Crystal structures of two camelid nanobodies raised against GldL, a component of the type IX secretion system from Flavobacterium johnsoniae.

GldL is an inner-membrane protein that is essential for the function of the type IX secretion system (T9SS) in Flavobacterium johnsoniae. The complex that it forms with GldM is supposed to act as a new rotary motor involved in the gliding motility of the bacterium. In the context of structural studies of GldL to gain information on the assembly and function of the T9SS, two camelid nanobodies were selected, produced and purified. Their interaction with the cytoplasmic domain of GldL was characterized and their crystal structures were solved. These nanobodies will be used as crystallization chaperones to help in the crystallization of the cytoplasmic domain of GldL and could also help to solve the structure of the complex using molecular replacement.

Flavobacterium inviolabile sp. nov. isolated from stream water.

A Gram-stain-negative, rod-shaped, aerobic and non-motile bacterium, designated P2-65T, was isolated from Moonsan stream water in the Republic of Korea. The temperature, NaCl concentration and pH ranges for growth of strain P2-65T were 10-37 °C, 0.0-3.0% (w/v) and 6.5-8.5 with optimum growth at 25-30 °C, 0.0-1.0% and 7.0-7.5, respectively. Comparison of 16S rRNA gene sequence showed that strain P2-65T was closely related to Flavobacterium cauense (95.4%) and Flavobacterium cheniae (95.3%). The major fatty acids were iso-C15:0, iso C17:0 3-OH, summed feature 3 (C16:1ω7c and/or C16:1ω6c), summed feature 9 (iso-C17:1 ω9c and/or 10-methyl C16:0) and iso-C15:0 3-OH. The predominant respiratory quinone was menaquinone-6 (MK-6). The major polar lipids detected in the strain were phosphatidylethanolamine, one aminophospholipid, one unidentified aminolipid and one unidentified polar lipid. The G + C content of the genomic DNA was 39.7%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values for strain P2-65T with closely related Flavobacterium species were below 74.8% and 20%, respectively. Based on polyphasic features, strain P2-65T is considered to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium inviolabile sp. nov. is proposed. The type strain is P2-65T (= KCTC 62055T = NBRC 112953T).

Large-Scale Vortices with Dynamic Rotation Emerged from Monolayer Collective Motion of Gliding Flavobacteria.

A collective motion of self-driven particles has been a fascinating subject in physics and biology. Sophisticated macroscopic behavior emerges through a population of thousands or millions of bacterial cells propelling itself by flagellar rotation and chemotactic responses. Here, we found a series of collective motions accompanying successive phase transitions for a nonflagellated rod-shaped soil bacterium, Flavobacterium johnsoniae, which was driven by a surface cell movement known as gliding motility. When we spotted the cells on an agar plate with a low level of nutrients, the bacterial community exhibited vortex patterns that spontaneously appeared as lattice and integrated into a large-scale circular plate. All patterns were exhibited with a monolayer of bacteria, which enabled us to two-dimensionally visualize an individual cell with high resolution within a wide-range pattern. The single cells moved with random orientation, but the cells that were connected with one another showed left-turn-biased trajectories in a starved environment. This feature is possibly due to the collision of cells inducing a nematic alignment of dense cells as self-propelled rods. Subsequently, each vortex oscillated independently and then transformed to the rotating mode as an independent circular plate. Notably, the rotational direction of the circular plate was counterclockwise without exception. The plates developed accompanying rotation with constant angular velocity, suggesting that the mode is an efficient strategy for bacterial survival. IMPORTANCE Self-propelled bacteria propelled by flagellar rotation often display highly organized dynamic patterns at high cell densities. Here, we found a new mode of collective motion in nonflagellated bacteria; vortex patterns spontaneously appeared as lattice and were integrated into a large-scale circular plate, comprising hundreds of thousands of cells, which exhibited unidirectional rotation in a counterclockwise manner and expanded in size on agar. A series of collective motions was driven by gliding motility of the rod-shaped soil bacterium Flavobacterium johnsoniae. In a low-nutrient environment, single cells moved with random orientation, while cells at high density moved together as a unitary cluster. This might be an efficient strategy for cells of this species to find nutrients.

