Sphingomonas yabuuchiae sp. nov. and Brevundimonas nasdae sp. nov., isolated from the Russian space laboratory Mir

Exploring space is a challenge for human beings. The space station is a special closed environment where astronauts are constantly faced with the unfavourable effects of space flight and where medical treatment facilities are extremely limited. Basic data on the bacterial microflora inside the space station are essential to manage the astronauts' health. As part of an international research effort between Japan and Russia, we investigated the bacterial microflora from condensation water and air on the Russian space laboratory Mir in 1997. The bacterial population in the space station has been reported previously by Kawamura et al. (2001). In this study, we determined the taxonomic status of two Gram-negative isolates that belong to the α-Proteobacteria. Polyphasic taxonomic study of the isolates showed that they belonged to the genera Sphingomonas and Brevundimonas, both affiliated to the α-Proteobacteria (Garrity & Holt, 2001), but neither isolate could be classified as any existing species.

The genus Sphingomonas was proposed by Yabuuchi et al. (1990). Members of the genus are non-fermentative, Gram-negative, non-motile or motile rods and are characterized by the presence of 2-hydroxymyristic acid (2-OH 14 : 0), by the absence of 3-hydroxy fatty acids and by the presence of ubiquinone 10 (Q10). The genus Brevundimonas was proposed by Segers et al. (1994). All strains are characterized by two major fatty acids, 16 : 0 and 18 : 1, and the G+C content ranges from 65 to 68 mol% (Segers et al., 1994).

In this study, we used morphological, biochemical, chemotaxonomic and genetic data to classify these two strains and propose two novel species, Sphingomonas yabuuchiae sp. nov. and Brevundimonas nasdae sp. nov.

Sample collection, strains, culture and DNA extraction.
Air samples from the living environment of the Mir station were collected by an air vacuum sampler 16 and 3 days before the end of the mission and trapped on Millipore HA filters (0·45 µm pore size). The filters were placed in TGE medium (l¹: 5·0 g beef extract, 10·0 g tryptone, 2·0 g glucose, 1 g triphenyl tetrazolium) and kept at room temperature under aerobic conditions for 7 days and then stored at 28 °C until the return to Earth. Three samples of condensation water were collected in different rooms 3 days before the end of the mission and stored at 28 °C. All samples were cultured directly on various selected media at 30 and 37 °C under aerobic, 5 % CO₂ and anaerobic conditions for 3 days to 4 weeks after transfer to the laboratory. Single colonies were transferred to brain heart infusion (BHI) agar plates and incubated at 30 or 37 °C.

The following type strains were cultured on BHI agar plates at 30 °C: Sphingomonas sanguinis GTC 829^T (=IFO 13937^T), Sphingomonas parapaucimobilis GTC 413^T (=JCM 7570^T), Sphingomonas paucimobilis Gifu 2395^T (=ATCC 29837^T), Sphingomonas trueperi EY 4281^T (=ATCC 12417^T), Sphingomonas roseiflava GTC 1968^T (=IAM 14823^T), Sphingomonas pituitosa EY 4370^T (=DSM 13101^T), Brevundimonas intermedia GTC 1682^T (=DSM 4732^T), Brevundimonas aurantiaca GTC 1681^T (=DSM 4731^T), Brevundimonas diminuta GTC 14^T (=NCTC 8548^T) and Brevundimonas vesicularis Gifu 2388^T (=ATCC 11426^T). DNA was extracted as described previously (Ezaki et al., 1994).

Sequencing of the 16S rRNA gene, phylogenetic analysis and determination of DNA composition.
The 16S rRNA gene was amplified with universal primers and PCR products for all isolates were sequenced in both directions. The sequences were analysed with the FASTA search system (Pearson & Lipman, 1988) on the DDBJ website () to find closely related bacterial 16S rDNA sequences. CLUSTAL W software (Thompson et al., 1994) was also used to determine the phylogenetic relationships of the isolates. Phylogenetic trees were produced according to the neighbour-joining method (Saitou & Nei, 1987) and visualized using TreeView (Page, 1996). G+C content was measured by HPLC as described previously (Ezaki et al., 1990). Escherichia coli was used as a standard (G+C content 51·19 mol%).

Determination of biochemical characteristics.
Biochemical characterization of these strains was performed using the Biolog GN2 MicroPlate assay, Nonfergram S-1 kit (Wako Chemical) and API 20 NE (API bioMérieux). Preparations were performed according to the manufacturers' instructions. Oxidase activity was determined with Oxidase test strips (Eiken Chemical). Catalase activity was estimated by the production of bubbles from 3 % hydrogen peroxide solutions.

