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1 S. N. Winogradsky Institute of Microbiology, Russian Academy of Sciences, Prospect 60-let Octyabrya 7/2, Moscow 117312, Russia
2 German Research Centre for Biotechnology, Chemical Microbiology, Mascheroder Weg 1, D-38124 Braunschweig, Germany
Correspondence
Svetlana N. Dedysh
dedysh{at}mail.ru
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7c and the major phospholipids were phosphatidylglycerols. The G+C content of the DNA was 60.4 mol%. On the basis of 16S rRNA gene sequence similarity, strain Z-0023T was most closely related to Asticcacaulis biprosthecium (98 % similarity), Asticcacaulis taihuensis (98 %) and Asticcacaulis excentricus (95 %). However, low levels of DNA–DNA relatedness to these organisms and a number of distinctive features of the tundra wetland isolate indicated that it represented a novel species of the genus Asticcacaulis, for which the name Asticcacaulis benevestitus sp. nov. is proposed. The type strain is Z-0023T (=DSM 16100T=ATCC BAA-896T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Z-0023T is AM087199.
Tables showing data for utilization of carbon compounds, glycolipid and phospolipid fatty acid content, and polar lipid content of Asticcacaulis species are available as supplementary material in IJSEM Online.
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; Poindexter, 1964, 1981a, 1999; Nikitin et al., 1966; Belyaev, 1968; Staley, 1968; Staley et al., 1987; Lapteva, 1987). An important characteristic of DPB is their ability to metabolize organic materials available in extremely low quantities and to tolerate prolonged periods of nutrient scarcity (Larson & Pate, 1975; Poindexter, 1981b; Vasilyeva & Zavarzin, 1995).
DPB are characterized by asymmetric cell reproduction. Each reproductive event in these bacteria produces two siblings: a sessile cell with a cellular stalk (a prostheca) and a motile cell with a polar flagellum. On the basis of the morphology of the reproductive stage (fission or budding), two fundamentally different types of DPB are distinguished: caulobacteria (Caulobacter, Brevundimonas, Maricaulis and Asticcacaulis) and hyphomicrobia (Hyphomicrobium, Pedomicrobium and Hyphomonas) (Poindexter, 1999; Abraham et al., 1999). Caulobacteria are especially widespread in environments where nutrient concentrations and ambient temperatures are low (Poindexter, 1981a, 1999; Staley et al., 1987). The taxonomy of caulobacteria has for many years relied primarily upon morphological criteria. Recently, a polyphasic approach, comprising 16S rRNA gene sequencing, lipid analysis and NaCl tolerance characterization, was used to analyse a large set of strains of these bacteria (Abraham et al., 1999, 2001). It has been shown that caulobacteria form two different phylogenetic lineages within the Alphaproteobacteria, one comprising the freshwater and brackish water representatives of the group Caulobacter–Brevundimonas–Asticcacaulis, and the other comprising the marine species of the genus Maricaulis.
All known representatives of the genus Asticcacaulis have been isolated from freshwater samples. Sessile cells of these bacteria develop subpolar or lateral prosthecae, which do not possess adhesive material at their tips. Instead, the cells bear holdfast material directly on their surfaces. At the time of writing, the genus comprised three recognized species, Asticcacaulis excentricus (Poindexter, 1964), Asticcacaulis biprosthecium (Pate et al., 1973) and Asticcacaulis taihuensis (Liu et al., 2005). Representatives of the genus Asticcacaulis are rarely observed and even more rarely isolated. In this report, we describe a novel species of the genus that has been isolated from tundra wetland peat.
