Biogeochemical transformation of mercury driven by microbes involved in anaerobic digestion of municipal wastewater.

Anaerobic digestion (AD) with municipal wastewater contained heavy metal mercury (Hg) highly affects the utilization of activated sludge, and poses severe threat to the health of human beings. However, the biogeochemical transformation of Hg during AD remains unclear. Here, we investigated the biogeochemical transformation and environmental characteristics of Hg and the variations of dominant microbes during AD. The results showed that Hg(II) methylation is dominant in the early stage of AD, while methylmercury (MeHg) demethylation dominates in the later stage. Dissolved total Hg (DTHg) in the effluent sludge decreased with time, while THg levels enhanced to varying degrees at the final stage. Sulfate significant inhibits MeHg formation, reduces bioavailability of Hg(II) by microbes and thus inhibits Hg(II) methylation. Microbial community analysis reveals that strains in Methanosarcina and Aminobacterium from the class of Methanomicrobia, rather than Deltaproteobacteria, may be directly related to Hg(II) methylation and MeHg demethylation. Overall, this research provide insights into the biogeochemical transformation of Hg in the anaerobic digestion of municipal wastewater treatment. This work is beneficial for scientific treatment of municipal wastewater and effluent sludge, thus reducing the risk of MeHg to human beings.

Bacteria and archaea involved in anaerobic mercury methylation and methane oxidation in anaerobic sulfate-rich reactors.

The identification of dominant microbes in anaerobic mercury (Hg) methylation, methylmercury (MeHg) demethylation, and methane oxidation as sulfate-reducing bacteria, methanogens or, probably, anaerobic methanotrophic archaea (ANMEs) is of great interest. To date, however, the interrelationship of bacteria and archaea involved in these processes remains unclear. Here, we demonstrated the dynamics of microorganisms participating in these processes. Anaerobic fixed-bed reactors were operated with swine manure and sludge to produce methane stably, and then, sulfate (reactor C), sulfate and Hg(II) (reactor H), and sulfate and MeHg (reactor M) were added, and the reactors were operated for 120 d, divided equally into four periods, P1-P4. The bacterial compositions changed nonsignificantly, whereas Methanosaeta in reactors H and M decreased significantly, revealing that it was irrelevant for Hg transformation. The abundances of Syntrophomonadaceae, Methanoculleus, Candidatus Methanogranum and Candidatus Methanoplasma increased continuously with time; these species probably functioned in these processes, but further evidence is needed. Desulfocella and Desulfobacterium dominated first but eventually almost vanished, while the dominant archaeal genera Methanogenium, Methanoculleus and Methanocorpusculum were closely related to ANME-1 and ANME-2. PLS-DA results indicated that both bacteria and archaea in different periods in the three reactors were clustered separately, implying that the microbial compositions in the same periods were similar and changed markedly with time.

Efficient expressions of reporter genes in the industrial filamentous fungus Sclerotium rolfsii mediated by Agrobacterium tumefaciens.

Sclerotium rolfsii (teleomorph Athelia rolfsii) is one of the plant pathogenic basidiomycetes, which causes severe stem-rot disease in hundreds of plants and produces important metabolites, such as scleroglucan and TF-specific lectin. However, further molecular biological research on this filamentous fungus is severely plateaued out due to the lack of genetic methods. In this study, the A. tumefaciens strain LBA4404 harboring a binary vector containing the basta resistance gene fused with three reporters (DsRed, tdTomato, and GUSPlus) respectively, driven by the SrGPD promoter, was used for genetic transformation of S. rolfsii. The results showed that the three reporter genes were all effectively expressed in S. rolfsii. This study also showed that the intron of the SrGPD promoter is not necessary for transgene expression in this fungus. Besides, we showed that these reporters' signals could be observed easily but in a short time window. The efficient Agrobacterium-mediated transformation system and the three reporter gene plasmids for S. rolfsii developed in this study are of significance in overcoming current limitations of no available transformation and genetic manipulation techniques in S. rolfsii, facilitating further genetic manipulations and gene function exploration.

Effects of high-level expression of A1-ATPase on H2 production in Thermococcus kodakarensis.

