The fish pathogen Flavobacterium columnare represents four distinct species: Flavobacterium columnare,Flavobacterium covae sp. nov., Flavobacterium davisii sp. nov. and Flavobacterium oreochromis sp. nov., and emended description of Flavobacterium columnare

Flavobacterium columnare is the causative agent of columnaris disease in freshwater fish and four discrete genetic groups exist within the species, suggesting that the species designation requires revision. The present study determined the taxonomic status of the four genetic groups of F. columnare using polyphasic and phylogenomic approaches and included five representative isolates from each genetic group (including type strain ATCC 23463^T; genetic group 1). 16S rRNA gene sequence analysis revealed genetic group 2 isolate AL-02-36^T, genetic group 3 isolate 90-106^T, and genetic group 4 isolate Costa Rica 04-02-TN^T shared less than <98.8 % sequence identity to F. columnare ATCC 23463^T. Phylogenetic analyses of 16S rRNA and gyrB genes using different methodologies demonstrated the four genetic groups formed well-supported and distinct clades within the genus Flavobacterium. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (GGDC) values between F. columnare ATCC 23463^T, genetic group 2 isolate AL-02-36^T, genetic group 3 isolate 90-106^T, and genetic group 4 isolate Costa Rica 04-02-TN^T were less than 90.84% and 42.7%, respectively. Biochemical and physiological characteristics were similar among the four genetic groups; however, quantitative differences in fatty acid profiles were detected and MALDI-TOF analyses demonstrated numerous distinguishing peaks unique to each genetic group. Chemotaxonomic, MALDI-TOF characterization and ANI/GGDC calculations afforded differentiation between the genetic groups, indicating each group is a discrete species. Herein, the names F. covae sp. nov. (AL-02-36^T), F. davisii sp. nov. (90-106^T), and F. oreochromis sp. nov. (Costa Rica 04-02-TN^T) are proposed to represent genetic groups 2, 3, and 4, respectively.     

Flavobacterium sp. strain 4221 and Pedobacter sp. strain 4236 beta-1,3-glucanases that are active at low temperatures

Secretion of beta-1,3-glucanases by the arctic bacterial isolates 4221 and 4236, related to the genera Flavobacterium and Pedobacter, was discovered. Escherichia coli and Lactococcus lactis expression of beta-1,3-glucanases Glc4221-1 and Glc4236-1 from the respective isolates was achieved. The enzymes hydrolyzed fungal cell walls and retained activity at low temperatures.     

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 1.1-56^T (98.3% 16S rRNA gene sequence similarity) while group II (n = 4) showed Flavobacterium hydatis DSM 2063^T 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 C_15:0 iso, C_15:1 iso G, C_15:0 iso 3-OH, C_17:0 iso 3OH and Summed Feature 3 (C_16:1 ω7c/C_16:1 ω6c). Strain CCM 8828^T (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 8825^T (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 8828^T = P5626^T = LMG 30617^T) and Flavobacterium cupreum sp. nov. (type strain CCM 8825^T = P2683^T = LMG 30614^T) are proposed.     

A novel meta-cleavage product hydrolase from Flavobacterium sp. ATCC27551

The organophosphate degrading (opd) gene cluster of plasmid pPDL2 of Flavobacterium sp. ATCC27551 contains a novel open-reading frame, orf243. This was predicted to encode an alpha/beta hydrolase distantly related to the meta-fission product (MFP) hydrolases such as XylF, PhnD, and CumD. By homology modeling Orf243 has most of the structural features of MFP hydrolases including the characteristic active site catalytic triad. The purified protein (designated MfhA) is a homotetramer and shows similar affinity for 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD), 2-hydroxymuconic semialdehyde (HMSA), and 2-hydroxy-5-methylmuconic semialdehyde (HMMSA), the meta-fission products of 3-methyl catechol, catechol, and 4-methyl catechol. The unique catalytic properties of MfhA and the presence near its structural gene of cis-elements required for transposition suggest that mfhA has evolved towards encoding a common hydrolase that can act on meta-fission products containing either aldehyde or ketone groups.     

