Functional Analyses of Novel Photolyases Extreme Microbes from High-Altitude Andean Lakes (HAAL).

SIMON, J.; ALBARRACÍN V. H.; DOUKI T.; FARIAS M.E.; GÄRTNER W.

Córdoba

Congreso; 16th International Congress on Photobiology; 2014

High-Altitude Andean Lakes (HAAL) are shallow lakes located in the South American Puna-High Andes region. In order to survive under the extreme environmental conditions of those habitats (high UV-exposure, wide temperature variations, high toxic element contamination), microorganisms had to develop highly efficient mechanisms. A major task for survival is taken over by photolyases, which are able to repair UV-induced damages in DNA. The complete genome sequence of Acinetobacter sp. Ver3, an extremophilic bacterium isolated from HAAL, revealed two genes encoding photolyases (PL1/Ver3Phr and PL2) [1]. During previous work these two genes had been cloned into E. coli BL21. Here, spectroscopic and functional characterization of these novel light-driven repair enzymes is presented [2]. E. coli strains harbouring the Ver3Phr gene were subjected to UV-B irradiation and then exposed to light (PR) and dark repair (DR). Survival was assessed by traditional UFC counting and measurement of DNA bipyrimidine photoproducts by HPLC coupled with ionization-tandem mass spectrometry (ESI-MS/MS) detection. All experiments were run in parallel with the ortholog E. coli photolyase (EcPhr) expressed in the same host cells to allow for a direct functional comparison of the novel photolyases from HAAL. Clear enzymatic function was assessed for Ver3Phr, both in vivo and in vitro. E. coli cells harbouring the recombinant plasmid carrying Ver3Phr survived after UV exposure while cells with the empty vector were completely depleted after the treatment. The recovery after PR in the cells carrying the Ver3Phr was three orders of magnitudes greater than observed in the control, prooving the functional and efficient activity of Ver3Phr. [1.] V. H. Albarracin, G. P. Pathak, T. Douki, J. Cadet, C. D. Borsarelli, W. Gärtner, M. E. Farias, Origins of Life and Evolution of Biospheres 2012, 42 201-221. [2.] V. H. Albarracin, J. Simon, G. P. Pathak, L. Valle, T. Douki, J. Cadet, C. D. Borsarelli, M. E. Farias, W. Gärtner, First characterization of CPD-class I photolyase from a UV-resistant extremophile isolated from High-Altitude Andean Lakes, Photochem. Photobiol. Sci. 2014, in press (DOI: 10.1039/c3pp50399b).