Insights into the chaotropic tolerance of the desert cyanobacterium Chroococcidiopsis sp. 029 (Chroococcidiopsales, Cyanobacteria).

The mechanism of perchlorate resistance of the desert cyanobacterium Chroococcidiopsis sp. CCMEE 029 was investigated by assessing whether the pathways associated with its desiccation tolerance might play a role against the destabilizing effects of this chaotropic agent. During 3 weeks of growth in the presence of 2.4 mM perchlorate, an upregulation of trehalose and sucrose biosynthetic pathways was detected. This suggested that in response to the water stress triggered by perchlorate salts, these two compatible solutes play a role in the stabilization of macromolecules and membranes as they do in response to dehydration. During the perchlorate exposure, the production of oxidizing species was observed by using an oxidant-sensing fluorochrome and determining the expression of the antioxidant defense genes, namely superoxide dismutases and catalases, while the presence of oxidative DNA damage was highlighted by the over-expression of genes of the base excision repair. The involvement of desiccation-tolerance mechanisms in the perchlorate resistance of this desert cyanobacterium is interesting since, so far, chaotropic-tolerant bacteria have been identified among halophiles. Hence, it is anticipated that desert microorganisms might possess an unrevealed capability of adapting to perchlorate concentrations exceeding those naturally occurring in dry environments. Furthermore, in the endeavor of supporting future human outposts on Mars, the identified mechanisms might contribute to enhance the perchlorate resistance of microorganisms relevant for biologically driven utilization of the perchlorate-rich soil of the red planet.

Enabling deep-space experimentations on cyanobacteria by monitoring cell division resumption in dried Chroococcidiopsis sp. 029 with accumulated DNA damage.

Cyanobacteria are gaining considerable interest as a method of supporting the long-term presence of humans on the Moon and settlements on Mars due to their ability to produce oxygen and their potential as bio-factories for space biotechnology/synthetic biology and other applications. Since many unknowns remain in our knowledge to bridge the gap and move cyanobacterial bioprocesses from Earth to space, we investigated cell division resumption on the rehydration of dried Chroococcidiopsis sp. CCMEE 029 accumulated DNA damage while exposed to space vacuum, Mars-like conditions, and Fe-ion radiation. Upon rehydration, the monitoring of the ftsZ gene showed that cell division was arrested until DNA damage was repaired, which took 48 h under laboratory conditions. During the recovery, a progressive DNA repair lasting 48 h of rehydration was revealed by PCR-stop assay. This was followed by overexpression of the ftsZ gene, ranging from 7.5- to 9-fold compared to the non-hydrated samples. Knowing the time required for DNA repair and cell division resumption is mandatory for deep-space experiments that are designed to unravel the effects of reduced/microgravity on this process. It is also necessary to meet mission requirements for dried-sample implementation and real-time monitoring upon recovery. Future experiments as part of the lunar exploration mission Artemis and the lunar gateway station will undoubtedly help to move cyanobacterial bioprocesses beyond low Earth orbit. From an astrobiological perspective, these experiments will further our understanding of microbial responses to deep-space conditions.

Identification of far-red light acclimation in an endolithic Chroococcidiopsis strain and associated genomic features: Implications for oxygenic photosynthesis on exoplanets.

Deserts represent extreme habitats where photosynthetic life is restricted to the lithic niche. The ability of rock-inhabiting cyanobacteria to modify their photosynthetic apparatus and harvest far-red light (near-infrared) was investigated in 10 strains of the genus Chroococcidiopsis, previously isolated from diverse endolithic and hypolithic desert communities. The analysis of their growth capacity, photosynthetic pigments, and apcE2-gene presence revealed that only Chroococcidiopsis sp. CCMEE 010 was capable of far-red light photoacclimation (FaRLiP). A total of 15 FaRLiP genes were identified, encoding paralogous subunits of photosystem I, photosystem II, and the phycobilisome, along with three regulatory elements. CCMEE 010 is unique among known FaRLiP strains by undergoing this acclimation process with a significantly reduced cluster, which lacks major photosystem I paralogs psaA and psaB. The identification of an endolithic, extremotolerant cyanobacterium capable of FaRLiP not only contributes to our appreciation of this phenotype's distribution in nature but also has implications for the possibility of oxygenic photosynthesis on exoplanets.

