A specific type II restriction endonuclease T.Smu451I has been purified to electrophoretic homogeneity from the frozen cells of soil bacterium Sphingobacterium multivorum 451 (formerly Flavobacterium multivorum 451), using ultrasonic grinding, nucleic acid removal by streptomycin sulfate, protein precipitation by ammonium sulfate and phosphocellulose P-11, DEAE-Cellulose DE-52, Hepharin-Sepharose CL-6B chromatography, and elucidated several characteristics of T.Smu451I. The molecular weight of the enzyme determined by gel filtration and SDS-polyacrylamide gel electrophoresis was calculated to be 45,000 ± 2000 D (dimer) and 23,000 ± 1000 D (monomer), respectively. The isoelectric point (pI) of T.Smu451I is 5.4. T.Smu451I recognizes pentanucleotide palindromic sequences 5'-GGNC↓C-3' and cleaves between C and C in position shown by arrow to produce 3'-cohesive terminus of trinucleotide. Therefore, T.Smu451I is a neoschizomer of T.AsuI.
In the present study, potentiality of endophytic microorganisms such as Rigidiporus vinctus AAU EF, Trichoderma reesei UH EF, and Sphingobacterium tabacisoli UH EB in the management of panama wilt and growth promotion of banana was assessed through artificial inoculation. During the study, a total of 220 bacterial and 110 fungal endophytes were isolated from root, pseudostem, and leaf samples of banana, and they were evaluated against Fusarium oxysporum f. sp cubense causing panama wilt. Out of total 330 bacterial and fungal endophytes, only five endophytes exhibited antagonism against Fusarium oxysporum f. sp cubense, out of which only three isolates, namely Trichoderma reesei UH EF, Rigidiporus vinctus AAU EF, and Sphingobacterium tabacisoli UH EB, produced indole acetic acid, siderophore, and hydrogen cyanide, except one bacterial strain Sphingobacterium tabacisoli UH EB which does not produce hydrogen cyanide. Furthermore, these three endophytes were identified through cultural and morphological characteristics as well as by the sequencing internal transcribed spacer (ITS) and 16S rRNA gene sequences analysis for bacteria, respectively. The response of host plant to endophyte inoculation was assessed by measuring the change in four growth parameters; plant height, pseudo stem girth (diameter), number of roots, and total number of leaves. The application of endophytes, irrespective of isolate and treatment type promoted the overall growth of the plant growth when compared with diseased plants with significant higher values recorded for all parameters assessed. The endophytes reported as growth promoters were found to have significant inhibition effect on Foc which can evidenced with lowest AUDPC values and epidemic rate at 99.09 units2 and 0.02 unit/day, respectively.
- MeSH
- banánovník * mikrobiologie MeSH
- endofyty * fyziologie MeSH
- Fusarium * fyziologie MeSH
- Hypocreales fyziologie MeSH
- mikrobiální interakce fyziologie MeSH
- nemoci rostlin * mikrobiologie prevence a kontrola MeSH
- Polyporales fyziologie MeSH
- RNA ribozomální 16S genetika MeSH
- Sphingobacterium fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
The composition and spatial variability of microbial communities that reside within the extensive (>200 000 km(2)) biologically active area encompassing the Greenland ice sheet (GrIS) is hypothesized to be variable. We examined bacterial communities from cryoconite debris and surface ice across the GrIS, using sequence analysis and quantitative PCR of 16S rRNA genes from co-extracted DNA and RNA. Communities were found to differ across the ice sheet, with 82.8% of the total calculated variation attributed to spatial distribution on a scale of tens of kilometers separation. Amplicons related to Sphingobacteriaceae, Pseudanabaenaceae and WPS-2 accounted for the greatest portion of calculated dissimilarities. The bacterial communities of ice and cryoconite were moderately similar (global R = 0.360, P = 0.002) and the sampled surface type (ice versus cryoconite) did not contribute heavily towards community dissimilarities (2.3% of total variability calculated). The majority of dissimilarities found between cryoconite 16S rRNA gene amplicons from DNA and RNA was calculated to be the result of changes in three taxa, Pseudanabaenaceae, Sphingobacteriaceae and WPS-2, which together contributed towards 80.8 ± 12.6% of dissimilarities between samples. Bacterial communities across the GrIS are spatially variable active communities that are likely influenced by localized biological inputs and physicochemical conditions.
