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1 Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung Marine University, No. 142 Hai-Chuan Road, Nan-Tzu, Kaohsiung City 811, Taiwan, ROC
2 Department of Soil and Environmental Sciences, College of Agriculture and Natural Resources, National Chung Hsing University, Taichung, Taiwan, ROC
3 Graduate School of Biotechnology and Bioinformatics, Yuan-Ze University, Chung-Li, Taoyuan 320, Taiwan, ROC
Correspondence
Wen-Ming Chen
p62365{at}ms28.hinet.net
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ABSTRACT |
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, containing lysine, glutamic acid and alanine. The isolate contained MK-7 as the major component of the quinone system. The predominant polar lipid consisted of phosphatidylglycerol and diphosphatidylglycerol, with some unknown phospho- and glycolipids as minor components. The major fatty acids were anteiso-15 : 0 (57.3 %), anteiso-17 : 0 (17.0 %) and 16 : 0 (9.3 %). The G+C content of the genomic DNA was 56.1 mol%. Hence, genotypic, chemotaxonomic and phenotypic data demonstrate that strain L-143T should be classified as a novel species in the genus Rothia, for which the name Rothia terrae sp. nov. is proposed. The type strain is L-143T (=BCRC 17588T=LMG 23708T).
A supplementary table of fatty acid composition data and a supplementary figure showing the polar lipid profile of strain L-143T are available with the online version of this paper.
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. Historically, this taxon was classified in the family Actinomycetaceae because of similarities in morphological characteristics (Schaal, 1992). Stackebrandt et al. (1997) further amended and transferred the genus to the Micrococcaceae based on phylogenetic and other analyses. Currently, Rothia encompasses five species with validly published names: Rothia aeria, Rothia amarae, Rothia dentocariosa, Rothia mucilaginosa and Rothia nasimurium. All five reported Rothia species are Gram-positive and form non-motile coccoid cells. R. dentocariosa and R. mucilaginosa were isolated from the human oral cavity and pharynx (Schaal, 1992; Collins et al., 2000), respectively. R. dentocariosa has been identified to be an opportunistic pathogen causing septicaemia, endocarditis and other serious infections (Schafer et al., 1979; Pape et al., 1979; Minato & Abiko, 1984; Pers et al., 1987; Schiff & Kaplan, 1987). R. nasimurium was isolated from the nose of a healthy mouse (Collins et al., 2000) while R. amarae was isolated from sludge of a foul water sewer (Fan et al., 2002). R. aeria was isolated from air samples collected from the Russian space laboratory Mir (Li et al., 2004).
In the course of a study aiming to isolate actinomycetes from subtropical fields, strain L-143T was isolated from a soil sample collected from Tainan County, Taiwan. This organism was isolated on an HV agar plate (Hayakawa & Nonomura, 1987) that had been incubated at 30 °C for 3 days following inoculation with a suspension of a soil sample. Strain L-143T was further maintained on nutrient agar (BD Difco). The strain was preserved at –80 °C in nutrient broth (BD Difco) with 20 % (v/v) glycerol or by lyophilization. The organism was the subject of a polyphasic taxonomic study which showed that it belonged to a new species of Rothia. The name proposed for this taxon is Rothia terrae sp. nov.
The morphology of the bacterial cells was observed during the lag, exponential and stationary phases of growth under a phase-contrast microscope. The motility of the cells was tested by a hanging drop method. Flagellum staining was performed by using the Spot Test Flagella Stain (BD Difco). The Gram reaction was performed using the Gram Stain Set (BD Difco) and the Ryu non-staining KOH method (Powers, 1995). Accumulation of poly-β-hydroxybutyrate granules was observed under a light microscope after staining cells with Sudan black. The optimum pH range for growth was examined in nutrient broth using appropriate biological buffers (pH 4–10 at intervals of 1.0 pH unit) (Chung et al., 1995). pH was adjusted prior to sterilization; post-sterilization controls revealed that only minor changes in pH values had occurred. The requirement for NaCl was determined using nutrient broth containing 0, 0.5 and 1.0–10.0 % (w/v) NaCl (at 1.0 % intervals). The temperature range for growth (4, 10, 15, 20, 25, 30, 35, 40 and 45 °C) was examined in nutrient broth adjusted to pH 7. Growth was examined by measuring the turbidity (OD600) of cultures grown at various pH values, NaCl concentrations and temperatures. Anaerobic cultivation was performed on nutrient agar using the Oxoid AnaeroGen system.
