Birgul Ozcan

Anoxybacillus salavatliensis sp. nov., an α-glucosidase producing, thermophilic bacterium isolated from Salavatli, Turkey

A novel moderately thermophilic, Gram‐positive staining, rod‐shaped, spore‐forming, motile, facultative anaerobic, and α‐glucosidase producing strain A343T, was isolated from a high temperature well‐pipeline sediment sample in Salavatli province of Aydin, Turkey. Growth was observed at 37–69 ºC (optimum 60 °C), at pH 5.5–9.5 (optimum 8.0–9.0) and at salinities from 0 to 4.5% (w/v) (optimum 2%). Strain A343T was able to grow on a wide range of carbon sources. Gelatin and starch utilization, amylase, catalase and oxidase activities, reduction of nitrate to nitrite were all positive. The G+C content of the genomic DNA was 45.1 mol%. The major menaquinone was MK‐7. The dominant cellular fatty acids were: iso‐C15:0, C16:0, and iso‐C17:0. The phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain A343T belonged to the genus Anoxybacillus. The 16S rRNA gene sequence similarity between strain A343T and the type strains of recognized Anoxybacillus species was ranged from 95.8 to 99.4%. DNA‐DNA hybridization revealed low homology with its closest relative Anoxybacillus kamchatkensis (49.7%). In addition to the total cell protein profiles, the Rep‐PCR and the intergenic 16S–23S rRNA gene fingerprinting profiles differentiated strain A343T from all of the reference Anoxybacillus species used. Based upon phenotypic, phylogenetic and chemotaxonomic evidence, strain A343T was assigned to a new species within the genus Anoxybacillus, A. salavatliensis sp. nov. (The type strain A343T= DSM 22626T = NCIMB 14579T). The 16S rRNA gene nucleotide sequence of strain A343T is available in the GenBank database under the accession number – EU326496. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Isolations of α-glucosidase-producing thermophilic bacilli from hot springs of Turkey

From 42 different hot springs in six provinces belonging to distinct geographical regions of Turkey, 451 thermophilic bacilli were isolated and 67 isolates with a high amylase activity were selected to determine the α-glucosidase production capacities by using pNPG as a substrate. α-Glucosidase production capacities of the isolates varied within the range from 77.18 to 0.001 U/g. Eleven of our thermophilic bacilli produced α-glucosidase at significant levels comparable with that of the reference strains tested; thus, five strains, F84b (77.18 U/g), A333 (48.64 U/g), F84a (36.64 U/g), E134 (32.09 U/g), and A343 (10.79 U/g), were selected for further experiments. 16S rDNA sequence analysis revealed that these selected isolates all belonged to thermophilic bacilli 16S rDNA genetic group 5, four of them representing the genus Geobacillus, while strain A343 had an uncultured bacterium as the closest relative. Changes in α-glucosidase levels in the intracellular and extracellular fractions were determined during 48-h cultivation of A333, A343, F84a, F84b, E134, and the reference strain G. stearothermophilus ATCC 12980. According to α-glucosidase production type and enzyme levels in intracellular and extracellular fractions, Geobacillus spp. A333, F84a, and F84b were defined as extracellular enzyme producers, whereas the thermophilic bacterium A343 was found to be an intracellular α-glucosidase producer, similar to ATCC 12980 strain. Geobacillus sp. E134 differed in α-glucosidase production type from all tested isolates and the reference strain; it was described as a membrane-associated cell-bound enzyme producer. In this study, apart from screening a great number of new thermophilic bacilli from the hot springs of Turkey, which have not yet been thoroughly studied, five new thermostable α-1,4-glucosidase-producing bacilli that have biotechnological potential with α-glucosidases located at different cell positions were obtained.

Characterization of extremely halophilic Archaea isolated from saline environment in different parts of Turkey

Ninety-five extremely halophilic strains were isolated from six distinct saline regions of Turkey by using complex medium containing 25% NaCl. The selected regions are Tuz Golu (salt lake), Ankara; Aci Lake, Denizli; Salda Lake, Denizli; Seyfe Lake, Kyrsherhir; Tuzla Lake, Kayseri; and Bolluk Lake, Konya. The isolated strains were tested for motility, gram reaction, cell and colony morphologies, pigmentation, biochemical characteristics, and antibiotic sensitivities. According to membrane glycerol diether moieties and antibiotic susceptibilities, all isolated strains were found to belong to the domain Archaea. All isolates were examined for the presence of plasmids by agarose gel electrophoresis and it was established that most isolates contained plasmids that varied in number and whose molecular sizes ranged from 1 to 36.9 kbp. Whole-cell protein profiles from isolates were analyzed by SDS-PAGE and a similarity dendogram was constructed using the UPGMA method. Significant similarities and differences were observed among the isolates. The strains were clustered in eight groups and ten of our isolates were placed in the same group with the standard strains. The current study represents the first isolation and characterization of such a large collection of archeal strains from Turkey.

