Antifungal activity of novel indole derivative from endophytic bacteria Pantoea ananatis 4G-9 against Mycosphaerella musicola

An endophytic bacterium isolated from banana G-9 (AAA genotype) leaves exhibited strong antagonistic activity against Mycosphaerella musicola. The isolate was identified as Pantoea ananatis 4G-9 by 16S rRNA sequence analysis. Secondary metabolite obtained from P. ananatis 4G-9 was found to have antifungal activity. The active compound was purified from crude extract using column chromatography. Purity of the active compound was assessed using high-performance liquid chromatography. Spectral analysis of compound using infrared, mass spectrometry and nuclear magnetic resonance indicated that the compound structure is an indole derivative. The compound showed strong and dose-dependent antifungal activity against M. musicola. This is the first report on P. ananatis isolated as an endophyte from banana leaves and its antifungal activity against M. musicola.     

Detection and quantitative analysis of zoospores of Pythium porphyrae, causative organism of red rot disease in Porphyra, by competitive PCR

The detection and quantitative analysis of Pythium porphyraezoospores was performed by PCR using PP-1 and PP-2 primers specific tothe internal transcribed spacer region of P. porphyrae. To estimatethe amount of fungal zoospores of P. porphyrae, an internal standardplasmid (pPPISC) containing a modified DNA fragment was constructed. Both ends of this fragment were complementary to the PCR primers. Amplification using primers PP-1 and PP-2 produced DNA fragments ofapproximately 700 and 400 bp from the target DNA of P. porphyraezoospores and from the pPPISC, respectively. To perform quantitativePCR, known quantities of pPPISC were added to reaction mixturescontaining the experimental DNAs extracted from zoospores. After aco-amplification reaction, the two different sized PCR products wereseparated by agarose gel electrophoresis and visualized by ethidium bromidestaining. The number of zoospores was estimated by comparing thefluorescence intensities of the PCR products using a charge-coupled deviceimage analyzer. The results show that competitive PCR using P.porphyrae specific primers and competitor pPPISC are useful tools for thequantitative analysis of P. porphyrae zoospores in seawater from Porphyra cultivation farms.     

Phylogenetic analysis of Syntrophobacter wolinii reveals a relationship with sulfate-reducing bacteria

A 16S rRNA sequence analysis of Syntrophobacter wolinii was done by using PCR amplification of the 16S rRNA-genes from DNA isolated from the S. wolinii-Desulfovibrio sp. coculture. Phylogenetic analysis using the obtained sequence revealed that S. wolinii was not related to bacteria growing syntrophically on other fatty acids than propionate, but was related to sulfate-reducing bacteria. The closest related bacteria are Desulfomonile tiedjei and Desulfoarculus baarsii.     

Purification and characterization of a novel O-methyltransferase from Flammulina velutipes

An enzyme catalyzing the methylation of phenolic hydroxyl groups in polyphenols was identified from mycelial cultures of edible mushrooms to synthesize O-methylated polyphenols. Enzyme activity was measured to assess whether methyl groups were introduced into (?)-epigallocatechin-3-O-gallate (EGCG) using SAM as a methyl donor, and (?)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3″Me), (?)-epigallocatechin-3-O-(4-O-methyl)-gallate (EGCG4″Me), and (?)-epigallocatechin-3-O-(3,5-O-dimethyl)-gallate (EGCG3″,5″diMe) peaks were detected using crude enzyme preparations from mycelial cultures of Flammulina velutipes. The enzyme was purified using chromatographic and two-dimensional electrophoresis. The purified enzyme was subsequently analyzed on the basis of the partial amino acid sequence using LC–MS/MS. Partial amino acid sequencing identified the 17 and 12 amino acid sequences, VLEVGTLGGYSTTWLAR and TGGIIIVDNVVR. In database searches, these sequences showed high identity with O-methyltransferases from other mushroom species and completely matched 11 of 17 and 9 of 12 amino acids from five other mushroom O-methyltransferases.     

Detection of the protozoan parasite Theileria annulata in Hyalomma ticks by the polymerase chain reaction

Adult Hyalomma ticks were examined for the presence of Theileria annulata infection using the Polymerase Chain Reaction (PCR). A 372 bp DNA fragment derived from the small ribosomal RNA gene of T. annulata was amplified from 45 out of 50 (90%) H. dromedarii ticks and from 36 out of 50 (72%) H. marginatum marginatum ticks. No product was amplified from non-infected control ticks. Restriction enzyme digestion with Sac II confirmed that the product was derived from the targeted T. annulata gene. As a further confirmation it was shown that both species of Hyalomma ticks were able to transmit T. annulata to experimental calves. PCR detection of Theileria parasites in ticks was compared with conventional staining of dissected salivary glands using methyl green pyronin and its comparative advantages are discussed.     