Revival of Anhydrobiotic Cyanobacterium Biofilms Exposed to Space Vacuum and Prolonged Dryness: Implications for Future Missions beyond Low Earth Orbit.

Dried biofilms of Chroococcidiopsis sp. CCMEE 029 were revived after a 672-day exposure to space vacuum outside the International Space Station during the EXPOSE-R2 space mission. After retrieval, they were air-dried stored for 3.5 years. Space vacuum reduced cell viability and increased DNA damage compared to air-dried storage for 6 years under laboratory conditions. Long exposure times to space vacuum and extreme dryness decrease the changes of survival that ultimately depend on DNA damage repair upon rehydration, and hence, an 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 that were over-expressed during 1 and 6 h rehydration of space-vacuum exposed biofilms. 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. Unravelling how long anhydrobiotic cyanobacteria can persist under space vacuum followed by prolonged air-dried storage is relevant to future astrobiological experiments that use space platforms and might require prolonged air-dried storage of the exposed samples before retrieval to Earth.

Over-Expression of UV-Damage DNA Repair Genes and Ribonucleic Acid Persistence Contribute to the Resilience of Dried Biofilms of the Desert Cyanobacetrium Chroococcidiopsis Exposed to Mars-Like UV Flux and Long-Term Desiccation.

The survival limits of the desert cyanobacterium Chroococcidiopsis were challenged by rewetting dried biofilms and dried biofilms exposed to 1.5 × 103 kJ/m2 of a Mars-like UV, after 7 years of air-dried storage. PCR-stop assays revealed the presence of DNA lesions in dried biofilms and an increased accumulation in dried-UV-irradiated biofilms. Different types and/or amounts of DNA lesions were highlighted by a different expression of uvrA, uvrB, uvrC, phrA, and uvsE genes in dried-rewetted biofilms and dried-UV-irradiated-rewetted biofilms, after rehydration for 30 and 60 min. The up-regulation in dried-rewetted biofilms of uvsE gene encoding an UV damage endonuclease, suggested that UV-damage DNA repair contributed to the repair of desiccation-induced damage. While the phrA gene encoding a photolyase was up-regulated only in dried-UV-irradiated-rewetted biofilms. Nucleotide excision repair genes were over-expressed in dried-rewetted biofilms and dried-UV-irradiated-rewetted biofilms, with uvrC gene showing the highest increase in dried-UV-irradiated-rewetted biofilms. Dried biofilms preserved intact mRNAs (at least of the investigated genes) and 16S ribosomal RNA that the persistence of the ribosome machinery and mRNAs might have played a key role in the early phase recovery. Results have implications for the search of extra-terrestrial life by contributing to the definition of habitability of astrobiologically relevant targets such as Mars or planets orbiting around other stars.

Dried Biofilms of Desert Strains of Chroococcidiopsis Survived Prolonged Exposure to Space and Mars-like Conditions in Low Earth Orbit.

Dried biofilms and dried multilayered planktonic counterparts obtained from three desert strains of Chroococcidiopsis were exposed to low Earth conditions by using the EXPOSE-R2 facility outside the International Space Station. During the space mission, samples in Tray 1 (space vacuum and solar radiation, from λ ≈ 110 nm) and Tray 2 (Mars-like UV flux, λ > 200 nm and Mars-like atmosphere) received total UV (200-400 nm) fluences of about 4.58 × 102 kJ/m2 and 4.92 × 102 kJ/m2, respectively, and 0.5 Gy of cosmic ionizing radiation. Postflight analyses were performed on 2.5-year-old samples due to the space mission duration, from launch to sample return to the lab. The occurrence of survivors was determined by evaluating cell division upon rehydration and damage to the genome and photosynthetic apparatus by polymerase chain reaction-stop assays and confocal laser scanning microscopy. Biofilms recovered better than their planktonic counterparts, accumulating less damage not only when exposed to UV radiation under space and Mars-like conditions but also when exposed in dark conditions to low Earth conditions and laboratory control conditions. This suggests that, despite the shielding provided by top-cell layers being sufficient for a certain degree of survival of the multilayered planktonic samples, the enhanced survival of biofilms was due to the presence of abundant extracellular polymeric substances and to additional features acquired upon drying.