Anoxybacillus calidus sp. nov., a thermophilic bacterium isolated from soil near a thermal power plant

A novel thermophilic, Gram-stain-positive, facultatively anaerobic, endospore-forming, motile, rod-shaped bacterium, strain C161ab(T), was isolated from a soil sample collected near Kizildere, Saraykoy-Buharkent power plant in Denizli. The isolate could grow at temperatures between 35 and 70 °C (optimum 55 °C), at pH 6.5-9.0 (optimum pH 8.0-8.5) and with 0-2.5 % NaCl (optimum 0.5 %, w/v). The strain formed cream-coloured, circular colonies and tolerated up to 70 mM boron. Its DNA G+C content was 37.8 mol%. The peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. Strain C161ab(T) contained menaquinones MK-7 (96 %) and MK-6 (4 %). The major cellular fatty acids were iso-branched fatty acids: iso-C15 : 0 (52.2 %) and iso-C17 : 0 (28.0 %,) with small amounts of C16 : 0 (7.4 %). Phylogenetic analysis based on the 16S rRNA gene revealed 94.6-96.8 % sequence similarity with all recognized species of the genus Anoxybacillus. Strain C161ab(T) showed the greatest sequence similarity to Anoxybacillus rupiensis DSM 17127(T) and Anoxybacillus voinovskiensis DSM 17075(T), both had 96.8 % similarity to strain C161ab(T), as well as to Anoxybacillus caldiproteolyticus DSM 15730(T) (96.6 %). DNA-DNA hybridization revealed low levels of relatedness with the closest relatives of strain C161ab(T), A. rupiensis (21.2 %) and A. voinovskiensis (16.5 %). On the basis of the results obtained from phenotypic, chemotaxonomic, genomic fingerprinting, phylogenetic and hybridization analyses, the isolate is proposed to represent a novel species, Anoxybacillus calidus sp. nov. (type strain C161ab(T) = DSM 25520(T) = NCIMB 14851(T)).

Antimicrobial and Antioxidant Activities of Various Extracts of Verbascum antiochium Boiss. (Scrophulariaceae)

Verbascum antiochium Boiss., a member of the Scrophulariaceae family, is endemic to Turkey. The extracts obtained from V. antiochium by increased polarity and direct methanol extraction were tested by the agar well diffusion method against various Gram-positive and Gram-negative bacteria and one fungus. The methanol/water extract exhibited a larger inhibition zone against both the Gram-negative and Gram-positive bacteria than the other extracts. Haemophilus influenzae was found to be the most sensitive bacterium among the bacteria tested. The antioxidant activities of the methanolic extract of V. antiochium were examined by two complementary test systems. The 50% inhibition activity of the methanolic extract of V. antiochium against the free radical 2,2-diphenyl-1-picrylhydrazyl was determined as 4.80 mg/mL. In the case of the linoleic acid system, oxidation of linoleic acid was inhibited by the methanolic extract of V. antiochium with 79.92% inhibition, which is close to the value of the synthetic antioxidant reagent, tert-butylated hydroxytoluene. The total phenolic components of V. antiochium were determined to be 92.71 mg of gallic acid equivalents/g. Iridoid glycosides, flavonoids, and saponins were detected as the major chemical constituents in the extract.

Phylogenetic analysis and characterization of lipolytic activity of halophilic archaeal isolates

Five isolates designated as B45, D83A, A206A, A85 and E49 found to possess lipolytic activities were taxonomically classified on the basis of their phylogenetic, phenotypic and chemotaxonomic characteristics. The isolates were determined to be gram-negative, catalase and oxidase positive, hydrolyzing Tween 80 and 60 but not starch, need 3.5–4 M NaCl for optimal growth and lack of anaerobic growth with arginine or DMSO. All isolates had the highest lipolytic activity at pH 8.5. Lipase and esterase activities increased with salt concentration up to 3–4.5 M NaCl, and decreased at 5 M NaCl. Esterase and lipase showed their maximal activities at 50–55°C and 60–65°C, respectively. The phylogenetic tree constructed by the neighbor-joining method indicated that the strain B45 and A85 were closely related to the members of genera Halovivax and Natrinema, respectively. The closest relative of the strain A206A and D83A were found to be Haloterrigena saccharevitans. The strain E49 displayed a more distant relationship to known strains.

A Novel Application for Determination of Germin Gene Products In Situ

ABSTRACT In situ nucleic acid hybridization is one of the most powerful techniques developed for localizing the expression site of a particular gene at the cell, tissue and organ levels. This method is especially useful in understanding the function of specific gene products in particular tissues and the relation between tissue function and its localization in the whole structure of an organ. In this system, labeled anti-sense RNA probes were used to hybridize with desired mRNA while labeled sense probes were used as negative control. In general, each of these labeled probes applied to successive sections rather then the same section. That might cause some uncertainty if there are any spatial differences in the expression of gene products. In the current study, in our knowledge for the first time, anti-sense and sense probes were applied to the same sample to overcome any spatial differentiation of gene expression. The technique was successfully used to localize a plant gene product by applying anti-sense probes to the one site of the section while the other site of the same section was reacted with sense probes for control hybridization.

Spatial Localization of Germin-Like Oxalate Oxidase Genes in Developing Wheat Shoots

ABSTRACT When quiescent embryos in dry grains of ripened wheat are isolated and provided with ample water and oxygen at an appropriate temperature, they begin to grow very rapidly. The onset of the growth is signaled by nascent synthesis of germin genes which encodes a relatively rare, water-soluble homop entameric glycoprotein. Germin is resistant to pepsin digestion under conditions that lead to hydrolysis of virtually all other proteins in wheat embryos. Germin proteins have oxalate oxidase activity, an activity that degrades oxalic acid to generate hydrogen peroxide which involves in many aspects of plant development. Following 48-hour imbibition on water, wheat embryos give rise to distinguishable shoot and roots. Shoots comprise coleoptile, leaf primordium and shoot apex. In the current study, non-radioactively labeled germin riboprobes were prepared by in vitro transcription. The riboprobes were used to search and localize germin mRNAs in sections taken from throughout of shoots. The results revealed that although leaf primordium and shoot apex did not show any signals of the presence of germin mRNAs, coleoptiles as a whole tissue displayed germin gene expression on epidermal cells and vascular bundle sheath cells. Among the sections taken from different parts of shoots, the sections from middle part gave the strongest signals on coleoptile cells.