Abstract
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains YM21-132T and YM27-005T are AB297805 and AB297806, respectively.
At present, the phylum Verrucomicrobia (Hedlund et al., 1997; Hugenholtz et al., 1998) is classified informally into six subdivisions (Hugenholtz et al., 1998; Vandekerckhove et al., 2000). Currently, subdivision 1 contains species with validly published names from either freshwater or human faeces, including Verrucomicrobium spinosum (Schlesner, 1987), Prosthecobacter spp. (Staley et al., 1976; Hedlund et al., 1996, 1997) and Akkermansia muciniphila (Derrien et al., 2004). More recently, Rubritalea marina Pol012T (Scheuermayer et al., 2006) and Rubritalea squalenifaciens HOact23T (Kasai et al., 2007), isolated from marine animals (sponges), have been reported as novel representatives of the family Verrucomicrobiaceae within the phylum Verrucomicrobia. The latter organism has also been described as a squalene-producing marine bacterium. However, in spite of the abundance of members of the phylum Verrucomicrobia in nature (Joseph et al., 2003; Rappé & Giovannoni, 2003; Kanokratana et al., 2004; Haukka et al., 2005, 2006; Dedysh et al., 2006), to date few representatives have been cultivated and most of them have yet to be cultured and described.
Strain YM21-132T was isolated from an unidentified marine sponge sample collected near the shore of Ohshima, Natsudomari Peninsula, Aomori, Japan (depth 0.5 m; GPS location: 4 ° 00' 14.5'' N 14 ° 52' 50.5'' E) in September 2005. Strain YM27-005T was isolated from the visceral specimen of an unidentified sea hare collected from Himetsu, Sado, Niigata, Japan (depth 0.5 m; GPS location: 3 ° 04' 42.6'' N 13 ° 14' 36.3'' E) in October 2006. The samples (0.5–1 cm3) were homogenized with a glass rod in 5 ml sterile seawater. A 50 µl sample of the homogenate was applied to the surface of an agar isolation medium (medium P; Yoon et al., 2007). Strains YM21-132T and YM27-005T appeared after incubation for 30 days at 25 °C. The bacteria were purified on marine broth 2216 (Difco) containing 1.5 % agar by cultivation for 7–10 days.
In the present study, we attempted to elucidate the phylogenetic positions of strains YM21-132T and YM27-005T by using a polyphasic taxonomic approach, including 16S rRNA gene sequence analysis. In parallel, we performed physiological, biochemical and chemotaxonomic analyses to characterize the two novel isolates. Based on these data, it is proposed that the isolates represent two novel species of the genus Rubritalea within the phylum Verrucomicrobia.
The temperature range and pH range for growth were determined by incubating the isolates on 1/2 strength R2A agar (Difco) with 75 % artificial seawater (Lyman & Fleming, 1940). The NaCl concentration for growth was determined on 1/2 R2A agar containing 0–10 % (w/v) NaCl. Gram-staining was performed as described by Murray et al. (1994). Cell morphology was observed using light microscopy (BX60; Olympus). Cells of strain YM21-132T on 1/2 strength R2A agar with 75 % artificial seawater were coccoid (0.6–1.0 µm in diameter) or rod-shaped (0.5–1.0 µm wide and 0.8–1.2 µm long). Cells of strain YM27-005T on the same medium as used for strain YM21-132T were coccoid (0.5–0.8 µm in diameter) or rod-shaped (0.5–0.8 µm wide and 1.0–1.5 µm long). No motility by flagella or gliding movement was observed for either strain. Cell division by binary fission was observed for both strains. Growth under anaerobic conditions was determined after 2 weeks incubation in an AnaeroPack (Mitsubishi Gas Chemical Co., Inc.) on 1/2 strength R2A agar with 75 % artificial seawater. Catalase activity was determined by the observation of bubble formation in a 3 % H2O2 solution. Oxidase activity was determined using cytochrome oxidase paper (Nissui Pharmaceutical Co., Ltd). API 20NE and API ZYM strips (bioMérieux) were used to determine physiological and biochemical characteristics. The API 20NE and API ZYM strips were read after 72 h incubation at 30 °C and 4 h incubation at 37 °C, respectively. The nutritional features of strains YM21-132T and YM27-005T were determined using Biolog MicroPlates. The strains were grown on 1/2 strength R2A agar with 75 % artificial seawater at 30 °C for 72 h and suspended in sterile saline medium (0.85 % NaCl, w/v) within the density range specified by the manufacturer with a Biolog photometer (model 21907). Immediately after the cells had been suspended in saline