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1 Marine Microbiology, Department of Biology and Environmental Sciences, University of Kalmar, SE-39182 Kalmar, Sweden
2 School of Agricultural Science, University of Tasmania, Private Bag 54, Hobart, Tasmania, 7001, Australia
3 Pacific Institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Academy of Sciences, Pr. 100 Let Vladivostoku 159, 690022, Vladivostok, Russia
4 Institut de Ciències del Mar-CMIMA (CSIC), Passeig Marítim de la Barceloneta 37-49, E-08003 Barcelona, Catalunya, Spain
Correspondence
Jarone Pinhassi
jarone.pinhassi{at}hik.se
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strain MED 217T are DQ294290 and DQ294291 for two copies of the gene. The GenBank/EMBL/DDBJ accession number for the genome sequence is AANC00000000.
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; Kirchman, 2002), frequently accounting for approximately one-third of the bacteria in water of the world's oceans and seas. The Flavobacteriaceae is one of the main families in the Bacteroidetes, and members of the family effectively account for a large proportion of the members of the Bacteroidetes abundant in the marine environment (Abell & Bowman, 2005; Kirchman et al., 2003).
Recent studies have suggested that bacteria belonging to the phylum Bacteroidetes play particularly important roles in the degradation of organic matter during and following algal blooms (Pinhassi et al., 1999; Riemann et al., 2000; Suzuki et al., 2001). In seawater mesocosms in which the decay of phytoplankton was simulated by the addition of protein, Pinhassi et al. (1999) found a rapid growth response by a limited number of species in the phylum Bacteroidetes. Subsequent work measuring the uptake of radioactively labelled protein corroborated that bacteria of this phylum are well adapted to consuming and degrading protein (Cottrell & Kirchman, 2000).
In a recent experimental study of the identity of bacteria associated with different phytoplankton communities, Pinhassi et al. (2004) showed that flavobacteria are particularly responsive to phytoplankton blooms. In order to investigate whether the response of flavobacteria was a general phenomenon, their phylogenetic analysis included 16S rRNA gene sequences of representatives of the phylum Bacteroidetes from all published studies on the diversity of marine bacterioplankton associated with natural or experimental algal blooms (see Pinhassi et al., 2004, and references therein). These studies encompassed blooms of diatoms, dinoflagellates as well as cyanobacteria. The analysis revealed that, even though the phylum Bacteroidetes is highly diverse, as many as 80 % of the Bacteroidetes sequences obtained (among a total of 63 sequences) belonged to one single family, the Flavobacteriaceae. Taken together, current data indicate that flavobacteria represent bacterial populations that are likely to mediate a substantial proportion of the carbon flow and nutrient turnover in the sea during and following algal blooms. An increased understanding of the diversity of the family Flavobacteriaceae is therefore important.
In the present study, we have characterized a novel flavobacterium, designated strain MED 217T, isolated from a seawater sample. On the basis of a polyphasic approach (i.e. phylogenetic, phenotypic and genotypic analyses), we describe a novel member of the genus Leeuwenhoekiella.
Strain MED 217T was isolated from a surface seawater sample from the Bay of Blanes, in the north-western Mediterranean Sea (41° 40' N 2° 48' E) on the coast of Spain, collected on 23 May 2001. The sample was enriched with 0.6 µM Na2HPO4 (final concentration), and was incubated for 48 h at 16 °C in the dark. For strain isolation, 0.1 ml of a 100x dilution of sample water was spread onto ZoBell agar plates prepared from seawater from the Bay of Blanes (indicating an abundance of approximately 1x103 c.f.u. ml–1). Strain MED 217T was one of the bacteria that exhibited an active growth response to phosphorus enrichment in this Mediterranean seawater, where typically bacterial growth is limited by the availability of phosphorus. After primary isolation and purification, strain MED 217T was cultivated at 23 °C on the same medium and stored at –80 °C in ZoBell's medium with 25 % (v/v) glycerol. For subsequent culturing of MED 217T, marine broth/agar 2216 (Difco) was used, unless stated otherwise.
