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1 Biotechnology Research Center, The University of Tokyo, Yayoi 1-1-1, Bunkyo-Ku, Tokyo 113-8657, Japan
2 SORST, JST, Chiyoda-Ku, Tokyo, Japan
3 National Agricultural Research Center, Park Road, Islamabad 45500, Pakistan
4 Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-Ku, Tokyo 113-0032, Japan
Correspondence
Toru Fujiwara
atorufu{at}mail.ecc.u-tokyo.ac.jp
Hiroki Miwa
umiwa{at}mail.ecc.u-tokyo.ac.jp
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ABSTRACT |
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7c (21.4 %) and C17 : 0 cyclo (19.0 %) as the major fatty acids. Based on phenotypic, chemotaxonomic and phylogenetic data, strain BAM-48T should be classified as a member of a novel species in the genus Variovorax, for which the name Variovorax boronicumulans sp. nov. is proposed. The type strain is BAM-48T (=NBRC 103145T =KCTC 22010T).
Details of boron uptake by strain BAM-48T and related type strains are available as supplementary material with the online version of this paper.
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) and possibly for animals (Rowe et al., 1998; Rowe & Eckhert, 1999), its essentiality to bacteria has been reported only recently (in the case of Bacillus boroniphilus; Ahmed et al., 2007a). In bacteria, boron also has a biological function in quorum sensing as a structural component of a receptor complex (Chen et al., 2002). On the other hand, boron is toxic to living cells when present above a certain threshold. Because of its toxicity, boron has long been used to kill Candida and Saccharomyces, the causal organisms of recurrent vulvovaginal candidiasis (Swate & Weed, 1974). However, recently, Bacillus boroniphilus, Gracilibacillus boraciitolerans, Chimaereicella boritolerans and Lysinibacillus boronitolerans have been reported to tolerate more than 450, 450, 300 and 150 mM boric acid, respectively (Ahmed et al., 2007a, b, c, d).
The genus Variovorax was proposed by reclassification of Alcaligenes paradoxus as Variovorax paradoxus (Willems et al., 1991). The type species of this genus is Variovorax paradoxus and, only recently, two other members have been characterized as Variovorax dokdonensis (Yoon et al., 2006a) and Variovorax soli (Kim et al., 2006).
In the course of screening for boron-accumulating bacteria, strain BAM-48T was isolated from soil sampled from an experimental field of the University of Tokyo (Yayoi campus), Tokyo, Japan. The soil samples (5 g) were incubated in sterilized PBS (pH 7.0) at 30 °C for several days, after which the supernatant was streaked on tryptic soya agar (TSA; Difco) and incubated at 25 °C. Purified cultures of the isolates recovered were maintained on TSA medium and also maintained as glycerol (35 %, w/v) stocks at –80 °C. Based upon the taxonomic characterization, we propose that strain BAM-48T represents a novel species of the genus Variovorax.
Intracellular boron concentrations were studied as follows. Individual colonies of the isolate grown in 1.5 ml tryptic soya broth (TSB; Difco) (pH 7.2) containing 100 µM boric acid at 25 °C for 7 days were harvested by centrifugation and analysed for their ability to accumulate boric acid by the Azomethine-H staining method (Gupta & Stewart, 1975). To determine the intracellular boron concentration more precisely, cells of candidate boron-accumulating strains, grown at 25 °C for 2 days in 5 ml TSB containing 100 µM boric acid, were harvested and washed with TSB. The pellet was boiled in MilliQ water and the supernatant was analysed for boron content by inductively coupled plasma-mass spectrometer (ICP-MS) (model SII SPQ-8000A; Seiko Instruments) after digestion with boron-free nitric acid (60–61 %, w/w) in Teflon tubes. The weight of the freeze-dried pellet was also measured to calculate the intracellular boron concentration. Strain BAM-48T contained 728±88.3 nmol boric acid (g dry weight)–1, which was higher than closely related species belonging to Variovorax (Supplementary Table S1, available in IJSEM Online). The ability to accumulate boric acid distinguished strain BAM-48T from closely related species and BAM-48T was therefore subjected to polyphasic characterization.
The morphology, sporangia and motility of the cells were examined under phase-contrast microscopy as described previously (Ahmed et al., 2007a). Gram-staining, performed according to Hucker's modified method, showed that cells of strain BAM-48T were Gram-negative. The temperature range for growth was determined by incubation for 2 weeks at various temperatures (4, 10, 17, 20, 25, 28, 30, 37, 45 and 50 °C) on TSA. The pH range for growth was determined in nutrient broth (Difco) adjusted to various pH values (pH 4.0–10.0 at intervals of 1.0 pH unit) using Na2CO3 or HCl prior to sterilization (Yoon et al., 2006a). Growth under anaerobic conditions was determined on TSA after 2 weeks incubation in an AnaeroPack (Mitsubishi Gas). Cells of strain BAM-48T were positive for catalase and oxidase activities, determined as described previously (Yoon et al., 2006b). API 20E, 20NE and API ZYM strips (bioMérieux) were used to examine physiological and biochemical characteristics according to the manufacturer's protocols. Strain BAM-48T showed many physiological characteristics that differentiated it from closely related species (Table 1); additional features are included in the species description.
