Extremophiles in a changing world.

Extremophiles and their products have been a major focus of research interest for over 40 years. Through this period, studies of these organisms have contributed hugely to many aspects of the fundamental and applied sciences, and to wider and more philosophical issues such as the origins of life and astrobiology. Our understanding of the cellular adaptations to extreme conditions (such as acid, temperature, pressure and more), of the mechanisms underpinning the stability of macromolecules, and of the subtleties, complexities and limits of fundamental biochemical processes has been informed by research on extremophiles. Extremophiles have also contributed numerous products and processes to the many fields of biotechnology, from diagnostics to bioremediation. Yet, after 40 years of dedicated research, there remains much to be discovered in this field. Fortunately, extremophiles remain an active and vibrant area of research. In the third decade of the twenty-first century, with decreasing global resources and a steadily increasing human population, the world's attention has turned with increasing urgency to issues of sustainability. These global concerns were encapsulated and formalized by the United Nations with the adoption of the 2030 Agenda for Sustainable Development and the presentation of the seventeen Sustainable Development Goals (SDGs) in 2015. In the run-up to 2030, we consider the contributions that extremophiles have made, and will in the future make, to the SDGs.

Crystal structure of a novel-type archaeal rubisco with pentagonal symmetry.

BACKGROUND: Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme of the Calvin-Benson cycle and catalyzes the primary reaction of CO2 fixation in plants, algae, and bacteria. Rubiscos have been so far classified into two types. Type I is composed of eight large subunits (L subunits) and eight small subunits (S subunits) with tetragonal symmetry (L8S8), but type II is usually composed only of two L subunits (L2). Recently, some genuinely active Rubiscos of unknown physiological function have been reported from archaea. RESULTS: The crystal structure of Rubisco from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-Rubisco) was determined at 2.8 A resolution. The enzyme is composed only of L subunits and showed a novel (L2)5 decameric structure. Compared to previously known type I enzymes, each L2 dimer is inclined approximately 16 degrees to form a toroid-shaped decamer with its unique L2-L2 interfaces. Differential scanning calorimetry (DSC), circular dichroism (CD), and gel permeation chromatography (GPC) showed that Tk-Rubisco maintains its secondary structure and decameric assembly even at high temperatures. CONCLUSIONS: The present study provides the first structure of an archaeal Rubisco, an unprecedented (L2)5 decamer. Biochemical studies indicate that Tk-Rubisco maintains its decameric structure at high temperatures. The structure is distinct from type I and type II Rubiscos and strongly supports that Tk-Rubisco should be classified as a novel type III Rubisco.

Peroxisomal and mitochondrial carnitine acetyltransferase isozymes of the n-alkane-assimilating yeast, Candida tropicalis, occurred by alternative initiation of translation from the transcripts of a single gene.

Carnitine acetyltransferase (CAT; EC 2.3.1.7) is localized in two subcellular organelles, peroxisomes and mitochondria, in an n-alkane-assimilating yeast, Candida tropicalis. The isozymes are synthesized from the first and second ATG codon of the open reading frame of one gene, CtCAT. Primer extension analysis and RNase protection assay (RPA) revealed that multiple transcription initiation sites were found upstream of the first ATG codon. 5' ends could not be detected between the first and second ATG codons. These results suggested that the peroxisomal CAT of C. tropicalis, initiating at the second AUG codon of the transcripts, was synthesized by a translational readthrough of the first AUG codon of the open reading frame. When CtCAT was introduced into the other yeast, Saccharomyces cerevisiae, 5' ends of transcripts and the protein products were similar to those observed in C. tropicalis. This suggested that the transcripts harbored sufficient information to bring about alternative initiation of translation in both yeasts. Using S. cerevisiae as the host cell, introduction of mutations into the sequence near the first AUG codon or a deletion in the region between the first and second AUG codons resulted in an increased ratio of translation from the first AUG codon, although initiation sites of transcription did not change. Moreover, replacing the 5' leader sequence to that of C. tropicalis isocitrate lyase promoter (UPR-ICL) eliminated the product initiating at the second AUG codon. The transcript from these cells was shorter than those detected from the native CtCAT-harboring cells. From these results, it was strongly suggested that peroxisomal and mitochondrial CAT isozymes occurred by the alternative initiation of translation mainly dependent on the structure and sequence context of the region from the 5' end to the second AUG codon, and not the insufficient length of the 5' leader.

