Cumhur Cokmus

Characterization of Staphylococcus species by SDS–PAGE of Whole-Cell and Extracellular Proteins

In this study, a total of fifteen staphylococcal strains belonging to different species were characterized by whole-cell and extracellular protein profiles using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The results are presented as dendrograms after quantitative analysis of the band patterns with a computer program. Visual inspection of protein bands and cluster analysis of protein patterns of to be used 15 strains, representing 10 Staphylococcus species, showed that whole-cell and extracellular protein profiles differed in several protein bands in Staphylococcus aureus, S. epidermidis, S. simulans, and other species of Staphylococcus; however, the differences were insufficient for reliable differentiation of Staphylococcus species by the SDS–PAGE method.

Anoxybacillus salavatliensis sp. nov., an α-glucosidase producing, thermophilic bacterium isolated from Salavatli, Turkey

A novel moderately thermophilic, Gram‐positive staining, rod‐shaped, spore‐forming, motile, facultative anaerobic, and α‐glucosidase producing strain A343T, was isolated from a high temperature well‐pipeline sediment sample in Salavatli province of Aydin, Turkey. Growth was observed at 37–69 ºC (optimum 60 °C), at pH 5.5–9.5 (optimum 8.0–9.0) and at salinities from 0 to 4.5% (w/v) (optimum 2%). Strain A343T was able to grow on a wide range of carbon sources. Gelatin and starch utilization, amylase, catalase and oxidase activities, reduction of nitrate to nitrite were all positive. The G+C content of the genomic DNA was 45.1 mol%. The major menaquinone was MK‐7. The dominant cellular fatty acids were: iso‐C15:0, C16:0, and iso‐C17:0. The phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain A343T belonged to the genus Anoxybacillus. The 16S rRNA gene sequence similarity between strain A343T and the type strains of recognized Anoxybacillus species was ranged from 95.8 to 99.4%. DNA‐DNA hybridization revealed low homology with its closest relative Anoxybacillus kamchatkensis (49.7%). In addition to the total cell protein profiles, the Rep‐PCR and the intergenic 16S–23S rRNA gene fingerprinting profiles differentiated strain A343T from all of the reference Anoxybacillus species used. Based upon phenotypic, phylogenetic and chemotaxonomic evidence, strain A343T was assigned to a new species within the genus Anoxybacillus, A. salavatliensis sp. nov. (The type strain A343T= DSM 22626T = NCIMB 14579T). The 16S rRNA gene nucleotide sequence of strain A343T is available in the GenBank database under the accession number – EU326496. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Isolations of α-glucosidase-producing thermophilic bacilli from hot springs of Turkey

From 42 different hot springs in six provinces belonging to distinct geographical regions of Turkey, 451 thermophilic bacilli were isolated and 67 isolates with a high amylase activity were selected to determine the α-glucosidase production capacities by using pNPG as a substrate. α-Glucosidase production capacities of the isolates varied within the range from 77.18 to 0.001 U/g. Eleven of our thermophilic bacilli produced α-glucosidase at significant levels comparable with that of the reference strains tested; thus, five strains, F84b (77.18 U/g), A333 (48.64 U/g), F84a (36.64 U/g), E134 (32.09 U/g), and A343 (10.79 U/g), were selected for further experiments. 16S rDNA sequence analysis revealed that these selected isolates all belonged to thermophilic bacilli 16S rDNA genetic group 5, four of them representing the genus Geobacillus, while strain A343 had an uncultured bacterium as the closest relative. Changes in α-glucosidase levels in the intracellular and extracellular fractions were determined during 48-h cultivation of A333, A343, F84a, F84b, E134, and the reference strain G. stearothermophilus ATCC 12980. According to α-glucosidase production type and enzyme levels in intracellular and extracellular fractions, Geobacillus spp. A333, F84a, and F84b were defined as extracellular enzyme producers, whereas the thermophilic bacterium A343 was found to be an intracellular α-glucosidase producer, similar to ATCC 12980 strain. Geobacillus sp. E134 differed in α-glucosidase production type from all tested isolates and the reference strain; it was described as a membrane-associated cell-bound enzyme producer. In this study, apart from screening a great number of new thermophilic bacilli from the hot springs of Turkey, which have not yet been thoroughly studied, five new thermostable α-1,4-glucosidase-producing bacilli that have biotechnological potential with α-glucosidases located at different cell positions were obtained.

Characterization of extremely halophilic Archaea isolated from saline environment in different parts of Turkey

Ninety-five extremely halophilic strains were isolated from six distinct saline regions of Turkey by using complex medium containing 25% NaCl. The selected regions are Tuz Golu (salt lake), Ankara; Aci Lake, Denizli; Salda Lake, Denizli; Seyfe Lake, Kyrsherhir; Tuzla Lake, Kayseri; and Bolluk Lake, Konya. The isolated strains were tested for motility, gram reaction, cell and colony morphologies, pigmentation, biochemical characteristics, and antibiotic sensitivities. According to membrane glycerol diether moieties and antibiotic susceptibilities, all isolated strains were found to belong to the domain Archaea. All isolates were examined for the presence of plasmids by agarose gel electrophoresis and it was established that most isolates contained plasmids that varied in number and whose molecular sizes ranged from 1 to 36.9 kbp. Whole-cell protein profiles from isolates were analyzed by SDS-PAGE and a similarity dendogram was constructed using the UPGMA method. Significant similarities and differences were observed among the isolates. The strains were clustered in eight groups and ten of our isolates were placed in the same group with the standard strains. The current study represents the first isolation and characterization of such a large collection of archeal strains from Turkey.

