Sabriye Canakci

Flavobacterium anatoliense sp. nov., isolated from fresh water, and emended description of Flavobacterium ceti

A Gram-staining-negative, catalase- and oxidase-positive, strictly aerobic, rod-shaped bacterial strain isolated from fresh water in Trabzon, Turkey and designated MK3(T) was characterized by phenotypic and molecular methods in order to determine its phylogenetic position. On the basis of 16S rRNA gene sequence similarity, strain MK3(T) was shown to belong to the genus Flavobacterium, being most closely related to Flavobacterium ceti CECT 7184(T) (93.6%). Sequence similarity with other species of the genus Flavobacterium with validly published names was less than 91.6%. Phenotypic and chemotaxonomic data supported the affiliation of strain MK3(T) 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 MK3(T) to be distinguished phenotypically from Flavobacterium ceti CECT 7184(T). Strain MK3(T), therefore, represents a novel species of the genus Flavobacterium, for which the name Flavobacterium anatoliense sp. nov. is proposed. The type strain is MK3(T) (=LMG 26441(T)=NCCB 100384(T)). An emended description of Flavobacterium ceti is also proposed.

Cloning, purification and characterization of a thermostable carboxylesterase from Anoxybacillus sp. PDF1

The gene encoding a carboxylesterase from Anoxybacillus sp., PDF1, was cloned and sequenced. The recombinant protein was expressed in Escherichia coli BL21, under the control of isopropyl-β-D-thiogalactopyranoside-inducible T7 promoter. The enzyme, designated as PDF1Est, was purified by heat shock and ion-exchange column chromatography. The molecular mass of the native protein, as determined by SDS-PAGE, was about 26 kDa. PDF1Est was active under a broad pH range (pH 5.0-10.0) and a broad temperature range (25-90 °C), and it had an optimum pH of 8.0 and an optimum temperature of 60 °C. The enzyme was thermostable carboxylesterase, and did not lose any activity after 30 min of incubation at 60 °C. The enzyme exhibited a high level of activity with p-nitrophenyl butyrate with apparent K(m), V(max), and K(cat) values of 0.348 ± 0.030 mM, 3725.8 U/mg, and 1500 ± 54.50/s, respectively. The effect of some chemicals on the esterase activity indicated that Anoxybacillus sp. PDF1 produce an carboxylesterase having serine residue in active site and -SH groups in specific sites, which are required for its activity.

Anoxybacillus kaynarcensis sp. nov., a moderately thermophilic, xylanase producing bacterium

A novel moderately thermophilic, Gram‐positive, endospore‐forming, rod‐shaped, motile bacteria and alkaline active xylanase producing strain D1021T, 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 D1021T 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 D1021T 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 D1021T were iso‐C15:0 (57.46%) and iso‐C17:0 (13.98%). The DNA G + C content was 42.9 mol %. DNA–DNA relatedness values between Anoxybacillus spp. and D1021T were lower than 70%. On the basis of phenotypic characteristics, rpoB analysis, phylogenetic and DNA–DNA hybridization data, it was proposed that the strain D1021T (= DSM 21706T = LMG 25303T) 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.

Brevibacillus aydinogluensis sp. nov., a moderately thermophilic bacterium isolated from Karakoc hot spring

Two Gram-positive, moderately thermophilic, endospore-forming, rod-shaped, motile bacteria, designated PDF25T and PDF30, were isolated from Karakoc hot spring in the province of Izmir, Turkey, and were characterized in order to determine their phylogenetic positions. 16S rRNA gene sequence analysis revealed that the two strains belonged to the genus Brevibacillus; strain PDF25T showed highest sequence similarity to strain PDF30 (99.4 %) and Brevibacillus thermoruber DSM 7064T (98.5 %). The major fatty acids of strain PDF25T were iso-C15:0 (39.30 %), anteiso-C15:0 (26.10 %) and iso-C16:0 (14.75 %). Polar lipid analysis revealed diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, and a variety of unidentified aminophospholipids, phospholipids and aminolipids. The major isoprenoid quinone was MK-7. The G+C content of the genomic DNA was 56.09 mol%. DNA-DNA hybridization experiments revealed 58 % relatedness between strain PDF25T and B. thermoruber DSM 7064T. Based on these data, the two strains are considered to represent a novel species of the genus Brevibacillus, for which the name Brevibacillus aydinogluensis sp. nov. is proposed. The type strain is PDF25T (=DSM 24395T=LMG 26289T).

