Azospirillum thiophilum sp. nov., a diazotrophic bacterium isolated from a sulfide spring

The genus Azospirillum was first described by Tarrand et al. (1978) to comprise two species: Azospirillum lipoferum and Azospirillum brasilense. At present, the genus Azospirillum comprises 13 recognized species, most of which have been described in the last 10 years using polyphasic taxonomic approaches (Eckert et al., 2001; Xie & Yokota, 2005; Peng et al., 2006; Mehnaz et al., 2007a, b). Azospirillum species are widely distributed, especially in the soils of tropical, subtropical and temperate regions all over the world, where they are frequently associated with grasses, cereals and crops (Döbereiner et al., 1976; Bally et al., 1983; Ladha et al., 1987; Kirchhof et al., 1997; Gunarto et al., 1998; Magalhães et al., 1983; Reinhold et al., 1987; Khammas et al., 1989; Ben Dekhil et al., 1997). Two free-living species of the genus Azospirillum, Azospirillum rugosum and Azospirillum picis, isolated from oil-contaminated soil and discarded road tar, respectively, have been described recently (Young et al., 2008; Lin et al., 2009). The present study includes the characterization of a novel strain of the genus Azospirillum, designated BV-S^T, which was isolated from a bacterial mat of a sulfide spring.

Strain BV-S^T was isolated from a bacterial mat of the sulfide spring ‘Shameless Baths’ in Stavropol Krai, North Caucasus, Russia (nearly 28 °C, pH 7.8, 1.4–1.6 g minerals ml⁻¹, 3.4 mg sulfide l⁻¹). The bacterial mat was dominated by sulfur-oxidizing representatives of Sphaerotilus natans and had developed on a slope alongside the water flflow (Gridneva et al., 2009). Isolation of strain BV-S^T was carried out in a semi-solid MPSS medium (Caraway & Krieg, 1974) with a freshly prepared FeS suspension (Kucera & Wolfe, 1957) of the following composition (l⁻¹): 1 g (NH₄)₂SO₄, 1 g MgSO₄, 0.03 g CaCl₂ . 2H₂O, 0.002 g FeCl₃ . 6H₂O, 0.002 g MnSO₄, 1 g sodium succinate, 1 g casein hydrolysate, 1 g agar (Difco); pH 7.5. Vitamins and trace elements (Pfennig & Lippert, 1966) were added before inoculation. Routine cultivation was carried out in liquid MPSS medium without FeS and with casein hydrolysate replaced with 2 g peptone l⁻¹.

The novel strain showed abundant growth in medium with an O₂/H₂S (FeS, CaS or Na₂S) gradient. Colonies on nutrient agar (NA) after 48 h of cultivation were white, smooth, round and flat, with a diameter of 4–6 mm. Morphology was observed by light microscopy (CX 4; Olympus) equipped with a phase-contrast device and by transmission electron microscopy (JEM-100C; JEOL). Cells of strain BV-S^T grown in semi-solid MPSS medium were spiral, curved rods, 1.1–2.0 μm wide and 3.6–7.0 μm long, and motile by a single flagellum. Cells grown in semi-solid nitrogen-free MPSS medium [without (NH₄)₂SO₄ and peptone] also showed the presence of cysts. A striking feature of the cells was the presence of numerous intracellular sulfur globules when grown in a medium containing sulfide (Fig. 1⇓) or in the natural environment.

Strain BV-S^T was examined for a range of phenotypic and biochemical properties using standard methods (Gerhardt et al., 1981). The physiological properties of strain BV-S^T and type strains of closely related species of the genus Azospirillum were determined in MPSS medium containing single carbon sources. Strain BV-S^T grew at 15–40 °C (optimum 37 °C), at pH 6.5–8.5 (optimum pH 7.5) and with 0–3 % (w/v) NaCl. A comparison of the morphological and physiological properties of strain BV-S^T and type strains of species of the genus Azospirillum is given in Table 1⇓ and in Supplementary Table S1 (available in IJSEM Online).

Strain BV-S^T grew in a medium without nitrogen and was capable of N₂ assimilation. The ability of the strain to fix nitrogen was tested using the acetylene-reduction technique of Stewart et al. (1968). Briefly, strain BV-S^T was grown in triplicate in Hungate tubes in a nitrogen-free medium containing succinate under microaerobic conditions (5 % O₂, v/v, in the headspace) to mid-exponential growth phase. Acetylene was added to a final concentration of about 10 % (v/v) in the gas phase and the cultures were incubated at 22 °C on a rotary shaker (200 r.p.m.) for 24 h. The amount of ethylene produced was measured with a Cristall 5000.1 gas chromatograph equipped with a flame-ionization detector. An uninoculated tube was used to determine the level of ethylene contamination in the acetylene. Acetylene-reduction activity ranged from 1000 to 2000 nmol ethylene h⁻¹ (mg protein)⁻¹ .

To create microaerobic conditions to determine metabolic characteristics, rubber-stoppered 50 ml vials with 10 ml MPSS medium were flushed with filter-sterilized argon and sterile air was injected to a final concentration of 5 % (v/v) oxygen in the gas phase. In contrast to known Azospirillum species, strain BV-S^T was capable of mixotrophic growth under microaerobic conditions with simultaneous utilization of organic substrates and thiosulfate as electron donors for energy metabolism (Lavrinenko et al., 2009). This was confirmed by using enzymic methods and polarographic analysis. Thiosulfate : ferricyanide oxidoreductase activity was tested as described by Petushkova & Ivanovsky (1976) and high activity [135.1–763.2 nmol min⁻¹ (mg protein)⁻¹] was found. The respiratory activity of the cell suspension was measured using a standard Clark-type oxygen electrode at 25 °C. Thiosulfate oxidation [67–300 nmol min⁻¹ (mg protein)⁻¹] was associated with the respiratory chain. Sulfide oxidation was accompanied by deposits of sulfur globules within the cells.

