Owenweeksia hongkongensis gen. nov., sp. nov., a novel marine bacterium of the phylum 'Bacteroidetes'

An aerobic, Gram-negative, non-fermentative, rod-shaped, motile, orange-pigmented bacterium, UST20020801^T, was isolated from sea-water samples collected from Port Shelter, Hong Kong, S.A.R., China, in August 2002. The full 16S rRNA gene sequence of this strain shared only 87·5 % similarity with its nearest relative, Crocinitomix catalasitica, a species of the family Cryomorphaceae. However, strain UST20020801^T possessed menaquinone-6, a major respiratory quinone of members of the family Flavobacteriaceae. This strain contains unique fatty acids such as i15 : 1G, i17 : 1ω9c, 2-OH 15 : 0, 15 : 1ω6c 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 UST20020801^T (=NRRL B-23963^T=JCM 12287^T).

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Published online ahead of print on 3 December 2004 as DOI 10.1099/ijs.0.63155-0.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of UST20020801^T is AB125062.

Micrographs of Owenweeksia hongkongensis UST20020801^T and a phylogenetic tree based on 16S rRNA gene sequences showing the relationship between strain UST20020801^T and related taxa are available as supplementary material in IJSEM Online.

Members of the phylum Bacteroidetes are important decomposers of high-molecular-mass organic matter in sea water (Cottrell & Kirchman, 2000). 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 UST20020801^T) 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 UST20020801^T represents a novel species of a new genus, Owenweeksia hongkongensis gen. nov., sp. nov., of the family Cryomorphaceae in the phylum Bacteroidetes.

Strain UST20020801^T 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 300700 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 3·010·0) 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). β-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 ml¹ 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 UST20020801^T 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 (). The sequences of UST20020801^T 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 () 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 UST20020801^T 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 ae). 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 UST20020801^T is MK-6, a major respiratory quinone detected in members of the family Flavobacteriaceae (Bernardet et al., 2002).

Table 1. Phenotypic features of strain UST20020801T This strain cannot form acid from or utilize glucose, dextran, glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol, methyl β-D-xylopyranoside, D-galactose, D-glucose, D-fructose, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl α-D-mannopyranoside, methyl α-D-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, aesculin, ferric citrate, salicin, D-cellobiose, D-maltose, D-lactose, D-melibiose, sucrose, D-trehalose, D-melezitose, D-raffinose, glycogen, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol or potassium gluconate. It is unable to ferment glucose, D-galactose, raffinose, sucrose, maltose, ()-D-mannitol, D-sorbitol, inositol or dextran.

The fatty acid profile of UST20020801^T and those of phylogenetically related members of the family Cryomorphaceae are given in Table 2. UST20020801^T 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 : 1ω9c and 3-OH i15 : 1. The fatty acid profile of UST20020801^T differed significantly from those of other members of the family Cryomorphaceae. The strain possessed several unique fatty acids: i15 : 1G, i17 : 1ω9c, 2-OH 15 : 0, 15 : 1ω6c and three unknown fatty acids with equivalent chain-lengths of 11·543, 13·565 and 16·582. Strain UST20020801^T had the highest level of 3-OH i17 : 0 among members of the family Cryomorphaceae. Compared with other members of the family Cryomorphaceae, UST20020801^T most resembled Cryomorpha ignava 1-22^T in terms of its relatively low content of straight-chain fatty acids and relatively high content of branched-chain hydroxy fatty acids.

Table 2. Whole-cell fatty acid profiles of strain UST20020801T and the members of the family Cryomorphaceae Strains: 1, UST20020801T; 2, Brumimicrobium glaciale IC156T; 3, Cryomorpha ignava 1-22T; 4, Crocinitomix catalasitica NCIMB 1418T. Values given are percentages of total fatty acids. Fatty acids are designated as follows: total number of carbon atoms : number of double bonds, followed by the position of the double bond from the aliphatic end of the molecule. Prefixes i, a and OH represent iso-branched, anteiso-branched and hydroxy fatty acids, respectively. The suffix c represents a cis isomer. ECL, equivalent chain-length; TR, less than 0·1 %. Data for UST20020801T are from this study; data for strains 24 are from Bowman et al. (2003).

The 16S rRNA gene sequence of strain UST20020801^T (1482 nt positions) showed 92·4 % similarity to an uncultured clone, ARKIA-105 (GenBank/EMBL/DDBJ accession no. AF468278), isolated from Arctic pack ice located at Northern Fram Strait (Brinkmeyer et al., 2003), 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 UST20020801^T and clone ARKIA-105 formed a distinct lineage within the family Cryomorphaceae, linked to Cryomorpha ignava 1-22^T 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 1-22^T, 87·2 %; Brumimicrobium glaciale IC156^T, 86·1 %), indicating that strain UST20020801^T is distantly related to them at the genus level. Phenotypically, strain UST20020801^T 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 UST20020801^T was 39·6±0·5 mol%, which is within the range of values for members of the family Cryomorphaceae (3540 mol%) and close to that of its nearest phylogenetic relative, Cryomorpha ignava (3637 mol%). Strain UST20020801^T differed from other members of the family Cryomorphaceae by possessing oxidase and gelatinase activities and growing well at 37 °C. Strain UST20020801^T 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 UST20020801^T should be placed in a new genus as a representative of a novel species, Owenweeksia hongkongensis gen. nov., sp. nov.

(29K): Fig. 1. Phylogenetic relationship of strain UST20020801T and related taxa within the phylum Bacteroidetes based on 16S rRNA gene sequences. The tree was created by using the neighbour-joining method and the numbers at the nodes represent levels of bootstrap support (as percentages) from 300 resampled datasets. Escherichia coli ATCC 11775T (GenBank/EMBL/DDBJ accession no. X80725) was used as the outgroup. Bar, 0·02 nt substitutions per position.

Table 3. Characteristics that differentiate strain UST20020801T from other related members in the phylum Bacteroidetes Strain/taxa:1, Brumimicrobium glaciale; 2, Cryomorpha ignava; 3, Crocinitomix catalasitica; 4, the genus Cellulophaga; 5, [Cytophaga] marinoflava; 6, Croceibacter atlanticus; 7, Tenacibaculum maritimum; 8, [Cytophaga] latercula; 9, Muricauda ruestringensis; 10, Tenacibaculum amylolyticum; 11, UST20020801T; 12, Vitellibacter vladivostokensis; 13, Arenibacter latericius; 14, the genus Zobellia; 15, Tenacibaculum mesophilum. Data are from Bowman et al. (2003), Johansen et al. (1999), Reichenbach (1989), Cho & Giovannoni (2003), Suzuki et al. (2001), Bruns et al. (2001), Nedashkovskaya et al. (2003), Ivanova et al. (2001), Barbeyron et al. (2001) and this study. ND, Not determined; +, positive; , negative; V, variable; W, weak; A, strict aerobe; F, facultative anaerobe, O, orange; R, red; Y, yellow.

Description of Owenweeksia gen. nov.
Owenweeksia (Ow.en.week'si.a. N.L. fem. n. Owenweeksia bacterial genus named after Owen B. Weeks, who did a lot of work in the 1950s, 1960s and 1970s on Flavobacterium, Cytophaga and related species).

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 : 1ω7c, i17 : 1ω9c, 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 0·30·5 µm in diameter and 0·54·0 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 437 °C and pH 5·29·0 with optimum growth at 2533 °C and pH 6·08·0. 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 UST20020801^T (=NRRL B-23963^T=JCM 12287^T), 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%.