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1 Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung City 811, Taiwan
2 School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK
3 Department of Soil Environmental Science, College of Agriculture and Natural Resources, National Chung Hsing University, Taichung, Taiwan
4 Department of Marine Biotechnology, National Kaohsiung Marine University, Kaohsiung, Taiwan
Correspondence
Wen-Ming Chen
p62365{at}ms28.hinet.net
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ABSTRACT |
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Fatty acid compositions and whole-cell protein profiles of strain Liujia-146T and related strains are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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, b, 2001; Subba-Rao et al., 1995). A variety of bacteria have been isolated from such nodules, particularly from Neptunia oleracea (syn. N. natans, N. prostrata; http://www.ildis.org), including a species closely related to Rhizobium, Allorhizobium undicola (de Lajudie et al., 1998), and the alphaproteobacterium Devosia neptuniae (Rivas et al., 2002, 2003). In addition to these ‘exotic’ bacterial species, more ‘conventional’ rhizobia have also been isolated from Neptunia nodules, for example Rhizobium tropici strains UPRM8033 and DUS239 from Neptunia plena (Zurdo-Piñeiro et al., 2004). In the present study, we report on a novel non-nodulating strain from the genus Labrys, Liujia-146T, isolated from root nodules of N. oleracea growing aquatically in Taiwan. Labrys is a genus of budding bacteria, first described by Vasilyeva & Semenov (1984). At present, it consists of two species, Labrys monachus (Vasilyeva & Semenov, 1984) and Labrys methylaminiphilus (Miller et al., 2005). Cells of L. monachus possess triangular radial symmetry (Vasilyeva & Semenov, 1984) and L. methylaminiphilus is a facultative methylotroph (Miller et al., 2005). Comparisons of their 16S rRNA gene sequences indicate that L. monachus and L. methylaminiphilus belong to the Alphaproteobacteria (Fritz et al., 2004; Miller et al., 2005).
Root nodules were collected from N. oleracea growing in a freshwater pond at Tainan County in southern Taiwan. They were immersed in 75 % ethanol for 10 s, sterilized in 0.1 % (w/v) mercuric chloride for 10 min and then washed six times with sterile distilled water. Individual nodules were crushed and streaked onto yeast extract-mannitol (YEM) agar (Vincent, 1970) and incubated at 28 °C. The majority of isolates (>95 %) were identified as A. undicola. However, a bacterial strain with a colony morphology different from that of A. undicola was also recovered. This was designated Liujia-146T. Type strains of L. monachus (DSM 5896T) and L. methylaminiphilus (DSM 16812T) were obtained from the DSMZ.
The pH range for growth was determined by measuring the optical density (wavelength 595 nm) of cultures grown in YEM medium with pH ranging from 4 to 10, adjusted with appropriate biological buffers (Chung et al., 1995). Anaerobic cultivation was performed on YEM medium under the Oxoid AnaeroGen system. Strain Liujia-146T formed visible colonies that were circular, convex, opaque and mucoid with entire edges. The colony diameter was approximately 1.0–2.0 mm on YEM agar after 48 h of incubation at 28 °C. Strain Liujia-146T grew well at temperatures ranging from 25 to 35 °C and at a pH between 4 and 10; optimal growth conditions were 28–30 °C and pH 7.0–8.0. Strain Liujia-146T did not require additional growth factors, although addition of yeast extract was found to stimulate growth. It did not grow over 120 h of incubation at 28 °C under anaerobic conditions, suggesting that it is an aerobic bacterium.
Cell morphology was observed under a light microscope and by scanning electron microscopy. Motility of cells was examined by the hanging drop method. Gram Stain Set S (Difco) and Indian ink (Difco) were used to perform Gram and capsule staining. Strain Liujia-146T is a Gram-negative, non-motile, non-spore-forming, capsulated, rod-shaped bacterium, 0.7–0.9 µm in diameter and 1.2–1.5 µm in length (Fig. 1). Cells are found singly or in pairs and were seen to reproduce by budding. Capsule formation, observed by electron microscopy, was also visualized with Indian ink by light microscopy.
