Synthetic phytochelatin surface display in Cupriavidus metallidurans CH34 for enhanced metals bioremediation.

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 ß-domain of the Neisseria gonorrhoeae IgA protease precursor (IgAß), which successfully targeted the hybrid protein toward the C. metallidurans outer membrane. The expression of the SS-EC20sp-IgAß 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.

A metal-repressed promoter from gram-positive Bacillus subtilis is highly active and metal-induced in gram-negative Cupriavidus metallidurans.

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.

Inhibition of enzymatic and pharmacological activities of some snake venoms and toxins by Mandevilla velutina (Apocynaceae) aqueous extract.

Phospholipases A(2) (PLA(2)) are multifunctional proteins which exhibit varied biological activities correlated to the structural diversities of the sub-classes. The crude aqueous extract from subterranean system of Mandevilla velutina, a plant found in Brazilian savanna, was assayed for its ability to inhibit biological activities of several snake venoms and isolated PLA(2)s. The extract induced total inhibition of the phospholipase activity of Crotalus durissus terrificus venom and only partial inhibition of Bothrops venoms. When assayed against purified toxins, the highest efficacy was detected against CB and crotoxin, while almost ineffective against PLA(2)s from the genus Bothrops. Although M. velutina crude extract significantly inhibited the myotoxic activity of C. d. terrificus venom and CB, it produced only partial inhibition of either Bothrops jararacussu venom or its main myotoxins BthTX-I (basic Lys49), BthTX-II (basic Asp49) and BthA-I-PLA(2) (acidic Asp49). The extract exhibited also full inhibition of hemorrhage caused by Bothrops alternatus, Bothrops moojeni and Bothrops pirajai snake venoms, but partial inhibition (90%) of that induced by B. jararacussu venom. The extract was ineffective to inhibit the fibrinogenolytic activity of B. moojeni, B. alternatus and B. pirajai crude venoms, while their caseinolytic activity was only partially inhibited. No inhibition of the anticoagulant activity, although partial reduction of the edema-inducing activity of C. d. terrificus and B. alternatus crude venoms, CB, PrTX-I, BthTX-I and crotoxin was observed. Besides extending survival of mice injected with lethal doses of C. d. terrificus and B. jararacussu venoms, M. velutina extract decreased to 50% the lethality of mice. Extracts of 18 month old micropropagated plants were able to partially neutralize the effect of the crude venoms and toxins.