Haruyuki Atomi

Description of Thermococcus kodakaraensis sp. nov., a well studied hyperthermophilic archaeon previously reported as Pyrococcus sp. KOD1

A hyperthermophilic archaeal strain, KOD1, isolated from a solfatara on Kodakara Island, Japan, has previously been reported as Pyrococcus sp. KOD1. However, a detailed phylogenetic tree, made possible by the recent accumulation of 16S rRNA sequences of various species in the order Thermococcales, indicated that strain KOD1 is a member of the genus Thermococcus. We performed DNA-DNA hybridization tests against species that displayed high similarity in terms of 16S ribosomal DNA sequences, including Thermococcus peptonophilus and Thermococcus stetteri. Hybridization results and differences in growth characteristics and substrate utilization differentiated strain KOD1 from T. peptonophilus and T. stetteri at the species level. Our results indicate that strain KOD1 represents a new species of Thermococcus, which we designate as Thermococcus kodakaraensis KOD1 sp. nov.

Insights into the evolution of Archaea and eukaryotic protein modifier systems revealed by the genome of a novel archaeal group

The domain Archaea has historically been divided into two phyla, the Crenarchaeota and Euryarchaeota. Although regarded as members of the Crenarchaeota based on small subunit rRNA phylogeny, environmental genomics and efforts for cultivation have recently revealed two novel phyla/divisions in the Archaea; the ‘Thaumarchaeota’ and ‘Korarchaeota’. Here, we show the genome sequence of Candidatus ‘Caldiarchaeum subterraneum’ that represents an uncultivated crenarchaeotic group. A composite genome was reconstructed from a metagenomic library previously prepared from a microbial mat at a geothermal water stream of a sub-surface gold mine. The genome was found to be clearly distinct from those of the known phyla/divisions, Crenarchaeota (hyperthermophiles), Euryarchaeota, Thaumarchaeota and Korarchaeota. The unique traits suggest that this crenarchaeotic group can be considered as a novel archaeal phylum/division. Moreover, C. subterraneum harbors an ubiquitin-like protein modifier system consisting of Ub, E1, E2 and small Zn RING finger family protein with structural motifs specific to eukaryotic system proteins, a system clearly distinct from the prokaryote-type system recently identified in Haloferax and Mycobacterium. The presence of such a eukaryote-type system is unprecedented in prokaryotes, and indicates that a prototype of the eukaryotic protein modifier system is present in the Archaea.

Improved and Versatile Transformation System Allowing Multiple Genetic Manipulations of the Hyperthermophilic Archaeon Thermococcus kodakaraensis

ABSTRACT We have recently developed a gene disruption system for the hyperthermophilic archaeon Thermococcus kodakaraensis by utilizing a pyrF-deficient mutant, KU25, as a host strain and the pyrF gene as a selectable marker. To achieve multiple genetic manipulations for more advanced functional analyses of genes in vivo, it is necessary to establish multiple host-marker systems or to develop a system in which repeated utilization of one marker gene is possible. In this study, we first constructed a new host strain, KU216 (ΔpyrF), by specific and almost complete deletion of endogenous pyrF through homologous recombination. In this refined host, there is no need to consider unknown mutations caused by random mutagenesis, and unlike in the previous host, KU25, there is little, if any, possibility that unintended recombination between the marker gene and the chromosomal allele occurs. Furthermore, a new host-marker combination of a trpE deletant, KW128 (ΔpyrF ΔtrpE::pyrF), and the trpE gene was developed. This system made it possible to isolate transformants through a more simple selection procedure as well as to deduce the transformation efficiency, overcoming practical disadvantages of the first system. The effects of the transformation conditions were also investigated using this system. Finally, we have also established a system in which repeated utilization of the counterselectable pyrF marker is possible through its excision by pop-out recombination. Both endogenous and exogenous sequences could be applied as tandem repeats flanking the marker pyrF for pop-out recombination. A double deletion mutant, KUW1 (ΔpyrF ΔtrpE), constructed with the pop-out strategy, was demonstrated to be a useful host for the dual markers pyrF and trpE. Likewise, a triple deletion mutant, KUWH1 (ΔpyrF ΔtrpE ΔhisD), could also be constructed. The transformation systems developed here now provide the means for extensive genetic studies in this hyperthermophilic archaeon.

