<Emphasis Type="Italic">Sulfolobus tokodaii</Emphasis> sp. nov. (f.<Emphasis Type="Italic"> Sulfolobus</Emphasis> sp. strain 7), a new member of the genus<Emphasis Type="Italic"> Sulfolobus</Emphasis> isolated from Beppu Hot Springs,Japan

The taxonomic position of a thermoacidophilic crenarchaeote Sulfolobus sp. strain 7, previously isolated from the Beppu Hot Springs in the geothermal area of Kyushu Island, Japan, was investigated by cloning and sequencing, by phylogenetic analysis of the 16S rRNA gene sequence, by DNA-DNA homology with similar species, and by biochemical characterization of the isolate. This isolate is an obligate aerobe and grows optimally at 80 degrees C and pH2.5-3 under aerobic and chemoheterotrophic growth conditions by aerobic respiration rather than simple fermentation. In conjunction with the phenotypic properties, the present phylogenetic analysis based on the 16S rRNA gene sequence and DNA-DNA hybridization experiments indicate that this isolate is related to the described Sulfolobus taxon and should be considered a novel species of the genus. We propose that this isolate is a novel species of the genus Sulfolobus that we name Sulfolobus tokodaii sp. nov. The type strain is strain 7 (JCM 10545).     

Continuous cell-free protein synthesis directed by messenger DNA and catalyzed by extract of Thermus thermophilus HB27

Polypeptide synthesis directed by DNA as the messenger in a cell-free system of Thermus thermophilus was investigated. Polypeptides were synthesized with the addition of neomycin in the presence of DNA catalyzed by the cell extract. The stability of messenger DNA was greater than that of messenger RNA. Continuous cell-free translation with messenger DNA produced polypeptides at the rate of more than 8 microg/h in the presence of spermine.     

A novel chiral thiol reagent for automated precolumn derivatization and high-performance liquid chromatographic enantioseparation of amino acids and its application to the aspartate racemase assay

A novel optically active thiol compound, N-(tert-butylthiocarbamoyl)-L-cysteine ethyl ester (BTCC), is synthesized as a chiral derivatization reagent. This compound and o-phthalaldehyde react with amino acid enantiomers to produce fluorescent diastereomers that are readily separable on a reverse-phase column by HPLC. Enantioseparation of acidic amino acids in particular is markedly improved using BTCC. In this study, the HPLC method for enantioseparation with the novel compound is applied to the aspartate (Asp) racemase assay. Derivatized D-Asp is eluted before the L-Asp derivative. Consequently, a small amount of D-Asp produced by the activity of racemase on a large quantity of L-Asp substrate may be quantified accurately, even at very low activity. Since the derivatization reaction proceeds rapidly at room temperature, a fully automated system is established for derivatization and sample injection. The automated method is practical and successfully applied to the archaeal Asp racemase assay. We presume that the procedure is additionally applicable to the enantioseparation of other amino acids, amino alcohols, and catecholamines.     

Selection of stabilized 3-isopropylmalate dehydrogenase of Saccharomyces cerevisiae using the host-vector system of an extreme thermophile, Thermus thermophilus

A leuB strain of Thermus thermophilus TTY1, was transformed with a plasmid vector that directed expression of 3-isopropylmalate dehydrogenase (IPMDH) of Saccharomyces cerevisiae encoded by the LEU2 gene. The original strain could not grow at 50 degrees C without leucine, probably because of the low stability of S. cerevisiae IPMDH. The mutants that could grow without leucine were selected at 50 degrees, 60 degrees, 62 degrees, 65 degrees, 67 degrees, and 70 degrees C, step by step. All the mutant strains except for one isolated at 50 degrees C accumulated mutations. Mutations were serially accumulated: Glu255Val, Asn43Tyr, Ala62Thr, Asn110Lys, and Alal 12Val, respectively, at each step. The analyses of residual activity after heat treatment and the denaturation profile as monitored by circular dichroism showed that thermal stability was increased with accumulation of the mutations. The kinetic parameters of most mutant enzymes were similar to those of the wild type. However, some mutant enzymes showed a reverse correlation between stability and activity: the enzymes with a large increase in thermal stability showed lower activity. Although the wild-type enzyme is unstable in the absence of glycerol, the stabilizing effect of glycerol was not observed for all the mutant enzymes containing the Glu255Val substitution, which is assumed to be located at the hydrophobic interface between two subunits.     