Avoidance of protein oxidation correlates with the desiccation and radiation resistance of hot and cold desert strains of the cyanobacterium Chroococcidiopsis.

To investigate the relationship between desiccation and the extent of protein oxidation in desert strains of Chroococcidiopsis a selection of 10 isolates from hot and cold deserts and the terrestrial cyanobacterium Chroococcidiopsis thermalis sp. PCC 7203 were exposed to desiccation (air-drying) and analyzed for survival. Strain CCMEE 029 from the Negev desert and the aquatic cyanobacterium Synechocystis sp. PCC 6803 were further investigated for protein oxidation after desiccation (drying over silica gel), treatment with H2O2 up to 1 M and exposure to γ-rays up to 25 kGy. Then a selection of desert strains of Chroococcidiopsis with different survival rates after prolonged desiccation, as well as Synechocystis sp. PCC 6803 and Chroococcidiopsis thermalis sp. PCC 7203, were analyzed for protein oxidation after treatment with 10 and 100 mM of H2O2. Results suggest that in the investigated strains a tight correlation occurs between desiccation and radiation tolerance and avoidance of protein oxidation.

Anti-herpes simplex virus 1 and immunomodulatory activities of a poly-γ- glutamic acid from Bacillus horneckiae strain APA of shallow vent origin.

Herpes simplex virus type 1 (HSV-1) is responsible of common and widespread viral infections in humans through the world, and of rare, but extremely severe, clinical syndromes in the central nervous system. The emergence of resistant strains to drugs actually in use encourages the searching for novel antiviral compounds, including those of natural origin. In this study, the recently described poly-γ-glutamic acid (γ-PGA-APA), produced by the marine thermotolerant Bacillus horneckiae strain APA, and previously shown to possess biological and antiviral activity, was evaluated for its anti-HSV-1 and immunomodulatory properties. The biopolymer hindered the HSV-1 infection in the very early phase of virus replication. In addition, the γ-PGA-APA was shown to exert low cytotoxicity and noticeable immunomodulatory activities towards TNF-α and IL-1ß gene expression. Moreover, the capacity to positively modulate the transcriptional activity of the cytokine genes was paired with increased level of activation of the transcription factor NF-kB by γ-PGA-APA. Overall, as non-cytotoxic biopolymer able to contribute in the antiviral defense against HSV-1, γ-PGA-APA could lead to the development of novel natural drugs for alternative therapies.

HSV-1-induced activation of NF-κB protects U937 monocytic cells against both virus replication and apoptosis.

The transcription factor nuclear factor-kappa B (NF-κB) is a crucial player of the antiviral innate response. Intriguingly, however, NF-κB activation is assumed to favour herpes simplex virus (HSV) infection rather than restrict it. Apoptosis, a form of innate response to viruses, is completely inhibited by HSV in fully permissive cells, but not in cells incapable to fully sustain HSV replication, such as immunocompetent cells. To resolve the intricate interplay among NF-κB signalling, apoptosis and permissiveness to HSV-1 in monocytic cells, we utilized U937 monocytic cells in which NF-κB activation was inhibited by expressing a dominant-negative IκBα. Surprisingly, viral production was increased in monocytic cells in which NF-κB was inhibited. Moreover, inhibition of NF-κB led to increased apoptosis following HSV-1 infection, associated with lysosomal membrane permeabilization. High expression of late viral proteins and induction of apoptosis occurred in distinct cells. Transcriptional analysis of known innate response genes by real-time quantitative reverse transcription-PCR excluded a contribution of the assayed genes to the observed phenomena. Thus, in monocytic cells NF-κB activation simultaneously serves as an innate process to restrict viral replication as well as a mechanism to limit the damage of an excessive apoptotic response to HSV-1 infection. This finding may clarify mechanisms controlling HSV-1 infection in monocytic cells.