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 (ϭIAM 15023TϭKCTC 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: ϩ82–42–869–5617, Fax: ϩ82–42–863–5617 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 (ϭIAM 15023T). 200 ml distilled water. Morphological and phenotypic characteristics. Determination of GϩC 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 (5Ј-GAGTTTGATCCTGGCTCAG-3Ј) Ј 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 [5Ј-GAGTTT- Identification System (MIDI, Inc., Newark, DE, USA). GATCCTGGCTCAG-3Ј positions 9–27 (Escherichia The fatty acids analyzed by a gas chromatograph coli 16S rRNA numbering)], 341F [5Ј-CCTACGGGAG- (Hewlett Packard 6890) were identified by the Micro- GCAGCAG-3Ј; positions 341–357], 519F [5Ј-CAGC- bial Identification software package (Sasser, 1990). AGCCGCGGTAATAC-3Ј; positions 519–536], 907F [5Ј- DNA extraction. Chromosomal DNA was extracted AAACTCAAAKGAATTGACGG-3Ј; positions 907–926], as described by Ausubel et al. (1995). Colonies grown 536R [5Ј-GTATTACCGCGGCTGCTG-3Ј; positions on an agar plate were collected in a 15 ml conical tube 536–519], 1100R [5Ј-GGGTTGCGCTCGTTG-3Ј; posi- and washed with distilled water, and suspended in tions 1114–1110], and 1512R (5Ј-ACGGHTACCTTGT- 1.8 ml of TE buffer. The suspension was supplemented TACGACTT-3Ј; 1512–1492). with 210 ml SDS (10%, w/v) and 30 ml proteinase K Phylogenetic analysis. The 16S rDNA sequences (10 mg mlϪ1), 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) mlϪ1 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 0.7ϫ0.9 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 GϩC 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 Ϫ 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 Ϫ Ϫ line, rhamnose, N-acetyl-glucosamine, D-ribose, inosi- Motility ϩ tol, D-sucrose, maltose, lactate, L-alanine, 5-ketoglu- Catalase ϩ conate, glycogen, and mannose as a sole carbon Oxidase Nitrogen fixation Ϫ source. However, the strain showed negative results Ϫ→ Ϫ ϩ NO3 NO2 for propionate, caprate, valerate, citrate, histidine, Ϫ→ Ϫ NO2 NO2 4-hydroxy-benzoate, itaconate, suberate, malonate, Urease ϩ acetate, 3-hydroxy-benzoate, L-serine, gluconate, adi- b-Glucosidase ϩ pate, malate, and phenyl-acetate.