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1 S. N. Winogradsky Institute of Microbiology, Russian Academy of Sciences, Prospect 60-letya Octyabrya 7/2, Moscow 117312, Russia
2 DSMZ – German Collection of Microorganisms and Cell Cultures, D-38124 Braunschweig, Germany
3 Max-Planck-Institut für terrestrische Mikrobiologie, D-35043 Marburg, Germany
Correspondence
Svetlana N. Dedysh
dedysh{at}mail.ru
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7c); the quinones are MK-7 and MK-6. Comparative 16S rRNA gene sequence analysis revealed that the novel strains share 97 % sequence similarity and belong to the family Sphingobacteriaceae; however, they are related only distantly to members of the genera Pedobacter (91.8–93.3 % similarity) and Sphingobacterium (89.6–91.2 % similarity). The DNA G+C content of strains TPT18T and TPT56T is 42.4 and 46.1 mol%, respectively. The low DNA–DNA hybridization value (42 %) and a number of phenotypic differences between strains TPT18T and TPT56T indicated that they represent two separate species. Since the two isolates are clearly distinct from all currently described members of the family Sphingobacteriaceae, we propose a novel genus, Mucilaginibacter gen. nov., containing two novel species, Mucilaginibacter gracilis sp. nov. and Mucilaginibacter paludis sp. nov. The type strains of Mucilaginibacter gracilis and Mucilaginibacter paludis are respectively TPT18T (=ATCC BAA-1391T =VKM B-2447T) and TPT56T (=ATCC BAA-1394T =VKM B-2446T).
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Mucilaginibacter gracilis TPT18T and M. paludis TPT56T are AM490402 and AM490403.
Detailed phenotypic characteristics of strains TPT18T and TPT56T are available as supplementary material with the online version of this paper.
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). However, in contrast to many other environments, little is known about members of the Bacteroidetes that inhabit acidic Sphagnum peat bogs. So far, taxonomically characterized members of the Bacteroidetes from this acidic habitat are represented by only four strains of gliding, xylanolytic and laminarinolytic bacteria that were identified as Chitinophaga arvensicola (Pankratov et al., 2006). Here, we describe two more polysaccharide-degrading representatives of the phylum Bacteroidetes from acidic peat.
Strains TPT18T and TPT56T were isolated from a sample collected from 10–20 cm below the surface of the Sphagnum peat bog Bakchar, in the Tomsk region of western Siberia (5 ° 51' N 8 ° 50' E), by spread-plating of serially diluted peat suspension onto the surface of peat extract medium solidified with gellan gum (a polysaccharide from Alteromonas sp.; Fluka) (Dedysh et al., 2006). Two highly distinctive types of either cream- or pink-coloured, raised, semi-transparent and slimy colonies developed on plates inoculated with the terminal dilutions of peat suspension. Both types of colonies were composed of rod-shaped bacteria that produced large amounts of an extracellular polymeric substance (EPS). Partial sequencing of the 16S rRNA genes from these two bacteria showed that they are affiliated with the Bacteroidetes and are only distantly related (89–91 % sequence similarity) to members of the genera Pedobacter and Sphingobacterium. Thus, the goal of our study was to determine the taxonomic position of the newly isolated strains and to examine their capacity to degrade various biopolymers under acidic conditions.
The isolates were maintained on tenfold-diluted agar medium R2A (Difco). All tests were carried out using medium MM1 containing (l–1 distilled water) 0.5 g glucose, 0.04 g MgSO4 . 7H2O, 0.02 g CaCl2 . 2H2O and 0.1 g yeast extract. Cell morphology and cell life cycle were examined using cultures grown on agar medium MM1. Preparation of ultrathin sections was performed as described before (Kulichevskaya et al., 2006). Growth of the novel strains under a variety of conditions, including temperatures of 2–37 °C, pH 4.0–9.0 and NaCl concentrations of 0.01–3.0 % (w/v), was examined using batch cultures grown in liquid medium MM1. The OD600 was measured in an Eppendorf BioPhotometer at 2 day intervals for 2 weeks. The range of potential growth substrates of the novel strains was examined by replacing glucose in medium MM1 with various carbon sources. The ability to degrade different biopolymers was examined by measuring the rate of CO2 production in tightly closed 120 ml serum bottles containing 20 ml liquid medium MM1 with 0.05 % (w/v) of the corresponding polymer substrate for 1 month at 20 °C. Control incubations were run in parallel under the same conditions but without substrate. Fermentative utilization of carbohydrates was investigated as described for the Hugh–Leifson test (Gerhardt et al., 1981). Susceptibility to antibiotics was determined on MM1 agar plates using discs (Oxoid) containing the following antibiotics: ampicillin (10 µg), gentamicin (10 µg), kanamycin (30 µg), neomycin (10 µg), novobiocin (30 µg), streptomycin (10 µg), chloramphenicol (30 µg) and lincomycin (10 µg).
