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Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Sector 39A, Chandigarh 160 036, India
Correspondence
T. Chakrabarti
tapan{at}imtech.res.in
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain GPTSA100-15T is AY904352.
An extended phylogenetic tree based on 16S rRNA gene sequences of members of the phylum Bacteroidetes is available as supplementary material in IJSEM Online.
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; Garrity & Holt, 2001) are widely distributed in nature, particularly in aquatic systems (Glöckner et al., 1999; Kirchman, 2002; Yoon et al., 2005), and are known to play important roles in biogeochemical cycling and degradation of complex biopolymers like cellulose, chitin (Cottrell & Kirchman, 2000) and agar (Nedashkovskaya et al., 2003). Recently, a member of this phylum associated with decay of a planktonic bloom, Aquiflexum balticum, was described (Brettar et al., 2004). Over the past few years taxonomic reclassification of many taxa has been reported (Bernardet et al., 1996; Bowman, 2000). In the second edition of Bergey's Manual of Systematic Bacteriology, the phylum Bacteroidetes includes three classes, ‘Bacteroides’, Flavobacteria and ‘Sphingobacteria’. The class ‘Sphingobacteria’ includes five families, of which ‘Flexibacteraceae’ is the largest, represented by ten genera (Garrity & Holt, 2001). Since then, a few more genera have been described. In this paper, taxonomic characterization is described of a novel bacterial strain belonging to the family ‘Flexibacteraceae’, GPTSA100-15T, isolated from Assam, India. Strain GPTSA100-15T was isolated on TSBA100 medium (normal strength tryptic soy broth diluted 100 times and solidified with 1.5 %, w/v, agarose) by dilution plating and incubating the plates at 37 °C. Since the strain could not grow on commonly used nutritionally rich media, its ability to grow on media of different compositions was checked. Media used in this study are as follows (compositions expressed in %, w/v): TSBA (tryptic soy broth, 3; agar, 1.5), YEA (yeast extract, 0.5; CaCl2, 0.1; agar, 1.5; pH 7.2), modified R2A (MR2A) agar (Casamino acids, 0.025; yeast extract, 0.05; peptone, 0.075; glucose, 0.05; soluble starch, 0.05; K2HPO4, 0.03; MgSO4.7H2O, 0.0024; sodium pyruvate, 0.03; agar, 1.5; pH 7.2), R3A agar, MHA (Mueller–Hinton agar), TSIA (triple-sugar iron agar), MAC (MacConkey agar), NA (nutrient agar), NB (nutrient broth), NA-NaCl (NA devoid of NaCl), CA (cytophaga agar), LY agar [1 mM phosphate buffer, pH 7.0; yeast extract, 0.01; (NH4)2SO4, 0.1; MgSO4.7H2O, 0.05; CaCl2.2H2O, 0.005; standard trace elements; agar, 1.5], LBA (Luria–Bertani agar), ZMA (ZoBell marine agar) and PCA (plate count agar). Media were made nutritionally poor by diluting these media with distilled water and solidifying them with 1.5 % agarose. Dilutions are indicated by the abbreviated name of the medium followed by 100. For acid production, modified MR2A broth (MR2A without starch, glucose and sodium pyruvate) supplemented with 0.5 % of the desired carbon source and phenol red (as pH indicator) was used; for methyl red/Voges–Proskauer tests, the same basal medium as above was used, supplemented with 0.5 % glucose. Sole carbon source utilization medium (pH 7.2) contained the following, supplemented with 1 % carbon source (%): (NH4)2SO4, 0.2; K2HPO4, 0.024; MgSO4.7H2O, 0.024; KCl, 0.01; yeast extract, 0.01; agarose, 1.5.
