J. Gen. Appl. Microbiol., 58, 245‒251 (2012) Short Communication Comamonas terrae sp. nov., an arsenite-oxidizing bacterium isolated from agricultural soil in Thailand Kitja Chitpirom,1 Somboon Tanasupawat,2,* Ancharida Akaracharanya,3 Natchanun Leepepatpiboon,4 Alexander Prange,5 Kyoung-Woong Kim,6 Keun Chul Lee,7 and Jung-Sook Lee7 1 Inter-department of Environmental Science, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand 2 Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand 3 Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand 4 Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand 5 Microbiology and Food Hygiene, Niederrhein University of Applied Sciences, Rheydter Strasse 277, D-41065 Möenchengladbach, Germany 6 Department of Environmental Science and Engineering, Gwangju Institute of Science and Technology, Gwangju 500‒712, Korea 7 Korean Collection for Type Cultures, Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong, Daejeon 305‒806, Republic of Korea (Received August 11, 2011; Accepted February 14, 2012) Key Words—arsenite-oxidizing bacterium; Comamonas terrae sp. nov.; β-Proteobacteria; 16S rRNA gene The genus Comamonas belonging to the family Co- 1987; Wauters et al., 2003; Young et al., 2008; Yu et al., mamonadaceae of the class Beta-Proteobacteria was 2011). Comamonas strains have been isolated from a proposed by De Vos et al. (1985) after a polyphasic phenol-activated-sludge process and soils contami- study. In the genus Comamonas, 13 species have nated with heavy metals (Kanazawa and Mori, 1996; been described at the time of writing, C. aquatica, C. Watanabe et al., 1999). During the course of screening badia, C. denitrifi cans, C. kerstersii, C. koreensis, C. of a high arsenic resistant bacteria from arsenic con- nitrativorans, C. odontotermitis, C. terrigena (type spe- taminated soils in Thailand, Comamonas strain A3-3T cies), C. testosteroni, C. composti, C. zonglianii, C. was isolated and reported (Chitpirom et al., 2009). This thiooxidans, and C. granuli (Chang et al., 2002; Chou paper, we propose Comamonas terrae sp. nov. to et al., 2007; De Vos et al.,1985; Etchebehere et al., strain A3-3T. 2001; Gumaelius et al., 2001; Kim et al., 2008; Narayan Strain A3-3T, an arsenite oxidizing bacterium was et al., 2010; Tago and Yokota, 2004; Tamaoka et al., isolated from the arsenic contaminated soil samples (< 5 mg/kg) in central of Thailand by an enrichment culture method using 0.1 g/L sodium arsenite (Chit- * Address reprint requests to: Dr. Somboon Tanasupawat, pirom et al., 2009). The MICs for arsenite and arsenate Department of Biochemistry and Microbiology, Faculty of Phar- of the A3-3T isolate were 40 and 400 mM, respectively maceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand. (Chitpirom et al., 2009). The capability to oxidize ar- T E-mail: [email protected] senite (+III) to arsenate (+V) of A3-3 was tested in The GenBank/EMBL/DDBJ accession number for the 16S situ by X-ray absorption near edge structure (XANES) rRNA gene sequence of strain A3-3T is GQ497244. analysis. The strain was phenotypically characterized 246 CHITPIROM et al. Vol. 58 by Gram staining, fl agella staining, cell morphology, and 50°C) were investigated in TYEG broth. Antibiotic colony appearance, spore formation and pigmenta- susceptibility was determined by disc diffusion assay tion on TYEG (tryptone-yeast extract-glucose) agar (Bauer et al., 1966). The antibiotic discs contained am- medium at 30°C for 24 h (Chitpirom et al., 2009). Cata- picillin (10 μg), bacitracin (10 U), carbenicillin (100 μg), lase, oxidase, hydrolysis of casein, starch, Tween 80, cephalothin (30 μg), clindamycin (2 μg), erythromycin nitrate reduction and deoxyribonuclease (DNase) ac- (15 μg), gentamicin (10 μg), imipenem (10 μg), kana- tivity were determined as described by Barrow and mycin (30 μg), novobiocin (5 μg), penicillin (20 U), Feltham (1993). The methyl red (MR)/Voges-Proskau- streptomycin (10 μg), sulphonamide (300 μg), tetracy- er (VP) reactions, indole test, citrate test, urease activ- cline (30 μg), tobramycin (10 μg) and vancomycin ity, amino acid decarboxylase (ADH, LDC, ODC), TDA (30 μg). Plates were incubated at 30°C for 24 h and the (tryptophan deaminase), ONPG (orthonitrophenyl-be- inhibition zone