A specific proteomic response of Sulfolobus solfataricus P2 to gamma radiations.

Sulfolobus solfataricus is an acidophilic hyperthermophilic crenarchaeon living at 80 °C in aerobic conditions. As other thermophilic organisms, S. solfataricus is resistant to gamma irradiation and we studied the response of this microorganism to this ionizing irradiation by monitoring cell growth, DNA integrity and proteome variations. In aerobic conditions, the S. solfataricus genome was fragmented due to the multiple DNA double strand breakages induced by γ-rays and was fully restored within a couple of hours. Comparison of irradiated and unirradiated cell proteomes indicated that only few proteins changed. The proteins identified by mass spectrometry are involved in different cellular pathways including DNA replication, recombination and repair. Interestingly, we observed that some proteins are irradiation dose-specific while others are common to the cell response regardless of the irradiation dose. Most of the proteins highlighted in these conditions seem to act together to allow an efficient cell response to γ-irradiation.

Extremophiles survival to simulated space conditions: an astrobiology model study.

In this work we investigated the ability of four extremophilic bacteria from Archaea and Bacteria domains to resist to space environment by exposing them to extreme conditions of temperature, UV radiation, desiccation coupled to low pressure generated in a Mars' conditions simulator. All the investigated extremophilic strains (namely Sulfolobus solfataricus, Haloterrigena hispanica, Thermotoga neapolitana and Geobacillus thermantarcticus) showed a good resistance to the simulation of the temperature variation in the space; on the other hand irradiation with UV at 254 nm affected only slightly the growth of H. hispanica, G. thermantarcticus and S. solfataricus; finally exposition to Mars simulated condition showed that H. hispanica and G. thermantarcticus were resistant to desiccation and low pressure.

Repair of DNA double-strand breaks induced by ionizing radiation damage correlates with upregulation of homologous recombination genes in Sulfolobus solfataricus.

The mechanisms used by members of the archaeal branch of life to repair DNA damage are not well understood. DNA damage responses have been of particular interest in hyperthermophilic archaea, since these microbes live under environmental conditions that constantly elevate the potential for DNA damage. The work described here focuses on the response of four Sulfolobus solfataricus strains to ionizing radiation (IR) damage. Cellular survival of three wild-type strains and a defined deletion mutant strain was examined following exposure to various IR doses. Using pulsed-field gel electrophoresis, we determined chromosomal DNA double-strand break persistence and repair rates. Among the strains, variable responses were observed, the most surprising of which occurred with the defined deletion mutant strain. This strain displayed higher chromosomal repair rates than the parent strain and was also found to have increased resistance to IR. Using quantitative real-time PCR, we found that transcript levels of homologous recombination-related genes were strongly upregulated following damage in all the strains. The mutant strain again had an enhanced response and dramatically upregulated expression of recombination genes above levels observed for the parent strain, suggesting that increased levels of recombinational repair could account for its increased radiation resistance phenotype. Our results demonstrate a transcriptional response to IR in S. solfataricus for the first time and describe a defined deletion mutant strain that may give the first insight into a damage-based archaeal control element.

Repair of DNA double-strand breaks following UV damage in three Sulfolobus solfataricus strains.

DNA damage repair mechanisms have been most thoroughly explored in the eubacterial and eukaryotic branches of life. The methods by which members of the archaeal branch repair DNA are significantly less well understood but have been gaining increasing attention. In particular, the approaches employed by hyperthermophilic archaea have been a general source of interest, since these organisms thrive under conditions that likely lead to constant chromosomal damage. In this work we have characterized the responses of three Sulfolobus solfataricus strains to UV-C irradiation, which often results in double-strand break formation. We examined S. solfataricus strain P2 obtained from two different sources and S. solfataricus strain 98/2, a popular strain for site-directed mutation by homologous recombination. Cellular recovery, as determined by survival curves and the ability to return to growth after irradiation, was found to be strain specific and differed depending on the dose applied. Chromosomal damage was directly visualized using pulsed-field gel electrophoresis and demonstrated repair rate variations among the strains following UV-C irradiation-induced double-strand breaks. Several genes involved in double-strand break repair were found to be significantly upregulated after UV-C irradiation. Transcript abundance levels and temporal expression patterns for double-strand break repair genes were also distinct for each strain, indicating that these Sulfolobus solfataricus strains have differential responses to UV-C-induced DNA double-strand break damage.

Reactions to UV damage in the model archaeon Sulfolobus solfataricus.

Mechanisms involved in DNA repair and genome maintenance are essential for all organisms on Earth and have been studied intensively in bacteria and eukaryotes. Their analysis in extremely thermophilic archaea offers the opportunity to discover strategies for maintaining genome integrity of the relatively little explored third domain of life, thereby shedding light on the diversity and evolution of these central and important systems. These studies might also reveal special adaptations that are essential for life at high temperature. A number of investigations of the hyperthermophilic and acidophilic crenarchaeote Sulfolobus solfataricus have been performed in recent years. Mostly, the reactions to DNA damage caused by UV light have been analysed. Whole-genome transcriptomics have demonstrated that a UV-specific response in S. solfataricus does not involve the transcriptional induction of DNA-repair genes and it is therefore different from the well-known SOS response in bacteria. Nevertheless, the UV response in S. solfataricus is impressively complex and involves many different levels of action, some of which have been elucidated and shed light on novel strategies for DNA repair, while others involve proteins of unknown function whose actions in the cell remain to be elucidated. The present review summarizes and discusses recent investigations on the UV response of S. solfataricus on both the molecular biological and the cellular levels.

