J. Gen. Appl. Microbiol., 50, 249–254 (2004)
Full Paper
Kaistia adipata gen. nov., sp. nov., a novel a-proteobacterium
Wan-Taek Im,1, 2 Akira Yokota,2 Myung-Kyum Kim,1 and Sung-Taik Lee1,*
1 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373–1, Guseong-dong, Yuseong-gu, Daejeon 305–701, Republic of Korea 2 Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113–0032, Japan
(Received July 25, 2003; Accepted August 6, 2004)
A taxonomic study was carried out on Chj404T †, a bacterial strain isolated from a soil sample collected in an industrial stream near the Chung-Ju industrial complex in Korea. The strain was a gram-negative, aerobic, short rod to coccus-shaped bacterium. It grew well on nutrient agar medium and utilized a broad spectrum of carbon sources. The G C content of the DNA was
67.4 mol% and the major composition of ubiquinone was Q-10. The major fatty acid was C18:1. Comparative 16S rDNA studies showed a clear affiliation of this bacterium to a-Proteobacteria. Comparison of phylogenetic data indicated that it was most closely related to Prosthecomicro- bium pneumaticum (92.7% similarity in 16S rDNA sequence). Since strain Chj404 is clearly distinct from closely related species, we propose the name Kaistia adipata gen. nov., sp. nov. for this strain Chj404T ( IAM 15023T KCTC 12095T).
Key Words——Kaistia adipata gen. nov., sp. nov.; polyphasic taxonomy; 16S rDNA sequence
Introduction been isolated simultaneously. Identification of the strain Chj404T, which was resistant to 1 mM phenol For several years, our laboratory has isolated and 4-chlorophenol concentrations for 1 week cultur- numerous bacterial strains from soils and wastewater ing, showed that Chj404T was phylogenetically distant contaminated with toxic compounds in order to find from other bacteria isolated thus far, and as such we useful bacteria that can degrade phenolic compounds have decided to classify this bacterium. such as phenol, chlorophenol, and nitrophenol. By A polyphasic approach, including phenotypic, enrichment, many phenolic compound-degrading bac- chemotaxonomic and molecular methods, was used to teria have been isolated (Bae et al., 1996a, b; Baek et determine the taxonomic position of this isolated al., 2001; Yoon et al., 1999, 2000). Furthermore, some strain. In this study, we report the morphological, bio- bacteria that could not degrade phenolic compounds, chemical, and phylogenetic characteristics of the novel but instead were resistant to such compounds have strain Chj404T.
* Address reprint requests to: Dr. Sung-Taik Lee, Department of Biological Sciences, Korea Advanced Institute of Science Materials and Methods and Technology, 373–1, Guseong-dong, Yuseong-gu, Daejeon Isolation of bacterial strain and culture condition. 305–701, Republic of Korea. T Tel: 82–42–869–5617, Fax: 82–42–863–5617 The bacterial strain Chj404 used in this study was E-mail: [email protected] isolated from a soil sample collected in an industrial † The NCBI GenBank accession number for the 16S rDNA se- stream near the Chung-Ju industrial complex in Korea. quence of strain Chj404T is AY039817. Strain Chj404T was isolated using nutrient agar con- 250 IM et al. Vol. 50 taining 50 mM phenol after enrichment for 3 weeks. centrifuged at 5,000 rpm for 10 min. The DNA in the After the strain Chj404T was isolated, it was conserved supernatant was precipitated by adding 0.6 vol. of iso- by transferring it onto nutrient agar every month, after propanol, collected by centrifugation, and washed with which it was deposited in the Korean Collection for 70% ethanol. The obtained DNA was suspended in Type Cultures as KCTC 12095T ( IAM 15023T). 200 ml distilled water. Morphological and phenotypic characteristics. Determination of G C content. DNA base compo- Motility was determined with a light microscope using sition was determined using the HPLC method. DNA the hanging drop technique. Morphology was deter- was enzymatically degraded into nucleosides as mined using a scanning electron microscope with described by Mesbah et al. (1989). The nucleoside 40,000 magnifications. Growth at a different tempera- mixture obtained was then separated by HPLC using a ture was observed in nutrient agar broth at 10, 25, 30, Cosmosil 5C18R column thermostatted at 40°C. The
