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1 College of Biological Sciences, China Agricultural University, Beijing 100094, People's Republic of China
2 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, ZhongGuanCun, Haidian, Beijing 100080, People's Republic of China
Correspondence
Shuang-Jiang Liu
liusj{at}sun.im.ac.cn
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ABSTRACT |
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A transmission electron micrograph of strain HY9T, two phylogenetic trees and a table of fatty acid compositions are available as supplementary material in IJSEM Online.
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; and references therein). For instance, members of the genus Cyclobacterium displaying the unique ring-like and horseshoe-shaped morphology are common constituents of marine environments. At the time of writing, the genus Cyclobacterium (Raj & Maloy, 1990) contains two species with validly published names: Cyclobacterium marinum, isolated from the deep sea in the Gulf of California (Raj & Maloy, 1990; Euzéby, 1998), and Cyclobacterium amurskyense, isolated from water in the Sea of Japan (Nedashkovskaya et al., 2005). Organisms sharing high levels of 16S rRNA gene sequence similarity with Cyclobacterium species have also been found in salt-marsh sediment (GenBank accession no. AY259502) and a soda lake (AF275712). In this study, we describe a rose-pigmented bacterial strain with ring-like and horseshoe-shaped cells, designated strain HY9T and isolated from sediment of the South China Sea. A polyphasic analysis of strain HY9T indicated that the isolate represents a novel species of the genus Cyclobacterium.
Strain HY9T was isolated from sediment of the Xijiang oilfield in the South China Sea, near Fujian Province, China; sampling was performed at a depth of about 100 m. For isolation, serially diluted sediment samples were spread onto low-organic marine agar 2216 plates [containing 0.5 g peptone l–1 and 0.1 g yeast extract l–1; the salt composition and concentration were the same as in marine agar 2216 (MA; Difco)] and incubated at 30 °C for 10 days. A colony of HY9T was collected and subcultured on MA. The temperature and pH ranges for growth, the tolerance of NaCl and the substrates used as sole carbon sources were determined according to the methods described by Cho & Giovannoni (2003). Strain HY9T was tested on API ZYM (bioMérieux) and Biolog GN2 MicroPlate systems, according to the manufacturers' instructions. All other biochemical tests were performed according to Dong & Cai (2001). Susceptibility to antibiotics was determined using filter-paper discs containing various antibiotics, as specified in the species description. The morphology of cells grown on MA for 2 days at 30 °C was studied using transmission electron microscopy (Zhang et al., 2002).
The novel isolate displayed the basic characteristics of members of the genus Cyclobacterium, e.g. the colonies were rose-pigmented and the cells were curved, ring-like or horseshoe-shaped (see Supplementary Fig. S1 available in IJSEM Online). Other phenotypic properties of strain HY9T are given in the species description and in Table 1.
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) and sequenced. Preliminary comparisons with sequences held in GenBank, performed using BLASTN searches, revealed that the isolate was related to C. marinum. Further analysis of 16S rRNA gene sequences was performed using MEGA, version 3.1 (Kumar et al., 2004). Strain HY9T showed highest sequence similarity with C. marinum DSM 745T (93.6 %) and C. amurskyense KMM 6143T (92.8 %), followed by members of the genera Aquiflexum (89.7 %), Belliella (89.7 %), Hongiella (87.8–90.3 %), Chimaereicella (88.4 %) and Algoriphagus (88.3–89.5 %). Phylogenetic trees were constructed with MEGA, version 3.1, using the neighbour-joining method (Saitou & Nei, 1987), maximum parsimony (Fitch, 1971) and minimum evolution (Felsenstein, 1997). The resulting tree topology was evaluated using the Kimura two-parameter calculation model (Kumar et al., 2004) based on 1000 replicates. As shown in Fig. 1 and Supplementary Fig. S2 (available in IJSEM Online), strain HY9T formed a monophyletic clade with C. marinum and C. amurskyense. The closest relatives of this clade were members of the genera Aquiflexum and Belliella (Brettar et al., 2004a, b).
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7c; 16.6 %), iso-C17 : 19c (10.3 %), iso-C17 : 0 3-OH (8.0 %) and anteiso-C15 : 0 (6.4 %), similar to the profiles reported for C. marinum and C. amurskyense (Nedashkovskaya et al., 2005). The complete fatty acid composition of strain HY9T is given in Supplementary Table S1 (available in IJSEM Online). The G+C content of the DNA was determined by thermal denaturation (Marmur & Doty, 1962; Seidler & Mandel, 1971) using DNA from Escherichia coli K-12 as a control. The G+C content of strain HY9T was 45.2 mol%, which is slightly higher than those of the type strains of C. marinum (41.9 mol%) and C. amurskyense (41.3 mol%) (Nedashkovskaya et al., 2005).
On the basis of these results, we concluded that strain HY9T represents a novel species within the genus Cyclobacterium, for which the name Cyclobacterium lianum sp. nov. is proposed. The properties that serve to differentiate strain HY9T, C. marinum and C. amurskyense from each other are listed in Table 1
. As some of the properties of C. lianum (from this study) and C. amurskyense (Nedashkovskaya et al., 2005) were not included in or are not compatible with the original description of the genus Cyclobacterium (Raj & Maloy, 1990), an emended description of the genus is also proposed.
Description of Cyclobacterium lianum sp. nov.
Cyclobacterium lianum (N.L. neut. adj. lianum pertaining to Li, named in honour of Professor Ji-Lun Li, who devotes himself to microbiological research and education in China).
