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Bacteroidetes

Arcicella aurantiaca sp. nov., isolated from stream water

Shih-Yi Sheu, Cho-Song Yang, Ming-Hui Chen, A. B. Arun, Chiu-Chung Young, Wen-Ming Chen

  • 1Department of Marine Biotechnology, National Kaohsiung Marine University, No. 142, Hai-Chuan Rd, Nan-Tzu, Kaohsiung City 811, Taiwan, ROC
  • 2Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung Marine University, No. 142, Hai-Chuan Rd, Nan-Tzu, Kaohsiung City 811, Taiwan, ROC
  • 3Yenepoya Research Center, Yenepoya University, University Rd, Deralakatee, Mangalore, Karnataka, India
  • 4Department of Soil Environmental Science, College of Agriculture and Natural Resources, National Chung Hsing University, Taichung, Taiwan, ROC
  • Correspondence
    Wen-Ming Chen
    p62365{at}ms28.hinet.net

    • International Journal of Systematic and Evolutionary Microbiology 2010; 60(12):2979–2983 · https://doi.org/10.1099/ijs.0.021527-0

    View at publisher PubMed

    Abstract

    A bacterial strain designated TNR-18T was isolated from stream water in southern Taiwan and characterized using a polyphasic taxonomic approach. Cells of strain TNR-18T were Gram-stain-negative, strictly aerobic, non-motile and vibrioid, and the strain formed orange colonies. Growth occurred at 10–30 °C, with 0–0.2 % NaCl and at pH 6.0–10.0. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain TNR-18T belonged to the genus Arcicella; its closest neighbours were Arcicella rosea TW5T and Arcicella aquatica NO-502T, with respective sequence similarities of 97.9 and 97.8 %. Predominant cellular fatty acids (>10 %) were summed feature 3 (C16 : 1ω7c/C16 : 1ω6c; 37.7 %), C16 : 1ω5c (13.0 %) and iso-C15 : 0 (11.1 %). The major respiratory quinone was MK-7. The polar lipid profile contained phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several uncharacterized polar lipids. The DNA G+C content was 39.9 mol%. On the basis of the phylogenetic and phenotypic data, strain TNR-18T should be classified as representing a novel species, for which the name Arcicella aurantiaca sp. nov. is proposed. The type strain is TNR-18T (=LMG 25207T =BCRC 17969T).

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain TNR-18T is FJ593908.

    • Results of 2D TLC of polar lipids of strain TNR-18T are available as supplementary material with the online version of this paper.

    The genus Arcicella, first proposed by Nikitin et al. (2004), belongs to the family Cytophagaceae of the order Cytophagales, class ‘Cytophagia’, phylum Bacteroidetes (Ludwig et al., 2008). At the time of writing, the genus contains only two species with validly published names, the type species Arcicella aquatica, isolated from a neuston film on a freshwater lake (Nikitin et al., 2004), and Arcicella rosea, from tap water (Kämpfer et al., 2009). The present study was carried out to clarify the taxonomic position of the Arcicella-like bacterial strain TNR-18T by a polyphasic taxonomic approach.

    Stream water (approx. 200 ml) was collected in a sterile bag from Maolin township (GPS location 2 ° 54′ 12.5″ N 12 ° 40′ 59.9″ E) in Kaohsiung Prefecture, southern Taiwan, stored at 4 °C and transported to the laboratory within 2 h. The water sample was spread-plated on R2A agar (BD Difco) following the standard dilution plating method. After incubation of the plates at 25 °C for 5 days, strain TNR-18T was purified as a single colony. The strain was preserved at −80 °C as a 20 % (v/v) glycerol suspension in R2A broth or by lyophilization with 20 % (w/v) skimmed milk. A. aquatica LMG 21963T and A. rosea CCUG 55942T were obtained from the Belgian Coordinated Collections of Microorganisms and the Culture Collection of the University of Göteborg, respectively, for detailed comparisons.

