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    ABSTRACT: The chitinase B (chiB) and C (chiC) genes and flanking regions from a local isolate of Serratia marcescens were cloned individually and sequenced. Results showed that these chiB and chiC genes have a 96 % maximum similarity with chiB and chiC from different S. marcescens species (GenBank numbers Z36295.1 and AJ630582.1, respectively). The amplified chiB fragment, including some upstream and downstream regions, is 1,689-bp long with an open reading frame of 1,500 bp. The amplified fragment of chiC is 1,844 bp with an open reading frame of 1,443 bp. These sequences were submitted to the GenBank with accession numbers JX847796 (chiB) and JX847797 (chiC). Putative promoter regions and Shine-Dalgarno sequences were identified in both genes. The genes were cloned into a shuttle vector and the constructs were designated as pHYSB and pHYSC, respectively. Both plasmids were introduced separately into kurstaki and israelensis strains of Bacillus thuringiensis and the insecticidal activities of the engineered B. thuringiensis strains were assayed in larvae of Galleria mellonella and adult of Drosophila melanogaster. Engineered B. thuringiensis strains showed higher insecticidal activity than parental strain and the parental S. marcescens. In addition, pHYSB and pHYSC were stable over 16 daily passages under non-selective conditions in transformed B. t. israelensis 5724 strain.
    Current Microbiology 06/2013;
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    ABSTRACT: A Gram-staining-negative, catalase- and oxidase- positive, strictly aerobic, rod-shaped bacterial strain isolated from fresh water in Trabzon, Turkey and designated MK3T was characterized by phenotypic and molecular methods in order to determine its phylogenetic position. On the basis of 16S rRNA gene sequence similarity, strain MK3T was shown to belong to the genus Flavobacterium, being most closely related to Flavobacterium ceti CECT 7184 (93.6 %). Sequence similarity with other validly named Flavobacterium species was less than 91.6%. Phenotypic and chemotaxonomic data supported the affiliation of strain MK3T to the genus Flavobacterium. The only menaquinone was MK-6; the major fatty acids were iso-C15: 0 (45.2%), summed feature 9 (C16:0 10-methyl and/or iso C17:1ω9c; 20.4%) and summed feature 3 (C16:1ω7c and/or C16:1ω6c; 13.3 %) and the major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid and two unidentified phospholipids. The G+C content of the genomic DNA was 38.6 %mol. The results of physiological and biochemical tests allowed strain MK3T to be distinguished phenotypically from Flavobacterium ceti CECT 7184T. Strain MK3T, therefore, represents a novel species of the genus Flavobacterium, for which the name Flavobacterium anatoliense sp. nov. is proposed. An emended description of Flavobacterium ceti is also proposed. The type strain is MK3T (=LMG 26441T =NCCB 100384T ).
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 10/2012;
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    ABSTRACT: This study concerns a new neogregarine parasitic in the great spruce bark beetle Dendroctonus micans (Kugelann) (Curculionidae, Scolytinae). The rate of infection was high, reaching 27.3%. There was no difference in the rate of infection of male and female beetles. The life-cycle stages of the pathogen were described by light and electron microscopy. Each gametocyst of the neogregarine included 8-16 actinocephalid oocysts measuring 11.19 ± 0.42 × 4.99 ± 0.25 μm. The described pathogen has the typical characteristics of members of the genus Menzbieria within the order Neogregarinida and it was identified as Menzbieria chalcographi. This is the first record of an infection of D. micans by M. chalcographi. Possibly, this pathogen could be useful for the biological control of this destructive bark beetle.
    Acta Parasitologica 09/2012; 57(3):216-20.
