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.

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 MT1(T)) 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 MT1(T) 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 MT1(T) 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 MT1(T) were iso-C(15:0) (43.0%) and iso-C(17:0) (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 MT1(T) was 69.6 mol%. On the basis of a taxonomic study using a polyphasic approach, strain MT1(T) 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 MT1(T) (=NCCB 100425(T) =LMG 26880(T)).

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 an alkali-stable endoxylanase from thermophilic Geobacillus sp. 71.

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.

Cloning, purification, and characterization of a thermostable alpha-L-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari.

The gene, AbfAC26Sari, encoding an alpha-L-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari, was isolated, cloned, sequenced, and characterized. On the basis of amino acid sequence similarities, this 57-kDa enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. Characterization of the purified recombinant alpha-L-arabinofuranosidase produced in Escherichia coli BL21 revealed that it is active at a broad pH range (pH 4.5 to 9.0) and at a broad temperature range (45-85 degrees C) and it has an optimum pH of 5.5 and an optimum temperature of 65 degrees C. Kinetic experiment at 65 degrees C with p-nitrophenyl alpha-L-arabinofuranoside as a substrate gave a Vmax and Km values of 1,019 U/mg and 0.139 mM, respectively. The enzyme had no apparent requirement of metal ions for activity, and its activity was strongly inhibited by 1 mM Cu2+ and Hg2+. The recombinant arabinofuranosidase released L-arabinose from arabinan, arabinoxylan, oat spelt xylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose. Endoarabinanase activity was not detected. These findings suggest that AbfAC26Sari is an exo-acting enzyme.

Evaluation of arabinofuranosidase and xylanase activities of Geobacillus spp. isolated from some hot springs in Turkey.

Some hot springs located in the west of Turkey were investigated with respect to the presence of thermophilic microorganisms. Based on phenotyping characteristics and 16S rRNA gene sequence analysis, 16 of the isolates belonged to the genus Geobacillus and grew optimally at about 60 degrees C on nutrient agar. 16S rRNA gene sequence analysis showed that these isolates resembled Geobacillus species by > or = 97%, but SDS-PAGE profiles of these 16 isolates differ from some of the other species of the genus Geobacillus. However, it is also known that analysis of 16S rRNA gene sequences may be insufficient to distinguish between some species. It is proposed that recN sequence comparisons could accurately measure genome similarities for the Geobacillus genus. Based on recN sequence analysis, isolates 11, IT3, and 12 are strains of G stearothermophilus; isolate 14.3 is a strain of G thermodenitrificans; isolates 9.1, IT4.1, and 4.5 are uncertain and it is required to make further analysis. The presence of xylanase and arabinofuranosidase activities, and their optimum temperature and pH were also investigated. These results showed that 7 of the strains have both xylanase and arabinofuranosidase activities, 4 of them has only xylanase, and the remaning 5 strains have neither of these activities. The isolates 9.1, 7.1, and 3.3 have the highest temperature optima (80 degrees C), and 7.2, 9.1, AO4, 9.2, and AO17 have the highest pH optima (pH 8) of xylanase. Isolates 7.2, AO4, AC15, and 12 have optimum arabinofuranosidase activities at 75 degrees C, and only isolate AC 15 has the lowest pH of 5.5.