The ATPase activity of the G2alt gene encoding an aluminium tolerance protein from Anoxybacillus gonensis G2

The G2ALT gene was cloned and sequenced from the thermophilic bacterium Anoxybacillus gonensis G2. The gene is 666 bp long and encodes a protein 221 amino acids in length. The gene was overexpressed in E. coli and purified to homogeneity and biochemically characterized. The enzyme has a molecular mass of 24.5 kDa and it could be classified as a member of the family of bacterial aluminium resistance proteins based on homology searches. When this fragment was expressed in E. coli, it endowed E. coli with Al tolerance to 500 μM. The purified G2ALT protein is active at a broad pH range (pH 4.0–10.0) and temperature range (25°C–80°C) with optima of 6.0 and the apparent optimal temperature of 73°C respectively. Under optimal conditions, G2ALT exhibited a low ATPase activity with Km− and Vmax− values of 10±0.55 μM and 26.81±0.13 mg Pi released/min/mg enzyme, respectively. The ATPase activity of G2ALT requires Mg2+ and Na+ ions, while Zn2+ and Al3+ stimulate the activity. Cd2+ and Ag+ reduced the activity and Li+, Cu2+, and Co2+ inhibited the activity. Known inhibitors of most ATPases, like such as β-mercaptoethanol and ouabain, also inhibited the activity of the G2ALT. These biochemical characterizations suggested that G2ALT belongs to the PP-loop ATPase superfamily and it can be responsible for aluminium tolerance in A. gonensis G2.

Characterization of a xylanase from a thermophilic strain of Anoxybacillus pushchinoensis A8

A facultatively anaerobic, thermophilic, xylanolytic bacterium was isolated from a sample collected from the Diyadin Hot Springs, Turkey. According to morphological, biochemical and molecular identification, this new strain was suggested to be representative of the Anoxybacillus pushchinoensis and it was designated as Anoxybacillus pushchinoensis strain A8. It exhibited 97% similarity to 16S rRNA gene sequence of A. pushchinoensis and 77% DNA homology by DNA-DNA hybridization studies. Q-sepharose and CM-sepharose chromatography was used to purify an extracellular xylanase to >90% purity from this species. The enzyme had a molecular mass of approximately 83 kDa. The enzyme showed optimum activity at pH 6.5 and it was 96% stable over a broad pH range of 6.5–11 for 24 hours. The enzyme had optimum activity at 55°C and it was 100% stable at temperature between 50–60°C up to 24 hours. Kinetic characterization of the enzyme was performed at temperature optima (55°C) and Vmax and Km were found to be 59.88 U/mg protein and 0.909 mg/mL, respectively. Oat spelt xylan but not xylooligosaccharides was degraded by the enzyme and xylose was the only product detected from oat xylan degradation. This suggested that the enzyme was an exo-acting xylanase.