Stimulation of adventitious rooting of Taxus species by thiamine

Summary Results obtained from using root inducing compounds on Taxus species cuttings suggested that rooting could be significantly enhanced by the presence of thiamine. This observation was verified using a root inducing solution containing a set concentration of IBA (0.2%), NAA (0.1%), and supplemented with various concentrations of thiamine. The best rooting response for Taxus cuspidata stem cuttings was found using this solution supplemented with 0.08% thiamine. Rooted cuttings were easily established and developed into vigorous plants. In addition, Taxus brevifolia shoots obtained from tissue cultures via in vitro organogenesis also responded favorably to this 0.08% thiamine supplemented rooting solution.     

Detection of Legionella pneumophila serogroup 1 antigen in respiratory samples using an immunochromatographic membrane test

The immunochromatographic membrane test (ICT) efficacy of Legionella antigen detection (Binax Now Legionella®) was evaluated using respiratory samples, including bronchial washings (44 cases) and sputum (128 cases), from suspected Legionella pneumonia patients. The ICT results using respiratory samples agreed well with isolation of L. pneumophila SG1 and ICT using urines.     

Biochemical characterization and molecular modeling of a unique lipase from Staphylococcus arlettae JPBW‐1

A three‐step purification of a unique lipase with halo‐, solvent‐, detergent‐, and thermo‐tolerance from Staphylococcus arlettae JPBW‐1 gave raise to a 27‐fold purification with a specific activity of 32.5 U/mg. The molecular weight of the purified lipase was estimated to be 45 kDa using SDS–PAGE, and its amino acid sequence was characterized using MALDI‐TOF‐MS analysis. The sequence obtained from MALDI‐TOF‐MS showed significant similarity with the capsular polysaccharide biosynthesis protein (CapD) of Staphylococcus aureus through comparative modeling approach using ROBETTA server. Identification of responsible fragments for homodimer formation was performed using comparative modeling and substrate binding domain through C‐terminus matching of this new lipase with the CapD of Staphylococcus aureus was executed. Thus, the experimental coupled molecular modeling postulated a structure–activity relationship of lipase from S. arlettae JPBW‐1, a potential candidate for detergent, leather, pulp, and paper industries.     

Use of entomopathogenic fungi and insecticide against some insect pests attacking peanuts and sugarbeet in Egypt

Larvae of Spodoptera littoralis, Spodoptera exigua and nymphs of Aphis craccivora were treated with four compounds, the entomopathogenic fungi (two commercial products Biosect, Biover) and the bacterium (Dipel 2x) and one insecticide (Lannet). These compounds were sprayed on the insects attacking peanut (Arachis hypogaea) weekly. A significant decrease in the population of S. littoralis larvae was recorded after one week from the application among first and second instars. The first instar reached zero in all treatments compared with control (309 control was 3.9 larvae/day on the same date). Larvae of S. exigua was markedly decreased by using Biosect, Biover, Dipel 2x or Lannet. Nymphs of A. craccivora decreased from 64.6 to 0.1 nymph/day after seven days from spraying Biosect, while it decreased from 72.0 to 0.6 nymph/day by using biover, and decreased from 69.8 to 0.0 nymph/day by using lannet while the population stayed between 60.8 before application and 72.8 nymph/day after seven days from spraying.

Suspensions of Metarhizium anisopliae was prepared by harvesting spores with distilled water. The concentration was calculated using hemocitometer slide (3.5 × 10⁷ spores/ml). Commercial (3.5 × 10⁷ spores/ml) suspension of Beauveria bassiana was used for dusting and spraying in distilled water. Studies were carried out in both the laboratory and field. The entomopathogenic fungi showed a high effect on P. mixta either in the laboratory and field experiments.     

