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School of Earth and Environmental Sciences, Seoul National University, Shillim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea
Correspondence
Byung C. Cho
bccho{at}snu.ac.kr
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ABSTRACT |
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5c (mean±SD, 26.9±10.8 %), iso-C15 : 0 2-OH and/or C16 : 17c (19.2±2.3 %) and iso-C15 : 0 (12.1±1.3 %). The DNA G+C content was found to be 38.3 mol%. Phenotypic, chemotaxonomic and phylogenetic analyses indicated that strain CL-GP79T could be assigned to the genus Flectobacillus, but could be distinguished from F. major. The strain CL-GP79T therefore represents a novel species, for which the name Flectobacillus lacus sp. nov. is proposed, with CL-GP79T (=KCCM 42271T=JCM 13398T) as the type strain.
Selected phenotypic characteristics of strain CL-GP79T and Flectobacillus major DSM 103T are available in a supplementary table in IJSEM Online.
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MAIN TEXT |
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. Later, two species ([Flectobacillus] marinus and [Flectobacillus] glomeratus) assigned to the genus Flectobacillus (Borrall & Larkin, 1978; McGuire et al., 1987) were reclassified as Cyclobacterium marinum (Raj & Maloy, 1990) and Polaribacter glomeratus (Gosink et al., 1998), respectively. At present, Flectobacillus major is the only accepted Flectobacillus species with a validly published name. The species was first described as ‘Microcyclus major’ by Gromov (1963) and is the type species of the genus. In this study, we isolated a strain from a highly eutrophic pond and found that the 16S rRNA gene sequence of the isolate was related to those of species of Flectobacillus. Here we describe its phenotypic, physiological and chemotaxonomic features. In November 2003, a surface freshwater sample was collected, using a sterile tube, from a highly eutrophic pond, Gongdae Pond, located in the campus of Seoul National University. The sample, at ambient temperature, was brought back to the laboratory within 15 min; 10–100 µl sample was spread on plates containing R2A agar [0.05 % (w/v) yeast extract, 0.05 % (w/v) peptone, 0.05 % (w/v) Casamino acids, 0.05 % (w/v) glucose, 0.05 % (w/v) starch, 0.03 % (w/v) sodium pyruvate, 0.03 % (w/v) K2HPO4, 0.005 % (w/v) MgSO4 and 1.5 % (w/v) agar, pH 7; Difco] and incubated at 20 °C for 1 week. Thirteen colonies were randomly selected and subsequently purified four times on R2A agar at 20 °C. The isolates were initially characterized on the basis of their 16S rRNA gene sequences. Two of the isolates showed relatively low levels of sequence similarity (94–96 %) to previously described species from different genera: one of these is strain CL-GP79T, colonies of which are pink on R2A agar. The strain was stored at –80 °C in R2A broth (Difco) supplemented with 30 % (v/v) glycerol until required for further analyses.
The temperature range for growth was tested on the basis of colony formation on R2A plates incubated at temperatures ranging from 5 to 45 °C, using increments of 5 °C. The pH range (pH 5–10, using increments of 1 pH unit) for growth was determined by assessing changes in OD600 over the incubation period (up to 7 days) in R2A broth. The final pH was adjusted using NaOH and HCl solutions. To test the salt tolerance, R2A agar (Difco) and nutrient agar [0.3 % (w/v) beef extract, 0.5 % (w/v) peptone, 1.5 % (w/v) agar, pH 7; Difco] containing various concentrations of NaCl (0, 0.1, 0.5, 1, 1.5, 2, 3, 5, 7 %, w/v) were used. Growth of the isolate occurred at temperatures between 10 and 35 °C, with optimal growth around 25–30 °C. The isolate did not grow on either R2A agar or nutrient agar supplemented with 0.5–7 % (w/v) NaCl. The isolate grew at pH values from 6 to 9; optimal growth occurred at pH 7.