The hyperthermophilic archaeon Thermococcus kodakarensis can grow on pyruvate or maltooligosaccharides through H2 fermentation. H2 production levels of members of the Thermococcales are high, and studies to improve their production potential have been reported. Although H2 production is primary metabolism, here we aimed to partially uncouple cell growth and H2 production of T. kodakarensis. Additional A1-type ATPase genes were introduced into T. kodakarensis KU216 under the control of two promoters; the strong constitutive cell surface glycoprotein promoter, Pcsg, and the sugar-inducible fructose-1,6-bisphosphate aldolase promoter, Pfba. Whereas cells with the A1-type ATPase genes under the control of Pcsg displayed only trace levels of growth, cells with Pfba (strain KUA-PF) displayed growth sufficient for further analysis. Increased levels of A1-type ATPase protein were detected in KUA-PF cells grown on pyruvate or maltodextrin, when compared to the levels in the host strain KU216. The growth and H2 production levels of strain KUA-PF with pyruvate or maltodextrin as a carbon and electron source were analyzed and compared to those of the host strain KU216. Compared to a small decrease in total H2 production, significantly larger decreases in cell growth were observed, resulting in an increase in cell-specific H2 production. Quantification of the substrate also revealed that ATPase overexpression led to increased cell-specific pyruvate and maltodextrin consumptions. The results clearly indicate that ATPase production results in partial uncoupling of cell growth and H2 production in T. kodakarensis.

The metal transporter CrNRAMP1 is involved in zinc and cobalt transports in Chlamydomonas reinhardtii.

Metal homeostasis is essential cellular progress for cell growth. Metal ion transporters play important roles in the first line of defense to cellular metal homeostasis perturbations. NRAMP transporter family was one of the most important classes in plant cells. However, functions and substrate specificities of the NRAMP family remain unknown in Chlamydomonas reinhardtii, a model unicellular plant. In this study, we identified CrNRAMP1 as an important transporter involved in zinc and cobalt transport. Heterologous and homologous functional analyses of CrNRAMP1 showed that CrNRAMP1 plays important roles in zinc and cobalt homeostasis. The expression of CrNRAMP1 correlated with zinc or cobalt concentrations, but excluding cadmium. These results help to understand the functions and specificities of NRAMP family members in C. reinhardtii.

Crystal structure of pantoate kinase from Thermococcus kodakarensis.

The coenzyme A biosynthesis pathways in most archaea involve two unique enzymes, pantoate kinase and phosphopantothenate synthetase, to convert pantoate to 4'-phosphopantothenate. Here, we report the first crystal structure of pantoate kinase from the hyperthermophilic archaeon, Thermococcus kodakarensis and its complex with ATP and a magnesium ion. The electron density for the adenosine moiety of ATP was very weak, which most likely relates to its broad nucleotide specificity. Based on the structure of the active site that contains a glycerol molecule, the pantoate binding site and the roles of the highly conserved residues are suggested.

Pol B, a Family B DNA Polymerase, in Thermococcus kodakarensis is Important for DNA Repair, but not DNA Replication.

Thermococcus kodakarensis possesses two DNA polymerases, Pol B and Pol D. We generated a T. kodakarensis strain (DPB1) in which polB was completely deleted and a derivative of DPB1 in which polB was overexpressed; neither of the generated strains exhibited any growth delay, indicating that the lack or overexpression of Pol B in T. kodakarensis did not affect cell growth. We also found that DPB1 showed higher sensitivity to four DNA-damaging agents (ultraviolet C irradiation, γ-ray irradiation, methyl methanesulfonate, and mitomycin C) than the parental strain. The sensitivity of DPB1 was restored to the level of the parent strain by the introduction of a plasmid harboring polB, suggesting that the DNA damage-sensitive phenotype of DPB1 was due to the loss of polB. Collectively, these results indicate that Pol B is involved in DNA repair, but not DNA replication, which, in turn, implies that Pol D is the sole replicative DNA polymerase in Thermococcus species.

The C-terminal flexible region of branched-chain polyamine synthase facilitates substrate specificity and catalysis.

