sequence.

In order to produce explants of Mandevilla illustris (Vell) Woodson for the "Cerrado in vitro", the Germplasm Bank of UNAERP, we carried out a micropropagation protocol using MS or MS/3 medium supplemented with different concentrations of 6-benzyladeninepurine (BA), Zeatin or 2-isopentenyladenine for nodal segment growth, and alpha-naphthaleneacetic acid, indole-3-butyric acid (IBA) or 1,4 dithiothreitol for rooting. For nodal segments, all the cytokinins tested yielded similar results. However, 2.22 micro M BA is more economical to use. MS/3 medium supplemented with 0.49 micro M IBA was the most appropriate medium for rooting, resulting in 29% rooted explants. The crude aqueous extract from the subterranean system (SS) of M. illustris was assayed for its inhibitory action on the enzymatic activity of Crotalus durissus terrificus snake venom, isolated basic phospholipase A2 (CB) and crotoxin. It totally inhibited the phospholipase activity of crude Cdt venom and CB toxin and inhibited the phospholipase activity of crotoxin by 49%. The toxic action of both the crude venom and crotoxin was partially inhibited-there was a prolonged survival time and a 40.0% decrease in lethality.

A synthetic version of the metal-regulated gene A (mrgA) promoter from Bacillus subtilis, which in this Gram-positive bacterium is negatively regulated by manganese, iron, cobalt, or copper turned out to promote high level of basal gene expression that is further enhanced by Co(II), Cd(II), Mn(II), Zn(II), Cu(II), or Ni(II), when cloned in the Gram-negative bacterium Cupriavidus metallidurans. Promoter activity was monitored by expression of the reporter gene coding for the enhanced green fluorescent protein (EGFP), and cellular intensity fluorescence was quantified by flow cytometry. Expression levels in C. metallidurans driven by the heterologous promoter, here called pan, ranged from 20- to 53-fold the expression level driven by the Escherichia coli lac promoter (which is constitutively expressed in C. metallidurans), whether in the absence or presence of metal ions, respectively. The pan promoter did also function in E. coli in a constitutive pattern, regardless of the presence of Mn(II) or Fe(II). In conclusion, the pan promoter proved to be a powerful tool to express heterologous proteins in Gram-negative bacteria, especially in C. metallidurans grown upon high levels of toxic metals, with potential applications in bioremediation.

Mandevilla velutina (Mart.) Woodson (Apocynaceae) is a medicinal plant species with antivenom properties, native from Brazilian Savanna regions (Cerrado), which due to overexploitation and habitat deforestation is in danger of extinction. As an initiative for conserving this endangered but economically important plant species, a micropropagation protocol was developed and genotypes were stored in the Germplasm Bank "Cerrado In vitro". For the in vitro propagation of M. velutina, nodal segments were inoculated on Murashige and Skoog (MS) medium supplemented with different concentrations of BA, Zeatin, 2ip, DTT and TDZ. Best multiplication ratio was achieved when to the medium 0.44 μM BA, ranging 1: 6.7, were added. Plantlets cultured on MS/2 medium supplemented with 26.85 μM NAA rooted successfully (50.5%). Although rooted and un-rooted plantlets acclimatized to soil conditions, great losses were observed within un-rooted plantlets, while the rooted presented 100 % survival. It was possible to maintain 43% of the M. velutina germplasm under healthy conditions for six months, with no subcultures, using the MS medium supplemented with 2% sucrose, 13.8 mM spermidine, 2% sorbitol and 2% dextrose.