In structure, archaeal RNA polymerases (RNAPs) and their promoters are most similar to their eukaryotic RNA polymerase II (Pol II) counterparts. However, as described, archaeal transcription initiation is most often regulated by repression and so resembles bacterial regulation, whereas archaeal transcription is terminated by oligo-T sequences reminiscent of intrinsic termination established for eukaryotic Pol III.

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In highly processive genomic DNA duplication, ring-shaped sliding clamps and clamp loader ATPases are essential factors. Proliferating cell nuclear antigen (PCNA) and replication factor C (RFC) play crucial roles as the clamp loader and the clamp, respectively. Pyrococcus furiosus RFC and PCNA were used to reconstruct a clamp loading complex with a synthetic template-primer DNA, and the electron microscopic structure of a P. furiosus clamp loading complex at 12 Å resolution was obtained. This complex exhibits excellent fitting of each atomic structure of RFC, PCNA, and the primed DNA. The complex appears to represent the intermediate, in which the PCNA ring is kept open with a distorted spring washer-like conformation. A model of the clamp loading mechanism is proposed, based on the 3D structure of the complex, along with information from biochemical analyses.

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A new gene from the hyperthermophilic archaeon Sulfolobus solfataricus MT4, reported to show sequence identity with members of the amidohydrolase superfamily, was cloned by means of the polymerase chain reaction from the S. solfataricus genomic DNA. In order to analyse the biochemical properties of this protein an overexpression system in Escherichia coli was established. The recombinant protein was purified to homogeneity and characterized. The S. solfataricus enzyme was demonstrated to have a low paraoxonase activity and also an even lower but detectable esterase activity. The paraoxonase activity was dependent from metal cations present in the E. coli culture with Cd²⁺>Ni²⁺=Co²⁺>Zn²⁺ and was thermophilic and thermostable. A 3D model of SsoPox was constructed on the basis of the Pseudomonas putida phosphotriesterase (PTE) structure and residues likely to be involved in substrate specificity and catalysis were identified.

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The thermostable enzymes pyroglutamyl carboxyl peptidase, L-aminoacylase and alcohol dehydrogenase have been cloned and over-expressed in Escherichia coli. The enzymes have potential for their use in industrial biocatalysis. Stuctural studies have aided our understanding of the mechanisms used for thermostability by these proteins. Site-specific mutations of the pyroglutamyl carboxyl peptidase where a cysteine involved in inter-subunit disulfide bond formation and a phenylalanine, leucine rich hydrophobic core have been removed or substituted, have provided some insight for the future engineering of more robust novel proteins for industrial bio-transformations.

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An amylolytic enzyme, designated as PFTA (Pyrococcus furiosus thermostable amylolytic enzyme), was cloned from Pyrococcus furiosus and expressed in Escherichia coli. The recombinant PFTA was extremely thermostable, with an optimal temperature of 90°C. PFTA hydrolyzed maltooligosaccharides and starch to mainly produce maltotriose and maltotetraose. Also, the enzyme was capable of degrading pullulan and β-cyclodextrin, which are generally resistant to the attack of α-amylase. The major products from these substrates (pullulan and β-cyclodextrin) were mostly panose and maltoheptaose, respectively. These results verified that PFTA possesses novel catalytic characteristics of both α-amylase and cyclodextrin-hydrolyzing enzymes. Interestingly, this enzyme showed a unique hydrolytic activity of opening cyclodextrin rings to create maltooligosaccharides without showing significant degrading activity toward the resulting maltooligosaccharides. By applying these interesting characteristics of PFTA, the production of valuable maltooligosaccharides, such as maltohexaose, maltoheptaose and maltooctaose, with high degree of purity could be possible at industrial scale.

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We studied cold-adaptation mechanisms of a psychrotroph, Shewanella livingstonensis Ac10, isolated from Antarctic seawater. We found that eicosapentaenoic acid (EPA) occurring in phospholipids is important for the growth at low temperatures by disrupting the genes essential for EPA synthesis. On the other hand, we carried out proteomic analysis of the soluble and membrane proteins of this bacterium grown at 4°C and 18°C and found that the amounts of 51 proteins were increased at 4°C (> 2-fold). We identified 28 of them by peptide mass fingerprinting. Disruption of the gene coding for one of these proteins, a homolog of OmpC, caused a growth defect at low temperatures, suggesting its role in cold-adaptation. Since S. livingstonensis Ac10 grows well at low temperatures, it is expected to be useful as the host for the production of thermolabile heterologous proteins. We constructed expression vectors for this psychrotroph and demonstrated their usefulness.