Thermoalkalophilic lipase of Bacillus thermocatenulatus. I. molecular cloning, nucleotide sequence, purification and some properties.

An expression library was generated by partial Sau3A digestion of genomic DNA from the thermophile Bacillus thermocatenulatus and cloning of DNA fragments in pUC18 in Escherichia coli DH5alpha. Screening for lipase activity identified a 4.5 kb insert in pUC18 which directed the production of lipase in E. coli DH5alpha. A subclone with a 2.2 kb insert was sequenced. The lipase gene codes for a mature lipase of 388 amino acid residues, corresponding to a molecular weight of 43 kDa. As in other Bacillus lipases, an Ala replaces the first Gly in the conserved pentapeptide Gly-X-Ser-X-Gly found in most lipases. The region upstream of the lipase gene contains a Bacillus promoter which directs the expression of lipase in E. coli DH5alpha. The expressed lipase was isolated and purified 312-fold to homogeneity. N-terminal sequencing of the purified lipase revealed a correct cleavage of the preprotein in E. coli DH5alpha. Maximum activity was found at pH 8.0-9.0 with tributyrin and olive oil as substrates and at 60-70 degrees C with p-NPP and olive oil as substrates. The lipase showed high stability at pH 9.0-11.0 and towards various detergents and organic solvents.

Functional expression of a mammalian acetylcholinesterase in Pichia pastoris: comparison to acetylcholinesterase, expressed and reconstituted from Escherichia coli.

The mature rat brain acetylcholinesterase gene (T subunit, AChE) was subcloned downstream of the temperature-inducible lambda promoter PL and fused to the signal peptide of the OmpA protein. Three different expression vectors were constructed: (i) pCompmA containing the mature AChE, (ii) pComp delta TA containing a truncated AChE and (iii) pComp delta TAH containing the truncated AChE C-terminal fused to a 6xHis-tag. With all expression vectors the overexpression of AChE in Escherichia coli resulted mainly in cytoplasmic inclusion bodies (IB). However, some activity was found in the periplasmic space. The inclusion bodies were refolded in vitro, yielding up to 1.42 U/mg IB of active AChE. The refolded AChE was partially purified (approx. 300-fold) by affinity chromatography with a specific activity of approx. 250 U/mg. Removing the cysteine residue near the C-terminus (truncated AChE, delta TAChE) assuming to affect the refolding, did not increase the amount of active enzyme obtained after refolding. Purification of denatured delta TAChE-6xHis prior to refolding by Ni-NTA-chromatography increased the refolding efficiency by a factor of 1.5. Functional expression and secretion of rat brain acetylcholinesterase into the medium was achieved in Pichia pastoris. By optimizing the culture conditions, 100 mU/ml AChE in the medium was produced. In this work we are describing the functional expression of a mammalian AChE in a microbial host in good yields for the first time. The physico-chemical properties of both, the bacterial and yeast expressed AChE were compared with those of the native AChE. The properties of the yeast expressed AChE and the native AChE were similar, whereas the E. coli expressed enzyme was found to be less stable and had different inhibition properties.

Analysis of carbon source-regulated gene expression by the upstream region of the Candida tropicalis malate synthase gene in Saccharomyces cerevisiae.