Flavobacterium agri sp. nov., a novel bacterial species isolated from rhizospheric soil of Coriandrum sativum.

A Gram-stain-negative, strictly aerobic, cream yellow colored, non-motile, rod-shaped bacterial strain, designated MAH-1T was isolated from rhizospheric soil of Coriandrum sativum. A polyphasic taxonomic study was performed on the isolated strain. Optimal growth occurred at 28-30 °C, pH 6.5 and 0% NaCl. The strain showed activity for both catalase and oxidase tests. Cell growth occurs on R2A agar, nutrient agar and Luria-Bertani agar. Cells were able to hydrolyze starch, aesculin, gelatin, and Tween 20. Alignment of 16S rRNA gene sequences indicated that strain MAH-1T was associated with the genus Flavobacterium and was most closely related to Flavobacterium longum YIT 12745T (94.5% sequence similarity) and Flavobacterium caeni LM5T (93.0%). Strain MAH-1T had a genome size of 3,975,600 bp. Genome contained 67 contigs encoded by 3,522 protein-coding genes with 38 tRNA and 6 rRNA genes. The genomic DNA G + C contents of strain MAH-1T was 47.1 mol %. The genomic ANI and dDDH values between strain MAH-1T and one of the close relatives F. caeni LM5T were 72.2 and 18.8%, respectively. The major fatty acids were C15:0 iso, C16:0 iso and C15:0 anteiso. The predominant respiratory quinone was menaquinone 6 (MK-6). Based on physiological, biochemical and phylogenetic data for this isolate, it was confirmed that strain MAH-1T was affiliated to the genus Flavobacterium and represented a novel species, for which the name Flavobacterium agri sp. nov. is proposed. The type strain is MAH-1T (= KACC 19300T = CGMCC 1.16617T).

Photochemical characterization of flavobacterial rhodopsin: The importance of the helix E region for heat stability.

Although many microbial rhodopsins have been discovered many of organisms in a variety of habitats, little is known about the property and diversity of rhodopsin in flavobacteria. Recent studies discovered that many proteorhodopsin (PR)-like proteins exist in genomes of flavobacteria. Following the isolation of a flavobacterial rhodopsins (FR) from the flavobacteria IMCC1997 from the East Sea of Korea, we characterized its photochemical features. We confirmed that the FR expression is induced by light in the IMCC1997 cell. Upon receiving light energy in vitro, the proton acceptor (D83) and donor (E94) of the FR translocate protons from intracellular to extracellular regions. Compared with proteorhodopsin (PR), the FR from IMCC 1997 cells is very unstable, which may be explained by their primary sequence differences. The ratio of all trans/13-cis retinal conformation does not influence this stability. To measure the stability of FR, we tested heat endurance at 70 °C and found that the heat endurance time of some FR mutants increased. Based upon these results, we found the helix E of this protein to be critical for the unstability of FR.

Flavobacterium sangjuense sp. nov. isolated from sediment.

A yellow-pigmented bacterial strain, GS03T, was isolated from sediment in a branch of the Nackdong River in Sangju, Korea. Cells were observed to be Gram-negative, aerobic and rod-shaped with gliding motility, and to be positive for catalase and oxidase. Growth was found to occur at 4-30 °C (optimum 25 °C), at pH 7.0-8.5 (optimum pH 7.5) and at NaCl 0% (optimum NaCl 0%, w/v). The major cellular fatty acids (> 10% of the total) were identified as iso C15:0, iso C15:1 G, C15:1ω6c, iso C15: 0 3-OH and iso C17: 0 3-OH. The major respiratory quinone was found to be menaquinone MK-6. The genome sequence of GS03T is 3.1 Mb with G+C content of 36.1 mol%. The major polar lipids of the isolate were identified as phosphatidylethanolamine, three unidentified aminolipids, two unidentified phospholipids, an unidentified lipid and an unidentified aminophospholipid. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain GS03T clusters with Flavobacterium paronense KNUS1TT, with similarity of 96.8%. The phenotypic, phylogenetic, and chemotaxonomic characteristics indicate that strain GS03T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium sangjuense sp. nov. is proposed. The type strain is GS03T (= FBCC 502459T = KCTC 62568T = JCM 32764T).