solution, the suspensions were transferred to sterile multichannel pipetter reservoirs (Biolog) and the Biolog GN2 MicroPlates were inoculated with 150 µl cell suspension per well by means of an eight-channel repeating pipetter (Biolog). The inoculated plates were incubated at 30 °C for 1 week, and the results were read with a MicroPlate Reader using Microlog 3.59 software. Determination of the respiratory quinone system and cellular fatty acid composition was carried out as described previously (Katsuta et al., 2005). DNA was prepared according to the method of Marmur (1961) from cells grown on 1/2 strength R2A agar with 75 % artificial seawater and the DNA base composition was determined by using the HPLC method of Mesbah et al. (1989). DNA–DNA hybridizations were carried out with photobiotin-labelled probes in microplate wells as described by Ezaki et al. (1989). The hybridization temperature was set at 47 °C. Hybridization was performed using five replications for each. The highest and lowest values for each sample were excluded and the means of the remaining three values are quoted as DNA–DNA relatedness values. Cell walls were prepared using the methods described by Schleifer & Kandler (1972), and the amino acids in an acid hydrolysate of the cell walls were identified by TLC (Harper & Davis, 1979) and by HPLC, as their phenylthiocarbamoyl derivatives, with a model LC-10AD HPLC apparatus (Shimazu) equipped with a Wakopak WS-PTC column (Wako Pure Chemical Industries) (Yokota et al., 1993). An approximately 1500 bp fragment of the 16S rRNA gene was amplified from the extracted DNA by using bacterial universal primers specific to the 16S rRNA gene: 27F and 1492R (Escherichia coli numbering system; Weisburg et al., 1991). To ascertain the phylogenetic position of the novel isolates, the 16S rRNA gene sequences of strains YM21-132T and YM27-005T were compared with sequences obtained from GenBank (National Center for Biotechnology Information, ). Multiple alignments of the sequences were performed using CLUSTAL_X (version 1.83) (Thompson et al., 1997). Alignment gaps and ambiguous bases were not taken into consideration when the 1224 bases of the 16S rRNA gene nucleotides were compared. Aligned sequences were analysed using MEGA3.1 software (Kumar et al., 2004). Evolutionary distances (distance options according to the Kimura two-parameter model; Kimura, 1983) and clustering with the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) methods were determined by using bootstrap values based on 1000 replications (Felsenstein, 1985). The similarity values were calculated using the same software.
Comparative analysis of the 16S rRNA gene sequences revealed that strains YM21-132T and YM27-005T were phylogenetically affiliated with the genus Rubritalea with bootstrap values of 99 % using the neighbour-joining method (Fig. 1) and 96 % using maximum-parsimony (data not shown). Analysis of the 16S rRNA gene sequences also showed that the sequence of strain YM21-132T had the highest similarity (97.7 %) to that of strain YM27-005T, followed by the marine bacteria R. marina strain Pol012T (96.4 %) and R. squalenifaciens strain HOact23T (93.4 %). Strain YM27-005T showed similarities of 96.9 % to R. marina Pol012T and 93.5 % to R. squalenifaciens HOact23T. R. marina Pol012T also showed a similarity of 94.1 % to R. squalenifaciens HOact23T. All other cultivated species of the phylum Verrucomicrobia with validly published names were more distantly related, possessing 16S rRNA gene sequence similarity levels of 90 % or less.
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DNA–DNA hybridization values between strains YM21-132T and YM27-005T were on average 5.1 %. These results strongly suggest that strains YM21-132T and YM27-005T should be classified as representing two separate species (Wayne et al., 1987).
Analysis of the red pigments produced by strains YM21-132T and YM27-005T was performed using HPLC/PAD (photodiode array detection)/APCI (atmospheric pressure chemical ionization)–MS (mass spectrometry) (TermoFinnigan) of crude acetone extracts of frozen cells. The profiles of the carotenoid in the extracts of both strains were identical to that of the carotenoid produced by R. squalenifaciens HOact23T (Shindo et al., 2007), based on the UV-VIS absorption spectra, an MS spectrum showing (M–H)– mass at m/z 801 and the HPLC retention time. Squalene was also detected in the two strains using the HPLC/PAD/APCI system.
