Catalases in Acinetobacter: KatG signal peptide leads functional folding and periplasmic localization

PALAVECINO, M. A.; CORTEZ, NÉSTOR; SARTORIO, MARIANA

San Migel de Tucumán

Congreso; XII Congreso Argentino de Microbiología General SAMiGe; 2017

Asociación Civil de Microbiología General SAMiGe

The genus Acinetobacter includes a broad group of physiologically versatile bacteria occupying different natural ecosystems. Some species of the genus are becoming emerging model organisms because of their genome plasticity and the resulting adaptability to environment. Acinetobacter sp. Ver3 is a gamma proteobacterium isolated from high altitude Andean wetlands. This polyextremophile was able togrow under hostile environmental conditions such as intense UV-B radiation, high salt concentration or the presence of arsenite up to 10mM. Interestingly, total catalase activity in Acinetobacter sp. Ver3 free cell extracts is about 15 times higher than those found in control collection strains. After a genome pyrosequencing strategy and annotation (http://rast.nmpdr.org), two genes were identified, correspondingto a monofunctional catalase katE and a bifunctional catalase-peroxidase katG. Although displaying a significant degree of structural conservation among all monofunctional catalases, Ver3 KatE exhibits one of the highest catalytic efficiencies reported up to date (kcat = 1,29.107 ± 0,25.107 s-1 ). Hydrogen peroxide sensitivity assays in the presence of the KatE inhibitor 3-amino-1,2,4-triazole revealed asignificant decrease of tolerance in Ver3 when compared to the response of several collection control strains. These results indicate a critical role of the cytosolic KatE in peroxide detoxification. The katG gene codifying a bifunctional catalase-peroxidase was cloned and overexpressed as 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), suggests the presence of a targeting signal peptide of 19 residues. Posterior subcellular fractionation of transformant E. coli cells expressing Ver3 KatG shows its periplasmic localization. When katG was overexpressed after removing the N-terminal leader sequence, the enzyme was unable to reach the periplasm. Moreover, the protein product accumulated in cytosol as an haem-bound inactive enzyme. These results reveal that polyextremophile Acinetobacter sp. Ver3 takes advantage of both, a cytosolic highly efficient monofunctional catalase and a bifunctional catalase-peroxidase as periplasmic antioxidant barrier.