Flavobacterium circumlabens sp. nov. and Flavobacterium cupreum sp. nov., two psychrotrophic species isolated from Antarctic environmental samples.

A taxonomic study of 24 Gram-stain-negative rod-shaped bacteria originating from the Antarctic environment is described. Phylogenetic analysis using 16S rRNA gene sequencing differentiated isolated strains into two groups belonging to the genus Flavobacterium. Group I (n=20) was closest to Flavobacterium aquidurense WB 1.1-56T (98.3% 16S rRNA gene sequence similarity) while group II (n=4) showed Flavobacterium hydatis DSM 2063T as its nearest neighbour (98.5-98.9% 16S rRNA gene sequence similarity). Despite high 16S rRNA gene sequence similarity, these two groups represented two distinct novel species as shown by phenotypic traits and low genomic relatedness assessed by rep-PCR fingerprinting, DNA-DNA hybridization and whole-genome sequencing. Common to representative strains of both groups were the presence of major menaquinone MK-6 and sym-homospermidine as the major polyamine. Common major fatty acids were C15:0 iso, C15:1 iso G, C15:0 iso 3-OH, C17:0 iso 3OH and Summed Feature 3 (C16:1ω7c/C16:1ω6c). Strain CCM 8828T (group I) contained phosphatidylethanolamine, three unidentified lipids lacking a functional group, three unidentified aminolipids and single unidentified glycolipid in the polar lipid profile. Strain CCM 8825T (group II) contained phosphatidylethanolamine, eight unidentified lipids lacking a functional group, three unidentified aminolipids and two unidentified glycolipids in the polar lipid profile. These characteristics corresponded to characteristics of the genus Flavobacterium. The obtained results showed that the analysed strains represent novel species of the genus Flavobacterium, for which the names Flavobacterium circumlabens sp. nov. (type strain CCM 8828T=P5626T=LMG 30617T) and Flavobacterium cupreum sp. nov. (type strain CCM 8825T=P2683T=LMG 30614T) are proposed.

Identification of a Novel Elastin-Degrading Enzyme from the Fish Pathogen Flavobacterium psychrophilum.

Hydrolytic extracellular enzymes degrading host tissues potentially play a role in bacterial pathogenesis. Flavobacterium psychrophilum is an important bacterial pathogen of salmonid fish reared in freshwater throughout the world. Diversity among isolates has been described at the phenotypic, serological, and genomic levels, but the links between these various traits remain poorly understood. Using a genome-wide association study, we identified a gene encoding a novel elastinolytic enzyme in F. psychrophilum To formally demonstrate enzymatic activity, this gene (FP0506 from strain JIP 02/86) was expressed in the elastinolysis-deficient strain OSU THCO2-90, resulting in proficient elastin-degrading cells. The encoded protein is predicted to be a cell-surface-exposed lipoprotein with no homology to previously reported elastases. FP0506 might belong to the zincin tribe and gluzincin clan of metalloproteases, and this new elastase-encoding gene seems to be present only in some members of the family FlavobacteriaceaeIMPORTANCE Elastin is an important proteinaceous component of vertebrate connective tissues (e.g., blood vessels, lung, and skin), to which it confers elasticity. Elastases have been identified in a number of pathogenic bacteria. They are thought to be required for tissue penetration and dissemination, acting as "spreading factors." Flavobacterium psychrophilum is a devastating bacterial pathogen of salmonid fish (salmon and trout) that is responsible for severe economic losses worldwide. This pathogen displays strong proteolytic activities. Using a variety of techniques, including genome comparisons, we identified a gene encoding a novel elastase in F. psychrophilum The encoded protein is predicted to be a cell-surface-exposed lipoprotein with no homology to previously reported elastases. In addition, this elastase likely belongs to a new family of proteases that seems to be present only in some members of this important group of bacteria.

Flavobacterium sharifuzzamanii sp. nov., Isolated from the Sediments of the East China Sea.