We investigated the regulation of expression of a gene encoding malate synthase (MS) of an n-alkane-utilizable yeast Candida tropicalis in the yeast Saccharomyces cerevisiae, where its expression is highly induced by acetate. By comparing levels of gene expression in cells grown on glucose, acetate, lactate, and oleic acid, we found that the increase in gene expression was due to a glucose repression-derepression mechanism. In order to obtain information concerning the regulation of the gene expression, a fusion gene which consists of the 5'-upstream region of MS-2 (UPR-MS-2) and the lacZ gene (encoding Escherichia coli beta-galactosidase), was introduced into S. cerevisiae, and beta-galactosidase activities were measured with cells grown on glucose or acetate. Deletion analysis of UPR-MS-2 revealed that the region between -777 and -448 (against the translation initiation codon) enhanced the level of gene expression in both glucose- and acetate-grown cells. In this region, sequences which resemble binding sites of Rap1p/Grf1p/Tufp, a global transcription activator, were found at seven locations and one was found for another pleiotropic activator Abf1p. The result also suggested the presence of multiple upstream repression sequences (URSs), which function specifically in glucose-grown cells, in the region between -368 and -126. In the repressing region, there were three tandem C(A/T)CTCCC sequences and also a putative binding site of Mig1p, a transcriptional repressor which mediates glucose repression of several other genes. When MIG1 gene of S. cerevisiae was disrupted, the expression of the UPR-MS-2-lacZ gene in glucose-grown cells increased approx. 10-fold. Furthermore, the effect of deletion of a putative Mig1p binding site was abolished in the MIG1-disrupted strain, suggesting Mig1p binds to this site and brings about glucose repression. When the SNF1 gene was disrupted, the high level gene expression observed in acetate-grown cells bearing UPR-MS-2 was abolished. This indicated that derepression of UPR-MS-2 -mediated gene expression was dependent on Snf1p, as is the case of genes encoding isocitrate lyase and gluconeogenic enzymes in S. cerevisiae.

Ribulose bisphosphate carboxylase/oxygenase from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 is composed solely of large subunits and forms a pentagonal structure.

We previously reported the presence of a highly active, carboxylase-specific ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. In this study, structural analysis of Pk -Rubisco has been performed. Phylogenetic analysis of Rubiscos indicated that archaeal Rubiscos, including Pk -Rubisco, were distinct from previously reported type I and type II enzymes in terms of primary structure. In order to investigate the existence of small subunits in native Pk -Rubisco, immunoprecipitation and native-PAGE experiments were performed. No specific protein other than the expected large subunit of Pk -Rubisco was detected when the cell-free extracts of P. kodakaraensis KOD1 were immunoprecipitated with polyclonal antibodies against the recombinant enzyme. Furthermore, native and recombinant Pk -Rubiscos exhibited identical mobilities on native-PAGE. These results indicated that native Pk -Rubisco consisted solely of large subunits. Electron micrographs of purified recombinant Pk -Rubisco displayed pentagonal ring-like assemblies of the molecules. Crystals of Pk -Rubisco obtained from ammonium sulfate solutions diffracted X-rays beyond 2.8 A resolution. The self-rotation function of the diffraction data showed the existence of 5-fold and 2-fold axes, which are located perpendicularly to each other. These results, along with the molecular mass of Pk -Rubisco estimated from gel filtration, strongly suggest that Pk -Rubisco is a decamer composed only of large subunits, with pentagonal ring-like structure. This is the first report of a decameric assembly of Rubisco, which is thought to belong to neither type I nor type II Rubiscos.

A unique DNase activity shares the active site with ATPase activity of the RecA/Rad51 homologue (Pk-REC) from a hyperthermophilic archaeon.

A RecA/Rad51 homologue from Pyrococcus kodakaraensis KOD1 (Pk-REC) is the smallest protein among various RecA/Rad51 homologues. Nevertheless, Pk-Rec is a super multifunctional protein and shows a deoxyribonuclease activity. This deoxyribonuclease activity was inhibited by 3 mM or more ATP, suggesting that the catalytic centers of the ATPase and deoxyribonuclease activities are overlapped. To examine whether these two enzymatic activities share the same active site, a number of site-directed mutations were introduced into Pk-REC and the ATPase and deoxyribonuclease activities of the mutant proteins were determined. The mutant enzyme in which double mutations Lys-33 to Ala and Thr-34 to Ala were introduced, fully lost both of these activities, indicating that Lys-33 and/or Thr-34 are important for both ATPase and deoxyribonuclease activities. The mutation of Asp-112 to Ala slightly and almost equally reduced both ATPase and deoxyribonuclease activities. In addition, the mutation of Glu-54 to Gln did not seriously affect the ATPase, deoxyribonuclease, and UV tolerant activities. These results strongly suggest that the active sites of the ATPase and deoxyribonuclease activities of Pk-REC are common. It is noted that unlike Glu-96 in Escherichia coli RecA, which has been proposed to be a catalytic residue for the ATPase activity, the corresponding residual Glu-54 in Pk-REC is not involved in the catalytic function of the protein.

