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1 Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, SAR, P. R. China
2 Department of Biology, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, SAR, P. R. China
Correspondence
Ken W. K. Lau
sslwk{at}ust.hk
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A figure showing a slot-blot DNA–DNA hybridization assay with DNA of strain UST010306-043T as the probe is available as supplementary material in IJSEM Online.
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showed that, based on rRNA gene sequences, the genus Alteromonas (Baumann et al., 1972) was composed of four distinct branches and reclassified the branch containing Alteromonas vaga and Alteromonas communis as the genus Marinomonas. At the time of writing, the genus comprises eight species with validly published names: Marinomonas vaga, Marinomonas communis (Baumann et al., 1972; Van Landschoot & De Ley, 1983), Marinomonas mediterranea (Solano & Sanchez-Amat, 1999), Marinomonas primoryensis (Romanenko et al., 2003), Marinomonas pontica (Ivanova et al., 2005), Marinomonas ushuaiensis (Prabagaran et al., 2005), Marinomonas aquimarina (Macián et al., 2005) and Marinomonas dokdonensis (Yoon et al., 2005). In this study, we report a novel strain, UST010306-043T, which was isolated from a sea-water sample from a pearl-oyster culture pond in the HKUST-CAS Joint Laboratory in Sanya City, Hainan Province, China, on 6 January 2001. Based on polyphasic taxonomy, we propose that strain UST010306-043T represents a novel species, Marinomonas ostreistagni sp. nov.
Sanya City is located at the southern tip of Hainan Island (18o N 109o E), with a tropical oceanic monsoon climate. About 500 ml sea water was sampled from the pearl-oyster culture pond at a depth of 3 m, and was transported in a sterile plastic container to the laboratory within 2 h.
The sea water was filtered through a 1 µm polycarbonate filter (Osmonics). Aliquots of 100 µl filtrate were spread on nutrient agar (Oxoid; prepared with 0.45 µm-filtered sea water) and incubated at room temperature for 2–3 days and then stored at 4 °C. Colonies were purified by being repeatedly restreaked on marine agar 2216 (MA; Difco) plates. Purified strains were stored at –80 °C in marine broth 2216 (MB) with 30 % (v/v) glycerol. Partial 16S rRNA genes were amplified by using the primers GM5F (5'-CCTACGGGAGGCAGCAG-3') and 907R (5'-CCGTCAATTCCTTTRAGTTT-3') (Muyzer et al., 1995) with Taq polymerase (Promega) and sequenced using ABI PRISM BigDye Terminator cycle sequencing ready reaction kit version 2.0 and the primer 341F. Strain UST010306-043T, a single novel isolate related to the genus Marinomonas, was selected for further analysis. Colony morphology was observed after incubation for 24 h at 30 °C. The temperature range for growth was evaluated in MB at 4, 16, 20, 25, 30, 33, 37, 40 and 42 °C. Growth at pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 was evaluated in YP-SW broth (0.4 % yeast extract, 0.2 % peptone, 75 % 0.45 µm-filtered sea water), adjusted with 1 M HCl or NaOH. The requirement for sodium ions was tested in artificial sea water (ASW; 0.1 % CaCl2.2H2O, 0.1 % KCl, 0.5 % MgSO4.7H2O, 2.5 % NaCl) (Lewin & Lounsbery, 1969), with NaCl replaced by equal molar KCl and addition of 0.4 % yeast extract. Tolerance to NaCl was tested in ASW supplemented with 0.4 % yeast extract and with NaCl added at final concentrations of 0, 1, 2, 3, 4, 5, 7.5, 10 and 15 %. Anaerobic growth was examined by using the Oxoid anaerobic system for cultures grown on MA supplemented with 0.1 % sodium nitrate or 0.1 % glucose. Cellular morphology was determined from a MB overnight culture using a Zeiss MC100 Spot microscope at a magnification of x1000. Flagella morphology was examined by using transmission electron microscopy (JOEL 100CXII at 80 kV), using cells placed on a carbon grid and stained with 1 % (w/v) phosphotungstic acid (pH 7.0).
