Arzu Coleri Cihan

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)

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

Determination of the biofilm production capacities and characteristics of members belonging to Bacillaceae family

The biofilm characteristics of many endospore-forming bacilli, especially the thermophiles are still unclear. In this study, a detailed identification and description of biofilm production characteristics of totally 145 isolates and reference strains belonging to Bacillaceae family, displaying thermophilic (n = 115), facultative thermophilic (n = 24) and mesophilic (n = 6) growth from genera Anoxybacillus, Geobacillus, Thermolongibacillus, Aeribacillus, Brevibacillus, Paenibacillus and Bacillus were presented. The incubation temperatures were adjusted to 37, 45 and 55–65 °C for mesophiles, facultative thermophiles, and thermophiles, respectively. The bacilli were evaluated based on their colony morphotypes on Congo red (CR) agar, their complex exopolysaccharide production on calcofluor supplemented tryptic soy agar, and as well as their pellicle formation at the liquid–air surface in tryptic soy broth cultures. Their biofilm production capabilities were also tested on abiotic surfaces of both polystyrene and stainless steel by crystal violet binding assay and viable biofilm cell enumerations, respectively. As a result, the biofilm production capacities of Bacillaceae members from genera to species level, the effects of osmolarity, temperature, incubation time and abiotic surfaces on biofilm formation as well as the CR morphotypes associated with the biofilm production were able to reveal in a wide group of bacilli. Besides, general enrichment-inoculation approaches and methodologies were also offered, which allow and facilitate the screening and determining the biofilm producing endospore forming bacilli.

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.

Introduction of novel thermostable α-amylases from genus Anoxybacillus and proposing to group the Bacillaceae related α-amylases under five individual GH13 subfamilies

Among the thermophilic Bacillaceae family members, α-amylase production of 15 bacilli from genus Anoxybacillus was investigated, some of which are biotechnologically important. These Anoxybacillus α-amylase genes displayed ≥ 91.0% sequence similarities to Anoxybacillus enzymes (ASKA, ADTA and GSX-BL), but relatively lower similarities to Geobacillus (≤ 69.4% to GTA, Gt-amyII), and Bacillus aquimaris (≤ 61.3% to BaqA) amylases, all formerly proposed only in a Glycoside Hydrolase 13 (GH13) subfamily. The phylogenetic analyses of 63 bacilli-originated protein sequences among 93 α-amylases revealed the overall relationships within Bacillaceae amylolytic enzymes. All bacilli α-amylases formed 5 clades different from 15 predefined GH13 subfamilies. Their phylogenetic findings, taxonomic relationships, temperature requirements, and comparisonal structural analyses (including their CSR-I-VII regions, 12 sugar- and 4 calcium-binding sites, presence or absence of the complete catalytic machinery, and their currently unassigned status in a valid GH13 subfamiliy) revealed that these five GH13 α-amylase clades related to familly share some common characteristics, but also display differentiative features from each other and the preclassified ones. Based on these findings, we proposed to divide Bacillaceae related GH13 subfamilies into 5 individual groups: the novel a2 subfamily clustered around α-amylase B2M1-A (Anoxybacillus sp.), the a1, a3 and a4 subfamilies (including the representatives E184aa-A (Anoxybacillus sp.), ATA (Anoxybacillus tepidamans), and BaqA,) all of which were composed from the division of the previously grouped single subfamily around α-amylase BaqA, and the undefinite subfamily formerly defined as xy including Bacillus megaterium NL3.

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.

Lentinus edodes, Lactarius delicious ve Ganoderma lucidum’un antibiyofilm ve antimikrobiyal etkinlikleri

Calismanin amaci: Bu calisma kapsaminda tibbi onemi olan uc makrofungus turunun [( Lentinus edodes (Berk.) Sing. (Shiitake), Lactarius deliciosus Fr. ve Ganoderma lucidum (Curtis) P. Karst.)] metanolik (%60) ve etanolik (%95) ozutlerinin Pseudomonas aeruginosa ve Salmonella Typhimurium’daki antibakteriyal ve biyofilm olusumunu engelleme (antibiyofilm) potansiyelleri arastirilmistir. Materyal ve Yontem: Antimikrobiyal aktivitelerin degerlendirilmesinde standart agar kuyu difuzyon, minimum inhibisyon konsantrasyon (MIK) ve minimum bakterisidal konsantrasyon (MBK) testleri kullanilmistir. Makrofungus ozutlerinin biyofilm olusumu uzerine (antibiyofilm) olan etkilerinin degerlendirmesinde kristal viyole baglanma yontemi esas alinmistir. Temel sonuclar: Makrofungus orneklerinin yalnizca metanolik ozutlerinde antimikrobiyal etkinlik saptanmistir. Antibiyofilm aktivite degerlendirmesindeyse en yuksek aktivite G . lucidum ’da gorulmustur. Arastirma vurgulari: Elde edilen bulgular, dogal antimikrobiyal ajanlarin arastirilmasinda siklikla tercih edilen tibbi makrofungus turlerinin, biyofilmlerle mucadele arastirmalarinda da yuksek potansiyel icermelerinden oturu tercih edilebileceklerini kanitlar mahiyettedir.Aim of study: In this study, the antimicrobial and antibiofilm potentials of methanolic (60%) and ethanolic (95%) extracts of three medicinal macrofungi species [(Lentinus edodes (Berk.) Sing. (Shiitake), Lactarius deliciosus Fr., and Ganoderma lucidum (Curtis) P. Karst.)] on Pseudomonas aeruginosa, and Salmonella Typhimurium were investigated. Material and Methods: Standart agar well diffusion, minimum inhibitory concentration, and minimum bactericidal concentration tests were performed to evaluate the antimicrobial activities. The effects of macrofungi extracts on biofilm formation (antibiofilm) were evaluated based on crystal violet binding