Branched-chain polyamine synthase (BpsA) catalyzes sequential aminopropyl transfer from the donor, decarboxylated S-adenosylmethionine (dcSAM), to the acceptor, linear-chain polyamine, resulting in the production of a quaternary-branched polyamine via tertiary branched polyamine intermediates. Here, we analyzed the catalytic properties and X-ray crystal structure of Tth-BpsA from Thermus thermophilus and compared them with those of Tk-BpsA from Thermococcus kodakarensis, which revealed differences in acceptor substrate specificity and C-terminal structure between these two enzymes. To investigate the role of the C-terminal flexible region in acceptor recognition, a region (QDEEATTY) in Tth-BpsA was replaced with that in Tk-BpsA (YDDEESSTT) to create chimeric Tth-BpsA C9, which showed a severe reduction in catalytic efficiency toward N4 -aminopropylnorspermidine, but not toward N4 -aminopropylspermidine, mimicking Tk-BpsA substrate specificity. Tth-BpsA C9 Tyr346 and Thr354 contributed to discrimination between tertiary branched-chain polyamine substrates, suggesting that the C-terminal region of BpsA recognizes acceptor substrates. Liquid chromatography-tandem mass spectrometry analysis on a Tk-BpsA reaction mixture with dcSAM revealed two aminopropyl groups bound to two of five aspartate/glutamate residues (Glu339 , Asp342 , Asp343 , Glu344 , and Glu345 ) in the C-terminal flexible region. Mutating each of these five amino acid residues to asparagine/glutamine resulted in a slight decrease in activity. The quadruple mutant D342N/D343N/E344Q/E345Q exhibited a severe reduction in catalytic efficiency, suggesting that these aspartate/glutamate residues function to receive aminopropyl chains. In addition, the X-ray crystal structure of the Tk-BpsA ternary complex bound to N4 -bis(aminopropyl)spermidine revealed that Asp126 and Glu259 interacted with the aminopropyl moiety in N4 -aminopropylspermidine.

Random mutagenesis of a hyperthermophilic archaeon identified tRNA modifications associated with cellular hyperthermotolerance.

Random mutagenesis for the hyperthermophilic archaeon Thermococcus kodakarensis was established by the insertion of an artificial transposon designed to allow easy identification of the transposon-inserted locus. The phenotypic screening was applied for the isolation of thermosensitive mutants of T. kodakarensis, which resulted in the isolation of 16 mutants showing defective growth at the supraoptimal temperature 93°C. The high occurrence of the mutants suggested that the high thermotolerance of hyperthermophiles was achieved by a combination of diverse gene functions. The transposon insertion sites in two-thirds of the mutants were identified in a group of genes responsible for tRNA modifications including 7-formamidino-7-deaza-guanosine (archaeosine), N1-methyladenosine/N1-methylinosine, N4-acetylcytidine, and N2-dimethylguanosine/N2,N2-dimethylguanosine. LC-MS/MS analyses of tRNA nucleosides and fragments exhibited disappearance of the corresponding modifications in the mutants. The melting temperature of total tRNA fraction isolated from the mutants lacking archaeosine or N1-methyladenosine/N1-methylinosine decreased significantly, suggesting that the thermosensitive phenotype of these mutants was attributed to low stability of the hypomodified tRNAs. Genes for metabolism, transporters, and hypothetical proteins were also identified in the thermosensitive mutants. The present results demonstrated the usefulness of random mutagenesis for the studies on the hyperthermophile, as well as crucial roles of tRNA modifications in cellular thermotolerance.

Pcal_0970: an extremely thermostable L-asparaginase from Pyrobaculum calidifontis with no detectable glutaminase activity.

The genome sequence of Pyrobaculum calidifontis contains two open reading frames, Pcal_0144 and Pcal_0970, exhibiting homology with L-asparaginases. In search of a thermostable L-asparaginase with no glutaminase activity, we have cloned and expressed the gene encoding Pcal_0970 in Escherichia coli. Recombinant Pcal_0970 was produced in insoluble and inactive form which was solubilized and refolded into enzymatically active form. The refolded Pcal_0970 showed the highest activity at or above 100 °C. Optimum pH for the enzyme activity was 6.5. Addition of divalent metal cations or EDTA had no significant effect on the activity. The enzyme was capable of hydrolyzing D-asparagine with a 20% activity as compared to 100% with L-asparagine. Pcal_0970 did not show any detectable activity when L-glutamine or D-glutamine was used as substrate. Pcal_0970 exhibited a Km value of 4.5 ± 0.4 mmol/L and Vmax of 355 ± 13 µmol min-1 mg-1 towards L-asparagine. The activation energy, from the linear Arrhenius plot, was determined as 39.9 ± 0.6 kJ mol-1. To the best of our knowledge, Pcal_0970 is the most thermostable L-asparaginase with a half-life of more than 150 min at 100 °C and this is the first report on characterization of an L-asparaginase from phylum Crenarchaeota.

Branched-Chain Polyamine Found in Hyperthermophiles Induces Unique Temperature-Dependent Structural Changes in Genome-Size DNA.

