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1 Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, S. A. R., P. R. China
2 Department of Biology, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, S. A. R., P. R. China
Correspondence
Madeline Wu
bomwu{at}ust.hk
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9c, 2-OH 15 : 0, 15 : 16c and three unknown fatty acids of equivalent chain-length of 11·543, 13·565 and 16·582. Further analysis of its ecophysiology and biochemistry suggests that this strain represents a new genus in the phylum ‘Bacteroidetes’. The name Owenweeksia hongkongensis gen. nov., sp. nov. is proposed. The type strain is UST20020801T (=NRRL B-23963T=JCM 12287T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of UST20020801T is AB125062.
Micrographs of Owenweeksia hongkongensis UST20020801T and a phylogenetic tree based on 16S rRNA gene sequences showing the relationship between strain UST20020801T and related taxa are available as supplementary material in IJSEM Online.
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). Recently, the family Cryomorphaceae was proposed as a member of the phylum ‘Bacteroidetes’. This family encompasses three genera, Brumimicrobium, Cryomorpha and Crocinitomix, which form a distinct clade in the class ‘Flavobacteria’, branching between the families Flavobacteriaceae and Bacteroidaceae (Bowman et al., 2003). Current members of the family Cryomorphaceae are psychrotolerant, rod- to filamentous-shaped and possess carotenoid pigments. They also require sea-water salts and complex organic compounds for growth (Bowman et al., 2003). In a study of the bacterial diversity in Hong Kong coastal sea water, a mesophilic, orange-pigmented, rod-shaped, moderately halophilic bacterium (strain UST20020801T) was isolated and characterized by polyphasic taxonomy. Analysis of the 16S rRNA gene sequence confirmed that this bacterium was affiliated to the family Cryomorphaceae in the ‘Bacteroidetes’; sequence similarity with its nearest phylogenetic relative, Crocinitomix catalasitica, was 87·5 %. On the basis of phenotypic and genotypic characteristics, fatty acid composition and phylogenetic position, it is proposed that strain UST20020801T represents a novel species of a new genus, Owenweeksia hongkongensis gen. nov., sp. nov., of the family Cryomorphaceae in the phylum ‘Bacteroidetes’.
Strain UST20020801T was isolated from a sea-water sample collected from the outlet of a tank storing sand-filtered sea water that was pumped from a depth of 5 m adjacent to the Coastal Marine Laboratory of the Hong Kong University of Science and Technology. Aliquots of 100 µl were spread onto agar plates containing YPS-SW medium (0·4 % yeast extract, 0·2 % peptone, 1 % starch, 75 % sea water filtered through a 0·45 µm filter and 1·5 % agar) and incubated at 30 °C for 3 days. The isolate was cultivated aerobically on YP-SW medium (YPS-SW without starch), marine agar 2216 (Difco) or artificial sea water (ASW; 0·1 % CaCl2.2H2O, 0·1 % KCl, 0·5 % MgSO4.7H2O, 0·25 % NaCl) (Lewin & Lounsbery, 1969) with 0·4 % yeast extract and stored at –80 °C in YP-SW supplemented with 20 % glycerol. Colony morphology was examined on YP-SW agar plates that had been incubated at 30 °C for 5 days. Cell morphology under various growth conditions and gliding motility were elucidated by using an Olympus light microscope at 1000x magnification. Cell surface morphology was examined by scanning electron microscopy (Philips XL30) using a conventional method (Robinson et al., 1987). Gram-reaction was assessed according to Collins et al. (1989). The absorption spectrum of pigment extracted with 7 : 3 (v/v) acetone : methanol was determined at 300–700 nm with a Beckman DU650 spectrophotometer. Bathochromatic shift test of flexirubin was performed by addition of 20 % KOH (Fautz & Reichenbach, 1980). Growth was evaluated at various temperatures (4, 16, 20, 25, 30, 33, 37, 40 and 42 °C) for up to 1 month in YP-SW broth. Growth at various pH values (pH 3·0–10·0) was evaluated in YP-SW broth adjusted with HCl or NaOH. Salinity tolerance was determined in YP-SW broth prepared with 0, 5, 15, 20, 40, 75 or 100 % filtered sea water. Salt requirement and tolerance were tested in modified ASW supplemented with 0·4 % yeast extract and with NaCl added at 0, 1, 2, 5, 7·5, 10 or 15 %. Anaerobic growth was examined by using the Oxoid anaerobic system. Acid production from carbohydrates was determined by using API 50CH strips (bioMérieux) and cells grown on a medium composed of 50 % CHB/E medium (bioMérieux) with 0·075 % CaCl2.2H2O, 1·875 % NaCl and 0·375 % MgCl2. Carbohydrate assimilation was determined using API 50CH strips and cells grown on ASW supplemented with 0·05 % yeast extract. Fermentation of (+)-D-glucose, D-galactose, raffinose, sucrose, maltose, (–)-D-mannitol, D-sorbitol, inositol and dextran and hydrolysis of alginate, chitin and Tween 20 were tested according to Baumann & Baumann (1981). Catalase, oxidase, alkaline phosphatase and lecithinase activities, nitrate reduction, indole production, H2S generation from thiosulfate or cysteine, and hydrolysis of cellulose, starch and gelatin were tested according to Smibert & Krieg (1994). 