UV-inducible cellular aggregation of the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by pili formation.

The hyperthermophilic archaeon Sulfolobus solfataricus has been shown to exhibit a complex transcriptional response to UV irradiation involving 55 genes. Among the strongest UV-induced genes was a putative pili biogenesis operon encoding a potential secretion ATPase, two pre-pilins, a putative transmembrane protein and a protein of unknown function. Electron microscopy and image reconstruction of UV-treated cells showed straight pili with 10 nm in diameter, variable in length, not bundled or polarized and composed of three evenly spaced helices, thereby clearly being distinguishable from archaeal flagella. A deletion mutant of SSO0120, the central type II/IV secretion ATPase, did not produce pili. It could be complemented by reintroducing the gene on a plasmid vector. We have named the operon ups operon for UV-inducible pili operon of Sulfolobus. Overexpression of the pre-pilins, Ups-A/B (SSO0117/0118) in Sulfolobus resulted in production of extremely long filaments. Pronounced cellular aggregation was observed and quantified upon UV treatment. This aggregation was a UV-dose-dependent, dynamic process, not inducible by other physical stressors (such as pH or temperature shift) but stimulated by chemically induced double-strand breaks in DNA. We hypothesize that pili formation and subsequent cellular aggregation enhance DNA transfer among Sulfolobus cells to provide increased repair of damaged DNA via homologous recombination.

Response of the hyperthermophilic archaeon Sulfolobus solfataricus to UV damage.

In order to characterize the genome-wide transcriptional response of the hyperthermophilic, aerobic crenarchaeote Sulfolobus solfataricus to UV damage, we used high-density DNA microarrays which covered 3,368 genetic features encoded on the host genome, as well as the genes of several extrachromosomal genetic elements. While no significant up-regulation of genes potentially involved in direct DNA damage reversal was observed, a specific transcriptional UV response involving 55 genes could be dissected. Although flow cytometry showed only modest perturbation of the cell cycle, strong modulation of the transcript levels of the Cdc6 replication initiator genes was observed. Up-regulation of an operon encoding Mre11 and Rad50 homologs pointed to induction of recombinational repair. Consistent with this, DNA double-strand breaks were observed between 2 and 8 h after UV treatment, possibly resulting from replication fork collapse at damaged DNA sites. The strong transcriptional induction of genes which potentially encode functions for pilus formation suggested that conjugational activity might lead to enhanced exchange of genetic material. In support of this, a statistical microscopic analysis demonstrated that large cell aggregates formed upon UV exposure. Together, this provided supporting evidence to a link between recombinational repair and conjugation events.

Responses of hyperthermophilic crenarchaea to UV irradiation.

BACKGROUND: DNA damage leads to cellular responses that include the increased expression of DNA repair genes, repression of DNA replication and alterations in cellular metabolism. Archaeal information processing pathways resemble those in eukaryotes, but archaeal damage response pathways remain poorly understood. RESULTS: We analyzed the transcriptional response to UV irradiation in two related crenarchaea, Sulfolobus solfataricus and Sulfolobus acidocaldarius. Sulfolobus species encounter high levels of DNA damage in nature, as they inhabit high temperature, aerobic environments and are exposed to sunlight. No increase in expression of DNA repair genes following UV irradiation was observed. There was, however, a clear transcriptional response, including repression of DNA replication and chromatin proteins. Differential effects on the expression of the three transcription factor B (tfb) genes hint at a mechanism for the modulation of transcriptional patterns in response to DNA damage. TFB3, which is strongly induced following UV irradiation, competes with TFB1 for binding to RNA polymerase in vitro, and may act as a repressor of transcription or an alternative transcription factor for certain promoters. CONCLUSION: A clear response to DNA damage was observed, with down-regulation of the DNA replication machinery, changes in transcriptional regulatory proteins, and up-regulation of the biosynthetic enzymes for beta-carotene, which has UV protective properties, and proteins that detoxify reactive oxygen species. However, unlike eukaryotes and bacteria, there was no induction of DNA repair proteins in response to DNA damage, probably because these are expressed constitutively to deal with increased damage arising due to high growth temperatures.

Equal rates of repair of DNA photoproducts in transcribed and non-transcribed strands in Sulfolobus solfataricus.

The nucleotide excision repair (NER) pathway removes bulky lesions such as photoproducts from DNA. In both bacteria and eukarya, lesions located in transcribed strands are repaired significantly faster than those located in non-transcribed strands due to damage signalling by stalled RNA polymerase molecules: a phenomenon known as transcription-coupled repair (TCR). TCR requires a mechanism for coupling the detection of stalled RNA polymerase molecules to the NER pathway, provided in bacteria by the Mfd protein. In the third domain of life, archaea, the pathway of NER is not well defined, there are no Mfd homologues and the existence of TCR has not been investigated. In this report we looked at rates of removal of photoproducts in three different operons of the crenarchaeon Sulfolobus solfataricus following UV irradiation. We found no evidence for significantly faster repair in the transcribed strands of these three operons. The rate of global genome repair in S. solfataricus is relatively rapid, and this may obviate the requirement for a specialized TCR pathway. Significantly faster repair kinetics were observed in the presence of visible light, consistent with the presence of a gene for photolyase in the genome of S. solfataricus.