Determination of isoprenoid quinones and cellular fatty acids.
Isoprenoid quinone analysis was performed as previously described (Yano et al., 1987). Fatty acid methyl esters were extracted and prepared using the standard protocol for the MIDI/Hewlett Packard Microbial Identification System. Fatty acid methyl ester extracts were analysed using a Hewlett Packard GC (model HP6890) equipped with a flame-ionization detector, an automatic sampler, an integrator and a computer, as described by Kämpfer & Kroppenstedt (1996).

DNADNA hybridization.
Quantitative microplate DNADNA hybridization for selected strains was carried out as described previously (Ezaki et al., 1989). Hybridization experiments were carried out under optimal and stringent temperatures calculated from the melting temperatures (T_m) based on the G+C content of each test strain.

Thirty-six bacterial colonies were isolated from air samples and bacteria were found at concentrations of 2·1x10⁶, 5·2x10² and 3·0x10¹ c.f.u. ml¹ in three condensation water samples (Kawamura et al., 2001). After performing morphological observations, we selected 16 and seven representative isolates from air samples and condensation water samples, respectively, to characterize further.

16S rDNA sequence similarity analysis showed that strain A1-18^T and W1-2B^T were affiliated with two genera within the α-Proteobacteria, Sphingomonas and Brevundimonas, respectively. Finally, A1-18^T and W1-2B^T were confirmed to represent distinct species after assessment of their taxonomic status by polyphasic taxonomic study.

Strain A1-18^T
Strain A1-18^T was isolated from an air sample. 16S rDNA sequence analysis showed that strain A1-18^T formed a coherent cluster with S. sanguinis, S. parapaucimobilis, S. paucimobilis and S. roseiflava (Fig. 1). 16S rDNA sequence similarity was determined to be 98·6, 98·4, 97·5 and 98·3 %, respectively, by CLUSTAL W analysis and the similarity to other members of genus Sphingomonas was lower than 97 %. The DNA G+C content of strain A1-18^T (66·1 mol%) was within the range of values (61·667·8 mol%) reported for established Sphingomonas species (Denner et al., 2001). Therefore, strain A1-18^T is affiliated phylogenetically with the genus Sphingomonas.

(57K): Fig. 1. Phylogenetic position of two Gram-negative isolates from the Mir space station and selected members of the genera Sphingomonas and Brevundimonas based on 16S rDNA sequences. Distances were calculated by the neighbour-joining method. Numbers at branch points are bootstrap values (based on 1000 samplings). GenBank accession numbers are given in parentheses. Bar, 0·01 estimated substitutions per nucleotide position. Arthrobacter oxydans was used as an outgroup.

Strain A1-18^T showed some similar morphological properties to Sphingomonas species (see species description). Some common biochemical properties with related species (shown in Table 1) of the genus Sphingomonas were also found: the strain was positive for catalase and β-galactosidase activities, hydrolysis of aesculin and assimilation of glucose, L-arabinose, maltose and DL-malate and was negative for arginine dihydrolase and urease activities and assimilation of D-mannitol and phenylacetate. However, some biochemical characteristics also differentiated strain A1-18^T biochemically from other related species (Table 1).

Table 1. Comparison of properties between Sphingomonas yabuuchiae sp. nov. A1-18T and closely related Sphingomonas species Strains: 1, S. yabuuchiae sp. nov. A1-18T; 2, S. paucimobilis ATCC 29837T; 3, S. parapaucimobilis JCM 7510T; 4, S. sanguinis IFO 13937T; 5, S. trueperi ATCC 12417T; 6, S. pituitosa DSM 13101T; 7, S. roseiflava IAM 14823T. W, Weakly positive reaction; DY, deep yellow; LY, light yellow; PY, pinkyellow; ND, not done. Data were taken from this study, Yabuuchi et al. (1990), Kämpfer et al. (1997), Denner et al. (2001) and Yun et al. (2000).

Reverse-phase TLC of isoprenoid quinones extracted from A1-18^T showed a major spot corresponding to Q10, the characteristic respiratory quinone of the genus Sphingomonas (Yabuuchi et al., 2002). Cellular fatty acid composition was unique and distinguished strain A1-18^T from its phylogenetic neighbours. The presence of 2-hydroxymyristic acid (2-OH 14 : 0) and the absence of 3-hydroxy fatty acids in strain A1-18^T are consistent with other related species of the genus Sphingomonas (Table 2), but quantitative differences in the fatty acid composition distinguished A1-18^T from related species (Table 2).