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). Multiple-sequence alignment comparing strain Liujia-146T and its closest relatives was performed using BioEdit software (Hall, 1999). Distances (distance options according to the Kimura two-parameter model) and clustering with the neighbour-joining and maximum-parsimony methods were determined by using bootstrap values based on 1000 replications. Comparison of the 16S rRNA gene sequence of strain Liujia-146T with available 16S rRNA gene sequences in public databases revealed that strain Liujia-146T belonged to the order Rhizobiales of the Alphaproteobacteria. The highest similarity values were obtained from the comparison with L. monachus VKM B-1479T (97.8 % similarity) and L. methylaminiphilus JLW10T (95.5 %). The two type strains of Labrys species, VKM B-1479T and JLW10T, and strain Liujia-146T were found to form a well-separated, monophyletic group (Fig. 2). The 16S rRNA gene sequence similarity of strain Liujia-146T to other established bacterial species within the Alphaproteobacteria was less than 95 %. Recently, Lee et al. (2005) proposed a novel family Xanthobacteraceae of the order Rhizobiales to include the genera Labrys, Azorhizobium, Ancylobacter, Starkeya and Xanthobacter. However, our phylogenetic analysis based on neighbour-joining and maximum-parsimony methods did not support the affiliation of Labrys species within the family Xanthobacteraceae, which was based on only one species, L. methylaminiphilus.
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, with data from experiments performed in triplicate indicating that the DNA–DNA binding level of strain Liujia-146T with L. monachus VKM B-1479T and L. methylaminiphilus JLW10T was only 18.6 (±5.1) % and 41.0 (±7.6) %, respectively. The DNA G+C content of strain Liujia-146T was determined in triplicate using the method described by Mesbah et al. (1989) and was found to be 62.7 (±1.7) mol%. For biochemical characterization, the API 20NE, API ZYM (bioMérieux) and Microlog GN2 (Biolog) systems were used according to the manufacturers' instructions. Additionally, sensitivity to antibiotics was examined by spreading cells (0.5 McFarland) onto YEM agar and placing onto them discs (Difco) containing the following individual antibiotics: 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) and tetracycline (30 µg). Results of biochemical characterization and antibiotic sensitivity are given in the species description.
Chemotaxonomic differentiation of strain Liujia-146T from its closest phylogenetic neighbours was examined using several approaches. For analysis of protein electrophoretic patterns, preparation of whole-cell proteins and SDS-PAGE were performed as described by Pot et al. (1994). The whole-cell protein profile of strain Liujia-146T could be differentiated clearly from those of L. monachus VKM B-1479T and L. methylaminiphilus JLW10T (see Supplementary Fig. S1 available in IJSEM Online). For fatty acid methyl ester analysis, cell culture was harvested after an incubation period of 48 h at 28 °C; fatty acid methyl esters were then prepared, separated and identified according to the instructions of the Microbial Identification System (MIDI; Microbial ID). The major cellular fatty acids of strain Liujia-146T were 16 : 0, 18 : 1
7c and 19 : 0 cyclo, as found with L. monachus VKM B-1479T and L. methylaminiphilus JLW10T (Miller et al., 2005). However, strain Liujia-146T could again be clearly distinguished from L. monachus VKM B-1479T and L. methylaminiphilus JLW10T by its significantly larger relative amount of 18 : 17c (68.8 %), coupled with the smaller relative amount of 19 : 0 cyclo (7.8 %) and the absence of detectable amounts of 20 : 19 (Table 1 and Supplementary Table S1 in IJSEM Online).
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). Plants of N. oleracea and Macroptilium atropurpureum were harvested at 45 days after inoculation with strain Liujia-146T, and no nodules had formed by that time. This indicates that bacteria isolated from nodules are not necessarily able to induce nodulation.