Cells are Gram-negative, aerobic and heterotrophic, non-motile, curved, ring-like or horseshoe-shaped, 0.4–0.5 µm wide, and the outer diameter of rings is 1.5–1.8 µm. Colonies grown for 3 days on MA are circular (2–3 mm in diameter), light rose in colour and shiny. Growth occurs at 15–42 °C (optimum 33 °C), at pH 6.5–9.0 (optimum pH 7.5–8.0) and with 0.1–12 % NaCl (optimum 1–4 %). Positive for oxidase and catalase activities, but negative for arginine dihydrolase, urease and lecithinase activities. Indole and H2S are not produced and nitrate is not reduced. Aesculin and Tween 20 are hydrolysed. Tweens 40 and 80 are hydrolysed weakly; agar, casein, gelatin, starch, DNA and carboxymethyl-cellulose are not hydrolysed. Glucose, sucrose, D-melibiose, ribose, lactose, galactose, maltose, melezitose, inulin, L-rhamnose, L-arabinose, D-raffinose, trehalose, cellobiose, methyl 
-D-glucoside and gluconate are utilized as sole carbon sources. Glycerol, mannitol, D-mannose, D-fructose, D-xylose, lactic acid, succinate, malate, pyruvate and L-glutamic acid are weakly utilized. L-Fucose, L-sorbose, dulcitol, adonitol, myo-inositol, citrate, malonate, L-lysine, L-alanine, formic acid, butyric acid and caprate are not utilized. Forms acid from glucose, ribose (weakly), sucrose, D-melibiose, lactose, galactose, maltose, melezitose, inulin, L-rhamnose, L-arabinose, D-raffinose, trehalose, cellobiose, D-xylose, glycerol (weakly) and methyl -D-glucoside. Shows strong activity in the API ZYM system for alkaline and acid phosphatases, leucine and valine arylamidases, naphthol-AS-BI-phosphohydrolase, 
-galactosidase, - and -glucosidases and N-acetyl--glucosaminidase. Shows weak activity for esterases C4 and C8, cystine arylamidase, -galactosidase and -mannosidase. No activity is shown for trypsin, -chymotrypsin, -glucuronidase, -fucosidase or lipase (C14). In GN2 MicroPlates, dextrin, N-acetyl-D-glucosamine, L-arabinose, D-cellobiose, D-fructose, D-galactose, gentiobiose, -D-glucose, -D-lactose, lactulose, maltose, D-mannose, D-melibiose, methyl -D-glucoside, D-raffinose, sucrose, D-trehalose, turanose, D-galacturonic acid, DL-lactic acid, glucuronamide, L-alaninamide, L-alanine, DL-carnitine, 2,3-butanediol, glycerol, DL--glycerol phosphate and glucose 1-phosphate are oxidized. Weak or variable results are detected with glycogen, N-acetyl-D-galactosamine, i-erythritol, D-mannitol, D-psicose, L-rhamnose, D-sorbitol, monomethyl succinate, D-gluconic acid, -ketovaleric acid, succinic acid, L-alanyl glycine, L-asparagine, L-aspartic acid, glutamic acid, L-ornithine, L-proline, L-pyroglutamic acid, DL-serine, L-threonine, 
-aminobutyric acid, uridine, 2-aminoethanol and glucose 6-phosphate. Resistant to the following antibiotics (µg): gentamicin (10), neomycin (30), polymyxin B (300), streptomycin (10) and tetracycline (30). Sensitive to the following antibiotics (µg): ampicillin (10), carbenicillin (100), vancomycin (30), ciprofloxacin (5), rifampicin (5), norfloxacin (10), chloramphenicol (30), benzyl penicillin (10), kanamycin (30) and erythromycin (15). The major cellular fatty acids (>5 %) are iso-C15 : 0 (28.3 %), summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 17c; 16.6 %), iso-C17 : 19c (10.3 %), iso-C17 : 0 3-OH (8.0 %) and anteiso-C15 : 0 (6.4 %). The molar G+C content of the DNA is 45.2 mol%.
The type strain, HY9T (=CGMCC 1.6102T=JCM 14011T), was isolated from sediment from the Xijiang oilfield in the South China Sea.
Emended description of the genus Cyclobacterium
Colonies on MA are pink-pigmented and shiny. Cells are curved, ring-like or horseshoe-shaped. Neutrophilic and mesophilic. Optimal growth temperature range is 25–30 °C. NaCl is required for growth. The major cellular fatty acids are iso-C15 : 0, summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 17c), iso-C17 : 19c, iso-C17 : 0 3-OH and anteiso-C15 : 0. The DNA G+C content is 41–45 mol%.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Brettar, I., Christen, R. & Höfle, M. G. (2004a). Belliella baltica gen. nov., sp. nov., a novel marine bacterium of the Cytophaga–Flavobacterium–Bacteroides group isolated from surface water of the central Baltic Sea. Int J Syst Evol Microbiol 54, 65–70.
Brettar, I., Christen, R. & Höfle, M. G. (2004b). Aquiflexum balticum gen. nov., sp. nov., a novel marine bacterium of the Cytophaga–Flavobacterium–Bacteroides group isolated from surface water of the central Baltic Sea. Int J Syst Evol Microbiol 54, 2335–2341.
Cho, J. C. & Giovannoni, S. J. (2003). Parvularcula bermudensis gen. nov., sp. nov., a marine bacterium that forms a deep branch in the -Proteobacteria. Int J Syst Evol Microbiol 53, 1031–1036.
Dong, X.-Z. & Cai, M.-Y. (2001). Determinative Manual for Routine Bacteriology. Beijing: Scientific Press.
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