Genomic DNA was extracted from cells of strain BV-S^T using the protocol of Ausubel et al. (1994) with slight modifications and using the freeze–thaw method (Bej et al., 1991). To determine the phylogenetic relationships between strain BV-S^T and type strains of species of the genus Azospirillum, the 16S rRNA gene was amplified using primers 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′-TACGGYTACCTTGTTACGACTT-3′) (Lane, 1991; Medlin et al., 1988) and the amplification products were sequenced using a CEQ2000 XL automatic sequencer (Beckman Coulter). The 16S rRNA gene sequence (1316 nt) of strain BV-S^T was compared with 16S rRNA gene sequences in the GenBank database using blast (Altschul et al., 1997) and an alignment was computed using clustal x (Thompson et al., 1997). A phylogenetic tree was constructed using treecon (Van de Peer & De Wachter, 1994) and the neighbour-joining method (Saitou & Nei, 1987), using an evolutionary-distance matrix that was calculated using the correction of Jukes & Cantor (1969). The robustness of the tree topology was evaluated by calculating bootstrap values for 1000 resamplings. The analysis showed that the isolate belonged to the cluster for the genus Azospirillum (Fig. 2⇓) and formed a distinct subclade with Azospirillum doebereinerae GSF71^T (90 % bootstrap value). The closest described relatives of strain BV-S^T were A. doebereinerae GSF71^T, A. picis IMMIB TAR-3^T and A. lipoferum ATCC 29707^T (97.7, 97.7 and 97.4 % 16S rRNA gene sequence similarity, respectively). 16S rRNA gene sequence similarities with type strains of other known species of the genus Azospirillum were ≤97.0 %.

DNA–DNA hybridization experiments were performed in triplicate between strain BV-S^T and A. doebereinerae DSM 13131^T, A. picis DSM 19922^T and A. lipoferum ATCC 29707^T using the optical renaturation method (De Ley et al., 1970) and a Pye Unicam SP 1800 spectrophotometer equipped with a thermoprogrammer and hermetically sealed thermocuvettes. The standard deviations of the triplicate experiments ranged between 2.0 and 3.7 %. DNA–DNA relatedness between strain BV-S^T and A. doebereinerae DSM 13131^T, A. picis DSM 19922^T and A. lipoferum ATCC 29707^T was 38, 55 and 42 %, respectively (Supplementary Table S2). DNA base composition was determined by the thermal denaturation method as described by Owen & Lapage (1976). Strain BV-S^T had a DNA G+C content of 67 mol%, which is consistent with the range of 64–71 mol% reported for the genus Azospirillum (Mehnaz et al., 2007a, b; Young et al., 2008; Lin et al., 2009).

Partial nifH sequences for strain BV-S^T and A. doebereinerae DSM 13131^T were determined using two pairs of primers, F1 (5′-TAYGGIAARGGIAARGGIGGIATIGGIAARTC-3′) plus nifH-3r (5′-TTGTTGGCIGCRTASAKIGCCATT-3′) and nifH-univ (5′-GCIWTITAYGGIAARGGIGG-3′) plus R6 (5′-GCCATCATYTCICCIGA-3′), and the protocols of Fedorov et al. (2008) and Marusina et al. (2001). Amplification products of the expected sizes (450 and 380 bp) were obtained from strain BV-S^T and A. doebereinerae DSM 13131^T, respectively. Amplification reactions with A. picis DSM 19922^T yielded no amplification products, probably because of mismatches at the 3′ ends of the primers. The amplification products of strain BV-S^T and A. doebereinerae DSM 13131^T were purified and sequenced and the sequences were compared with nifH sequences retrieved from the GenBank database for A. lipoferum ATCC 29707^T and other members of the genus Azospirillum. Strain BV-S^T had the highest nifH sequence similarity with A. doebereinerae DSM 13131^T (96.8 %). Similarities between nifH sequences of strain BV-S^T and other members of the genus Azospirillum ranged from 94.5 to 96.7 %.

The fatty acids of strain BV-S^T, A. doebereinerae DSM 13131^T, A. picis DSM 19922^T and A. lipoferum ATCC 29707^T were determined using the Sherlock Microbial Identification System (MIDI), according to the manufacturer's protocol (Stead et al., 1992). The fatty acid profiles are compared in Table 2⇓. The major fatty acid in strain BV-S^T was C_18 : 1ω7c. The isoprenoid quinones of strain BV-S^T and A. doebereinerae DSM 13131^T were extracted and purified as described by Collins & Jones (1981). Strains BV-S^T and A. doebereinerae DSM 13131^T contained Q-10 as the major isoprenoid quinone of the respiratory chain.

In conclusion, the results of the present study, including morphological, chemotaxonomic and phylogenetic evidence and physiological and biochemical differences, indicate that strain BV-S^T represents a novel species of the genus Azospirillum, for which the name Azospirillum thiophilum sp. nov. is proposed.