A novel thermophilic, Gram-stain-positive, facultatively anaerobic, endospore-forming, motile, rodshaped 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 6C (optimum 55 6C), 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 mesodiaminopimelic 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-C 15 : 0 (52.2 %) and iso-C 17 : 0 (28.0 %,) with small amounts of C 16 : 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 5DSM 25520 T 5NCIMB 14851 T ).
Thermophilic 32 isolates and 20 reference bacilli were subjected to Rep-PCR and ITS-PCR fingerprinting for determination of their genotypic diversity, before screening lipase activities. By these methods, all the isolates and references could easily be differentiated up to subspecies level from each other. In screening assay, 11 isolates and 7 references were found to be lipase producing. Their extracellular lipase activities were measured quantitatively by incubating in both tributyrin and olive oil broths at 60 °C and pH 7.0. During the 24, 48 and 72-h period of incubation, the changes in the lipase activities, culture absorbance, wet weight of biomass and pH were all measured. The activity was determined by using pNPB in 50 mM phosphate buffer at pH 7.0 at 60 °C. The lipase production of the isolates in olive oil broths varied between 0.008 and 0.052, whereas these values were found to be 0.002-0.019 (U/mL) in the case of tyributyrin. For comparison, an index was established by dividing the lipase activities to cell biomass (U/mg). The maximum thermostable lipase production was achieved by the isolates F84a, F84b, and G. thermodenitrificans DSM 465T (0.009, 0.008 and 0.008 U/mg) within olive oil broth, whereas G. stearothermophilus A113 displayed the highest lipase activity than its type strain in tyributyrin. Therefore, as some of these isolates displayed higher activities in comparison to references, new lipase producing bacilli were determined by presenting their genotypic diversity with DNA fingerprinting techniques.
Two novel endospore-forming, aerobic bacilli, strains E173aT and E265T, were isolated from soil and sediment samples from Kozakli and Altinsu hot springs, Nevsehir (Turkey). Their young cells in the exponential phase of growth were motile, Gram-stain-positive, straight rods, 0.6–1.1×3.0–8.0 µm in size, but they became strikingly long, approximately 0.6–1.2 by 9.0–35.0 µm, after the stationary phase of growth. Cells varied in tests for oxidase, and had a weakly positive reaction for catalase. Both strains could grow between 40 and 70 °C, with optimal growth at 60 °C (E173aT) and 55 °C (E265T). Growth occurred within the range pH 5.0–11.0 with optimal growth at pH 9.0 (E173aT) and pH 8.5 (E265T). Strain E173aT grew within a salinity range from 0 to1.5 % (w/v) NaCl with optimal growth at 0.5 %, while strain E265T grew within the range 0–5.0 % (w/v), with an optimum at 3.0 %. The new isolates differed from each other in some phenotypic and chemotaxonomic characters as well as repetitive extragenic palindromic element PCR (rep-PCR) fingerprints. 16S rRNA gene sequence similarities suggested distant relationships with other members of the family Bacillaceae (<95.8 %), although the two strains showed 97.5 % sequence similarity between them, and had 55 % relatedness by DNA–DNA hybridization. The DNA G+C contents were 44.8 (E173aT) and 43.5 mol% (E265T). Moreover, the chemotaxonomic data of E173aT and E265T [presence of low amounts of meso-diaminopimelic acid, A1γ to A1γ′ cross-linkage types in peptidoglycan, fatty acids including iso-C15 : 0 (>60 %), iso-C17 : 0 and C16 : 0] supported the consideration of these isolates as members of a novel genus. Based upon phenotypic, phylogenetic and chemotaxonomic characteristics, it is proposed that new isolates represent a novel genus, Thermolongibacillus gen. nov., with two novel species: Thermolongibacillus altinsuensis sp. nov. (type strain E265T = DSM 24979T = NCIMB 14850T) and Thermolongibacillus kozakliensis sp. nov. (type strain E173aT = DSM 24978T = NCIMB 14849T).
The type strain of Anoxybacillus calidus sp. nov. was displayed incorrectly in this paper and is actually C161ab T (5DSM 25220 T 5NCIMB 14851 T ).
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