ARTICLE IN PRESS Systematic and Applied Microbiology 28 (2005) 415–420 www.elsevier.de/syapm Caldimonas taiwanensis sp. nov., a amylase producing bacterium isolated from a hot spring Wen-Ming Chena,Ã, Jo-Shu Changb, Ching-Hsiang Chiua, Shu-Chen Changc, Wen-Chieh Chena, Chii-Ming Jianga aDepartment of Seafood Science, National Kaohsiung Marine University, No. 142, Hai-Chuan Rd. Nan-Tzu, Kaohsiung City 811, Taiwan bDepartment of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan cTajen Institute of Technology, Yen-Pu, Pingtung, Taiwan Received 22 February 2005 Abstract During screening for amylase-producing bacteria, a strain designated On1T was isolated from a hot spring located in Pingtung area, which is near the very southern part of Taiwan. Cells of this organism were Gram-negative rods motile by means of a single polar flagellum. Optimum conditions for growthwere 55 1C and pH 7. Strain On1T grew well in minimal medium containing starchas thesole carbon source, and its extracellular products expressed amylase activity. The 16S rRNA gene sequence analysis indicates that strain On1T is a member of b-Proteobacteria. On the basis of a phylogenetic analysis of 16S rDNA sequences, DNA–DNA similarity data, physiological and biochemical characteristics, as well as fatty acid compositions, the organism belonged to the genus Caldimonas and represented a novel species within this genus. The predominant cellular fatty acids of strain On1T were 16:0 (about 30%), 18:1 o7c (about 20%) and summed feature 3 (16:1o7c or 15:0 iso 2OH or both[about 31%]). Its DNA base ratio was 65.9 mol% G+C. We propose to classify strain On1T ( ¼ BCRC 17405T ¼ LMG 22827T)asCaldimonas taiwanensis sp. nov. r 2005 Elsevier GmbH. All rights reserved. Keywords: Caldimonas taiwanensis; b-Proteobacteria; Hot spring; Amylase; Taxonomy Introduction including dextrins and progressively smaller polymers composed of glucose units [1,7,10]. Amylolytic enzymes Starchis a major polysaccharidereserve in plants. play an important role in the biogeochemical cycle of Starchhydrolysis creates a lot of important products carbon and they are also among the most important with different physical and chemical properties for food enzymes in current biotechnological-based food, deter- and industries [13]. Amylases are enzymes which gent, and pharmaceutical industries [16]. Due to the hydrolyse starch molecules to give diverse products extensive industrial importance of amylase, there is ongoing interest in the isolation of effective bacterial ÃCorresponding author. Tel.: +886 8 733 06 04; strains producing amylases well suited to new industrial fax: +886 8 733 86 00. applications. Since hydrolysis of starch is known to E-mail address: [email protected] (W.-M. Chen). proceed at a much faster rate at high temperatures, the 0723-2020/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2005.02.008 ARTICLE IN PRESS 416 W.-M. Chen et al. / Systematic and Applied Microbiology 28 (2005) 415–420 thermophilic amylases are apparently preferable to was performed on LB medium under the Oxoid practical applications. Several efforts were made on AnaeroGen system. identifying thermophilic bacteria producing extracellu- lar amylases, which are shown to have significant 16S rDNA sequencing and phylogenetic analysis industrial importance [2,3,14]. In the present study, a novel bacterium was isolated Amplification and sequence analysis of the 16S rRNA from a hot spring located in southern Taiwan. The gene was performed as described previously [5].The characteristics of this isolate were compared with its sequence was compared withothers available in Gen- nearest neighbor. Bank and Ribosomal Database Project II. The multiple- sequence alignment including strain On1T and its closest relatives were performed using the BioEdit software [12]. Materials and methods The phylogenetic reconstruction was inferred by using the neighbor-joining method [20]. A bootstrap analysis Isolation and growth condition (confidence values estimated from 1000 replications of eachsequence) was performed using theCLUSTAL w In May 2004, a sample (1000 ml) of water was 1.7 program [23]. A phylogenetic tree was drawn using collected from a Sih-Chong-Si hot spring at Pingtung, the TREEVIEW program [18]. Sequence identities were located in the southern part of Taiwan. The temperature calculated using the BioEdit software [12]. of the spring water ranged from 50 to 60 1C, and the pH was approximately 8.0. The water sample was diluted Determination of the DNA base composition withsterile distilled water (121 1C, 15 min) and spread onto plates of SD medium incubated at 55 1C. The SD DNA was prepared as described by Pitcher et al. [19] À1 medium (l ) consisted of 10 g starch, 2 g (NH4)2SO4, and degraded enzymatically into nucleosides as de- 0.7 g K2HPO4, 0.3 g KH2PO4, 0.5 g MgSO4 Á 7H2O, scribed by Mesbahet al. [15]. The obtained nucleoside 0.01 g FeSO4 Á 7H2O and 15 g agar. Amylase production mixture was then separated by high-performance liquid was determined by the development of a colorless chromatography