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1 Institute of Biological Resources and Function, Hokkaido Center, National Institute of Advanced Industrial Science and Technology, Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan
2 Department of Bioscience and Technology, School of Engineering, Hokkaido Tokai University, Minaminosawa, Minami-ku, Sapporo 005-8601, Japan
3 Laboratory of Electron Microscopy, School of Dentistry, Hokkaido University, Kita-ku, Sapporo 060-8586, Japan
4 Institute of Biological Resources and Function, Tsukuba Center, National Institute of Advanced Industrial Science and Technology, Higashi 1-1, Tsukuba, Ibaraki 305-8566, Japan
Correspondence
Isao Yumoto
i.yumoto{at}aist.go.jp
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Published online ahead of print on 21 March 2003 as DOI 10.1099/ijs.0.02596-0.
The GenBank accession number for the 16S rRNA sequence of strain AM31DT is AB086897.
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), but also in ordinary soils and faeces (Horikoshi, 1991). Alkaliphilic bacteria can be either facultative or obligate alkaliphiles (Krulwich & Guffanti, 1989). Numerous alkaliphilic micro-organisms have been isolated and more than 16 species of alkaliphilic Bacillus spp., including facultative and obligate types, have been identified to date (Vedder, 1934; Spanka & Fritze, 1993; Nielsen et al., 1995; Agnew et al., 1995; Fritze, 1996; Switzer Blum et al., 1998; Yumoto et al., 1998).
The microbial degradation of aromatic compounds is important not only for the industrial applications of the respective enzymes involved, but also for studying structural, functional and genetic aspects of aromatic compound oxygenases. However, there is little information concerning alkaliphiles that degrade aromatic compounds, or concerning aromatic compound oxygenases isolated from them. Isolation of aromatic-compound-degrading alkaliphiles might enable the acquisition of new information not only on the taxonomy, physiology and enzymology of alkaliphiles, but also on aromatic compound oxygenases (Gibson & Subramanian, 1984; Mason & Cammack, 1992).
In the present study, obligately alkaliphilic micro-organisms that can degrade benzoate and m-hydroxybenzoate were isolated to study the degradation of aromatic compounds in alkaline environments and the aromatic compound oxygenases that are active at high pH values. Phenotypic characterization and phylogenetic analysis based on 16S rRNA gene sequences showed that the new isolates merited classification as a novel Bacillus species.
Aromatic-compound-contaminated garden soil samples obtained from Tsukuba (36° 7' N, 140° 13' E), Ibaraki, Japan, were added to 10 ml alkaline mineral basal salt (AMBS) medium (pH 10) containing 0·2 g yeast extract, 3 g hydroxybenzoate, 2·5 g NH4NO3, 1·5 g K2HPO4, 1·5 g Na2HPO4, 0·5 g MgSO4.7H2O, 10 mg FeSO4.7H2O, 20 mg CaCl2.2H2O, 1 mg MnSO4.nH2O, 0·5 mg ZnSO4.7H2O and 10 g Na2CO3 per litre in large tubes (25x200 mm) and incubated aerobically at 30 °C for 48 h. Enrichments (0·5 ml) were transferred to fresh medium and cultured for 24 h, then plated onto AMBS agar plates. The isolates were reisolated five times and maintained in PYA (peptone/yeast extract/alkaline) agar medium consisting of 8 g peptone (Kyokuto), 3 g yeast extract (Merck), 15 g agar, 1 g K2HPO4, 3·5 mg EDTA, 3 mg ZnSO4.7H2O, 10 mg FeSO4.7H2O, 2 mg MnSO4.nH2O, 1 mg CuSO4.5H2O, 2 mg Co(NO3)2.6H2O and 1 mg H3BO3 in 1 l NaHCO3/Na2CO3 buffer (100 mM in deionized water; pH 10) at 27 °C. Cells for chemotaxonomic analysis were harvested in the late exponential phase after cultivation with reciprocal shaking (140 r.p.m.) at 27 °C in PYA medium. In addition to these isolates, Bacillus alcalophilus JCM 5262T and Bacillus pseudalcaliphilus DSM 8725T were used as reference strains for DNA–DNA hybridization. These micro-organisms were cultivated using PYA broth at 27 °C.
For the phenotypic characterization, PYA medium was used as the basal medium. The culture was incubated at 27 °C for 2 weeks and the experiment was performed more than twice. Acid production from carbohydrate was determined by the method of Hugh & Leifson (1953) using thymol blue instead of bromothymol blue at pH 10. Growth experiments at pH 7–10 were performed using PYA media containing 100 mM NaH2PO4/Na2HPO4 buffer (pH 7–8) or 100 mM NaHCO3/Na2CO3 buffer (pH 9–10). Anaerobic growth was tested in PYA broth (pH 10) by substituting air with N2 gas. Other physiological and biochemical characteristics were examined according to the methods of Yumoto et al. (1998) and as described in Barrow & Feltham (1993).
For observation of negatively stained cells by transmission electron microscopy (TEM) and for platinum- and palladium-coated cells by scanning electron microscopy (SEM), cells were grown on a PYA agar slant. The procedure for TEM and SEM preparations and observations were performed as described previously (Yumoto et al., 2002).
Analyses of whole-cell fatty acids and isoprenoid quinones were performed as described previously (Yumoto et al., 2001).
Bacterial DNA was prepared according to the method of Marmur (1961). DNA base composition was determined by the method of Tamaoka & Komagata (1984). The level of DNA–DNA relatedness was determined fluorometrically by the method of Ezaki et al. (1989) using photobiotin-labelled DNA probes and black microplates.