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Neopullulanase catalyzes the hydrolysis of α-1,4- and α-1,6-glucosidic linkages, as well as transglycosylation to form α-1,4- and α-1,6-glucosidic linkages. Based on the series of experimental results using the neopullulanase, we pointed out the same catalytic machinery and the common catalytic mechanism of the enzymes that catalyze these four reactions, and thus, proposed and defined the concept of the α-amylase family. Mutational and structural analyses provided the conclusive proof that one active center of neopullulanase participate in all four reactions. We have been trying to interconvert glucanohydrolases/glucanotransferases, and their specificities and create tailor-made industrially useful enzymes based on the concept of the α-amylase family. We engineered Thermus amylomaltase to essentially erase hydrolytic activity and created perfect 4-α-glucanotransferase for the industrial production of cycloamylose.

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We have found many novel and useful enzyme-producing microorganisms from the deep-sea and deep-subsurface. Alkaline mannnanase is useful for detergents and bleaching of kraft pulp. A novel species of alkaliphilic Bacillus isolates JAMB-750 produced alkaline mannanase extracellularly. The optimal pH of the enzyme is the highest among mannanases reported to date. Agaro- or neoagaro-oligosaccharides are known to show various physiological activities. We isolated many β-agarases, which were produced by deep-sea microorganisms. A β-agarase from an Agarivorans sp. produced neoagarobiose as the final product from agar. Neoagarobiose is useful for cosmetic additive. An alkaline and thermostable α-glucosidase was found in a Geobacillus sp. that was isolated from the sediment of the Mariana Trench. The enzyme possessed a strong transglycosylation activity toward non-sugar molecules with maltose as a sugar donor. A highly alkaline protease was found in culture broth of an extremely anaerobic alkaliphile isolated from a mine water containment dam at 3,200 m below land surface. The enzyme showed the maximal activity greater than pH 12.6 at 40°C. According to the deduced amino acid sequence of the mature enzyme, it forms a new cluster in a phyrogenetic tree of subtilase family A.

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Cold-adapted enzymes produced by microorganisms living in permanently cold habitats are characterized by a high specific activity at low and moderate temperatures. Typically they display a high thermosensitivity related to a high flexibility of a part or of the whole molecular edifice. Indeed, this flexibility is apparently essential in counteracting the freezing effect of the low temperature environment on the three dimensional structure, thereby securing an appropriate plasticity as required for catalysis. One of the main strategies selected by extracellular enzymes interacting with large size substrates (α-amylase, proteases etc..) consists of increasing the overall flexibility of the molecular structure by weakening of numerous intra-molecular interactions in all domains of the molecule. However, in the case of enzymes which interact with small size substrates such as for example, phosphoglycerate kinase and chitobiase, only one part of the protein, presumably that related directly or indirectly to the catalytic site, appears to be flexible, whereas the other parts are even more rigid than their mesophilic counterparts, as demonstrated by microcalorimetry and quenching of intrinsic fluorescence using acrylamide. In the case of a cold-adapted cellulase, isolated from an Antarctic bacterium, recent investigations have demonstrated that the flexibility, required for a good accommodation of the large size substrate at low temperatures, is mainly provided by an unusually long and flexible linker connecting the catalytic and cellulose binding modules. This has been demonstrated by x-ray crystallography and small angle x-ray scattering coupled to protein engineering experiments leading to a reduction of the length and to an increased rigidity of the linker. Cold-adapted enzymes such as β-galactosidase and xylanase have been found to be very efficient in milk and baking industry respectively.

