The Atacama Rover Astrobiology Drilling Studies (ARADS) Project.

With advances in commercial space launch capabilities and reduced costs to orbit, humans may arrive on Mars within a decade. Both to preserve any signs of past (and extant) martian life and to protect the health of human crews (and Earth's biosphere), it will be necessary to assess the risk of cross-contamination on the surface, in blown dust, and into the near-subsurface (where exploration and resource-harvesting can be reasonably anticipated). Thus, evaluating for the presence of life and biosignatures may become a critical-path Mars exploration precursor in the not-so-far future, circa 2030. This Special Collection of papers from the Atacama Rover Astrobiology Drilling Studies (ARADS) project describes many of the scientific, technological, and operational issues associated with searching for and identifying biosignatures in an extreme hyperarid region in Chile's Atacama Desert, a well-studied terrestrial Mars analog environment. This paper provides an overview of the ARADS project and discusses in context the five other papers in the ARADS Special Collection, as well as prior ARADS project results.

Salt shield: intracellular salts provide cellular protection against ionizing radiation in the halophilic archaeon, Halobacterium salinarum NRC-1.

The halophilic archaeon Halobacterium salinarum NRC-1 was used as a model system to investigate cellular damage induced by exposure to high doses of ionizing radiation (IR). Oxidative damages are the main lesions from IR and result from free radicals production via radiolysis of water. This is the first study to quantify DNA base modification in a prokaryote, revealing a direct relationship between yield of DNA lesions and IR dose. Most importantly, our data demonstrate the significance of DNA radiation damage other than strand breaks on cell survival. We also report the first in vivo evidence of reactive oxygen species scavenging by intracellular halides in H. salinarum NRC-1, resulting in increased protection against nucleotide modification and carbonylation of protein residues. Bromide ions, which are highly reactive with hydroxyl radicals, provided the greatest protection to cellular macromolecules. Modified DNA bases were repaired in 2 h post irradiation, indicating effective DNA repair systems. In addition, measurements of H. salinarum NRC-1 cell interior revealed a high Mn/Fe ratio similar to that of Deinococcus radiodurans and other radiation-resistant microorganisms, which has been shown to provide a measure of protection for proteins against oxidative damage. The work presented here supports previous studies showing that radiation resistance is the product of mechanisms for cellular protection and detoxification, as well as for the repair of oxidative damage to cellular macromolecules. The finding that not only Mn/Fe but also the presence of halides can decrease the oxidative damage to DNA and proteins emphasizes the significance of the intracellular milieu in determining microbial radiation resistance.

Rad50 is not essential for the Mre11-dependent repair of DNA double-strand breaks in Halobacterium sp. strain NRC-1.

The genome of the halophilic archaeon Halobacterium sp. strain NRC-1 encodes homologs of the eukaryotic Mre11 and Rad50 proteins, which are involved in the recognition and end processing of DNA double-strand breaks in the homologous recombination repair pathway. We have analyzed the phenotype of Halobacterium deletion mutants lacking mre11 and/or rad50 after exposure to UV-C radiation, an alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine), and gamma radiation, none of which resulted in a decrease in survival of the mutant strains compared to that of the background strain. However, a decreased rate of repair of DNA double-strand breaks in strains lacking the mre11 gene was observed using pulsed-field gel electrophoresis. These observations led to the hypothesis that Mre11 is essential for the repair of DNA double-strand breaks in Halobacterium, whereas Rad50 is dispensable. This is the first identification of a Rad50-independent function for the Mre11 protein, and it represents a shift in the Archaea away from the eukaryotic model of homologous recombination repair of DNA double-strand breaks.

The RadB protein from Pyrococcus does not complement E. coli recA mutations in vivo.

A previous publication claimed that the radB gene called Pk-REC from Pyrococcus furiosus complemented an E. coli recA mutation. We found that a sequencing error had led to the test of a mutant form of Pk-REC. The wild-type radB gene from P. furiosus cloned in a similar expression vector to the mutant Pk-REC also appeared to complement an E. coli recA mutation. However, the cloned P. furiosus gdh (glutamate dehydrogenase) gene showed the same activity. We therefore concluded that overexpression of any protein can produce an artificial growth inhibition or stationary phase in recA mutant cells, which allows cells to recover from UV damage due to the action of repair systems that do not require RecA-like activity.

Evidence of recent lateral gene transfer among hyperthermophilic archaea.