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    ABSTRACT: A novel moderately thermophilic, Gram-positive, endospore-forming, rod-shaped, motile bacteria and alkaline active xylanase producing strain D1021(T) , was isolated from Kaynarca hot spring in the province of Izmir, Turkey and was characterized in order to determine its phylogenetic position. Growth was observed at 35-70 °C (optimum 60 °C), at pH 6.0-10.0 (optimum pH 7.0). The strain D1021(T) grew on a wide range of carbon sources including ribose, xylose, glucose, maltose, sucrose, arabinose, and melibiose. The major isoprenoid quinone was MK-7. 16S rRNA gene sequence analysis showed that strain D1021(T) is related to members of genus Anoxybacillus, with similarities to Anoxybacillus spp. varying from 94.7 to 98.7. The major fatty acids of strain D1021(T) were iso-C(15:0) (57.46%) and iso-C(17:0) (13.98%). The DNA G + C content was 42.9 mol %. DNA-DNA relatedness values between Anoxybacillus spp. and D1021(T) were lower than 70%. On the basis of phenotypic characteristics, rpoB analysis, phylogenetic and DNA-DNA hybridization data, it was proposed that the strain D1021(T) (= DSM 21706(T) = LMG 25303(T) ) should be placed in the genus Anoxybacillus as the type strain of a novel species, Anoxybacillus kaynarcensis sp. nov. The GenBank accession number for the 16S rRNA sequence is EU926955. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).
    Journal of Basic Microbiology 06/2012;
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    ABSTRACT: Chilo iridescent virus (CIV) is an insect virus belonging to the Iridoviridae. The DNA genome (212,482 base pairs) is entirely sequenced, however very little is known about viral gene regulation, expression and function. The structure and transcriptional regulation of the CIV 012L gene is investigated in this study. Infection of Bombyx mori SPC-BM-36 cells in the presence of Ara-C (inhibits DNA replication) or cycloheximide (inhibits protein synthesis), followed by RT-PCR on isolated total RNA, showed that CIV 012L is transcribed as an immediate-early gene. Detecting the RNA transcript of the CIV 012L early in infection confirmed the data about the temporal class of the gene obtained with the inhibitors. Time course transcription of the gene showed that the transcription starts immediately after infection and reach up to maximum level at 4h p.i. 5' RACE analysis on RNA isolated from CIV-infected BM cells showed that the transcription initiation site is located 30 nucleotides upstream of the translational start codon. To map the limits of the putative promoter of this gene, upstream sequences of various lengths were cloned in front of a firefly luciferase reporter gene. The resulting plasmid constructs were tested in a transfection assay, in which the baculovirus IE-l promoter fused to Renilla luciferase was used as an internal control for transfection efficiency. A gradual reduction in luciferase expression occurred as the deletions extended from -200 to -10, relative to the transcription start site. It is clearly shown that sequences between -20 and -10 relative to the transcription start site have key promoter activity for CIV 012L gene. However this key sequence could not be found at the upstream region of CIV's other potential immediate early genes.
    Virus Research 06/2012; 167(2):353-7.
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    ABSTRACT: The gene encoding a xylanase from Geobacillus sp. 71 was isolated, cloned, and sequenced. Purification of the Geobacillus sp 7.1 xylanase, XyzGeo71, following overexpression in E. coli produced an enzyme of 47 kDa with an optimum temperature of 75°C. The optimum pH of the enzyme is 8.0, but it is active over a broad pH range. This protein showed the highest sequence identity (93%) with the xylanase from Geobacillus thermodenitrificans NG80-2. XyzGeo71 contains a catalytic domain that belongs to the glycoside hydrolase family 10 (GH10). XyzGeo71 exhibited good pH stability, remaining stable after treatment with buffers ranging from pH 7.0 to 11.0 for 6 h. Its activity was partially inhibited by Al(3+) and Cu(2+) but strongly inhibited by Hg(2+). The enzyme follows Michaelis-Menten kinetics, with K(m) and V(max) values of 0.425 mg xylan/ml and 500 μmol/min.mg, respectively. The enzyme was free from cellulase activity and degraded xylan in an endo fashion. The action of the enzyme on oat spelt xylan produced xylobiose and xylotetrose.
    MIRCEN Journal of Applied Microbiology and Biotechnology 05/2012; 28(5):1981-8.
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    ABSTRACT: A novel cry2Ab gene was cloned and sequenced from the indigenous isolate of Bacillus thuringiensis subsp. kurstaki. This gene was designated as cry2Ab25 and its sequence revealed an open reading frame of 1,902 bp encoding a 633 aa protein with calculated molecular mass of 70 kDa and pI value of 8.98. The amino acid sequence of the Cry2Ab25 protein was compared with previously known Cry2Ab toxins, and the phylogenetic relationships among them were determined. The deduced amino acid sequence of the Cry2Ab25 protein showed 99% homology to the known Cry2Ab proteins, except for Cry2Ab10 and Cry2Ab12 with 97% homology, and a variation in one amino acid residue in comparison with all known Cry2Ab proteins. The cry2Ab25 gene was expressed in Escherichia coli BL21(DE3) cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the Cry2Ab25 protein is about 70 kDa. The toxin expressed in BL21(DE3) exhibited high toxicity against Malacosoma neustria and Rhagoletis cerasi with 73% and 75% mortality after 5 days of treatment, respectively.