Cell biomass for cellular fatty acid and isoprenoid quinone analyses and for DNA extraction was obtained from batch cultures grown in liquid MM1 medium at 24 °C for 1 week. Cellular fatty acid analysis was performed by the DSMZ Identification Service. Isoprenoid quinones were extracted according to Collins (1985) and analysed using an LCQ ADVANTAGE MAX tandem-type mass spectrometer and a Finnigan Mat 8430 ionization mass spectrometer. The DNA base composition of strains was determined by thermal denaturation using a Unicam SP1800 spectrophotometer at a heating rate of 0.5 °C min–1. The G+C content was calculated according to Owen et al. (1969). PCR-mediated amplification of the 16S rRNA gene was performed using primers 9f and 1492r and reaction conditions described by Weisburg et al. (1991). 16S rRNA gene amplicons were purified using QIAquick spin columns (Qiagen) and sequenced on an ABI Prism 377 DNA sequencer (PE Applied Biosystems). Phylogenetic analysis was carried out using the ARB program package (Ludwig et al., 2004).
On solid media made with agar or gellan gum, strains TPT18T and TPT56T formed large (up to 1 cm in diameter), convex, circular, semi-transparent colonies of slimy consistency. The colony colour varied from cream to light orange for strain TPT18T and from light pink to reddish for strain TPT56T. Cells of these strains were Gram-negative, non-spore-forming, non-motile rods that occurred singly, in pairs or in short chains and were covered with large capsules of EPS. Young (2–3 days old) cultures contained long cells (6–15 µm) (Fig. 1a, b); cells up to 40 µm in length were occasionally observed. On ageing, long cells divided by binary fission into several non-motile shorter (1.5–5 µm) cells. Cells of strain TPT56T were slightly thicker (0.5–0.8 µm) than cells of strain TPT18T (0.4–0.5 µm). Old cultures of both strains contained L-forms of cells (Fig. 1c). Morphologically, the L-forms appeared as spherical bodies of varying sizes (0.5–3 µm); some of them were granular and some contained vacuoles (Fig. 1d). After transfer to fresh medium, the L-forms reverted to normal vegetative cells that continued to grow as usual. Ultrathin sections of vegetative cells revealed the presence of numerous spherical outer-membrane vesicles, ranging in size from 50 to 240 nm (Fig. 1e, f). Also, small globular bodies bounded by bilayered membranes and resembling mesosome-like structures were observed in many cells of the novel isolates (Fig. 1e, f). The nature of these cell structures remains unclear.
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Strains TPT18T and TPT56T grew in the pH range 4.2–8.2, with an optimum at pH 6.0–6.5. The temperature range for growth was 2–33 °C, with an optimum at 20 °C. Growth was inhibited completely at NaCl concentrations above 1.0 % (w/v). Both strains were resistant to ampicillin, gentamicin, kanamycin, neomycin and novobiocin and susceptible to streptomycin. Strain TPT18T differed from strain TPT56T in its susceptibility to lincomycin and resistance to chloramphenicol.
Both novel isolates contained menaquinone-7 (MK-7) as the predominant isoprenoid quinone and MK-6 as a minor component. The cellular fatty acid profiles of strains TPT18T and TPT56T had significant similarity to those of members of the family Sphingobacteriaceae. Similar to representatives of the genera Pedobacter and Sphingobacterium, the major fatty acids of the novel strains were iso-C15 : 0, iso-C17 : 0 3-OH, iso-C15 : 0 2-OH and C16 : 17c (Table 1). However, the cellular fatty acid profiles of the novel isolates differed from those of Pedobacter and Sphingobacterium species by the presence of significant amounts of anteiso-C15 : 0 (up to 21 % of the total fatty acids).
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). The two strains shared a 16S rRNA gene sequence similarity of 97 %. The DNA G+C content of strains TPT18T and TPT56T was 42.4 and 46.1 mol%, respectively. A low DNA–DNA hybridization value (42 %) and a number of phenotypic differences between them indicated that they represent two separate species.
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), we propose a novel genus, Mucilaginibacter gen. nov., and two novel species, Mucilaginibacter gracilis sp. nov. and Mucilaginibacter paludis sp. nov., for strains TPT18T and TPT56T.