Isolation of a methyl parathion-degrading Pseudomonas sp. that possesses DNA homologous to the opd gene from a Flavobacterium sp.

Two mixed bacterial cultures isolated by soil enrichment were capable of utilizing methyl parathion (O,O-dimethyl O-p-nitrophenylphosphorothioate) and parathion (O,O-diethyl O-p-nitrophenylphosphorothioate) as a sole source of carbon. Four isolates from these mixed cultures lost their ability to utilize the pesticides independently in transfers subsequent to the initial isolation. One member of the mixed cultures, a Pseudomonas sp., however, hydrolyzed the pesticides to p-nitrophenol but required glucose or another carbon source for growth. The crude cell extracts prepared from this bacterium showed an optimum pH range from 7.5 to 9.5 for the enzymatic hydrolysis. Maximum enzymatic activity occurred between 35 and 40 degrees C. The enzyme activity was not inhibited by heavy metals, EDTA, or NaN3. Another isolate from the mixed cultures, a Flavobacterium sp., used p-nitrophenol for growth and degraded it to nitrite. Nitrite was assimilated into the cells under conditions during which the nitrogen source was excluded from the minimal growth medium. The hybridization data showed that the DNAs from a Pseudomonas sp. and from the mixed culture had homology with the opd (organophosphate degradation) gene from a previously reported parathion-hydrolyzing bacterium, Flavobacterium sp. The use of the opd gene as a probe may accelerate progress toward understanding the complex interactions of soil microorganisms with parathions.     

Reconstitution of Pronase-Susceptible Flocs of Flavobacterium sp.

A component responsible for the aggregation of cells was extracted from Flavobacterium strain B by treatment of cells with 5 m guanidine hydrochloride and partially purified by gel filtration. The guanidine hydrochloride-extracted cells were reaggregated with the component after dialysis against 0.3mm of CaCl₂. Various divalent cations were effective in place of Ca²⁺, but Ca²⁺ was most effective for reconstitution. The reconstituted flocs were deflocculated by the treatment of Pronase or ethylenediaminetetraacetic acid indicating that reconstituted flocs closely resemble natural flocs.     

Isolation of a methyl parathion-degrading Pseudomonas sp. that possesses DNA homologous to the opd gene from a Flavobacterium sp

Two mixed bacterial cultures isolated by soil enrichment were capable of utilizing methyl parathion (O,O-dimethyl O-p-nitrophenylphosphorothioate) and parathion (O,O-diethyl O-p-nitrophenylphosphorothioate) as a sole source of carbon. Four isolates from these mixed cultures lost their ability to utilize the pesticides independently in transfers subsequent to the initial isolation. One member of the mixed cultures, a Pseudomonas sp., however, hydrolyzed the pesticides to p-nitrophenol but required glucose or another carbon source for growth. The crude cell extracts prepared from this bacterium showed an optimum pH range from 7.5 to 9.5 for the enzymatic hydrolysis. Maximum enzymatic activity occurred between 35 and 40 degrees C. The enzyme activity was not inhibited by heavy metals, EDTA, or NaN3. Another isolate from the mixed cultures, a Flavobacterium sp., used p-nitrophenol for growth and degraded it to nitrite. Nitrite was assimilated into the cells under conditions during which the nitrogen source was excluded from the minimal growth medium. The hybridization data showed that the DNAs from a Pseudomonas sp. and from the mixed culture had homology with the opd (organophosphate degradation) gene from a previously reported parathion-hydrolyzing bacterium, Flavobacterium sp. The use of the opd gene as a probe may accelerate progress toward understanding the complex interactions of soil microorganisms with parathions.     

Purification and characterization of a cyclodextrin-degrading enzyme from Flavobacterium sp.