-Galactosidase activity and casein hydrolysis were examined according to Gosink et al. (1998) and Norris et al. (1985), respectively. Haemolytic activity was investigated using defibrinated rabbit blood (5 %, v/v) agar prepared with blood agar base (BBL). Degradation of dead yeast cells was tested on VY/2 agar prepared with 20 % filtered sea water (Reichenbach, 1989). DNA hydrolysis was examined by culturing cells in YP-SW broth supplemented with 100 µg salmon sperm DNA ml–1 for 36 h and monitoring DNA degradation by agarose-gel electrophoresis. Susceptibility to antibiotics was tested by the disc-diffusion plate method on YP-SW agar. Isoprenoid quinone analysis was performed by the HPLC method (Collins, 1994) using menaquinones extracted from Cytophaga lytica (Nakagawa & Yamasato, 1993) and Sphingobacterium heparinum (Steyn et al., 1998) as the MK-6 and MK-7 references, respectively. The whole-cell fatty acid methyl ester profile was determined by using the MIDI Sherlock Microbial Identification system (Microbial ID) with cells of UST20020801T grown at 10 °C for 5 days in marine agar 2216 (Difco). Genomic DNA was extracted by using the Invisorb spin tissue kit (Invitek) and DNA base composition was determined by the HPLC method (Johnson, 1985), calibrated and calculated according to Mesbah et al. (1989). The 16S rRNA gene was amplified by using primer pair 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1525R (5'-AAGGAGTGWTCCARCC-3') (Lane, 1991) with Taq polymerase (Promega) and the amplicon was purified with the ConceRT-PCR purification kit (Life Technologies), treated with the ABI PRISM BigDye Terminator cycle sequencing ready reaction kit version 3.0 and sequenced using an Applied Biosystems 3100 automated DNA sequencer. The sequences were aligned and assembled before being compared with sequences from GenBank using the programs BLASTN (Altschul et al., 1997) and WU-BLAST 2.0 (http://blast.wustl.edu). The sequences of UST20020801T and related species were then aligned by using CLUSTAL_X (Thompson et al., 1997) and the alignment file was edited with the BIOEDIT sequence alignment editor V5.0.9 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) with gaps and missing nucleotide positions removed. Evolutionary distances of 1139 aligned positions were computed using the Kimura two-parameter model (Kimura, 1980) and the phylogenetic tree was generated by TREECON v1.3b (Van de Peer & De Wachter, 1994) using the neighbour-joining method (Saitou & Nei, 1987) and evaluated by bootstrap analyses (Felsenstein, 1985) based on 300 resamplings. Maximum-parsimony analysis was conducted by using MEGA version 2.1 (Kumar et al., 2001).
Morphological properties of UST20020801T are listed in the genus and species descriptions. A photograph of its colony morphology and phase-contrast and scanning electron micrographs are available as supplementary material in IJSEM Online (Figs a–e). Table 1 lists all the physiological and biochemical properties that were analysed. The more conserved characteristics of this strain were also investigated. The isoprenoid quinone of UST20020801T is MK-6, a major respiratory quinone detected in members of the family Flavobacteriaceae (Bernardet et al., 2002).
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. UST20020801T contained a high percentage of the following branched-chain fatty acids (77·8 % total fatty acids): i15 : 1G, i15 : 1, 3-OH i17 : 0, i17 : 19c and 3-OH i15 : 1. The fatty acid profile of UST20020801T differed significantly from those of other members of the family Cryomorphaceae. The strain possessed several unique fatty acids: i15 : 1G, i17 : 19c, 2-OH 15 : 0, 15 : 16c and three unknown fatty acids with equivalent chain-lengths of 11·543, 13·565 and 16·582. Strain UST20020801T had the highest level of 3-OH i17 : 0 among members of the family Cryomorphaceae. Compared with other members of the family Cryomorphaceae, UST20020801T most resembled Cryomorpha ignava 1-22T in terms of its relatively low content of straight-chain fatty acids and relatively high content of branched-chain hydroxy fatty acids.