Strain L-143T grew well aerobically in complex media, such as trypticase soy, nutrient and Luria–Bertani media. Strain L-143T could grow under anaerobic conditions, but the growth was poor.
Extraction of genomic DNA, PCR amplification and sequencing of the 16S rRNA gene were carried out as described by Chen et al. (2001). Sequence reaction fragments were separated using a DNA sequencer (ABI PRISM 310; Applied Biosystems) and sequences were assembled by using the Fragment Assembly System program from the Wisconsin Package 9.1 (Genetics Computer Group, Madison, WI, USA). The resultant sequence was compared with available 16S rRNA gene sequences from the Ribosomal Database Project and GenBank databases. The multiple-sequence alignment including strain L-143T and its closest relatives was performed using BioEdit software (Hall, 1999) and MEGA version 3.1 (Kumar et al., 2004). Phylogenetic trees were inferred using the maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Kluge & Farris, 1969) and neighbour-joining (Saitou & Nei, 1987) tree-making algorithms. An evolutionary distance matrix was generated for the neighbour-joining algorithm using the Jukes & Cantor (1969) distance model and bootstrap analysis (1000 resamplings).
The nearly complete 16S rRNA gene sequence (1436 nt) of strain L-143T was obtained. A comparison of the sequence with those of representatives of the genera classified in the family Micrococcaceae of the Actinobacteria showed that the organism fell within the evolutionary radiation occupied by the genus Rothia (Fig. 1). In the tree based on the neighbour-joining algorithm, strain L-143T formed a coherent cluster with R. nasimurium CCUG 35957T and R. amarae JCM 11375T; the branching order was supported further by higher bootstrap values. A similar tree topology was also obtained with the phylogenetic trees generated using maximum-parsimony and maximum-likelihood algorithms (data not shown). Sequence similarity calculations based on pairwise alignment obtained using the EzTaxon database (Chun et al., 2007) showed the highest degree of similarity to R. nasimurium CCUG 35957T and R. amarae JCM 11375T, sharing a 16S rRNA gene sequence similarity of 98.3 and 97.6 %, respectively. Strain L-143T shared much lower 16S rRNA gene sequence similarity with other type strains: R. dentocariosa (96.0 %), R. aeria (96.0 %) and R. mucilaginosa (95.8 %).
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. Additionally, reciprocal analysis was carried out to avoid any inconsistency during DNA binding. DNA–DNA hybridization results indicated low levels of relatedness between strain L-143T and its closest phylogenetic neighbours R. nasimurium CCUG 35957T [21.0±2.8 and 12.7±1.1 % (reciprocal)] and R. amarae JCM 11375T [15.3±5.0 and 10.7±2.1 % (reciprocal)]. These values are clearly below the 70 % cut-off point recommended for the assignment of strains to the same genomic species (Wayne et al., 1987), indicating that strain L-143T warrants novel species status in the genus Rothia.
The DNA G+C content of strain L-143T was determined in triplicate as described by Mesbah et al. (1989) and was found to be 56.1±0.5 mol% (Table 1).
