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Laboratorium voor Microbiologie, Vakgroep Biochemie, Fysiologie en Microbiologie, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
Correspondence
Stefanie Van Trappen
stefanie.vantrappen{at}UGent.be
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ABSTRACT |
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Published online ahead of print on 24 January 2003 as DOI 10.1099/ijs.0.02583-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains LMG 21619 and LMG 21477T are AJ507151 and AJ440996.
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MAIN TEXT |
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).
Several novel species, added to the genus since 1996, were derived from Antarctic habitats, and several new genera containing polar organisms have recently been described within the family Flavobacteriaceae (Gelidibacter, Psychroserpens, Polaribacter, Psychroflexus, Salegentibacter). So far, only one species, Flavobacterium tegetincola, has been isolated from a cyanobacterial mat, collected from the Antarctic saline Ace Lake, located in the Vestfold Hills (McCammon & Bowman, 2000).
During the MICROMAT project (November 1998 to February 2001), 746 bacterial strains were isolated under heterotrophic conditions from microbial mat samples, collected from 10 Antarctic lakes in the Vestfold Hills (lakes Ace, Druzhby, Grace, Highway, Pendant, Organic and Watts), the Larsemann Hills (Lake Reid) and the McMurdo Dry Valleys (lakes Hoare and Fryxell) (Van Trappen et al., 2002). Numerical analysis of their fatty acid composition revealed 41 clusters, and 16S rRNA gene sequence analysis, performed on representative strains, showed that they belong to the 
-, 
- and 
-subclasses of the Proteobacteria, the high- and low-G+C-content Gram-positives and to the Cytophaga–Flavobacterium–Bacteroides branch (Van Trappen et al., 2002). The results of fatty acid analysis and 16S rRNA gene sequence analysis showed that the diversity of heterotrophic bacteria in microbial mats from Antarctic lakes is very high. Moreover, many fatty acid clusters contain multiple taxa, as defined by repetitive extragenic palindromic DNA-PCR (rep-PCR) fingerprinting, a technique used to investigate the genomic diversity of each fatty acid cluster in more detail (Van Trappen et al., 2001).
In the present work, we studied further the taxonomic relationships of 22 strains from fatty acid cluster 10 (as delineated by Van Trappen et al., 2002), related to the genus Flavobacterium, by genomic and phenotypic characterization. Van Trappen et al. (2002) found less than 96 % 16S rRNA gene sequence similarity to the closest relatives within the genus Flavobacterium, indicating that these strains constitute a novel species (Stackebrandt & Goebel, 1994).
The isolates investigated, together with their sources, are listed in Table 1. The strains were routinely cultivated on R2A medium (Difco) at 20 °C for 48 h or, for strain LMG 8328T, on TSA medium (BBL) at 20 °C for 48 h, except where mentioned otherwise.
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, and rep-PCR fingerprinting was performed on all strains of fatty acid analysis cluster 10 of Van Trappen et al. (2002), using the primer GTG5 (Versalovic et al., 1991), as described by Rademaker & de Bruijn (1997) and Rademaker et al. (2000). The 22 strains listed in Table 1
showed similar profiles (data not shown). Numerical analysis (data not shown) was carried out using the BIONUMERICS software package (Applied Maths), as described by the same authors, and these 22 strains could be divided into three clusters according to their profile type, hereafter referred to as rep-PCR profile type I (with 9 strains), rep-PCR profile type II (with 12 strains) and rep-PCR profile type III (containing the single strain LMG 21620) (Table 1). Versalovic et al. (1994) have shown that strains with the same rep-PCR profile are always closely related, and this has been confirmed by several authors (e.g. Rademaker & De Bruijn, 1997).
