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Flavobacterium aquidurense sp. nov. and Flavobacterium hercynium sp. nov., from a hard-water creek

DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany

Correspondence
Sylvie Cousin
sylvie.cousin{at}dsmz.de

	ABSTRACT

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Ten new Flavobacterium-like strains were isolated from freshwater of the hard-water creek Westerhöfer Bach, northern Germany. These strains formed two phylogenetic groups: strains WB 1.1-56^T, WB 1.1-04, WB 1.1-14, WB 1.1-57 and WB 1.1-63; and strains WB 4.2-33^T, WB 4.1-86, WB 4.2-34, WB 4.2-32 and WB 4.2-78. Cells were Gram-negative, yellow-pigmented, chemoheterotrophic rods. Their major fatty acid profiles were similar, consisting of iso-C_{15 : 0}, iso-C_{15 : 0} 3-OH, iso-C_{17 : 0} 3-OH and summed feature 3 (C_{16 : 1}7c and/or iso-C_{15 : 0} 2-OH). DNA G+C contents for strains WB 1.1-56^T and WB 4.2-33^T were 33.5 and 37.5 mol%, respectively. Phylogenetic analysis based on almost complete 16S rRNA gene sequences indicated that strain WB 1.1-56^T was phylogenetically most closely related to Flavobacterium frigidimaris KUC-1^T, and that strain WB 4.2-33^T was related most closely to F. frigidimaris KUC-1^T and Flavobacterium saccharophilum DSM 1811^T. Levels of 16S rRNA gene sequence similarity between strains WB 1.1-56^T and WB 4.2-33^T and the type strains of recognized members of the genus Flavobacterium were below 98 %. DNA–DNA hybridization experiments confirmed the separate genomic status of strains WB 1.1-56^T and WB 4.2-33^T. Strains WB 1.1-56^T and WB 4.2-33^T and their respective relatives differed from phylogenetically related Flavobacterium species based on several phenotypic characteristics. On the basis of their phenotypic and phylogenetic distinctiveness, the two groups of strains are considered to represent two novel species, for which the names Flavobacterium aquidurense sp. nov. (type strain WB 1.1-56^T=DSM 18293^T=CIP 109242^T) and Flavobacterium hercynium sp. nov. (type strain WB 4.2-33^T=DSM 18292^T=CIP 109241^T) are proposed.

A table giving the fatty acid compositions of the novel strains and related members of the genus Flavobacterium and a dendrogram based on these results are available as supplementary material in IJSEM Online.

	MAIN TEXT

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The genus Flavobacterium Bergey et al. 1923 belongs to the family Flavobacteriaceae in the phylum Bacteroidetes. With emendation of the description of the genus (Bernardet et al., 1996), several species previously belonging to the genus were reclassified and placed in new or different genera. Recently, Bernardet & Bowman (2005) provided an excellent overview of the taxonomy and ecology of members of the genus Flavobacterium. Species of the genus have been isolated worldwide from habitats such as marine and freshwater environments, soil, sediment, sea-ice biofilms and diseased fish. In the past few years, many novel species have been added to the genus, isolated from freshwater sediments (Tamaki et al., 2003; Wang et al., 2006), glacier ice (Zhu et al., 2003), soil (Yoon et al., 2006; Kim et al., 2006), Antarctic habitats (McCammon & Bowman, 2000; Humphry et al., 2001; Van Trappen et al., 2003, 2004, 2005; Yi et al., 2005; Nogi et al., 2005; Yi & Chun, 2006), the gut of an earthworm (Horn et al., 2005) and bacterial aggregates of a wastewater treatment plant (Aslam et al., 2005). A rich diversity of flavobacteria has also been reported from river epilithon (O'Sullivan et al., 2006). In a recent study on the diversity of freshwater bacteria, hundreds of Gram-negative, rod-shaped Flavobacterium-like bacteria were isolated from water of a hard-water creek from the western slopes of the Harz Mountains, Germany. Following a pre-screening by MALDI-TOF mass spectrometry (Brambilla et al., 2007) and partial 16S rRNA gene sequence analysis, several groups of isolates were found to cluster separately from the type strains of recognized members of the genus Flavobacterium. Two of these groups were subjected to a polyphasic taxonomic study.

