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.     

Ca2+-dependent modulation of intracellular Mg2+ concentration with amiloride and KB-R7943 in pig carotid artery

It has long been recognized that magnesium is associated with several important diseases, including diabetes, hypertension, cardiovascular, and cerebrovascular diseases. In the present study, we measured the intracellular free Mg2+ concentration ([Mg2+]i) using 31P nuclear magnetic resonance (NMR) in pig carotid artery smooth muscle. In normal solution, application of amiloride (1 mm) decreased [Mg2+]i by approximately 12% after 100 min. Subsequent washout tended to further decrease [Mg2+]i. In contrast, application of amiloride significantly increased [Mg2+]i (by approximately 13% after 100 min) under Ca2+-free conditions, where passive Mg2+ influx is facilitated. The treatments had little effect on intracellular ATP and pH (pHi). Essentially the same Ca2+-dependent changes in [Mg2+]i were produced with KB-R7943, a selective blocker of reverse mode Na+-Ca2+ exchange. Application of dimethyl amiloride (0.1 mM) in the presence of Ca2+ did not significantly change [Mg2+]i, although it inhibited Na+-H+ exchange at the same concentration. Removal of extracellular Na+ caused a marginal increase in [Mg2+]i after 100-200 min, as seen in intestinal smooth muscle in which Na+-Mg2+ exchange is known to be the primary mechanism of maintaining a low [Mg2+]i against electrochemical equilibrium. In Na+-free solution (containing Ca2+), neither amiloride nor KB-R7943 decreased [Mg2+]i, but they rather increased it. The results suggest that these inhibitory drugs for Na+-Ca2+ exchange directly modulate Na+-Mg2+ exchange in a Ca2+-dependent manner, and consequently produce the paradoxical decrease in [Mg2+]i in the presence of Ca2+.     

Characteristic domain motion in the ribosome recycling factor revealed by 15N NMR relaxation experiments and molecular dynamics simulations

The backbone dynamics of ribosome recycling factor (RRF) from Escherichia coli in water were characterized by (15)N NMR relaxation analysis and molecular dynamics (MD) simulation. RRF is composed of two domains connected by a joint region that consists of two peptide chains, such that the overall structure seems to mimic that of tRNA. MD trajectories indicated that the relative orientation of domains varies on the nanosecond time scale. We analyzed the observed (15)N T(1), T(2), and NOE using an extended model-free spectral density function in which the domain motions with a nanosecond time scale were considered. At 30 degrees C, the order parameters of slow motion () were determined to be approximately 0.9 for domain I and 0.7 for domain II, respectively. These values indicate that domain I is nearly fixed on the molecular diffusion frame, and domain II is wobbling in a cone for which the semi-angle is about 30 degrees.     

Characterization of a cytosolic NiFe-hydrogenase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

We have identified an NiFe-hydrogenase exclusively localized in the cytoplasm of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (T. kodakaraensis hydrogenase). A gene cluster encoding T. kodakaraensis hydrogenase was composed of four open reading frames (hyhBGSL(Tk)), where the hyhS(Tk) and hyhL(Tk) gene products corresponded to the small and the large subunits of NiFe-hydrogenase, respectively. A putative open reading frame for hydrogenase-specific maturation endopeptidase (hybD(Tk)) was found downstream of the cluster. Polyclonal antibodies raised against recombinant HyhL(Tk) were used for immunoaffinity purification of T. kodakaraensis hydrogenase, leading to a 259-fold concentration of hydrogenase activity. The purified T. kodakaraensis hydrogenase was composed of four subunits (beta, gamma, delta, and alpha), corresponding to the products of hyhBGSL(Tk), respectively. Each alphabetagammadelta unit contained 0.8 mol of Ni, 22.3 mol of Fe, 21.1 mol of acid-labile sulfide, and 1.01 mol of flavin adenine dinucleotide. The optimal temperature for the T. kodakaraensis hydrogenase was 95 degrees C for H(2) uptake and 90 degrees C for H(2) production with methyl viologen as the electron carrier. We found that NADP(+) and NADPH promoted high levels of uptake and evolution of H(2), respectively, suggesting that the molecule is the electron carrier for the T. kodakaraensis hydrogenase.     

Oleomonas sagaranensis gen. nov., sp. nov., represents a novel genus in the alpha-Proteobacteria

A Gram-negative bacterium was previously isolated from an oil field in Shizuoka, Japan, and designated strain HD-1. Here we have performed detailed characterization of the strain, and have found that it represents a novel genus. The 16S rRNA sequence of strain HD-1 displayed highest similarity to various uncultured species (86.7-99.7%), along with 86.2-88.2% similarity to sequences from Azospirillum, Methylobacterium, Rhizobium, and Hyphomicrobium, all members of the alpha-Proteobacteria. Phylogenetic analysis revealed that HD-1 represented a deep-branched lineage among the alpha-Proteobacteria. DNA-DNA hybridization analysis with Azospirillum lipoferum and Hyphomicrobium vulgare revealed low levels of similarity among the strains. We further examined the biochemical properties of the strain under aerobic conditions. Among carbon sources, ethanol, n-propanol, n-butanol, and n-tetradecanol were the most preferred, while acetate, propionate, and pyruvate also supported high levels of growth. The strain could also grow on aromatic compounds such as toluene, benzene and phenol, and aliphatic hydrocarbons such as n-octane and n-tetradecane. In contrast, glycerol and various sugars, including glucose, fructose, maltose, and lactose, failed to support growth of HD-1. Under an anaerobic gas phase with butanol as the carbon source, little increase in cell weight was observed with the addition of several possible electron acceptors. As strain HD-1 represents a novel genus in the alpha-Proteobacteria, we designated the strain as Oleomonas sagaranensis gen. nov., sp. nov., strain HD-1.     