A pentavalent branched-chain polyamine, N4 -bis(aminopropyl)spermidine 3(3)(3)4, is a unique polycation found in the hyperthermophilic archaeon Thermococcus kodakarensis, which grows at temperatures between 60 and 100 °C. We studied the effects of this branched-chain polyamine on DNA structure at different temperatures up to 80 °C. Atomic force microscopic observation revealed that 3(3)(3)4 induces a mesh-like structure on a large DNA (166 kbp) at 24 °C. With an increase in temperature, DNA molecules tend to unwind, and multiple nano-loops with a diameter of 10-50 nm are generated along the DNA strand at 80 °C. These results were compared to those obtained with linear-chain polyamines, homocaldopentamine 3334 and spermidine, the former of which is a structural isomer of 3(3)(3)4. These specific effects are expected to neatly concern with its role on high-temperature preference in hyperthermophiles.

An ornithine ω-aminotransferase required for growth in the absence of exogenous proline in the archaeon Thermococcus kodakarensis.

Aminotransferases are pyridoxal 5'-phosphate-dependent enzymes that catalyze reversible transamination reactions between amino acids and α-keto acids, and are important for the cellular metabolism of nitrogen. Many bacterial and eukaryotic ω-aminotransferases that use l-ornithine (Orn), l-lysine (Lys), or γ-aminobutyrate (GABA) have been identified and characterized, but the corresponding enzymes from archaea are unknown. Here, we examined the activity and function of TK2101, a gene annotated as a GABA aminotransferase, from the hyperthermophilic archaeon Thermococcus kodakarensis We overexpressed the TK2101 gene in T. kodakarensis and purified and characterized the recombinant protein and found that it displays only low levels of GABA aminotransferase activity. Instead, we observed a relatively high ω-aminotransferase activity with l-Orn and l-Lys as amino donors. The most preferred amino acceptor was 2-oxoglutarate. To examine the physiological role of TK2101, we created a TK2101 gene-disruption strain (ΔTK2101), which was auxotrophic for proline. Growth comparison with the parent strain KU216 and the biochemical characteristics of the protein strongly suggested that TK2101 encodes an Orn aminotransferase involved in the biosynthesis of l-Pro. Phylogenetic comparisons of the TK2101 sequence with related sequences retrieved from the databases revealed the presence of several distinct protein groups, some of which having no experimentally studied member. We conclude that TK2101 is part of a novel group of Orn aminotransferases that are widely distributed at least in the genus Thermococcus, but perhaps also throughout the Archaea.

Pcal_0632, a phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Pyrobaculum calidifontis.

Genome sequence of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0632, annotated as glyceraldehyde-3-phosphate dehydrogenase, which is partially overlapped with phosphoglycerate kinase. In the phylogenetic tree, Pcal_0632 clustered with phosphorylating glyceraldehyde-3-phosphate dehydrogenases characterized from hyperthermophilic archaea and exhibited highest identity of 54% with glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus tokodaii. To examine biochemical function of the protein, Pcal_0632 gene was expressed in Escherichia coli and the gene product was purified. The recombinant enzyme catalyzed the conversion of glyceraldehyde 3-phosphate and inorganic phosphate into 1,3-bisphosphoglycerate utilizing both NAD and NADP as cofactor with a marked preference for NADP. The enzyme was highly stable against temperature and denaturants. Half-life of the enzyme was 60 min at 100 °C. It retained more than 60% of its activity even after an incubation of 72 h at room temperature in the presence of 6 M urea. High thermostability and resistance against denaturants make Pcal_0632 a novel glyceraldehyde-3-phosphate dehydrogenase.

Identification of the glucosamine kinase in the chitinolytic pathway of Thermococcus kodakarensis.

Although the chitinolytic pathway of the hyperthermophilic archaeon Thermococcus kodakarensis is well-studied, the genome does not contain genes homologous to previously identified glucosamine kinase genes. As some ADP-dependent glucokinases in the order Thermococcales exhibit phosphorylation activities for both glucose and glucosamine in vitro, the homolog in T. kodakarensis, encoded by TK1110, was selected as a candidate for the missing glucosamine kinase gene. The purified, recombinant TK1110 enzyme exhibited phosphorylation activities for not only glucose but also glucosamine and N-acetylglucosamine. Kinetic analysis indicated that activity towards glucosamine was as significant as that towards glucose. In order to determine the physiological role of TK1110 in the chitinolytic pathway of T. kodakarensis, a gene disruption strain of TK1110 was constructed. When grown in chitin-containing medium, the TK1110 disruption resulted in almost complete impairment in chitin degradation, and a complete loss of chitin-dependent H2 production. As H2 production is tightly linked to cell growth in T. kodakarensis, the present results strongly suggest that TK1110 functions as the glucosamine kinase responsible for the chitin degradation in T. kodakarensis.