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). Polar lipids were extracted and analysed by two-dimensional TLC according to Embley & Wait (1994). Purified cell-wall preparations were obtained by the method of Schleifer & Kandler (1972). The amino acids in peptidoglycan hydrolysates were analysed by TLC as outlined in Schleifer (1985) and the composition of amino acids was determined by HPLC using the Pico Tag system (Waters Associates). Menaquinones were extracted and analysed by HPLC as described by Collins (1994). Strain L-143T shared chemotaxonomic characteristics consistent with the genus Rothia. The major cellular fatty acid composition of strain L-143T showed the presence of anteiso-15 : 0 (57.3 %), anteiso-17 : 0 (17.0 %) and 16 : 0 (9.3 %) fatty acids. Details of the cellular fatty acid composition with other closely related Rothia species are presented in Table S1 (available with the online version of the paper). In general, all these organisms had similar whole-cell fatty acid profiles (Collins et al., 2000; Fan et al., 2002; Li et al., 2004). The predominant polar lipids of strain L-143T were phosphatidylglycerol and diphosphatidylglycerol, with some unknown phospho- and glycolipids as minor components (Fig. S1, available with the online version of the paper). The TLC analysis indicated that strain L-143T lacks diaminopimelic acid in the cell wall. Further HPLC analysis of the peptidoglycan showed the presence of amino acids glutamic acid, alanine and lysine in a molar ratio of 1 : 3 : 1. These results confirmed that the peptidoglycan type of strain L-143T was A3 (Schleifer & Kandler, 1972), which is also consistent with R. aeria (Li et al., 2004) and R. amarae (Fan et al., 2002). The predominant menaquinone of strain L-143T was MK-7 (Table 1
).
Strain L-143T was examined for a broad range of phenotypic properties. Catalase, oxidase, acid production from carbohydrate, triple-sugar iron agar test, Voges–Proskauer test, motility test and hydrolysis of starch, casein, Tween 20, Tween 40, Tween 60 and Tween 80 were determined using standard methods (Gerhardt et al., 1994; Lanyi, 1987; MacFaddin, 2000). Additional biochemical tests were performed by using commercially available API Coryne and API ZYM (bioMérieux), and Biolog GP2 microtest systems according to the methods outlined by the manufacturers. Sensitivity of strain L-143T to different antibiotics was analysed by the disc diffusion method on nutrient agar plates. The following antibiotic discs were used: ampicillin (10 µg), chloramphenicol (30 µg), erythromycin (15 µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid (30 µg), novobiocin (30 µg), rifampicin (5 µg), penicillin G (10 U), streptomycin (10 µg) and tetracycline (30 µg). The effect of antibiotics on cell growth was assessed after 3 days incubation at 30 °C.
Detailed results of the phenotypic study are provided in Table 1 and in the species description below. The phenotypic properties of strain L-143T were similar to those reported for other species in the genus Rothia, such as positive results for nitrate reduction, aesculin hydrolysis and acid production from D-glucose, sucrose and maltose, but negative results for urease, and acid production from xylose and D-mannitol. These characteristics strongly support the inclusion of strain L-143T in the genus Rothia. However, a few characteristics that are unique to strain L-143T differentiate it from other type strains of Rothia species (Table 1).
Phenotypic, chemotaxonomic and phylogenetic data support the description of strain L-143T as a novel species in the genus Rothia. The name Rothia terrae sp. nov. is proposed for this taxon.
Description of Rothia terrae sp. nov.
Rothia terrae (ter'rae. L. gen. n. terrae, of the earth, referring to the organism being isolated from soil).
