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Screening and Functional Analyses of Photoreceptors
in Extremophilic Microbial Communities

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 north-western part of Argentina. A classification approach for microorganisms from this environment (mostly bacteria, Archaea, and diatomees) has been initiated and is still in progress. Special emphasis has been given to recently identified stromatolites, the only ones found on earth at such high altitudes and under such environmental conditions. Collection of information has been accomplished by high-throughput metagenome sequencing and by genome sequencing of selected microbial strains. High-throughput metagenome sequence was completed during this project for the HAAL stromatolites, yielding by 16s ribonucleic acid (RNA) analysis a species classification and - thanks to the wide data set - allowed for functional analysis of the deoxyribonucleic acid (DNA)-derived putative gene products (functional characterisation of putative gene products / proteins).

Selection for full genome determination has been performed for two stress factors:
(i) the high heavy-metal contamination of these shallow lakes, partly due to volcanic activity; and
(ii) the exposition to the extremely intense ultraviolet (UV) irradiation due to the high altitude and low clouding of this region.

Out of the established strain culture collection at the home research unit in Tucuman, several bacterial strains have been selected for a genome-sequence determination and a more detailed functional analysis, including Acinetobacter Ver3 that exhibited unexpected survival capability.

Acinetobacter Ver3 is 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 UVB irradiation. Survival experiments performed in the laboratory under in vivo conditions confirmed a blue light-driven survival strategy. Full genome sequencing revealed the presence of two genes encoding for photolyases light-driven DNA-repair enzymes (PL1 and PL2). For characterisation of the enzyme properties, both genes were cloned and heterologously expressed in Escherichia coli. Functional identification for PL1 could be completed. Both chromophores, a methylenetetrahydrofolate (MTHF) antenna and a flavin adenine dinucleotide (FAD) chromophore, could unambiguously be identified by absorption and fluorescence spectroscopy and by high-performance liquid chromatography (HPLC). The photochemical reactivity upon blue light irradiation showed a conversion between two stable states. The UV damage repair capability in vivo had been determined using the well-characterised photolyase from E. coli as reference: a photolyase-deficient E. coli strain was transformed with either its endogeneous PL or with the enzyme from Acinetobacter Ver3 under various irradiation conditions, unambiguously demonstrating the high activity of the HAAL-derived enzyme in E. coli. Light driven repair functions in vitro applying chemically synthesised DNA are still under investigation.

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