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Department of Microbiology, Oregon State University, Corvallis, OR 97331, USA
Correspondence
Stephen J. Giovannoni
steve.giovannoni{at}oregonstate.edu
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ABSTRACT |
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The GenBank accession numbers for the 16S rRNA gene sequences of strains HTCC2501T and HTCC2514 are AY424899 and AY424900.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Bernardet et al., 2002) is one of the main phylogenetic lineages within the phylum ‘Bacteroidetes’ (Garrity & Holt, 2001), members of which generally comprise 6–30 % of total bacterial communities in sea water, as determined by fluorescence in-situ hybridization (Glöckner et al., 1999; Eilers et al., 2000; Cottrell & Kirchman, 2000). According to the emended description of the family Flavobacteriaceae by Bernardet et al. (2002), members of the family are Gram-negative, non-spore-forming, short to moderately long rods that cannot degrade crystalline cellulose. Their major cellular fatty acids are branched or hydroxy fatty acids, with high levels of i15 : 0 and 3-OH i17 : 0, and the DNA G+C contents range from 27 to 44 mol%. In the last few years, polyphasic approaches (Vandamme et al., 1996) powered by 16S rRNA gene sequence-based phylogeny have rapidly expanded the discovery and description of members of the family Flavobacteriaceae, including Gelidibacter and Psychroserpens (Bowman et al., 1997), Polaribacter (Gosink et al., 1998), Psychroflexus (Bowman et al., 1998), Salegentibacter (McCammon & Bowman, 2000), Cellulophaga (Johansen et al., 1999), Arenibacter (Ivanova et al., 2001), Muricauda (Bruns et al., 2001), Aequorivita (Bowman & Nichols, 2002), Gillisia (Van Trappen et al., 2004), Vitellibacter, Ulvibacter and Mesonia (Nedashkovskaya et al., 2003a, b, 2004) and Croceibacter (Cho & Giovannoni, 2003a), most of which were cultivated from marine environments. We have isolated and characterized several novel marine strains within the family Flavobacteriaceae from a series of high-throughput culturing (HTC) experiments. In this study, we describe two of these strains, HTCC2501T and HTCC2514, as members of a new genus in the family Flavobacteriaceae. The two strains were isolated from the Sargasso Sea (Atlantic Ocean), using HTC, and were maintained on Marine agar 2216 (Difco) at 30 °C as described previously (Cho & Giovannoni, 2003a).
Phylogeny
16S rRNA gene fragments were generated by a PCR and directly sequenced as described by Cho & Giovannoni (2003a). Nearly full-length sequences of the 16S rRNA gene, 1434 and 1454 bp for strains HTCC2501T and HTCC2514, respectively, were aligned using the ARB software package (Ludwig et al., 1998) and 1169 unambiguously aligned nucleotide positions were used for phylogenetic analyses in PAUP* 4.0 beta 10. Phylogenetic trees were inferred by neighbour-joining with the Kimura two-parameter model, maximum parsimony and maximum likelihood (tree bisection–reconnection-branching; a transition/transversion ratio of 1·326). The tree topologies from neighbour-joining and maximum parsimony were evaluated by bootstrap analyses based on 1000 resamplings. The sequence similarity between strains HTCC2501T and HTCC2514 was 99·99 % (only 2 nucleotide differences), so they were considered to be the same species using this criterion as well as on the basis of their DNA relatedness (94·6±3·9 %; for methods, see Cho & Giovannoni, 2003b). None of the taxa with validly published names showed more than 90 % 16S rRNA gene sequence similarity to the strains. The two HTCC isolates formed a distinct lineage within the family Flavobacteriaceae (Fig. 1), the most closely related genera being Zobellia (88·6–89·7 %), Arenibacter (88·3–88·4 %), Aequorivita (88·3–89·2 %) and Vitellibacter (87·8–88·0 %), and the lineage did not associate significantly with any of the described genera in the family. The branching orders and phylogenetic relationships between the strains and Muricauda–Zobellia–Arenibacter–Aequorivita–Vitellibacter were well conserved in all three phylogenetic trees (Fig. 1). The robustness of the phylogenetic relationships and the low sequence similarities between the strains and the other genera demonstrate that the two novel HTCC isolates represent a new genus in the family Flavobacteriaceae.
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, b), except that the incubation temperature used was 30 °C. Biochemical tests were carried out using API 20NE strips (bioMérieux). Custom-made 48-well microplates containing 47 different carbon compounds at a final concentration of 0·2 % (w/v or v/v) were used to test sole-carbon-source utilization. Cell morphology varied clearly with the growth stage of the cells. Cells in early exponential phase (1–2 days incubation) were predominantly straight rods, 1·6–5·6 µm long and 0·3–0·7 µm wide, while those in stationary phase (5–8 days incubation) tended to transform into coccoid cells that were 0·6–1·2 µm in diameter (Fig. 2). This pleomorphic property has been reported for other genera of the family Flavobacteriaceae, including Gelidibacter and Psychroserpens (Bowman et al., 1997). Colonies on Marine agar 2216 were 1·0–2·0 mm in diameter, rusty orange-coloured, uniformly circular, pulvinate, opaque and possessed a smooth surface. Gliding motility and flagella were not observed. They did not grow under either strictly anaerobic conditions or microaerobic conditions. The temperature range for growth was 10–44 °C, with optimum growth at 30 °C. No growth was observed at 4 or 48 °C. The pH range for growth was pH 6·0–9·0 (optimum 8·0–8·5). The isolates were moderately halophilic, showing good growth at NaCl concentrations of 0·25–10 % (w/v; optimum 2·5 %). Both strains produced carotenoid pigments with wavelength absorbance spectral peaks at 339 and 457 nm, with a major peak at 457 nm. The bathochromic shift test results indicated that flexirubin pigments were absent. Other phenotypic results for biochemical characteristics, sole-carbon-source utilization, antibiotic susceptibility and degradation of macromolecules are given in Table 1 and in the species description.
