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1 Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China
2 Department of Biology, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China
3 Department of Microbiology, Faculty of Medicine, The University of Hong Kong, Queen Mary Hospital Compound, Pokfulam Road, Hong Kong SAR, China
Correspondence
Ken W. K. Lau
sslwk{at}cuhk.edu.hk
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ABSTRACT |
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Photomicrographs of strain UST040201-002T are available as a supplementary figure with the online version of this paper.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Seawater was sampled in February 2004 from the outlet of a tank storing sand-filtered seawater that was pumped from a depth of 5 m adjacent to the Coastal Marine Laboratory of Hong Kong University of Science and Technology. Aliquots of 100 µl were spread onto YPS-SW agar (Lau et al., 2005
) and incubated at 30 °C for 3 days. A pale yellow colony was selected and purified by repeated restreaking on YPS-SW agar. It was cultivated in marine broth 2216 (MB) and stored in MB supplemented with 50 % (v/v) glycerol at –80 °C.
Colony morphology was observed on YP-SW (YPS-SW without starch) agar plates that had been incubated at 30 °C for 3 days. Cell morphology was examined using a Zeiss MC100 Spot microscope at 1000xmagnification. Cell motility was determined in motility agar [0.5 % marine agar (MA) with 0.005 % 2,3,5-triphenyltetrazolium chloride]. Gram-reaction was assessed according to Collins et al. (1989). Growth was evaluated at various temperatures (4, 16, 20, 25, 30, 33, 37, 40 and 42 °C) in YP-SW medium (Lau et al., 2005). Growth at various pH values (3.0–9.0 in single unit steps) was evaluated in artificial seawater (ASW; Lewin & Lounsbery, 1969) containing 0.1 % yeast extract and buffered with 10 mM sodium acetate (pH 3.0–4.0), 10 mM MES (pH 5.0–6.0) or 10 mM Tris/HCl (pH 7.0–9.0). pH values of the media were measured before and after autoclaving and only slight changes of 0.0–0.2 units were observed. Salinity requirements were determined in YP-SW prepared with 0, 5, 15, 20, 40, 75 or 100 % filtered seawater. Salt tolerance was tested in ASW containing 0.4 % yeast extract plus 0, 1, 2, 3, 4, 5, 7.5, 10 or 15 % (w/v) NaCl. NaCl and KCl requirements were tested in a medium containing 0.1 % yeast extract supplemented with NaCl or KCl at 0, 0.2, 0.4, 0.6, 0.8 or 1.0 %. The requirement for magnesium ions was tested in ASW with 0.2 % yeast extract, with magnesium sulfate replaced by equimolar levels of potassium sulfate. Anaerobic growth was examined in the Oxoid anaerobic system on YP-SW agar supplemented with 0.1 % NaNO3, 0.1 % glucose or 0.1 % meat extract. Acid production from glucose, sucrose, mannose, maltose and lactose was determined in Leifson's modified O/F medium with 0.1 % cysteine-HCl (Smibert & Krieg, 1994). Carbohydrate assimilation was determined with API 50 CH strips using ASW supplemented with 0.05 % yeast extract for 2 weeks at 30 °C. Fermentation of (+)-D-glucose, (–)-D-mannitol and sucrose, and hydrolysis of chitin and Tweens 20, 40, 60 and 80 were carried out according to Baumann & Baumann (1981). Catalase, oxidase, lecithinase (observed after 2 weeks) and nitrate reductase activities, indole production, H2S generation from cysteine or thiosulfate, and hydrolysis of casein, cellulose, starch and gelatin were performed according to Smibert & Krieg (1994). DNA hydrolysis was performed according to Lau et al. (2005). Haemolytic activity was investigated using defibrinated rabbit blood (5 %, v/v) prepared with blood agar base (BBL) dissolved in filtered seawater. Degradation of dead yeast cells was tested on VY/2 agar (Reichenbach, 1989) prepared with 100 % filtered seawater. Other biochemical characteristics were determined with the API ZYM, API 