The peat sample was collected from a depth of 3–6 cm of a shrub tundra wetland, north-east of Vorkuta in the Polar Urals of Russia (68° N 52° E). One gram of peat was homogenized in 10 ml sterile water, and 1 ml of this suspension was used to inoculate 100 ml of a liquid dilute peptone–yeast extract (PY) medium containing 0.005 % peptone and 0.005 % yeast extract, pH 6.8, supplemented with 1 % (v/v) vitamin stock solution and 2 % (v/v) modified Hutner's basal salts as described by Staley (1968). For enrichment of psychrophilic bacteria, incubation was carried out for 2 months at 6 °C. The resultant enriched cell suspension was spread-plated onto the surface of the PY medium supplemented with sodium acetate (0.1 %, w/v) and solidified with Difco agar (2 %, w/v), and the plates were then further incubated at 6 °C. As colonies became visible, they were successively restreaked onto fresh plates with the same agar medium until a culture with uniform colonies was obtained. This isolate, designated Z-0023T, was maintained both on agar slants and in liquid cultures using the above-described PY medium with acetate (PY-A) and PYG medium (0.1 % peptone, 0.1 % yeast extract, 0.1 % glucose, pH 6.7). Cell morphology and cell life cycle, as well as culture purity, were examined by using phase-contrast and electron microscopy. In the latter case, the samples were stained with 1 % (w/v) uranyl acetate. For preparation of thin sections, cells were collected from agar plates and prefixed 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 a graded series of ethanol, the samples were embedded in a Spurr epoxy resin. Thin sections were cut on an LKB-4800 microtome, and stained with 3 % (w/v) uranyl acetate in 70 % (v/v) ethanol. The specimen samples were examined with a JEM-100C transmission electron microscope. Growth of the isolate was monitored by nephelometry at 600 nm for 2–4 weeks in PY-A and PYG liquid media under a variety of conditions, including temperatures of 4–37 °C, pH 4.5–8.3 and NaCl concentrations of 0.1–5.0 % (w/v). Variations in acidity level were achieved by mixing 0.05 M solutions of Na2HPO4 and KH2PO4 to create media with the same ionic strength. To determine the range of potential growth substrates of strain Z-0023T, the following carbon sources were tested using liquid PY medium with addition of the respective compounds at a concentration of 0.1 % (w/v): D-glucose, L-arabinose, D-ribose, D-xylose, D-galactose, D-fructose, lactose, D-maltose, sucrose, D-cellobiose, D-mannose, D-melibiose, raffinose, L-rhamnose, D-trehalose, D-mannitol, D-sorbitol, starch, acetate, propionate, butyrate, pyruvate, malate, fumarate, succinate, citrate, methanol, ethanol, propanol, butanol, L-arginine, DL-alanine, L-phenylalanine, L-glutamate, L-lysine, L-proline, L-hydroxyproline, L-serine and L-tryptophan. Growth was examined after 8 days incubation at 20 °C. Sensitivity to antibiotics was tested by spreading 2-day-old cell suspension onto PYG agar medium and applying filter discs containing the following antibiotics: polymixin M (300 U), benzylpenicillin (10 U), ristomycin (30 µg), canomycin (30 µg), monomycin (30 µg), tetracycline (30 µg), erythromycin (15 µg), streptomycin (30 µg) and levomycetin (30 µg). Growth was assessed after 2 days incubation at 20 °C.
For fatty acid analyses, cells of strain Z-0023T were grown on PYG agar plates at 10 and 20 °C. Cells were saponified [15 % (w/v) NaOH, 30 min, 100 °C], methylated to fatty acid methyl esters (FAMEs) (methanolic HCl, 10 min, 80 °C) and extracted [hexane/methyl tert-butyl ether (1 : 1, v/v)] as described in detail by Osterhout et al. (1991). FAMEs were analysed on a gas chromatograph equipped with a flame-ionization detector and an autosampler. Separation of FAMEs was achieved using a fused-silica capillary column (25 mx0.2 mm) with cross-linked 5 % phenyl methyl silicone (film thickness 0.33 µm; HP Ultra 2). Injection temperature was 250 °C and detector temperature was 300 °C. The oven programme was 150 °C for 2 min, then increased from 150 to 289 °C at 4 °C min–1, followed by an isothermal period of 11 min. The instrument was equipped with a flame-ionization detector and an autosampler; H2 served as the carrier gas. FAMEs were identified by comparison with standards or by GC-MS (Abraham et al., 1998). For polar lipid fatty acid analysis, lipids were extracted using a modified Bligh–Dyer procedure (Bligh & Dyer, 1959) as described previously by Abraham et al. (1997). The extract of the total lipids was analysed by electrospray ionization using a quadrupole time-of-flight mass spectrometer (Yakimov et al., 2004).
