Rapid purification, characterization and substrate specificity of heparinase from a novel species of Sphingobacterium.
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A type of heparinase (heparin lysase, no EC number) was isolated from the periplasmic space of a novel species of Sphingobacterium by three-step osmotic shock. It was further purified to apparent homogeneity by a combination of SP-sepharose and Source 30S chromatographies with a final specific activity of 17.6 IU/mg protein and purification factor of 13-fold. MALDI-TOF mass spectrum of the purified heparinase gave a molecular mass of 75,674 Da of the native enzyme. Peptide mass spectrum showed poor homogeneity with the database in the peptide bank. Inhibition of the enzyme activity by N-acetylimidazole indicated that tyrosine residues were necessary for enzyme activity. K(m) and V(max) of the heparinase for de-o-sulfated-N-acetyl heparin were 42 micro M and 166 microM/min/mg protein, respectively. The heparinase showed similar activity on both heparin and heparan sulfate, except for the heparin from bovine lung. The heparinase exhibited only 8.3% of the activity when de-N-sulfated heparin was used as the substrate, but N-acetylation of the de-N-sulfated heparin restored the activity to 78.4%. Thus modification of N-site in heparin structure was favorable for heparinase activity. On the other hand, de-o-sulfation in heparin showed positive effects on the heparinase activity, since the enzyme activity for N-acetyl-de-o-sulfated heparin was increased by 150%. Based on the present findings, the sphingobacterial heparinase differed from flavobacterial and other reported heparinases in molecular mass, composition, charge properties, active site, substrate specificities and other important characteristics, suggesting that it a novel heparin lysase distinct from those from other sources. (+info)
Structural analysis of sphingophospholipids derived from Sphingobacterium spiritivorum, the type species of genus Sphingobacterium.
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The unique feature of the genus Sphingobacterium is the presence of sphingophospholipids and ceramides, besides diacylglycerophospholipids. As major cellular lipid components, five kinds of sphingophospholipids were purified from Sphingobacterium spiritivorum ATCC 33861(T), the type species of genus Sphingobacterium. They were identified as ceramide phosphorylethanolamines (CerPE-1 and CerPE-2), ceramide phosphoryl-myo-inositols (CerPI-1 and CerPI-2), and ceramide phosphorylmannose (CerPM-1). The ceramide of CerPE-1, CerPI-1, and CerPM-1 was composed of 15-methylhexadecasphinganine (isoheptadeca sphinganine, iso-C17:0) and 13-methyltetradecanoic acid (isopentadecanoic acid, iso-C15:0), whereas that of CerPE-2 and CerPI-2 was composed of isoheptadeca sphinganine and 2-hydroxy-13-methyltetradecanoic acid (2-hydroxy isopentadecanoic acid, 2-OH iso-C15:0). These sphingophospholipids were also found in cellular lipids of Sphingobacterium multivorum ATCC 33613(T), Sphingobacterium mizutaii ATCC 33299(T), Sphingobacterium faecium IFO 15299(T), Sphingobacterium thalpophilum ATCC 43320(T), and Sphingobacterium antarcticum ATCC 51969(T). To our knowledge, the existence of CerPM-1 is a novel sphingophospholipid through eukaryotic and prokaryotic cells. (+info)
Extrinsic allergic alveolitis (hypersensitivity pneumonitis) caused by Sphingobacterium spiritivorum from the water reservoir of a steam iron.
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A case of extrinsic allergic alveolitis (EAA) caused by Sphingobacterium spiritivorum is described. The symptoms were associated with the use of a steam iron. The water reservoir was heavily contaminated with S. spiritivorum (10(6) CFU ml(-1)). This is the first report of S. spiritivorum as a causative agent of EAA. (+info)
Pedobacter sandarakinus sp. nov., isolated from soil.
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A Gram-negative, non-motile, rod-shaped bacterial strain, designated DS-27(T), was isolated from a soil sample, and its taxonomic position was investigated by using a polyphasic approach. The organism grew optimally at 30 degrees C and in the presence of 0-0.5 % (w/v) NaCl. Strain DS-27(T) contained MK-7 as the predominant menaquinone and iso-C(15 : 0), C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH and iso-C(17 : 0) 3-OH as the major fatty acids. The DNA G + C content was 39.7 mol%. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain DS-27(T) is most closely related to the genus Pedobacter of the family Sphingobacteriaceae. Similarity values between the 16S rRNA gene sequences of strain DS-27(T) and the type strains of recognized Pedobacter species ranged from 90.6 to 95.5 %. Differential phenotypic properties, together with the phylogenetic distinctiveness, were sufficient to categorize strain DS-27(T) as representing a species that is separate from recognized Pedobacter species. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain DS-27(T) (=KCTC 12559(T) = CIP 108922(T)) was classified in the genus Pedobacter as a member of a novel species, for which the name Pedobacter sandarakinus sp. nov. is proposed. (+info)
Sphingobacterium daejeonense sp. nov., isolated from a compost sample.