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Gram-negative, non-spore-forming, non-motile rods that occur singly, in pairs or in short chains and produce large amounts of EPS. Cells undergo a cyclic shape change during culture development. Young cultures contain long cells (6–15 µm, sometimes up to 40 µm in length). On ageing, long cells divide by binary fission into several non-motile shorter (1.5–5 µm) cells. Old cultures contain L-forms of cells, which revert to normal vegetative cells after transfer to fresh medium. Cells produce numerous outer-membrane vesicles. Colonies are large (3–10 mm in diameter), convex, circular, semi-transparent and slimy. The colony colour on tenfold-diluted medium R2A varies from cream to light orange and reddish. Flexirubin-type pigments are absent. Oxidase- and catalase-positive. Chemo-organotrophic facultative aerobes. Sugars are the preferred growth substrates. Capable of fermenting glucose and sucrose. Cannot utilize melibiose, melezitose, cellulose, chitin, heparin or aesculin. Hydrolyse pectin, xylan, laminarin, chondroitin 6-sulfate, gellan gum, pullulan and starch. Do not produce H2S from thiosulfate or indole from tryptophan. Capable of growth at pH 4.2–8.2 and at 2–33 °C. NaCl inhibits growth at concentrations above 1 % (w/v). Major fatty acids are iso-C15 : 0, anteiso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 17c); the quinones are MK-7 and MK-6. The G+C content of the DNA varies between 42.4 and 46.1 mol%. Member of the family Sphingobacteriaceae. Strains have been isolated from acidic wetlands, specifically Sphagnum peat bogs. The type species is Mucilaginibacter paludis.
Description of Mucilaginibacter paludis sp. nov.
Mucilaginibacter paludis (pa.lu'dis. L. gen. n. paludis of a swamp, of a marsh, of a bog).
The description is as for the genus with the following additional traits. Cells are 0.5–0.8 µm wide. Colony colour varies from light pink to reddish. Utilizes D-cellobiose, D-fructose, D-galactose, sodium D-galacturonate, sodium D-gluconate, D-glucose, D-lactose, leucrose, maltose, D-rhamnose, sucrose, trehalose, D-xylose, D-mannose, myo-inositol and N-acetylglucosamine. Does not utilize acetate, adonitol, arabitol, arbutin, butyrate, capronate, citrate, D-arabinose, D-melezitose, D-melibiose, D-raffinose, dulcitol, fumarate, inulin, malate, mannitol, propionate, pyruvate, salicin, sorbitol, succinate or valerate. Capable of fermenting glucose and sucrose. Hydrolyses chondroitin 6-sulfate, gellan gum, laminarin, pectin, pullulan, starch and xylan but not alginate, CM-cellulose, cellulose, chitin, aesculin, fucoidan, heparin or lichenan. Produces acid from cellobiose, fructose, galactose, glucose, lactose, maltose, rhamnose, sucrose, xylose, mannose, laminarin and xylan. Optimal growth at 20–25 °C and at pH 6.0–6.5.
The type strain is strain TPT56T (=ATCC BAA-1394T =VKM B-2446T), which was isolated from the Sphagnum peat bog Bakchar in the Tomsk region of western Siberia.
Description of Mucilaginibacter gracilis sp. nov.
Mucilaginibacter gracilis (gra'ci.lis. L. masc. adj. gracilis slender or thin).
The description is as for the genus with the following additional traits. Cells are 0.4–0.5 µm wide. Colony colour varies from creamy to yellowish or light orange. Utilizes D-arabinose, D-galactose, D-glucose, D-xylose, D-lactose, maltose, D-rhamnose, D-raffinose, salicin, sucrose, trehalose, D-fructose, D-cellobiose and pyruvate but not N-acetylglucosamine, D-melibiose, D-melezitose, L-sorbose, acetate, butyrate, valerate, gluconate, capronate, malate, propionate, succinate, fumarate, citrate, adonitol, arabitol, arbutin, dulcitol, inulin, mannitol, myo-inositol, sorbitol, mannose or sodium D-galacturonate. Capable of fermenting glucose and sucrose. Hydrolyses chondroitin 6-sulfate, gellan gum, laminarin, pectin, pullulan, starch and xylan but not alginate, CM-cellulose, cellulose, chitin, aesculin, fucoidan, heparin or lichenan. Produces acid from D-galactose, D-glucose, D-xylose, maltose, sucrose, trehalose, D-fructose, D-cellobiose, laminarin and xylan but not from mannose. Optimal growth at 18–22 °C and at pH 5.8–6.2.
The type strain is strain TPT18T (=ATCC BAA-1391T =VKM B-2447T), which was isolated from the Sphagnum peat bog Bakchar in the Tomsk region of western Siberia.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Dedysh, S. N., Pankratov, T. A., Kulichevskaya, I. S., Belova, S. E. & Liesack, W. (2006). Phylogenetic analysis and in situ identification of bacteria community composition in an acidic Sphagnum peat bog. Appl Environ Microbiol 72, 2110–2117.
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