A total of 153 nucleotide differences were found over a contiguous 16 kb region between two hyperthermophilic Archaea, Pyrococcus furiosus and Thermococcus litoralis. The 16 kb region in P. furiosus is flanked by insertion sequence (IS) elements with inverted and direct repeats. Both IS elements contain a single open reading frame (ORF) encoding a putative protein of 233 amino acids identified as a transposase. This 16 kb region has the features of a typical bacterial composite transposon and represents a possible mechanism for lateral gene transfer between Archaea or possibly between Archaea and Bacteria. A total of 23 homologous IS elements was found in the genome sequence of P. furiosus, whereas no full-length IS elements were identified in the genomes of Pyrococcus abyssi and Pyrococcus horikoshii. Only one IS element was found in T. litoralis. In P. furiosus and T. litoralis, the 16 kb region contains an ABC transport system for maltose and trehalose that was characterized biochemically for T. litoralis. Regulation of expression studies showed that the malE gene, located on the transposon, and the encoded trehalose/maltose-binding protein (TMBP) are induced in the presence of maltose and trehalose in both P. furiosus and T. litoralis. The implications of transposition as a mechanism for lateral gene transfer among Archaea are discussed.

Both RadA and RadB are involved in homologous recombination in Pyrococcus furiosus.

RecA and Rad51 proteins are essential for homologous recombination in Bacteria and Eukarya, respectively. Homologous proteins, called RadA, have been described for Archaea. Here we present the characterization of two RecA/Rad51 family proteins, RadA and RadB, from Pyrococcus furiosus. The radA and radB genes were not induced by DNA damage resulting from exposure of the cells to gamma and UV irradiation and heat shock, suggesting that they might be constitutively expressed in this hyperthermophile. RadA had DNA-dependent ATPase, D-loop formation, and strand exchange activities. In contrast, RadB had a very weak ATPase activity that is not stimulated by DNA. This protein had a strong binding affinity for DNA, but little strand exchange activity could be detected. A direct interaction between RadA and RadB was detected by an immunoprecipitation assay. Moreover, RadB, but not RadA, coprecipitated with Hjc, a Holliday junction resolvase found in P. furiosus, in the absence of ATP. This interaction was suppressed in the presence of ATP. The Holliday junction cleavage activity of Hjc was inhibited by RadB in the absence, but not in the presence, of ATP. These results suggest that RadB has important roles in homologous recombination in Archaea and may regulate the cleavage reactions of the branch-structured DNA.

Cloning and sequence analysis of the mercury resistance operon of Streptomyces sp. Strain CHR28 reveals a novel putative second regulatory gene.

A DNA library of pRJ28, a large linear plasmid encoding mercury resistance, was constructed, and the mercury resistance genes were cloned. The 5,921-bp sequence was analyzed and showed a high degree of similarity to the Streptomyces lividans 1326 mercury resistance operon. Genes merR, merT, merP, and orfIV were found in a similar order and in a single transcription unit. merA and merB were found to be transcribed in the opposite direction to genes merR, merT, merP, and orfIV, as in S. lividans 1326. A novel putative regulatory gene, orfX, was found 22 bp downstream of merA. orfX encodes a 137-amino acid protein with a potential helix-turn-helix motif in the N-terminal domain, characteristic of the MerR family of transcriptional regulators. Transcriptional studies showed that orfX is cotranscribed with merA and merB. It is hypothesized that orfX plays a role in the regulation of the mercury resistance operon, probably by binding at the MerR operator site.

DNA repair systems in archaea: mementos from the last universal common ancestor?

DNA repair in the Archaea is relevant to the consideration of genome maintenance and replication fidelity in the last universal common ancestor (LUCA) from two perspectives. First, these prokaryotes embody a mix of bacterial and eukaryal molecular features. Second, DNA repair proteins would have been essential in LUCA to maintain genome integrity, regardless of the environmental temperature. Yet we know very little of the basic molecular mechanisms of DNA damage and repair in the Archaea in general. Many studies on DNA repair in archaea have been conducted with hyperthermophiles because of the additional stress imposed on their macromolecules by high temperatures. In addition, of the six complete archaeal genome sequences published so far, five are thermophilic archaea. We have recently shown that the hyperthermophile Pyrococcus furiosus has an extraordinarily high capacity for repair of radiation-induced double-strand breaks and we have identified and sequenced several genes involved in DNA repair in P. furiosus. At the sequence level, only a few genes share homology with known bacterial repair genes. For instance, our phylogenetic analysis indicates that archaeal recombinases occur in two paralogous gene families, one of which is very deeply branched, and both recombinases are more closely related to the eukaryotic RAD51 and Dmc1 gene families than to the Escherichia coli recA gene. We have also identified a gene encoding a repair endo/exonuclease in the genomes of several Archaea. The archaeal sequences are highly homologous to those of the eukaryotic Rad2 family and they cluster with genes of the FEN-1 subfamily, which are known to be involved in DNA replication and repair in eukaryotes. We argue that there is a commonality of mechanisms and protein sequences, shared between prokaryotes and eukaryotes for several modes of DNA repair, reflecting diversification from a minimal set of genes thought to represent the genome of the LUCA.