Genomic DNA from strain Z-0023T was extracted using an SDS-based assay as described by Dedysh et al. (1998). The DNA base composition of strain Z-0023T was determined by thermal denaturation using a Unicam SP1800 spectrophotometer at a heating rate of 0.5 °C min–1. The G+C value was calculated by using the equation of Owen et al. (1969): G+C (%)=Tmx2.08–106.4. The DNA of Escherichia coli K-12 was used as the standard. DNA–DNA hybridization experiments between strain Z-0023T and two species of the genus Asticcacaulis, i.e. A. excentricus (DSM 4724T) and A. biprosthecium (DSM 4723T), were carried on nitrocellulose membrane filters (Hybond-N; Amersham International) according to Lysenko et al. (1988). The genome size of strain Z-0023T was calculated by using the equation of De Ley et al. (1970): M=[98.37–0.91xG+C (%)]/k, where k is the DNA reassociation rate. PCR-mediated amplification of the 16S rRNA gene from positions 28 to 1491 (numbering according to the International Union of Biochemistry nomenclature for E. coli 16S rRNA) was performed using primers Eub9f and Eub1492r and reaction conditions described by Lane (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).
Following growth on PY-A agar for 2 weeks at 6 °C, small (1–2 mm in diameter), smooth, circular, convex with an entire edge, opaque, white colonies were observed. Single colonies were successively selected from plates for restreaking until a pure culture, designated Z-0023T, was obtained. Microscopic examination revealed that cells of strain Z-0023T were Gram-negative, motile rods, 0.5–0.7 µm in width and 1.4–2.0 µm in length (Fig. 1a). These cells reproduced by binary fission and underwent a dimorphic life cycle during which a sessile cell with a cellular stalk (a prostheca) divided to give rise to a motile cell with a polar flagellum (Fig. 1b). A single, non-adhesive prostheca of these bacteria was 0.10–0.15 µm in diameter and had an excentral, subpolar location on the cell (Fig. 1c, d), which is characteristic of representatives of the genus Asticcacaulis (Poindexter, 1964; Pate et al., 1973). Prostheca length ranged from 0.5 to 5.0 µm depending on cultivation conditions; maximal length was attained in defined minimal media. In cells grown on rich complex media (e.g. PYG medium), prosthecae were short or were not observed, and the cells themselves appeared significantly elongated (up to 10-fold) and misshapen.
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). Cell division occurred within this sheath (Fig. 1d). Interestingly, these clothes-like sheaths appeared on cells grown at low temperatures (below 10 °C) but not on cells grown at moderate temperatures (15–25 °C). In stationary liquid cultures, most of the growth of strain Z-0023T occurred in the form of a pellicle that developed at the surface of the medium. In liquid cultures incubated on a rotary shaker, cells were evenly dispersed throughout the culture.
Similar to other known representatives of the genus Asticcacaulis, strain Z-0023T was not capable of growth in a mineral medium with glucose or another compound as the only growth substrate. It had an absolute requirement for the presence of growth factors in cultivation media. Thus, utilization of a given carbon compound was assumed to have occurred when growth was distinctly improved in its presence compared with on basal PY medium alone. The carbon compounds tested and the results are presented in Supplementary Table S1 (available in IJSEM Online). Most sugars, ethanol and several organic acids (acetate, malate, fumarate and succinate) were the preferred growth substrates. Strain Z-0023T differed from A. excentricus and A. biprosthecium by its inability to utilize pyruvate, and it could also be differentiated from A. taihuensis by its inability to utilize D-cellobiose, D-mannose and D-melibiose.
Strain Z-0023T grew in the pH range 4.5–8.3 with the optimum at pH 5.6–6.0. The temperature range for growth was 4–28 °C with the optimum at 15–20 °C. The culture generation time under optimal temperature conditions, calculated from increases in OD600 in the exponential phase of growth, was in the range 30–35 h. Both the specific growth rate and the growth yield of the culture at 25–28 °C were significantly lower than those attained at 15–20 °C. No growth occurred at 37 °C.
Compared with A. excentricus DSM 4724T and A. biprosthecium DSM 4723T (Abraham et al., 2001), NaCl was not required for growth of strain Z-0023T and this isolate had less tolerance of dissolved salts. Growth inhibition of 50 % was observed in the presence of NaCl in the medium at concentrations of 1–1.5 % (w/v), whereas NaCl at concentrations above 2.0 % completely inhibited growth. Strain Z-0023T was susceptible to antibiotics that inhibit prokaryotic protein synthesis, i.e. streptomycin, tetracycline, erythromycin, levomycetin and monomycin.