Gram staining was performed according to Collins et al. (1989). Tests for requirement of organic growth factors, utilization of various carbon sources and chitin, fermentation of glucose, sucrose or D-mannitol and accumulation of poly-
-hydroxybutyrate were performed according to Baumann & Baumann (1981). Alkaline phosphatase activity, H2S generation from cysteine or thiosulfate, oxidation of glucose and hydrolysis of starch, casein, gelatin and cellulose were determined according to Smibert & Krieg (1994). Fatty acid methyl ester analysis was performed as described by Svetashev et al. (1995), except that the bacterial strains were cultured at 30 °C for 24 h. DNA hydrolysis test and sequencing of the nearly complete16S rRNA gene were performed as described by Lau et al. (2005). Related sequences were identified by MEGABLAST search (Zhang et al., 2000), retrieved from the NCBI nucleotide database, aligned using CLUSTAL_X (Thompson et al., 1997), edited with BioEdit version 5.0.9 (Hall, 1999) and analysed using MEGA version 2.1 (Kumar et al., 2001). A phylogenetic tree was generated by using the neighbour-joining method (Saitou & Nei, 1987) with the Jukes–Cantor substitution model (Jukes & Cantor, 1969) and was evaluated by bootstrap analysis (Felsenstein, 1985) based on 1000 resamplings. M. aquimarina CECT 5080T and M. communis CECT 5003T were obtained from the Spanish Type Culture Collection.
For DNA–DNA hybridization experiments, genomic DNA was extracted according to Maloy (1990) with modifications: 1.5 ml overnight culture was centrifuged and resuspended in 467 µl TE buffer, containing 30 µl SDS (10 %, w/v) and 3 µl proteinase K (20 mg ml–1), incubated at 37 °C for 1 h and then extracted twice with equal amounts of phenol/chloroform/isoamyl alcohol (25 : 24 : 1, by vol.). DNA was precipitated with 0.1 vol. 3 M sodium acetate (pH 5.2) and 0.6 vol. 2-propanol. The precipitate was washed with 0.5 ml 70 % ethanol, dried in a vacuum, dissolved in 100 µl TE buffer with RNase (100 µg ml–1) and incubated at 37 °C for 1 h. DNA was diluted in TE buffer to 0.5–1.0 ng µl–1 and run in an agarose gel (1 %) with a 
HindIII marker (100 ng; TaKaRa). Quantification of DNA was done by comparing the band intensities of isolated DNA and that of the marker using the Spot Denso function of the IS-1000 Digital Imaging System (Alpha Innotech). Slot-blot analysis of DNA was performed according to Brown (2005) with Hybond-N+ membrane (Millipore). One or 10 ng genomic DNA of each strain was loaded per well. One hundred nanogram probe DNA was labelled using the Gene Image random prime labelling module (Amersham), according to the manufacturer's instructions. Overnight hybridization was performed at 60 °C. The membrane was washed three times with 1x SSC/0.1 % SDS and three times with 0.5x SSC/0.1 % SDS at 60 °C (each washing step lasted for 10 min), detected using a Gene Image CDP-Star detection module (Amersham) according to the manufacturer's instructions and exposed to a 100NIF Xray film (Fuji) for 15 min. Levels of hybridization were determined with EagleSight software version 3.2 in the Eagle Eye II system (Stratagene). The DNA G+C content was determined by using the HPLC method (Mesbah et al., 1989), with genomic DNA extracted with a MiniBEST Bacterial Genomic DNA Extraction kit (TaKaRa). Susceptibility to antibiotics was tested by using the disc-diffusion plate method on MA. The following antibiotics were tested: ampicillin (10 µg), polymyxin B (300 U), chloramphenicol (30 µg), tetracycline (30 µg), streptomycin (10 µg), rifampicin (10 µg) and penicillin G (2 U).
Morphological, physiological and biochemical features of UST010306-043T are given in the species description.