    Bacterial cells were observed by phase-contrast microscopy (DM 2000; Leica) in the lag, exponential and stationary phases of growth to ascertain their morphology. Flagellar and gliding motility were tested using the hanging drop method. Spot Test flagella stain (BD Difco) was used to stain any flagella that might be present. The Gram stain set (BD Difco) kit and the Ryu non-staining KOH method (Powers, 1995) were used to test Gram-staining behaviour. Colony morphology was examined on R2A agar using a stereoscopic microscope (SMZ 800; Nikon). The presence of flexirubin-type pigments and carotenoids was investigated as described by Reichenbach (1992), Bernardet et al. (2002) and Schmidt et al. (1994).

    The pH range for growth was determined by measuring the OD600 of R2A broth cultures (BD Difco). The medium was adjusted to pH 4.0–10.0 (at intervals of 0.5 pH units) using appropriate biological buffers (Chung et al., 1995). To investigate the tolerance of NaCl, R2A broth was prepared according to the formula of the BD Difco medium with the NaCl concentration adjusted to 0–1.0 % (w/v) (at intervals of 0.1 %). The temperature range for growth was determined in R2A broth at 4 and 10–45 °C (at intervals of 5 °C). Growth under anaerobic conditions was determined after incubating strain TNR-18T in the Oxoid AnaeroGen system.

    Strain TNR-18T was examined for a broad range of phenotypic properties. Activities of catalase, oxidase and lipase (corn oil) and hydrolysis of starch, casein and Tweens 20, 40, 60 and 80 were determined according to standard methods (Gerhardt et al., 1994). Additional biochemical tests were performed using the API ZYM and API 20NE kits (bioMérieux) and carbon source utilization was evaluated using the Biolog GN2 microplate. Phenotypic tests using commercial kits were performed according to the manufacturers' recommendations.

    Sensitivity of strain TNR-18T to antibiotics was tested by the disc diffusion method after spreading cell suspensions (0.5 McFarland) on R2A agar (BD Difco) plates. The discs (Oxoid) contained the following antibiotics: ampicillin (10 μg), chloramphenicol (30 μg), gentamicin (10 μg), kanamycin (30 μg), nalidixic acid (30 μg), novobiocin (30 μg), penicillin G (10 μg), rifampicin (5 μg), streptomycin (10 μg), sulfamethoxazole plus trimethoprim (23.75/1.25 μg) and tetracycline (30 μg). The effect of antibiotics on cell growth was assessed after 3 days of incubation at 25 °C. The strain was considered susceptible when the diameter of the inhibition zone was >13 mm, intermediate at 10–12 mm and resistant at <10 mm. Detailed results from the phenotypic and biochemical analyses of strain TNR-18T are provided in Table 1⇓ and the species description. Phenotypic characteristics that serve to differentiate strain TNR-18T from the type strains of the genus Arcicella are presented in Table 1⇓.

    Table 1.

    Differential characteristics of type strains of Arcicella species

    Strains: 1, A. aurantiaca sp. nov. TNR-18T; 2, A. aquatica LMG 21963T; 3, A. rosea CCUG 55942T. Data were acquired in this study. +, Positive; −, negative; w, weakly positive. All strains stain Gram-negative and are positive for C8 esterase lipase, leucine arylamidase, trypsin, naphthol-AS-BI-phosphohydrolase, α-glucosidase and N-acetyl-β-glucosaminidase activities and negative for indole production, glucose fermentation, arginine dihydrolase, urease, C14 lipase, β-glucuronidase, α-mannosidase and lipase (corn oil) activities and hydrolysis of Tweens 20, 40, 60 and 80.