Metabarcoding of PalEnDNA as An Efficient Tool to Recover Ancient Bacterial Diversity

Past bacterial diversity of a paleosol was reconstructed using metabarcoding of paleo environmental DNA (PalEnDNA). The paleosol was subsampled from a sediment core which was excavated from a palaeo beach-ridge located 2.6?km away from present sea shore and identified that it was deposited under marine influence ～6000?years ago, using geological proxies. The bacterial community contained 37 bacterial phyla and dominated by Proteobacteria, followed by Bacteroidetes, Firmicutes, Actinobacteria, Verrucomicrobia, and Chloroflexi. The bacterial community was a mix-up of marine and terrestrial population, and thereby diversity was higher than marine populations. The result shows metabarcoding of PalEnDNA can effectively reconstruct past bacterial community structure.     

Predicting distributions of seven bitterling fishes in northern Kyushu,Japan

The distributions of seven bitterling species and subspecies—Tanakia lanceolata, T. limbata, Acheilognathus tabira nakamurae, A. rhombeus, Rhodeus ocellatus kurumeus, R. ocellatus ocellatus, and R. atremius atremius—in northern Kyushu were predicted using generalized linear models (GLMs) in order to provide information helpful for conserving native bitterlings and preventing the expansion of alien bitterling species. Predictions were made according to the following procedure: (1) a set of GLMs for each species was formulated using environmental data from 710 sites that were derived using digital maps and GIS software, from which the best fit model for each species was selected using the Akaike information criterion for predicting the fish occurrence, (2) model performance was evaluated based on the receiver-operating characteristics (ROC) analysis using occurrence and environmental data from 362 sites, and (3) potential distributions of the bitterling were analyzed using the best fit models and environmental data for 1,272 sites, of which 200 data points without occurrence data were prepared. The best fit models revealed that 4–6 environmental factors were important in predicting seven bitterling distributions, which was supported by the area under the ROC curve (AUC) values of these fishes ranging from 0.753 to 0.927. The AUC values in model evaluation were significantly greater than 0.5 for six fishes, suggesting the moderate accuracies of these best fit models for predicting the fish distributions. These predictive models can be used for evaluating potential native bitterling richness and the potential distribution expansion of an alien subspecies.     

Intra-hospital dissemination of clinical and environmental isolates of Stenotrophomonas maltophilia from Tehran

Stenotrophomonas maltophilia isolates are responsible for various hospital-acquired infections and are particularly increasing in the immunocompromised patients. The aim of this study was to determine the clonal relatedness between S. maltophilia isolates originating from the clinic and environment. A total of 150 S. maltophilia isolates from patients and 1108 environmental samples obtained in three hospitals from Tehran. Following molecular identification targeting 23S rRNA gene, the clonal relatedness of the environmental and clinical isolates was determined using pulsed field gel electrophoresis (PFGE). Of the 150 clinical and 18 environmental isolates identified using phenotypic tests, the speciation of 120 and 15 was confirmed by targeting the 23S rRNA gene. The 24 common pulsotypes (PTs) and 32 single PTs were identified by PFGE. Only a small cluster was shared among the clinic and environment within a hospital; therefore, the intra-hospital dissemination of certain isolates of S. maltophilia among the clinic and environment was demonstrated.     

Hydroxylation of daidzein by CYP107H1 from Bacillus subtilis 168

CYP107H1, from Bacillus subtilis 168 known as fatty acid hydroxylase, showed the ortho-specific hydroxylation activity to daidzein, when coupled to the putidaredoxin reductase (camA) and putidaredoxin (camB) from Pseudomonas putida as the redox partners. The electron transfer system of the three proteins was constructed in Escherichia coli BL21 (DE3) system using the two plasmids containing different selection markers. The daidzein hydroxylation was demonstrated with recombinant whole cell and in vitro system using the artificial redox partner for electron transfer. The identification of the hydroxylation reaction yielding 7,3′,4′-trihydroxyisoflavone was elucidated using gas chromatography mass spectrometry (GC–MS). This oxidizing activity of CYP107H1 towards daidzein represents the new hydroxylation of aromatic compound as substrate.     

Glucose-induced production of a Penicillium purpurogenum xylanase by Aspergillus nidulans

The heterologous secretion of xylanase B from Penicillium purpurogenum using glucose as inducer was performed in Aspergillus nidulans. For this purpose, plasmid pEVXB, containing the xylanase B cDNA (including its own signal peptide) under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter, was constructed and used to transform A. nidulans. Analysis of transformed clones showed that several of them secreted extracellular xylanase activity when grown in a medium containing glucose. The clone showing the highest xylanase activity was chosen for further work. When this clone was grown on glucose, xylanase activity (0.72 U/ml), was detected in the culture supernatant. This was confirmed by a zymogram analysis and by the amplification of xynB cDNA from this clone. To our knowledge, this is the first example of the production of a xylanase from Penicillium in heterologous fungal hosts using glucose as inducer.     