Strain L-143T forms visible cream-white colonies, circular and convex in shape with entire edges. Cells are Gram-positive, non-spore-forming, non-motile, ovoid to spherical, 0.5–1.0 µm in width and 0.5–1.5 µm in length. Cells occur either singly or in pairs or tetrads. Non-pigmented. Facultatively anaerobic and catalase-positive. Grows at 15–40 °C, in the presence of 0–7.0 % NaCl and at pH 5–10. Optimum growth occurs at 35 °C, with 0–0.5 % NaCl and at pH 7.0–8.0. No accumulation of poly-β-hydroxybutyrate granules. Acid is produced from fructose, mannose, glycerol and trehalose. Acid is not produced from adonitol or salicin. Voges–Proskauer-positive. Starch, casein, Tween 20, Tween 40, Tween 60 and Tween 80 are not hydrolysed. Hydrogen sulfide production is not detected on triple-sugar iron agar. Positive (API Coryne) for nitrate reduction, pyrazinamidase, alkaline phosphatase, β-galactosidase, -glucosidase, aesculin hydrolysis, gelatin hydrolysis and acid production from D-glucose, D-maltose, D-lactose and D-sucrose. Negative (API Coryne) for pyrrolidonyl arylamidase, β-glucuronidase, N-acetyl-β-glucosaminidase, urease and acid production from D-ribose, D-xylose, D-mannitol and glycogen. Positive (API ZYM) for alkaline phosphatase, C4 esterase, C8 lipase, leucine arylamidase, naphthol-AS-BI-phosphohydrolase, β-galactosidase, -glucosidase and β-glucosidase. Negative (API ZYM) for C14 lipase, valine arylamidase, cysteine arylamidase, trypsin, -chymotrypsin, acid phosphatase, -galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, -mannosidase and -fucosidase. The following carbon sources are utilized (Biolog GP2 system): dextrin, glycogen, D-fructose, -D-glucose, maltose, D-mannose, 3-methyl-D-glucose, D-psicose, sucrose, D-trehalose, turanose, D-lactic acid methyl ester, L-lactic acid, pyruvic acid methyl ester, L-serine and glycerol. The following carbon sources are not utilized (Biolog GP2 system): -cyclodextrin, β-cyclodextrin, inulin, mannan, Tween 40, Tween 80, N-acetyl-D-glucosamine, N-acetyl-β-D-mannosamine, amygdalin, L-arabinose, D-arabitol, arbutin, D-cellobiose, L-fucose, D-galactose, D-galacturonic acid, gentiobiose, m-inositol, -D-lactose, lactulose, maltotriose, D-mannitol, D-melezitose, D-melibiose, -methyl-D-galactoside, β-methyl-D-galactoside, -methyl-D-glucoside, β-methyl-D-glucoside, -methyl-D-mannoside, palatinose, D-raffinose, L-rhamnose, D-ribose, salicin, sedoheptulosan, D-sorbitol, stachyose, D-tagatose, xylitol, D-xylose, acetic acid, -hydroxybutyric acid, β-hydroxybutyric acid, 
-hydroxybutyric acid, p-hydroxyphenylacetic acid, -ketoglutaric acid, -ketovaleric acid, lactamide, D-malic acid, L-malic acid, succinic acid monomethyl ester, propionic acid, pyruvic acid, succinamic acid, succinic acid, N-acetyl-L-glutamic acid, L-alaninamide, D-alanine, L-alanine, L-alanylglycine, L-asparagine, L-glutamic acid, glycyl-L-glutamic acid, L-pyroglutamic acid, putrescine, 2,3-butanediol, adenosine, 2'-deoxyadenosine, inosine, thymidine, uridine, adenosine-5'-monophosphate, thymidine-5'-monophosphate, uridine-5'-monophosphate, D-fructose 6-phosphate, -D-glucose 1-phosphate, -D-glucose 6-phosphate and DL--glycerol phosphate. Resistant to gentamicin, kanamycin and nalidixic acid; sensitive to ampicillin, chloramphenicol, erythromycin, penicillin G, rifampicin, novobiocin, streptomycin and tetracycline. The peptidoglycan type is A3 containing lysine, glutamic acid and alanine. The predominant polar lipids are phosphatidylglycerol and diphosphatidylglycerol, with some unknown phospho- and glycolipids as minor components. Predominant fatty acids are anteiso-15 : 0, anteiso-17 : 0 and 16 : 0. The major menaquinone is MK-7. DNA G+C content is 56.1 mol%.
The type strain, L-143T (=BCRC 17588T=LMG 23708T), was isolated from wasteland soil in Tainan County, Taiwan.
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ACKNOWLEDGEMENTS |
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