Pcal_1311, an alcohol dehydrogenase homologue from Pyrobaculum calidifontis, displays NADH-dependent high aldehyde reductase activity.

Genome sequence of Pyrobaculum calidifontis, a hyperthermophilic archaeon, harbors three open-reading frames annotated as alcohol dehydrogenases. One of them, Pcal_1311, does not display a significantly high homology with any of the characterized alcohol dehydrogenases. Highest homology of 38% was found with the characterized counterpart from Geobacillus stearothermophilus. To examine the biochemical properties of Pcal_1311, we have cloned and functionally expressed the gene in Escherichia coli. Purified recombinant Pcal_1311 catalyzed the NAD(H)-dependent oxidation of various alcohols and reduction of aldehydes, with a marked preference for substrates with functional group at the terminal carbon. Highest activity for the oxidation reaction (3 µmol min-1 mg-1) was found with 1,4-butanediol and for the reduction reaction (150 µmol min-1 mg-1) with glutaraldehyde. Both the oxidation and reduction activities increased with the increase in temperature up to 80 °C. Recombinant Pcal_1311 was highly stable and retained more than 90% activity even after incubation of 180 min at 90 °C. In addition to the thermostabilty, Pcal_1311 was highly stable in the presence of known denaturants including urea and guanidine hydrochloride. The high stability, particularly thermostability, and the NADH-dependent aldehyde reduction activity make Pcal_1311 a unique member in the alcohol dehydrogenase family.

Active site geometry of a novel aminopropyltransferase for biosynthesis of hyperthermophile-specific branched-chain polyamine.

Branched-chain polyamines are found exclusively in thermophilic bacteria and Euryarchaeota and play essential roles in survival at high temperatures. In the present study, kinetic analyses of a branched-chain polyamine synthase from the hyperthermophilic archaeon Thermococcus kodakarensis (Tk-BpsA) were conducted, showing that N4 -bis(aminopropyl)spermidine was produced by sequential additions of decarboxylated S-adenosylmethionine (dcSAM) aminopropyl groups to spermidine, through bifunctional catalytic action. Tk-BpsA catalyzed the aminopropylation of the linear-chain polyamines spermidine, spermine, norspermidine, and the tertiary-branched polyamines N4 -aminopropylspermidine and N4 -aminopropylnorspermidine, but not of short-chain diamines, putrescine, and cadaverine, suggesting that Tk-BpsA does not catalyze the aminopropylation of primary amino groups of diamines. X-ray structural analyses of Tk-BpsA in the presence or absence of the substrates spermidine and dcSAM revealed that a large, negatively charged cavity is responsible for the binding of branched-chain substrates. The binding is different from that in the active site of linear polyamine spermidine/spermine synthases, and loop-closures occur upon the binding of spermidine. Based on structural analyses, further kinetic studies were carried out for various mutants, revealing that Asp159, positioned between the reactive secondary amino group of the substrate polyamine and a sulfur atom of the product 5'-methylthioadenosine and in a Gly-Asp-Asp-Asp motif, functions as a catalytic center, with reactions proceeding via a ping-pong mechanism. Our study provides a novel aminopropyltransfer reaction mechanism, distinct from the SN 2 displacement mechanism found in other known linear spermidine/spermine synthases. DATABASE: Atomic coordinates and structure factors have been deposited in the Protein Data Bank with PDB codes 5XNF for apo-Tk-BpsA, 5XNH for the binary complex, and 5XNC for the ternary complex.

Escherichia coli Signal Peptidase Recognizes and Cleaves Archaeal Signal Sequence.

Tk1884, an open reading frame encoding α-amylase in Thermococcus kodakarensis, was cloned with the native signal sequence and expressed in Escherichia coli. Heterologous gene expression resulted in secretion of the recombinant protein to the extracellular culture medium. Extracellular α-amylase activity gradually increased after induction. Tk1884 was purified from the extracellular medium, and its molecular mass determined by electrospray ionization mass spectrometry indicated the cleavage of a few amino acids. The N-terminal amino acid sequence of the purified Tk1884 was determined, which revealed that the signal peptide was cleaved between Ala26 and Ala27 by E. coli signal peptidase. To the best of our knowledge, this is the first report describing an archaeal signal sequence recognized and cleaved by E. coli signal peptidase.

