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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 M. V. Lomonosov Moscow State University, Faculty of Soil Science, GSP-2, Leninskie Gory, Moscow 119992, Russia
3 Max Planck Institute for Terrestrial Microbiology, D-35043 Marburg, Germany
Correspondence
Svetlana N. Dedysh
dedysh{at}mail.ru
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ABSTRACT |
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7c and 18 : 17c; the major quinones are Q-10 and Q-9. The DNA G+C content of strain RST is 62.6 mol%. Analysis of the 16S rRNA gene sequence revealed that the novel isolate is most closely related (97.3 % sequence similarity) to the type strain ATCC 25092T of the moderately acidophilic purple non-sulfur bacterium Rhodoblastus acidophilus, formerly named Rhodopseudomonas acidophila. However, in contrast to Rbl. acidophilus, strain RST is not capable of aerobic growth in the dark, has no spirilloxanthin among the carotenoids and differs in the pattern of substrate utilization. The value for DNA–DNA hybridization between strain RST and Rbl. acidophilus ATCC 25092T is only 22 %. Thus, strain RST represents a novel species of the genus Rhodoblastus, for which the name Rhodoblastus sphagnicola sp. nov. is proposed. Strain RST (=DSM 16996T=VKM B-2361T) is the type strain.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain RST is AM040096.
The absorption spectrum of a cell homogenate of phototrophically grown strain RST and a graph showing the influence of pH on growth of strain RST are available as supplementary material in IJSEM Online.
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). These phototrophic micro-organisms belong to the Alphaproteobacteria and Betaproteobacteria but do not form monophyletic groups within these phyla (Woese et al., 1984; Stackebrandt et al., 1988; Kawasaki et al., 1993; Imhoff & Trüper, 1992; Imhoff, 2001a). Instead, many representatives of the PNSB are closely related to non-phototrophic, strictly chemotrophic bacteria.
PNSB are widely distributed in various aquatic ecosystems as well as in sediments, moist soils, natural wetlands and paddy fields (Pratt & Gorham, 1970; Burke et al., 1974; Imhoff, 2001a). Stagnant water bodies with significant amounts of soluble organic matter and low oxygen tension are the preferred habitats for these bacteria. Sphagnum peat bogs exemplify this kind of environment. However, in contrast to other freshwater habitats, peat bogs are rarely reported as sources of PNSB. The low pH (3.5–5.5) and extremely low mineral salt contents (5–50 mg l–1 in peat water) are possible reasons for this. So far, taxonomically characterized PNSB from this acidic habitat are represented by only one strain, strain 3251, which was isolated from a Sphagnum peat bog near Kolshorn/Hannover (Germany) and subsequently described as a strain of Rhodopseudomonas acidophila (Pfennig, 1969). Later, this taxon was reclassified as Rhodoblastus acidophilus (Imhoff, 2001b). Here, we describe another example of PNSB isolation from acidic Sphagnum peat.
In the course of a study on cellulose degradation in Sphagnum peat, we detected intensive development of some phototrophic purple bacteria in anaerobic cellulolytic enrichments incubated under light. Several strains of PNSB that were morphologically and phenotypically similar to Rbl. acidophilus were isolated from these enrichments. These strains had the same morphology and physiological traits and possessed identical 16S rRNA gene sequences: thus, only one of these isolates was studied in detail. These phenotypic and genotypic studies revealed a number of features that distinguished Rbl. acidophilus from the newly isolated strain. Thus, we conclude that it represents a novel species of the genus Rhodoblastus.
The sample used in our study was collected from 5–10 cm below the surface of an acidic peat (pH 3.5–4.2) underlying the Andromeda–Eriophorum–Sphagnum plant community in the raised centre of the Sosvyatskoe ombrotrophic bog located in Tver Region, West Dvinskiy district, at the field station of the Institute of Forestry, Russian Academy of Sciences (56° 10' N 32° 12' E).
