[in Japanese]

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Extreme thermophiles produce unique polyamines, especially long polyamines and branched polyamines. The author and his colleagues investigated the roles of these polyamines at high temperature. Reverse genetic studies suggested that the unique polyamines are essential for life at high temperature extremes. In an extreme thermophile, Thermus thermophilus, polyamines are synthesized by a new pathway. Polyamines stabilize nucleic acids. Double stranded structure was stabilized more efficiently by the presence of long polyamines, whereas stem-and-loop structures are more stabilized by the addition of a branched polyamine, tetrakis(3-aminopropyl)ammonium.

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Polyphosphates (polyP) are ubiquitous molecules present in most organisms including bacteria, archaea and eukaryotes. Although several physiological functions have been attributed to polyP in addition to being a reservoir of phosphate, nothing is known about the possible role of polyP in archaea. A model for the participation of polyP in the tolerance to heavy metals has been proposed in bacteria. The intracellular concentrations of these ions would stimulate polyP degradation and the hydrolyzed phosphate obtained by the action of PPX could be transported out of the cell along with the metal cations. To study if such a system exists in Archaea, the presence of polyP was determined by the electron energy loss spectroscopy (EELS) procedure and quantified by using specific enzymatic methods in S. acidocaldarius, S. metallicus and S. solfataricus. All three microorganisms synthesized polyP during growth, but only S. metallicus greatly accumulated polyP granules. The differences in the capacity to accumulate polyP between these archaeons may reflect adaptive responses to their natural environment. Thus, S. metallicus was able to grow and tolerate up to 200 mM copper sulfate, with a concomitant decrease in its polyP levels with increasing copper concentrations. On the other hand, S. solfataricus could not grow in or tolerate more than 1-5 mM copper sulfate, most likely due to its low levels of polyP. Shifting S. metallicus cells to copper sulfate concentrations up to 100 mM showed a rapid increase in their PPX activity which was concomitant in time with a decrease in their polyP levels and a stimulation of phosphate efflux. Furthermore, copper in the range of 10 μM greatly stimulated PPX activity in cell-free extracts from S. metallicus. The results strongly suggest that a metal tolerance mechanism mediated through polyP is also functional in members of the Sulfolobus group.

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The high hydrostatic pressure (HHP) response and adaptation was studied of Lactobacillus sanfranciscensis used in food biotechnology. Changes were characterized in the membrane physiology with fluorescent techniques, the proteome with 2-D electrophoresis and the transcriptome with microarracys and real time PCR. The up-regulated proteins and genes included representatives of heat and cold shock corroborating the hypothesis that the cell tries to compensate for pressure induced impairing of membrane transport and translation. Overexpression of ssrA (transfer mRNA) in a barotolerant mutant suggests a role of the ribosome as primary thermodynamic HHP sensor determining the adaptive capacity of the cell. Thus, we propose trans-translation and peptide tagging, processes that promote recycling of stalled ribosomes and prevent accumulation of abortively synthesised polypeptides, to be involved in combating high pressure damage and conferring moderate barotolerance.

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There have been growing interests in understanding microbial lives under deep-sea high-pressure environments as well as biological responses to changes in hydrostatic pressure. While molecular adaptation to high-pressure environments has been extensively analyzed in various marine prokaryotes, little is known about eukaryotic microbes in deep-sea environments. To establish the molecular basis for piezoadaptation, we have introduced advanced techniques of yeast cell biology into high-pressure research using Saccharomyces cerevisiae, and propose a research field “piezophsiology”. This paper presents phenomena occurred in yeast cells at high pressure.

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Actinomycetes are present in great diversity over the complete range of ocean depths and represent increasing numbers of authentic indigenous marine microorganisms. The biotechnology resource provided by actinomycetes, particularly marine actinomycetes, is far from being exhausted and recent search and discovery programmes have revealed many new chemical entities, bioactive metabolites, and biocatalytic activities.

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We have developed techniques to clone the entire microbial metagenome; viruses, prokaryotes and eukaryotes. Established approaches were used to isolate environmental DNA and make prokaryotic gene libraries. These libraries contain genes encoding enzymes; cellulases and esterases have been functionally expressed. PCR amplification of environmental DNA has been used to identify integron associated gene cassettes encoding unidentified ORFs. Sequence independent DNA amplification was used to make a faecal virus metagenomic library. Analysis of this library showed the presence of protein ORFS especially enzymes involved in nucleic acid metabolism. Most virus ORFs were unrelated to known sequences. Finally metagenomic microbial eukaryotic RNA was reverse transcribed to select for mRNA; the cDNA was used to make libraries containing many eukaryotic ORFs.

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The microbial community in salt-saturated crystallizer ponds of solar salterns is dominated by square red halophilic Archaea, Salinibacter (Bacteria), and the primary producer Dunaliella. Thanks to their often very high community densities, much information can be gained about these halophiles by analysis of samples collected directly from the field. This is illustrated by lipid and pigment studies in which specific compounds may serve as biomarkers, studies using inhibitors to differentiate between activities performed by the different groups, and studies in which utilization and metabolism of selected carbon sources is monitored to obtain information on the mode of life of the organisms in situ. These studies prove that saltern crystallizer ponds form perfect outdoor laboratories for the study of extremely halophilic microorganisms.

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In a search for new alkaline enzymes for laundry and dishwashing detergents, we identified the novel enzymes cellulase Egl-237, protease KP43, and α-amylase AmyK38 from alkaliphilic Bacillus strains isolated from soil samples. These enzymes possessed useful properties, such as higher stability to heat or various ingredients of detergent and/or greater detergency than conventional enzymes for detergents. . Enzymatic properties and structural features of these new enzymes are presented here. Based on the structural analysis, we also succeeded in improvement of thermal stability of AmyK38 by protein engineering.

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Glucose sensing is used as a model to explore the advantages deriving from the use of sugar - binding proteins isolated from thermophilic organisms to develop stable fluorescence biosensors. The gene of a putative thermostable sugar-binding protein was cloned and expressed in E. coli. The recombinant protein was purified to homogeneity and used as a probe for the development of a fluorescence biosensor for the detection of glucose. Fluorescence spectroscopy experiments demonstrated that the recombinant protein binds glucose with a dissociation constant of about 10 mM, a concentration of sugar close to the concentration of glucose present in the human blood. A docking simulation on the modeled structure of the protein confirmed its ability to bind glucose and proposed possible modifications to improve the affinity for glucose and/or its detection. The obtained results suggest the use of this thermostable protein as a probe for a stable glucose biosensor.

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