Anthranilate phosphoribosyltransferase from the hyperthermophilic archaeon Thermococcus kodakarensis shows maximum activity with zinc and forms a unique dimeric structure.

Anthranilate phosphoribosyltransferase (TrpD) is involved in tryptophan biosynthesis, catalyzing the transfer of a phosphoribosyl group to anthranilate, leading to the generation of phosphoribosyl anthranilate. TrpD belongs to the phosphoribosyltransferase (PRT) superfamily and is the only member of the structural class IV. X-ray structures of TrpD from seven species have been solved to date. Here, functional and structural characterization of a recombinant TrpD from hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkTrpD) was carried out. Contrary to previously characterized Mg2+-dependent TrpD enzymes, TkTrpD was found to have a unique divalent cation dependency characterized by maximum activity in the presence of Zn2+ (1580 µmol·min-1·mg-1, the highest reported for any TrpD) followed by Ca2+ (948 µmol·min-1·mg-1) and Mg2+ (711 µmol·min-1·mg-1). TkTrpD displayed an unusually low thermostability compared to other previously characterized proteins from T. kodakarensis KOD1. The crystal structure of TkTrpD was determined in free form and in the presence of Zn2+ to 1.9 and 2.4 Å resolutions, respectively. TkTrpD structure displayed the typical PRT fold similar to other class IV PRTs, with a small N-terminal α-helical domain and a larger C-terminal α/ß domain. Electron densities for Zn2+ were identified at the expected zinc-binding motif, DE(217-218), of the enzyme in each subunit of the dimer. Two additional Zn2+ were found at a new dimer interface formed in the presence of Zn2+. A fifth Zn2+ was found bound to Glu118 at crystal lattice contacts and a sixth one was ligated with Glu235. Based on the TkTrpD-Zn2+ structure, it is suggested that the formation of a new dimer may be responsible for the higher enzyme activity of TkTrpD in the presence of Zn2+ ions.

A single-molecule assessment of the protective effect of DMSO against DNA double-strand breaks induced by photo-and γ-ray-irradiation, and freezing.

Dimethyl sulfoxide (DMSO) is widely used as a cryoprotectant for organs, tissues, and cell suspension in storage. In addition, DMSO is known to be a useful free radical scavenger and a radio-protectant. To date, many in vitro assays using cultured cells have been performed for analysing the protective effect of DMSO against genomic DNA damage; however, currently it has been rather difficult to detect DNA double strand breaks (DSBs) in a quantitative manner. In the present study, we aimed to observe the extent of DNA damage by use of single molecular observation with a fluorescence microscope to evaluate DSBs induced by photo- and γ-ray-irradiation, or freeze/thawing in variable concentrations of DMSO. As a result, we found that 2% DMSO conferred the maximum protective effect against all of the injury sources tested, and these effects were maintained at higher concentrations. Further, DMSO showed a significantly higher protective effect against freezing-induced damage than against photo- and γ-ray-irradiation-induced damage. Our study provides significant data for the optimization of DNA cryopreservation with DMSO, as well as for the usage of DNA as the protective agent against the injuries caused by active oxygen and radiations.

Gene regulation of two ferredoxin:NADP+ oxidoreductases by the redox-responsive regulator SurR in Thermococcus kodakarensis.

The redox-responsive regulator SurR in the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus kodakarensis binds to the SurR-binding consensus sequence (SBS) by responding to the presence of elemental sulfur. Here we constructed a surR gene disruption strain (DTS) in T. kodakarensis, and identified the genes that were under SurR control by comparing the transcriptomes of DTS and parent strains. Among these genes, transcript levels of ferredoxin:NADP+ oxidoreductases 1 and 2 (FNOR1 and FNOR2) genes displayed opposite responses to surR deletion, indicating that SurR repressed FNOR1 transcription while enhancing FNOR2 transcription. Each promoter region contains an SBS upstream (uSBS) and downstream (dSBS) of TATA. In addition to in vitro binding assays, we examined the roles of each SBS in vivo. In FNOR1, mutations in either one of the SBSs resulted in a complete loss of repression, indicating that the presence of both SBSs was essential for repression. In FNOR2, uSBS indeed functioned to enhance gene expression, whereas dSBS functioned in gene repression. SurR bound to uSBS2 of FNOR2 more efficiently than to dSBS2 in vitro, which may explain why SurR overall enhances FNOR2 transcription. Further analyses indicated the importance in the distance between uSBS and TATA for transcriptional activation in FNOR2.