Morphological and physiological tests were also performed. Gram-staining was performed as described by Smibert & Krieg (1994). The cellular morphology and the motility of the isolate were observed by phase-contrast microscopy. Transmission electron microscopy (EX2; JEOL) was used to establish whether flagella were present. Anaerobic growth was checked on R2A agar using the GasPak anaerobic system (BBL). The pigment absorption spectrum between 200 and 800 nm was determined by using an Ultraspec 2000 spectrophotometer (Pharmacia Biotech) after ethanol extraction (Gosink et al., 1998). The sample was further alkalized with 0.1 vol. 0.1 M NaOH, and the absorption spectrum was obtained again to check for the presence of flexirubin pigments (Gosink et al., 1998). The cells were Gram-negative, aerobic, straight to slightly curved, non-motile rods with no flagella (Fig. 1). Other morphological characteristics are shown in Table 1. Pigment extracts of strain CL-GP79T showed an absorption maximum at 477–481 nm and a spectrum similar to that of F. major DSM 103T (i.e. the maximum absorption peak at 480–482 nm determined in this study; data not shown). Flexirubin pigments were not detected in either strain.
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; gelatinase, amylase and nitrate reductase activities and the Voges–Proskauer test were investigated as described by Hansen & Sørheim (1991). The utilization of organic compounds as sole carbon sources was tested on the basis of colony formation on a minimal medium (Nikitin et al., 2004) to which NaCl and CaCl2 had not been added. Growth was scored as negative when it was equal to, or less than, that in the negative control (i.e. lacking a carbon source) after incubation at 25 °C for 1 month. Additional biochemical tests were performed by using API 20NE, API ZYM and API 50 CH strips (bioMérieux) according to the manufacturer's instructions. Sensitivities to antibiotics were assessed using disc-diffusion methodology (Bauer et al., 1966). All phenotypic tests for strain CL-GP79T were performed simultaneously with F. major DSM 103T as a reference strain. Strain CL-GP79T was positive for catalase, oxidase, gelatinase and amylase, but negative in the Voges–Proskauer test. In the API tests, strain CL-GP79T showed a physiological profile that was very similar to that of F. major DSM 103T, i.e. they gave the same results for 25 out of 27 tests of enzyme activities, 39 out of 49 tests for acid production, and 8 out of 12 tests for carbon assimilation (Table 1
; see also Supplementary Table S1 available in IJSEM Online). Features that differentiated strain CL-GP79T from F. major DSM 103T were as follows: acid was produced from D-arabitol and 5-ketogluconate, but not from DL-arabinose, D-xylose, L-rhamnose, amygdalin, arbutin, D-lyxose or L-fucose. Strain CL-GP79T assimilated adipate, but did not assimilate L-arabinose, D-mannose, N-acetylglucosamine, D-maltose or gluconate. Strain CL-GP79T was not capable of utilizing DL-cysteine or succinate as a sole carbon source. The other results of the biochemical and physiological tests are given in Table 1, Supplementary Table S1 and the species description.
The DNA G+C content of strain CL-GP79T, determined by HPLC analysis (Mesbah et al., 1989), was 38.3 mol%. This is similar to the values obtained for F. major (39.5–40.3 mol%; Larkin et al., 1977). The whole-cell fatty acid methyl esters in strains CL-GP79T and F. major DSM 103T, grown under the same conditions (on R2A plates at 25 °C for 2 days), were analysed by gas chromatography according to the instructions of the Microbial Identification System (MIDI) at the Korean Culture Center of Microorganisms (Seoul, Korea). The fatty acid composition of strain CL-GP79T was similar to that of F. major DSM 103T (Table 2). The major fatty acids of strain CL-GP79T were found to be C16 : 15c (mean±SD, 26.9±10.8 %), iso-C15 : 0 2-OH and/or C16 : 17c (19.2±2.3 %) and iso-C15 : 0 (12.1±1.3 %). However, a significant amount (14.7±0.1 %) of an unknown fatty acid with a carbon chain length equivalent to 15.731 was found in F. major DSM 103T but not in strain CL-GP79T (Table 2). These results support the affiliation of strain CL-GP79T to the genus Flectobacillus. It is noteworthy that the fatty acid profile of F. major DSM 103T obtained in this study is quite distinct from that of F. major reported by Urakami & Komagata (1986), possibly because of differences in the cultivation conditions and techniques used.