Genes encoding peroxisomal enzymes are not necessarily assigned on the same chromosome of an n-alkane-utilizable yeast Candida tropicalis.

We have resolved eight chromosomal bands from an n-alkane-assimilating yeast, Candida tropicalis pK 233, by using contour-clamped homogeneous electric field gel electrophoresis (CHEF). From the results of hybridization of DNA probes of yeast peroxisomal enzymes--catalase, acyl-CoA oxidase, carnitine acetyltransferase, isocitrate lyase, malate synthase, acetoacetyl-CoA thiolase, and 3-ketoacyl-CoA thiolase--to Southern transfers of CHEF gels, these genes were proven not necessarily to be located on the same chromosome. This fact shows that the genes encoding the enzymes tested were not distributed to be cistronic, although simultaneous and inducible synthesis of peroxisomal enzymes occurred in harmony with the proliferation of peroxisomes, suggesting that their co-ordinated expression might be mainly regulated by certain trans-acting factors.

Expression of acetoacetyl-CoA thiolase isozyme genes of n-alkane-assimilating yeast, Candida tropicalis: isozymes in two intracellular compartments are derived from the same genes.

In the n-alkane-assimilating yeast Candida tropicalis, there are two isozymes of acetoacetyl-CoA thiolase, peroxisomal acetoacetyl-CoA thiolase (peroxisomal Thiolase I), and cytosolic acetoacetyl-CoA thiolase (cytosolic Thiolase I). We have previously isolated two genes (CT-T1A and CT-T1B) which encode Thiolase I. In order to compare the expressed products of Thiolase I isozyme-encoding genes in C. tropicalis, cytosolic Thiolase I was first purified from glucose-grown C. tropicalis in which the proliferation of peroxisomes and the expression of peroxisomal Thiolase I were repressed. Cytosolic Thiolase I was virtually identical to peroxisomal Thiolase I in molecular mass, kinetic and immunochemical properties, and primary structure at the N-terminus. Amino acid sequence analysis revealed that cytosolic Thiolase I was the mixture of products of two genes (CT-T1A and CT-T1B), as in the case of the peroxisomal enzyme. CT-T1A and CT-T1B were expressed independently in the yeast Saccharomyces cerevisiae and the recombinant proteins were purified. Recombinant Thiolase IA and IB exhibited practically identical enzymatic properties to cytosolic and peroxisomal Thiolase Is from C. tropicalis. These results revealed that cytosolic Thiolase I and peroxisomal Thiolase I were encoded not by different genes, but by the same genes (CT-T1A and CT-T1B) and are present as a mixture of products expressed by both genes, although their subcellular localizations are different.

Presence of two transcribed malate synthase genes in an n-alkane-utilizing yeast, Candida tropicalis.

The presence of two genomic DNA regions encoding malate synthase (MS) was shown by Southern blot analysis of the genomic DNA from an n-alkane-assimilating yeast, Candida tropicalis, using a partial MS cDNA probe, in accordance with the fact that two types of partial MS cDNAs have previously been isolated. This was also confirmed by the restriction mapping of the two genes screened from the yeast lambda EMBL library. Nucleotide sequence analysis of the respective genomic DNAs, named MS-1 gene and MS-2 gene, revealed that both regions encoding MS had the same length of 1,653 base pairs, corresponding to 551 amino acids (molecular mass of MS-1, 62,448 Da; MS-2, 62,421 Da). Although 29 nucleotide pairs differed in the sequences of the coding regions, the number of amino acid replacements was only one: 159Asn (MS-1)----159Ser (MS-2). In the 5'-flanking regions, there were replacements of four nucleotide pairs, deletion of one pair, and insertion of four pairs. In spite of the fact that two genomic genes were present and transcribed, RNA blot analysis demonstrated that only one band (about 2 kb) was observable even when the carbon sources in the cultivation medium were changed. A comparison of the amino acid sequences was made with MSs of rape (Brassica napus L.), cucumber seed, pumpkin seed, Escherichia coli, and Hansenula polymorpha. A high homology was observed among these enzymes, the results indicating that the protein structure was relatively well conserved through the evolution of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Peroxisomal isocitrate lyase of the n-alkane-assimilating yeast Candida tropicalis: gene analysis and characterization.