Isolation of strains from the Westerhöfer Bach has been described by Brambilla et al. (2007). Strains WB 1.1-56^T, WB 1.1-04, WB 1.1-14, WB 1.1-57 and WB 1.1-63 were isolated from spring water, and strains WB 4.2-33^T, WB 4.1-86, WB 4.2-34, WB 4.2-32 and WB 4.2-78 were isolated from creek water about 320 m downstream from this spring (station 4). Following isolation on R2A medium (Difco) strains were transferred to and maintained on medium 67 (M67; DSMZ, 2001) at 25 °C for at least 24 h. The same medium was also used to cultivate the reference strains Flavobacterium saccharophilum DSM 1811^T, Flavobacterium frigidimaris DSM 15937^T, Flavobacterium flevense DSM 1076^T and Flavobacterium pectinovorum DSM 6368^T.

DNA extraction and PCR amplification of the 16S rRNA genes were carried out as described by Rainey et al. (1996). The PCR amplificate was purified by using the QIAquick PCR purification kit (Qiagen) according to the manufacturer's instructions. Sequencing of the PCR products, manual alignment of the sequences with those of recognized Flavobacterium species and determination of similarity coefficients were performed as described by Rainey et al. (1996). The algorithm of De Soete (1983) and the neighbour-joining algorithm (Felsenstein, 1993) were used to generate tree topologies. Bootstrap values were calculated according to Felsenstein (1985).

All strains were first subjected to partial 16S rRNA gene sequence analysis (436 bp, 5' terminus). The five strains isolated from the spring water showed 100 % sequence similarity to each other. The partial 16S rRNA gene sequences of the five strains isolated from station 4 showed between 98.6 and 100 % similarity to each other.

The almost complete sequences of the 16S rRNA gene were determined for strains WB 1.1-56^T, WB 4.2-33^T and WB 4.2-78. The sequences of the type strains of F. saccharophilum and F. pectinovorum were reanalysed because of sequence ambiguity in the data deposited in public databases. A sequence similarity value of 97.9 % was found between strains WB 1.1-56^T and WB 4.2-33^T. These two novel strains showed 16S rRNA gene sequence similarity values of less than 98 % to the type strains of recognized members of the genus Flavobacterium. Strain WB 1.1-56^T was related most closely to F. frigidimaris DSM 15937^T (97.9 %), and WB 4.2-33^T to F. saccharophilum DSM 1811^T (96.7 %). None of the other recognized type strains of the genus Flavobacterium clustered closely to strains WB 1.1-56^T or WB 4.2-33^T. A neighbour-joining tree showing the phylogenetic position of strains WB 1.1-56^T, WB 4.2-33^T and WB 4.2-78 among related members of the genus Flavobacterium is shown in Fig. 1. The topologies of the trees generated with the neighbour-joining method and the algorithm of De Soete (1983) were almost identical. Only a few branch points of the dendrogram were supported by high bootstrap values, which is consistent with published data (Brambilla et al., 2007). The 16S rRNA gene sequences of strains WB 4.2-33^T and WB 4.2-78 were identical.

View larger version (43K): [in this window] [in a new window]   Fig. 1. Relationship between strains WB 1.1-56T, WB 4.2-33T and WB 4.2-78 and the type strains of neighbouring Flavobacterium species. The dendrogram is based on 16S rRNA gene sequence comparisons using the neighbour-joining algorithm of Felsenstein (1993). Numbers at branch points are bootstrap values >50 % (500 resamplings). Bar, 5 % sequence divergence, as determined by measuring the length of the horizontal lines connecting any two species. The sequences of less closely related Flavobacterium species served as a root.