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Xylanases are generally classified into glycoside hydrolase families 10 and 11 yet we have isolated eight novel cold-adapted xylanases from Antarctic and Arctic bacteria that belong to glycoside hydrolase family 8. One of these enzymes, pXyl from an Antarctic Pseudoalteromonas sp., was further studied and is found to be a very unique enzyme, displaying an (α/α)₆ barrel fold and catalysing hydrolysis with inversion of the anomeric configuration. Indeed this is the first xylanase shown to display such a structure and mechanism of action and highlights the importance of extremophilic environments as sources of novel enzymes. This enzyme is specific for xylan, is most active on long-chain xylo-oligosaccharides and in contrast to most other xylanases studied to date it is not active on aryl-β-glycosides of xylobiose or xylotriose. It displays typical characteristics for a cold-adapted enzyme: high activity at low temperatures, a reduced thermal and chemical stability and an increased flexibility as compared to a thermophilic homologous enzyme. Structural analysis indicates the basis for this adaptation as being a reduced number of salt bridges and an increased exposure of hydrophobic residues. Finally, baking trials were used to clearly demonstrate the effectiveness of all eight of the isolated xylanases as technological aids in the baking industry. Indeed, the psychrophilic characteristics (high activity at low to moderate temperatures and low stability) of this enzyme, and of psychrophilic enzymes in general, should be of much benefit in those biotechnological processes carried out at low to moderate temperatures and in particular in the food industry.

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Clostridium thermocellum produces a large cellulase complex consisting of an extracellular, multi-protein aggregate termed the cellulosome and many free biomass-degrading enzymes. It is not well understood how the bacterium coordinates the expression of a large number of genes encoding its complex cellulase system. We submitted the C. thermocellum genomic DNA to the U.S. Department of Energy (US DOE) Joint Genome Institute (JGI) for sequencing and a draft sequence is now available. We searched the sequence for clustered cellulase genes that may signal the existence of potential cellulase operons. A new cellulase gene cluster consisting of five previously unidentified genes was discovered. To gain insight on the regulatory mechanism, we also searched the genome sequence for putative transcription regulatory elements. BLAST search revealed three genes highly homologous to the lacI gene of Escherichia coli, which codes for the repressor protein of the Lac operon.

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Several gram-negative bacterial species use N-acyl homoserine lactone (AHL) molecules as quorum sensing (QS) signals to regulate various biological functions. Similarly various bacteria can stimulate, inhibit or inactivate quorum sensing signals in other bacteria by producing molecules called quorum sensing inhibitors (QSI). In the present study, QSI screenings were conducted on several halophilic and halotolerant bacteria isolated from marine environments and saltpans, using Serratia rubidaea N-1 as an indicator organism. S. rubidaea controls the production of red pigment, prodigiosin by acyl homoserine lactone quorum sensing. We have found that S. rubidaea N-1 produces high amount of pigment when growth medium contains 0.5 to 1 M NaCl and hence we decided this strain as a suitable indicator for finding QSI. Among several bacteria which were isolated from salt pans of South India, 5 strains showed sufficient growth in the wide concentrations of NaCl, 0.5 to 3 M. Results showed that some marine bacteria have the ability to produce QSI which was observed by the lack of pigmentation of S. rubidaea N-1 in the vicinity of the test sample. This simple screening strategy using the pigment inhibition of S. rubidaea N-1 strain allows the rapid and sensitive detection of potential QS blockers from large number of bacteria from marine environments.

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Group II chaperonins and prefoldins take important roles in protein folding and maintenance in hyperthermophilic archaea since only four types of molecular chaperones are encoded on their genomes. In this paper, we will describe recent results of our studies on protein folding cycles of a group II chaperonin and cooperation mechanism between a prefoldin and a group II chaperonin from hyperthermophilic archaea.

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Thermophilic bacteria belonging to Bacillus genetic group 5 have been reclassified as being members of Geobacillus gen. nov., with G. stearothermophilus as the type strain. Geobacillus species, literally meaning earth, or soil, bacillus, are widely distributed and readily isolated from natural and man-made thermophilic biotopes. At present there is an abundance of genomic information available for this genera with sequencing projects of the isolates G. stearothermophilus, and G. kaustophilus HTA426.Within this paper we wish to present the pertinent features of the proteome of this microorganism and thus hope to widen our knowledge and understanding of how these microorganisms are able to adapt and survive in extreme environments.