    The 16S rRNA gene sequence was analysed as described previously by Chen et al. (2001). Analysis of the sequence was performed by using the software BioEdit (Hall, 1999) and mega version 3.1 (Kumar et al., 2004) after multiple alignment of the data by clustal_x (Thompson et al., 1997). Distances (corrected according to Kimura's two-parameter model; Kimura, 1983) were calculated and clustering was performed with the neighbour-joining method (Saitou & Nei, 1987). Maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Kluge & Farris, 1969) trees were generated by using the treeing algorithms contained in the phylip software package (Felsenstein, 1993). In each case, bootstrap values were calculated based on 1000 replications. An almost-complete 16S rRNA gene sequence (1411 bp) of strain TNR-18T was compared against 16S rRNA gene sequences available from the EzTaxon server (Chun et al., 2007), the Ribosomal Database Project (Maidak et al., 2001) and the GenBank database (). 16S rRNA gene sequence analysis indicated that strain TNR-18T belonged to the family Cytophagaceae (order Cytophagales, class ‘Cytophagia’, phylum Bacteroidetes). Strain TNR-18T formed a distinct subline within the genus Arcicella in the neighbour-joining tree (Fig. 1⇓). The overall topologies of the phylogenetic trees retrieved by the maximum-likelihood and maximum-parsimony methods were similar. Sequence similarity calculations (over 1400 bp) indicated that strain TNR-18T was closely related to A. rosea TW5T (97.9 % 16S rRNA gene sequence similarity) and A. aquatica NO-502T (97.8 %). Lower sequence similarities (<93.1 %) were found with representative members of all other genera listed in Fig. 1⇓.

    Figure image not available in archive
    Fig. 1.

    Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the position of Arcicella aurantiaca sp. nov. TNR-18T and related taxa in the class ‘Cytophagia’. Numbers at nodes are bootstrap percentages (>70 %) based on the neighbour-joining (above nodes) and maximum-parsimony (below nodes) tree-making algorithms. Filled circles indicate branches of the tree that were also recovered using the maximum-likelihood and maximum-parsimony algorithms. Flexibacter litoralis ATCC 23117T was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.

    Whole genome DNA–DNA hybridization experiments were carried out with photobiotin-labelled probes as described by Ezaki et al. (1989). The degree of hybridization was calculated as the mean of triplicate experiments. Separate species status of strain TNR-18T was confirmed by the hybridization values obtained with A. aquatica LMG 21963T and A. rosea CCUG 55942T (59.5±3.1 and 23.1±2.4 %, respectively). The DNA–DNA relatedness between strain TNR-18T and its closest phylogenetic neighbours was well below the 70 % cut-off point recommended for the assignment of strains to the same genomic species (Wayne et al., 1987). Based on the above data, strain TNR-18T warrants separate species status in the genus Arcicella.

    Fatty acid methyl esters were prepared, separated and identified according to the instructions of the Microbial Identification System (Microbial ID; Sasser, 1990). The fatty acid profiles of strain TNR-18T, A. aquatica LMG 21963T and A. rosea CCUG 55942T were determined using cells grown on R2A agar at 25 °C for 3 days. The fatty acid profile of strain TNR-18T was similar to those of the other two Arcicella type strains, although there were differences in the proportions of some components (Table 2⇓). Cellular fatty acids present in strain TNR-18T at ≥1 % included summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 1ω5c, iso-C15 : 0, iso-C15 : 0 3-OH, C16 : 0, C16 : 0 3-OH, iso-C17 : 0 3-OH, C14 : 0, anteiso-C15 : 0, C15 : 1ω6c, iso-C17 : 1ω9c and C18 : 0. Isoprenoid quinones were extracted and purified according to the method of Collins (1985) and analysed by HPLC; the major respiratory quinone of strain TNR-18T was MK-7. The DNA G+C content of strain TNR-18T, determined by HPLC according to Mesbah et al. (1989), was 39.9±1.0 mol%.

    Table 2.

    Cellular fatty acid compositions of the type strains of Arcicella species

    Strains: 1, A. aurantiaca sp. nov. TNR-18T; 2, A. aquatica LMG 21963T; 3, A. rosea CCUG 55942T. Data were acquired in this study. Values are percentages of total fatty acids. Fatty acids that made up <1 % of the total in all strains are not shown; −, <1 %. For unsaturated fatty acids, the position of the double bond is located by counting from the methyl (ω) end of the carbon chain; cis isomers are indicated by the suffix c.

    Polar lipids were extracted and analysed by two-dimensional TLC according to Lechevalier et al. (1977). Strain TNR-18T exhibited a complex polar lipid profile consisting of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several uncharacterized polar lipids (see Supplementary Fig. S1, available in IJSEM Online). Strain TNR-18T exhibited a polar lipid profile very similar to that of its closest relative, A. rosea TW5T (Kämpfer et al., 2009), in which the predominant compounds were also phosphatidylethanolamine and an unknown aminophospholipid.