Anaerobic cellulolytic communities were enriched using screw-cap 120 ml serum bottles containing 30 ml liquid mineral medium of the following composition [g (l distilled water)–1]: KH2PO4, 0.04; NH4Cl, 0.1; MgCl2.6H2O, 0.01; CaCl2.2H2O, 0.05, with the addition of 0.1 % (w/v) cellulose, 0.5 % (v/v) trace element stock solution (Pfennig & Lippert, 1966) and 0.1 % (v/v) vitamin stock solution (Wolin et al., 1963). The initial pH of the medium was 5.5. The inoculation was done with 100 mg peat, and the bottles were closed with silicone-rubber septa, flushed with sterile N2 and incubated at 25 °C in the light (2000 lx). As soon as visual development of purple phototrophic bacteria in these enrichments occurred, an aliquot was taken for pure-culture isolation. This was achieved by means of repeated deep-agar (0.8 %) dilution series on succinate mineral medium (Pfennig, 1969). An isolate obtained was designated strain RST. Culture purity was ensured by examination under phase-contrast microscopy and by the absence of colourless colonies in a column of agar succinate mineral medium. Physiological tests were performed in liquid medium no. 27 recommended by the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) under anoxic conditions in the light (2000 lx). Rbl. acidophilus ATCC 25092T was used as a reference strain. Growth of strain RST was monitored by nephelometry at 600 nm for 7–10 days under a variety of conditions, including temperatures of 10–37 °C, pH 4.0–7.5 and NaCl concentrations of 0.01–3.0 % (w/v). Variations in the pH were achieved by mixing 0.1 M solutions of HCl and KOH. The range of potential growth substrates for strain RST was examined using 0.02–0.05 % (w/v) concentrations of the following carbon sources: acetate, arginine, benzoate, butyrate, caproate, citrate, ethanol, formate, fructose, glucose, glutamate, glycerol, lactate, malate, malonate, mannitol, propionate, pyruvate, succinate, tartrate and valerate. The capacity to utilize methanol was determined at a range of concentrations from 0.01 to 3 % (v/v). Tests for photolithotrophic growth with molecular hydrogen and carbon dioxide were performed in screw-capped 120 ml serum bottles containing 30 ml mineral medium no. 27 with a gas phase of H2/CO2 (80 : 20, v/v). Growth was examined after 7–10 days incubation under light by comparison with a negative control. Tests for the ability to grow in the dark under aerobic conditions were performed using both liquid and agar succinate mineral medium (Pfennig, 1969). The capacity to develop in nitrogen-free media was tested using liquid mineral medium no. 27 without yeast extract or NH4Cl.
For the preparation of ultrathin sections, cells of exponentially growing cultures were collected by centrifugation and pre-fixed with 1.5 % (w/v) glutaraldehyde in 0.05 M cacodylate buffer (pH 6.5) for 1 h at 4 °C and then fixed in 1 % (w/v) OsO4 in the same buffer for 4 h at 20 °C. After dehydration in an ethanol series, the samples were embedded in a Spurr epoxy resin. Thin sections were cut on an LKB-4800 microtome and then stained with 3 % (w/v) uranyl acetate in 70 % (v/v) ethanol. The specimen samples were examined with a JEM-100C transmission electron microscope (JEOL).