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Thermococcus kodakaraensis KOD1 possesses two histone molecules, HpkA and HpkB. In vitro study revealed that HpkB possessed higher affinity to DNA and more extensive ability to compact DNA than HpkA. It seems likely that DNA compaction is achieved by tetramerization of dimeric units. Based on structural modeling, tetramer formation by dimer-dimer interaction is considered to require two intermolecular ion pairs formed between histidine and aspartate. In order to examine the role of the ion pairs on DNA compaction, mutant histones were constructed and analyzed using HpkB as a model protein. The mutant histones, HpkB-H50A, HpkB-H50V, and HpkB-H50G were constructed by replacing conserved surface His50 with Ala, Val, and Gly, respectively. Gel mobility shift assays showed that all mutants possessed DNA binding ability, like wild-type HpkB, however all mutants compacted DNA less efficiently than the wild-type. Moreover, all mutants could not maintain the compacted structure of DNA above 80 °C. These results suggest that surface ion pairs between His and Asp play an important role in maintenance of nucleosome structure and DNA stabilization at high temperature.

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Here, we describe our research on the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. Recently, we have determined the complete genome sequence of the archaeon. The genome (2,088,737-base) contained a total of 2,306 coding DNA sequences (CDSs), among which half (1,165 CDSs) are annotatable whereas the functions of 41% (936 CDSs) cannot be predicted from the primary structures. To analyze the physiological function of each gene, we have developed a system for specifically disrupting (or replacing) genes in the genome, which is the first system in the hyperthermophiles. The availability of both genome information and gene exchange technique make the archaeon an attractive organism for analyzing and manipulating its metabolic pathways. Therefore, we have applied these features to improve the hydrogen(H₂)-producing potential of the archaeon.

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An optimization of microbially-facilitated metal ore leaching could benefit from understanding the genetics and biochemistry of the iron- and sulfur- oxidative metabolism of key organisms. We used Sulfobacillus acidophilus as a model for gram-positive biomining organisms and applied a subtractive hybridization of PCR-amplified cDNA to identify genes upregulated in cells grown heterotrophically under ferrous iron- or sulfur-oxidizing conditions. Among the obtained cDNA fragments, those coding for genes involved in redox processes were sequenced. Two different trimeric DMSO-reductase family operons were shown to be upregulated in iron- and sulfur-grown cells, respectively. The catalytic subunits of both operons showed close homologies to DMSO-reductases involved in the reductive cleavage of sulfur-sulfur bonds.

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The mechanism of intein-mediated protein splicing was first described using an intein from a Pyrococcus species. Inteins are sporadically dispersed amongst the various phyla. Inteins are often encoded by mobile genetic elements that allow them to be horizontally transferred. Inteins naturally lacking the standard amino-terminal nucleophile can still splice by a slightly different mechanism. Understanding the mechanism of protein splicing has allowed scientists to harness inteins for numerous bioorganic chemistry and biotechnology application

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We have recently developed the way of designing substitutions that can improve thermostability of proteins. The method uses phylogenetic analysis of sequences of family enzymes. It is possible to infer ancestral residues possessed by Commonote, the common ancestor of all the living organisms. The ancestral residues are then introduced to the sequence of contemporary enzyme. The mutant enzymes were analyzed and compared with original enzymes. The results suggested that the ancestral residues tend to improve tharmostablity of proteins. In this report we discuss the results reported on the 3-isopropylmalate dehydrogenase and isocitrate dehydrogenase. The results suggest that the method is applicable to wider variety of proteins.

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Escherichia coli, a mesophilic bacterium, acquired viability above its permissive temperature with the aid of broad-host range gene transfer particle (VP) originating from Aquifex sp and Thermococcus kodakaraensis B41. Gene transfer capable VPs have likely mediated the transfer of a functional thermo-resistance gene. VPs are already known to transfer enzyme genes to rescue auxotrophic recipients. VP particles, as part of a group of ubiquitously distributing virus-like particles (VLPs), are likely to take part in gene flux that enables horizontal gene transfer that enhances biodiversity and evolution.

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Proteins, from deep-sea adapted microorganisms ‘piezophiles’, could be active under high-pressure conditions in general. Actually normal proteins can be inactive under higher-pressure conditions, ca, 500 MPa and more. For bio-processing under pressure, people are looking for pressure-tolerant enzymes, thus, ‘piezophilic proteins’ would be focus on such industrial applications. We have done the comparative studies of the same functional proteins and/or enzymes between from Escherichia coli and deep-sea piezophiles. In the case of cell divisional protein FtsZ, RNA polymerase subunit structure, and dihydrofolate reductase (DHFR), piezophilic proteins were much more stable and/or active under higher pressure conditions than E. coli's proteins.