Extremely Stable and Versatile Carboxylesterase from a Hyperthermophilic Archaeon

ABSTRACT We have found that the hyperthermophilic archaeon Pyrobaculum calidifontis VA1 produced a thermostable esterase. We isolated and sequenced the esterase gene (estPc) from strain VA1. estPc consisted of 939 bp, corresponding to 313 amino acid residues with a molecular mass of 34,354 Da. As estPc showed significant identity (30%) to mammalian hormone-sensitive lipases (HSLs), esterase of P. calidifontis (Est) could be regarded as a new member of the HSL family. Activity levels of the enzyme were comparable or higher than those of previously reported enzymes not only at high temperature (6,410 U/mg at 90°C), but also at ambient temperature (1,050 U/mg at 30°C). The enzyme displayed extremely high thermostability and was also stable after incubation with various water-miscible organic solvents at a concentration of 80%. The enzyme also exhibited activity in the presence of organic solvents. Est of P. calidifontis showed higher hydrolytic activity towards esters with short to medium chains, with p-nitrophenyl caproate (C6) the best substrate among the p-nitrophenyl esters examined. As for the alcoholic moiety, the enzyme displayed esterase activity towards esters with both straight- and branched-chain alcohols. Most surprisingly, we found that this Est enzyme hydrolyzed the tertiary alcohol ester tert-butyl acetate, a feature very rare among previously reported lipolytic enzymes. The extreme stability against heat and organic solvents, along with its activity towards a tertiary alcohol ester, indicates a high potential for the Est of P. calidifontis in future applications.

Low-Temperature Lipase from Psychrotrophic Pseudomonas sp. Strain KB700A

ABSTRACT We have previously reported that a psychrotrophic bacterium,Pseudomonas sp. strain KB700A, which displays sigmoidal growth even at −5°C, produced a lipase. A genomic DNA library of strain KB700A was introduced into Escherichia coli TG1, and screening on tributyrin-containing agar plates led to the isolation of the lipase gene. Sequence analysis revealed an open reading frame (KB-lip) consisting of 1,422 nucleotides that encoded a protein (KB-Lip) of 474 amino acids with a molecular mass of 49,924 Da. KB-Lip showed 90% identity with the lipase fromPseudomonas fluorescens and was found to be a member of Subfamily I.3 lipase. Gene expression and purification of the recombinant protein were performed. KB-Lip displayed high lipase activity in the presence of Ca2+. Addition of EDTA completely abolished lipase activity, indicating that KB-Lip was a Ca2+-dependent lipase. Addition of Mn2+ and Sr2+ also led to enhancement of lipase activity but to a much lower extent than that produced by Ca2+. The optimal pH of KB-Lip was 8 to 8.5. The addition of detergents enhanced the enzyme activity. When p-nitrophenyl esters and triglyceride substrates of various chain-lengths were examined, the lipase displayed highest activity towards C10 acyl groups. We also determined the positional specificity and found that the activity was 20-fold higher toward the 1(3) position than toward the 2 position. The optimal temperature for KB-Lip was 35°C, lower than that for any previously reported Subfamily I.3 lipase. The enzyme was also thermolabile compared to these lipases. Furthermore, KB-Lip displayed higher levels of activity at low temperatures than did other enzymes from Subfamily I.3, indicating that KB-Lip has evolved to function in cold environments, in accordance with the temperature range for growth of its psychrotrophic host, strain KB700A.

