Final Report Summary - EXTREMOPHIL (Screening and Functional Analyses of Photoreceptors in Extremophilic Microbial Communities) This project addressed the biodiversity and adaptation capability of the outstandingly complex and variable microbial community found in the “High Altitude Andean Lakes (HAAL)” in the Northwestern part of Argentina. The results obtained in this return phase complete a three-year bilateral research project between Argentinean and German researchers. In this period, special emphasis has been given to study the UV resistance mechanisms in a model extremophile Acinetobacter sp. Ver3 which full genome sequence was available during the incoming phase. Acinetobacter sp. Ver3 was considered as a model organism from HAAL for the study of many aspects of adaptation, showing amongst other properties an outstanding resistance and survival against high UV-B irradiation. As in-vivo experiments pointed to a strong photo-induced repair activity for Ver3-significantly greater than regular strain collection-derived reference strains- we performed a functional complementation of a photolyase knock-out Escherichia coli strain using the gene coding for a class-I photolyase (Ver3Phr) found within the genome of Ver3. This was assessed by traditional UFC counting and measurement of DNA bipyrimidine photoproducts by HPLC coupled with electrospray ionisation-tandem mass spectrometry detection. The results identified strong photoreactivation ability in vivo of Ver3Phr while its non-photoreactivation function, probably related with the stimulation of nucleotide excision repair (NER), was not as manifest as for EcPhr (E. coli photolyase). To unravel the complex genetic and physiological systems of adaptation and resistance to UV in Ver3, herein called UV-resistome, we use both, genomics and proteomics approaches. Genomics. The draft genome of Acinetobacter sp. Ver3 consists of 3,345,299 bp, with a GC content of 38.9%. NCBI PGAP annotation shows 3,241 genes, including 3,080 coding sequences, 66 RNAs and 95 pseudogenes. RAST annotation server was used to have a broad view of genome features. 1445 genes (46.91%) were included in the Subsytems classification, revealing some of the extremophilic features of this organism. Consistent with the extreme environment, Acinetobacter sp. Ver3 presented genes for resistance to UV irradiation (e.g. photolyases and catalase), metals, salinity, and even some antibiotics. Proteomics. Protein expression of Acinetobacter sp. Ver3 cells in the presence or absence of UV radiation was assessed by two-dimensional protein gel analyses and mass spectrometry identification of the differential spots. Overexpressed proteins were identified as related with oxidative damage response, quorum sensing, and protein synthesis. Down-regulated proteins belong to pathways related to energy homeostasis, carbohydrate metabolism and protein synthesis. Knockout mutants on photoreceptors (photolyases, BLUF domains and cryptochromes) are currently under construction and will be subjected to functional analyses (proteomics, transcriptomics) to compare their behavior with the wild Ver3 strain in relation with its UV resistance and biofilm formation ability.Research on biofilm formation by Ver3, as part of the UV-resistome, and its regulation by blue-light photoreceptors was initiated. Results so far indicated that Ver3 is able to form biofilm on synthetic surfaces and that its formation is up-regulated upon UV and arsenic exposure. The results obtained in this project contribute to the understanding of the complex molecular basis of Acinetobacter sp. Ver3 UV-resistome. These findings have implications for several diverse fields, ranging from throwing light on the evolution of microbial life in early habitats to the discovery of novel metabolites and molecules with potential biotechnological applications on energy conversion, biomedicine or industry. Finally, the implementation of the project attracted more attention to the biological uniqueness of these environments - and in this sense collect more data to support an intensive protection program of the HAAL whole biological diversity.