The presence of flexirubin pigments in strain MED 217T was determined by the method of Fautz & Reichenbach (1980). Determination of growth at different temperatures, requirement for NaCl for growth, production of acid from carbohydrates, nitrate reduction, production of hydrogen sulfide, indole and acetoin (Voges–Proskauer reaction), hydrolysis of casein, gelatin, starch, Tweens 20, 40 and 80, agar (1.5 %, w/v), DNA, urea and cellulose [carboxymethylcellulose (CM-cellulose) and filter paper] and oxidase, catalase, 
-galactosidase and alkaline phosphatase activities were carried out according to standard procedures in bacteriology (Gerhardt, 1994). Utilization of carbon sources was determined as previously described (Nedashkovskaya et al., 2003b). Hydrolysis of chitin (1 %, w/v) was determined by the appearance of clear zones around colonies on chitin agar. Gliding motility was evaluated as described by Bowman (2000). Susceptibility to antibiotics was examined by the disc-diffusion plate method, following the method of Nedashkovskaya et al. (2003a); in addition, discs containing chloramphenicol (30 µg), doxycycline (10 µg) and erythromycin (15 µg) were used. The API ZYM (bioMérieux) gallery was used for testing of enzyme activities of the strain according to the manufacturer's instructions. For the study of cell morphology, cells were grown at 21 °C in marine broth until early exponential phase (24 h incubation), when cells were fixed with glutaraldehyde and filtered onto polycarbonate filters of 0.2 µm pore size (Nuclepore). Samples were treated by sequential ethanol dehydration steps and critical-point drying with CO2 and silver coating and viewed with a Hitachi S-3500N scanning electron microscope.
For analysis of the fatty acid composition, isolate MED 217T, Leeuwenhoekiella aequorea LMG 22550T and Leeuwenhoekiella marinoflava LMG 1345T were grown on marine agar at 20 °C for 5 days until dense growth had developed. Cells were harvested and fatty acid composition was determined by quantitative GC and GC-MS procedures as described by Nichols et al. (1986, 1993).
Genomic DNA was extracted using the Marmur technique (Marmur & Doty, 1962). The DNA G+C content was determined by the thermal denaturation procedure (Sly et al., 1986), using Cellulophaga lytica ATCC 23178T (DNA G+C content 33.0 mol%) and Escherichia coli K-12 ATCC 10798 (51.0 mol%) as reference strains. DNA–DNA hybridization was carried out by the spectrophotometric renaturation kinetics approach (Huß et al., 1983), as later modified (Bowman et al., 1998).
Whole-genome sequencing was carried out by the J. Craig Venter Institute through the Gordon and Betty Moore foundation initiative in Marine Microbiology (https://research.venterinstitute.org/moore/). The complete 16S rRNA gene sequences of MED 217T were 1515 and 1527 nt in length for two copies of the gene. A phylogenetic tree was constructed with reference sequences of members of the family Flavobacteriaceae as described by Bowman & McCuaig (2003), using Jukes–Cantor distances with the PHYLIP package (version 3.5) (Felsenstein, 1989) and the sequence of Flexibacter flexilis ATCC 23079T as the outgroup.
Strain MED 217T was a Gram-negative and chemo-organotrophic bacterium with respiratory-type metabolism and cells were motile by means of gliding. Cells appeared as single rods 0.4–0.7 µm in diameter and 1.5–4 µm in length; short chains were also observed. Cells of strain MED 217T divided by binary fission, although some showed polar constrictions resembling coccoid forms (Fig. 1), suggesting that cells may also form through budding fission, as previously reported for Formosa agariphila KMM 3901T (Nedashkovskaya et al., 2006). The cell surface structures seen in Fig. 1 are most likely remnants of exopolysaccharides and/or precipitated salts. Other physiological and biochemical characteristics are given in the species description below and in Tables 1 and 2. Strain MED 217T is similar to recognized species in the genus Leeuwenhoekiella with respect to several phenotypic characteristics (Nedashkovskaya et al., 2005). Nevertheless, specific features, including maximum temperature for growth, oxidation of D-galactose, D-glucose and DL-xylose, utilization of sucrose and mannitol and susceptibility to benzylpenicillin and tetracycline, distinguish MED 217T from its closest relatives (Table 1). The notably higher temperature for optimum growth of strain MED 217T may reflect that it was isolated from surface waters of the north-western Mediterranean Sea, which frequently reach high temperatures (25 °C or more during the summer), whereas L. aequorea and L. marinoflava strains were recovered from the colder Antarctic and north-western Pacific Ocean marine environments, and from the North Sea, respectively (Colwell et al., 1966; Tan et al., 1999). In addition, the salinity optimum of MED 217T matches the range of salinities found at the site of its isolation in the Mediterranean Sea (36–39 
).