Genomic relatedness between strains WB 1.1-56^T and WB 4.2-33^T and their most closely related phylogenetic neighbours, i.e. the type strains of F. frigidimaris and F. saccharophilum, was determined according to the spectrophotometric DNA–DNA reassociation method. DNA was isolated using a French pressure cell (Thermo Spectronic) and was purified by hydroxyapatite chromatography as described by Cashion et al. (1977). DNA–DNA hybridization was carried out as described by De Ley et al. (1970), with the modifications of Huß et al. (1983) and Escara & Hutton (1980), by using a model Cary 100 Bio UV/VIS-spectrophotometer equipped with a Peltier-thermostatted 6x6 multicell changer and a temperature controller with in-situ temperature probe (Varian). Hybridization values were determined at least twice for any given strain pair. Strain WB 1.1-56^T was moderately related to F. saccharophilum DSM 1811^T (60.5 %), while reassociation values with F. frigidimaris DSM 15937^T (26.2 %) and with WB 4.2-33^T and WB 4.2-78 (5.2 and 8.1 %, respectively) were significantly lower. Strain WB 4.2-33^T shared 100 % DNA–DNA relatedness with strains WB 4.2-78 and WB 4.1-86, i.e. two other members of the same 16S rRNA gene cluster. Reassociation values for strains WB 4.2-33^T and WB 4.2-78 with F. frigidimaris DSM 15937^T and F. saccharophilum DSM 1811^T were low, ranging between 31.4–41.8 and 44.0 %. These values indicate that the two new strain groups represent two distinct novel Flavobacterium genospecies.

The ten novel strains were subjected to fatty acid methyl ester analysis in order to determine their chemotaxonomic coherence. Also included were their closest phylogenetic neighbours, F. saccharophilum DSM 1811^T and F. frigidimaris DSM 15937^T, as well as the type strains of Flavobacterium denitrificans, F. pectinovorum, F. flevense and Flavobacterium hibernum. Fatty acids were extracted and analysed (Miller, 1982) according to the standard protocol of the Microbial Identification System (MIDI Microbial ID Inc.) by using the TSBA40 method. Extracts were analysed by using a Hewlett Packard model HP6890A gas chromatograph equipped with a flame-ionization detector as described by Kämpfer & Kroppenstedt (1996). The major compounds identified in the novel strains were iso-C_{15 : 0}, iso-C_{15 : 0} 3-OH, iso-C_{17 : 0} 3-OH and summed feature 3 (C_{16 : 1}7c and/or iso-C_{15 : 0} 2-OH) (Table 1; the complete data set is given in Supplementary Table S1 in IJSEM Online). These fatty acids are generally present in recognized members of the genus Flavobacterium. WB 1.1-56^T and related strains contained larger amounts of C_{15 : 0} 3-OH, C_{15 : 0}, C_{15 : 1}6c, iso-C_{17 : 1}9c and C_{17 : 1}6c fatty acids than strain WB 4.2-33^T and its relatives. The latter strain group possessed larger amounts of C_{16 : 0} and C_{16 : 0} 3-OH, a feature also found for F. frigidimaris DSM 15937^T. The main fatty acid differences between the novel strains and their closest described relatives consist of variations in the relative amounts of less abundant and minor components. Supplementary Fig. S1 (see IJSEM Online) provides a dendrogram of fatty acid similarities in which the Euclidean distances reveal the coherence of the two new strain clusters (<6 %). On the basis of fatty acid similarities, F. denitrificans DSM 15936^T and F. pectinovorum DSM 6368^T group with F. saccharophilum DSM 1811^T, while F. flevense DSM 1076^T and F. hibernum DSM 12611^T group with F. frigidimaris DSM 15937^T. None of these type strains cluster closely with the novel strains.

The optimal pH for growth was tested in buffered M67 at 25 °C between pH 5.8 and 8.4, with steps of 0.2 pH units. Salt tolerance was tested on R2A medium supplemented with 1–6 % NaCl, in 1 % step increases. Reading was after 1 day of incubation.