A cell-bound cyclodextrin-degrading enzyme with a relative molecular mass (M_r) of around 62 000 and an isoelectric point (pI) near 8.0 was isolated and purified to 94% homogeneity from Flavobacterium sp. The enzyme hydrolysed maltooligosaccharides and cyclodextrins to glucose, maltose, and maltotriose. Less glucose, but larger amounts of the line of maltooligosaccharides from maltose to (in case of cyclodextrins) the linearized substrates were found in short-term digests. Digestion of maltotriose yielded glucose, maltose, and some maltotetraose to maltohexaose, i.e. the enzyme catalysed both hydrolysis and transglycosylation. Starch was a poorer substrate, and was hydrolysed to mainly glucose and maltose, presumably by a kind of exo-attack. Pullulan was slightly digested, the products being glucose, panose/isopanose, and larger saccharides containing -1,6-glucosidic bonds. Since maltohexaose to maltooctaose were hydrolysed at higher rates than the cyclodextrins of corresponding lengths, the enzyme of Flavobacterium sp. was proposed to be classified as a decycling maltodextrinase. Correspondence to: H. Bender     

Isolation and characterization of Flavobacterium strains that degrade pentachlorophenol.

Bacteria able to mineralize 100 to 200 ppm of pentachlorophenol (PCP) were isolated by selective enrichment from PCP-contaminated soils from three geographic areas of Minnesota. Although differing somewhat in their responses to various biochemical and biophysical tests, all strains were assigned to the genus Flavobacterium. Five representative strains were examined in detail. All strains metabolized PCP as a sole source of carbon and energy; 73 to 83% of all carbon in the form of [U-14C]PCP was returned as 14CO2, with full liberation of chlorine as chloride. A comparison between strains in their ability to metabolize PCP showed some strains to be more efficient than others. Guanine-plus-cytosine contents of DNA ranged from 58.8 to 63.8%, and DNA/DNA hybridization studies with total DNA digests suggested substantial genetic homology between strains. All strains were shown to possess an 80- to 100-kilobase plasmid, and evidence suggested the presence of a larger plasmid (greater than 200 kilobases).     

Flavobacterium frigidimaris sp. nov., isolated from Antarctic seawater

We described the polyphasic characterization of the psychrotolerant isolated from Antarctic seawater. The strain was closely related to Flavobacterium hydatis, F. pectinovorum, and F. saccharophilum on the basis of the 16S rDNA sequence analysis. However, DNA–DNA hybridization experiments showed that the DNA-similarities between strain KUC-1^T and the reference strains of Flavobacterium were less than 30%. Therefore, we can definite a new species of Flavobacterium phylogenetically, and strain KUC-1^T can be considered to be a new species of Flavobacterium. i.e. F. frigidimaris (KUC-1^T: JCM 12218^T and DSM 15937^T; mol% G+C of DNA of the type strain is 34.5 mol%). Useful phenotypical features for discrimination of F. frigidimaris from other Flavobacterium species, such as a resistance to NaCl, optimum growth temperature, and cellular fatty acid composition, were also determined.     

A comparative study on phyllosphere nitrogen fixation by newly isolated Corynebacterium sp. & Flavobacterium sp. and their potentialities as biofertilizer

A number of nitrogen fixing bacteria has been isolated from forest phyllosphere on the basis of nitrogenase activity. Among them two best isolates are selected and identified as Corynebacterium sp. AN1 & Flavobacterium sp. TK2 able to reduce 88 and 132 n mol of acetylene (10(8)cells(-1)h(-1)) respectively. They were grown in large amount and sprayed on the phyllosphere of maize plants as a substitute for nitrogenous fertilizer. Marked improvements in growth and total nitrogen content of the plant have been observed by the application of these nitrogen-fixing bacteria. An average 30-37% increase in yield was obtained, which is nearer to chemical fertilizer treatment. Comparatively better effect was obtained by application of Flavobacterium sp.     

