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.

Association between uncooperativeness and the glucose metabolism of patients with chronic behavioral disorders after severe traumatic brain injury: a cross-sectional retrospective study.

BAKGROUND: Patients with behavioral disorders following severe traumatic brain injury (sTBI) often have disorders of consciousness that make expressing their emotional distress difficult. However, no standard method for assessing the unsettled and unforeseen responses that are associated with behavioral disorders has yet to be established. Because the thalamus is known to play a role in maintaining consciousness and cognition, we used 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG-PET/CT) to examine the association between brain glucose metabolism in the thalamus and behavioral disorders. METHODS: We retrospectively analyzed 70 consecutive patients with sTBI who had been involved in motor vehicle accidents. To assess behavioral disorders, we evaluated 18 symptoms using the Brief Psychiatric Rating Scale (BPRS): Emotional Withdrawal, Conceptual Disorganization, Tension, Mannerisms and Posturing, Motor Retardation, Uncooperativeness, Blunted Affect, Excitement, Somatic Concern, Anxiety, Feeling of Guilt, Grandiosity, Depressive Mood, Hostility, Suspiciousness, Hallucinatory Behavior, Unusual Thought Content, and Disorientation. First, we identified clinical characteristics of sTBI patients with behavioral disorders. Next, we retrospectively analyzed 18F-FDG-PET/CT data to assess how thalamic activity was related with abnormal behaviors. RESULTS: Twenty-six patients possessed the minimum communicatory ability required for psychiatric interview. Among them, 15 patients (57.7%) were diagnosed with behavioral disorder, 14 of whom had reached a stable psychiatric state after about 426.6 days of treatment. Excitement (13 patients) and uncooperativeness (10 patients) were the most frequently observed symptoms. Available 18F-FDG-PET/CT data indicated that thalamic glucose metabolism was imbalanced and lateralized (p = 0.04) in 6 patients who exhibited uncooperativeness. CONCLUSIONS: Behavioral symptoms of excitement and uncooperativeness were common in patients with sTBI, although most symptoms improved as the chronic stage continued. Our data support the idea that imbalanced laterality of glucose metabolism in the thalamus might be related to behavioral disorders characterized by uncooperativeness. TRIAL REGISTRATION: UMIN 000029531. Registered 27 March 2017, retrospectively registered.

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.

Crystal structure of a Ca2+-dependent regulator of flagellar motility reveals the open-closed structural transition.

Sperm chemotaxis toward a chemoattractant is very important for the success of fertilization. Calaxin, a member of the neuronal calcium sensor protein family, directly acts on outer-arm dynein and regulates specific flagellar movement during sperm chemotaxis of ascidian, Ciona intestinalis. Here, we present the crystal structures of calaxin both in the open and closed states upon Ca2+ and Mg2+ binding. The crystal structures revealed that three of the four EF-hands of a calaxin molecule bound Ca2+ ions and that EF2 and EF3 played a critical role in the conformational transition between the open and closed states. The rotation of α7 and α8 helices induces a significant conformational change of a part of the α10 helix into the loop. The structural differences between the Ca2+- and Mg2+-bound forms indicates that EF3 in the closed state has a lower affinity for Mg2+, suggesting that calaxin tends to adopt the open state in Mg2+-bound form. SAXS data supports that Ca2+-binding causes the structural transition toward the closed state. The changes in the structural transition of the C-terminal domain may be required to bind outer-arm dynein. These results provide a novel mechanism for recognizing a target protein using a calcium sensor protein.

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.

Crystal structure of dibenzothiophene sulfone monooxygenase BdsA from Bacillus subtilis WU-S2B.