A novel bacterial strain A7.6T was isolated from the sediments collected near the Zhairuo Island located in the East China Sea and characterized using a polyphasic approach. Cells were Gram-stain-negative, rod-shaped, non-spore forming, non-flagellated but motile by gliding. The strain was aerobic, positive for oxidase and catalase activities. The strain can grow at 4-35 °C, pH 5.5-9.0, and 0-3% (w/v) NaCl concentration. The major polar lipid was phosphatidylethanolamine, the predominant fatty acids (> 10%) were iso-C15:0 and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c). The genomic G+C content was 33.6 mol% and the major respiratory quinone was menaquinone 6. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain A7.6T belonged to the genus Flavobacterium and was closely related to Flavobacterium tistrianum GB 56.1T (98.4% similarity), F. nitrogenifigens NXU-44T (98.4%), F. ginsenosidimutans THG 01T (98.0%) and F. anhuiense D3T (97.7%). Average nucleotide identities and digital DNA-DNA hybridizations values for genomes ranged from 75.9 to 91.4% and 21.4 to 43.9% between strain A7.6T and its closest phylogenetic neighbors. The polyphasic characterization indicated that strain A7.6T represented a novel species of the genus Flavobacterium, for which the name Flavobacterium sharifuzzamanii is proposed. The type strain is A7.6T (= KCTC 62405T = MCCC 1K03485T). The NCBI GenBank accession number for the 16S rRNA gene of A7.6T is MH396692, and for the genome sequence is QJGZ00000000. The digital protologue database (DPD) Taxon Number is TA00643.

Type 9 secretion system structures reveal a new protein transport mechanism.

The type 9 secretion system (T9SS) is the protein export pathway of bacteria of the Gram-negative Fibrobacteres-Chlorobi-Bacteroidetes superphylum and is an essential determinant of pathogenicity in severe periodontal disease. The central element of the T9SS is a so-far uncharacterized protein-conducting translocon located in the bacterial outer membrane. Here, using cryo-electron microscopy, we provide structural evidence that the translocon is the T9SS protein SprA. SprA forms an extremely large (36-strand) single polypeptide transmembrane ß-barrel. The barrel pore is capped on the extracellular end, but has a lateral opening to the external membrane surface. Structures of SprA bound to different components of the T9SS show that partner proteins control access to the lateral opening and to the periplasmic end of the pore. Our results identify a protein transporter with a distinctive architecture that uses an alternating access mechanism in which the two ends of the protein-conducting channel are open at different times.

Novel Enzyme Actions for Sulphated Galactofucan Depolymerisation and a New Engineering Strategy for Molecular Stabilisation of Fucoidan Degrading Enzymes.

Fucoidans from brown macroalgae have beneficial biomedical properties but their use as pharma products requires homogenous oligomeric products. In this study, the action of five recombinant microbial fucoidan degrading enzymes were evaluated on fucoidans from brown macroalgae: Sargassum mcclurei, Fucus evanescens, Fucus vesiculosus, Turbinaria ornata, Saccharina cichorioides, and Undaria pinnatifida. The enzymes included three endo-fucoidanases (EC 3.2.1.-GH 107), FcnA2, Fda1, and Fda2, and two unclassified endo-fucoglucuronomannan lyases, FdlA and FdlB. The oligosaccharide product profiles were assessed by carbohydrate-polyacrylamide gel electrophoresis and size exclusion chromatography. The recombinant enzymes FcnA2, Fda1, and Fda2 were unstable but were stabilised by truncation of the C-terminal end (removing up to 40% of the enzyme sequence). All five enzymes catalysed degradation of fucoidans containing α(1→4)-linked l-fucosyls. Fda2 also degraded S. cichorioides and U. pinnatifida fucoidans that have α(1→3)-linked l-fucosyls in their backbone. In the stabilised form, Fda1 also cleaved α(1→3) bonds. For the first time, we also show that several enzymes catalyse degradation of S. mcclurei galactofucan-fucoidan, known to contain α(1→4) and α(1→3) linked l-fucosyls and galactosyl-ß(1→3) bonds in the backbone. These data enhance our understanding of fucoidan degrading enzymes and their substrate preferences and may assist development of enzyme-assisted production of defined fuco-oligosaccharides from fucoidan substrates.

Flavobacterium amnigenum sp. nov. Isolated from a River.