Divergence of the hyperthermophilic archaea Pyrococcus furiosus and P. horikoshii inferred from complete genomic sequences.

Divergence of the hyperthermophilic Archaea, Pyrococcus furiosus and Pyrococcus horikoshii, was assessed by analysis of complete genomic sequences of both species. The average nucleotide identity between the genomic sequences is 70-75% within ORFs. The P. furiosus genome (1.908 mbp) is 170 kbp larger than the P. horikoshii genome (1.738 mbp) and the latter displays significant deletions in coding regions, including the trp, his, aro, leu-ile-val, arg, pro, cys, thr, and mal operons. P. horikoshii is auxotrophic for tryptophan and histidine and is unable to utilize maltose, unlike P. furiosus. In addition, the genomes differ considerably in gene order, displaying displacements and inversions. Six allelic intein sites are common to both Pyrococcus genomes, and two intein insertions occur in each species and not the other. The bacteria-like methylated chemotaxis proteins form a functional group in P. horikoshii, but are absent in P. furiosus. Two paralogous families of ferredoxin oxidoreductases provide evidence of gene duplication preceding the divergence of the Pyrococcus species.

Molecular and biochemical analysis of MalK, the ATP-hydrolyzing subunit of the trehalose/maltose transport system of the hyperthermophilic archaeon Thermococcus litoralis.

We report the cloning, sequencing, and expression of malK encoding the ATP-hydrolyzing subunit of the maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis. According to the deduced amino acid sequence, MalK consists of 372 amino acids with a calculated molecular weight of 41,787. It shows 47% identity with the MalK protein of Escherichia coli and high sequence conservation in important regions. C-terminal His-tagged MalK was purified. The soluble protein appeared monomeric by molecular sieve chromatography and showed ATPase activity. Enzymatic activity was highest at 80 degrees C with a Km of 150 microM and a Vmax of 0.55 micromol of ATP hydrolyzed/min/mg of protein. ADP was not a substrate but a competitive inhibitor (Ki 230 microM). GTP and CTP were also hydrolyzed. ATPase activity was inhibited by N-ethylmaleimide but not by vanadate. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the ABC transporters in these two phylogenetic branches.

Archaeal binding protein-dependent ABC transporter: molecular and biochemical analysis of the trehalose/maltose transport system of the hyperthermophilic archaeon Thermococcus litoralis.

We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis that is homologous to the malEFG cluster encoding the Escherichia coli maltose transport system. The deduced amino acid sequence of the malE product, the trehalose/maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine. The T. litoralis malE gene was expressed in E. coli under control of an inducible promoter with and without its natural signal sequence. In addition, in one construct the endogenous signal sequence was replaced by the E. coli MalE signal sequence. The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose. The protein bound both sugars at 85 degrees C with a Kd of 0.16 microM. Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from T. litoralis membranes as well as the products from all other DNA constructs expressed in E. coli. Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the E. coli MalF protein are missing in the T. litoralis MalF. MalG is homologous throughout the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches.

Repair of extensive ionizing-radiation DNA damage at 95 degrees C in the hyperthermophilic archaeon Pyrococcus furiosus.

We investigated the capacity of the hyperthermophile Pyrococcus furiosus for DNA repair by measuring survival at high levels of 60Co gamma-irradiation. The P. furiosus 2-Mb chromosome was fragmented into pieces ranging from 500 kb to shorter than 30 kb at a dose of 2,500 Gy and was fully restored upon incubation at 95 degrees C. We suggest that recombination repair could be an extremely active repair mechanism in P. furiosus and that it might be an important determinant of survival of hyperthermophiles at high temperatures.

Characterization of the reverse gyrase from the hyperthermophilic archaeon Pyrococcus furiosus.

The reverse gyrase gene rgy from the hyperthermophilic archaeon Pyrococcus furiosus was cloned and sequenced. The gene is 3,642 bp (1,214 amino acids) in length. The deduced amino acid sequence has relatively high similarity to the sequences of the Methanococcus jannaschii reverse gyrase (48% overall identity), the Sulfolobus acidocaldarius reverse gyrase (41% identity), and the Methanopynrus kandleri reverse gyrase (37% identity). The P. furiosus reverse gyrase is a monomeric protein, containing a helicase-like module and a type I topoisomerase module, which resembles the enzyme from S. acidocaldarius more than that from M. kandleri, a heterodimeric protein encoded by two separate genes. The control region of the P. furiosus rgy gene contains a typical archaeal putative box A promoter element which is located at position -26 from the transcription start identified by primer extension experiments. The initiating ATG codon is preceded by a possible prokaryote-type ribosome-binding site. Purified P. furiosus reverse gyrase has a sedimentation coefficient of 6S, suggesting a monomeric structure for the native protein. The enzyme is a single polypeptide with an apparent molecular mass of 120 kDa, in agreement with the gene structure. The sequence of the N terminus of the protein corresponded to the deduced amino acid sequence. Phylogenetic analysis indicates that all known reverse gyrase topoisomerase modules form a subgroup inside subfamily IA of type I DNA topoisomerases (sensu Wang [J. C. Wang, Annu. Rev. Biochem. 65:635-692, 1996]). Our results suggest that the fusion between the topoisomerase and helicase modules of reverse gyrase occurred before the divergence of the two archaeal phyla, Crenoarchaeota and Euryarchaeota.