A genomic DNA clone encoding isocitrate lyase, a key enzyme of the glyoxylate cycle and a peroxisomal enzyme of the n-alkane-assimilating yeast Candida tropicalis has been isolated with a cDNA probe from the yeast lambda EMBL library. Nucleotide sequence analysis of the genomic DNA clone disclosed that the region coding isocitrate lyase had a length of 1,650 base pairs, corresponding to 550 amino acids (61,602 Da). RNA blot analysis demonstrated that only one kind of mRNA (2 kb) supposed to be transcribed from this gene was present in the cells. A comparison of the amino acid sequences was made with the isocitrate lyase of castor bean, one of the glyoxysomal enzymes, and the enzyme of E. coli. The isocitrate lyases of C. tropicalis and castor bean had high homology, and the presence of some amino acid stretches conserved in all three enzymes suggests that these might be involved in the catalysis of the common reaction. There was an insertion common to the isocitrate lyases of C. tropicalis and castor bean, which is of interest concerning their evolution. In the C-terminal region, a characteristic sequence similar to that previously proposed as the import signal to peroxisomes was present.

Individual expression of Candida tropicalis peroxisomal and mitochondrial carnitine acetyltransferase-encoding genes and subcellular localization of the products in Saccharomyces cerevisiae.

In an n-alkane assimilating yeast, Candida tropicalis, carnitine acetyltransferase (CAT; EC 2.3.1.7) was localized in both peroxisomes and mitochondria. Both CATs were encoded by one gene, CT-CAT, although the initiation sites of translation were suggested to be different. In the present study, the genes corresponding to the supposed C. tropicalis peroxisomal and mitochondrial CATs, which were truncated from the CT-CAT gene, were individually expressed in Saccharomyces cerevisiae, using the C. tropicalis isocitrate lyase promoter (UPR-ICL), which is inducible by oleic acid in concert with proliferation of peroxisomes in S. cerevisiae [Umemura, K., Atomi, H., Kanai, T., Teranishi, Y., Ueda, M., and Tanaka, A. (1995) Appl. Microbiol. Biotechnol. 43, 489-492]. The 71 kDa precursor of mitochondrial CAT, initiating at the first Met, was found to be processed to the mature size (66 kDa) in S. cerevisiae and immunoelectronmicroscopical observation revealed that this enzyme was localized in mitochondria. On the other hand, 68 kDa CAT, initiating at the second Met (residue No. 19), had no cleavable signal and was translocated into peroxisomes and cytosol, but not into mitochondria. The amino-terminal amino acid sequences of individually expressed CATs were identical to those of CATs isolated from alkane-grown C. tropicalis cells, respectively. These results demonstrated that only the 71 kDa protein yielded the 66 kDa protein and that peroxisomal and mitochondrial CATs arose from the difference in the initiation sites of translation.

Presence of carnitine acetyltransferase in peroxisomes and in mitochondria of oleic acid-grown Saccharomyces cerevisiae.

Activity of carnitine acetyltransferase was detected in glucose- and oleic acid-grown Saccharomyces cerevisiae. Oleic acid-grown cells showed a ten-fold higher activity than glucose-grown cells. Subcellular fractionation of oleic acid-grown cells showed that carnitine acetyltransferase was present in peroxisomes, mitochondria, and cytosol. The results suggested the plausible presence of an 'acetylcarnitine shuttle' in this yeast, as in the case of an n-alkane-assimilating yeast, Candida tropicalis.

Metal-binding properties of phytochelatin-related peptides.