Tests for phenotypic properties were carried out by standard methods (Smibert & Krieg, 1994; Murray et al., 1994; Powers, 1995; Cowan & Steel, 1965). Growth at different temperatures, pH and NaCl concentrations was checked on basal TSBA100 medium. Growth on various media (TSBA, CA, LY agar, YEA, R2A agar, R3A agar, MR2A agar, MHA, TSIA, NA, NA-NaCl, ZMA, PCA, NA100, LBA100, ZMA100, MAC100, MHA100 and TSI100) was also checked at 37 °C. Determination of gliding motility was made essentially according to Bowman (2000) but on MR2A agar. Flexirubin pigment production was determined by exposing bacterial cell mass to 20 % (w/v) KOH solution according to Reichenbach (1989). All biochemical tests were done at 37 °C. Phenotypic properties of the strain are given in the genus and species descriptions. Hydrolysis of cellulose (filter paper) and chitin was tested by overlaying the respective substrates on MR2A agar solidified with 1.2 % agar. Hydrolysis of carboxymethylcellulose (CMC) was checked on modified MR2A agar supplemented with 0.5 % (w/v) CMC (Sigma) for up to 7 days. Plates were flooded with 0.2 % aqueous Congo red dye solution followed by removal of extra dye and flooding the plate with 1 M NaCl solution. A clear zone around bacterial growth indicated positive hydrolysis. For determining hydrolysis of various other substrates, MR2A agar supplemented with substrates at concentrations normally recommended in standard methods (Smibert & Krieg, 1994; Cowan & Steel, 1965) was used. Sensitivity to antibiotics was tested on MR2A agar using antibiotic susceptibility discs (Hi-Media) of ampicillin (10 µg), bacitracin (8 U), chloramphenicol (30 µg), erythromycin (15 µg), gentamicin (10 µg), kanamycin (30 µg), lincomycin (2 µg), neomycin (30 µg), novobiocin (30 µg), norfloxacin (10 µg), penicillin G (10 U), polymyxin B (300 U), rifampicin (2 µg), streptomycin (10 µg), sulfasomidine (300 µg) and tetracycline (3 µg).
For cellular fatty acid analysis, the isolate was grown on TSBA100 medium at 30 °C for 48 h. Extraction and analysis of cellular fatty acids were done according to procedures of the SHERLOCK Microbial Identification system (MIDI), as described previously (Pandey et al., 2002). Major fatty acids were iso-C15 : 0 (42.9 %), iso-C17 : 0 3-OH (12.5 %), anteiso-C15 : 0 (12.2 %), summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1
7c, 11.3 %), iso-C15 : 0 3-OH (5.6 %), C15 : 16c (5.5 %) and C16 : 15c (3.8 %). The presence of iso-C15 : 0 fatty acid has been reported for many members of the family ‘Flexibacteraceae’. Genomic DNA of GPTSA100-15T was isolated according to Marmur (Johnson, 1994). The G+C content of genomic DNA was determined spectrophotometrically as described previously (Saha et al., 2005). Genomic G+C content of strain GPTSA100-15T was 36.9 mol%. Amplification of the 16S rRNA gene from the isolate was done using primers 27f (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492r (5'-TACGGYTACCTTGTTACGACTT-3'). Amplification, purification of the amplicon and sequencing of the 16S rRNA gene were done as described by Pandey et al. (2002). A nearly complete (1385 nt continuous stretch) 16S rRNA gene sequence of the isolate was used to search for similar sequences from GenBank and the RDP. Various online sequence analysis tools available from RDP release 9.1 (http://rdp.cme.msu.edu/index.jsp, RDP analysis tools) were also used to get an idea of the systematic position of the strain. Sequence analysis revealed that GPTSA100-15T is a member of the phylum Bacteroidetes showing closest similarity (94.0 %) with an uncultured bacterial clone, HP1A92, which was a representative sequence of a sludge microbial community (McMahon et al., 2002). Among the culturable bacteria, it showed closest similarity to Leadbetterella byssophila 4M15T (87.8 %). Sequence similarities with other members of the phylum Bacteroidetes were less than 85 %. Since 16S rRNA gene sequence similarity of 95 % or less is generally regarded as a borderline for describing a new genus (Ludwig et al., 1998), low sequence similarity (87.8 % and less) with cultured bacterial relatives clearly indicated that GPTSA100-15T should be treated as a representative of a new genus. For determination of the phylogenetic position of GPTSA100-15T, the sequence of its closest uncultured bacterial relative as well as 73 strains from different representative families within the phylum Bacteroidetes were retrieved (RDP analysis tools Hierarchy Browser; NCBI taxonomy browser), aligned by the program CLUSTAL_X (Thompson et al., 1997) and edited manually. Aligned sequences were analysed by the TREECON software package (Van de Peer & De Wachter, 1997). Neighbour-joining (NJ) analysis (Saitou & Nei, 1987) with the correction of Jukes & Cantor (1969) was carried out with Bacillus subtilis as an outgroup. The stability of the tree was estimated by bootstrap analysis of 1000 replications. The analyses indicated phylogenetic positioning of the strain in the family ‘Flexibacteraceae’ (see Supplementary Fig. S1 in IJSEM Online). Using the parameters and software mentioned above and 16S rRNA gene sequences from 22 representative culturable genera of the family ‘Flexibacteraceae’ and the sequence of uncultured bacterial clone HP1A92, an NJ tree was constructed to reveal the position of strain GPTSA100-15T within this family. A phylogenetic tree was also constructed by the maximum-parsimony method using DNAPARS from the PHYLIP package (Felsenstein, 1993). Irrespective of the tree-generating method used, strain GPTSA100-15T always formed a clade with the uncultured bacterial clone HP1A92 that was separate from the Leadbetterella clade within the family ‘Flexibacteraceae’ with a very high bootstrap value (Fig. 1). Strain GPTSA100-15T could also be differentiated from its closest relatives on the basis of phenotypic properties and G+C content (Table 1). Moreover, the similarity of strain GPTSA100-15T with its closest cultured bacterial relatives at the 16S rRNA gene sequence level was low enough (87.8 % and less) to justify it as a member of a new genus. Thus, on the basis of polyphasic characterization, it is concluded that GPTSA100-15T represents a novel species in a new genus for which the name Emticicia oligotrophica gen. nov., sp. nov. is proposed.