was interpreted. Ubiquinones (Q) were ta-D-galactopyranoside), assimilation of carbohydrates, extracted from freeze-dried cells with chloroform: metha- hydrolysis of esculin were determined with commer- nol (2：1) and purifi ed with a silica gel TLC (Merck cially product kit systems (API 20E, API 20NE and API No.1.05744). The purifi ed quinones were analyzed by 50CH, bioMérieux). Growth in 3, 4, 4.5 and 5% (w/v) HPLC (Komagata and Suzuki, 1987). Total cellular fatty NaCl, at different starting pH (3, 4, 5, 6, 7, 8, 9 and 10) acid analysis of cells grown on tryptic soy agar (TSA, and at different temperatures (4°C, 30°C, 37°C, 40°C Difco) for 48 h at 30°C was performed by Gas Liquid Table 1. Differential characteristics of strain A3-3T and related Comamonas species. Characteristics 1 2 3 4 5 6 7 b 8 c 9 d Cell shape Rd Rd Rd Rd Rd Rd Rd Rd Rd Flagella P P P P A P P P P Growth at 40°C++--w --++ Growth at pH 5 + - +++---- Urease + ----+ - ++ Nitrate reduction - +++++++- Tween 80 hydrolysis - ++++- ++- Citrate utilization + + + + --ww- Assimilation of : Arbutin ---+ ----- Glucose ---++--+ - Inositol ----+ ---- Mannitol ----+ ---- Tagatose ----+ ---- Caprate ---+ ----- Malate + + + + + - + - + Citrate ---w+- ++- Gluconate + - +++++-- Susceptibility to Ampicillin R R S R R S R R S Bacitracin R R S R S S R R S Cephalothin R S S R S S R R S Novobiocin S R S S R S S S R Penicillin G R R S R S S R R S Streptomycin S S S R R S R R S aG+C content (mol%) 69.6 61.0 64.0 62.5 66.0 64.0 61.6 61.4 68.4 Abbreviations: 1, A3-3T; 2, C. kerstersii LMG 3475T; 3, C. aquatica LMG 2370T; 4, C. testosteroni KCTC 2990T; 5, C. koreensis KCTC 12005T; 6, C. terrigena KCTC 2989T; 7, C. odontotermitis LMG 23579T; 8, C. thiooxidans DSM 17888T; 9, C. granuli KCTC 12199T. +, positive; w, weakly positive; -, negative; R, resistant; S, sensitive; Rd, rods; P, polar fl agellum; A, absent; ND, no data. aData for the other type strains were obtained from Chang et al. (2002), De Vos et al. (1985), Tomaoka et al. (1987) and Young et al. (2008). bChou et al. (2007) and this study; cNarayan et al. (2010) and this study; dKim et al. (2008) and this study. 2012 Comamonas terrae sp. nov. 247 Table 2. Zone diameter (mm) a of antimicrobial disk susceptibility test for strain A3-3 and Comamonas species. Strain LMG LMG KCTC KCTC KCTC Antibiotics A3-3T 3475T 2370T 12005T 2990T 2989T Ampicillin (10 μg) 0 0 34 ± 1.4 0 0 39 ± 1.4 Bacitracin (10 U) 0 0 13 ± 1.4 14 ± 1.4 0 16 ± 1.4 Carbenicillin (100 μg) 21 ± 1.4 26.5 ± 0.7 35 ± 1.4 33 ± 1.4 29.5 ± 0.7 35 ± 1.4 Cephalothin (30 μg) 0 29 ± 1.4 32.5 ± 0.7 13.5 ± 0.7 0 25 ± 1.4 Clindamycin (2 μg) 0 0 8.5 ± 0.7 0 0 0 Erythromycin (15 μg) 17.5 ± 0.7 21.5 ± 0.7 26.5 ± 0.7 23 ± 1.4 10.5 ± 0.7 13.5 ± 0.7 Gentamicin (10 μg) 18.5 ± 0.7 19.5 ± 0.7 19.5 ± 0.7 19 ± 1.4 8.5 ± 0.7 24 ± 1.4 Imipenem (10 μg) 34.5 ± 0.7 33 ± 1.4 32 ± 2.8 35 ± 1.4 32.5 ± 0.7 39 ± 1.4 Kanamycin (30 μg) 24.5 ± 0.7 20.5 ± 0.7 19.5 ± 0.7 24.5 ± 0.7 21 ± 1.4 21 ± 1.4 Novobiocin (5 μg) 7.5 ± 0.7 0 14.5 ± 0.7 0 11.5 ± 0.7 16.5 ± 0.7 Penicillin G (20 U) 0 0 32.5 ± 0.7 19 ± 1.4 0 39 ± 1.4 Streptomycin (10 μg) 10.5 ± 0.7 12.5 ± 0.7 10.5 ± 0.7 0 0 9.5 ± 0.7 Sulphonamide (300 μg) 31 ± 1.4 32.5 ± 0.7 35 ± 1.4 29 ± 1.4 30.5 ± 0.7 23 ± 1.4 Tetracycline (30 μg) 25.5 ± 0.7 31 ± 1.4 34 ± 1.4 33 ± 1.4 26 ± 1.4 31 ± 1.4 Tobramycin (10 μg) 19.5 ± 0.7 19 ± 1.4 18.5 ± 0.7 21 ± 1.4 11.5 ± 0.7 19 ± 1.4 Vancomycin (30 μg)000000 aValues are expressed as the means of two determinations. Chromatography (GLC) according to the instructions Simmon citrate, urease, VP, hydrolysis of esculin, of the Microbial Identifi cation System (MIDI) Sherlock oxidation of glucose, assimilation of potassium glu- version 6.0 (Sasser, 1990) with the RTSBA6 MIDI data- conate, adipate and malate were positive. It grew at base. 40°C, in the presence of 4.5% (w/v) NaCl and at pH 5 DNA of strain A3-3T was extracted from 18‒24 h cells to 10 but it could not grow at 4°C and 50°C, in the pres- grown on TYEG agar and purifi ed by the method of ence of 5% (w/v) NaCl or at pH 4. Strain A3-3T was Saito and Miura (1963). DNA base composition was differentiated from the closest type strain, C. kerstersii determined by reversed-phase HPLC (Tamaoka and LMG 3475T by the growth at pH 5, urease, nitrate re- Komagata, 1984).