Five strains (LMG 21477T, LMG 21618, LMG 21619, LMG 21620 and LMG 21621) representing the three rep-PCR profile types and chosen on the basis of their isolation source were used for DNA–DNA hybridizations to investigate their genomic relatedness. DNA–DNA hybridizations were carried out with photobiotin-labelled probes in microplate wells, as described by Ezaki et al. (1989), using a HTS7000 Bio Assay Reader (Perkin Elmer) for the fluorescence measurements. The hybridization temperature was 30 °C and reciprocal experiments were performed for every pair of strains. The DNA–DNA binding values among the five strains were high, ranging from 87 to 97 %, and differences between reciprocal experiments were less than 13 %. These DNA–DNA binding values confirm that the 22 strains belong to a single species (Wayne et al., 1987).
The G+C content of the DNAs from strains LMG 21477T, LMG 21618, LMG 21619, LMG 21620 and LMG 21621 was determined using an HPLC method. DNA was enzymically degraded into nucleosides as described by Mesbah et al. (1989). The nucleoside mixture obtained was then separated by HPLC using a Waters Symmetry Shield C8 column thermostatted at 37 °C. The solvent was 0·02 M NH4H2PO4, pH 4·0, with 1·5 % acetonitrile. Non-methylated 
-phage DNA (Sigma) was used as the calibration reference. The G+C contents of the novel strains were 30·0–30·4 mol%, which is slightly below the range (32–37 mol% G+C) mentioned by Bernardet et al. (1996) for the genus Flavobacterium.
Almost-complete 16S rRNA gene sequences (1467–1468 bp) of strains LMG 21477T (rep-profile type I) and LMG 21619 (rep-profile type II) were obtained as described previously (Mergaert et al., 2001). The most closely related sequences were found using the FASTA program. Phylogenetic analysis was performed using the BIONUMERICS software package, taking into account homologous nucleotide positions after discarding all unknown bases and gaps. A neighbour-joining dendrogram (Saitou & Nei, 1987) with the nearest phylogenetic relatives was constructed on the basis of global alignment of the sequences, using the same software package (Fig. 1). Dendrograms obtained using maximum-parsimony and maximum-likelihood analyses showed essentially the same topography. The 16S rRNA gene sequences of strains LMG 21477T and LMG 21619 differed by only one base, and showed 95·1 % similarity to that of F. flevense, 95·0 % to that of F. tegetincola, less than 95 % to sequences of other Flavobacterium species and less than 90 % to sequences of other genera, indicating that they belong to a novel Flavobacterium species.
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, or were determined as described by the same authors. The 22 novel strains yielded very similar fatty acid profiles. The mean composition was 4 % 14 : 0 iso, 10 % 15 : 0, 1 % 15 : 0 3-OH, 8 % 15 : 0 anteiso, 12 % 15 : 0 iso, 6 % 15 : 0 iso 3-OH, 1 % 15 : 1 anteiso, 10 % 15 : 1 iso, 6 % 15 : 1
6c, 8 % 16 : 0 iso, 10 % 16 : 0 iso 3-OH, 4 % 16 : 1 iso, 6 % 17 : 0 iso 3-OH, 3 % 17 : 16c, 2 % 17 : 1 iso 9c, 1 % 18 : 15c and 2 % 15 : 0 iso 2-OH and/or 16 : 16c. Other fatty acids each accounted for less than 1 %. The fatty acid profiles of the novel strains resemble those determined for other Flavobacterium species (Bernardet et al., 1996), but differ in terms of the relative amounts of 15 : 0 anteiso, 15 : 0 iso, 16 : 0 iso and 16 : 0 iso 3-OH.