The 16S rRNA gene sequence corresponding to position 27–1519 in the 16S rRNA gene sequence of Escherichia coli (Brosius et al., 1978) was amplified by PCR. The approximately 1·5 kb PCR product was sequenced directly by the dideoxynucleotide chain-termination method using a DNA sequencer (PRISM 377; Applied Biosystems). Multiple alignments of the sequence were performed and the nucleotide substitution rate (Knuc) was calculated. A phylogenetic tree was constructed by the neighbour-joining method (Kimura, 1980; Saitou & Nei, 1987) using the CLUSTAL W program (Thompson et al., 1994). Similarity values for sequences were calculated using the GENETYX program (Software Development).
Two strains were isolated on AMBS medium (pH 10). These isolates grew better in AMBS broth containing either 0·3 % m-hydroxybenzoate or benzoate than in the same medium devoid of the aromatic compounds. Strain AM31T grew strongly with both m-hydroxybenzoate (OD650=0·87) and benzoate (OD650=0·86) as the substrate. In contrast, AM11D grew better with benzoate (OD650=0·67) than with m-hydroxybenzoate (OD650=0·32). These results demonstrated that the two isolates utilized aromatic compounds such as benzoate and m-hydroxybenzoate as the substrate. To the best of our knowledge, this is the first observation of alkaliphilic Bacillus strains utilizing aromatic compounds.
Colonies of strain AM31DT and AM11D on PYA agar were circular and colourless; cells were Gram-positive, peritrichously flagellated rods measuring 0·5–0·7x1·5–2·6 µm (Fig. 1) and produced subterminally located ellipsoidal spores. Spores did not cause swelling of the sporangia.
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The isoprenoid quinones extracted from isolates using TLC were analysed by HPLC. Analysis revealed that menaquinone-5, -6 and -7 are the major quinones of the isolates. Similar menaquinone contents occur in Bacillus halodurans (Aono, 1995; Takami & Horikoshi, 1999). The ratio of menaquinone-5/-6/-7 was 1 : 11 : 27. GLC analysis revealed that the fatty acids of these strains consisted of isoC15 : 0, anteisoC15 : 0, C16 : 1, anteisoC17 : 0 and anteisoC17 : 1. Among these, anteisoC15 : 0 was the major component, comprising 45·6–49·0 % of the total fatty acid content. Results of the analysis of the two isolates are shown in Table 1. Clejan et al. (1986) reported that obligate alkaliphilic Bacillus spp. contain a high concentration of unsaturated fatty acids. The isolates also contained a larger amount of unsaturated fatty acids than Bacillus subtilis, the faculatively alkaliphilic Bacillus cohnii and the obligately alkaliphilic Bacillus alcalophilus. The menaquinone and fatty acid contents of the new isolates are characteristic of the genus Bacillus.
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) and 16S rRNA gene sequence similarity (data not shown) showed that strain AM31DT was a member of the Bacillaceae. Strain AM31DT was placed in group 6 (alkaliphiles) (Nielsen et al., 1994). The highest similarity value was observed with the obligate alkaliphiles (Nielsen et al., 1995) Bacillus alcalophilus (94·7 %) and Bacillus pseudalcaliphilus (94·8 %). These results and the demonstrated obligate alkaliphilic nature of AM31DT were consistent with the phylogenetic placement of the isolates.
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According to the results of the 16S rRNA gene sequence analysis, strain AM31DT was closely related to Bacillus alcalophilus and Bacillus pseudalcaliphilus. Therefore, the level of DNA–DNA relatedness between strains AM31DT and AM11D, and the closely related strains given above were estimated. DNA–DNA relatedness results indicated that the two isolates were closely related to each other (more than 94·7 % similarity) and different (9·5–19·3 % similarity) from Bacillus alcalophilus and Bacillus pseudalcaliphilus (Table 2).
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).
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Description of Bacillus krulwichiae sp. nov.
Bacillus krulwichiae (krul.wich.i'ae N.L. fem. gen. n. krulwichiae of Krulwich; named after American microbiologist Terry A. Krulwich who made fundamental contributions to the study of alkaliphilic bacteria).
Cells are Gram-positive peritrichously flagellated straight rods (0·5–0·7x1·5–2·6 µm) and produce subterminally located ellipsoidal spores. Spores do not cause swelling of sporangia. Both aerobic and anaerobic growth is observed. Colonies are circular and colourless. Catalase and oxidase reactions are positive. Negative for indole production, ONPG hydrolysis and H2S production. Growth occurs at pH 8–10 at almost equal intensity, but not at pH 7. Grows at 14 % NaCl, but not at higher concentrations. Nitrate is reduced to nitrite. Acid, but no gas, is produced from D-xylose, D-glucose, D-fructose, D-galactose, D-ribose, maltose, sucrose, trehalose, glycerol and mannitol when grown at pH 10. No acid is produced from D-arabinose, L-rhamnose, D-mannose, lactose, cellobiose, melibiose, raffinose, myo-inositol and sorbitol. Positive for hydrolysis of starch, DNA, hippurate and Tween 20, 40, 60 and 80. Hydrolysis of casein and gelatin is variable among strains. Utilizes benzoate and m-hydroxybenzoate as sole carbon sources. The major isoprenoid quinones are menaquinone-5, -6 and -7. isoC15 : 0 (17·1–19·2 %) and anteisoC15 : 0 (45·6–49·0 %) represent the main fatty acids produced during growth in an alkaline medium (pH 10). The DNA G+C content is 40·6–41·5 mol%, as determined by HPLC. Strains AM31DT and AM11D were isolated from a soil sample obtained from Tsukuba, Ibaraki, Japan. Type strain is AM31DT (=NCIMB 13904T=JCM 11691T=IAM 15000T).
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