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1 Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, SAR, People's Republic of China
2 Department of Biology, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, SAR, People's Republic of China
Correspondence
Ken W. K. Lau
sslwk{at}ust.hk
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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A photograph showing colony morphology and phase-contrast and scanning electron micrographs of strain UST040201-001T are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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established the family Cryomorphaceae in the phylum Bacteroidetes based on a polyphasic taxonomic approach. The Cryomorphaceae encompasses four marine genera, Brumimicrobium, Cryomorpha, Crocinitomix and Owenweeksia, and a recently described freshwater genus, Fluviicola, which form a distinct clade in the class Flavobacteria, branching between the families Flavobacteriaceae and Bacteroidaceae (Bowman et al., 2003; Lau et al., 2005; O'Sullivan et al., 2005). Members of the Cryomorphaceae cannot utilize carbohydrates and require complex organic compounds for growth (Bowman et al., 2003; Lau et al., 2005; O'Sullivan et al., 2005). Molecular phylogenetic studies have found that phylotypes related to the Cryomorphaceae are associated with phytoplankton blooms (Pinhassi et al., 2004; Grossart et al., 2005).
A seawater sample was collected in February 2004 from the outlet of a tank storing sand-filtered seawater that was pumped from 5 m depth 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. Pigmented colonies were selected and then purified by repeatedly restreaking on YPS-SW agar. These isolates were grown in marine broth 2216 (MB; Difco) and stored in MB supplemented with 50 % (v/v) glycerol at –80 °C. The isolates were subjected to partial 16S rRNA gene sequencing and one isolate, UST040201-001T, shared only 90 % sequence similarity with its nearest relative, Crocinitomix catalasitica NCIMB 1418T. We further characterized this novel bacterium and determined its taxonomic position by using the polyphasic approach and propose that strain UST040201-001T represents a novel species of a new genus in the family Cryomorphaceae.
We observed colony morphology on plates of marine agar 2216 (MA; Difco) that had been incubated at 30 °C for 5 days. Cell size and gliding motility were examined using a Zeiss MC100 Spot microscope at x1000 magnification. To study surface morphology, we collected cells from an overnight culture that were then washed in PBS, fixed overnight in 2.5 % glutaraldehyde, dehydrated in ethanol, dried in a critical-point drier and coated with gold before being examined under a scanning electron microscope (JOEL JSM 6300F). The Gram-reaction was assessed according to Collins et al. (1989). Growth under different conditions was monitored by measuring the optical density at 600 nm with a spectrophotometer and the range giving the highest cell yield was taken as the optimum. Growth was evaluated at various temperatures (4, 16, 20, 25, 30, 33, 37, 40 and 42 °C) in artificial seawater (ASW; Lewin & Lounsbery, 1969) containing 0.4 % yeast extract (ASWY). Growth at various pH values (pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10.0) was evaluated in YP-SW medium (Lau et al., 2005) adjusted with 1 M HCl or NaOH. Salinity tolerance was determined in YP-SW prepared with 0, 5, 15, 20, 40, 75 or 100 % filtered seawater. Salt requirement and tolerance were tested in ASWY with NaCl added at 0, 1, 2, 3, 4, 5, 7.5, 10 or 15 % (w/v). 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 carbohydrates was determined using API 50CH strips (bioMérieux), using 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, using ASW supplemented with 0.05 % yeast extract for resuspension of cells, and the strips were incubated at 30 °C for 2 weeks. Tests for fermentation of D(+)-glucose, D(–)-mannitol and sucrose and hydrolysis of alginate, chitin and Tweens 20, 40, 60 and 80 were carried out according to Baumann & Baumann (1981). The activities of catalase, oxidase, lecithinase and nitrate reductase, indole production, H2S generation from cysteine or thiosulfate and hydrolysis of casein, cellulose, starch and gelatin were examined according to Smibert & Krieg (1994). 
-Galactosidase activity was examined according to Gosink et al. (1998). DNA hydrolysis was determined according to Lau et al. (2005). Haemolytic activity was investigated using defibrinated rabbit blood (5 %, v/v) prepared with blood agar base (BBL) using filtered seawater or double-distilled water. Degradation of dead yeast cells was tested on VY/2 agar (Reichenbach, 1989) prepared with 100 % filtered seawater.
The absorption spectrum was determined by extraction of a 48 h YP-SW culture with absolute ethanol and scanning the extract from 300 to 700 nm with a Beckman DU650 spectrophotometer. The bathochromatic shift test for flexirubin was performed by addition of 20 % KOH (Fautz & Reichenbach, 1980). Isoprenoid quinone analysis was performed by the HPLC method (Collins, 1994), using menaquinones extracted from Cytophaga lytica (Nakagawa & Yamasato, 1993) as the MK-6 reference. Fatty acid methyl ester analysis was determined by the MIDI Sherlock Microbial Identification System (Microbial ID) with cells grown on MA in 12 °C for 5 days. Genomic DNA was extracted using the TaKaRa MiniBEST bacterial genomic DNA extraction kit and the DNA base composition was determined by the HPLC method (Mesbah et al., 1989). The 16S rRNA gene was amplified by 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 MEGABLAST search (Zhang et al., 2000). Sequences of strain UST040201-001T and related strains were aligned by using CLUSTAL X (Thompson et al., 1997) and the alignment was edited by using the BioEdit sequence alignment editor version 5.0.9 (Hall, 1999; http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Evolutionary distances were computed using Kimura's 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 maximum-parsimony algorithm and evaluated by bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings.