The following morphological, physiological and biochemical tests were performed. Colony morphology was determined on R2A medium after 6 days. In addition, growth and adherence of colonies on marine and nutrient agars, TSA and Anacker & Ordal's agar (Anacker & Ordal, 1955) were tested after 14 days growth. Cells were tested for their reaction to the Gram stain and for catalase and oxidase activity. Tests in the commercial systems API ZYM, API 20NE and API 20E (bioMérieux) were performed according to the instructions of the manufacturer. API ZYM tests were read after 4 h incubation at 20 °C; other API tests were read after 48 h at 20 °C. Congo red absorption (Bernardet et al., 2002), production of flexirubin-type pigments (Reichenbach, 1989), the presence of gliding motility, degradation of casein and chitin (Reichenbach & Dworkin, 1981), alginate (West & Colwell, 1984), DNA (using DNA agar from Difco, supplemented with 0·01 % toluidine blue from Merck), pectin (Paton, 1959), starch and L-tyrosine (Barrow & Feltham, 1993), the production of a brown diffusible pigment on L-tyrosine agar and the precipitation of egg-yolk agar (Barrow & Feltham, 1993) were also investigated; reactions were read after 5 days. Hydrolysis of carboxymethylcellulose was tested in Anacker & Ordal's broth gelified with 3 % carboxymethylcellulose sodium salt (high viscosity; Sigma). This medium was stab-inoculated, and liquefaction of the medium within 7 days was scored as a positive reaction. Growth at different temperatures was assessed after 5 days incubation. Salt tolerance was tested on R2A medium supplemented with 1–10 % NaCl after 14 days incubation.
The strains showed morphological characteristics typical of Flavobacterium (Bernardet et al., 2002) and were almost identical in their physiological and biochemical characteristics (see Description). The novel species can be clearly differentiated from other Flavobacterium species by means of several phenotypic characteristics (Table 2).
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Description of Flavobacterium gelidilacus sp. nov.
Flavobacterium gelidilacus (ge.li.di.la'cus. L. adj. gelidus ice-cold; L. n. lacus lake; N.L. gen. n. gelidilacus of the ice-cold lake, referring to the isolation source, microbial mats in Antarctic lakes).
Gram-negative rods, <1x2–4 µm, that exhibit gliding motility on nutrient-poor medium (R2A), except for strains LMG 21477T and LMG 21619, for which no gliding motility is detected. Strains grow at 5–25 °C, with optimal growth at 20 °C; no growth at 30 °C. Yellow to orange, convex, translucent colonies, 1–4 mm in diameter and with entire margins, are formed on R2A plates after 6 days at 20 °C. Colonies on Anacker & Ordal's agar are flat, round with entire margins and 0·5–1 mm in diameter after 14 days incubation. Growth also occurs on TSA, nutrient agar and marine agar, and colonies do not adhere to the agar. Degrades casein and starch. Gelatinase activity is observed, except in the case of strain LMG 21619. Catalase- and oxidase-positive. No growth is observed on glucose, arabinose, mannose, mannitol, N-acetylglucosamine, maltose, gluconate, caprate, adipate, malate, citrate or phenylacetate. Acid is not produced from glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin or arabinose. Agar, alginate, pectin, chitin, aesculin, carboxymethylcellulose, DNA, tyrosine and urea are not degraded. Congo red is not absorbed and no flexirubin-type pigments are present. There is no production of a brown diffusible pigment on L-tyrosine agar and no precipitate is formed on egg-yolk agar. The Voges–Proskauer reaction and tests for indole production, citrate utilization, nitrate reduction and hydrogen sulfide production are negative. None of the strains shows activity for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, lipase (C14), -chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -mannosidase or -fucosidase. Weak enzymic activity is observed for cystine arylamidase, medium activity is found for acid phosphatase, esterase lipase (C8), phosphohydrolase and -glucosidase and strong activity is found for alkaline phosphatase, leucine arylamidase and valine arylamidase. No -glucosidase or N-acetyl--glucosaminidase activity is detected, except for strain LMG 21621. Different reactions are obtained for esterase (C4) and trypsin. The cells contain the fatty acids 15 : 0 iso, 16 : 0 iso 3-OH, 15 : 1 iso, 15 : 0, 15 : 0 anteiso and 16 : 0 iso as the main constituents. Growth occurs in the absence of NaCl and in the presence of 1–5 % NaCl, but not 10 % NaCl, indicating that the strains are not halophilic but merely halotolerant. The G+C content is 30·0–30·4 mol%.
The type strain is LMG 21477T (=DSM 15343T). Twenty-two strains were isolated from microbial mats from freshwater and saline lakes in eastern Antarctica (Table 1).
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ACKNOWLEDGEMENTS |
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