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). The G+C content of the DNA of MED 217T was 42.5 mol%, as determined by the thermal denaturation method, a value higher than those of L. aequorea or L. marinoflava (Table 1). Whole-genome sequencing resulted in a G+C content of the DNA of 40 mol%. The former value was chosen for the species description, as comparative whole-genome sequence data on G+C content are still lacking for the majority of taxonomically described bacteria.
Genome sequencing showed that strain MED 217T has an annotated genome size of approximately 4.24 Mbp (3735 putative ORFs), which is relatively large compared with the genomes of other members of the Flavobacteriaceae that have been sequenced. These range in size from 2.74 Mbp (2557 putative ORFs) for Polaribacter irgensii to 3.87 Mbp (3498 putative ORFs) for the uncharacterized strain HTCC2170 (https://research.venterinstitute.org/moore/). The genome size of L. marinoflava was previously determined to be 2.26x109 Da (Callies & Mannheim, 1980), which corresponds to approximately 4.20 Mbp. Thus, it seems that Leeuwenhoekiella species have a larger genome size than some other members of the Flavobacteriaceae.
Strain MED 217T has two copies of the 16S rRNA gene that share a sequence similarity of 98.3 % over the first 555 bp, after which the copies are identical (resulting in an overall sequence similarity of 99.5 %). Phylogenetic analysis of 16S rRNA gene sequence data showed that strain MED 217T clustered with bacteria belonging to the genus Leeuwenhoekiella (Fig. 2). Similarities of the two 16S rRNA gene sequences from strain MED 217T to the sequence of L. aequorea LMG 22550T were 96.1 and 96.7 % and similarities to the sequence of L. marinoflava LMG 1345T were 95.7 and 96.7 %. Sequence similarities to members of other genera of the Flavobacteriaceae, such as Pibocella and Cellulophaga, were consistently below 92 %. The level of DNA–DNA hybridization between strain MED 217T and L. marinoflava LMG 1345T averaged 21 % (±11 %, n=5).
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Description of Leeuwenhoekiella blandensis sp. nov.
Leeuwenhoekiella blandensis (blan.den'sis. L. fem. adj. blandensis pertaining to Blande or Blanda, the name the Romans used for the city of Blanes, which has given its name to the Bay of Blanes, where the type strain was isolated).
Characteristics are as given for the genus by Nedashkovskaya et al. (2005). In addition, cells range from 0.4 to 0.7 µm in diameter and from 1.5 to 4 µm in length. On marine agar colonies are round, 2–3 mm in diameter and yellow-pigmented with a shiny surface. Growth is observed at 10–41 °C. Optimal temperature for growth is 28–30 °C. Growth occurs at 0–17 % NaCl, with optimal growth at 2–4 % NaCl. -Galactosidase-positive. Nitrate is not reduced. Indole, H2S and acetoin (Voges–Proskauer reaction) production are negative. Flexirubin-type pigments are not detected. Decomposes casein, gelatin, starch and Tweens 20, 40 and 80. Does not hydrolyse agar, DNA, urea, cellulose (CM-cellulose and filter paper) or chitin. Forms acid from D-glucose, DL-xylose and glycerol, but not from L-arabinose, D-cellobiose, L-fucose, D-galactose, D-lactose, D-maltose, D-melibiose, L-raffinose, L-rhamnose, L-sorbose, sucrose, N-acetylglucosamine, adonitol, dulcitol, inositol or mannitol. Utilizes L-arabinose, D-lactose, D-mannose and sucrose, but not inositol, mannitol, sorbitol, malonate or citrate. According to the API ZYM gallery (bioMérieux), it produces 
- and -galactosidases, acid and alkaline phosphatases, esterase lipase (C8), leucine and valine arylamidases, trypsin, - and -glucosidases, N-acetyl--glucosaminidase, naphthol-AS-BI-phosphohydrolase, esterase (C4), lipase (C14), cystine arylamidase, -chymotrypsin, -glucuronidase and -mannosidase, but not naphthol-AS-BI-glucuronidase or -fucosidase. Susceptible to lincomycin, doxycycline, erythromycin, chloramphenicol and tetracycline; resistant to benzylpenicillin, gentamicin, kanamycin, neomycin and polymyxin B. The G+C content of the DNA is 42.5 mol%, a value that expands the range of G+C content of members of the genus.
The type strain, MED 217T (=CECT 7118T=CCUG 51940T), was isolated from a surface seawater sample enriched with inorganic phosphate, from the Bay of Blanes in the north-western Mediterranean Sea on the coast of Spain.
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ACKNOWLEDGEMENTS |
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