20E, VITEK ANI and VITEK NHI systems (bioMérieux). Isoprenoid quinone analysis was performed by the HPLC method (Collins, 1994) using ubiquinone-8 extracted from Escherichia coli (strain XL1 Blue) as a reference. Fatty acid methyl ester analysis was determined by the MIDI Sherlock Microbial Identification System (Microbial ID) with cells grown on heart-infusion blood agar supplemented with 3 % NaCl at 35 °C for 24 h. Genomic DNA was extracted using a TaKaRa MiniBEST Bacterial Genomic DNA Extraction kit and DNA base composition was determined by using the HPLC method (Mesbah et al., 1989). The 16S rRNA gene was amplified using the primer pair 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1525R (5'-AAGGAGTGWTCCARCC-3') (Lane, 1991) with Vent DNA polymerase (NEB) and sequenced using an Applied Biosystems 3100 automated DNA sequencer. Related 16S rRNA gene sequences were retrieved from the NCBI nucleotide database after searching with BLAST (Altschul et al., 1997). The sequences of strain UST040201-002T and related species were aligned with CLUSTAL_X (Thompson et al., 1997) and edited with the BioEdit sequence alignment editor V5.0.9 (Hall, 1999; www.mbio.ncsu.edu/BioEdit/bioedit.html). 16S rRNA gene sequence similarity values were calculated with 1255 aligned nucleotides, after removal of columns containing gaps or ambiguous nucleotides [positions 29–1419; Escherichia coli numbering (Brosius et al., 1978)], using BioEdit. Evolutionary distances were computed using the Kimura two-parameter model (Kimura, 1980) and phylogenetic trees were generated by MEGA version 2.1 (Kumar et al., 2001) using the neighbour-joining method (Saitou & Nei, 1987) or the minimum-evolution algorithm and evaluated by bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings.
At 30 °C, strain UST040201-002T grew on MA, YP-SW agar, heart-infusion agar with 5 % rabbit blood (HIAB; prepared with or without filtered seawater) and medium with 2.5 % KCl or NaCl plus 0.1 % yeast extract, but not on nutrient agar. At 25 °C, the bacterium grew on horse blood agar and chocolate agar, but not MacConkey agar. It did not grow on blood agar, chocolate agar or MacConkey agar, with or without CO2, at 37 °C. Colonies were 0.5–2.0 mm in diameter, pale yellow, circular, convex, smooth, glistening, translucent and mucoid with entire margins on YP-SW after 3 days incubation at 30 °C. On horse blood agar or chocolate agar, colonies appeared grey in colour. On HIAB agar (prepared in seawater), 
-haemolysis was observed after 4 days incubation at 30 °C. In MB overnight cultures, cells of strain UST040201-002T were short rods or coccobacilli (0.35–0.70x0.70–1.50 µm), occurring singly or in pairs. In the stationary phase, spherical cells (2.5–5.7 µm) were occasionally seen. Cells were non-motile. No flagella were observed when cells were stained with 1 % phosphotungstic acid and examined by TEM. Colony morphology and phase-contrast and SEM micrographs are available as Supplementary Fig. S1a–c in IJSEM Online. Strain UST040201-002T was mesophilic and grew at 16–40 °C with optimal growth at 30–33 °C. It grew at pH 5.0–8.8, with optimal growth around pH 4.9–6.8. The strain required either Na+ or K+, but not Mg2+, for growth. It grew in 0.4–7.5 % (w/v) NaCl, with optimal growth between 2 and 3 % (w/v) NaCl. The minimum amount of KCl that supported growth was about 0.6 %. The isoprenoid quinone of strain UST040201-002T was ubiquinone-8. The DNA G+C content of strain UST040201-002T was 53.9±0.4 mol%. Table 1 lists the phenotypic characteristics of strain UST040201-002T that were analysed.
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). Strain UST040201-002T contained a-17 : 0, a-15 : 0, 18 : 1
9c and 14 : 0 3-OH/16 : 1 ISO I as major fatty acids, whereas members of the genus Francisella contained 10 : 0, 14 : 0, 16 : 0, 18:0, 18 : 0 3-OH and 18 : 19c as major fatty acids.
