Characterization and application of a family B DNA polymerase from the hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans.

Thermococcus gammatolerans is anaerobic euryarchaeon which grows optimally at 88 °C and its genome encodes a family B DNA polymerase (Tga PolB). Herein, we cloned the gene of Tga PolB, expressed and purified the gene product, and characterized the enzyme biochemically. The recombinant Tga PolB can efficiently synthesize DNA at high temperature, and retain 93% activity after heated at 95 °C for 1.0 h, suggesting that the enzyme is thermostable. Furthermore, the optimal pH for the enzyme activity was measured to be 7.0-9.0. Tga PolB activity is dependent on a divalent cation, among which magnesium ion is optimal. NaCl at low concentration stimulates the enzyme activity but at high concentration inhibits enzyme activity. Interestingly, Tga PolB is able to efficiently bypass uracil in DNA, which is distinct from other archaeal family B DNA pols. By contrast, Tga PolB is halted by an AP site in DNA, as observed in other archaeal family B DNA polymerases. Furthermore, Tga PolB extends the mismatched ends with reduced efficiencies. The enzyme possesses 3'-5' exonuclease activity and this activity is inhibited by dNTPs. The DNA binding assays showed that Tga PolB can efficiently bind to ssDNA and primed DNA, and have a marked preference for primed DNA. Last, Tga PolB can be used in routine PCR.

Increase of positive supercoiling in a hyperthermophilic archaeon after UV irradiation.

Diverse DNA repair mechanisms are essential to all living organisms. Some of the most widespread repair systems allow recovery of genome integrity in the face of UV radiation. Here, we show that the hyperthermophilic archaeon Thermococcus nautili possesses a remarkable ability to recovery from extreme chromosomal damage. Immediately following UV irradiation, chromosomal DNA of T. nautili is fragmented beyond recognition. However, the extensive UV-induced double-stranded breaks (DSB) are repaired over the course of several hours, allowing restoration of growth. DSBs also disrupted plasmid DNA in this species. Similar to the chromosome, plasmid integrity was restored during an outgrowth period. Intriguingly, the topology of recovered pTN1 plasmids differed from control strain by being more positively supercoiled. As reverse gyrase (RG) is the only enzyme capable of inducing positive supercoiling, our results suggest the activation of RG activity by UV-induced stress. We suggest simple UV stress could be used to study archaeal DNA repair and responses to DSB.

Conditional Protein Splicing Switch in Hyperthermophiles through an Intein-Extein Partnership.

Inteins are intervening proteins that undergo an autocatalytic splicing reaction that ligates flanking host protein sequences termed exteins. Some intein-containing proteins have evolved to couple splicing to environmental signals; this represents a new form of posttranslational regulation. Of particular interest is RadA from the archaeon Pyrococcus horikoshii, for which long-range intein-extein interactions block splicing, requiring temperature and single-stranded DNA (ssDNA) substrate to splice rapidly and accurately. Here, we report that splicing of the intein-containing RadA from another archaeon, Thermococcus sibericus, is activated by significantly lower temperatures than is P. horikoshii RadA, consistent with differences in their growth environments. Investigation into variations between T. sibericus and P. horikoshii RadA inteins led to the discovery that a nonconserved region (NCR) of the intein, a flexible loop where a homing endonuclease previously resided, is critical to splicing. Deletion of the NCR leads to a substantial loss in the rate and accuracy of P. horikoshii RadA splicing only within native exteins. The influence of the NCR deletion can be largely overcome by ssDNA, demonstrating that the splicing-competent conformation can be achieved. We present a model whereby the NCR is a flexible hinge which acts as a switch by controlling distant intein-extein interactions that inhibit active site assembly. These results speak to the repurposing of the vestigial endonuclease loop to control an intein-extein partnership, which ultimately allows exquisite adaptation of protein splicing upon changes in the environment.IMPORTANCE Inteins are mobile genetic elements that interrupt coding sequences (exteins) and are removed by protein splicing. They are abundant elements in microbes, and recent work has demonstrated that protein splicing can be controlled by environmental cues, including the substrate of the intein-containing protein. Here, we describe an intein-extein collaboration that controls temperature-induced splicing of RadA from two archaea and how variation in this intein-extein partnership results in fine-tuning of splicing to closely match the environment. Specifically, we found that a small sequence difference between the two inteins, a flexible loop that likely once housed a homing endonuclease used for intein mobility, acts as a switch to control intein-extein interactions that block splicing. Our results argue strongly that some inteins have evolved away from a purely parasitic lifestyle to control the activity of host proteins, representing a new form of posttranslational regulation that is potentially widespread in the microbial world.

Radiation resistance in thermophiles: mechanisms and applications.