37 and 42°C. The growth experiment was performed solvent was 0.2 M NH4H2PO4 with 2.5% acetonitrile. using a cap tube containing 3 ml nutrient broth at pH Unmethylated l phage DNA (Sigma, St. Louis, MO, 4.0–10.0 and temperature at 37°C. Growth was esti- USA) was used as the calibration reference. mated by monitoring the OD600. Carbon-source utiliza- PCR amplification and 16S rRNA gene sequencing. tion and some enzyme activities were tested by using The 16S rDNA was amplified from the chromosomal the API 20NE gallery methods, API 20E and API ID32 DNA of strain Chj404T by using a universal eubacterial (bioMérieux, Co., Paris, France). Catalase activity was primer set, 9F (5 -GAGTTTGATCCTGGCTCAG-3 ) determined by bubble production in 3% (v/v) H2O2 and and 1512R (5 -ACGG(H)TACCTTGTTACGACTT) as oxidase activity was determined using 1% (w/v) described by William et al. (1991). After purification of tetramethyl p-phenylenediamine. the PCR product with a GFXTM PCR DNA and Gel Chemotaxonomic characteristics. Ubiquinones were Band Purification Kit (Amersham Biosciences, Corp., analyzed as described previously (Komagata and Piscataway, NJ, USA), the resulting PCR product was Suzuki, 1987). Cellular fatty acids were analyzed in the sequenced with an ABI Prism BigDye Terminator cycle strain Chj404T grown on a trypticase soy agar (TSA; sequencing ready reaction kit (Applied Biosystems, Difco, Detroit, MI, USA) for 24 h and 48 h. The cellular Foster City, CA, USA) and an automatic DNA se- fatty acids were saponified, methylated, and extracted quencer (model 310; Applied Biosystems). The according to the protocol of the Sherlock Microbial primers used for sequencing were 9F [5 -GAGTTT- Identification System (MIDI, Inc., Newark, DE, USA). GATCCTGGCTCAG-3 positions 9–27 (Escherichia The fatty acids analyzed by a gas chromatograph coli 16S rRNA numbering)], 341F [5 -CCTACGGGAG- (Hewlett Packard 6890) were identified by the Micro- GCAGCAG-3 ; positions 341–357], 519F [5 -CAGC- bial Identification software package (Sasser, 1990). AGCCGCGGTAATAC-3 ; positions 519–536], 907F [5 - DNA extraction. Chromosomal DNA was extracted AAACTCAAAKGAATTGACGG-3 ; positions 907–926], as described by Ausubel et al. (1995). Colonies grown 536R [5 -GTATTACCGCGGCTGCTG-3 ; positions on an agar plate were collected in a 15 ml conical tube 536–519], 1100R [5 -GGGTTGCGCTCGTTG-3 ; posi- and washed with distilled water, and suspended in tions 1114–1110], and 1512R (5 -ACGGHTACCTTGT- 1.8 ml of TE buffer. The suspension was supplemented TACGACTT-3 ; 1512–1492). with 210 ml SDS (10%, w/v) and 30 ml proteinase K Phylogenetic analysis. The 16S rDNA sequences (10 mg ml 1), and then incubated at 37°C for 1 h. To were edited by combining 16S rDNA partial sequences remove the RNA, 30 ml of Ribonuclease A and T1 solu- using the BioEdit program (Hall, 1999). The 16S rDNA tion (10 mg and 2,000 units) ml 1 was added and incu- sequences of related taxa were obtained from the bated for 30 min at room temperature. A 360 ml of 5 M GenBank. Those obtained sequences including the NaCl and 270 ml of CTAB/NaCl solution was added, 16S rDNA sequence of Chj404T and related taxa were and incubated at 65°C for 10 min. An equal volume of analyzed. For the multiple alignments, Clustal X pro- phenol/chloroform/isoamyl alcohol (25 : 24 : 1, v/v/v) gram (Thompson et al., 1997) was used. Gaps were was added to the reaction mixture and centrifuged at edited with the BioEdit program. The evolutionary dis- 5,000 rpm for 10 min. The experiment was performed tance was computed based on the no-gap option and twice, then an equal volume of chloroform/isoamyl using the Kimura two-parameter model (Kimura, alcohol (24 : 1, v/v) was added to the supernatant and 1983). A phylogenic tree was constructed by using 2004 Kaistia adipata gen. nov., sp. nov. 251 neighbor-joining method (Saitou and Nei, 1987) in MEGA2 Program (Kumar et al., 2001). The bootstrap neighbor-joining method was used to obtain the confi- dence level of neighbor-joining analysis with a 1,000 bootstrap data set (Felsenstein, 1985).