Bacteriophage resistance and flocculation deficiency of Flavobacterium sp. are phenotypically interrelated

Summary An analysis of Flavobacterium sp. strain 304 variants selected for flocculation deficiency and phage resistance has been carried out. The results show that flocculation is efficiently hindered by overexpressed protein in the outer membrane or carbohydrate on the cell surface. The floc-forming protein is suggested to be a minor component of the cell surface. The 12 flocculation-deficient and five phage-insensitive variants obtained were grouped into ten classes based on nine characteristics. In addition, bacteriophage 304 resistance and the pure culture flocculation deficiency of this strain are interrelated. Offsprint requests to: D. H. Bamford     

Isolation and Characterization of a Cold-Active Xylanase Enzyme from Flavobacterium sp.

Xylan is the major component of hemicellulose, and xylan should be fully utilized to improve the efficiencies of a biobased economy. There are a variety of industrial reaction conditions in which an active xylanase enzyme would be desired. As a result, xylanase enzymes with different activity profiles are of great interest. We isolated a xylanase gene (xyn10) from a Flavobacterium sp. whose sequence suggests that it is a glycosyl hydrolase family 10 member. The enzyme has a temperature optimum of 30°C, is active at cold temperatures, and is thermolabile. The enzyme has an apparent K_m of 1.8 mg/ml and k_cat of 100 sec⁻¹ for beechwood xylan, attacks highly branched native xylan substrates, and does not have activity against glucans.     

Defluviitalea phaphyphila sp. nov., a Novel Thermophilic Bacterium That Degrades Brown Algae

Brown algae are one of the largest groups of oceanic primary producers for CO₂ removal and carbon sinks for coastal regions. However, the mechanism for brown alga assimilation remains largely unknown in thermophilic microorganisms. In this work, a thermophilic alginolytic community was enriched from coastal sediment, from which an obligate anaerobic and thermophilic bacterial strain, designated Alg1, was isolated. Alg1 shared a 16S rRNA gene identity of 94.6% with Defluviitalea saccharophila LIND6LT2^T. Phenotypic, chemotaxonomic, and phylogenetic studies suggested strain Alg1 represented a novel species of the genus Defluviitalea, for which the name Defluviitalea phaphyphila sp. nov. is proposed. Alg1 exhibited an intriguing ability to convert carbohydrates of brown algae, including alginate, laminarin, and mannitol, to ethanol and acetic acid. Three gene clusters participating in this process were predicted to be in the genome, and candidate enzymes were successfully expressed, purified, and characterized. Six alginate lyases were demonstrated to synergistically deconstruct alginate into unsaturated monosaccharide, followed by one uronic acid reductase and two 2-keto-3-deoxy-d-gluconate (KDG) kinases to produce pyruvate. A nonclassical mannitol 1-phosphate dehydrogenase, catalyzing d-mannitol 1-phosphate to fructose 1-phosphate in the presence of NAD⁺, and one laminarase also were disclosed. This work revealed that a thermophilic brown alga-decomposing system containing numerous novel thermophilic alginate lyases and a unique mannitol 1-phosphate dehydrogenase was adopted by the natural ethanologenic strain Alg1 during the process of evolution in hostile habitats.     

Substrate specificity of endo-beta-galactosidases from Flavobacterium keratolyticus and Escherichia freundii is different from that of Pseudomonas sp

