Simple and Rapid Detection of ESBL blaSHV gene from an Urban River in Tokyo by Loop-Mediated Isothermal Amplification.

Extended-spectrum ß-lactamases (ESBLs) are produced mainly by gram-negative bacteria in Enterobacteriaceae. One of the major types of ESBLs is sulfhydryl variable (SHV) -type ESBL. Herein, we attempted to develop a simple and rapid method for the detection of the ESBL blaSHV gene by loop-mediated isothermal amplification (LAMP) . The five-primer set designed could amplify blaSHV gene at an isothermal temperature of 65℃. The detection limit of the LAMP method with the LF loop primer was 1 copy/tube, which was 10,000-fold more sensitive than that of the conventional PCR. The LAMP assay could also detect the direct amplification of blaSHV gene from a single river water sample in Tokyo. The LAMP method has great potential for applications in hospital, soil and water environment, food, and livestock.

Draft Genome Sequences of the Lipid-Degrading Bacteria Moritella sp. Strains F1 and F3, Isolated from Mesopelagic Seawater from the Sagami Trough, in Japan.

Moritella sp. strains F1 and F3 are lipid-degrading bacteria that were isolated from intermediate water from the Sagami Trough, in Japan. We present the draft genome sequences of these two strains, which have 4,983,334 bp and 4,967,310 bp, respectively.

Comparison of Benzoï¼»aï¼½pyrene-Degrading Activities between Olleya sp. ITB9 Isolated from Tokyo Bay and Other Type Strains of the Genus Olleya.

Benzoï¼»aï¼½pyrene (BaP) is one of the strongest carcinogenic compounds among polycyclicaromatic hydrocarbons (PAHs) .We previously identified the ITB9 strain of Olleya species, which shows BaP-degrading activity; our report was the first about BaP degradation by the genus Olleya. In this study, BaP-degradation efficiency by ITB9 was about 50% when the strain was suspended in 20 ml of L9 liquid medium with 100 µg/ml BaP and 0.2 M NaCl, with pH 8.0, and incubated at 25℃ for 5 days. Under the same conditions, all four type strains (O. marilimosa CIP108537, O. aquimaris KCTC22661, O. namhaensis KCTC23673, and O. algicola KCTC22024) also showed BaP-degrading activities, at efficiencies ranging from 49% to 63%. Olleya sp. ITB9 and O. aquimaris KCTC22661 were found to be in the same clade in the phylogenetic tree of the genus Olleya, given that the homology of 16S rRNA gene sequences between ITB9 and KCTC22661 was 99.77%.

Q301P mutant of Vibrio PR protease affects activities under low-temperature and high-pressure conditions.

We characterized a protease of the M4 family from the cold-adapted Vibrio sp. Pr21 that was isolated from seawater at 320-m deep in Sagami Bay, Japan, and named it as PR protease based on the strain name Pr21. The PR protease had activities at 10-60 °C and 0.1-350 MPa, with the optimal temperature and pressure at 40 °C and 250 MPa. The mutant 10C9 (Q301P) obtained by error-prone PCR had higher activities than the wild-type enzyme at 10-60 °C, and the Q301P mutation contributed to the increase of the activity. The specific activity value of 10C9 was also higher than that of the wild-type enzyme at 0.1-200 MPa, but the specific activity ratios (1.28-1.59) of 10C9/wild-type enzyme at 50-200 MPa at 30 °C were smaller than those at 10-60 °C (1.73-4.39) at 0.1 MPa. The catalytic efficiency value of 10C9 was lower than that of the wild-type enzyme at 200 MPa. The homology models of PR protease suggested that the side chain of Q301 was hydrogen-bonded with the carbonyl oxygen atom of the main chain of N234 in the wild-type enzyme, and P301 had no contact with N234 in 10C9. The break of the hydrogen bond in 10C9 might strengthen the increase of the flexibility of the ß-sheet near the substrate binding pocket under high-temperature conditions, whereas the flexibility of the ß-sheet in 10C9 might be moderately increased compared to that in the wild-type enzyme under high-pressure conditions.

Antibiotic-resistance of Fecal Coliforms at the Bottom of the Tama River, Tokyo.