Phytochelatins (PCs, (gamma Glu-Cys)(n)-Gly, n=2-11) are produced by higher plants, algae and some fungi in order to detoxify Cd(2+) by sequestration to form Cd-PCs complexes. In order to investigate what chemical structures of PCs are responsible for their metal-binding ability, various cysteine-rich peptides ((X-Cys)(7)-Gly, X=Glu, Asp, Lys, Gly, Ser and Gln) were chemically synthesized. Water-solubility, metal-binding property, and detoxification effect toward Cd(2+) were analyzed and compared with those of (gamma EC)(7)G. (SC)(7)G and (QC)(7)G were insoluble at pH below 10, and (GC)(7)G was not soluble at any pH between 1 and 12, indicating that charged side chains were at least required for the molecules to be solubilized in aqueous solution. By spectroscopic analyses using DTNB method and UV method, we found that (EC)(7)G and (DC)(7)G had almost equivalent abilities of Cd(2+)-binding as PC ((gamma EC)(7)G), indicating that the distance between each thiol group was not a major factor for the binding to Cd(2+). (beta DC)(7)G and (KC)(7)G interacted to Cd(2+) with fourth coordination as in the case of other soluble PC-related peptides. However, compared to (gamma EC)(7)G, (beta DC)(7)G displayed a slightly weaker binding to Cd(2+), and (KC)(7)G showed a drastic decrease in binding ability. The affinities of PC-related peptides toward Cd(2+) were evaluated as below; (gamma EC)(7)G=(EC)(7)G=(DC)(7)G>(beta DC)(7)G>>(KC)(7)G=weak binding. The results of Cd(2+)-detoxification assays were consistent with the affinity between Cd(2+) and the peptides. We concluded that the structure consisting of thiol and carboxyl groups were essential for the formation of a tight Cd-peptides complex such as Cd-PCs.

Isolation of bacterial strains colonizable in mosquito larval guts as novel host cells for mosquito control.

We screened for microorganisms that can be utilized as new host cells for mosquito larvicides. As long persistence in the environment is required of host cells, we examined the bacterial populations in the guts of mosquito larvae collected from natural breeding habitats. Larvae of Aedes aegypti and Culex quinquefasciatus were examined, and Bacillus species, particularly Bacillus cereus, were found to be the dominant bacterial species in their guts. To investigate the relationship between these Bacillus strains and the mosquito larvae, we re-introduced the bacteria into larvae of Aedes aegypti, C. quinquefasciatus and another common mosquito strain, Anopheles dirus. The cell numbers of Bacillus cereus strains Ae10 and Cx5 in the guts were consistent throughout a 7-d period without food supplementation, suggesting that these strains were able to colonize in the guts of the larvae. To confirm this, we introduced a plasmid containing a kanamycin resistance marker into Ae10 and Cx5 and fed these recombinant strains to C. quinquefasciatus larvae. Even when food was supplemented for 7 d, the recombinant strains, particularly Ae10, were still found in the guts. Under similar conditions, B. thuringiensis serovar israelensis c4Q2-72 was hardly detectable after 2 d, while Escherichia coli could not be detected at all. Their stable retention in mosquito larvae guts and the feasibility of genetic manipulation indicates these strains possess high potential as novel host cells for application in mosquito control.

High-level production of erythritol by strain 618A-01 isolated from pollen.

We have isolated a microorganism (strain 618A-01) from pollen which has the ability to produce erythritol when grown in the presence of glucose as the carbon source. When cultivated in a medium consisting of 20% glucose and 1% dried bouillon in a shake flask, 75 g/l erythritol was produced after 950 h, corresponding to a 37.5% yield against glucose consumption. No other polyols, including glycerol, were detected in the medium. Positive-ion fast atom bombardment mass spectrometry and 1H- and 13C-NMR analyses confirmed that the fermentation product was erythritol. Scanning electron microscopic analysis clearly demonstrated that the cells grown on YPD medium at 30 degrees C showed yeast-like morphology, while they appeared like hyphae at 37 degrees C. The complete 18S rRNA sequence of the isolate was determined, which showed high identity (99.5%) with the genus Ustilago of the phylum Basidiomycota. The data strongly suggest that strain 618A-01 belongs to the class Ustilaginomycetes. The culture conditions for the production of erythritol by the isolate were examined. The use of medium containing 1% tryptone, 0.5% yeast extract and 0.5% NaCl yielded the highest cell growth and erythritol productivity among the media tested. Continuous glucose feeding at 6-7% to the fermentor further increased the production of erythritol, and we obtained a maximal 100 g/l erythritol after 530 h, with a 39.3% yield.

Isolation and characterization of psychrotrophs from subterranean environments.

