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1 Department of Soil & Environmental Sciences, College of Agriculture and Natural Resources, National Chung Hsing University, Taichung, Taiwan, ROC
2 Department of Marine Biotechnology, National Kaohsiung Marine University, Kaohsiung, Taiwan, ROC
3 Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung Marine University, No. 142 Hai-Chuan Road, Nan-Tzu, Kaohsiung City 811, Taiwan, ROC
Correspondence
Wen-Ming Chen
p62365{at}ms28.hinet.net
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ABSTRACT |
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7c (13 %) and summed feature 3 (16 : 17c and/or 15 : 0 iso 2-OH; 41 %). The DNA G+C content of the genomic DNA was 62.8 mol%. It is evident from the genotypic and phenotypic data that strain YY287T represents a novel species in the genus Comamonas, for which the name Comamonas composti sp. nov. is proposed. The type strain is YY287T (=BCRC 17659T=LMG 24008T).
A phylogenetic tree based on 16S rRNA gene sequences and an antibiogram of strain YY287T and type strains of other Comamonas species are available with the online version of this paper.
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belongs to the family Comamonadaceae of the class Betaproteobacteria. At the time of writing the genus Comamonas encompasses nine species with validly published names: Comamonas aquatica, C. badia, C. denitrificans, C. kerstersii, C. koreensis, C. nitrativorans, C. odontotermitis, C. terrigena and C. testosteroni. The aim of the present study was to determine the taxonomic position of a Comamonas-like isolate, YY287T, that was isolated from food waste compost.
During the characterization of micro-organisms from food waste compost collected from Kinmen County, Taiwan, strain YY287T was isolated and maintained on nutrient agar (BD Difco) after incubating at 32 °C for 3 days. Subcultivation was performed on nutrient agar at 25 °C. Type strains of C. badia, C. denitrificans, C. koreensis, C. nitrativorans and C. testosteroni were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and the type strains of C. aquatica, C. kerstersii and C. terrigena were obtained from the Laboratorium voor Microbiologie-Bacteriënverzameling (LMG) for comparison. The type strain of C. odontotermitis was from our laboratory (Chou et al., 2007).
Cultural and morphological characteristics were observed on nutrient agar. The morphology of bacterial cells was observed during the lag, exponential and stationary phases of growth under a phase-contrast microscope (Leica DM 2000). Flagellar staining was performed using Spot Test flagella stain (BD Difco). The Gram reaction was performed using a Gram stain set (BD Difco) and the Ryu non-staining KOH method (Powers, 1995). Accumulation of poly-β-hydroxybutyrate granules was observed by light microscopy after staining cells with Sudan black. Colony morphology was examined by using a stereoscopic microscope (Nikon SMZ 800). The optimum growth pH, temperature and tolerance to various NaCl levels were examined on tryptic soy broth (BD Difco) and nutrient broth (BD Difco) (Chung et al., 1995; Chou et al., 2007). Anaerobic cultivation was performed on nutrient agar using the Oxoid AnaeroGen system.
Cells of strain YY287T were Gram-negative, motile, non-spore-forming rods, 0.5 µm in diameter and 1.0–2.0 µm in length. Strain YY287T formed visible semi-transparent and irregularly edged colonies with umbonate elevation. The colony diameter was approximately 3.0 mm on nutrient agar after 48 h incubation at 25 °C. Strain YY287T grew well at temperatures of 15–35 °C and 0–3 % NaCl with pH ranging from 6 to 9. Optimum growth was observed at 25–35 °C, 0–1 % NaCl and pH 6–8. Strain YY287T was able to grow at 25 °C after 24 h incubation under anaerobic conditions.
Extraction of genomic DNA and PCR amplification and sequencing of the 16S rRNA gene were carried out as described by Chen et al. (2001). Sequence reaction fragments were separated by using a DNA sequencer (ABI PRISM 310 instrument; Applied Biosystems). DNA sequences were assembled by using the Fragment Assembly System program from the Wisconsin Package 9.1 (GCG, 1995). The resulting sequence was compared with available 16S rRNA gene sequences from the Ribosomal Database Project II and GenBank databases. Multiple sequence alignments including strain YY287T and its closest relatives were performed using the BioEdit software (Hall, 1999) and MEGA version 3.1 (Kumar et al., 2004). Phylogenetic trees were inferred by using the maximum-parsimony (Kluge & Farris, 1969) and neighbour-joining (Saitou & Nei, 1987) tree-making algorithms. An evolutionary distance matrix was generated for neighbour-joining algorithm with the help of the Kimura two-parameter distance model (Kimura, 1980) and bootstrap analysis (1000 resamplings).