As shown in Table 1, the predominant cellular fatty acids of the two novel strains were iso-C14 : 0 (35.5–40.6 %), iso-C16 : 0 (12.2–16.1 %) and C16 : 1ω7c (11.8–12.7 %), similar to other members of the genus Rubritalea. On the other hand, strains YM21-132T and YM27-005T could be distinguished from other species of the genus Rubritalea by the different amounts of C16 : 0 and anteiso-C15 : 0. In addition, strains YM21-132T and YM27-005T could be distinguished from other species of the genus Rubritalea by the phenotypic characteristics given in Table 2. The cell walls of the two novel isolates were prepared by disrupting cells, followed by heating with 3 % SDS, washing and centrifugation. Amino acid analysis of the cell wall hydrolysates indicated the presence of muramic acid and meso-diaminopimelic acid in the cell wall peptidoglycan of both isolates.
Table 1. Cellular fatty acid contents (%) of strains YM21-132T (Rubritalea spongiae sp. nov.) and YM27-005T (Rubritalea tangerina sp. nov.) and other species of the genus Rubritalea Strains: 1, strain YM21-132T; 2, strain YM27-005T; 3, R. marina Pol012T (data from Scheuermayer et al., 2006); 4, R. squalenifaciens HOact23T (Kasai et al., 2007). Data are expressed as percentages of total fatty acids. Fatty acids representing less than 1 % are not shown. ND, Not determined; –, not detected; tr, trace.
Table 2. Differential characteristics of strains YM21-132T (Rubritalea spongiae sp. nov.) and YM27-005T (Rubritalea tangerina sp. nov.) and related Rubritalea species Strains: 1, strain YM21-132T; 2, strain YM27-005T; 3, R. marina Pol012T (data from Scheuermayer et al., 2006); 4, R. squalenifaciens HOact23T (Kasai et al., 2007). +, Positive; –, negative. All strains are oxidase positive.
Based on the results of the phylogenetic analysis and the biochemical and physiological properties, the novel strains YM21-132T and YM27-005T should be classified as representing two independent members of the genus Rubritalea. We propose the names Rubritalea spongiae sp. nov. (type strain YM21-132T) and Rubritalea tangerina sp. nov. (type strain YM27-005T).
Description of Rubritalea spongiae sp. nov.
Rubritalea spongiae (spon.gi'ae. L. gen. n. spongiae of a sponge, referring to the isolation source of the micro-organism).
Cells are Gram-negative, non-motile, obligately aerobic and coccoid (0.6–1.0 µm in diameter) or rod-shaped (0.5–1.0x0.8–1.2 µm). Neither cellular gliding movement nor swarming growth is observed. Colonies on 1/2 strength R2A agar with 75 % artificial seawater are circular, convex and reddish pink in colour. The temperature range for growth is 4–37 °C, with optimum growth at 30–37 °C. No growth occurs at 45 °C. The pH range for growth is 6.5–8.0. NaCl is required for growth; tolerates up to 7 % (w/v) NaCl. Catalase- and oxidase-positive. Nitrate is not reduced. Alkaline phosphatase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and α-fucosidase are positive, but esterase (C4), esterase lipase (C8), lipase (C4), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase and α-mannosidase are negative. D-Fructose, L-fucose, D-glucose, D-mannitol, D-melibiose, D-sorbitol, acetic acid, cis-aconitic acid, α-ketobutyric acid, α-ketoglutaric acid, inosine and α-D-glucose 1-phosphate are oxidized, but cyclodextrin, dextrin, glycogen, Tween 40, Tween 80, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, adonitol, L-arabinose, D-arabitol, cellobiose, i-erythritol, D-galactose, gentiobiose, myo-inositol, α-D-lactose, lactulose, maltose, D-mannose, methyl β-D-glucoside, D-psicose, D-raffinose, L-rhamnose, sucrose, trehalose, furanose, xylitol, pyruvic acid methyl ester, succinic acid monomethyl ester, citric acid, formic acid, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, α-hydroxybutyric acid, β-hydroxybutyric acid, γ-hydroxybutyric acid, p-hydroxyphenylacetic acid, itaconic acid, α-ketovaleric acid, DL-lactic acid, malonic acid, propionic acid, quinic acid, D-saccharic acid, sebacic acid, succinic acid, bromosuccinic acid, succinamic acid, glucuronamide, alaninamide, D-alanine, L-alanine, L-alanyl glycine, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-histidine, hydroxy-L-proline, L-leucine, L-ornithine, L-phenylalanine, L-proline, L-pyroglutamic acid, D-serine, L-serine, L-threonine, DL-carnitine, γ-aminobutyric acid, urocanic acid, uridine, thymidine, phenylethylamine, putrescine, 2-aminoethanol, 2,3-butanediol, glycerol, DL-α-glycerol phosphate and D-glucose 6-phosphate are not oxidized. Major respiratory menaquinones are MK-8 and MK-9. The cell wall peptidoglycan contains muramic acid and meso-diaminopimelic acid. Major fatty acid components (>1.0 %) include iso-C14 : 0 (40.6 %), anteiso-C15 : 0 (12.9 %), C15 : 1ω6c (2.0 %), C15 : 0 (2.6 %), iso-C16 : 0 (16.1 %), C16 : 1ω7c (11.8 %), C16 : 0 (7.3 %), anteiso-C17 : 0 (2.0 %) and C17 : 0 (2.8 %). The G+C content of the genomic DNA of the type strain is 48.0 mol%.
