Saccharibacillus kuerlensis sp. nov., isolated from a desert soil

The genus Saccharibacillus of the family ‘Paenibacillaceae’ was first proposed by Rivas et al. (2008) to include a Gram-variable, facultatively anaerobic, motile and rod-shaped bacterium that was negative for catalase and oxidase activities and that had anteiso-C_15 : 0 as the main fatty acid and menaquinone-7 as the major respiratory menaquinone. At present, the genus comprises only one species, Saccharibacillus sacchari.

In our search for organisms capable of growing in desert ecosystems, a bacterial strain, designated HR1^T, was isolated from a soil sample collected from the southern desert of Kuerle (4 ° 46′ N 8 ° 07′ E), a city at the heart of Xinjiang, the most westerly province of China. The combination of phenotypic and chemotaxonomic characteristics, phylogenetic analysis based on 16S rRNA gene sequences and DNA–DNA hybridization showed that strain HR1^T is a representative of a novel species of the genus Saccharibacillus.

Strain HR1^T was isolated using the standard dilution-plating technique on Luria–Bertani (LB) agar medium. The isolate was cultivated routinely on LB agar at 30 °C under aerobic conditions and maintained as a glycerol suspension (20 %, w/v) at −70 °C. Biomass for molecular systematic and chemotaxonomic studies was obtained by culturing strain HR1^T in liquid LB medium and harvesting the cells by centrifugation.

Amplification and sequencing of the 16S rRNA gene was performed as described by Cui et al. (2001). The sequence obtained was compared with available 16S rRNA gene sequences retrieved from GenBank using the blast program () to determine an approximate phylogenetic affiliation. Phylogenetic analysis was performed using the software packages phylip (Felsenstein, 1993) and mega version 3.1 (Kumar et al., 2001) after multiple alignment of the data by clustal_x (Thompson et al., 1997). Distances (distance options according to Kimura's two-parameter model; Kimura, 1980, 1983) and clustering were based on the neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Kluge & Farris, 1969) methods. Bootstrap analysis (1000 replications) was used to evaluate the topology of the neighbour-joining tree (Felsenstein, 1985).

The almost-complete 16S rRNA gene sequence of strain HR1^T (1513 bp) revealed the highest similarity to S. sacchari LMG 24085^T (97.9 %); the sequence similarities to members of other genera of the family ‘Paenibacillaceae’ with validly published names were lower than 93.4 %. Phylogenetic trees obtained with different algorithms were identical with respect to the position of strain HR1^T, which formed a distinct branch with its closest relative S. sacchari LMG 24085^T. A phylogenetic tree based on the neighbour-joining algorithm is shown in Fig. 1⇓.

Genomic DNA was extracted and purified according to Yoon et al. (1996) and the DNA G+C content was determined using the thermal denaturation method (Mandel & Marmur, 1968). The G+C content of the DNA was 50.5 mol%, which is slightly lower than the value recorded for S. sacchari LMG 24085^T (57.8 mol%; Rivas et al., 2008). DNA–DNA hybridization was carried out to evaluate the genomic DNA relatedness between strain HR1^T and S. sacchari LMG 24085^T by applying the optical renaturation rate method as described by De Ley et al. (1970) with the modifications described by Huß et al. (1983) and Escara & Hutton (1980). Renaturation rates were computed with the program transfer.bas (Jahnke, 1992). The DNA–DNA relatedness between strain HR1^T and its closest relative S. sacchari LMG 24085^T was 29.6 %. According to Stackebrandt & Goebel (1994) and Wayne et al. (1987), the values for 16S rRNA gene similarity (<97 %) and DNA–DNA relatedness (<70 %) support the genomic distinction of strain HR1^T from S. sacchari LMG 24085^T.