Results and Discussion
Morphological and physiological characteristics Strain Chj404T is an aerobic, gram-negative, non- motile and short rod to coccus-shaped bacterium of 0.7 0.9 mm lengths (Fig. 1). Colonies formed on a nu- T trient agar plate (Difco) were ivory-pigmented and Fig. 1. An electron micrograph of the strain Chj404 . round, raised with a greasy surface. The strain gave positive results for catalase and oxidase. This strain Table 1. Major characteristics of strain Chj404T. could grow at 10–37°C but not at 42°C. The optimum temperature for growth was 37°C. The optimum pH Characteristic was within pH 6.0–7.0. As shown in Table 1, strain G C content 67.4 mol% Chj404T showed positive results for urease, b-glucosi- Major ubiquinone Q-10 dase, and b-galactosidase from the API NE test. This Morphology Short rod to coccus strain could grow by uptake of a broad range of carbon Gram-staining sources: mannitol, D-glucose, salicin, D-melibiose, L-fu- Size (length, mm) 0.7–0.9 cose, D-sorbitol, L-arabinose, 2-ketogluconate, L-pro- Flagella line, rhamnose, N-acetyl-glucosamine, D-ribose, inosi- Motility tol, D-sucrose, maltose, lactate, L-alanine, 5-ketoglu- Catalase conate, glycogen, and mannose as a sole carbon Oxidase Nitrogen fixation source. However, the strain showed negative results → NO3 NO2 for propionate, caprate, valerate, citrate, histidine, → NO2 NO2 4-hydroxy-benzoate, itaconate, suberate, malonate, Urease acetate, 3-hydroxy-benzoate, L-serine, gluconate, adi- b-Glucosidase pate, malate, and phenyl-acetate. It did not utilize b-Galactosidase starch or lipids and could not hydrolyze gelatin. In the Carbon assimilation API 20E test, strain Chj404T showed positive results Adipate for b-galactosidase and urease. It also showed nega- Citrate Fucose tive results for the following: arginine dihydrolase, D-Glucose lysine decarboxylase, ornithine decarboxylase, gelatin Lactate hydrolysis, citrate utilization, H2S production, indole Lactose production, Voges-Proskauer test, tryptophane dea- Malate minase, and acid production from glucose, mannitol, Maltose inositol, sorbitol, rhamnose, sucrose, melibiose, amyg- Propionate dalin, and arabinose. This strain was resistant to 1 mM Rhamnose phenol and 4-chlorophenol concentrations for 1 week Sucrose culturing.
and C16:0 (11.04%) for 24 h growth on TSA agar. Whole Chemotaxonomic characteristics cell fatty acid composition changed with time. How-
The major composition of ubiquinone was Q-10. As ever, the total amount of C19:0 cyclo w8c and C18:1 shown in Table 2, the major fatty acids of strain w7c/w9t/w12t was similar. Thus we could infer that T Chj404 had a summed feature of 7 (C18:1 w7c/w9t/ C18:1 w7c/w9t/w12t was transformed to C19:0 cyclo w8c w12t; 47.70%), C19:0 cyclo w8c (19.73%), C18:0 (16.04%) with time. 252 IM et al. Vol. 50 Table 2. Cellular fatty acid composition of strain Chj404T in time 24 h and 48 h. constructed. The phylogenetic tree based Knuc values shown in Fig. 2 indicated that the strain Chj404T is dis- Fatty acid 24 h 48 h tinctively different from related genera and from all other known bacteria in the a-Proteobacteria group. Saturated According to phylogenetic analyses based on 16S C16:0 11.04 22.17 rDNA sequence, the closest genus, Prosthecomicro- C 1.64 3.47 T 17:0 bium pneumaticum ATCC 23633 (AB017203), is dis- C 16.04 14.80 18:0 tant enough (92.6% similarity) to warrant placing Unsaturated Chj404T in a new genus, which was also supported by C18:1 w9c 0.77 1.10 Cyclo the phenotypic characteristics (Table 1). On the basis of morphological, physiological and chemotaxonomic C19:0 cyclo w8c 19.73 42.21 Summed feature characteristics, together with data from 16S rDNA T 7; C18:1 w7c/w9t/w12t 47.70 10.33 sequence comparison described above, strain Chj404 should be placed into a new genus containing a single Values are percentage of total fatty acids; values less than species, for which we propose the name, Kaistia adi- 0.5% are not shown. pata gen. nov., sp. nov.