We investigated the midstream bottom of the Tama River, which flows through Tokyo, to evaluate the occurrence and degree of antibiotic-resistant fecal coliforms including multidrug-resistant fecal coliforms. The genera Klebsiella and Escherichia were the major isolates among the fecal coliforms. For the genus Klebsiella, the highest antibiotic resistance was observed for ampicillin (100%) , followed by kanamycin, tetracycline, cefotaxime, and cefoxitin. The highest resistance to E. coli was found for kanamycin (44.4%) , followed by ampicillin, tetracycline, chloramphenicol, amoxicillin-clavulanate, cefotaxime, ceftazidime, and aztreonam. Multidrug resistance （MDR） was observed in three E. coli isolates. A double disc synergy test confirmed the production of extended-spectrum ß-lactamases by the six-antibiotic-resistant isolate E. coli hfa7, and the strain had CTX-M-1 group gene. Assessments of antibiotic-resistant fecal coliforms at the bottom of the Tama River are important toward the goals of preventing the spread of antibiotic-resistant fecal coliforms in humans, animals, and the environment.

Enzymatic properties and the gene structure of a cold-adapted laminarinase from Pseudoalteromonas species LA.

We isolated a laminarin-degrading cold-adapted bacterium strain LA from coastal seawater in Sagami Bay, Japan and identified it as a Pseudoalteromonas species. We named the extracellular laminarinase LA-Lam, and purified and characterized it. LA-Lam showed high degradation activity for Laminaria digitata laminarin in the ranges of 15-50°C and pH 5.0-9.0. The major terminal products degraded from L. digitata laminarin with LA-Lam were glucose, laminaribiose, and laminaritriose. The degradation profile of laminarioligosaccharides with LA-Lam suggested that the enzyme has a high substrate binding ability toward tetrameric or larger saccharides. Our results of the gene sequence and the SDS-PAGE analyses revealed that the major part of mature LA-Lam is a catalytic domain that belongs to the GH16 family, although its precursor is composed of a signal peptide, the catalytic domain, and three-repeated unknown regions.

Neutralization of acidic drainage by Cryptococcus sp. T1 immobilized in alginate beads.

We isolated Cryptococcus sp. T1 from Lake Tazawa's acidic water in Japan. Cryptococcus sp. T1 neutralized an acidic casamino acid solution (pH 3.0) and released ammonia from the casamino acids to aid the neutralization. The neutralization volume was estimated to be approximately 0.4 mL/h. The casamino acids' amino acids decreased (1.24→0.15 mM); ammonia increased (0.22→0.99 mM). We neutralized acidic drainage water (1 L) from a Tamagawa River neutralization plant, which was run through the column with the T1-immobilized alginate beads at a flow rate of 0.5 mL/min, and observed that the viscosity, particle size and amounts of the alginate beads affected the acidic drainage neutralization with an increase of the pH value from 5.26 to 6.61 in the last fraction. An increase in the Al concentration decreased Cryptococcus sp. T1's neutralization ability. After 48 h, the pH of acidic water with 50 mg/L Al was apparently lower than that without Al. Almost no pH increase was observed at 75 mg/L.

Isolation of aquatic yeasts with the ability to neutralize acidic media, from an extremely acidic river near Japan's Kusatsu-Shirane Volcano.

The Yukawa River is an extremely acidic river whose waters on the east foot of the Kusatu-Shirane Volcano (in Gunma Prefecture, Japan) contain sulfate ions. Here we isolated many acid-tolerant yeasts from the Yukawa River, and some of them neutralized an acidic R2A medium containing casamino acid. Candida fluviatilis strain CeA16 had the strongest acid tolerance and neutralizing activity against the acidic medium. To clarify these phenomena, we performed neutralization tests with strain CeA16 using casamino acid, a mixture of amino acids, and 17 single amino acid solutions adjusted to pH 3.0, respectively. Strain CeA16 neutralized not only acidic casamino acid and the mixture of amino acids but also some of the acidic single amino acid solutions. Seven amino acids were strongly decomposed by strain CeA16 and simultaneously released ammonium ions. These results suggest strain CeA16 is a potential yeast as a new tool to neutralize acidic environments.

Citeromyces matritensis M37 is a salt-tolerant yeast that produces ethanol from salted algae.