Colony morphology was determined on M67, R2A, nutrient agar (NA) and trypticase soy agar (TSA) (both Difco), MacConkey and DNA agar (Merck) and Anacker & Ordal agar (AOA) (Anacker & Ordal, 1955) after 5 days. Cells were tested for flexirubin pigments (Bernardet et al., 2002), Gram staining using the aminopeptidase and KOH reactions and for oxidase and catalase (H₂O₂ test) activities. Results are given in the species descriptions below and in Table 2. As determined by electron microscopy, cells of strains WB 1.1-56^T and WB 4.2-33^T lacked flagella (not shown). Physiological properties were determined by using the commercial API ZYM and API 20NE systems (bioMérieux), as well as the GN MicroPlate system (Biolog), according to the manufacturers' instructions. API ZYM tests were read after 6 h incubation while API 20NE and GN MicroPlate tests were read after 48 h. Incubation was at 25 °C. Also investigated were gliding motility (hanging drop technique; Bernardet et al., 2002), formation of coccoid degenerative cells (spheroplasts; phase-contrast microscopy), degradation of casein (Reichenbach & Dworkin, 1981), DNA (by using DNA agar from Difco), cellulose (strip of Whatman paper No. 1 in M67 broth), starch and L-tyrosine (Barrow & Feltham, 1993) and production of diffusible pigments on L-tyrosine agar. Reactions were read after 3–10 days. Microaerophily was tested by the candle jar method (Gerhardt et al., 1981).

The two novel strain clusters differed from each other in several physiological reactions, and both could clearly be distinguished from the type strains of F. saccharophilum and F. frigidimaris (Table 2). Identical reactions for the strains investigated are listed in the legend to Table 2. For the following properties, strains related to WB 4.2-33^T showed a larger number of positive reactions (at least 80 % of strains) in the substrate test panel supplied than those strains related to strain WB 1.1-56^T: -glucosidase, glycogen, gelatin, trisodium citrate, D-arabitol, D-galactose, D-mannitol, methyl -D-glycoside and D-trehalose. Positive reactions for at least 80 % of the strains related to WB 1.1-56^T and negative reactions for the strains related to WB 4.2-33^T were found for cystine arylamidase, N-acetyl--glucosaminidase, -galactosidase and L-arabinose (Table 2).

In conclusion, the novel strains have been shown to be phylogenetically related to members of the genus Flavobacterium (Fig. 1; Brambilla et al., 2007) and based on similarities in Gram-negative staining behaviour, chemoheterotrophy, fatty acid profiles, the presence of flexirubin pigments and base composition of DNA. They also show the typical morphological and cultural characteristics of members of the genus Flavobacterium as given by Bernardet et al. (1996) in their emended description of the genus. Strains WB 1.1-56^T are WB 4.2-33^T show less than 98.0 % 16S rRNA gene sequence similarity to their closest phylogenetic neighbours and have DNA–DNA relatedness values distinctly lower than 70 %, the threshold value considered for species delineation (Wayne et al., 1987). As the two genospecies show sufficient intra-cluster coherence with respect to phenotype, fatty acid profiles and 16S rRNA gene sequence similarities, while showing sufficient differences among each other and their closest recognized neighbours in phenotypic properties to allow their phenotypic identification, we propose the description of two novel species, Flavobacterium aquidurense sp. nov. and Flavobacterium hercynium sp. nov.

Description of Flavobacterium aquidurense sp. nov.
Flavobacterium aquidurense (a.qui.du.ren'se. L. fem. n. aqua water; L. fem. adj. durus hard; N.L. neut. adj. aquidurense pertaining to hard water).

Gram-negative, aerobic and microaerobic rods (0.9x2.8 µm). Growth occurs between 13 and 30 °C with an optimal temperature between 19 and 28 °C; no growth occurs below 8 °C or above 31.5 °C. Growth occurs between pH 6.0 and 6.6 with an optimal pH of 6.4. Growth occurs at NaCl concentrations between 0 and 1 %. No gliding motility; no flagella. Non-swarming; local spreading around colonies (maximum 6 mm) is observed on some growth media. Spheroplasts form after 3–4 days in R2A broth. Colonies formed on M67 agar after 5 days incubation are 3–8 mm in diameter, yellowish, spreading, irregular, flat with a slight rise at the centre but not umbonate, smooth, shiny, glistening, transparent and butyrous with filamentous margins. Colonies on AOA are zinc yellow, spreading, punctiform, flat with a central rise and with undulated margins fading to transparent. Colonies on R2A are 1–2 mm in diameter (mean 1.8 mm), zinc yellow, spreading, circular, convex, entire, smooth, translucent and butyrous with filamentous margins. Colonies on DNA agar are 0.9–1.5 mm in diameter (mean 1 mm), maize yellow, circular, non-spreading, convex-pyramidal, smooth and translucent to slightly opaque in the centre with undulated margins. Colonies on TSA are 0.7–1 mm in diameter, honey yellow, non-spreading, circular, convex, smooth, translucent and butyrous with undulate margins. Colonies on NA are 2.2 mm in diameter, maize yellow, have a central bump, are umbonate, smooth and translucent with undulate margins; irregular swarming is observed. No growth on MacConkey agar. Colonies do not adhere to the agar. Non-diffusible flexirubin pigments are present. Catalase- and oxidase-positive. Starch, L-tyrosine and casein are degraded. A brown pigment is produced on L-tyrosine agar. Cellulose, DNA and agar are not hydrolysed. Other physiological reactions are given in Table 1. Cells contain iso-C_{15 : 0}, iso-C_{17 : 0} 3-OH, iso-C_{15 : 0} 3-OH, iso-C_{17 : 1}9c, C_{15 : 0} and summed feature 3 (C_{16 : 1}7c and/or iso-C_{15 : 0} 2-OH) as main fatty acids. The DNA G+C content of the type strain is 33.5 mol%.