Whole-cell fatty acid content and comparison of glycolipid and phospholipid fatty acid profiles of strains Z-0023T, A. biprosthecium DSM 4723T, A. excentricus DSM 4724T and A. taihuensis T3-B7T are shown in Table 1 and in Supplementary Table S2 in IJSEM Online, respectively. As with recognized members of the genus Asticcacaulis, 18 : 17c was the major fatty acid in strain Z-0023T. The distinguishing feature of the fatty acid profile of strain Z-0023T was the absence of 12 : 1 3-OH, which was considered to be a common feature of the FAME profiles in Asticcacaulis (Abraham et al., 2001). Glycolipids present in recognized Asticcacaulis species are 
-D-glucopyranosyl- and -D-glucopyranuronosyl-diacylglycerols, which are also common in Caulobacter, Brevundimonas and some other members of the Alphaproteobacteria. In strain Z-0023T, four different glycolipids were detected, among them a compound of 796 Da previously detected in A. biprosthecium DSM 4723T but lacking in A. excentricus DSM 4724T. The electrospray ionization mass spectra of the phospholipids of strain Z-0023T showed clear differences from those of the other type strains (Supplementary Table S3 in IJSEM Online). Phospholipids of this bacterium were of the phosphatidylglycerol type and six different phospholipids could be identified, analogous to A. biprosthecium DSM 4723T and A. excentricus DSM 4724T. Interestingly, most of the phospholipids detected in strain Z-0023T were also found in A. excentricus DSM 4724T but not in A. biprosthecium DSM 4723T.
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). The novel isolate from tundra wetland peat was most closely related to the three known representatives of the genus Asticcacaulis, i.e. A. excentricus DSM 4724T (95 % 16S rRNA gene sequence similarity), A. taihuensis T3-B7T (98 %) and A. biprosthecium DSM 4723T (98 %). The DNA G+C content of strain Z-0023T was 60.4 mol% and the genome size was 2.5x109 Da. Levels of DNA–DNA hybridization between strain Z-0023T and A. biprosthecium DSM 4723T and A. excentricus DSM 4724T were 40 and 35 %, respectively.
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) and the absence of fatty acid 12 : 1 3-OH in the FAME profile. Thus we propose a novel species, Asticcacaulis benevestitus sp. nov., to accommodate this prosthecate bacterium from tundra wetland peat. The major characteristics differentiating this novel species from recognized species of the genus Asticcacaulis are summarized in Table 2.
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Cells are rod-shaped, Gram-negative, 0.5–0.7 by 1.4–2.0 µm. Reproduces by binary fission. Undergoes a dimorphic life cycle during which a sessile cell with one excentral, subpolar prostheca divides to give rise to a motile cell with a single, polar flagellum. A single, non-adhesive prostheca 0.10–0.15 µm in diameter arises from a subpolar site. At temperatures below 10 °C most prosthecate cells are embedded in a conical bell-shaped sheath, which is attached to the cell at the point of juncture of prostheca and cell. Colonies are opaque, white, smooth, circular, convex with an entire edge, and 1–2.5 mm in diameter after 7 days growth at 20 °C on PYG medium. Chemo-organotrophic aerobe. Carbon sources include glucose, sucrose, xylose, maltose, galactose, arabinose, lactose, fructose, ethanol, acetate, malate, fumarate, succinate, raffinose, rhamnose and trehalose. Yeast extract is required for growth. Temperature range for growth is 4–28 °C (optimum at 15–20 °C). Growth occurs between pH 4.5 and 8.3, with an optimum at pH 5.6–6.0. NaCl is not required for growth and is tolerated up to a concentration of 2.0 % (w/v). Susceptible to antibiotics that inhibit prokaryotic protein synthesis. The major fatty acid is 18 : 17c and the major phospholipids are phosphatidylglycerols. The DNA G+C content is 60.4 mol%. Genome size is 2.5x109 Da.
The type strain, Z-0023T (=DSM 16100T=ATCC BAA-896T), was isolated from a tundra wetland in the Vorkuta region, northern Russia.
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ACKNOWLEDGEMENTS |
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