DNA base composition Description of Kaistia gen. nov. The G C content of the DNA of strain Chj404T was Kaistia (Ka.ist.i’a. M.L. dim. fem. ending-ia; M.L. 67.4 mol%. fem. n. Kaistia arbitrary name formed from the acronym of the Korea Advanced Institute of Science Phylogenetic analysis and Technology, KAIST, where taxonomic studies of Nearly complete 16S rDNA sequences (nucleotide this taxon were performed). positions 9–1512, E. coli numbering) of strain Chj404T The cells are rods and cocci-shaped with size vary- were determined. Comparison of the Chj404T 16S ing between 0.7 and 0.9 mm long. Colonies formed on rDNA sequence with the GenBank database using the a nutrient agar plate are ivory-pigmented and round, BLASTN program (Altshul et al., 1990) and RDP (Ri- raised with greasy surface. The cells are gram-nega- bosomal Data Project) (Maidak et al., 1997) revealed tive and non-motile. Spore is not observed. It grows that the most closely related bacterial species be- aerobically. Does not produce acid from glucose. longed to Rhizobiales in a-Proteobacteria. With a sim- Major composition of quinone is Q-10 and the residues ple blast search, we observed that the closest species are composed of Q-7, Q-8, and Q-9. The major fatty T was Chelatococcus asaccharovorans DSM 6450 acid is C18:1 cultured on TSA agar for 24 h, but C19:0 (AJ011762), with 92.8% identity between positions 28 cyclo w8c for 48 h. According to the 16S rDNA and 1448 (E. coli numbering). sequence, the genus Kaistia belongs to the a subclass It has been suggested that in bacterial strains with of the Proteobacteria. less than 97% 16S rDNA sequence identity, the DNA- DNA hybridization level is less than 70% (Stacke- Description of Kaistia adipata gen. nov., sp. nov. brandt and Goebel, 1994), which defines a genomic Kaistia adipata (a.di.pa’ta. M.L. adj. adipatus fatty, species (Wayne et al., 1987). Thus, based on the 16S greasy). rDNA sequence analyses, a new taxon could be Optimum temperature for growth is 37°C. It grows detected. In the absence of any close homologous with many variable carbon sources such as mannitol, sequence in the GenBank database and the RDP D-glucose, salicin, D-melibiose, L-fucose, D-sorbitol, (92.8% or less sequence identity), phylogenetic rela- L-arabinose, 2-ketogluconate, L-proline, rhamnose, tionships were investigated using nearly all sequences N-acetyl-glucosamine, D-ribose, inositol, D-sucrose, of the generic type species of the order Rhizobiales. maltose, lactate, L-alanine, 5-ketogluconate, glycogen, Thirty representative a-Proteobacteria were chosen for and mannose. However, it showed negative results for the phylogenetic analysis and obtained from GenBank. propionate, caprate, valerate, citrate, histidine, 4-hy- The 16S rDNA sequence of Chj404T and related droxy-benzoate, itaconate, suberate, malonate, ac- organisms were aligned and a phylogenetic tree was etate, 3-hydroxy-benzoate, L-serine, gluconate, adi- 2004 Kaistia adipata gen. nov., sp. nov. 253
Fig. 2. Rooted phylogenetic tree based on the 16S rDNA sequences of strain Chj404T and related bacteria of Rhi- zobiales in a-Proteobacteria. This tree was made using the neighbor-joining method (Saitou and Nei, 1987) with a Kimura (1983) two-parameter distance matrix and pairwise deletion. Bootstrap values (expressed as percentages of 1,000 replications) greater than 50% are shown at the branch points. The bar represents 20 nucleotide substitutions per 1,000 nucleotides. pate, malate, and phenyl-acetate. It does not utilize the at GenBank as AY039817. Type strain is strain starch and lipid and can not hydrolyze gelatin. Urea Chj404T ( KCTC 12095T IAM 15023T). and esculin are hydrolyzed. It shows negative results for the following: arginine dihydrolase, lysine decar- Acknowledgments boxylase, ornithine decarboxylase, gelatin hydrolysis, citrate utilization, H S production, indole production, This work was supported by the Brain Korea 21 Project in 2 2002. Voges-Proskauer test, tryptophane deaminase, and acid production from glucose, mannitol, inositol, sor- bitol, rhamnose, sucrose, melibiose, amygdalin, and References arabinose. The cells were resistant to 1 mM phenol and Altshul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, 4-chlorophenol concentrations for 1 week culturing. D. J. (1990) Basic local alignment search tool. J. Mol. Biol., The G C content of the DNA is 67.7 mol%. The 16S 215, 403–410. rDNA sequence of strain Chj404T has been deposited Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seid- 254 IM et al. Vol. 50
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