Table 2. Fatty acid composition of selected Sphingomonas and Brevundimonas species Values are percentages of total fatty acids. Strains: 1, S. yabuuchiae sp. nov. A1-18T; 2, S. paucimobilis ATCC 29837T; 3, S. parapaucimobilis JCM 7510T; 4, S. sanguinis IFO 13937T; 5, S. trueperi ATCC 12417T; 6, B. nasdae sp. nov. W1-2BT; 7, B. aurantiaca DSM 4731T; 8, B. intermedia DSM 4732T; 9, B. diminuta NCTC 8548T; 10, B. vesicularis ATCC 11426T. ECL, Equivalent chain length; tr, trace (<1·0 %); , not detected. Data were from the present study, Abraham et al. (1999) and Denner et al. (2001). Summed features represent groups of two or three fatty acids that could not be separated by GLC with the MIDI system (Microbial ID). Summed feature 4 contained one or more of the following fatty acids: 16 : 1ω7t, 15 : 0 iso 2-OH and 16 : 1ω7c. Summed feature 7 contained one or more of the following fatty acids: 18 : 1ω7c, 18 : 1ω9t and/or 18 : 1ω12t. In addition, B. nasdae contained 15 : 0 iso (1·54 %) and 17 : 0 iso (1·05 %).

It is necessary to perform DNADNA hybridization to judge whether a novel isolate belongs to the same species when they share more than 97 % 16S rDNA sequence similarity (Stackebrandt & Goebel, 1994). Thus, the four species S. sanguinis, S. parapaucimobilis, S. paucimobilis and S. roseiflava were selected for DNADNA hybridization. DNADNA hybridization rates at optimal temperatures among these related species were only 46·4, 51·7, 40·7 and 24·7 % (Supplementary Table A in IJSEM Online). All hybridization rates were below the threshold value (approximately 70 %) under optimal hybridization conditions (T_m25 °C) that has been suggested as delineating a bacterial species (Grimont, 1999; Wayne et al., 1987). This confirmed that this strain represents a genetically independent species.

In summary, morphological, biochemical, chemotaxonomic and phylogenetic study demonstrated that strain A1-18^T (=GTC 868^T) represents a novel species within the genus Sphingomonas, for which we propose the name Sphingomonas yabuuchiae sp. nov.

Strain W1-2B^T
Strain W1-2B^T was isolated from condensed water. 16S rDNA sequence analysis indicated that strain W1-2B^T forms a distinct lineage within the genus Brevundimonas, forming a coherent cluster with B. vesicularis, B. aurantiaca and B. intermedia (Fig. 1). 16S sequence similarity was respectively 99·5, 98·8, 98·6 % and the similarities to other species of genus Brevundimonas were less than 97 % by CLUSTAL W analysis. The G+C content was 66·5 mol%, which is within the characteristic range of the genus Brevundimonas (6568 mol%; Segers et al., 1994). These genetic results indicated that strain W1-2B^T is affiliated with the genus Brevundimonas.

Some similar phenotypic and biochemical characteristics to the Brevundimonas species also supported the assignment of strain W1-2B^T as a member of the genus Brevundimonas. Cells were short, neatly arrayed rods arranged in a rosette pattern, Gram-negative, 1·54 µm long, 0·5 µm in diameter and motile in 0·3 % semi-solid agar. Colonies were slightly yellow, circular and smooth. No growth was observed at 4 °C. Like other species of genus Brevundimonas (Segers et al., 1994), strain W1-2B^T is oxidase- and catalase-positive and the strain does not form indole, produce lipase (Tween 80 hydrolysis) or liquefy gelatin. However, some biochemical characteristics differentiate strain W1-2B^T from closely related species of the genus Brevundimonas (Table 3).

Table 3. Comparison of properties between Brevundimonas nasdae sp. nov. W1-2BT and related species of the genus Brevundimonas Strains: 1, B. nasdae sp. nov. W1-2BT; 2, B. aurantiaca DSM 4731T; 3, B. intermedia DSM 4732T; 4, B. diminuta NCTC 8548T; 5, B. vesicularis ATCC 11426T. All strains were positive for catalase activity and negative for reduction of nitrate and nitrite, production of indole, acid from glucose and assimilation of L-arabinose, potassium gluconate, adipic acid, sodium citrate and phenyl acetate. W, Weakly positive.