The absorption spectra of living cells were recorded with a LOMO SPh-56 spectrophotometer. For these measurements, the cells were suspended in 50 % glycerol. In addition, pigments were extracted with acetone/methanol (1 : 1, v/v) and the absorption spectra of these extracts were recorded. For estimation of the pigment content, cells were harvested at approximately early stationary growth phase by centrifugation. Carotenoids were analysed by HPLC as described previously (Sidorova et al., 1998). Satisfactory separation of the pigments was achieved using a Spherisorb ODS2 column (250x4.6 mm, 5 µm; Waters). The solvent flow rate was 1.0 ml min–1. The column was equilibrated with a mixture composed of 77 % acetonitrile/water (9 : 1, v/v) and 23 % ethyl acetate for 5 min. The mixture was then linearly substituted by ethyl acetate. The total separation time varied from 25 to 30 min depending on the carotenoid composition. Pigment fractions were collected automatically and their absorption spectra were recorded on a Shimadzu UV-160 spectrophotometer (Toropygina et al., 2003). Quantitative estimations of the individual carotenoids were made using the absorbance coefficients given by Davies (1976) and Heinemeyer & Schmidt (1983). For fatty acid and respiratory quinone analyses, cells of strains RST and Rbl. acidophilus ATCC 25092T were grown on succinate mineral medium and harvested in the late exponential growth phase. Analyses were performed by the Identification Service of the DSMZ. Genomic DNA from strain RST was extracted by using the method of Marmur (1961). The DNA G+C content was determined by means of thermal denaturation using a Unicam SP1800 spectrophotometer (at a heating rate of 0.5 °C min–1) and calculated according to Owen et al. (1969). DNA–DNA hybridization of strain RST and Rbl. acidophilus ATCC 25092T was performed as described by De Ley et al. (1970). PCR-mediated amplification of the 16S rRNA gene from position 28 to 1491 (numbering according to the International Union of Biochemistry nomenclature for Escherichia coli 16S rRNA) was performed using primers Eub9f and Eub1492r and the reaction conditions described by Weisburg et al. (1991). The 16S rRNA gene amplicons were purified using QIAquick spin columns (Qiagen) and sequenced on an ABI Prism 377 DNA sequencer using BigDye terminator chemistry, as specified by the manufacturer (PE Applied Biosystems). Phylogenetic analysis was carried out using the ARB program package (Ludwig et al., 2004).
Photosynthetically grown liquid cultures of strain RST were purplish red in colour. Cells of this isolate were Gram-negative, large rods, 0.8–1.0 µm in width and 2.0–6.0 µm in length (Fig. 1a, b). They showed polar growth and they reproduced by budding. No tube or filament was formed between mother and daughter cells. Young cells were motile by means of a single polar flagellum, while in older cultures cells were non-motile and had a tendency to form rosette-like clusters. Electron microscopy of ultrathin sections revealed the presence of internal photosynthetic membranes appearing as lamellae underlying, and parallel to, the cytoplasmic membrane (Fig. 1c).
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). Lycopene was the next greatest component (12.9 %), followed by rhodopinol (10.9 %), lycopenal (6.4 %), rhodopinal glucoside (5.6 %) and lycopenal glucoside (5.1 %). A high content of carotenoid glucosides (38–40 %) was shown to be characteristic of Rbl. acidophilus (Schmidt, 1971; Heinemeyer & Schmidt, 1983). However, in contrast to Rbl. acidophilus ATCC 25092T, our isolate did not contain spirilloxanthin.
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The isolate grew in the pH range 4.8–7.0, with a pH optimum of 5.2–5.5 (Supplementary Fig. S2). The temperature optimum for growth was 25–30 °C. Growth inhibition of 50 % was observed in the presence of 1 % (w/v) NaCl in the medium, whereas 2 % NaCl inhibited growth completely.
The cellular fatty acid composition of strain RST is shown in Table 2. Similarly to Rbl. acidophilus, the major components of the phospholipid fatty acid profile of strain RST were straight-chain, monounsaturated 9-cis-hexadecenoic acid (16 : 17c) and 11-cis-octadecenoic acid (18 : 17c), which comprised 45.4 and 42.2 %, respectively, of the total phospholipid fatty acid content. However, in comparison with that of Rbl. acidophilus ATCC 25092T, the phospholipid fatty acid profile of the novel isolate contained a smaller percentage of 16 : 0 fatty acids and contained minor amounts (<1 %) of some other saturated fatty acids, such as 15 : 0 and 17 : 0. Cells of strain RST contained ubiquinones Q-10 and Q-9, which comprised 92 and 8 %, respectively, of the total quinone content. In contrast to Rbl. acidophilus, strain RST did not contain any menaquinones or rhodoquinones (Table 3).