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and Ahmed et al. (2007a). The 16S rRNA gene sequence of strain BAM-48T was compared with sequences obtained from GenBank. The sequences were aligned with CLUSTAL_X software (version 1.8msw; Thompson et al., 1997) and evolutionary distances and Knuc values (Kimura, 1980) were calculated. Alignment gaps and ambiguous bases were not taken into consideration in the comparison. Phylogenetic trees were constructed using the neighbour-joining and maximum-parsimony methods by MEGA version 3.1 (Kumar et al., 2004). The stability of relationships was assessed by bootstrap analysis by performing 1000 resamplings for the tree topology.
The 16S rRNA gene sequence (1416 nt) of strain BAM-48T showed the highest similarity to the sequences of V. paradoxus IAM 12373T (99.3 %), V. soli GH9-3T (98.3 %), V. dokdonensis DS-43T (97.0 %), Xylophilus ampelinus DSM 7250T (97.0 %) and Curvibacter gracilis IAM 15033T (96.3 %), but low sequence similarity (<96 %) to members of other genera. The phylogenetic analysis (Fig. 1) demonstrated that strain BAM-48T was clustered in the genus Variovorax with 91 % bootstrap support and occupied a position with V. paradoxus (99 % bootstrap value) in the neighbour-joining tree. The topology of the phylogenetic tree constructed using the maximum-parsimony algorithm (not shown) was similar to that of the neighbour-joining tree, especially for the clade comprising the novel strain (79 % bootstrap value), indicating that this strain is a member of the genus Variovorax.
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) for 2 days at 30 °C and the cellular fatty acid profile was determined using the GC-based Microbial Identification system (MIDI) according to the manufacturer's instructions. Since summed feature 3 represented a significant proportion of the total fatty acids in the profile, it was thought worthwhile to attempt to identify this compound. Fatty acids were therefore separated by TLC, and summed feature 3 was identified as C16 : 17c on the MIDI system. The major fatty acids of strain BAM-48T were C16 : 0 (36.1 %), C16 : 17c (21.4 %) and C17 : 0 cyclo (19.0 %). This profile is similar to those of other members of the genus Variovorax (Table 2), supporting the affiliation of the novel strain to this genus. Ubiquinone-8 (Q-8) was determined as the predominant quinone system in the novel strain using the method described previously (Xie & Yokota, 2003). These chemotaxonomic properties provided further evidence to confirm the phylogenetic results.
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. The DNA G+C content of strain BAM-48T was 71.2 mol%. DNA–DNA hybridizations were performed at 53 °C using photobiotin-labelled DNA probes and microplates, as described by Ezaki et al. (1989). DNA–DNA hybridization values between strain BAM-48T and V. paradoxus NBRC 15149T, V. dokdonensis KCTC 12544T and V. soli KACC 11579T were 49.1, 24.6 and 25.4 %, respectively. These results indicate that strain BAM-48T represents a separate species of the genus Variovorax (Stackebrandt & Goebel, 1994). On the basis of these morphological, physiological, biochemical, phylogenetic and genotypic results, we suggest that strain BAM-48T belongs to the genus Variovorax and represents a novel species, for which we propose the name Variovorax boronicumulans sp. nov.
Description of Variovorax boronicumulans sp. nov.
Variovorax boronicumulans (N.L. n. boron, -onis boron; L. part. adj. cumulans accumulating; N.L. part. adj. boronicumulans boron-accumulating).
Cells are Gram-negative, motile, non-spore-forming and rod-shaped (0.5–0.7 µm long and 1.0–2.0 µm in diameter). Colonies are yellow-coloured and circular with entire margins after 2 days on TSA plates at 30 °C. The temperature range for growth is 4–37 °C, with optimum growth at 30 °C; no growth at 
45 °C and little growth at 4 °C after 2 weeks of incubation. Growth is observed at pH 5–9, with optimum growth at pH 7; no growth at pH 4 or 10. NaCl is tolerated up to 1 % (w/v). Grows on TSA (Difco) and nutrient agar (Difco). Positive for catalase and oxidase activities, citrate utilization and the Voges–Proskauer test and weakly positive for assimilation of L-arabinose, mannitol, malic acid and capric acid. Negative for nitrate reduction, β-galactosidase, arginine dihydrolase, lysine and ornithine decarboxylases, H2S production, urease, tryptophan deaminase, assimilation of tryptophan, gelatin hydrolysis and utilization of inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin, D-glucose, D-mannose, N-acetyl-D-glucosamine, maltose, potassium gluconate, adipic acid and phenylacetic acid. Strong enzyme activity is observed for alkaline phosphatase, esterase lipase (C8) and leucine arylamidase and weak activity is observed for acid phosphatase; the other reactions of the API ZYM strip are negative. Major cellular fatty acids are C16 : 0 (36.1 %), C16 : 17c (21.4 %) and C17 : 0 cyclo (19.0 %). The G+C content of the type strain is 71.2 mol%.
The type strain, strain BAM-48T (=NBRC 103145T =KCTC 22010T), was isolated from soil collected in the experimental area of the University of Tokyo (Yayoi campus), Tokyo, Japan.
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ACKNOWLEDGEMENTS |
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