Detailed comparison of the physiological and biochemical characteristics of strain Liujia-146T, L. monachus VKM B-1479T and L. methylaminiphilus JLW10T is shown in Table 1
. Strain Liujia-146T could be distinguished clearly from L. monachus VKM B-1479T by many characters, especially regarding the capability for nitrite reduction, vitamin requirement for growth and assimilation of a variety of carbon substrates. Strain Liujia-146T could also be distinguished clearly from L. methylaminiphilus JLW10T by some enzyme activities (such as oxidase, catalase, arginine dihydrolase, 
-galactosidase and aesculin hydrolysis) and assimilation of some carbon substrates. In addition to physiological and biochemical evidence, fatty acid compositions, whole-cell protein profiles and DNA–DNA hybridization tests could also be used to distinguish strain Liujia-146T from the nearest species, L. monachus and L. methylaminiphilus. Therefore, based on phenotypic and phylogenetic criteria, we are of the opinion that strain Liujia-146T merits assignment to a novel species within the genus Labrys, for which the name Labrys neptuniae sp. nov. is proposed.
Description of Labrys neptuniae sp. nov.
Labrys neptuniae (nep.tu'ni.ae. N.L. gen. n. neptuniae of Neptunia, named because the type strain was isolated from Neptunia oleracea).
Cells are Gram-negative, non-motile, non-spore-forming, capsulated, rod-shaped and multiply by budding. They are 0.7–0.9 µm in diameter and 1.2–1.5 µm in length. Growth is evident at temperatures between 15 and 35 °C and at pH 4.0–10.0; optimum growth is displayed at 28–30 °C and pH 7.0–8.0. The following characters (API 20NE) are positive for the type strain: nitrate reduction, urease, aesculin hydrolysis, -galactosidase and assimilation of glucose, arabinose, mannose, mannitol, N-acetylglucosamine, gluconate and malate. Negative results are displayed for oxidase, indole production, glucose fermentation, arginine dihydrolase, gelatin hydrolysis and assimilation of maltose, caprate, adipate, citrate and phenylacetate. Positive results (API ZYM) are seen for activities of alkaline phosphatase, C4 esterase, leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase and negative results are obtained for C8 lipase, C14 lipase, valine arylamidase, cystine arylamidase, trypsin, 
-chymotrypsin, -galactosidase, -glucosidase, -glucuronidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase. The following carbon sources are oxidized (positive result with the Biolog GN2 system): glycogen, Tween 40, Tween 80, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, adonitol, L-arabinose, D-arabitol, cellobiose, i-erythritol, D-fructose, L-fucose, D-galactose, gentiobiose, myo-inositol, lactulose, D-mannitol, D-mannose, D-melibiose, methyl -D-glucoside, D-psicose, D-raffinose, L-rhamnose, D-sorbitol, D-trehalose, xylitol, methyl pyruvate, monomethyl succinate, acetic acid, cis-aconitic acid, formic acid, D-galactonic acid lactone, D-galacturonic acid, -hydroxybutyric acid, -hydroxybutyric acid, p-hydroxyphenylacetic acid, -ketobutyric acid, DL-lactate, malonic acid, propionic 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-glutamic acid, L-histidine, hydroxy-L-proline, L-leucine, L-ornithine, L-proline, L-pyroglutamic acid, L-serine, L-threonine, DL-carnitine, 
-aminobutyric acid, urocanic acid, inosine, uridine, thymidine, putrescine, 2-aminoethanol, glycerol, DL--glycerol phosphate, glucose 1-phosphate and glucose 6-phosphate. The type strain can not oxidize -cyclodextrin, dextrin, -D-lactose, maltose, sucrose, turanose, citrate, D-glucosaminic acid, D-glucuronic acid, -hydroxybutyric acid, itaconic acid, -ketoglutaric acid, -ketovaleric acid, quinic acid, D-saccharic acid, sebacic acid, glycyl L-aspartic acid, L-phenylalanine, D-serine, phenylethylamine or 2,3-butanediol. The type strain is resistant to ampicillin, chloramphenicol, nalidixic acid, penicillin G and rifampicin and sensitive to gentamicin, kanamycin, novobiocin, streptomycin and tetracycline. The major fatty acid components are 18 : 17c, 16 : 0 and 19 : 0 cyclo. The DNA G+C content of the type strain is 62.7 (±1.7) mol%.
The type strain, Liujia-146T (=BCRC 17578T=LMG 23578T), was isolated from root nodules of Neptunia oleracea, an aquatic legume, growing in a freshwater pond located in Tainan County in southern Taiwan.
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ACKNOWLEDGEMENTS |
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