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), and 87·5 % similarity to ‘Microscilla aggregans var. catalatica’ strain IFO 15977, which has been reclassified as the type strain of Crocinitomix catalasitica (Bowman et al., 2003). The 16S rRNA gene sequences of relatives of Crocinitomix catalasitica and other members of the phylum ‘Bacteroidetes’ were retrieved from GenBank. Phylogenetic analysis showed that strain UST20020801T and clone ARKIA-105 formed a distinct lineage within the family Cryomorphaceae, linked to Cryomorpha ignava 1-22T with bootstrap support of 85 % by the neighbour-joining method (Fig. 1) and 62 % by maximum-parsimony analysis (data not shown). Sequence similarities to other members of the family Cryomorphaceae were less than 90 % (Cryomorpha ignava 1-22T, 87·2 %; Brumimicrobium glaciale IC156T, 86·1 %), indicating that strain UST20020801T is distantly related to them at the genus level. Phenotypically, strain UST20020801T displayed some traits in common with members of the family Cryomorphaceae: rod- to filamentous-shaped cells; possession of carotenoid pigments; the ability to perform aerobic respiration; the requirement of sea-salts and complex organic compounds such as yeast extract or peptone for growth; no acid production from carbohydrate; and an inability to utilize starch, casein or chitin. The DNA G+C content of UST20020801T was 39·6±0·5 mol%, which is within the range of values for members of the family Cryomorphaceae (35–40 mol%) and close to that of its nearest phylogenetic relative, Cryomorpha ignava (36–37 mol%). Strain UST20020801T differed from other members of the family Cryomorphaceae by possessing oxidase and gelatinase activities and growing well at 37 °C. Strain UST20020801T can be distinguished from related genera of the ‘Bacteroidetes’ by several phenotypic tests (Table 3). On the basis of all characteristics described above, it is proposed that strain UST20020801T should be placed in a new genus as a representative of a novel species, Owenweeksia hongkongensis gen. nov., sp. nov.
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Cells are Gram-negative, halophilic, non-flagellated, motile, flexible, short rods with slightly tapered ends. Cells do not form spores and are strictly aerobic heterotrophs requiring Na+, Mg2+, sea-salts and either yeast extract or peptone for growth. Cells contain oxidase, catalase and alkaline phosphatase. Cellular fatty acids include large amounts of branched-chain fatty acids: i15 : 0G, i15 : 0, 3-OH i17 : 0, 2-OH i15 : 0/16 : 17c, i17 : 19c, 3-OH i15 : 0 and an unknown fatty acid of equivalent chain-length 13·565. The major respiratory quinone is MK-6. Phylogenetically, the genus Owenweeksia is a member of the family Cryomorphaceae in the phylum ‘Bacteroidetes’.
The type species is Owenweeksia hongkongensis.
Description of Owenweeksia hongkongensis sp. nov.
Owenweeksia hongkongensis (hong.kong.en'sis. N.L. fem. adj. hongkongensis pertaining to Hong Kong, S. A. R., P. R. China).
Exhibits the following properties in addition to those given in the genus description. Cells are 0·3–0·5 µm in diameter and 0·5–4·0 µm in length. Filamentous cells detected in cultures supplemented with 0·1 % Tween 20. In YP-SW broth incubated at 4 °C or room temperature for 1 week, cells with one end, both ends or the whole body greatly enlarged are observed. Colonies are orange, smooth, convex, glistening and translucent with an entire margin. Growth occurs at 4–37 °C and pH 5·2–9·0 with optimum growth at 25–33 °C and pH 6·0–8·0. Cells possess carotenoid pigments with maximum absorption at 470 nm and two shoulder peaks around 448 nm and 501 nm. Physiological and biochemical properties are listed in Table 1
. The fatty acid profile is given in Table 2. Sensitive to ampicillin (10 µg), chloramphenicol (30 µg), erythromycin (10 µg), penicillin G (2 U), rifampicin (10 µg), streptomycin (10 µg), tetracycline (30 µg) and polymyxin B (300 U), but resistant to kanamycin (10 µg), gentamicin sulfate (10 µg) and spectinomycin (10 µg).
The type strain is UST20020801T (=NRRL B-23963T=JCM 12287T), which was isolated from a sea-water sample collected in Port Shelter, Hong Kong, S. A. R., P. R. China. DNA G+C content of the type strain is 39·6±0·5 mol%.
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ACKNOWLEDGEMENTS |
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-D-mannopyranoside, methyl 