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). The PCR product was purified by using the AccuPrep PCR purification kit (Bioneer) and direct sequence determination of the purified 16S rRNA gene was performed with an Applied Biosystems automatic sequencer (ABI3730XL) at Macrogen Corp. (Seoul, Korea). The 16S rRNA gene sequence of strain CL-GP79T was compared with 16S rRNA gene sequences available in GenBank, using a BLASTN (Altschul et al., 1990) search. The sequence of strain CL-GP79T was aligned manually with representative sequences of the family Flexibacteraceae obtained from the GenBank and Ribosomal Database Project (Cole et al., 2003) databases, using known 16S rRNA secondary structure information. Phylogenetic trees were obtained by using the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) methods. An evolutionary distance matrix for the neighbour-joining method was generated according to the model of Jukes & Cantor (1969). The robustness of tree topologies was assessed by using bootstrap analyses based on 1000 replications (for the neighbour-joining and maximum-parsimony methods) or 100 replications (for the maximum-likelihood method). Alignment analysis was carried out using the jPHYDIT program (Jeon et al., 2005) and phylogenetic analyses were carried out using MEGA 3 (Kumar et al., 2004) and PAUP* 4.0 (Swofford, 1998). Likelihood parameters were estimated by using the hierarchical ratio tests in MODELTEST, version 3.04 (Posada & Crandall, 1998). An almost complete 16S rRNA gene sequence of strain CL-GP79T (1369 bp) was obtained. Strain CL-GP79T was closely related to F. major (95.7 % similarity). The tree topologies inferred from the three tree-making algorithms showed that strain CL-GP79T formed a robust cluster with species of the genus Flectobacillus (Fig. 2). This grouping was supported by high bootstrap values (neighbour-joining and maximum-likelihood methods, 100 %; maximum-parsimony method, 98 %). Thus, it is clear that our isolate belongs to the genus Flectobacillus. However, the relatively low level of similarity (95.7 %) between the 16S rRNA gene sequences of strain CL-GP79T and F. major ATCC 29496T indicated that strain CL-GP79T represents a novel species in the genus (Stackebrandt & Goebel, 1994; Rosselló-Mora & Amann, 2001).
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Description of Flectobacillus lacus sp. nov.
Flectobacillus lacus (la'cus. L. gen. n. lacus of a lake or pond, referring to the isolation of the type strain).
Gram-negative, aerobic, almost straight rods, approximately 0.3–0.6 µm wide and 4.7–10.0 µm long. Cells are non-motile and do not possess flagella. Colonies on R2A agar are pale pink to rose in colour and are round (2–6 mm in diameter), convex and smooth with entire margins. Growth occurs at 10–35 °C and pH 6–9; optimal temperature and pH for growth are 25–30 °C and pH 7, respectively. Grows well on R2A and nutrient agar without NaCl or with 0.1 % (w/v) NaCl; no growth occurs in the presence of 
0.5 % (w/v) NaCl. Grows on R2A medium and nutrient medium, but not on MacConkey medium. Flexirubin pigments are absent. Positive for catalase, oxidase, gelatinase and amylase activities; negative in the Voges–Proskauer test. Positive for 
-galactosidase, 
-galactosidase, aesculin hydrolysis, alkaline phosphatase, acid phosphatase, esterase (C4), esterase lipase (C8), leucine, valine and cystine arylamidases, trypsin, -chymotrypsin, naphthol-phosphohydrolase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase. Negative for indole production, arginine dihydrolase, lipase (C14) and -glucuronidase. Other phenotypic properties (e.g. acid production, utilization of carbon, sensitivity to antibiotics) are shown in Table 1 and Supplementary Table S1. The DNA G+C content is 38.3 mol%. The major fatty acids are C16 : 15c (mean±SD, 26.9±10.8 %), iso-C15 : 0 2-OH and/or C16 : 17c (19.2±2.3 %) and iso-C15 : 0 (12.1±1.3 %).
The type strain, CL-GP79T (=KCCM 42271T=JCM 13398T), was isolated from a highly eutrophic pond, Gongdae Pond, located in the campus of Seoul National University, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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