using a Waters Symmetry Shield C8 halo zone surrounding bacterial colonies when Lugol column thermostated at 37 1C. The solvent was 0.02 M solution (1% iodine in 2% potassium iodide [w/v]) NH4H2PO4 (pH 4.0) with1.5% acetonitrile. Non- was added to the plates. A bacterial strain, designated methylated lambda phage DNA (Sigma) was used as On1T, was obtained and shown to express amylase the calibration reference. activities. DNA–DNA hybridizations Morphological and physiological tests DNA–DNA hybridizations were performed with Strain On1T was grown on Luria-Bertani Broth(LB photobiotin-labeled probes as described by Ezaki et al. 1 Broth, Miller; Difco) for 24 h at 551C. The cell [8]. The hybridization temperature was 50 C and the morphology was observed by a light microscope (Zeiss reaction was carried out in 50% formamide. Eachvalue Axioskop) at 1000 Â magnification. The motility of was the mean of two hybridization experiments. exponential-phase cells was examined by the hanging drop method. The flagella stain was performed using Fatty acid methyl ester analysis SoptTest Flagella Stain (Difco). The Gram stain was performed using the Gram Stain Set S (Difco). Poly-b- Cells were grown on Tryptic Soy Agar (TSA) (Difco) hydroxybutyrate granule accumulation was observed by at 55 1C. Cellular fatty acids were analyzed as methyl light microscopy after being stained with Sudan black [9]. esters by GC according to the instructions of Microbial The pH range for growth was determined by Identification System (MIDI; Microbial ID) [21]. measuring optical densities of the cultures grown in LB medium, whose pH was adjusted to values between Biochemical analysis 3.0 and 11.0 withappropriate biological buffer [6]. For the determination of the temperature range for growth, Characteristics presented in the API 20NE and API cells were incubated in LB medium at temperatures ZYM (bioMe´ rieux) microtest systems were determined between 15 and 80 1C and the cell growth was according to the recommendations of the manufac- determined by measuring optical density of the culture turers. For carbon substrate metabolisation tests, Biolog withrespect to time. Tolerance to NaCl was determined GNII microtiter test plates were used. Early log-phase by adjusting the salinity of LB medium to values cultures were used as inoculum for the test plate between 0% and 3.0% NaCl. Anaerobic cultivation (150 ml wellÀ1). The plates were incubated at 55 1Cand ARTICLE IN PRESS W.-M. Chen et al. / Systematic and Applied Microbiology 28 (2005) 415–420 417 examined after 24 hand 48 hto allow for thedevelop- in public databases (GenBank and Ribosomal Database ment of a purple color indicative of substrate oxidation. Project II) revealed that strain On1T belonged to the Burkholderiales group of the b-Proteobacteria. The Antimicrobial susceptibility testing highest similarity values were obtained towards the genus Caldimonas, Leptothrix, Schlegelella and Tepidi- The susceptibility to antimicrobial agents was deter- monas. In order to determine the phylogenetic position, T mined by disk diffusion. The density of the bacterial the 16S rDNA sequence of strain On1 was compared suspension from exponential-phase culture was adjusted with those related species within the b-Proteobacteria. T withsterile saline to theturbidity corresponding to 0.5 of The 16S rDNA sequence of strain On1 clustered T the McFarland standard and then spread onto LB together with C. manganoxidans strain HS (97.0% T medium for further incubation at 55 1C. The following similarity) [22], T. aquatica strain CLN-1 (95.1% T antimicrobial disks (Dispens-O-Susceptibility Test Disks, similarity) [11] and T. ignava strain SPS-1037 (95.0% Difco) were used: ampicillin (10 mg), cefotaxime (30 mg), similarity) [17] (Fig. 1). The similarity levels towards chloramphenicol (30mg), gentamicin (10 mg), kanamycin other bacterial species belonging to the Burkholderiales (30 mg), nalidixic acid (30 mg), novobiocin (30 mg), peni- group of the b-Proteobacteria were lower than 95%. cillin G (10 mg), rifampin (5 mg), streptomycin (10 mg) and Subsequently, whole-genome DNA–DNA hybridization T tetracycline (30 mg). Susceptibility was defined as zones experiment was preformed between strain On1 and the 43 mm, resistance of zones of 1 mm, and moderate type strain of its nearest phylogenetic neighbor, C. o T resistance of zones of 1–3 mm to the edge of a disk. manganoxidans strain HS . The binding level of strain On1T towards C. manganoxidans strain HST was 40.3%. The guanine-plus-cytosine (G+C) content of strain On1T DNA was 65.9 mol%. Results and discussion Phenotypic and morphological characteristics Fatty acid compositions A bacterial strain, designated as On1T,wasisolated The major cellular fatty acid components of strain T from water of a hot spring in southern Taiwan. Strain On1 were 16:0 (30.4%), 18:1 o7c (20.0%) and summed On1T grew well aerobically in a minimal medium contain- feature 3 (31.3%) which comprised 16:1o7c or 15:0 iso T ing starchas thesole carbon source, and its extracellular 2OH or both.