A yellowish, flexirubin-pigment-producing strain I3-3T was isolated from river water in Iksan, the Republic of Korea. The strain was gram-negative, aerobic, non-motile, showed catalase and oxidase activities, and could grow at a temperature range of 10-35°C, pH 5.0-10 and 0-2.0% (w/v) of NaCl. The major fatty acids were iso-C15:0, iso-C17:0 3-OH and summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c). The isolate contained phosphatidylethanolamine, one aminolipid, and two unidentified lipids as the major polar lipids. Menaquinone-6 (MK6) was the major respiratory quinone. The G+C content of the genomic DNA of strain I3-3T was 35.6%. Comparison of the 16S rRNA gene sequence with the sequences of the closely related type strains showed highest sequence similarity of 96.95% and 96.93% to Flavobacterium nitrogenifigens NXU-44T and Flavobacterium compostarboris 15C3T, respectively. Based on phenotypic and phylogenetic distinctiveness, strain I3-3T is considered as a member of novel species within the genus Flavobacterium, for which Flavobacterium amnigenum sp. nov. is proposed. The type strain is I3-3T (=KCTC 52884T =NBRC 112871T).

Flavobacterium knui sp. nov., a novel member of the family Flavobacteriaceae.

A Gram-stain negative, non-motile, rod-shaped bacterial strain, designated 2-56T, was isolated from water and characterized taxonomically using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strain 2-56T belongs to the family Flavobacteriaceae in the phylum Bacteroidetes and is closely related to Flavobacterium paronense KNUS1T (98.4%) and Flavobacterium collinsense 4-T-2T (96.7%). The G + C content of the genomic DNA of strain 2-56T was 33.4 mol%. The isolate contained MK-6 as the predominant respiratory quinone, and iso-C15:1 G (15.9%), iso-C15:0 (15.8%), iso-C17:0 3-OH (10.7%), and iso-C15:0 3-OH (9.6%) were the major fatty acids. The major polar lipids were phosphatidylethanolamine and an unidentified lipid. The phenotypic and chemotaxonomic data support the affiliation of strain 2-56T with the genus Flavobacterium. However, the DNA-DNA relatedness between the isolate and F. paronense and F. collinsense were 35.7 and 21.5%, respectively, clearly showing that strain 2-56T is not related to them at the species level. Strain 2-56T could be clearly differentiated from its close neighbours on the basis of its phenotypic, genotypic and chemotaxonomic features. Therefore, strain 2-56T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium knui sp. nov. is proposed. The type strain is 2-56T (= KCTC 62061T = JCM 32247T).

Structural Basis for Superoxide Activation of Flavobacterium johnsoniae Class I Ribonucleotide Reductase and for Radical Initiation by Its Dimanganese Cofactor.

A ribonucleotide reductase (RNR) from Flavobacterium johnsoniae ( Fj) differs fundamentally from known (subclass a-c) class I RNRs, warranting its assignment to a new subclass, Id. Its ß subunit shares with Ib counterparts the requirements for manganese(II) and superoxide (O2-) for activation, but it does not require the O2--supplying flavoprotein (NrdI) needed in Ib systems, instead scavenging the oxidant from solution. Although Fj ß has tyrosine at the appropriate sequence position (Tyr 104), this residue is not oxidized to a radical upon activation, as occurs in the Ia/b proteins. Rather, Fj ß directly deploys an oxidized dimanganese cofactor for radical initiation. In treatment with one-electron reductants, the cofactor can undergo cooperative three-electron reduction to the II/II state, in contrast to the quantitative univalent reduction to inactive "met" (III/III) forms seen with I(a-c) ßs. This tendency makes Fj ß unusually robust, as the II/II form can readily be reactivated. The structure of the protein rationalizes its distinctive traits. A distortion in a core helix of the ferritin-like architecture renders the active site unusually open, introduces a cavity near the cofactor, and positions a subclass-d-specific Lys residue to shepherd O2- to the Mn2II/II cluster. Relative to the positions of the radical tyrosines in the Ia/b proteins, the unreactive Tyr 104 of Fj ß is held away from the cofactor by a hydrogen bond with a subclass-d-specific Thr residue. Structural comparisons, considered with its uniquely simple mode of activation, suggest that the Id protein might most closely resemble the primordial RNR-ß.