Algae are referred to as a third-generation biomass for ethanol production. However, salinity treatment is a problem that needs to be solved, because algal hydrolysates often contain high salt. Here, we isolated the salt-tolerant ethanol-producing yeast Citeromyces matritensis M37 from the east coast of Miura Peninsula in Japan. This yeast grew under osmotic stress conditions (20% NaCl or 60% glucose). It produced 6.55 g/L ethanol from YPD medium containing 15% NaCl after 48 h, and the ethanol accumulation was observed even at 20% NaCl. Using salted Undaria pinnatifida (wakame), we obtained 6.33 g/L glucose from approx. 150 g/L of the salted wakame powder with acidic and heat pretreatment followed by enzymatic saccharification, and the ethanol production reached 2.58 g/L for C. matritensis M37. The ethanol concentration was 1.4 times higher compared with that using the salt-tolerant ethanol-producing yeast Zygosaccharomyces rouxii S11.

Draft Genome Sequence of a Benzo[a]pyrene-Degrading Bacterium, Olleya sp. Strain ITB9.

Olleya sp. ITB9 is a benzo[a]pyrene-degrading bacterium, isolated from surface water near a waste treatment plant at Tokyo Bay, Japan. Here, we present the draft genome sequence of this strain, which consists of 58 contigs corresponding to 3.4 Mb and a G+C content of 31.2%.

Isolation and characterization of benzo[a]pyrene-degrading bacteria from the Tokyo Bay area and Tama River in Japan.

Benzo[a]pyrene (BaP) is one of the polycyclic aromatic hydrocarbons, and has serious detrimental effects on human health and aquatic environments. In this study, we isolated nine bacterial strains capable of degrading BaP from the Tokyo Bay area and Tama River in Japan. The isolated bacteria belonged to the phyla Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes, indicating that the BaP-degrading bacteria were widely present in the hydrosphere. ITB11, which shared 100% 16S rRNA identity with Mesoflavibacter zeaxanthinifaciens in the phylum Bacteroidetes, showed the highest degradation of BaP (approximately 86%) among the nine isolated strains after 42 days. Moreover, it was found that three of the nine isolated strains collectively removed 50-55% of BaP during the first 7 days. Growth measurement of M. zeaxanthinifaciens revealed that the strain utilized BaP as a sole carbon and energy source and salicylate acted only as an inducer of BaP degradation.

Strategy for cold adaptation of the tryptophan synthase α subunit from the psychrophile Shewanella frigidimarina K14-2: crystal structure and physicochemical properties.

To investigate the molecular basis of cold adaptation of enzymes, we determined the crystal structure of the tryptophan synthase α subunit (SfTSA) from the psychrophile Shewanella frigidimarina K14-2 by X-ray analysis at 2.6-Å resolution and also examined its physicochemical properties. SfTSA was found to have the following characteristics: (i) The stabilities against heat and denaturant of SfTSA were lower than those of an α subunit (EcTSA) from Escherichia coli. This lower equilibrium stability originated from both a faster unfolding rate and a slower refolding rate; (ii) the heat denaturation of SfTSA was completely reversible at pH 7.0 and the solubility of denatured SfTSA was higher than that of denatured EcTSA. The two-state transition of denaturation for SfTSA was highly cooperative, whereas the denaturation process of EcTSA was considerably more complex and (iii) the global structure of SfTSA was quite similar to those of α subunits from other species. Relative to those other proteins, SfTSA exhibited an increase in cavity volume and a decrease in the number of ion pairs. SfTSA also lacks a hydrogen bond near loop B, related to catalytic function. These characteristics of SfTSA might provide the conformational flexibility required for catalytic activity at low temperatures.

Potential of carotenoids in aquatic yeasts as a phylogenetically reliable marker and natural colorant for aquaculture.

Apart from Xanthophyllomyces dendrorhous, pink colony-forming yeasts have not been examined as a pigmentation source in captive animals. In this study, aquatic yeasts were screened with a view to abundances of carotenoids. Phylogenetic analyses of these caroetnoid-rich yeasts based on large subunit ribosomal RNA gene (LSU rDNA) partial sequences showed that all belonged to the order Sporidiobolales. Both the qualitative and the quantitative differences in carotenoids between the yeasts appeared to be consistent with their phylogenetic affiliations. This information might be useful in the selection of pigment-rich yeasts containing specific carotenoids from a large number of strains. We also found, for the first time, the potential of a pigment-rich Rhodotorula strain as a colorant for aquaculture. The integuments of tilapia and carp fed the alkali-treated cells of strain Rhodotorula dairenensis Sag 17 were pigmented after 3 months of cultivation. The fish integuments retained the yeast carotenes shortly after the start of feeding, and were converted to the fish-specific xanthophylls in vivo.