Effects of UV-light on Bacillus sphaericus and its protection by chemicals.

Although when UV‐irradiated seven most toxic strains of Bacillus sphaericus lost their viabilities between 2.5–4.5 min, their larvicidal activity was protected for longer periods. Benzaldehyde, cin‐namaldehyde, salicylaldehyde, methylene blue and yeast extract showed good protective effect for spore viability and larvicidal activity from UV inactivation in B. sphaericus. This protective effect has also been confirmed by SDS‐PAGE analyses whereby the 42 kDa and 51 kDa toxic proteins bands did not disappear following UV treatment.

Anoxybacillus calidus sp. nov., a thermophilic bacterium isolated from soil near a thermal power plant

A novel thermophilic, Gram-stain-positive, facultatively anaerobic, endospore-forming, motile, rod-shaped 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 °C (optimum 55 °C), 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 meso-diaminopimelic 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-C15 : 0 (52.2 %) and iso-C17 : 0 (28.0 %,) with small amounts of C16 : 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) = DSM 25520(T) = NCIMB 14851(T)).

Phylogenetic analysis and characterization of lipolytic activity of halophilic archaeal isolates

Five isolates designated as B45, D83A, A206A, A85 and E49 found to possess lipolytic activities were taxonomically classified on the basis of their phylogenetic, phenotypic and chemotaxonomic characteristics. The isolates were determined to be gram-negative, catalase and oxidase positive, hydrolyzing Tween 80 and 60 but not starch, need 3.5–4 M NaCl for optimal growth and lack of anaerobic growth with arginine or DMSO. All isolates had the highest lipolytic activity at pH 8.5. Lipase and esterase activities increased with salt concentration up to 3–4.5 M NaCl, and decreased at 5 M NaCl. Esterase and lipase showed their maximal activities at 50–55°C and 60–65°C, respectively. The phylogenetic tree constructed by the neighbor-joining method indicated that the strain B45 and A85 were closely related to the members of genera Halovivax and Natrinema, respectively. The closest relative of the strain A206A and D83A were found to be Haloterrigena saccharevitans. The strain E49 displayed a more distant relationship to known strains.

Purification and characterization of intracellular and extracellular α-glucosidases from Geobacillus toebii strain E134

Two different α‐glucosidase‐producing thermophilic E134 strains were isolated from a hot spring in Kozakli, Turkey. Based on the phenotypic, phylogenetic and chemotaxonomic evidence, the strain was proposed to be a species of G. toebii. Its thermostable exo‐α‐1,4‐glucosidases also were characterized and compared, which were purified from the intracellular and extracellular fractions with estimated molecular weights of 65 and 45 kDa. The intracellular and extracellular α‐glucosidases showed optimal activity at 65 °C, pH 7·0, and at 70 °C, pH 6·8, with 3·65 and 0·83 Km values for the pNPG substrate, respectively. Both enzymes remained active over temperature and pH ranges of 35–70 °C and 4·5–11·0. They retained 82 and 84% of their activities when incubated at 60 °C for 5 h. Their relative activities were 45–75% and 45–60% at pH 4·5 and 11·0 values for 15 h at 35 °C. They could hydrolyse the α‐1,3 and α‐1,4 bonds on substrates in addition to a high transglycosylation activity, although the intracellular enzyme had more affinity to the substrates both in hydrolysis and transglycosylation reactions. Furthermore, although sodium dodecyl sulfate behaved as an activator for both of them at 60 °C, urea and ethanol only increased the activity of the extracellular α‐glucosidase. By this study, G. toebii E134 strain was introduced, which might have a potential in biotechnological processes when the conformational stability of its enzymes to heat, pH and denaturants were considered. Copyright

The genotypic diversity and lipase production of some thermophilic bacilli from different genera

Abstract 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.

A Comparative Study: Taxonomic Grouping of Alkaline Protease Producing Bacilli

Alkaline proteases have biotechnological importance due to their activity and stability at alkaline pH. 56 bacteria, capable of growing under alkaline conditions were isolated and their alkaline protease activities were carried out at different parameters to determine their optimum alkaline protease production conditions. Seven isolates were showed higher alkaline protease production capacity than the reference strains. The highest alkaline protease producing isolates (103125 U/g), E114 and C265, were identified as Bacillus licheniformis with 99.4% and Bacillus mojavensis 99.8% based on 16S rRNA gene sequence similarities, respectively. Interestingly, the isolates identified as Bacillus safensis were also found to be high alkaline protease producing strains. Genotypic characterizations of the isolates were also determined by using a wide range of molecular techniques (ARDRA, ITS-PCR, (GTG)5-PCR, BOX-PCR). These different techniques allowed us to differentiate the alkaliphilic isolates and the results were in concurrence with phylogenetic analyses of the 16S rRNA genes. While ITS-PCR provided the highest correlation with 16S rRNA groups, (GTG)5-PCR showed the highest differentiation at species and intra-species level. In this study, each of the biotechnologically valuable alkaline protease producing isolates was grouped into their taxonomic positions with multi-genotypic analyses.