The type strain, WB 1.1-56^T (=DSM 18293^T=CIP 109242^T), was isolated from the spring of the Westerhöfer Bach, Westerhof, 40 km north of Göttingen, Germany.

Description of Flavobacterium hercynium sp. nov.
Flavobacterium hercynium (her.cy'ni.um. L. neut. adj. hercynium pertaining to Hercynia, the Roman name for a mountain range, including the Harz Mountains, that forms the southern border of the North German plain).

Gram-negative, aerobic and microaerobic rods (0.7–0.8x5.8–6.3 µm). Growth occurs between 12 and 29 °C with an optimal growth temperature of 20–27 °C; no growth occurs below 7 °C or above 30.5 °C. Growth occurs between pH 6.4 and 8.0 with an optimum of pH 6.4–7.8. Growth occurs at NaCl concentrations between 0 and 1 %. Motile by gliding; no flagella are observed. Cells glide on M67, AOA, NA and R2A media but not on DNA agar or TSA. Spheroplasts form after 3–4 days in R2A broth. On M67 agar growth is brown beige on plates and orange-yellow on loops; colony surface is effuse, irregular, glistening, spreading and with irregular lobate margins. Growth on AOA is brown beige; the surface is smooth, flat, translucent to transparent, and no margin is observed. Growth on R2A is dark orange to yellow; the surface is flat, glistening, translucent to transparent, with thinly lobate margins. Colonies on DNA agar are 4–8 mm in diameter, maize yellow, circular, non-spreading, umbonate, glistening, slightly irregular and translucent with undulate margins. Colonies on TSA are 4–7 mm in diameter, maize-yellow, non-spreading, circular, slightly umbonate, smooth, glistening and slightly irregular with undulate margins. Growth on NA is maize-yellow, glistening, irregular, flat and translucent. No growth is observed on MacConkey agar. Colonies do not adhere to the agar. Non-diffusible flexirubin pigments are present. Catalase- and oxidase-positive. Starch, L-tyrosine and casein are degraded. A brown pigment is produced on L-tyrosine agar. Cellulose, DNA and agar are not hydrolysed. Cells contain iso-C_{15 : 0}, iso-C_{17 : 0} 3-OH, iso-C_{15 : 0} 3-OH and summed feature 3 (C_{16 : 1}7c and/or iso-C_{15 : 0} 2-OH) as main fatty acids. The DNA G+C content of the type strain is 37.5 mol%.

The type strain, WB 4.2-33^T (=DSM 18292^T=CIP 109241^T), was isolated from a site about 320 m downstream of the spring of the Westerhöfer Bach, Westerhof, 40 km north of Göttingen, Germany.

	ACKNOWLEDGEMENTS

 
This project forms part of the Research Unit 571 ‘Geobiology of Organo- and Biofilms’, funded by the German Research Foundation (Sta 184/19-2; DFG-FOR 571; publication no. 6). We thank Hans Trüper for his advice with etymology. We thank Bettina Sträubler and Peter Schumann (DNA–DNA reassociation), Anja Frühling (phenotypic tests), Gabriele Pötter (fatty acid analysis) for technical advice and Manfred Rohde for help with electron microscopy.

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