For quantitative analysis of the cellular fatty acid content, cells were harvested after 2 days growth at 30 °C on tryptic soy agar (Merck) and fatty acid methyl esters were prepared and identified by following the instructions of the Microbial Identification system (MIDI) as described by Sasser (1990). The type strain of the closely related species S. sacchari (LMG 24085^T) was also cultured under the same nutrient and temperature conditions and characterized concurrently with strain HR1^T. Determination of the cell-wall peptidoglycan was performed using the methods of Schleifer & Kandler (1972). Polar lipids were extracted, examined by two-dimensional TLC and identified following Minnikin et al. (1984). Menaquinones were analysed as described by Komagata & Suzuki (1987) using reversed-phase HPLC. The cellular fatty acids of strain HR1^T are listed in Table 1⇓ and compared with S. sacchari LMG 24085^T. Anteiso-C_15 : 0, the major fatty acid found in S. sacchari LMG 24085^T (Rivas et al., 2008), was also the major fatty acid component of strain HR1^T, comprising 50.3 % of the total. The peptidoglycan type was meso-diaminopimelic acid, in line with all other members of the family ‘Paenibacillaceae’, which includes the genus Saccharibacillus. The predominant isoprenoid quinone was MK-7, which supports the affiliation of strain HR1^T to the genus Saccharibacillus; in addition, a small amount of MK-6 was detected. This profile is slightly different from that of S. sacchari LMG 24085^T, in which small amounts of both MK-6 and an unidentified menaquinone were detected (Rivas et al., 2008). The polar lipids of strain HR1^T are diphosphatidylglycerol, phosphatidylglycerol and an unknown phosphoglycolipid. Glycolipids and aminophospholipids, which occur in S. sacchari LMG 24085^T, and phosphatidylethanolamine, which occurs in members of other genera of the family ‘Paenibacillaceae’, were not detected. However, strain HR1^T contained the unknown phosphoglycolipid that is present in S. sacchari LMG 24085^T but has not been reported in other genera. This polar lipid profile provided decisive evidence that strain HR1^T belongs to the genus Saccharibacillus and is different from other related species.

Morphological features were examined by using light microscopy (model BH 2; Olympus) and transmission electron microscopy (Hitachi-7650) after 24 h growth on LB agar at 30 °C. For transmission electron microscopy observation, cells were negatively stained with 1 % (w/v) phosphotungstic acid and, after air drying, Gram-staining and endospore formation were investigated as described by Smibert & Krieg (1994). Growth at different temperatures and pH values was investigated as described by Xu et al. (2005), using LB broth as the basal medium. NaCl tolerance was tested using LB broth supplemented with 0, 1, 2, 3, 5 and 6 % NaCl, respectively. Growth was determined on cetrimide agar, Simmons' citrate agar, MacConkey agar and nutrient agar. All tests were incubated at 30 °C for 2 days. Antibiotic sensitivity tests were performed using the diffusion method on LB agar at 30 °C with filter-paper discs (8 mm diameter) containing one of the following antibiotics: amoxicillin (10 μg), ampicillin (10 μg), carbenicillin (100 μg), cefamezin (30 μg), ceftazidime (30 μg), chloramphenicol (30 μg), erythromycin (15 μg), gentamicin (10 μg), kanamycin (30 μg), ofloxacin (5 μg), penicillin G (10 IU), polymyxin B (30 μg), rifampicin (5 μg), streptomycin (10 μg), tetracycline (30 μg) and vancomycin (30 μg). After 2 days of incubation, the diameters of the inhibition zones were measured. Acid production from carbohydrates was determined using the medium and methods described by Yamaguchi & Yokoe (2000). Single carbon-source utilization was determined as described by Liu et al. (2008). Oxidase activity was tested by determining the oxidation of 1 % (w/v) tetramethyl-p-phenylenediamine (Merck) and catalase activity was evaluated by determining the production of oxygen bubbles in a 5 % (v/v) aqueous hydrogen peroxide solution. Phenylalanine deaminase activity and hydrolysis of Tweens 80 and 20, aesculin, DNA and CM-cellulose were investigated as described by Smibert & Krieg (1994). Urease activity and hydrolysis of casein, gelatin, tyrosine and starch were determined as described by Cowan & Steel (1965). Some other biochemical characteristics were tested using API 20E kits (bioMérieux) according to the instructions of the manufacturer. Cells of strain HR1^T stained Gram-positive and were motile, sporulating rods with a single lateral flagellum, about 0.4–0.5 μm wide and 0.8–1.4 μm long (Fig. 2⇓). Colonies were convex, almost-circular, opaque, non-glossy and pink after 48 h of incubation at 30 °C. Phenotypic features of strain HR1^T are given in the species description and in Table 2⇓.

In the light of the combined phylogenetic, biochemical and morphological data, strain HR1^T should be classified as the type strain of a novel species within the genus Saccharibacillus, for which the name Saccharibacillus kuerlensis sp. nov. is proposed.