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) and 89–90 % similarity with Francisella tularensis subsp. tularensis (Olsufiev et al., 1959; Larsson et al., 2005), Francisella tularensis subsp. mediasiatica and Francisella tularensis subsp. holarctica (Olsufjev & Mescheryakova, 1983), and Francisella novicida and Francisella philomiragia (Hollis et al., 1989). The 16S rRNA gene sequence of strain UST040201-002T shared similarities of only 84–85 % and 83–84 % with members of the families Piscirickettsiaceae and Thiotrichaceae, respectively.
Phylogenetic analysis using the neighbour-joining algorithm showed that strain UST040201-002T formed a distinct lineage within the order Thiotrichales and clustered with C. taeniospiralis with a 100 % bootstrap value; this clade was coherently linked to the lineage of Francisellaceae at 100 % bootstrap value (Fig. 1). This tight coherent clustering of strain UST040201-002T with C. taeniospiralis and members of the family Francisellaceae was confirmed in phylogenetic trees generated from minimum-evolution or maximum-parsimony algorithms (data not shown) and agreed with the findings of Beier et al. (2002) that C. taeniospiralis is closely affiliated with members of the family Francisellaceae.
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-lactamase-positive, urease-negative, and susceptible to tetracycline and chloramphenicol. However, strain UST040201-002T differed from current members of the genus Francisella by its higher G+C content and its inability to produce H2S from cysteine (Table 3). In particular, strain UST040201-002T could be distinguished from F. tularensis/F. novicida by its production of oxidase and gelatinase and from F. philomiragia by its inability to produce indole. On the basis of all characteristics described above, it is proposed that strain UST040201-002T be placed in a new genus in the order Thiotrichales as a representative of a novel species, Fangia hongkongensis gen. nov., sp. nov.
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Gram-negative, short rods to coccobacilli, occurring singly or in pairs, non-motile. Non-sporulating and non-flagellated. Divides by binary fission. Strictly aerobic, chemoheterotrophic, requiring sodium or potassium ions and organic growth factors such as yeast extract for growth. Produces acid from glucose. Catalase-positive; positive or weakly positive for oxidase. Major respiratory quinone is Q-8. Predominant fatty acids are a-17 : 0, a-15 : 0 and 18 : 19c. Phylogenetically, Fangia is a member of the order Thiotrichales. The type species is Fangia hongkongensis.
Description of Fangia hongkongensis sp. nov.
Fangia hongkongensis (hong.kong.en'sis. N.L. fem. adj. hongkongensis pertaining to Hong Kong SAR, PR China, where the bacterium was first isolated).
In addition to the characteristics given in the genus description, exhibits the following properties. In MB, cells are short rods about 0.35–0.70 µm in diameter and 0.7–1.5 µm in length. In half-strength MB, spherical and pleomorphic cells of 0.6–4.8x0.9–4.8 µm are seen. Colonies are circular, pale yellow, translucent, smooth, shiny, convex and mucoid with entire margins. Colonies are about 0.5–2.0 mm in diameter after 3 days culture on YP-SW agar at 30 °C. Cells are mesophilic. Grows at 16–40 °C and pH 4.9–8.8, with optimal growth at 30–33 °C and around pH 4.9–6.8. Requires sodium or potassium ions for growth. Grows in 0.4–7.5 % (w/v) NaCl, with optimal growth at 2–3 % (w/v) NaCl. Physiological and biochemical properties are listed in Table 1
. Fatty acid profile is given in Table 2. Resistant to ampicillin (10 µg), polymyxin B (300 U) and penicillin G (2 U) and sensitive to chloramphenicol (3 µg), tetracycline (30 µg), streptomycin (10 µg), gentamicin sulfate (10 µg) and kanamycin (20 µg).
The type strain is UST040201-002T (=JCM 14605T=NRRL B-41860T), which was isolated from a seawater sample collected from sand-filtered seawater, in the Port Shelter adjacent to the Coastal Marine Laboratory, Hong Kong University of Science and Technology.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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-glutamyl arylamidase, acid phosphatase, naphthol-AS-Bl-phosphorylase, 