Direct production of 4-hydroxybenzoic acid from cellulose using cellulase-displaying Pichia pastoris

4-hydroxybenzoic acid (4-HBA) is an industrially important aromatic compound, and there is an urgent need to establish a bioprocess to produce this compound in a sustainable and environmentally friendly manner from renewable feedstocks such as cellulosic biomass. Here, we developed a bioprocess to directly produce 4-HBA from cellulose using a recombinant Pichia pastoris strain that displays heterologous cellulolytic enzymes on its cell surface via the glycosylphosphatidylinositol (GPI)-anchoring system. β-glucosidase (BGL) from Aspergillus aculeatus, endoglucanase (EG) from Trichoderma reesei, and cellobiohydrolase (CBH) from Talaromyces emersonii were co-displayed on the cell surface of P. pastoris using an appropriate GPI-anchoring domain for each enzyme. The cell-surface cellulase activity was further enhanced using P. pastoris SPI1 promoter- and secretion signal sequences. The resulting strains efficiently hydrolyzed phosphoric acid swollen cellulose (PASC) to glucose. Then, we expressed a highly 4-HBA-resistant chorismate pyruvate-lyase (UbiC) from Providencia rustigianii in the cellulase-displaying strain. This strain produced 975 mg/L of 4-HBA from PASC, which corresponding to 36.8% of the theoretical maximum yield, after 96 h of batch fermentation without the addition of commercial cellulase. This 4-HBA yield was over two times higher than that obtained from glucose (12.3% of the theoretical maximum yield). To our knowledge, this is the first report on the direct production of an aromatic compound from cellulose using cellulase-displaying yeast.     

Species status of Incisitermes spp. (Isoptera: Kalotermitidae) in Japan

The species status of Japanese populations of Incisitermes immigrans from Iwo and Minami Daito Islands was examined using mitochondrial 16S molecular barcode sequences. The molecular sequences of these two populations were compared to those of other Incisitermes spp. deposited in the GenBank database using a maximum likelihood phylogenetic analysis. This analysis suggested that the Minami Daito population is indeed I. schwarzi, as suspected previously, while the sequence of the Iwo Island population was identical to that of authentic I. immigrans. In addition to I. minor, which is recorded from middle and southern Japan, the presence of three Incisitermes species in Japan was confirmed.     

Transgenic Brassica carinata as a vehicle for the production of recombinant proteins in seeds

Hirudin, a blood anticoagulant protein from leeches, and β-glucuronidase were produced in Brassica carinata Braun (Ethiopian mustard) seeds using oleosin as a carrier. Cotyledonary petioles were infected with Agrobacterium strains containing oleosin-glucuronidase (pCGNOBPGUS-A) or oleosin-hirudin (pCGN-OBHIRT) constructs. Polymerase chain reaction and neomycin phosphotransferase II enzyme assays confirmed the presence of the fusion genes in plants regenerating under selection. The fusion polypeptides were correctly expressed and targeted to the oil-bodies of the seeds with high fidelity (ca. 90%). Recombinant protein was purified from all other cellular protein by a simple flotation process and cleaved from oil-bodies using the endoprotease, Factor Xa. Hirudin activity was measured using a colorimetric thrombin inhibition assay and an activity in the range of 0.2–0.4 antithrombin units per milligram of oil-body protein was detected. B. carinata offers an attractive alternative for the production of recombinant proteins using oleosin technology. Received: 20 March 1997 / Revision received: 5 June 1997 / Accepted: 30 July 1997     

Isolation and physical characterization of streptomycete plasmids

Summary Covalently closed circular DNA was isolated from a strain of Streptomyces coelicolor ATCC 10147 and from a strain of Streptomyces coelicolor subspecies flavus ATCC 19894, using two different methods. The two plasmids were of uniform monomer size: 8.9 kb for pS 10147, the plasmid from S. coelicolor ATCC 10147, and around 125 kb for the plasmid from S. coelicolor ATCC 19894.A restriction enzyme map was constructed for pS 10147, using seven enzymes. Four of the enzymes, (BamHI, Bgl,II, PvuII, and XhoI) cut pS 10147 once while PstI made two cuts. The GC content of this plasmid was calculated to be 72%. The possible utilisation of pS 10147 as a cloning vector in Streptomyces is discussed.