The study of prokaryotic life in high temperature environments viz., geothermal areas, hot, acidic geysers and undersea hydrothermal vents has revealed the existence of thermophiles (or hyperthermophiles). These microorganisms possess various stress adaptation mechanisms which enable them to bypass multiple physical and chemical barriers for survival. The discovery of radiation resistant thermophile Deinococcus geothermalis has given new insights into the field of radiation microbiology. The ability of radiation resistant thermophiles to deal with the lethal effects of ionizing radiations like DNA damage, oxidative bursts and protein damage has made them a model system for exobiology and interplanetary transmission of life. They might be an antiquity of historical transport process that brought microbial life on Earth. These radiation resistant thermophiles are resistant to desiccation as well and maintain their homeostasis by advance DNA repair mechanisms, reactive oxygen species (ROS) detoxification system and accumulation of compatible solutes. Moreover, engineered radioresistant thermophilic strains are the best candidate for bioremediation of radionuclide waste while the extremolytes produced by these organisms may have predicted therapeutic uses. So, the present article delineate a picture of radiation resistance thermophiles, their adaptive mechanisms to evade stress viz., radiation and desiccation, their present applications along with new horizons in near future.

Dynamics and energetics of cyanobacterial photosystem I:ferredoxin complexes in different redox states.

Fast turnover of ferredoxin/Fd reduction by photosystem-I/PSI requires that it dissociates rapidly after it has been reduced by PSI:Fd intracomplex electron transfer. The rate constants of Fd dissociation from PSI have been determined by flash-absorption spectroscopy with different combinations of cyanobacterial PSIs and Fds, and different redox states of Fd and of the terminal PSI acceptor (FAFB). Newly obtained values were derived firstly from the fact that the dissociation constant between PSI and redox-inactive gallium-substituted Fd increases upon (FAFB) reduction and secondly from the characterization and elucidation of a kinetic phase following intracomplex Fd reduction to binding of oxidized Fd to PSI, a process which is rate-limited by the foregoing dissociation of reduced Fd from PSI. By reference to the complex with oxidized partners, dissociation rate constants were found to increase moderately with (FAFB) single reduction and by about one order of magnitude after electron transfer from (FAFB)- to Fd, therefore favoring turnover of Fd reduction by PSI. With Thermosynechococcus elongatus partners, values of 270, 730 and >10000s-1 were thus determined for (FAFB)Fdoxidized, (FAFB)-Fdoxidized and (FAFB)Fdreduced, respectively. Moreover, assuming a conservative upper limit for the association rate constant between reduced Fd and PSI, a significant negative shift of the Fd midpoint potential upon binding to PSI has been calculated (< -60mV for Thermosynechococcus elongatus). From the present state of knowledge, the question is still open whether this redox shift is compatible with a large (>10) equilibrium constant for intracomplex reduction of Fd from (FAFB)-.

Importance and determinants of induction of cold-induced DEAD RNA helicase in the hyperthermophilic archaeon Thermococcus kodakarensis.

Thermococcus kodakarensis, which grows optimally at 85°C, expresses cold stress-inducible DEAD box RNA helicase (Tk-deaD) when shifted to 60°C. A DDA1 deletion (ΔTk-deaD) mutant exhibited decreased cell growth, and cells underwent lysis at 60°C in nutrient broth in the absence of elemental sulfur. In contrast, cells in medium containing elemental sulfur at 60°C did not undergo lysis, suggesting that Tk-deaD is necessary for cell growth in sulfur-free medium. To identify the element responsible for the cold response, a pTKR expression probe plasmid was constructed using thermostable catalase from Pyrobaculum calidifontis as a reporter. The plasmid pTKRD, which contained the transcription factor B recognition element, TATA region, and Shine-Dalgarno (SD) region, including the initiation codon of the Tk-deaD gene, exhibited cold inducibility. We also constructed a series of deletion and chimeric constructs with the glutamate dehydrogenase (gdh) promoter, whose expression is constitutive independent of culture temperatures and catalase expression. Reporter assay experiments indicated that the regulatory element is located in the region between the SD region and the initiation codon (ATG). Nucleotide sequences in the upstream regions of Tk-deaD and gdh were compared and revealed a five-adenosine (AAAAA) sequence between SD and ATG of Tk-deaD that was not present in gdh. Replacement of the repeated adenosine sequence with other sequences revealed that the AAAAA sequence is important for cold induction. This sequence-specific mechanism is unique and is one that has not been identified in other known cold-inducible genes.

Recovery of ionizing-radiation damage after high doses of gamma ray in the hyperthermophilic archaeon Thermococcus gammatolerans.