Subterranean environments are potential sources for the isolation of novel microorganisms. Water and soil samples were collected at depths ranging from 10 to 1800 meters below the surface, and screening was carried out with aerobic rich and anaerobic minimal media. Two psychrotrophic and three chemoautotrophic strains were isolated. One of the psychrotrophic isolates, designated SN16A, grew at temperatures between -5 and 37 degrees C with optimal growth between 25 and 30 degrees C. The other psychrotroph, designated KB700A, grew between -10 and 30 degrees C. Little difference in growth rate could be observed between 20 and 30 degrees C; however, this strain did not grow at 37 degrees C. KB700A utilized CO2 chemoautotrophically at 30 degrees C, using hydrogen as an energy source. Both strains were characterized biochemically. The complete 16S rRNA sequence of KB700A was 98.7% homologous with that of Pseudomonas marginalis. However, the 16S rRNA of SN16A showed only 95.4% identity at maximum-with the corresponding gene of Arthrobacter globiformis-suggesting that this strain may belong to a novel genus. Both strains exhibited the ability to produce hydrolytic enzymes on plate assays. Our results suggest that subterranean environments are promising sources for the isolation of psychrotrophic microorganisms.

Gene analysis and enzymatic properties of thermostable beta-glycosidase from Pyrococcus kodakaraensis KOD1.

A beta-glycosidase with broad substrate specificity was identified from a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. The gene encoding beta-glycosidase (Pk-gly) consists of 1449 nucleotides corresponding to a polypeptide of 483 amino acids. The protein showed similarity with other beta-glycosidases from family-1 glycosyl hydrolases, in particular, it showed high identity to beta-mannosidase from P. furiosus (55.7%), beta-glycosidase from Sulfolobus solfataricus (42.7%) and beta-glucosidase from P. furiosus (41.9%). The cloned gene was expressed in Escherichia coli and the recombinant protein was purified. The beta-glycosidase showed optimal activity at pH 6.5 and at an extremely high temperature of 100 degrees C, and had a half-life of 18 h at 90 degrees C. The beta-glycosidase hydrolyzed various pNp-beta-glycopyranosides, with kcat K(m) values in the order of pNp-beta-glucopyranoside = pNp-beta-mannopyranoside > pNp-beta-galactopyranoside > pNp-beta-xylopyranoside. pNp-beta-mannopyranoside was the substrate exhibiting the lowest K(m) value [0.254 mM] with a kcat K(m) ratio comparable to that of pNp-beta-glucopyranoside. This substrate specificity was distinct from previously reported beta-glycosidases. We observed that the region in PK-Gly corresponding to the fifth alpha-helix and beta-strand region of beta-glycosidase from S. solfataricus, which constitutes a large portion of the channel for substrate incorporation, displayed a chimeric structure, with the N-terminal region similar to beta-glycosidases and the C-terminal region similar to beta-mannosidases. An exo-type hydrolytic activity and transglycosylation activity were also observed towards cellooligomers.

Rapid method for detection and detoxification of heavy metal ions in water environments using phytochelation.

Phytochelatins (PCs, (gammaGlu-Cys)n-Gly (n = 2-11)) are produced by higher plants, algae, and some fungi in response to heavy metal ion exposure. A rapid and convenient method for quantifying heavy metal ion concentrations in water environments was developed using a chemically synthesized PC as a mediator. The chelating ability of the PC and quantification of the thiol group were utilized to measure heavy metal ions at low concentrations. The method requires only ten minutes for measurement and only 1 ml of a liquid sample. A range of homogeneous PCs (n = 4-7) were chemically synthesized using a peptide synthesizer. These, especially PC7, exhibited higher sensitivity and consistency of measurement than the native PC from Silene cucubalus, which produced a mixture of PC2, PC3, and PC4. Detoxification of heavy metal ions in vitro by PC was also investigated. Using the paper disc method, the cell growth inhibition zone caused by cadmium ion against Salmonella typhimurium TA1538 was significantly decreased by addition of PC. Furthermore, at the minimum inhibitory concentration of cadmium ion (200 microM) in a nutrient broth culture of S. typhimurium, cell growth was almost completely recovered by addition of PC to the medium.