Examination of the reverse-phase, thin-layer chromatogram of isoprenoid quinones extracted from strain W1-2B^T indicated that only ubiquinone was present. Strain W1-2B^T produced a major spot corresponding to Q10, characteristic of the genus Brevundimonas, members of which are aerobic with a respiratory type metabolism and Q10 is an intermediate electron carrier (Segers et al., 1994; Abraham et al., 1999). Cellular fatty acid analysis showed that summed feature 7 (one or more of 18 : 1ω9t, 18 : 1ω7c and 18 : 1ω12t; 51·6 %) and 16 : 0 (19·7 %) were the major fatty acids. This strain contained no 2-hydroxy acids, but did contain 3-hydroxy fatty acids (Table 2). These cellular fatty acid profiles are consistent with the description of the genus Brevundimonas: all strains are characterized by two major fatty acids, 16 : 0 and 18 : 1 (18 : 1ω7c, 18 : 1ω9t and 18 : 1ω12t) (Segers et al., 1994). Quantitative differences in the fatty acid composition, however, distinguished strain W1-2B^T from the most closely related species B. vesicularis and B. intermedia (Table 2).

The DNADNA hybridization data showed that strain W1-2B^T had relatedness values of only 51·6, 54·8 and 50·2 %, respectively, with its closely related neighbours, B. vesicularis, B. aurantiaca and B. intermedia (Supplementary Table B in IJSEM Online). All hybridization rates were below the threshold value (approximately 70 %) that has been suggested as delineating a bacterial species (Wayne et al., 1987). The quantitative hybridization results indicate that this strain genetically represents an independent species.

In summary, the characteristics of strain W1-2B^T are consistent with the description of genus Brevundimonas with regard to morphology, biochemical and chemotaxonomic properties. They also support the conclusion from 16S rDNA alignment that strain W1-2B^T belongs to the genus Brevundimonas. However, some biochemical reactions and cellular fatty acid composition differentiate it from existing species of the genus. DNADNA hybridization further confirmed that strain W1-2B^T taxonomically represents an independent species. On the basis of these results, strain W1-2B^T is proposed as the type strain of a novel species of the genus Brevundimonas, Brevundimonas nasdae sp. nov.

Description of Sphingomonas yabuuchiae sp. nov.
Sphingomonas yabuuchiae (ya.bu.u'chi.ae. N.L. gen. n. yabuuchiae of Yabuuchi, in honour of Eiko Yabuuchi, a Japanese bacteriologist, who proposed the genus name Sphingomonas).

Grows well under aerobic conditions at 30 °C on BHI agar plates. Gram-negative, has rod-shaped cells with rounded ends, 14 µm long and 0·5 µm in diameter. Colonies are deep yellow, circular and smooth. Hydrolyses glycerol, starch and Tween 80 but not gelatin or ornithine. Does not reduce nitrate. Utilizes L-arabinose, cellobiose, fructose, galactose, glucose, lactose, maltose, mannose, melibiose, raffinose, sucrose and trehalose but not adonitol, inositol, mannitol, rhamnose or sorbitol. Catalase activity is positive. Q10 is the major isoprenoid quinone. 18 : 1 is the major cellular fatty acid and the major 2-hydroxy acid is 2-OH 14 : 0, while 3-hydroxy fatty acids are absent. The G+C content is 66·1 mol%.

The type strain, A1-18^T (=GTC 868^T=JCM 11416^T=DSM 14562^T), was isolated from an air sample from the Russian space station Mir.

Description of Brevundimonas nasdae sp. nov.
Brevundimonas nasdae (nas'dae. N.L. gen. n. nasdae of NASDA, arbitrary name referring to the National Space Development Agency of Japan, which contributed to this project).

Grows well under aerobic conditions at 30 °C on BHI agar plates. Gram-negative, has short rod-shaped cells and produces creamy white, circular and smooth colonies. No growth at 4 °C or in media containing 4 % NaCl. Positive for oxidase and catalase activity and negative for reduction of nitrate and nitrite, production of phenylalanine deaminase, urease, indole and acylamidase, hydrolysis of DNA, starch and arginine, decarboxylation of lysine and acid from mannitol. Utilizes acetate, pyruvate, methyl pyruvate, succinate and amino acids, including L-alanine, L-aspartic acid, glutamate and L-proline, but not arabinose, mannose, fructose or lactose. Produces acid from glucose, galactose, maltose and sucrose but is not able to hydrolyse Tween 80 or glycerol. Q10 is the major isoprenoid quinone. 18 : 1 (51·6 %) and 16 : 0 (19·7 %) are the most abundant cellular fatty acids and the G+C content is 66·5 mol%.

The type strain, W1-2B^T (=GTC 1043^T=JCM 11415^T =DSM 14572^T), was isolated from a condensation water sample from the Russian space station Mir.

This work was supported by the first Japanese and Russian collaboration on the Mir Utilization Project and a grant from the Japan Space Utilization Promotion Center (JSUP).