Small RNA deep sequencing‐based detection and further evidence of DNA viruses infecting sweetpotato plants in Tanzania

Small interfering RNA deep sequencing (SRDS) was used to detect viruses in 23 sweetpotato plants, collected from various locations in Tanzania. Alignment of small RNA reads using a MAQ program recovered genomes of viruses from five families, namely Geminiviridae (2), Closteroviridae (1), Betaflexiviridae (1), Caulimoviridae (1) and Potyviridae (1). This was in agreement with the variation of symptoms observed on sweetpotato plants in fields and screen house, which included leaf curl, vein yellowing, chlorosis, stunted growth and brown blotches. PCR was also used to confirm the occurrence of viruses associated with leaf curl and symptomless infections. A complete genome (2768 nucleotides) was obtained for a sweepovirus that was 89.9% identical to the strain of Sweet potato leaf curl Sao Paulo virus (SPLCSPV; Begomovirus) reported in South Africa. Sweepoviruses are known to undergo frequent recombinations and evidence for this was found in the SPLCSPV sequence studied. The SRDS‐based results indicated occurrence of the poorly studied Sweet potato badnavirus B (SPBV‐B) and Sweet potato badnavirus A (collectively known as Sweet potato pakakuy virus; SPPV; Caulimoviridae) in sweetpotato plants in Tanzania. A 5′‐end partial sequence (3065 nucleotides), encoding hypothetical, movement and coat proteins, was obtained and found to be 86.3% and 73.1% identical to SPBV‐B and SPBV‐A, respectively. A survey for the distribution of SPPV and Sweet potato symptomless mastrevirus 1 (SPSMV‐1) showed that these viruses were wide spread and co‐infecting sweetpotato plants in Tanzania. The importance of East Africa as a hot spot for the diversity and evolution of sweet potato viruses is discussed.     

Complete Polar Lipid Composition of Thermoplasma acidophilum HO-62 Determined by High-Performance Liquid Chromatography with Evaporative Light-Scattering Detection

Polar ether lipids of Thermoplasma acidophilum HO-62 were purified by high-performance liquid chromatography with an evaporative light-scattering detector. Structures of purified lipids were investigated by capillary gas chromatography, mass spectrometry, and nuclear magnetic resonance. Three types of ether lipids were found: phospholipids, glycolipids, and phosphoglycolipids. The two phospholipids had glycerophosphate as the phosphoester moiety. The seven glycolipids had different combinations of gulose, mannose, and glucose, which formed mono- or oligosaccharides. The eight phosphoglycolipids with two polar head groups contained glycerophosphate as the phosphoester moiety and gulose alone or gulose and mannose, which formed mono- or oligosaccharides, as the sugar moiety. Although gulose is an unusual sugar in nature, several glyco- and phosphoglycolipids contained gulose as one of the sugar moieties in Thermoplasma acidophilum. All the ether lipids had isopranoid chains of C(40) or C(20) with zero to three cyclopentane rings. The structures of these lipids including four new glycolipids and three new phosphoglycolipids were determined, and a glycosylation process for biosynthesis of these glycolipids was suggested.     

Diversity of bacteria and archaea from two shallow marine hydrothermal vents from Vulcano Island

Extremophiles - To obtain new insights into community compositions of hyperthermophilic microorganisms, defined as having optimal growth temperatures of 80&nbsp;°C and above, sediment and...     

Cloning and sequencing of a gene encoding 16S ribosomal RNA from a novel hyperthermophilic archaebacterium NC12

A hyperthermophile NC12 was newly isolated from Noboribetsu hot spring. To characterize this organism, a gene coding for 16S rRNA was cloned and sequenced. The 16S rRNA sequence from NC12 shows the highest similarity with those from Pyrodictium occultum and Desulfurococcus mobilis among the sequences in the database, indicating that NC12 belongs to a cluster of extreme thermophiles (Crenarchaeota) in the archaeal domain. However, since the highest identity score was only 91.2%, it is suggested that NC12 may constitute a new genus.     

Occurrence of sym-homospermidine in extremely thermophilic bacteria

Triamines produced by an extreme thermophile, Thermus thermophilus, were isolated and their chemical structures were determined. It was found that two novel triamines, norspermidine (1,7-diamino-4-azaheptane, NH₂(CH₂)₃· NH(CH₂)₃NH₂) and sym-homospermidine (1,9-diamino-5-azanonane, NH₂(CH₂)₄NH· (CH₂)₄NH₂) are present in the thermophile cells in addition to spermidine (1,8-diamino-4-azaoctane, NH₂(CH₂)₃NH(CH₂)₄NH₂).