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The Mrp family of monovalent cation/proton antiporters is widespread among prokaryotes, and its members possess characteristics that make this class of antiporters unique. These include a requirement for 6-7 genes for full function, in comparison to all previously characterized bacterial monovalent cation/proton antiporters which are single gene products. Characterization of the unusual properties of Mrp-dependent antiport will lead to an understanding of how this system is able to support growth at extremely high pH. We are completing a comparative analysis of the activity profiles of the alkaliphile Mrp system and that of B. subtilis and other non-extremophiles. The results suggest significant commonalities among several members of this conserved family of antiporters.

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A DNA methyltransferase, M.PabI, of a hyperthermophilic archaeon Pyrococcus abyssi was purified and characterized. Some restriction-modification genes behave as “selfish” mobile genetic elements, just as transposons, and they are often found associated with genomic rearrangements or polymorphisms. This nature helped us to identify a putative restriction-modification gene pair on P. abyssi genome through its comparative analysis with that of another Pyrococcal species. Its constituent restriction enzyme was identified by our previous work as a hyper-thermoresistant DNA endonuclease, PabI, which catalyzes cleavage at 5'-GTA/C-3'. In this study, M.PabI, the DNA methyltransferase of PabI restriction-modification system, was over-expressed in Escherichia coli and purified. We describe detailed enzymatic characterization of this enzyme, which includes measurements of thermodynamic and kinetic parameters. It generates 5'-GTm6AC-3' and is inhibited by Zn²⁺. It turned out to be the most thermophilic of all the reported DNA methyltransferases.

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Why and how the eukaryotic chromatin structure has evolved remains one of the intriguing questions in understanding life. It is absent from the eubacteria but is present in archaea in a variety of forms. A restriction enzyme (PabI) from archaeon Pyrococcus turned out to have a novel three-dimensional structure. Its homologue is found in eubacterium Helicobacter genomes, which carry extremely few PabI sites, likely through selection through past attacks by the PabI homologue. We hypothesize that Pyrococcus genomes are more resistant to this restriction enzyme family than Helicobacter. From this and several other lines of evidence, we further hypothesize that the chromatin structure may have evolved in the archaea to defend genome against attack by restriction enzymes. This hypothesis can explain the absence of restriction enzymes from the eukaryotes and is in harmony with the roles of DNA double-strand breakage in their programmed cell death and meiotic recombination.

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A unique CGTase producing microorganism was successfully isolated from soil in Japan and identified as Bacillus clarkii, a kind of alkalophilic bacterium. The CGTase from this bacterium was purified by affinity chromatography on a γ-CD-immobilized column. The purified enzyme converted pre-gelatinized potato starch into CDs and the majority of the CDs product was γ-form. This property is quite unique and thus, we named this enzyme as γ-CGTase. The entire gene sequence amplified by PCR was determined and cloned into Escherichia coli. The comparison of the deduced amino acid sequence of γ-CGTase with those of other known CGTases that show different product specificities revealed the importance of subsites -3 and -7 for its preferential γ-cyclization activity.

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Here we describe recent progress in our genetic system with the hyperthermophilic archaeon, Thermococcus kodakaraensis. We have developed a system that allows us to disrupt genes in a specific manner, which is the first of its kind in hyperthermophiles. This has been utilized to examine the functions of specific enzymes/genes in vivo. An example is described that identifies the physiologically relevant fructose 1,6-bisphosphatase. The gene disruption system has recently been improved, and it is now possible to isolate strains with multiple gene disruptions. As gene insertion can also be performed with this system, the technology described here provides new alternatives in future examination and application of hyperthermophiles.

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In order to explore the effect of high pressure on the cultivation of microorganisms from a petroleum reservoir, systems for sampling and high-pressure high-temperature (HPHT) cultivation were developed. The procedures for sampling from oil wells and the transfer of samples to HPHT cultivation cells was designed to avoid loosing any constituents in the original sample and exposing microorganisms present in the sample to rapid pressure changes. The concept was tested using samples from different oil wells from a North Sea oil reservoir. High pressure was found to have a positive effect on the growth of microorganisms found in the oil reservoir. HPHT cultures were found to contain bacteria of the genera Microbacterium, Micrococcus, Bacillus and Paenibacillus and archaea of the genus Thermococcus.

