Acinetobacter mesopotamicus sp. nov., Petroleum-degrading Bacterium, Isolated from Petroleum-Contaminated Soil in Diyarbakir, in the Southeast of Turkey.

A new petroleum-degrading bacterium, designated strain GC2T, was isolated from Bozkus 1 petroleum station in Diyarbakir, located in the southeast of Turkey. Cells were Gram-negative staining, aerobic, coccoid-rods, non-motile, non-spore-forming. The bacterium was found to degrade 100% of n-alkanes ranging from C11 to C34 presented in the 1% crude oil after incubation of 7 days. The membrane phospholipids were 1,2 diacylglycero-3-phosphorylethanolamine (PEA), phosphatidylglycerol (PG), dipalmitoyl-sn-glycerol 1- phosphocholine (PC1), 1,2 dipalmitoyl-sn-glycero-3-phosphocholine monohydrate (PC3), cardiolipin also called diphosphatidylglycerol (CL) and l-α- phosphatidic acid, dipalmitoyl (AP); predominant respiratory ubiquinone was Q-8 and C16:0, C18:1ω9c and C16:1 were the major cellular fatty acids. The 16S rRNA sequence analysis revealed that the strain GC2T was a member of genus Acinetobacter and was most closely related to Acinetobacter lwoffii DSM 2403 T (99.79%), Acinetobacter pseudolwoffii ANC 5318 T (98.83%) and Acinetobacter harbinensis HITLi 7 T (98.14%). The rpoB and gyrB gene sequence analysis confirmed that the strain GC2T was a member of genus Acinetobacter and that the closest relative was Acinetobacter lwoffii DSM 2403 T (99.08% and 100% similarity, respectively). DNA-DNA hybridization values between GC2T and its closest relatives ranged from 65.6% (with A. lwoffii) to 5.1% (with A. venetianus). The whole genome sequence of strain GC2T was obtained. The DNA G + C content of this strain was determined to be 42.9 mol %. ANI indexes, in silico estimations of DDH values and wet lab DDH values demonstrated that strain GC2T represents an independent genomospecies. On the basis of phenotypic characteristics, chemotaxonomic, phylogenetic data and DNA-DNA hybridization and whole genome analysis, we propose to assign strain GC2T as a new species of the genus Acinetobacter, for which the name Acinetobacter mesopotamicus sp. nov. is proposed. The type strain of this species is GC2T (DSM 26953 T = JCM 31073 T). The whole genome of strain GC2T has been deposited at DDBJ/ENA/GenBank under the accession JAALFF010000000.

Purification and Characterization of Thermostable and Detergent-Stable α-Amylase from Anoxybacillus sp. AH1.

A thermostable and detergent-stable α-amylase from a newly isolated Anoxybacillus sp. AH1 was purified and characterized. Maximum enzyme production (1874.8 U/mL) was obtained at 24 h of incubation. The amylase was purified by using Sephadex G-75 gel filtration, after which an 18-fold increase in specific activity and a yield of 9% were achieved. The molecular mass of the purified enzyme was estimated at 85 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature values of the enzyme were 7.0 and 60 °C, respectively. The enzyme was highly stable in the presence of 30% glycerol, retaining 85% of its original activity at 60 °C within 120 min. Km and vmax values were 0.102 µmol and 0.929 µmol/min, respectively, using Lineweaver-Burk plot. The enzyme activity was increased by various detergents, but it was significantly inhibited in the presence of urea. Mg2+ and Ca2+ also significantly activated α-amylase, while Zn2+, Cu2+ and metal ion chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline (phen) greatly inhibited the enzyme activity. α-Amylase activity was enhanced by ß-mercaptoethanol (ß-ME) and dithiothreitol (DTT) to a great extent, but inhibited by p-chloromercuribenzoic acid (PCMB). Iodoacetamide (IAA) and N-ethylmaleimide (NEM) had a slight, whereas phenylmethylsulfonyl fluoride (PMSF) had a strong inhibitory effect on the amylase activity.

Geobacillus subterraneus subsp. aromaticivorans subsp. nov., a novel thermophilic and alkaliphilic bacterium isolated from a hot spring in Sirnak, Turkey.

A new thermophilic spore-forming strain Ge1(T) was isolated from the Guclukonak hot spring in Sirnak, Turkey. The strain was identified by using a polyphasic taxonomic approach. Strain Ge1(T) was Gram-positive, spore-forming, alkaliphilic rod-shaped, motile, occurring in pairs or filamentous. Growth was observed between 30 and 65°C (optimum 60°C) and at pH 5.5-10.0 (optimum pH 9.0). It was capable of utilizing starch, growth was observed at 0-3% NaCl (w/v) and was positive for catalase and urease. The major cellular fatty acids were iso-C(15:0) and iso-C(17:0), and the predominant lipoquinone found was menaquinone MK7 type. The DNA G+C content of the genomic DNA of strain Ge1(T) was 52.0%. Comparative 16S rRNA gene sequence studies showed that the isolate belonged to the genus Geobacillus. The DNA-DNA hybridization mean values between the representative strain Ge1(T) and the closely related species G. subterraneus, G. thermodenitrificans, G. thermocatenulatus, G. vulcani and G. thermoleovorans were 69.3%, 57%, 37%, 27% and 26%, respectively. The results of DNA-DNA hybridization, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain Ge1(T). Based on these results, we propose assigning a novel subspecies of Geobacillus subterraneus, to be named as Geobacillus subterraneus subsp. aromaticivorans subsp. nov. with the type strain Ge1(T) (DSM 23066 (T)= CIP 110341(T)).