Discovery of serum L-amino acid oxidase in the rockfish Sebastes schlegeli: isolation and biochemical characterization.

Fish have a complex innate defense mechanism against microbial invasion. In particular, epidermal mucus and serum in fish play important roles in innate immunity and contain a variety of bioactive substances such as complements, lectins and lysozymes, involved in host defense. Recently, L-amino acid oxidases (LAOs) with antibacterial activity were isolated from the skin and/or gill mucous secretions of rockfish, great sculpin and flounder, and were identified to be a novel type of antibacterial protein in the integument of fish. In the present study, we found LAO activity in the serum of rockfish Sebastes schlegeli. The LAO was isolated from the serum by sequential column chromatography of Con-A lectin affinity chromatography, anion exchange HPLC, hydroxyapatite HPLC and gel filtration HPLC, and characterized. The LAO (a molecular mass of 160 kDa) comprised subunits with a molecular mass of 53 kDa and showed strict substrate specificity, catalyzing only L-lysine with Km 0.37 mM and kcat 57.1s(-1). The serum LAO exhibited a broad antibacterial activity against Gram positive and negative bacteria, most potently against Aeromonas hydrophila and Aeromonas salmonicida with a minimum inhibitory concentration of 0.078 µg/mL. This is the first report of LAO in the serum of fish and its involvement in innate immunity in the rockfish body.

Isolation and identification of dibutyl phthalate-degrading bacteria from hydrospheres in Tokyo.

Dibutyl phthalate (DBP) is used widely as a plasticizer and is thought to negatively affect various organisms. To isolate and investigate DBP-degrading bacteria from hydrospheres in Tokyo, strains were selected on YNB medium containing DBP as the sole carbon source, and candidate strains were identified by zones of clearing around the colonies. Degradation of DBP by the strains was subsequently measured with HPLC, and bacterial identification was accomplished using 16S rDNA sequences. Nineteen strains of DBP degraders were isolated from activated sludge in a sewage treatment plant, from Tokyo Bay, and from the Takahama Canal. These strains degraded 16.8%-88.0% of DBP (0.1%, v/v) for 2 weeks and were identified as several species of Acinetobacter, as well as Tsukamurella tyrosinosolvens, Ochrobactrum anthropi, and Staphylococcus saprophyticus. Commercially available strains of Acinetobacter were also found to degrade DBP.

Purification and molecular cloning of SE-cephalotoxin, a novel proteinaceous toxin from the posterior salivary gland of cuttlefish Sepia esculenta.

Cephalopods contain toxins in their salivary glands, presumably to paralyze prey animals such as crabs and bivalves. Proteinaceous toxins (called cephalotoxins) with crab lethality have previously been purified from three species of octopodiform cephalopods (octopuses) but their detailed properties and primary structures have remained unknown. In this study, salivary glands of six species of decapodiform cephalopods were newly found to be toxic; three species of cuttlefish were lethal only to crabs and three species of squid to both mice and crabs. A proteinaceous toxin (named SE-cephalotoxin) in the salivary gland of cuttlefish Sepia esculenta was soluble only in high-salt solvents. This unique solubility enabled us to purify SE-cephalotoxin by gel filtration HPLC and hydroxyapatite HPLC. SE-cephalotoxin was shown to be a 100 kDa monomeric glycoprotein with an LD(50) (against crabs) of 2 microg/kg. Based on the determined partial amino acid sequence, a full-length cDNA (3402 bp) coding for SE-cephalotoxin was cloned by RT-PCR and RACE. The SE-cephalotoxin precursor protein (1052 amino acid residues) is composed of a signal peptide (region 1-21), propeptide (region 22-29) and mature protein (region 30-1052). A database search failed to find any proteins sharing homology with SE-cephalotoxin.

Kunitz-type protease inhibitors from acrorhagi of three species of sea anemones.