A nearly complete 16S rRNA gene sequence (1472 nt) was obtained for strain YY287T. Comparison of the sequence with the representatives of the genera classified in the family Comamonadaceae of the class Betaproteobacteria showed that the organism fell within the evolutionary radiation occupied by the genus Comamonas (Fig. 1 and Supplementary Fig. S1, available with the online version of this paper). A tree depicting the phylogenetic relationships of this isolate within the genus Comamonas is shown in Fig. 1. Treeing analysis demonstrated that strain YY287T represents a hitherto unknown subline of the genus Comamonas. Although the organism displayed a loose association with C. testosteroni, C. terrigena, C. koreensis and C. odontotermitis, bootstrap resampling showed that it did not possess a statistically significant association with any recognized species. Phylogenetically, the closest relatives of strain YY287T were C. testosteroni DSM 50244T (96.5 %), C. terrigena DSM 7099T (95.4 %), C. odontotermitis Dant 3-8T (95.2 %) and C. koreensis KCTC 12005T (94.6 %). Strain YY287T shared lower 16S rRNA gene sequence similarity with C. nitrativorans 23310T (94.7 %), C. aquatica LMG 2370T (94.7 %), C. denitrificans 123T (94.4 %), C. kerstersii LMG 3475T (94.2 %) and C. badia IAM 14839T (93.8 %). The similarity of strain YY287T with all described bacterial species within the class Betaproteobacteria were less than 97 %. However, sequence divergence values of 
3 % with recognized Comamonas species show unequivocally that isolate YY287T represents a hitherto unknown species (Stackebrandt & Goebel, 1994).
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. The degree of hybridization was calculated by means of triplicate experiments. The separate species status of strain YY287T was demonstrated by the hybridization values obtained when it was hybridized with C. terrigena DSM 7099T, C. koreensis KCTC 12005T, C. testosteroni DSM 50244T and C. odontotermitis Dant 3-8T which showed only 46.0±7.2, 36.0±5.5, 26.2±4.1 and 20.5±5.0 % DNA–DNA relatedness, respectively. The DNA–DNA relatedness of strain YY287T with its closest phylogenetic neighbours was well below the 70 % cut-off point recommended for the assignment of the strains to the same genomic species (Wayne et al., 1987). Based on the above DNA–DNA relatedness data, isolate YY287T warrants separate species status in the genus Comamonas.
For G+C content calculations, the DNA sample was prepared in duplicate and degraded enzymically into nucleosides as described by Mesbah et al. (1989). The obtained nucleoside mixture was then separated with a HPLC system. The G+C content of strain YY287T was 62.8±1.0 mol%, which was within the range of DNA G+C contents previously reported for Comamonas species (60.8–66.3 mol%; Table 2).
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, Tago &Yokota (2004) and Chou et al. (2007). Fatty acid methyl esters were prepared, separated and identified according to the instructions of Microbial Identification System (MIDI; Microbial ID) (Sasser, 1990). Predominant fatty acids of strain YY287T were 16 : 0 (33.3 %), 18 : 17c (12.9 %) and summed feature 3 (16 : 17c and/or 15 : 0 iso 2-OH; 40.8 %). The fatty acid pattern of the strain YY287T is shown in Table 1 in comparison with other representative Comamonas species. The fatty acid profile of strain YY287T was in good agreement with data obtained for other members of the genus Comamonas (Chang et al., 2002; Wauters et al., 2003; Tago &Yokota, 2004; Chou et al., 2007) (Table 1).
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). The antibiotic discs contained ampicillin (10 µg), chloramphenicol (30 µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid (30 µg), novobiocin (30 µg), rifampicin (5 µg), penicillin G (10 U), streptomycin (10 µg), tetracycline (30 µg) and sulfamethoxazole (23.25 µg) plus trimethoprim (1.25 µg). The effect of antibiotics on cell growth was assessed after 3 days of incubation.