- MeSH
- biodiverzita MeSH
- DNA bakterií genetika MeSH
- ledový příkrov mikrobiologie MeSH
- mikrobiota genetika MeSH
- polymerázová řetězová reakce MeSH
- RNA ribozomální 16S genetika MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA MeSH
- Sphingobacterium genetika izolace a purifikace MeSH
- Synechococcus genetika izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Grónsko MeSH
A mannanase-coding gene was cloned from Sphingobacterium sp. GN25 isolated from the feces of Grus nigricollis. The gene encodes a 371-residue polypeptide (ManAGN25) showing less than 74 % identity with a number of hypothetical proteins and putative glucanases and mannanases. Before experiment's performance, ManAGN25 was predicted to be a low-temperature active mannanase based on the molecular characterization, including (1) ManAGN25 shared the highest identity of 41.1 % with the experimentally verified low-temperature active mannanase (ManAJB13) from Sphingomonas sp. JB13; (2) compared with their mesophilic and thermophilic counterparts, ManAGN25 and ManAJB13 had increased number of amino acid residues around their catalytic sites; (3) these increased number of amino acid residues built longer loops, more α-helices, and larger total accessible surface area and packing volume. Then the experiments of biochemical characterization verified that the purified recombinant ManAGN25 is a low-temperature active mannanase: the enzyme showed apparently optimal activity at 35-40 °C and retained 78.2, 44.8, and 15.0 % of its maximum activity when assayed at 30, 20, and 10 °C, respectively; the half-life of the enzyme was approximately 60 min at 37 °C; the enzyme presented a K m of 4.2 mg/ml and a k cat of 0.4/s in McIlvaine buffer (pH 7.0) at 35 °C using locust bean gum as the substrate; and the activation energy for hydrolysis of locust bean gum by the enzyme was 36.0 kJ/mol. This study is the first to report the molecular and biochemical characterizations of a mannanase from a strain.
- MeSH
- bakteriální proteiny chemie genetika izolace a purifikace metabolismus MeSH
- beta-mannosidasa chemie genetika izolace a purifikace metabolismus MeSH
- kinetika MeSH
- konformace proteinů MeSH
- molekulární sekvence - údaje MeSH
- nízká teplota MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- Sphingobacterium chemie enzymologie genetika MeSH
- stabilita enzymů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
An enzyme's substrate specificity is one of its most important characteristics. The quantitative comparison of broad-specificity enzymes requires the selection of a homogenous set of substrates for experimental testing, determination of substrate-specificity data and analysis using multivariate statistics. We describe a systematic analysis of the substrate specificities of nine wild-type and four engineered haloalkane dehalogenases. The enzymes were characterized experimentally using a set of 30 substrates selected using statistical experimental design from a set of nearly 200 halogenated compounds. Analysis of the activity data showed that the most universally useful substrates in the assessment of haloalkane dehalogenase activity are 1-bromobutane, 1-iodopropane, 1-iodobutane, 1,2-dibromoethane and 4-bromobutanenitrile. Functional relationships among the enzymes were explored using principal component analysis. Analysis of the untransformed specific activity data revealed that the overall activity of wild-type haloalkane dehalogenases decreases in the following order: LinB~DbjA>DhlA~DhaA~DbeA~DmbA>DatA~DmbC~DrbA. After transforming the data, we were able to classify haloalkane dehalogenases into four SSGs (substrate-specificity groups). These functional groups are clearly distinct from the evolutionary subfamilies, suggesting that phylogenetic analysis cannot be used to predict the substrate specificity of individual haloalkane dehalogenases. Structural and functional comparisons of wild-type and mutant enzymes revealed that the architecture of the active site and the main access tunnel significantly influences the substrate specificity of these enzymes, but is not its only determinant. The identification of other structural determinants of the substrate specificity remains a challenge for further research on haloalkane dehalogenases.
- MeSH
- Agrobacterium tumefaciens enzymologie genetika metabolismus MeSH
- aktivace enzymů MeSH
- biologické modely MeSH
- Bradyrhizobium enzymologie genetika metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- fylogeneze MeSH
- hydrolasy klasifikace genetika metabolismus fyziologie MeSH
- mutantní proteiny klasifikace genetika metabolismus MeSH
- Mycobacterium bovis enzymologie genetika metabolismus MeSH
- Mycobacterium smegmatis genetika metabolismus MeSH
- Rhodococcus enzymologie genetika metabolismus MeSH
- Sphingobacterium enzymologie genetika metabolismus MeSH
- substrátová specifita MeSH
- Xanthobacter enzymologie genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