Rapid differentiation of hyperthermophilic Archaea by restriction mapping of the intergenic spacer regions of the ribosomal RNA operons.

We report a sensitive and convenient method for rapid differentiation of new isolates of hyperthermophilic Archaea. Polymerase chain reaction (PCR) was used to amplify the intergenic spacer regions of the ribosomal RNA operons of eight Archaea. Spacer regions from one Euryarcheote, Pyrococcus furiosus, and one Crenarcheote, Pyrodictium brockii, were sequenced completely. Restriction fragment length polymorphism (RFLP) analyses were performed on the spacer regions from eight hyperthermophilic Archaea, and the restriction patterns were used as fingerprints for six known strains and two isolates. The PCR-RFLP method used in this study allowed the differentiation of seven of the eight strains tested and could be generally applicable to all the Archaea.

Expression and in vitro assembly of recombinant glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.

The gdhA gene, encoding the hexameric glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus furiosus, was expressed in Escherichia coli by using the pET11-d system. The recombinant GDH was soluble and constituted 15% of the E. coli cell extract. The N-terminal amino acid sequence of the recombinant protein was identical to the sequence of the P. furiosus enzyme, except for the presence of an initial methionine which was absent from the enzyme purified from P. furiosus. By molecular exclusion chromatography we showed that the recombinant GDH was composed of equal amounts of monomeric and hexameric forms. Heat treatment of the recombinant protein triggered in vitro assembly of inactive monomers into hexamers, resulting in increased GDH activity. The specific activity of the recombinant enzyme, purified by heat treatment and affinity chromatography, was equivalent to that of the native enzyme from P. furiosus. The recombinant GDH displayed a slightly lower level of thermostability, with a half-life of 8 h at 100 degrees C, compared with 10.5 h for the enzyme purified from P. furiosus.

Regulation of ribosomal RNA transcription by growth rate of the hyperthermophilic Archaeon, Pyrococcus furiosus.

We have studied the single rRNA gene cluster from the Archaeon, Pyrococcus furiosus. This isolate grows optimally at 100 degrees C and is thus a hyperthermophile. In P. furiosus, transcription of 16S rRNA is subject to regulation over a 7.5-fold range in response to a 20-fold increase in growth rate. The single cluster encoding the 16S and 23S rRNA genes of P. furiosus was cloned and the 1.9 kb region upstream of the 16S rRNA gene was sequenced.

Characterization, cloning, and in vitro expression of the extremely thermostable glutamate dehydrogenase from the hyperthermophilic Archaeon, ES4.

Glutamate dehydrogenase (GDH) from the hyperthermophilic Archaeon ES4 (optimal growth temperature 98 degrees C and maximum growth temperature 110 degrees C) was purified to homogeneity. The purified native enzyme had an M(r) of 270,000 +/- 5,000 and was shown by gel filtration and SDS-polyacrylamide gel electrophoresis to be a hexamer with identical subunits of M(r) = 46,000 +/- 3,000. The hexameric subunit composition was also evident from electron micrographs, which show a triangular antiprism structure very similar to that of bovine GDH. The enzyme is exceptionally thermostable, with a half-time of inactivation of 3.5 h at 105 degrees C. Differential scanning calorimetry revealed a tm for denaturation of 113 degrees C, and a tm for activation at 60 degrees C. Antigenic cross-reaction with ES4 GDH was observed with the purified GDH from the thermophilic Archaea, Pyrococcus furiosus and Thermococcus litoralis as well as with bovine and yeast GDHs. The genome of ES4 was shown to contain a single copy of the gdhA gene, and this was cloned and sequenced. The deduced amino acid sequence of the GDH from ES4 corresponded to the NH2-terminal amino acid sequence obtained from the pure protein. From the nucleotide sequence the ES4 protein is composed of 420 residues. It has a relatively high hydrophobicity and a low number of sulfur-containing residues compared with mesophilic GDHs. Relatively high homology (52%) exists between the deduced amino acid sequence of ES4 GDH and Clostridium difficile GDH. Of the two distinct families of GDH sequences known, ES4 GDH belongs to the same family as vertebrates, C. difficile, and other Archaea. The gdhA gene of ES4 was expressed in vitro in a rabbit reticulocyte cell-free lysate, thus providing a system for structural studies of the mechanisms of thermostability in hyper-thermophilic proteins.