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Gram-negative, strictly aerobic, non-motile rods occurring mostly singly, sometimes in pairs. Positive for oxidase and nitrate reduction; weakly positive for catalase. Major whole-cell fatty acids are iso-C15 : 0, iso-C17 : 0 3-OH, anteiso-C15 : 0, summed feature 3 (iso-C15 : 2-OH and/or C16 : 17c), iso-C15 : 0 3-OH and C15 : 16c. On the basis of 16S rRNA gene sequence analysis, the genus is affiliated to the family ‘Flexibacteraceae’ in the phylum Bacteroidetes. The type species is Emticicia oligotrophica.
Description of Emticicia oligotrophica sp. nov.
Emticicia oligotrophica (o.li.go.tro'phi.ca. Gr. adj. oligos few; Gr. adj. trophikos nursing, tending or feeding; N.L. fem. adj. oligotrophica eating little, referring to a bacterium living on low-nutrient media).
In addition to properties described for the genus, the species has the following characteristics. Colonies on TSBA100 medium after 2 days growth at 37 °C appear round, mucoid, light pink and convex with almost entire margins; with prolonged incubation, the colony colour deepens, it produces slime and appears glistening. Cells are usually 2–5 µm long and 0.3–0.4 µm wide; in an aged culture, some cells may be longer. No gliding motility observed. No flexirubin pigment detected. Grows at 15–42 °C and pH 5.0–11.0; cannot grow in 1 % NaCl. Positive for methyl red, but negative for Voges–Proskauer test. Cannot grow on most of the common nutritionally rich culture media (e.g. TSBA, NA, LBA, PCA, MHA, TSIA and ZMA), but grows on R2A, R3A and 100-fold dilutions of all these media. It can hydrolyse gelatin, starch, Tween 60 and urea, but not casein, ONPG, hypoxanthine, chitin, cellulose (CMC or filter paper), xylan, tyrosine or Tween 20, 40 or 80. Can utilize D-cellobiose, D-lactose, D-melezitose, D-raffinose and arbutin; weakly utilizes L-arabinose, L-arabitol, D-amygdalin, D-fructose, D-galactose, D-glucose, glycerol, myo-inositol, D-melibiose and sucrose as sole carbon sources. Cannot utilize adonitol, dulcitol, D-mannitol, D-sorbitol, L-sorbose or xylitol as sole carbon sources. Produces acids from arbutin, D-cellobiose, D-glucose, D-lactose, methyl 
-D-glucoside, salicin, D-trehalose and D-xylose and weakly from D-galactose, D-maltose, D-mannose, D-melibiose, D-melezitose and sucrose, but not from adonitol, D-amygdalin, L-arabinose, L-arabitol, dulcitol, aesculin, erythritol, D- or L-fucose, D-fructose, glycerol, glycogen, myo-inositol, inulin, 2-ketogluconate, D-mannitol, D-raffinose, D-ribose, D-sorbitol, L-sorbose or xylitol. Sensitive to ampicillin, bacitracin, chloramphenicol, erythromycin, gentamicin, kanamycin, lincomycin, neomycin, novobiocin, norfloxacin, penicillin G, polymyxin B, rifampicin, streptomycin, sulfasomidine and tetracycline. The genomic DNA G+C content of the type strain is 36.9 mol%.
The type strain is GPTSA100-15T (=MTCC 6937T=DSM 17448T), isolated from a warm spring water sample from Assam, India.
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ACKNOWLEDGEMENTS |
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