A bioinformatic approach to unravel the endurance of a desert cyanobacterium under Mars-like conditions in low Earth orbit

Despite the increasing interest in using microbial-based technologies to support human space exploration, many unknowns remain on microbial survivability and genetic stability after travel through space and reactivation under non-Earth conditions. Desert isolates of the cyanobacterium Chroococcidiopsis are characterized by an extraordinary desiccation and radiation tolerance, a prerequisite needed for astrobiological experiments performed in low Earth orbit (Billi et al., 2019a). During the BIOlogy and Mars EXperiment (BIOMEX) space experiment performed using the EXPOSE-R2 installed outside the International Space Station (July 2014-June 2016), dried cells of Chroococcidiopsis sp. CCMEE 029, mixed with Martian regolith simulant, were exposed to 0.5 Gy of ionizing cosmic radiation and 2.19 x 10^2 kJ/m2 of UV radiation (200-400 nm) combined to a Mars-like atmosphere. Post-flight analysis showed that dried Chroococcidiopsis accumulated DNA damage during the 1.5-year space that was repaired upon rehydration after retrieval back to Earth. Unravelling the mechanisms underlying the survivability of dried Chroococcidiopsis under space and Mars-like conditions and assessing if the accumulated damages are efficiently repaired represent a key step in unraveling the limit of life as we know it, but also to exploit Chroococcidiopsis in space biotechnologies/synthetic biology. Therefore two main approaches were followed in this thesis. First, genomic variants in a strain of Chroococcidiopsis sp. 029 obtained after exposure to Mars-like conditions in low Earth orbit were compared with the ground-reference strain. Second, a genome mining approach was used to identify the in the genome of Chroococcidiopsissp. 029, DNA repair genes, SOD-coding genes, and genes encoding enzymes for the synthesis of trehalose and sucrose. The comparative analysis of whole-genome sequences showed no increased variant numbers in the space-derivate compared to triplicates of the reference strain maintained on the ground. All the variants (single nucleotide variants, 4 insertion and/or deletions) found in the space-derivate occurred in at least one of the ground-reference triplicates. The alignment between the groundreference-genome and the longest-assembled scaffold of the space-derivate showed the absence of structural rearrangements. In the space-derivate the number of variants potentially affecting protein function, like stop codons or missense mutations, was comparable to that of each ground-reference replicate. As observed for each stop-codon gain found in the ground-reference replicates, the two predicted stop-codon gains occurring in the space-derivate did not involve any known proteins of DNA repair pathways. This result advanced cyanobacteria-based life support technologies to support human space exploration. In silico analysis of Chroococcidiopsis sp. CCMEE 029’s genome was performed with a focus on DNA repair pathways. The analysis identified a high number of genes that encode proteins of the homologous recombination RecF pathway and base excision repair. This suggests that Chroococcidiopsis developed a survival strategy against desiccation, with DNA repair playing a key role, which allowed the revival of biofilms exposed to space vacuum. Because trehalose and sucrose stabilize dried-desiccation tolerant cells, an in silico survey of the genome of the desert cyanobacterium Chroococcidiopsis sp. CCMEE 029 was performed to identify pathways for trehalose and sucrose biosynthesis. Finally, since desiccation causes oxidative stress, the first line of antioxidant defense given by superoxide dismutase, against oxidative stress was investigated. Three genes coding superoxide dismutases (SODs) were annotated as MnSODs and Cu/ZnSOD (rare among cyanobacteria). A signalpeptide bioinformatics prediction identified a Tat signal peptide at the Nterminus of the SodA2.1 that highlighted its transport across the thylakoid/cytoplasmic membranes and release in the periplasm/thylakoid lumen. Homologs of the Tat transport system were identified in Chroococcidiopsis sp. CCMEE 029. No signal peptide was predicted for the MnSOD (SodA2.2) and Cu/ZnSOD, thus suggesting their occurrence as cytoplasmic proteins. No FeSOD homologs were identified in 5 Chroococcidiopsis sp. CCMEE 029, a feature that might contribute to its desiccation tolerance since iron produces hydroxyl radical via the Fenton reaction. The periplasmic MnSOD protected the cell envelope against oxidative damage, the MnSOD localized in the thylakoid lumen scavengered superoxide anion radical produced during the photosynthesis, while the cytoplasmic MnSOD and Cu/ZnSOD reinforced the defense against reactive oxygen species generated at the onset of desiccation. Results contribute to deciphering the desiccation-tolerance mechanisms of this cyanobacterium, a key prerequisite for using the current configuration of the EXPOSE hardware that allows the exposure of cells in the dried state. Implications are also dealing with the transport of dried Chroococcidiopsis cells for exploitation for life support systems for space settlements.