The recently discovered hyperthermophilic and radioresistant archaeon Thermococcus gammatolerans is of great interest to compare and contrast the impact of its physiology on radioresistance and its ability to repair damaged chromosomes after exposure to gamma irradiation with radioresistant bacteria. We showed that, in contrast to other organisms, cell survival was not modified by the cellular growth phase under optimal growth conditions but nutrient-limited conditions did affect the T. gammatolerans radioresistance. We determined the first kinetics of damaged DNA recovery in an archaeon after exposure to massive doses of gamma irradiation and compared the efficiency of chromosomal DNA repair according to the cellular growth phase, nutrient availability and culture conditions. Chromosomal DNA repair kinetics showed that stationary phase cells reconstitute disrupted chromosomes more rapidly than exponential phase cells. Our data also revealed that this radioresistant archaeon was proficient to reconstitute shattered chromosomes either slowly or rapidly without any loss of viability. These results suggest that rapid DNA repair is not required for the extreme radioresistance of T. gammatolerans.

Thermococcus marinus sp. nov. and Thermococcus radiotolerans sp. nov., two hyperthermophilic archaea from deep-sea hydrothermal vents that resist ionizing radiation.

Enrichments for anaerobic, organotrophic hyperthermophiles were performed with hydrothermal chimney samples collected from the Mid-Atlantic Ridge at a depth of 3,550 m (23 degrees 22'N, 44 degrees 57'W) and the Guaymas Basin (27 degrees 01'N, 111 degrees 24'W) at a depth of 2,616 m. Positive enrichments were submitted to gamma-irradiation at doses of 20 and 30 kGy. Two hyperthermophilic, anaerobic, sulfur-metabolizing archaea were isolated. Strain EJ1T was isolated from chimney samples collected from the Mid-Atlantic Ridge after gamma-irradiation at 20 kGy, and strain EJ2T was isolated from the Guaymas Basin after gamma-irradiation at 30 kGy. Only strain EJ2T was motile, and both formed regular cocci. These new strains grew between 55 and 95 degrees C with the optimal temperature being 88 degrees C. The optimal pH for growth was 6.0, and the optimal NaCl concentration for growth was around 20 g l(-1). These strains were obligate anaerobic heterotrophs that utilized yeast extract, tryptone, and peptone as a carbon source for growth. Ten amino acids were essential for the growth of strain EJ1), such as arginine, aspartic acid, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tyrosine, and valine, while strain EJ2T was unable to grow on a mixture of amino acids. Elemental sulfur or cystine was required for EJ2T growth and was reduced to hydrogen sulfide. Rifampicin inhibited growth for both strains EJ1T and EJ2T. The G + C contents of the genomic DNA were 52.3 and 54.5 mol% for EJ1T and EJ2T, respectively. As determined by 16S rRNA gene sequence analysis, these strains were more closely related to Thermococcus gorgonarius, T. celer, T. guaymasensis, T. profundus, and T. hydrothermalis. However, no significant homology was observed between them with DNA-DNA hybridization. These novel organisms also possess phenotypic traits that differ from those of its closest phylogenetic relatives. Therefore, it is proposed that these isolates, which are amongst the most radioresistant hyperthermophilic archaea known to date with T. gammatolerans (Jolivet et al. 2003a), should be described as novel species T. marinus sp. nov. and T. radiotolerans sp. nov. The type strain of T. marinus is strain EJ1T (= DSM 15227T = JCM 11825T) and the type strain of T. radiotolerans is strain EJ2T (= DSM 15228T = JCM 11826T).

Thermococcus gammatolerans sp. nov., a hyperthermophilic archaeon from a deep-sea hydrothermal vent that resists ionizing radiation.

Enrichments for anaerobic organotrophic hyperthermophiles were performed with hydrothermal chimney samples collected at the Guaymas Basin (27 degrees 01' N, 111 degrees 24' W). Positive enrichments were submitted to gamma-irradiation at a dose of 30 kGy. One of the resistant strains, designated strain EJ3(T), formed regular motile cocci. The new strain grew between 55 and 95 degrees C, with an optimum growth temperature of 88 degrees C. The optimal pH for growth was 6.0, and the optimum NaCl concentration for growth was around 20 g l(-1). Strain EJ3(T) was an obligately anaerobic heterotroph that utilized yeast extract, tryptone and peptone. Elemental sulfur or cystine was required for growth and reduced to hydrogen sulfide. The G + C content of the genomic DNA was 51.3 mol%. As determined by 16S rRNA gene sequence analysis, the organism was most closely related to Thermococcus celer, Thermococcus guaymasensis, Thermococcus hydrothermalis, Thermococcus profundus and Thermococcus gorgonarius. However, no significant homology was observed between them by DNA-DNA hybridization. The novel organism also possessed phenotypic traits that differ from those of its closest phylogenetic relatives. Therefore, it is proposed that this isolate, which constitutes the most radioresistant hyperthermophilic archaeon known to date, should be described as the type strain of a novel species, Thermococcus gammatolerans sp. nov. The type strain is EJ3(T) (= DSM 15229(T) = JCM 11827(T)).