Sea anemones are rich in biologically active polypeptides such as toxins and protease inhibitors. These polypeptides have so far been isolated from whole bodies, tentacles or secreted mucus. Recently, two novel peptide toxins with crab lethality have been isolated from acrorhagi (specialized aggressive organs elaborated by only certain species of sea anemones belonging to the family Actiniidae) of Actinia equina. This prompted us to survey biologically active polypeptides in the acrorhagi of two species of sea anemones, Anthopleura aff. xanthogrammica and Anthopleura fuscoviridis. No potent crab lethality was displayed by the acrorhagial extracts of both species. However, significantly high protease inhibitory activity was instead detected in the acrorhagial extracts of the two species and also in that of A. equina. From the acrorhagi of A. equina, A. aff. xanthogrammica and A. fuscoviridis, one (AEAPI), one (AXAPI) and two (AFAPI-I and AFAPI-III) protease inhibitors were isolated, respectively. The complete amino acid sequences of the four inhibitors were elucidated by N-terminal sequencing and sequencing of the C-terminal peptide fragment produced upon asparaginylendopeptidase digestion. The determined amino acid sequences revealed that all the four inhibitors are new members of the Kunitz-type protease inhibitor family.

Novel peptide toxins from the sea anemone Stichodactyla haddoni.

Four peptide toxins, SHTX I-III with crab-paralyzing activity and SHTX IV with crab lethality, were isolated from the sea anemone Stichodactyla haddoni and their primary structures elucidated by protein sequencing and cDNA cloning. SHTX I (new toxin, 28 residues), II (analogue of SHTX I, 28 residues) and III (Kunitz-type protease inhibitor, 62 residues) are potassium channel toxins and SHTX IV (48 residues) is a member of the type 2 sea anemone sodium channel toxins. The precursor protein of SHTX IV is composed of a signal peptide, propart and mature peptide, while the propart is missing in that of SHTX III. In addition to these four toxins, an epidermal growth factor-like peptide was detected in S. haddoni by RT-PCR.

Specific and rapid analysis of ubiquinones using Craven's reaction and HPLC with postcolumn derivatization.

A new method for the analysis of ubiquinones in various samples was developed using an HPLC system with postcolumn derivatization. Craven's reaction, a specific color reaction for the analysis of ubiquinones, was used in the system. Because the reaction progressed in organic solvents that contained ubiquinones and ethylcyanoacetate under an alkaline condition, the selectivity for ubiquinone detection was higher than that for ubiquinone detection using the nonderivatized ultraviolet detection system at 275 nm, a system widely used for the analysis of ubiquinones. The new detection system can avoid the adverse effects of impurities. Furthermore, it can confirm specificity by stopping the color reaction under a neutral condition. The detection limit for ubiquinone-10 was 1 ng (1.2 pmol). A good linearity for the calibration curve was observed in the range of 11.7 pmol to 11.7 nmol. To investigate the possible application of this method, various samples, such as soybean capsules used as a dietary supplement and biological materials (rice as well as bovine plasma and liver samples), were applied to the system and their ubiquinone contents were quantified. This method is thought to be widely and conveniently applicable for determining the level of ubiquinones because of its high selectivity for ubiquinone detection.

Phosphorylation of Rho-associated kinase (Rho-kinase/ROCK/ROK) substrates by protein kinases A and C.

Rho-associated kinase (Rho-kinase/ROCK/ROK) is a serine/threonine kinase and plays an important role in various cellular functions. The cAMP-dependent protein kinase (protein kinase A/PKA) and protein kinase C (PKC) are also serine/threonine kinases, and directly and/or indirectly take part in the signal transduction pathways of Rho-kinase. They have similar phosphorylation site motifs, RXXS/T and RXS/T. The purpose of this study was to identify whether sites phosphorylated by Rho-kinase could be targets for PKA and PKC and to find peptide substrates that are specific to Rho-kinase, i.e., with no phosphorylation by PKA and PKC. A total of 18 substrates for Rho-kinase were tested for phosphorylation by PKA and PKC. Twelve of these sites were easily phosphorylated. These results mean that Rho-kinase substrates can be good substrates for PKA and/or PKC. On the other hand, six Rho-kinase substrates showing no or very low phosphorylation efficiency (<20%) for PKA and PKC were identified. Kinetic parameters (K(m) and k(cat)) showed that two of these peptides could be useful as substrates specific to Rho-kinase phosphorylation.