Formate-driven growth coupled with H2 production

Although a common reaction in anaerobic environments, the conversion of formate and water to bicarbonate and H2 (with a change in Gibbs free energy of ΔG° = +1.3 kJ mol−1) has not been considered energetic enough to support growth of microorganisms. Recently, experimental evidence for growth on formate was reported for syntrophic communities of Moorella sp. strain AMP and a hydrogen-consuming Methanothermobacter species and of Desulfovibrio sp. strain G11 and Methanobrevibacter arboriphilus strain AZ. The basis of the sustainable growth of the formate-users is explained by H2 consumption by the methanogens, which lowers the H2 partial pressure, thus making the pathway exergonic. However, it has not been shown that a single strain can grow on formate by catalysing its conversion to bicarbonate and H2. Here we report that several hyperthermophilic archaea belonging to the Thermococcus genus are capable of formate-oxidizing, H2-producing growth. The actual ΔG values for the formate metabolism are calculated to range between −8 and −20 kJ mol−1 under the physiological conditions where Thermococcus onnurineus strain NA1 are grown. Furthermore, we detected ATP synthesis in the presence of formate as a sole energy source. Gene expression profiling and disruption identified the gene cluster encoding formate hydrogen lyase, cation/proton antiporter and formate transporter, which were responsible for the growth of T. onnurineus NA1 on formate. This work shows formate-driven growth by a single microorganism with protons as the electron acceptor, and reports the biochemical basis of this ability.

Archaeal type III RuBisCOs function in a pathway for AMP metabolism

The type III ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) present in the archaeon Thermococcus kodakaraensis was found to participate in adenosine 5′-monophosphate (AMP) metabolism, a role that is distinct from that of classical RuBisCOs of the Calvin-Benson-Bassham cycle. Genes annotated as thymidine phosphorylase (deoA) and eucaryal translation initiation factor 2B (e2b2) were found to encode AMP phosphorylase and ribose-1,5-bisphosphate isomerase, respectively. These enzymes supplied the RuBisCO substrate, ribulose-1,5-bisphosphate, from AMP and phosphate. Archaea with type III RuBisCOs all harbor both DeoA and the corresponding E2b2 homologs. In this pathway, adenine was released from AMP and the phosphoribose moiety entered central-carbon metabolism.

Model organisms for genetics in the domain Archaea: methanogens, halophiles, Thermococcales and Sulfolobales

The tree of life is split into three main branches: eukaryotes, bacteria, and archaea. Our knowledge of eukaryotic and bacteria cell biology has been built on a foundation of studies in model organisms, using the complementary approaches of genetics and biochemistry. Archaea have led to some exciting discoveries in the field of biochemistry, but archaeal genetics has been slow to get off the ground, not least because these organisms inhabit some of the more inhospitable places on earth and are therefore believed to be difficult to culture. In fact, many species can be cultivated with relative ease and there has been tremendous progress in the development of genetic tools for both major archaeal phyla, the Euryarchaeota and the Crenarchaeota. There are several model organisms available for methanogens, halophiles, and thermophiles; in the latter group, there are genetic systems for Sulfolobales and Thermococcales. In this review, we present the advantages and disadvantages of working with each archaeal group, give an overview of their different genetic systems, and direct the neophyte archaeologist to the most appropriate model organism.

Reverse Gyrase Is Not a Prerequisite for Hyperthermophilic Life

We disrupted the reverse gyrase gene from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. An apparent positive supercoiling activity that was observed in the host strain was not found in the disruptant strain. We found that a lack of reverse gyrase led to a retardation in growth that was more striking at higher temperatures. However, the disruption of the reverse gyrase gene did not lead to a lethal phenotype at 90 degrees C. This study provides experimental evidence that reverse gyrase is not a prerequisite for hyperthermophilic life.

Disruption of a Sugar Transporter Gene Cluster in a Hyperthermophilic Archaeon Using a Host-Marker System Based on Antibiotic Resistance

We have developed a gene disruption system in the hyperthermophilic archaeon Thermococcus kodakaraensis using the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmg(Tk)) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apu(Tk)) or a gene cluster which includes apu(Tk) and genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-type T. kodakaraensis KOD1 cells, and transformants exhibiting resistance to 4 microM simvastatin were isolated. The transformants exhibited growth in the presence of 20 microM simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at the hmg(Tk) locus when the endogenous hmg(Tk) gene was used. This could be avoided by using the corresponding gene from Pyrococcus furiosus (hmg(Pf)) or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The Deltaapu(Tk) strain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that Apu(Tk) is a major polysaccharide-degrading enzyme in T. kodakaraensis.