The substrate specificity of endo-beta-galactosidase of Pseudomonas sp. was found to differ from that of Flavobacterium keratolyticus or Escherichia freundii, based on the following experimental results. The endo-beta-galactosidases from these three bacteria released 6-O-sulfo-GlcNAc beta 1-3Gal as one of the major products from keratan sulfates from different sources. In addition to the sulfated disaccharide, Flavobacterium and Escherichia enzymes produced GlcNAc beta 1-3Gal, which is also an integral repeating unit of keratan sulfate, whereas the Pseudomonas enzyme did not release any non-sulfated disaccharide. Tetrasaccharides were prepared from the teleost skin keratan sulfate by digestion with Pseudomonas enzyme followed by gel filtration on Sephadex G-50 chromatography. A part of the tetrasaccharide fraction was hydrolyzed by Flavobacterium enzyme to produce 6-O-sulfo-GlcNAc beta 1-3Gal and GlcNAc beta 1-3Gal, whereas the fraction was completely resistant to retreatment with the Pseudomonas enzyme. Endo-beta-galactosidases from F. keratolyticus and E. freundii hydrolyzed the internal beta-1,4-galactosyl linkage of various neolacto-type glycosphingolipids to produce glucosylceramides. However, these glycosphingolipids were completely resistant to the Pseudomonas enzyme. These findings clearly show that the sulfation on the N-acetylglucosamine adjacent to galactose in the lactosaminoglycans is essential for expression of the Pseudomonas enzyme, but not for that of the Flavobacterium or Escherichia enzyme.     

Flavobacterium ginsengiterrae sp. nov., isolated from a ginseng field

A novel strain of Flavobacterium, DCY55(T), a Gram-negative, yellow-pigmented, rod-shaped, non-spore-forming and gliding-motile bacterium, was isolated from the soil of a ginseng field in South Korea. Phylogenetic analysis, based on the 16S rRNA sequence, demonstrated that strain DCY55(T) belongs to the genus Flavobacterium within the family Flavobacteriaceae. Strain DCY55(T) showed the highest similarity with F. johnsoniae UW101(T) (97.1%), F. ginsenosidimutans THG 01(T) (96.8%), F. defluvii EMB 117(T) (96.6%), F. banpakuense 15F3(T) (96.3%) and F. anhuiense D3(T) (95.8%). Chemotaxonomic results showed that strain DCY55(T) predominantly contains menaquinone MK-6, that its DNA G+C content is 36.1mol%, and that its major cellular fatty acids are iso-C(15:0), summed feature 3 (comprising iso-C(15:0) 2-OH and/or C(16:1) ω 7c) and C(16:0). The chemotaxonomic and genotypic characteristics support the taxonomic classification of strain DCY55(T) to the genus Flavobacterium. The results of physiological and biochemical tests confirmed that strain DCY55(T) is distinct from previously validated species. We conclude that strain DCY55(T) should be classified as a novel species of the genus Flavobacterium, for which the name Flavobacterium ginsengiterrae sp. nov. is proposed, with the type strain DCY55(T) (=KCTC 23319(T) = JCM 17337(T)).     

Pseudomonas sp. M174 inhibits the fish pathogen Flavobacterium psychrophilum

Aims: To study the antagonic affect of probiotic Pseudomonas M174 on the fish pathogen Flavobacterium psychrophilum. Methods and Results: The ability of Pseudomonas M174 to inhibit the growth of Fl. psychrophilum was examined in iron‐sufficient and ‐deficient media. Possible siderophore production was also investigated. Antagonistic activity was confirmed in disease challenge experiments using a rainbow trout (Oncorhynchus mykiss) model. Adhesion of Pseudomonas M174 to fish surfaces and its ability to stimulate innate immunity was also investigated in vivo. Pseudomonas M174 antagonized Fl. psychrophilum and produced siderophores in vitro. In challenge experiments with Fl. psychrophilum, fish fed with Pseudomonas M174 had lower levels of mortalities than the controls. It was possible to find Pseudomonas M174 in the intestinal content of these fish after feeding and bathing with the probiotic, but probiotic was obtained from the gills only after feeding. Respiratory burst activity was also found to be enhanced in the M174 fed fish. Conclusions: These results suggest that M174 is a potential probiotic against Fl. psychrophilum and has several modes of action. Significance and Impact of the Study: Probiotics are a promising alternative to the use of antibiotics in aquaculture and could be a more sustainable disease control method.