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The goal of this investigation was to isolate, characterize and identify novel alkaliphiles from the soda lakes in Tanzania, Africa. The most phylogenetically unique alkaliphilic bacterium belonging to the genus Bacillus was further examined for its production of extracellular proteases. The biochemical properties of the natural and recombinant protease enzymes are compared in this study and its potential for large scale industrial use are reported. Another protease from a novel hyperthermophile belonging to the Thermococcus genus was expressed and studied.

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The living stromatolites of Shark Bay, Western Australia, represent analogues of one of the oldest life on Earth located in a hyersaline environment. This is an ideal biological system for studying survival strategies of complex microbial communities in an environmental condition suggested to be similar to that of early Earth. The aim of this study was to study osmoprotective mechanisms of cyanobacteria and archaea isolated previously [1] from the stromatolite. The presence of several osmolytes/compatible solutes in the microbial communities were detected by nuclear magnetic resonance spectroscopy (NMR). DNA molecular methods such as Panhandle PCR was also employed to identify candidate genes for the uptake of osmoprotectants. We identified a glycine betaine uptake system, OpuD gene homologues, as well as several genes encoding regulated salinity proteins in the halophilic archaea. This study provides an excellent platform for the identification of potentially novel osmolytes/compatible solutes that might have been produced by these microorganisms. Exploitation of the osmolyte accumulation mechanisms discovered could also lead to the genetic enhancement of drought and salinity tolerance in crop plants.

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One metagenomic cosmid library with an average inserted DNA about 35kb was constructed using the deep-sea sediment sample collected from 5274m Pacific nodule province ES0303 core. The biodiversity survey result showed that majority (60%) of the microbes detected in the library belonged to undetermined microbes and contain abundant unknown novel microbes. Besides the unknown microbes, Gemmatimonadetes, Chloroflexi, Proteobacteria and Planctomycetes were also enclosed. Endocellular and extrocellular crude extract of the cosmid clones were prepared for screening the antibiotics together with anticancer bioactive compounds. Lots of clones were proved to have the abilities of producing bioactive compounds.

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Alkaline pH homeostasis at pH ≥ 10 has been most extensively studied in extremely alkaliphilic Bacillus pseudofirmus OF4. Comparisons between B. pseudofirmus OF4 and neutrophile Bacillus subtilis show that the capacity for alkaline pH homeostasis is more robust in the alkaliphile and that it sets the upper pH limit for alkaliphile growth. Active pH homeostasis mechanisms are essential, including a Na⁺ cycle in which cytoplasmic accumulation of H⁺ relative to the outside is mediated by Na⁺/H⁺ antiporters. Additional transporters have a role in H⁺ capture and/or retention, e.g. a specially adapted H⁺-ATP synthase and an ammonium transporter of alkaliphiles. Several alkaliphile adaptations in support of pH homeostasis disadvantage their growth at pH ≤ 7.5, i.e. alkaliphiles are “hard-wired” for alkaliphily. At pH values at which their capacity for pH homeostasis no longer maintains a cytoplasmic pH < 8, extreme alkaliphiles still exhibit growth. They grow well when the cytoplasmic pH is 8.3-9.6, values that preclude most neutrophile growth. This indicates that there are adaptations of cytoplasmic processes of alkaliphiles.

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In alkaliphilic Bacillus, Na⁺-dependent pH homeostasis involves Na⁺/H⁺ antiport activities as well as Na⁺ re-entry routes. The MotPS flagellar stator proteins that are homologous to MotAB are required for the Na⁺-dependent motility of alkaliphilic B. pseudofirmus OF4. Purified and reconstituted MotPS support amiloride analogue-sensitive Na⁺ flux. Mutants lacking functional MotPS display no deficit in pH homeostasis in pH shift experiments conducted with sub-optimal added [Na⁺] in the absence of solutes whose uptake is coupled to Na⁺ re-entry. By contrast, a role was evident for the recently described Na_vBP (voltage-dependent) Na⁺ channel at sub-optimal [Na⁺]. Na_vBP mutants exhibit a significant loss of pH homeostasis capacity. Na_vBP has an additional role in chemotaxis; mutants in Na_vBP exhibited inverse chemotaxis as did the wild type alkaliphile when treated with the Na_vBP channel inhibitor nifedipine.

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