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). This novel phototrophic isolate is most closely related to the acidophilic phototrophic non-sulfur bacterium Rbl. acidophilus ATCC 25092T (97.3 % sequence similarity). Other closely related micro-organisms, exhibiting overall 16S rRNA gene sequence similarity of 94 % with respect to strain RST, are acidophilic methanotrophic bacteria of the genera Methylocella and Methylocapsa and acidophilic heterotrophic bacteria of the genus Beijerinckia. Interestingly, most known representatives of the genera Methylocella and Methylocapsa were also isolated from acidic Sphagnum peatlands (Dedysh et al., 2000, 2002, 2004). Together, these phototrophic, heterotrophic and methanotrophic bacteria of the genera Rhodoblastus, Beijerinckia, Methylocella and Methylocapsa form a monophyletic cluster, which is supported by a bootstrap value of 97 % (Fig. 2). All members of this phylogenetic cluster are moderately acidophilic organisms that inhabit acidic, boggy waters and soils and have a pH optimum for growth of about 5.5. A metabolic trait common to all organisms of this phylogenetic group, including those in the genus Beijerinckia (Dedysh et al., 2005), is the capacity for methylotrophic growth.
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) and also determined in our experiments, is 65.3 mol%, while the DNA G+C contents of other strains of this species range from 62.6 to 66.8 mol% (Pfennig, 1969; Imhoff, 2001b). The DNA–DNA hybridization value for strain RST and Rbl. acidophilus ATCC 25092T was only 22 %. This low level of genomic DNA relatedness suggests a differentiation of the two strains at the species level, which is also supported by the results of some physiological tests. Strain RST should therefore be assigned to a novel species of the genus Rhodoblastus, for which the name Rhodoblastus sphagnicola is proposed. The characteristics that differentiate this novel species from Rbl. acidophilus are summarized in Table 3
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Description of Rhodoblastus sphagnicola sp. nov.
Rhodoblastus sphagnicola (sphag.ni'co.la. N.L. n. Sphagnum generic name of sphagnum moss; L. suff. -cola from L. n. incola inhabitant, dweller; N.L. n. sphagnicola inhabitant of Sphagnum).
Cells are rod-shaped, straight or slightly curved, 0.8–1.0 µm wide and 2.0–6.0 µm long, motile by polar flagella in young cultures and have a tendency to form rosette-like clusters in older cultures. Cells reproduce by budding. No tube or filament is formed between mother and daughter cells. Cells contain lamellar intracytoplasmic photosynthetic membranes underlying, and parallel to, the cytoplasmic membrane. The colour of anaerobic liquid cultures is purplish red. Absorption spectra of living cells show maxima at 377, 463, 492, 527, 592, 806 and 867 nm. The photosynthetic pigments are bacteriochlorophyll a and carotenoids. Rhodopin and rhodopinal are major carotenoids, followed by lycopene, rhodopinol, lycopenal, rhodopinal glucoside and lycopenal glucoside. Spirilloxanthin is absent from the carotenoid composition. Preferred mode of growth is photoheterotrophic under anoxic conditions in the light with formate, acetate, butyrate, pyruvate, lactate, propionate, malate, malonate, succinate, glycerol, methanol, ethanol, caproate, lactate and malonate. Best growth occurs on butyrate and propionate. Methanol is utilized at a wide range of concentrations from 0.01 to 1 % (v/v). No growth occurs with benzoate, glucose, fructose, tartrate, citrate or glutamate. Nitrogen sources are N2 and ammonia. Photoautotrophic growth is possible with hydrogen as electron donor; sulfide and thiosulfate cannot be used. Growth factors are not required, though yeast extract increases the growth rate. No aerobic growth occurs in the dark. The major phospholipid fatty acids are 16 : 17c and 18 : 17c. Contains ubiquinones Q-10 and Q-9. Mesophilic, moderately acidophilic, with optimum growth at 25–30 °C and pH 5.2–5.5. NaCl inhibits growth at concentrations above 1 % (w/v). The DNA G+C content is 62.6 mol%.
The type strain, RST (=DSM 16996T=VKM B-2361T), was isolated from an acidic Sphagnum peat bog (Sosvyatskoe), Tver Region, Russia.
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ACKNOWLEDGEMENTS |
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Davies, B. H. (1976). Carotenoids. In Chemistry and Biochemistry of Plant Pigments, 2nd edn, pp. 38–160. Edited by T. W. Goodwin. London: Academic Press.
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