Unique presence of a manganese catalase in a hyperthermophilic archaeon,Pyrobaculum calidifontis VA1

We had previously isolated a facultatively anaerobic hyperthermophilic archaeon, Pyrobaculum calidifontis strain VA1. Here, we found that strain VA1, when grown under aerobic conditions, harbors high catalase activity. The catalase was purified 91-fold from crude extracts and displayed a specific activity of 23,500 U/mg at 70 degrees C. The enzyme exhibited a K(m) value of 170 mM toward H(2)O(2) and a k(cat) value of 2.9 x 10(4) s(-1).subunit(-1) at 25 degrees C. Gel filtration chromatography indicated that the enzyme was a homotetramer with a subunit molecular mass of 33,450 Da. The purified catalase did not display the Soret band, which is an absorption band particular to heme enzymes. In contrast to typical heme catalases, the catalase was not strongly inhibited by sodium azide. Furthermore, with plasma emission spectroscopy, we found that the catalase did not contain iron but instead contained manganese. Our biochemical results indicated that the purified catalase was not a heme catalase but a manganese (nonheme) catalase, the first example in archaea. Intracellular catalase activity decreased when cells were grown anaerobically, while under aerobic conditions, an increase in activity was observed with the removal of thiosulfate from the medium, or addition of manganese. Based on the N-terminal amino acid sequence of the purified protein, we cloned and sequenced the catalase gene (kat(Pc)). The deduced amino acid sequence showed similarity with that of the manganese catalase from a thermophilic bacterium, Thermus sp. YS 8-13. Interestingly, in the complete archaeal genome sequences, no open reading frame has been assigned as a manganese catalase gene. Moreover, a homology search with the sequence of kat(Pc) revealed that no orthologue genes were present on the archaeal genomes, including those from the "aerobic" (hyper)thermophilic archaea Aeropyrum pernix, Sulfolobus solfataricus, and Sulfolobus tokodaii. Therefore, Kat(Pc) can be considered a rare example of a manganese catalase from archaea.     

Tk-PTP,protein tyrosine/serine phosphatase from hyperthermophilic archaeon Thermococcus kodakaraensis KOD1: enzymatic characteristics and identification of its substrate proteins

The Tk-ptp gene encoding a protein tyrosine phosphatase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 was cloned and biochemical characteristics of the recombinant protein (Tk-PTP) were examined. A series of mutants, D63A (replacing Asp-63 with Ala), C93S, C93A, R99K, and R99M, were also constructed and analyzed. Two unique features were found. First, the Tk-PTP showed the phosphatase activity not only toward phosphotyrosine but also toward phosphoserine. Second, the conserved Asp-63, which corresponds to a critical residue among other known PTPs, was not essential for catalysis. Cys-93 and Arg-99 residues played a crucial role in substrate binding and catalysis. To know a specific substrate for Tk-PTP, C93S mutant was used to trap substrate proteins from cell extract of KOD1. Phenylalanyl-tRNA synthetase subunit beta-chain, one of the gene products of RNA terminal phosphate cyclase operon and phosphomannomutase, was identified, suggesting that they functioned for phosphate donation.     

Isolation and characterization of a halotolerant <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">subtilis</Emphasis> BBK-1 which produces three kinds of lipopeptides: bacillomycin L,plipastatin, and surfactin

Twenty-three halotolerant and biosurfactant producing strains were collected from salty conditions in central Thailand. One of the strains designated BBK-1 produced the biosurfactants with the highest activity. BBK-1 was isolated from fermented foods and was identified as B. subtilis based on its physiological characteristics and 16S rRNA gene sequence. We show that the strain grows in media containing NaCl up to 16% (w/v) and produces biosurfactants in NaCl up to 8%. We found that B. subtilis BBK-1 produces three kinds of surface-active lipopeptides simultaneously. By their respective molecular weights and amino acid compositions, it is indicated that these lipopeptides are bacillomycin L, plipastatin, and surfactin. In order to analyze the production mechanism of lipopeptides further in the strain, a generally important biosynthetic gene encoding 4'-phosphopantetheinyl transferase was cloned and sequenced. The gene existed in a single copy in the genome and the deduced amino acid sequence was almost identical to that of Lpa-14 from B. subtilis strain RB14, which co-produces iturin A and surfactin.     

Probenecid-induced changes in the clearance of pranoprofen enantiomers

Probenecid is known to inhibit the elimination of several acidic drugs. Its influence on the pharmacokinetics of pranoprofen was investigated in rabbit after a single intravenous injection of racemic mixture (5 mg/kg). Levels of (-)-(R)- and (+)-(S)-pranoprofen and their glucuronide (after hydrolysis with sodium hydroxide) were determined in plasma, urine, and several tissues. The plasma concentration of the (+)-(S)-isomer was higher than that of the (-)-(R)-form. Oral coadministered probenecid (100 mg/kg) resulted in an increased plasma concentration of both enantiomers. Probenecid reduced the apparent total clearance and excretion of pranoprofen enantiomers in urine. It had a slight effect on the tissue distribution of pranoprofen at the dose used, but significantly reduced the formation of glucuronide for both enantiomers to the same extent in kidney microsomes. The differences caused by probenecid were significant with respect to its ability to inhibit glucuronidation in the kidney and subsequent excretion into urine, but enantioselective effects were negligible.