Kadriye Inan

Identification and characterization of thermophilic bacteria isolated from hot springs in Turkey

The present study was conducted to identify and characterize the thermophilic bacteria isolated from various hot springs in Turkey by using phenotypic and genotypic methods including fatty acid methyl ester and rep-PCR profilings, and 16S rRNA sequencing. The data of fatty acid analysis showed the presence of 17 different fatty acids in 15 bacterial strains examined in this study. Six fatty acids, 15:0 iso, 15:0 anteiso, 16:0, 16:0 iso, 17:0 iso, and 17:0 anteiso, were present in all strains. The bacterial strains were classified into three phenotypic groups based on fatty acid profiles which were confirmed by genotypic methods such as 16S rRNA sequence analysis and rep-PCR genomic fingerprint profiles. After evaluating several primer sets targeting the repetitive DNA elements of REP, ERIC, BOX and (GTG)(5), the (GTG)(5) and BOXA1R primers were found to be the most reliable technique for identification and taxonomic characterization of thermophilic bacteria in the genera of Geobacillus, Anoxybacillus and Bacillus spp. Therefore, rep-PCR fingerprinting using the (GTG)(5) and BOXA1R primers can be considered as a promising genotypic tool for the identification and characterization of thermophilic bacteria from species to strain level.

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).

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.

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.

Algoriphagus trabzonensis sp. nov., isolated from freshwater, and emended description of Algoriphagus alkaliphilus

A Gram-staining-negative, non-motile, catalase- and oxidase-positive strain, designated MS7(T), was isolated from freshwater of a river near Trabzon, Turkey. Its taxonomy was investigated using a polyphasic approach. The strain grew optimally at 28 °C and pH 7.5 and in the presence of 2.0% NaCl. 16S rRNA gene sequence analysis revealed that the strain belonged to the genus Algoriphagus; strain MS7(T) showed highest sequence similarity to the type strains of Algoriphagus alkaliphilus (97.3%), Algoriphagus terrigena (96.8%), Algoriphagus jejuensis (96.2%), Algoriphagus boritolerans (96.1%) and Algoriphagus aquatilis (95.8%). The major fatty acids of strain MS7(T) were iso-C15 : 0 (30.14%) and summed future 9 (10-methyl C16 : 0 and/or iso-C17 : 1ω9c 18.75%). Polar lipid analysis revealed phosphatidylethanolamine, a variety of unidentified lipids, an unidentified aminophospholipid, an unidentified phospholipid and an unidentified aminolipid. The major isoprenoid quinone was MK-7.The DNA G+C content of MS7(T) was 41.6 mol%, a value consistent with that of members of the genus Algoriphagus. The level of DNA-DNA relatedness between strain MS7(T) and A. alkaliphilus LMG 22694(T) was 41%, which is clearly below the 70% threshold accepted for species delineation. Thus, our results support the placement of strain MS7(T) within a separate and previously unrecognized species. On the basis of these data, the strain is considered to represent a novel species of the genus Algoriphagus, for which the name Algoriphagus trabzonensis sp. nov. is proposed. The type strain is MS7(T) ( = NCCB 100372(T) = LMG 26290(T)). An emended description of A. alkaliphilus is also provided.

Brevibacillus gelatini sp.nov., isolated from hot spring in Turkey.

Two Gram-stain-positive, moderately thermophilic, endospore-forming, rod-shaped, motile bacteria designated PDF4T and PDF10, were isolated from Camkoy hot spring in the provinces of Aydın, Turkey and were characterized in order to determine their phylogenetic position. 16S rRNA gene sequence analysis revealed that the two strains belonged to the genus Brevibacillus. Strain PDF4T showed highest 16S rRNA gene sequence similarity to strain PDF10 (99.5 %), Brevibacillus brevis DSM 30T (98.9 %), Brevibacillus parabrevis DSM 8376T (98.6 %) and Brevibacillus formosus DSM 9885T (98.5 %); similarities to other species of the genus Brevibacillus were less than 98.5 %. The predominant fatty acids of strain PDF4T were anteiso-C15 : 0 (60.0 %) and iso-C15 : 0 (22.3 %). The polar lipids of strain PDF4T consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, an unknown phospholipid, two unknown lipid, an unknown aminophospholipid and two unknown aminolipids. MK-7 was detected as a sole respiratory quinone, and the cell wall of strain PDF4T contained meso-diaminopimelic acid. The DNA G+C content of strain PDF4T was 51.7 mol%. DNA-DNA hybridization showed less than 60 % relatedness between strain PDF4T and type strains of the most closely related species given above. Based on these data, the two strains are considered to represent a novel species of the genus Brevibacillus, for which the name Brevibacillus gelatini sp. nov. is proposed. The type strain is PDF4T ( = NCCB 100559T = DSM 100115T).

Thermus anatoliensis sp. nov., a thermophilic bacterium from geothermal waters of Buharkent, Turkey.

A Gram‐stain‐negative, lack of motility, catalase‐ and oxidase‐ positive bacterium (strain MT1T) was isolated from Buharkent hot spring in Aydin, Turkey. Its taxonomy was investigated using a polyphasic approach. The strain was able to grow at 45–80 °C, pH 5.5–10.5 and with a NaCI tolerance up to 2.0% (w/v). Strain MT1T was able to utilize d‐mannitol and l‐arabinose, not able to utilize d‐cellobiose as sole carbon source. 16S rRNA gene sequence analysis revealed that the strain belonged to the genus Thermus; strain MT1T detected low‐level similarities of 16S rRNA gene sequences (below 97%) compared with all other species in this genus. The predominant fatty acids of strain MT1T were iso‐C15:0 (43.0%) and iso‐C17:0 (27.4%). Polar lipid analysis revealed a major phospholipid, one major glycolipid, one major aminophospholipid, two minor aminolipids, one minor phospholipid, and several minor glycolipids. The major isoprenoid quinone was MK‐8. The DNA G+C content of MT1T was 69.6 mol%. On the basis of a taxonomic study using a polyphasic approach, strain MT1T is considered to represent a novel species of the genus Thermus, for which the name Thermus anatoliensis sp. nov. is proposed. The type strain is MT1T (=NCCB 100425T =LMG 26880T).