The type strain is YM21-132T (=MBIC08281T=KCTC 12906T), which was isolated from an unidentified marine sponge.
Description of Rubritalea tangerina sp. nov.
Rubritalea tangerina (tan'ge.ri.na. N.L. fem. adj. tangerina tangerine, referring to the reddish-orange colour of colonies).
Cells are Gram-negative, non-motile, facultatively anaerobic and coccoid (0.5–0.8 µm in diameter) or rod-shaped (0.5–0.8x1.0–1.5 µm). Neither cellular gliding movement nor swarming growth is observed. Colonies grown on 1/2 strength R2A agar with 75 % artificial seawater are circular, convex and reddish orange in colour. Temperature range for growth is 15–37 °C, with optimum growth at 30–37 °C. No growth occurs at 4 or 45 °C. pH range for growth is 6.5–8.5. NaCl is not required for growth, but can tolerate up to 9 % (w/v) NaCl. Catalase-negative but oxidase-positive. Nitrate is reduced to nitrite. Alkaline phosphatase, leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, N-acetyl-β-glucosaminidase are positive, but α-galactosidase, β-galactosidase, α-glucosidase, valine arylamidase, trypsin, esterase (C4), esterase lipase (C8), lipase (C4), cystine arylamidase, chymotrypsin, β-glucuronidase, β-glucosidase, α-mannosidase and α-fucosidase are negative. L-Fucose, D-mannose, cis-aconitic acid, citric acid, succinic acid, α-D-glucose 1-phosphate and D-glucose 6-phosphate are oxidized, but cyclodextrin, dextrin, glycogen, Tween 40, Tween 80, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, adonitol, L-arabinose, D-arabitol, cellobiose, i-erythritol, D-fructose, D-galactose, gentiobiose, D-glucose, myo-inositol, α-D-lactose, lactulose, maltose, D-mannitol, D-melibiose, methyl β-D-glucoside, D-psicose, D-raffinose, L-rhamnose, D-sorbitol, sucrose, trehalose, furanose, xylitol, pyruvic acid methyl ester, succinic acid monomethyl ester, acetic acid, formic acid, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, α-hydroxybutyric acid, β-hydroxybutyric acid, γ-hydroxybutyric acid, p-hydroxyphenylacetic acid, itaconic acid, α-ketobutyric acid, α-ketoglutaric acid, α-ketovaleric acid, DL-lactic acid, malonic acid, propionic acid, quinic acid, D-saccharic acid, sebacic acid, bromosuccinic acid, succinamic acid, glucuronamide, alaninamide, D-alanine, L-alanine, L-alanyl glycine, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-histidine, hydroxy-L-proline, L-leucine, L-ornithine, L-phenylalanine, L-proline, L-pyroglutamic acid, D-serine, L-serine, L-threonine, DL-carnitine, γ-aminobutyric acid, urocanic acid, inosine, uridine, thymidine, phenylethylamine, putrescine, 2-aminoethanol, 2,3-butanediol, glycerol and DL-α-glycerol phosphate are not oxidized. Major respiratory menaquinones are MK-8 and MK-9. The cell wall peptidoglycan contains muramic acid and meso-diaminopimelic acid. Major fatty acid components (>1.0 %) include iso-C14 : 0 (35.5 %), C14 : 0 (2.9 %), anteiso-C15 : 0 (4.7 %), C15 : 0 (1.4 %), iso-C16 : 0 (12.2 %), C16 : 1ω7c (12.7 %), C16 : 0 (23.1 %), C17 : 0 (1.1 %), C18 : 1ω7c (1.5 %) and C18 : 0 (2.4 %). The G+C content of the genomic DNA of the type strain is 50.3 mol%.
The type strain is YM27-005T (=MBIC08282T=KCTC 12907T), which was isolated from the visceral specimen of an unidentified sea hare.