    Journal of Microbiology and Biotechnology 01/2012; 22(1):133-40.
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    ABSTRACT: Leaf rolling is known as a typical response to water deficit in numerous species such as rice, maize, wheat and sorghum. However, it results not only from the water deficit but also from other abiotic stress factors such as salt, temperature, heavy metals and UV radiation. In addition to the abiotic factors, herbivores, viruses, bacteria and fungi are biotic factors of leaf rolling. Leaf rolling is an effective protective mechanism from the effects of high light levels in agricultural fields and protects leaves of unirrigated plants from photodamage. The rolling reduces effective leaf area and transpiration, and thus is a potentially useful drought avoidance mechanism in dry areas. The current review focuses on the recent progress in understanding leaf rolling in relation to abiotic and biotic stress factors, the role of signal molecules, and the mechanisms of gene regulation.
    Plant Science 01/2012; 182:42-8.
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    ABSTRACT: Genome predictions based on selected genes would be a very welcome approach for taxonomic studies. We analyzed three genes, recN, flaA, and ftsY, for determining if these genes are useful tools for systematic analyses in the genus Anoxybacillus. The genes encoding a DNA repair and genetic recombination protein (recN), the flagellin protein (flaA), and GTPase signal docking protein (ftsY) were sequenced for ten Anoxybacillus species. The sequence comparisons revealed that recN sequence similarities range between 61% and 99% in the genus Anoxybacillus. Comparisons to other bacterial recN genes indicated that levels of similarity did not differ from the levels within genus Anoxybacillus. These data showed that recN is not a useful marker for the genus Anoxybacillus. A 550-600-bp region of the flagellin gene was amplified for all Anoxybacillus strains except for Anoxybacillus contaminans. The sequence similarity of flaA gene varies between 61% and 76%. Comparisons to other bacterial flagellin genes obtained from GenBank (Bacillus, Pectinatus, Proteus, and Vibrio) indicated that the levels of similarity were lower (3-42%). Based on these data, we concluded that the variability in this single gene makes it a particularly useful marker. Another housekeeping gene ftsY suggested to reflect the G+C (mol/mol) content of whole genome was analyzed for Anoxybacillus strains. A mean difference of 1.4% was observed between the G+C content of the gene ftsY and the G+C content of the whole genome. These results showed that the gene ftsY can be used to represent whole G+C content of the Anoxybacillus species.
    Folia Microbiologica 01/2012; 57(1):61-9.
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    ABSTRACT: The gypsy moth, Lymantria dispar L. (Lepidoptera, Lymantriidae) is a common pest of forests and fruit trees throughout the world. This insect is also a major serious pest in Turkey. Nowadays L. dispar can be managed by biological control methods especially, using entomopathogenic viruses. The aim of this study is to characterize entomopathogenic viruses and is the first record of nucleopolyhedrovirus isolated from the gypsy moth in Turkey. PIBs obtained from infected larvae were measured and photographed using an Olympus BX51 microscope with a DP-25 digital camera and a DP2-BSW Soft Imaging System and examined with a Philips 208 electron microscope (TEM). The virus had the typical characteristics of nucleopolyhedroviruses. The dimension of the polyhedral inclusion bodies (PIBs) was 2.03±0.25 µm. PIBs varied in size from 1.65 to 2.21 µm and were usually polygonal in shape. Virions in PIBs contained 1 to 8 nucleocapsids per virion. The size of the viral particles was 366.67±54.72 (312-500) x 42.95±6.12 (30-47) nm. The isolation and characterization of a pure isolate of Lymantria dispar multinucleopolyhedrovirus (LdMNPV-TR) from Turkey is presented for the first time.
    Turkiye parazitolojii dergisi / Turkiye Parazitoloji Dernegi = Acta parasitologica Turcica / Turkish Society for Parasitology 01/2012; 36(2):92-5.
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