Cells of strain UST040201-001T were rod-shaped (0.3–0.5x0.5–3.8 µm) and non-flagellated. Enlarged cells and filamentous cells were occasionally seen in stationary phase in broth culture. Colonies were 1.0–2.4 mm in diameter, light orange, circular, convex, smooth, glistening and translucent, with entire margins in MA after 5 days incubation at 30 °C. A photomicrograph showing colony morphology and phase-contrast and scanning electron micrographs are available as Supplementary Fig. S1 in IJSEM Online. Cells were motile by gliding in liquid culture but did not swarm on MA or YP-SW agar. UST040201-001T was mesophilic, growing between 4 and 37 °C with optimum growth at 27–30 °C. Growth occurred between pH 5.0 and 9.0 with optimum growth at around pH 7.0. The strain was halophilic, growing between 1.0 and 7.5 % (w/v) NaCl, with optimum growth at 1–3 %. Cells contained carotenoid pigments with major absorption peaks at 446, 471 and 502 nm. The isoprenoid quinone of UST040201-001T was MK-6, which matches the major respiratory quinone of members of the family Flavobacteriaceae (Bernardet et al., 2002). The DNA G+C content of UST040201-001T was 35.8±0.5 mol%, which is within the range of values for members of the family Cryomorphaceae (35–40 mol%). Table 1 lists the phenotypic features analysed.
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. Strain UST040201-001T contained three major fatty acids: i15 : 0 (44.0 %), i15 : 1 (34.8 %) and 3-OH i17 : 0 (9.3 %), which accounted for 89 % of the total fatty acids. Similar to other members of the family Cryomorphaceae (Bowman et al., 2003; O'Sullivan et al., 2005), UST040201-001T possessed high levels of C13–C17 branched-chain fatty acids (79.7 %). The fatty acid profile of UST040201-001T showed more similarity to that of Owenweeksia hongkongensis UST20020801T, but differed in the content of 14 : 0, i15 : 0, 3-OH i15 : 0 and 3-OH i17 : 0. In summary, the fatty acid profile of strain UST040201-001T differed significantly from those of other members of the family Cryomorphaceae (Table 2).
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). Phylogenetic analysis showed that strain UST20020801T formed a distinct lineage within the family Cryomorphaceae and linked to Brumimicrobium glaciale IC156T with bootstrap support of 91 % by the neighbour-joining algorithm (Fig. 1) and 80 % by the maximum-parsimony analysis (data not shown). Sequence similarities to other members of the family Cryomorphaceae were less than 91 % (Brumimicrobium glaciale IC156T, 90.1 %; Fluviicola taffensis RW262T, 89.6 %; Owenweeksia hongkongensis UST20020801T, 87.3 %; Cryomorpha ignava 1-22T, 86.3 %), indicating that strain UST040201-001T is distantly related to them at the genus level.
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): from Owenweeksia hongkongensis UST20020801T by its caseinase activity and its lower DNA G+C content; from Fluviicola taffensis RW262T by its requirement for Na+ ions, the presence of oxidase, absence of DNase and susceptibility to chloramphenicol; and from Brumimicrobium glaciale IC156T, Crocinitomix catalasitica NCIMB 1418T and Cryomorpha ignava 1-22T by its ability to grow at 35 °C, tolerance of 7.5 % NaCl and presence of oxidase.
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Description of Lishizhenia gen. nov.
Lishizhenia [Li.shi.zhe'ni.a. N.L. fem. n. Lishizhenia after Li Shizhen (1518–1593), the famous Chinese naturalist].
Cells are Gram-negative, halophilic, heterotrophic, non-flagellated and motile flexible short rods, which divide by binary fission. Cells do not form spores, are strictly aerobic and require Na+, sea salts and organic growth factors such as yeast extract or peptone for growth. Cells do not utilize carbohydrates. Cells contain oxidase, catalase and alkaline phosphatase. Major respiratory quinone is MK-6. Predominant fatty acids are i15 : 0, i15 : 1, 3-OH i17 : 0 and 2-OH i15 : 0/16 : 1
7c. Phylogenetically, Lishizhenia is a member of the family Cryomorphaceae in the phylum Bacteroidetes. The type species is Lishizhenia caseinilytica.
Description of Lishizhenia caseinilytica sp. nov.
Lishizhenia caseinilytica (ca.sei.ni.ly'ti.ca. N.L. n. caseinum casein; N.L. fem. adj. lytica dissolving from Gr. v. lyein to dissolve; N.L. fem. adj. caseinilytica casein-dissolving).
Exhibits the following properties in addition to those given in the genus description. Cells are about 0.3–0.5 µm in diameter and 0.5–3.8 µm in length. Enlarged cells and filamentous cells are seen occasionally in stationary phase in broth culture. Colonies are light orange, circular, convex, smooth, glistening and translucent with an entire margin. Cells are motile by gliding but do not swarm on MA. Mesophilic, growing between 4 and 37 °C with optimum growth at 27–30 °C. Growth occurs between pH 5.0 and 9.0 with optimum growth at around pH 7.0. Halophilic, growing between 1.0 and 7.5 % (w/v) NaCl with optimum growth at 1–3 %. Cells possess carotenoid pigments. Other physiological and biochemical properties are listed in Table 1
. The fatty acid profile of the type strain is given in Table 2. Sensitive to ampicillin (10 µg), chloramphenicol (30 µg), penicillin G (2 U), rifampicin (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 DNA G+C content of the type strain is 35.8±0.5 mol%.
The type strain is UST040201-001T (=NRRL B-41434T=JCM 13821T), which was isolated from a sample of sand-filtered seawater collected at 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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TOPABSTRACTMAIN TEXTREFERENCES |
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-D-mannopyranoside, methyl 