The dibenzothiophene (DBT) sulfone monooxygenase BdsA from Bacillus subtilis WU-S2B catalyzes the conversion of DBT sulfone to 2'-hydroxybiphenyl 2-sulfinate. We report the crystal structures of BdsA at a resolution of 2.80 Å. BdsA exists as a homotetramer with a dimer-of-dimers configuration in the crystal, and the interaction between E288 and R296 in BdsA is important for tetramer formation. A structural comparison with homologous proteins shows that the orientation and location of the α9-α12 helices in BdsA are closer to those of the closed form than those of the open form in the EDTA monooxygenase EmoA. Proteins 2017; 85:1171-1177. © 2017 Wiley Periodicals, Inc.

Insight into the transition between the open and closed conformations of Thermus thermophilus carboxypeptidase.

Carboxypeptidase cleaves the C-terminal amino acid residue from proteins and peptides. Here, we report the functional and structural characterizations of carboxypeptidase belonging to the M32 family from the thermophilic bacterium Thermus thermophilus HB8 (TthCP). TthCP exhibits a relatively broad specificity for both hydrophilic (neutral and basic) and hydrophobic (aliphatic and aromatic) residues at the C-terminus and shows optimal activity in the temperature range of 75-80 °C and in the pH range of 6.8-7.2. Enzyme activity was significantly enhanced by cobalt or cadmium and was moderately inhibited by Tris at 25 °C. We also determined the crystal structure of TthCP at 2.6 Å resolution. Two dimer types of TthCP are present in the crystal. One type consists of two subunits in different states, open and closed, with a Cα RMSD value of 2.2 Å; the other type consists of two subunits in the same open state. This structure enables us to compare the open and closed states of an M32 carboxypeptidase. The TthCP subunit can be divided into two domains, L and S, which are separated by a substrate-binding groove. The L and S domains in the open state are almost identical to those in the closed state, with Cα RMSD values of 0.84 and 0.53 Å, respectively, suggesting that the transition between the open and closed states proceeds with a large hinge-bending motion. The superimposition between the closed states of TthCP and BsuCP, another M32 family member, revealed that most putative substrate-binding residues in the grooves are oriented in the same direction.

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.

A new target region for changing the substrate specificity of amine transaminases.

(R)-stereospecific amine transaminases (R-ATAs) are important biocatalysts for the production of (R)-amine compounds in a strict stereospecific manner. An improved R-ATA, ATA-117-Rd11, was successfully engineered for the manufacture of sitagliptin, a widely used therapeutic agent for type-2 diabetes. The effects of the individual mutations, however, have not yet been demonstrated due to the lack of experimentally determined structural information. Here we describe three crystal structures of the first isolated R-ATA, its G136F mutant and engineered ATA-117-Rd11, which indicated that the mutation introduced into the 136(th) residue altered the conformation of a loop next to the active site, resulting in a substrate-binding site with drastically modified volume, shape, and surface properties, to accommodate the large pro-sitagliptin ketone. Our findings provide a detailed explanation of the previously reported molecular engineering of ATA-117-Rd11 and propose that the loop near the active site is a new target for the rational design to change the substrate specificity of ATAs.

L-allo-threonine aldolase with an H128Y/S292R mutation from Aeromonas jandaei DK-39 reveals the structural basis of changes in substrate stereoselectivity.

L-allo-Threonine aldolase (LATA), a pyridoxal-5'-phosphate-dependent enzyme from Aeromonas jandaei DK-39, stereospecifically catalyzes the reversible interconversion of L-allo-threonine to glycine and acetaldehyde. Here, the crystal structures of LATA and its mutant LATA_H128Y/S292R were determined at 2.59 and 2.50 Šresolution, respectively. Their structures implied that conformational changes in the loop consisting of residues Ala123-Pro131, where His128 moved 4.2 Šoutwards from the active site on mutation to a tyrosine residue, regulate the substrate specificity for L-allo-threonine versus L-threonine. Saturation mutagenesis of His128 led to diverse stereoselectivity towards L-allo-threonine and L-threonine. Moreover, the H128Y mutant showed the highest activity towards the two substrates, with an 8.4-fold increase towards L-threonine and a 2.0-fold increase towards L-allo-threonine compared with the wild-type enzyme. The crystal structures of LATA and its mutant LATA_H128Y/S292R reported here will provide further insights into the regulation of the stereoselectivity of threonine aldolases targeted for the catalysis of L-allo-threonine/L-threonine synthesis.