Strain UST010306-043T shared many similarities with other Marinomonas species. Strains were all Gram-negative, isolated from marine habitats, had rod-shaped cells, were halophilic, strictly aerobic, heterotrophic, non-spore-forming and non-fermentative, utilized glucose and acetate and did not require complex organic growth factors. Strain UST010306-043T shared many similarities with M. aquimarina; in particular, their similarity in habitat (the former was found in sea water of a pearl-oyster pond and the latter was found in oyster flesh and sea water), growth up to 40 °C but not at 4 °C, oxidative response to glucose and inability to utilize maltose and D-sorbitol. The G+C content of the DNA of strain UST010306-043T was 49.8±0.5 mol%, which was close to the highest G+C content of members of the genus Marinomonas (Sanchez-Amat & Solano, 2005). The fatty acids of strain UST010306-043T and closely related species are shown in Table 1. The fatty acid profile of strain UST010306-043T was very similar to that of M. aquimarina CECT 5080T, but differed from that of M. communis CECT 5003T by having a slightly higher percentage of straight-chain fatty acids. Overall, the fatty acid profiles of strain UST010306-043T and members of the genus Marinomonas had a similar pattern; namely, with over 60 % fatty acids distributed among 18 : 1
7c, 16 : 0/16 : 0 iso and 16 : 17c/15 : 0 iso 2-OH (Table 1; Ivanova et al., 2000, 2005; Yoon et al., 2005; Prabagaran et al., 2005). Characteristics that differentiated strain UST010306-043T from other species of Marinomonas are shown in Table 2.
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). The 16S rRNA gene sequence of strain UST010306-043T shared 94.1–97.7 % similarity with other members of the genus Marinomonas, being most closely related to M. aquimarina (97.5–97.7 %) and M. communis (97.1 %). Its similarity to other species of Marinomonas was less than 97 %, indicating that they were different species (Stackebrandt & Goebel, 1994). As the 16S rRNA gene similarity of strain UST010306-043T with M. aquimarina and M. communis was at the borderline for discrimination of species by sequence analysis (Stackebrandt & Goebel, 1994), DNA–DNA hybridization, which has significantly higher resolution power than sequence analysis (Amann et al., 1992), was performed. The results of DNA–DNA hybridization (available as Supplementary Fig. S1 in IJSEM Online) showed that the levels of relatedness of strain UST010306-043T with M. aquimarina CECT 5080T and M. communis CECT 5003T were below 23 %. This was well below the 70 % threshold value for species discrimination (Wayne et al., 1987), suggesting that strain UST010306-043T represents a species that is different from M. aquimarina or M. communis. On the basis of all characteristics described above, a novel species with the name Marinomonas ostreistagni sp. nov. is proposed.
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Cells are Gram-negative rods, 0.6–3.6 µm in length and 0.4–0.6 µm in diameter and motile by a single polar flagellum. Cells divide by binary fission. Heterotrophic, strictly aerobic and requires sodium ions for growth. Cell aggregates are detected when cultured in MB. Colonies are non-pigmented, circular, opaque, convex, 1–2 mm in diameter with an entire margin after 24 h incubation at 30 °C on MA. Grows in 1–10 % NaCl but not in 15 % NaCl; optimum NaCl for growth is approximately 3 %. Grows at 16–40 °C, but not at 4 or 42 °C; optimum temperature for growth is 33–37 °C. Grows at pH 5.0–9.0, with optimum growth occurring around pH 7.0. Oxidase-, catalase- and alkaline phosphatase-positive. Oxidizes glucose. Does not produce sulfide from cysteine or thiosulfate and does not degrade extracellular DNA. Does not form endospores and does not accumulate poly--hydroxybutyrate. Utilizes glucose, sucrose, D-mannitol, aesculin, L-alanine, L-asparagine, L-serine, L-glutamic acid, acetate, citrate, pyruvate, propionate, lactate, ethanol and Tween 20, but not galactose, maltose, L-arabinose, trehalose, rhamnose, lactose, melibiose, D-sorbitol, methanol, glycerol, succinate, malate, glycine, L-leucine, L-arginine, L-lysine, L-histidine, L-methionine, L-threonine, valine, starch, gelatin, casein, agar, DNA, Tween 40, Tween 60, Tween 80 or cellulose. Does not ferment glucose, sucrose or D-mannitol. Sensitive to ampicillin (10 µg), polymyxin B (300 U), chloramphenicol (30 µg), tetracycline (30 µg), streptomycin (10 µg) and rifampicin (10 µg), but resistant to penicillin G (2 U). Predominant fatty acids are 18 : 17c, 16 : 0 and 16 : 17c/15 : 0 iso 2-OH. The DNA G+C content is 49.8±0.5 mol%.
The type strain is UST010306-043T(=JCM 13672T=NRRL B-41433T), which was isolated from the pearl-oyster culture pond of HKUST-CAS Joint Laboratory, Sanya, Hainan Province, China.
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ACKNOWLEDGEMENTS |
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