A novel DNA repair enzyme from Acinetobacter sp. Ver3 isolated from an extreme environment

PATHAK, G.,; ALBARRACIN VIRGINIA H.; FARIAS MARIA EUGENIA; DOUKI, T; CADET, J.; BORSARELLI, C.,; GÄRTNER, W.

Colonia

Workshop; 11th European Workshop on Astrobiology, Planets and life: evolution and distribution; 2011

European Astrobiology Network Association

Background: The Acinetobacter ver3 strain isolated from the High-Altitude Andean Lakes (HAAL) in the northwest of Argentina (from 3,000 to 6,000 m asl) was found in our previous investigation to be highly resistant to UV-B radiation. We have now identified from the genome of this strain a gene putatively encoding a protein with DNA repair activity. Phylogenetic analysis and 3D structure modeling of the predicted protein confirms the encoded protein as a DNA photolyase.   Materials and Methods: A cosmid genomic library was constructed from the Acinetobacter sp. Ver3 and the putative photolyase coding gene was identified by screening the cosmid clones using degenerate primers targeting conserved residues. The photolyase gene was fully sequenced from the detected clone using a primer walking approach. Phylogenetic analysis and 3D structure modeling was performed using the translated amino acid sequences. The gene was cloned in pET expression vectors and the protein was over-expressed in E. coli cells.   Results: Upon BLAST analysis the nearest match of the identified gene (HQ443199) was found to be the photolyase (ZP06062937) from A. johnsonii SH046 (66% identity). Based on homology modelling, a strong three-dimensional similarity to the photolyase from E. coli (PDB 1DNPA) was observed. Furthermore, the photolyase coding gene from Ver3 (HQ443199) was cloned into expression vector and over-expressed in E. coli BL21 cells. The UV-B resistance profile of the cells transformed with recombinant photolyase (HQ443199) was compared to the E. coli BL21 (without recombinant plasmid). When exposed to UV-B radiation for 15 minutes, more than 99% of the E. coli cells without the recombinant photolyase could not survive the radiation, while 48% of the cells expressing the recombinant photolyase did survive the same dose of UV-B exposure. Functional characterization of the purified photolyase protein is ongoing.   Conclusion: A novel photolyase coding gene from Acinetobacter ver3 (HQ443199) has been fully sequenced from a cosmid genomic library. The photolyase coding gene shows strong DNA repair activity in vivo in E. coli. Determination of the repair activity and the reason for the strong activity, as well as the photochemical properties is under way.