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A Gram-negative, strictly aerobic, rod-shaped, non-motile, non-spore-forming bacterial strain, designated TR6-04(T), was isolated from compost and characterized taxonomically by using a polyphasic approach. The organism grew optimally at 30 degrees Celsius and at pH 6.5-7.0. The isolate was positive for catalase and oxidase tests but negative for gelatinase, indole and H(2)S production. Comparative 16S rRNA gene sequence analysis showed that strain TR6-04(T) fell within the radiation of the cluster comprising Sphingobacterium species and clustered with Sphingobacterium mizutaii ATCC 33299(T) (96.7 % sequence similarity); the similarity to sequences of other species within the family Sphingobacteriaceae was less than 92.0 %. The G+C content of the genomic DNA was 38.7 mol%. The predominant respiratory quinone was MK-7. The major fatty acids were iso-C(15 : 0), iso-C(17 : 0) 3-OH and summed feature 4 (iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c). These chemotaxonomic data supported the affiliation of strain TR6-04(T) to the genus Sphingobacterium. However, on the basis of its phenotypic properties and phylogenetic distinctiveness, strain TR6-04(T) (=KCTC 12579(T)=LMG 23402(T)=CCUG 52468(T)) should be classified as the type strain of a novel species, for which the name Sphingobacterium daejeonense sp. nov. is proposed. (+info)
Emendation of the genus Flammeovirga and Flammeovirga aprica with the proposal of Flammeovirga arenaria nom. rev., comb. nov. and Flammeovirga yaeyamensis sp. nov.
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The taxonomic positions of five bacterial strains isolated from the Yaeyama Islands of Japan and 'Microscilla arenaria' NBRC 15982 were determined using a polyphasic taxonomic approach. 16S rRNA gene sequence analyses placed all of the strains close to the genus Flammeovirga. DNA-DNA hybridization studies, biochemical and physiological characterizations and chemotaxonomic analyses suggested that 'M. arenaria' NBRC 15982 and the five novel isolates represented two separate species of the genus Flammeovirga. Emendation of the genus Flammeovirga Nakagawa et al. 1997 and the species Flammeovirga aprica (Reichenbach 1989) Nakagawa et al. 1997 is proposed. In addition, 'Microscilla arenaria' Lewin 1969 is proposed as Flammeovirga arenaria nom. rev., comb. nov. (with the type strain NBRC 15982(T)=CIP 109101(T)) and the novel isolates are proposed as Flammeovirga yaeyamensis sp. nov. (type strain IR25-3(T)=NBRC 100898(T)=CIP 109099(T)). (+info)
Pedobacter suwonensis sp. nov., isolated from the rhizosphere of Chinese cabbage (Brassica campestris).
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A mesophilic bacterium, strain 15-52(T), was isolated from the rhizosphere of Chinese cabbage (Brassica campestris). On the basis of phenotypic and genotypic characteristics, the bacterium was identified as representing a novel species belonging to the genus Pedobacter. The strain is non-flagellated, non-spore-forming and grows at temperatures in the range 1-37 degrees C. Physiological tests of the strain showed the presence of oxidase, catalase, protease (gelatin and casein hydrolysis), beta-glucosidase and beta-galactosidase activities. The highest levels of 16S rRNA gene sequence similarity were found with respect to Pedobacter roseus CL-GP80(T) (97.3 %) and Pedobacter sandarakinus DS-27(T) (97.2 %). A phylogenetic analysis based on 16S rRNA gene sequence data indicated that strain 15-52(T) is a member of the genus Pedobacter. DNA-DNA hybridization analysis revealed low levels of relatedness (<42.3 %) between the isolate and two phylogenetically related type strains, P. roseus KCCM 42272(T) and P. sandarakinus KCTC 12559(T). The DNA G+C content is 44.2 mol% and the predominant fatty acids are iso-C(15 : 0) (35.4 %), iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c (27.8 %) and iso-C(17 : 0) 3-OH (15.8 %). On the basis of these data, strain 15-52(T) represents a novel species of the genus Pedobacter, for which the name Pedobacter suwonensis sp. nov. is proposed. The type strain is 15-52(T) (=KACC 11317(T)=DSM 18130(T)). (+info)
Structural investigations of the ferredoxin and terminal oxygenase components of the biphenyl 2,3-dioxygenase from Sphingobium yanoikuyae B1.
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BACKGROUND: The initial step involved in oxidative hydroxylation of monoaromatic and polyaromatic compounds by the microorganism Sphingobium yanoikuyae strain B1 (B1), previously known as Sphingomonas yanoikuyae strain B1 and Beijerinckia sp. strain B1, is performed by a set of multiple terminal Rieske non-heme iron oxygenases. These enzymes share a single electron donor system consisting of a reductase and a ferredoxin (BPDO-FB1). One of the terminal Rieske oxygenases, biphenyl 2,3-dioxygenase (BPDO-OB1), is responsible for B1's ability to dihydroxylate large aromatic compounds, such as chrysene and benzo[a]pyrene. RESULTS: In this study, crystal structures of BPDO-OB1 in both native and biphenyl bound forms are described. Sequence and structural comparisons to other Rieske oxygenases show this enzyme to be most similar, with 43.5 % sequence identity, to naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. While structurally similar to naphthalene 1,2-dioxygenase, the active site entrance is significantly larger than the entrance for naphthalene 1,2-dioxygenase. Differences in active site residues also allow the binding of large aromatic substrates. There are no major structural changes observed upon binding of the substrate. BPDO-FB1 has large sequence identity to other bacterial Rieske ferredoxins whose structures are known and demonstrates a high structural homology; however, differences in side chain composition and conformation around the Rieske cluster binding site are noted. CONCLUSION: This is the first structure of a Rieske oxygenase that oxidizes substrates with five aromatic rings to be reported. This ability to catalyze the oxidation of larger substrates is a result of both a larger entrance to the active site as well as the ability of the active site to accommodate larger substrates. While the biphenyl ferredoxin is structurally similar to other Rieske ferredoxins, there are distinct changes in the amino acids near the iron-sulfur cluster. Because this ferredoxin is used by multiple oxygenases present in the B1 organism, this ferredoxin-oxygenase system provides the structural platform to dissect the balance between promiscuity and selectivity in protein-protein electron transport systems. (+info)