Detailed results of biochemical characterization and antibiotic sensitivity are provided in Table 2
, Supplementary Table S1 and in the species description. It is clear from Table 2 that there are several phenotypic characters that readily distinguish strain YY287T from other phylogenetically related species. Based on the 16S rRNA gene sequence data, DNA–DNA hybridization and chemotaxonomic analyses, it is evident that strain YY287T should be classified as the type strain of a novel species in the genus Comamonas, for which the name Comamonas composti sp. nov. is proposed.
Description of Comamonas composti sp. nov.
Comamonas composti (com.pos'ti.N.L. gen. n. composti of compost).
Aerobic, Gram-negative, non-spore-forming, motile and rod-shaped. After 24 h of growth on nutrient agar at 25 °C, the mean cell size is 0.5 µm in width and 1.0–2.0 µm in length. Optimum growth occurs at 25–35 °C, 0–1 % NaCl and pH 6–8. In API 20NE tests, strain YY287T shows positive results for oxidase (weak), catalase, nitrate reduction and assimilation of gluconate, adipate and malate reactions, and negative results for indole production, hydrolysis of aesculin and gelatin, glucose fermentation, arginine dihydrolase, urease, β-galactosidase and assimilation of glucose, arabinose, mannose, maltose, N-acetylglucosamine, caprate, citrate and phenyl acetate. In API ZYM tests, positive results are recorded for alkaline phosphatase, C4 esterase, C8 lipase, C14 lipase, leucine arylamidase, valine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase, while negative results are recorded for cystine arylamidase, trypsin, 
-chymotrypsin, -galactosidase, β-glucuronidase, -glucosidase, β-glucosidase, -mannosidase, N-acetyl-β-glucosaminidase and -fucosidase reactions. The following compounds are oxidized in the Biolog GN2 microtitre test system: i-erythritol, acetic acid, bromosuccinic acid, urocanic acid, cis-aconitic acid, itaconic acid, succinamic acid, hydroxy-L-proline, citric acid, -ketobutyric acid, L-leucine, glycogen, formic acid, -ketoglutaric acid, Tween 40, Tween 80, DL-lactic acid, L-pyroglutamic acid, propionic acid, L-glutamic acid, -hydroxybutyric acid, pyruvic acid methyl ester, β-hydroxybutyric acid, sebacic acid, succinic acid monomethyl ester, 
-hydroxybutyric acid, succinic acid, -ketovaleric acid and L-asparagine. It cannot oxidize melibiose, p-hydroxyphenylacetic acid, L-histidine, -cyclodextrin, D-fructose, methyl β-D-glucoside, inosine, dextrin, L-fucose, D-psicose, glucuronamide, uridine, D-galactose, raffinose, L-alaninamide, L-ornithine, thymidine, gentiobiose, L-rhamnose, D-galactonic acid lactone, D-alanine, L-phenylalanine, phenyethylamine, -D-glucose, D-sorbitol, D-galacturonic acid, L-alanine, L-proline, putrescine, N-acetyl-D-galactosamine, myo-inositol, sucrose, D-gluconic acid, malonic acid, L-alanylglycine, 2-aminoethanol, N-acetyl-D-glucosamine, -D-lactose, trehalose, D-glucosaminic acid, D-serine, 2,3-butanediol, adonitol, lactulose, turanose, D-glucuronic acid, quinic acid, L-aspartic acid, L-serine, glycerol, L-arabinose, maltose, xylitol, D-saccharic acid, L-threonine, DL--glycerol phosphate, D-arabitol, D-mannitol, glycyl L-aspartic acid, DL-carnitine, -D-glucose 1-phosphate, D-cellobiose, D-mannose, glycyl L-glutamic acid, D-glucose 6-phosphate and -aminobutyric acid. Strain YY287T is resistant to streptomycin and gentamicin but sensitive to ampicillin, chloramphenicol, rifampicin, penicillin G, sulfamethoxazole plus trimethoprim, kanamycin, nalidixic acid, novobiocin and tetracycline. The major fatty acids are 16 : 0 (33.3 %), 18 : 17c (12.9 %) and summed feature 3 (16 : 17c and/or 15 : 0 iso 2-OH; 40.8 %). The DNA G+C content is 62.8 mol%.
The type strain YY287T (=BCRC 17659T=LMG 24008T) was isolated from food waste compost, Kinmen County, Taiwan.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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