    On the basis of 16S rRNA gene sequence similarity, strain TNR-18T occupies a distinct position within the genus Arcicella. This result was supported by the unique combination of chemotaxonomic characteristics and biochemical traits of the strain (Table 1⇑). Based on 16S rRNA gene sequence similarity, DNA–DNA relatedness, fatty acid profile, major respiratory quinone, DNA G+C content and phenotypic properties, it is evident that strain TNR-18T should be classified as the type strain of a novel species in the genus Arcicella, for which the name Arcicella aurantiaca sp. nov. is proposed.

    Description of Arcicella aurantiaca sp. nov.

    Arcicella aurantiaca (au.ran.ti.a′ca. N.L. fem. adj. aurantiaca orange-coloured).

    Cells are Gram-stain-negative, strictly aerobic, vibrioid and non-motile. After 72 h of incubation on R2A agar at 25 °C, mean cell dimensions are 0.5–0.6 μm diameter and 1.0–3.0 μm length. Colonies on R2A agar are orange, non-mucous, circular, smooth and convex with entire edges. Colonies are approximately 1.0–1.5 mm in diameter on R2A agar after 72 h of incubation at 25 °C. Growth occurs at 10–30 °C, with 0–0.2 % NaCl and at pH 6.0–10.0. Optimum growth occurs at 25 °C, in the absence of NaCl and at pH 7.0. Carotenoid/flexirubin pigments are absent. Positive for catalase and oxidase activities and hydrolysis of casein and starch. Negative for lipase activity and hydrolysis of Tweens 20, 40, 60 and 80. In API 20NE tests, positive for nitrate reduction, aesculin hydrolysis, gelatin hydrolysis and β-galactosidase activity and negative for indole production, d-glucose fermentation and arginine dihydrolase and urease activities. In API ZYM tests, alkaline phosphatase, C8 esterase lipase, leucine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-galactosidase, α-glucosidase and N-acetyl-β-glucosaminidase activities are present and C4 esterase, C14 lipase, valine arylamidase, cystine arylamidase, α-chymotrypsin, α-galactosidase, β-glucuronidase, β-glucosidase, α-mannosidase and α-fucosidase activities are absent. The following compounds in the Biolog GN2 microplate are utilized as sole carbon sources: α-cyclodextrin, dextrin, glycogen, N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, cellobiose, d-fructose, l-fucose, d-galactose, gentiobiose, α-d-glucose, lactose, lactulose, maltose, d-mannose, melibiose, methyl β-d-glucoside, d-psicose, raffinose, l-rhamnose, d-sorbitol, sucrose, trehalose, turanose, pyruvic acid methyl ester, succinic acid monomethyl ester, acetic acid, d-galactonic acid lactone, d-galacturonic acid, d-gluconic acid, d-glucosaminic acid, d-glucuronic acid, α-ketobutyric acid, α-ketoglutaric acid, α-ketovaleric acid, dl-lactic acid, propionic acid, quinic acid, succinic acid, l-alaninamide, l-alanine, l-alanyl glycine, l-aspartic acid, l-glutamic acid, glycyl l-aspartic acid, glycyl l-glutamic acid, hydroxy-l-proline, l-serine, inosine, uridine, 2,3-butanediol, α-d-glucose 1-phosphate and d-glucose 6-phosphate. Other substrates in the GN2 microplate are not utilized. Sensitive to tetracycline, sulfamethoxazole, rifampicin, novobiocin, streptomycin, chloramphenicol, nalidixic acid, penicillin G and ampicillin and resistant to gentamicin and kanamycin. The predominant cellular fatty acids (>10 %) are summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), C16 : 1ω5c and iso-C15 : 0. The major respiratory quinone is MK-7. The polar lipid profile consists of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several uncharacterized polar lipids; the predominant polar lipids are phosphatidylethanolamine and an unknown aminophospholipid. The DNA G+C content of the type strain is 39.9 mol%.

    The type strain is TNR-18T (=LMG 25207T =BCRC 17969T), isolated from stream water in Kaohsiung Prefecture, southern Taiwan.

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