CONICET | Buscador de Institutos y Recursos Humanos

The Acinetobacter genusincludes an extensive group of physiologicallyversatile bacteria occupying various natural ecosystems. Some species areemerging as model organisms due to their genome plasticity and the resultingadaptability to diverse environments. Acinetobacter sp. Ver3 is a polyextremophileisolated from high altitude Andean wetlands, able to grow under hostileenvironmental conditions such as intense UV radiation, high salt concentrationsor extreme pH. Interestingly, total catalase activity in Ver3 cell-freeextracts is about 15 times higher than those found in control collectionstrains.After genome pyrosequencing and gene annotation(http://rast.nmpdr.org), two related ORFs were identified, encoding amonofunctional catalase KatE anda bifunctional catalase-peroxidase KatG. While all monofunctional catalases display asignificant degree of structural conservation, Ver3-KatE exhibits one of thehighest catalytic efficiencies reported to date (kcat = 1,29.107± 0,25.107 s-1). Hydrogen peroxide sensitivity assays inthe presence of the KatE inhibitor 3-amino-1,2,4-triazole revealed asignificant decrease of tolerance in Ver3 when compared to the response ofseveral collection control strains. These results suggest a critical role ofthe cytosolic KatE in peroxide detoxification.The katGgene encoding a bifunctional catalase-peroxidase was cloned and overexpressedas recombinant protein employing chaperone helpers in the bacterial host E.coli BL21 [pKJE7]. Protein sequence analysis using the algorithms JGI(img.jgi.doe.gov), PSORT (psort.hgc.jp) and SignalP (www.cbs.dtu.dk), suggeststhe presence of a targeting signal peptide of 19 residues. Subsequentsubcellular fractionation of transformed E.coli cells expressing Ver3-KatG confirmed a periplasmic localization. When katG was expressed after removing theN-terminal leader sequence, the enzyme was unable to reach the periplasm.Moreover, the resulting protein product accumulated in the cytosol as anhaem-bound inactive enzyme.The results indicate that polyextremophile Acinetobacter sp. Ver3takes advantage of both a cytosolic, highly efficient monofunctional catalase,and a bifunctional catalase-peroxidase as a periplasmic antioxidant barrier.