Genetic manipulation of structural color in bacterial colonies.

Naturally occurring photonic structures are responsible for the bright and vivid coloration in a large variety of living organisms. Despite efforts to understand their biological functions, development, and complex optical response, little is known of the underlying genes involved in the development of these nanostructures in any domain of life. Here, we used Flavobacterium colonies as a model system to demonstrate that genes responsible for gliding motility, cell shape, the stringent response, and tRNA modification contribute to the optical appearance of the colony. By structural and optical analysis, we obtained a detailed correlation of how genetic modifications alter structural color in bacterial colonies. Understanding of genotype and phenotype relations in this system opens the way to genetic engineering of on-demand living optical materials, for use as paints and living sensors.

A simple and efficient method for the extraction and separation of menaquinone homologs from wet biomass of Flavobacterium.

Menaquinone homologs (MK-n), that is, MK-4, MK-5, and MK-6, can be produced by the fermentation of Flavobacterium. In this study, we proposed a simple and efficient method for the extraction of menaquinones from wet cells without the process of drying the biomass. Meanwhile, a rapid and effective solution for the separation of menaquinone homologs was developed using a single organic solvent, which was conducive to the recovery of the solvent. The results showed that the highest yield was obtained with pretreatment using absolute ethanol at a ratio of 6:1 (v/m) for 30 min and then two extractions of 30 min each using methanol at a ratio of 6:1 (v/m). The recovery efficiency of the menaquinones reached to 102.8% compared to the positive control. The menaquinone homologs were effectively separated using methanol as eluent at a flow rate of 0.52 mL/min by a glass reverse-phase C18 silica gel column with a height-to-diameter ratio of 5.5:1. The recovery of menaquinones achieved was 99.6%. In conclusion, the methods for extraction and separation of menaquinone homologs from wet Flavobacterium cells were simple and efficient, which makes them suitable not only on a laboratory scale but also for application on a large scale.

Improving thermal hysteresis activity of antifreeze protein from recombinant Pichia pastoris by removal of N-glycosylation.

To survive in a subzero environment, polar organisms produce ice-binding proteins (IBPs). These IBPs prevent the formation of large intracellular ice crystals, which may be fatal to the organism. Recently, a recombinant FfIBP (an IBP from Flavobacterium frigoris PS1) was cloned and produced in Pichia pastoris using fed-batch fermentation with methanol feeding. In this study, we demonstrate that FfIBP produced by P. pastoris has a glycosylation site, which diminishes the thermal hysteresis activity of FfIBP. The FfIBP expressed by P. pastoris exhibited a doublet on SDS-PAGE. The results of a glycosidase reaction suggested that FfIBP possesses complex N-linked oligosaccharides. These results indicate that the residues of the glycosylated site could disturb the binding of FfIBP to ice molecules. The findings of this study could be utilized to produce highly active antifreeze proteins on a large scale.

Flavobacterium jejuensis sp. nov., isolated from marine brown alga Ecklonia cava.

A bacterial strain, designated EC11(T) was isolated from brown alga Ecklonia cava collected from Jeju Island, Korea. EC11(T) was identified as a Gram-negative, rod-shaped and yellow-pigmented bacterial strain. The strain EC11(T) grew over a temperature range of 10 °C to 30 °C (optimally at 25 °C), and a pH range of 6.0-10.5 (optimally at pH 7.5). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain EC11(T) belongs to the genus Flavobacterium. Strain EC11(T) shared close similarity with Flavobacterium jumunjinense HME7102(T) (96.4%), Flavobacterium dongtanense LW30(T) (95.8%), Flavobacterium haoranii LQY-7(T) (95.3%), and Flavobacterium urocaniciphilum (95.1%). The major fatty acids (> 5%) were iso-C17:0 3-OH (22.4%), iso-C15:0 3-OH (19.0%), C15:0 (12.4%), summed feature 3 (comprising C16:1 ω7c/ C16:1 ω6c; 9.78%), iso-C15:1 G (9.6%), and iso-C16:0 3-OH (9.0%). The DNA G+C content was 28.1 mol% and the strain contained MK-6 as the predominant menaquinone. The major polar lipids were phosphatidylethanolamine, two unknown aminolipids and three unknown polar lipids. Based on phenotypic, chemotaxonomic and phylogenetic analysis, strain EC11T represents a novel species of the Flavobacterium genus, for which the name Flavobacterium jejuensis sp. nov. is proposed. The type strain of F. jejuensis is EC11(T) (=KCTC 42149(T) = JCM 30735(T)).