Cloning, expression, purification and characterisation of a thermostable chitinase fromBacillus licheniformis A1

The chitinase B gene (chiB65) ofBacillus licheniformis A1 (BlicA1) isolated from Diyadin hotspring in Turkey was cloned and sequenced. The gene is 1779 bp long and encodes a protein 592 amino acids with a 35-amino acid signal peptide at N-terminal. The gene has 99% percent similarity tochiB gene ofBacillus licheniformis under the GenBank number AY205293. The gene without signal peptide was overexpressed inEscherichia coli and the recombinant protein purified by nickel affinity chromatography. The activity of enzyme was shown on SDS-PAGE with the flourogenic substrate 4-methylumbelliferyl β-D-N,N′-diacetylchitobioside. Kinetic characterisation of the enzyme was performed at 65 °C by using chromogenic substratep-nitrophenylN,N′-diacetyl-β-D-chitobioside, andKm and Vmax were found to be 0.02 μM and 1017 U/mg protein, respectively. Enzyme has maximal activity at pH 6.0 and was stable over a broad pH range of 5.0–9.0 for 24 h at room temperature and 4 h at 65 °C. Enzyme was 60% stable at 65 °C for 1.5 h. The inhibition or activation of some substances on the activity of enzyme was determined. High kinetic properties of enzyme open the possibility of an extensive structural and enzyme-substrate interaction studies.

Intraspecific variation of Bufo bufo , based on 16S Ribosomal RNA sequences

More than half of the species within the family Bufonidae are contained within the genus Bufo. The 450 species of this genus are distributed throughout most major land masses of the world, including the Americas, Eurasia, Africa and excluding Australo-Papuan Realm and Madagascar (Frost, 2002). Because of their cosmopolitan distribution and variable life histories, these toads are of interest to investigators researching evolutionary history and biogeography (Pramuk et al., 2001). The Common toad Bufo bufo is a wideranging species, with a distribution encompassing much of Europe (except in northern Scandinavia), parts of northern Africa and western Asia (Luscher et al., 2001). Three subspecies are recognised: B. b. gredosicola from central Spain, B. b. spinosus from the Mediterranean region including southern Spain, southern France, southern Switzerland, Italy, several southeastern countries and northern Africa, and B. b. bufo from the other parts of Europe, including the north of the Swiss Alps (Luscher et al., 2001). The taxonomic status of Bufo bufo in Turkey is still disputed. It is believed to be represented by 3 subspecies. Bodenheimer (1944) identified

Use of rpoB sequences and rep-PCR for phylogenetic study of Anoxybacillus species

This study was conducted to investigate the applicability of rpoB, which encodes the β subunit of RNA polymerase, to be used as an alternative to 16S rRNA gene sequence similarity analysis in the thermophilic genus Anoxybacillus. Partial rpoB sequences were generated for the 14 type strains of Anoxybacillus species and 6 other strains of four Anoxybacillus species. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The rpoB gene was found to provide a better resolution for Anoxybacillus species, with lower interspecies sequence similarities. The rpoB sequence similarity analysis permitted a more accurate discrimination of the species within the Anoxybacillus genus than the more commonly used 16S rRNA gene. Furthermore, rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP-, ERIC-, and BOX-PCR) were employed for the specimens of genus Anoxybacillus. Through comparison of the three methods, it was found that the BOX-PCR method generated more informative results than REP-PCR for the studied strains; BOX-PCR profiles were more distinct for the different strains, including a higher number of bands. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (rep-PCR) constitute a suitable molecular approach for the validation and maintenance of taxonomy within the Anoxybacillus genus. The results of this study show that rpoB and rep-PCR provide rapid and reliable methods for molecular typing of Anoxybacillus species.

Complete genome of the potential thermozyme producer Anoxybacillus gonensis G2T isolated from the Gönen hot springs in Turkey

Anoxybacillus gonensis type strain G2(T) (=NCIMB 13,933(T) =NCCB 100040(T)) has been isolated from the Gönen hot springs in Turkey. This strain produces a number of well-studied, biotechnologically important enzymes, including xylose isomerase, carboxylesterase, and fructose-1,6-bisphosphate aldolase. In addition, this strain is an excellent candidate for the bioremediation of areas with heavy metal pollution. Here, we present a high-quality, annotated, complete genome of A. gonensis G2(T). Furthermore, this report provides insights into several novel enzymes of strain G2(T) and their potential industrial applications.