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, using a Platinum EPS reverse-phase C18 column (Alltech Associates), and phage 
DNA was used as a standard throughout the analyses. The DNA G+C contents of strains HTCC2501T and HTCC2514 were respectively 56·4±0·2 and 54·7±0·4 mol%. The G+C content of the strains was at least 10 mol% higher than that of other members of the family Flavobacteriaceae (Table 2). Cellular fatty acid methyl esters were prepared and analysed using GC according to the instructions of the Microbial Identification System (MIDI). The predominant fatty acids in the strains were i15 : 0 (24–28 %), i15 : 1 (14–21 %) and 3-OH i17 : 0 (25–27 %) (Table 1
). The major fatty acid types were branched acids and hydroxy acids, which comprised 75·7–77·9 % of total fatty acids. The fatty acid profiles of the two strains were almost identical to each other. Although the fatty acid profile of the HTCC isolates was similar to that of Muricauda ruestringensis, it differed significantly from those of the other phylogenetically related genera in the family Flavobacteriaceae (Table 2).
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). Polyphasic taxonomy based on 16S rRNA gene sequence analyses (Fig. 1) and phenotypic characteristics together with chemotaxonomic data (Tables 1 and 2) demonstrated that the two HTCC strains represent a coherent and novel genus within the family Flavobacteriaceae. The phenotypic and genotypic characteristics of the strains generally met the minimal standards for the family Flavobacteriaceae (Bernardet et al., 2002). Furthermore, comparisons of phenotypic and chemotaxonomic characteristics between the phylogenetically associated genera indicated clearly that the isolates could not be placed in any of the previously described genera (Table 2). The property of the strains that best differentiated them from other members of the family Flavobacteriaceae was the DNA G+C content, which was more than 10 mol% higher than those of the other genera within the family. In view of all the data presented in this study, the novel strains belong to a new genus and species within the family Flavobacteriaceae; therefore, we propose for them the name Robiginitalea biformata gen. nov., sp. nov.
Description of Robiginitalea gen. nov.
Robiginitalea (Ro.bi.gi.ni.tal'e.a. L. gen. n. robiginis of rust; L. fem. n. talea a rod; N.L. fem. n. Robiginitalea a rust-coloured rod).
Gram-negative. Morphology varies from straight rods, in exponential phase, to coccoid cells, in stationary phase. Cells do not exhibit motility or gliding motility. Oxidase- and catalase-positive. Carotenoid pigments are produced, but flexirubin pigments are not produced. Obligately aerobic and chemoheterotrophic. NaCl is required for growth. Starch and aesculin are degraded. The major fatty acid types are branched acids and hydroxy acids. The predominant fatty acids are i15 : 0 (24–28 %), i15 : 1 (14–21 %) and 3-OH i17 : 0 (25–27 %). DNA G+C content is 55–56 mol%. Phylogenetically, a novel member of the family Flavobacteriaceae. The type and only species of the genus is Robiginitalea biformata.
Description of Robiginitalea biformata sp. nov.
Robiginitalea biformata (bi.for.ma'ta. L. fem. adj. biformata double-formed, two-shaped, pertaining to the different cell morphology in different growth phases).
Description is the same as that for the genus with the following additional properties. Cells in exponential phase are straight rods, 1·6–5·6 µm long and 0·3–0·7 µm wide. Cells in stationary phase are coccoid, 0·6–1·2 µm in diameter. Endospores and poly-
-hydroxybutyrate granules are not formed. Colonies are rusty-orange-coloured, circular, pulvinate and opaque. Growth occurs at 10–44 °C, but not at 4 or 48 °C. The pH and salinity ranges for growth are 6·0–9·0 and 0·25–10 % (w/v), respectively. Moderately halophilic. Maximum absorption spectral peak occurs at 457 nm. No denitrification activity detected. A variety of carbon compounds are used as sole carbon sources, including pentoses, hexoses, oligosaccharides, sugar acids and amino acids. Cellular fatty acid composition, biochemical characteristics, sole-carbon-source utilization, antibiotic susceptibility and degradation of macromolecules are detailed in Table 1. The DNA G+C content of the type strain, HTCC2501T, is 56·4±0·2 mol% (HPLC method). Strains have been isolated from the western Sargasso Sea, Atlantic Ocean.
The type strain is HTCC2501T (=ATCC BAA-864T=KCTC 12146T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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