Cyanobacteria of the genus Chroococcidiopsis are highly resistant to desiccation, being in nature able to colonize lithic habitats in extremely dry environments on Earth, such as the McMurdo Dry Valleys (Antarctica) and the Atacama Desert (Chile), and such a feature that makes this cyanobacterium a proper model organism in astrobiology [1]. In order to withstand lethal damages induced by entry into, and exit from, the fully desiccated state, desert strains of Chroococcidiopsis must have evolved mechanisms to protect and/or repair their sub-cellular components. Evidences on the existence in these cyanobacteria of an interplay between DNA protection and repair mechanisms arise from: i) their capability to repair the genome fragmentation induced by ionizing radiation [2]; ii) the occurrence of desiccation-survivors lacking DNA fragmentation [3]; and iii) their survival to short exposure to unattenuated Martian UV flux [4]; iv) presence and absence of DNA damage in dried cells exposed - shielded by 3 mm of Antarctic arenaria -, to prolonged simulated space and Martian conditions [5]. In order to unravel the DNA repair systems in Chroococcidiopsis sp. CCMEE 029 and overcome the impairments due to the lack of its genome sequence, two genetic approaches were developed. The first one aimes to the screening a prey genomic library of Chroococcidiopsis by using DNA repair baits obtained from a sequenced cyanobacterium. The rationale relays on the homo/heterodimerization occurring between DNA repair proteins and validation tests were carried out according to the two hybrid assay in Escherichia coli. This approach identified proper baits for the mismatch repair system (MMR) and allowed the in vivo characterization the interactions between MMR proteins of a cyanobacterium. The second approach aimed to identify components of the DNA repair systems of Chroococcidiopsis by using evolutionary PCR. In addition, to localize DNA repair factories in Chroococcidiopsis, a GFP-tagging system was developed which allowed the localization of RecA in this cyanobacterium. Finally, a shuttle plasmid able to replicate in Chroococcidiopsis and Escherichia coli was developed to evaluate the mutation rate of Chroococcidiopsis cells exposed to desiccation and/or extraterrestrial conditions, by assessing the reversions of leucine mutants of E. coli. The outcome of these experiments will validate the employment of Chroococcidiopsis as a gene space cargo. References: 1 Grilli Caiola M, Billi D. 2007. Chroococcidiopsis from desert to Mars. In COLE Book Series Vol. 11. (ed J. Seckbach) Springer-Verlag, pp. 553-68. 2 Billi D, Friedmann EI, Hofer KG et al. 2000. Ionizing-radiation resistance in the desiccation-tolerant cyanobacterium Chroococcidiopsis. Appl. Environm. Microbiol. 66:1489-92. 3. Billi D. 2009. Subcellular integrities in Chroococcidiopsis sp. CCMEE 029 survivors after prolonged desiccation revealed by molecular probes and genome stability assays. Extremophiles 13:49-57. 4 Cockell CS, Schuerger AC, Billi D et al. Effects of a simulated martian UV flux on the cyanobacterium Chroococcidiopsis sp. 029. Astrobiology 5:127-40. 5 Billi D, Ghelardini P, Onofri S et al. 2008. Desert Cyanobacteria under simulated space and Martian conditions. EPSC Abstracts Vol. 3, EPSC2008-A-00474. This work was supported by the Italian Ministry of Foreign Affairs (Directorate General for Cultural Promotion and Collaboration) and by Italian Space Agency.
Unravelling the DNA protection and repair mechanisms in desert strains of Chroococcidiopsis under extreme terrestrial and extraterrestrial environments
P Ghelardini;
2009
Abstract
Cyanobacteria of the genus Chroococcidiopsis are highly resistant to desiccation, being in nature able to colonize lithic habitats in extremely dry environments on Earth, such as the McMurdo Dry Valleys (Antarctica) and the Atacama Desert (Chile), and such a feature that makes this cyanobacterium a proper model organism in astrobiology [1]. In order to withstand lethal damages induced by entry into, and exit from, the fully desiccated state, desert strains of Chroococcidiopsis must have evolved mechanisms to protect and/or repair their sub-cellular components. Evidences on the existence in these cyanobacteria of an interplay between DNA protection and repair mechanisms arise from: i) their capability to repair the genome fragmentation induced by ionizing radiation [2]; ii) the occurrence of desiccation-survivors lacking DNA fragmentation [3]; and iii) their survival to short exposure to unattenuated Martian UV flux [4]; iv) presence and absence of DNA damage in dried cells exposed - shielded by 3 mm of Antarctic arenaria -, to prolonged simulated space and Martian conditions [5]. In order to unravel the DNA repair systems in Chroococcidiopsis sp. CCMEE 029 and overcome the impairments due to the lack of its genome sequence, two genetic approaches were developed. The first one aimes to the screening a prey genomic library of Chroococcidiopsis by using DNA repair baits obtained from a sequenced cyanobacterium. The rationale relays on the homo/heterodimerization occurring between DNA repair proteins and validation tests were carried out according to the two hybrid assay in Escherichia coli. This approach identified proper baits for the mismatch repair system (MMR) and allowed the in vivo characterization the interactions between MMR proteins of a cyanobacterium. The second approach aimed to identify components of the DNA repair systems of Chroococcidiopsis by using evolutionary PCR. In addition, to localize DNA repair factories in Chroococcidiopsis, a GFP-tagging system was developed which allowed the localization of RecA in this cyanobacterium. Finally, a shuttle plasmid able to replicate in Chroococcidiopsis and Escherichia coli was developed to evaluate the mutation rate of Chroococcidiopsis cells exposed to desiccation and/or extraterrestrial conditions, by assessing the reversions of leucine mutants of E. coli. The outcome of these experiments will validate the employment of Chroococcidiopsis as a gene space cargo. References: 1 Grilli Caiola M, Billi D. 2007. Chroococcidiopsis from desert to Mars. In COLE Book Series Vol. 11. (ed J. Seckbach) Springer-Verlag, pp. 553-68. 2 Billi D, Friedmann EI, Hofer KG et al. 2000. Ionizing-radiation resistance in the desiccation-tolerant cyanobacterium Chroococcidiopsis. Appl. Environm. Microbiol. 66:1489-92. 3. Billi D. 2009. Subcellular integrities in Chroococcidiopsis sp. CCMEE 029 survivors after prolonged desiccation revealed by molecular probes and genome stability assays. Extremophiles 13:49-57. 4 Cockell CS, Schuerger AC, Billi D et al. Effects of a simulated martian UV flux on the cyanobacterium Chroococcidiopsis sp. 029. Astrobiology 5:127-40. 5 Billi D, Ghelardini P, Onofri S et al. 2008. Desert Cyanobacteria under simulated space and Martian conditions. EPSC Abstracts Vol. 3, EPSC2008-A-00474. This work was supported by the Italian Ministry of Foreign Affairs (Directorate General for Cultural Promotion and Collaboration) and by Italian Space Agency.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