Crystal structure of the novel haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 reveals a special halide-stabilizing pair and enantioselectivity mechanism.

A novel haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 belongs to the HLD-II subfamily and hydrolyzes brominated and iodinated compounds, leading to the generation of the corresponding alcohol, a halide ion, and a proton. Because DatA possesses a unique Asn-Tyr pair instead of the Asn-Trp pair conserved among the subfamily members, which was proposed to keep the released halide ion stable, the structural basis for its reaction mechanism should be elucidated. Here, we determined the crystal structures of DatA and its Y109W mutant at 1.70 and 1.95 Å, respectively, and confirmed the location of the active site by using its novel competitive inhibitor. The structural information from these two crystal structures and the docking simulation suggested that (i) the replacement of the Asn-Tyr pair with the Asn-Trp pair increases the binding affinity for some halogenated compounds, such as 1,3-dibromopropane, mainly due to the electrostatic interaction between Trp109 and halogenated compounds and the change of substrate-binding mode caused by the interaction and (ii) the primary halide-stabilizing residue is only Asn43 in the wild-type DatA, while Tyr109 is a secondary halide-stabilizing residue. Furthermore, docking simulation using the crystal structures of DatA indicated that its enantioselectivity is determined by the large and small spaces around the halogen-binding site.

Crystal structure and site-directed mutagenesis analyses of haloalkane dehalogenase LinB from Sphingobium sp. strain MI1205.

The enzymes LinB(UT) and LinB(MI) (LinB from Sphingobium japonicum UT26 and Sphingobium sp. MI1205, respectively) catalyze the hydrolytic dechlorination of ß-hexachlorocyclohexane (ß-HCH) and yield different products, 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL), respectively, despite their 98% identity in amino acid sequence. To reveal the structural basis of their different enzymatic properties, we performed site-directed mutagenesis and X-ray crystallographic studies of LinB(MI) and its seven point mutants. The mutation analysis revealed that the seven amino acid residues uniquely found in LinB(MI) were categorized into three groups based on the efficiency of the first-step (from ß-HCH to PCHL) and second-step (from PCHL to TCDL) conversions. Crystal structure analyses of wild-type LinB(MI) and its seven point mutants indicated how each mutated residue contributed to the first- and second-step conversions by LinB(MI). The dynamics simulation analyses of wild-type LinB(MI) and LinB(UT) revealed that the entrance of the substrate access tunnel of LinB(UT) was more flexible than that of LinB(MI), which could lead to the different efficiencies of dehalogenation activity between these dehalogenases.

Calaxin drives sperm chemotaxis by Ca²âº-mediated direct modulation of a dynein motor.

Sperm chemotaxis occurs widely in animals and plants and plays an important role in the success of fertilization. Several studies have recently demonstrated that Ca(2+) influx through specific Ca(2+) channels is a prerequisite for sperm chemotactic movement. However, the regulator that modulates flagellar movement in response to Ca(2+) is unknown. Here we show that a neuronal calcium sensor, calaxin, directly acts on outer-arm dynein and regulates specific flagellar movement during sperm chemotaxis. Calaxin inhibition resulted in significant loss of sperm chemotactic movement, despite normal increases in intracellular calcium concentration. Using a demembranated sperm model, we demonstrate that calaxin is essential for generation and propagation of Ca(2+)-induced asymmetric flagellar bending. An in vitro motility assay revealed that calaxin directly suppressed the velocity of microtubule sliding by outer-arm dynein at high Ca(2+) concentrations. This study describes the missing link between chemoattractant-mediated Ca(2+) signaling and motor-driven microtubule sliding during sperm chemotaxis.