Flavobacterium buctense sp. nov., isolated from freshwater.

A gram-negative, non-gliding motile, aerobic bacterium, designated as strain T7(T), was isolated from freshwater of Chishui River flowing through Maotai town, Guizhou Province, southwest of China. Based on the 16S rRNA gene sequence analysis, the isolate was identified as a member of the genus Flavobacterium and that shared less than 97 % sequence similarities with recognized Flavobacterium species. Its closest phylogenetic relative was Flavobacterium dankookense (96.9 %), followed by Flavobacterium cheonhonense (96.8 %) and Flavobacterium macrobrachii (96.7 %). The strain formed smooth yellow colonies on R2A plates, and cells were observed to be short rods. Strain T7(T) was found to be able to grow at 15-30 °C (optimum 25 °C), at NaCl concentration of 0-0.5 % (optimum 0 %) and at pH 6.5-9.5 (optimum pH 7.5). Catalase and oxidase tests were positive. Polar lipids of strain T7(T) included phosphatidylethanolamine, four unidentified polar lipids, one unidentified phospholipid and one unidentified aminolipid. Chemotaxonomic analysis revealed menaquinone-6 as the dominant respiratory quinone and C(15:0), iso-C(15:0) and iso-C(15:1) as the major fatty acids. The DNA G+C content of strain T7(T) was determined to be 38.2 mol%. On the basis of phylogenetic, phenotypic and genetic data obtained in this study, strain T7(T) represents a novel species of the genus Flavobacterium, for which the name Flavobacterium buctense sp. nov. is proposed. The type strain is T7(T) (=JCM 30750=CGMCC 1.15216).

Identification of a 4-deoxy-L-erythro-5-hexoseulose uronic acid reductase, FlRed, in an alginolytic bacterium Flavobacterium sp. strain UMI-01.

In alginate-assimilating bacteria, alginate is depolymerized to unsaturated monosaccharide by the actions of endolytic and exolytic alginate lyases (EC 4.2.2.3 and EC 4.2.2.11). The monosaccharide is non-enzymatically converted to 4-deoxy-L-ery thro-5-hexoseulose uronic acid (DEH), then reduced to 2-keto-3-deoxy-D-gluconate (KDG) by a specific reductase, and metabolized through the Entner-Doudoroff pathway. Recently, the NADPH-dependent reductase A1-R that belongs to short-chain dehydrogenases/reductases (SDR) superfamily was identified as the DEH-reductase in Sphingomonas sp. A1. We have subsequently noticed that an SDR-like enzyme gene, flred, occurred in the genome of an alginolytic bacterium Flavobacterium sp. strain UMI-01. In the present study, we report on the deduced amino-acid sequence of flred and DEH-reducing activity of recombinant FlRed. The deduced amino-acid sequence of flred comprised 254 residues and showed 34% amino-acid identities to that of A1-R from Sphingomonas sp. A1 and 80%-88% to those of SDR-like enzymes from several alginolytic bacteria. Common sequence motifs of SDR-superfamily enzymes, e.g., the catalytic tetrad Asn-Lys-Tyr-Ser and the cofactor-binding sequence Thr-Gly-x-x-x-Gly-x-Gly in Rossmann fold, were completely conserved in FlRed. On the other hand, an Arg residue that determined the NADPH-specificity of Sphingomonas A1-R was replaced by Glu in FlRed. Thus, we investigated cofactor-preference of FlRed using a recombinant enzyme. As a result, the recombinant FlRed (recFlRed) was found to show high specificity to NADH. recFlRed exhibited practically no activity toward variety of aldehyde, ketone, keto ester, keto acid and aldose substrates except for DEH. On the basis of these results, we conclude that FlRed is the NADH-dependent DEH-specific SDR of Flavobacterium sp. strain UMI-01.