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 , 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-. 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;

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] 1 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 7H2O, scribed by Mesbahet al. [15]. The obtained nucleoside 0.01 g FeSO4 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 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 well1). 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 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. Strain On1 had a fatty acid profile T products displayed amylase activity. Vitamins and yeast similar to that of C. manganoxidans strain HS which extract were not needed for growth. The strain also grew also contained the predominant fatty acid 16:0, 18:1 o7c T well on complex medium (LB, TSB and NB) and formed and summed feature 3. However, strain On1 contained light-yellow pigmented colonies on the surface of agar a less amount of fatty acid 17:0 cyclo (1.9%), and thus T plates. The colony morphology was circular, entire, and clearly differentiate from C. manganoxidans strain HS . convex shape. The size of colonies was approximately Table 1 shows the details of the fatty acid composition T T 1.0–2.5 mm in diameter on LB agar after 48 hof of C. manganoxidans strain HS and strain On1 grew incubation at 55 1C. Strain On1T grew at temperatures under identical growthconditions (TSA and 55 1C). ranging from 35 to 60 1C, pH values between 6.0 and 8.0, and NaCl concentrations between 0.2% and 1.0%. Biochemical analysis Optimal growthcondition (as determined by measuring optical densities) was around 55 1C, pH 7.0 and 0.5% Strain On1T exhibited following biochemical char- NaCl. The strain could not grow under anaerobic acteristics as determined using the results of API 20NE, condition by using the Oxoid AnaeroGen system. API ZYM and Biolog GNII microtest galleries: Light microscopic examination revealed that cells of catalase, nitrate reduction; indole production; gelatin T strain On1 were Gram-negative rods. Cells were non- hydrolysis, alkaline phosphatase, C4 esterase, C8 lipase, spore-forming and motile by means of single polar leucine arylamidase, acid phosphatase, naphthol-AS-BI- flagella. The sizes of rods were approximately phosphohydrolase, a-glucosidase; assimilation of 0.6–0.8 mm in diameter and 1.2–2.2 mm in length. Poly- glucose, mannitol, maltose, gluconate, adipate, citrate, b-hydroxybutyrate granules were present. D-fructose, methyl pyruvate, b-hydroxybutyric acid, D,L-lactate, L-aspartic acid, L-proline, D-sorbitol, D- Phylogenetic analysis trehalose, turanose, mono-methyl succinate, a-keto glutaric acid, urocanic acid, glycerol, arabitol, L- Comparison of the 16S rRNA gene sequence of strain glutamic acid, acetic acid, cis-aconitic acid, formic acid, T On1 (GenBank nucleotide sequence accession number a-hydroxybutyric acid, a-keto valeric acid, D-saccharic is AY845052) withavailable 16S rRNA gene sequences acid, succinic acid, D-alanine, glycyl-L-glutamic acid, ARTICLE IN PRESS 418 W.-M. Chen et al. / Systematic and Applied Microbiology 28 (2005) 415–420

Chitinimonas taiwanensis cf T(AY323827) Wautersia taiwanensis LMG 19424T (AF300324) Schlegelella thermodepolymerans K14T (AY152824) Leptothrix mobilis Feox-1T (X97071) Leptothrix cholodnii CCM 1827 (X97070) 66 Roseateles depolymerans 61AT (AB003623) Leptothrix discophora AT CC 43182T (L33975) 67 Sphaerotilus natans 565 (Z18534) Ideonella dechloratans CCUG 30898T(X72724) 96 Alcaligenes latus IAM 12599T (D88007) 67 T 86 Rubrivivax gelatinosus AT CC 17011 (D16213) Aquabacterium parvum B6T (AF035052) T 69 98 Aquabacterium citratiphilum B4 (AF035050) Aquabacterium commune B8T(AF035054) 100 Caldimonas taiwanensis On1T (AY 845052) T 100 Caldimonas manganoxidans JC M 10698 (AB008801) 100 Tepidimonas ignava SPS-1037T (AF177943) Tepidimonas aquatica CLN-1T (AY324139) Brachymonas denitrificans AS-P1T (D14320) Hydrogenophagaflava DSM 619T (AB021420) 75 T 99 Comamonastestosteroni AT CC 11996 (M11224) Delftia acidovorans ACM 489T (AF078774) T 90 Polaromonas vacuolata 34-P (U14585) Rhodoferax fermentans FR 2T (D16211) Acidovorax facilis CCUG 2113T (AF078765) 69 Variovorax paradoxus DSM 66T (AJ420329) 1% Xylophilus ampelinus′ AT CC 33914T (AF078758)

Fig. 1. Neighbor-joining phylogenetic tree of Caldimonas taiwanensis strain On1T and related bacteria of b-Proteobacteria based on 16S rRNA sequence comparisons. Scale bar indicates 1% sequence dissimilarity (one substitution per 100 nt). Bootstrap values (%) are indicated at the branches from 1000 replications. Only bootstrap values greater than 50% are shown. Representative sequences in the dendrogram obtained from GenBank with almost complete sequences were used in the phylogenetic analysis (GenBank accession numbers are shown in parentheses). The sequences of Chitinimonas taiwanensis cfT [4] and Wautersia taiwanensis LMG 19424T [5] were used as the outgroup.

Table 1. Fatty acid composition of the strains studieda The following biochemical characteristics were absent: oxidase, glucose fermentation, arginine dihydrolase, urease, Compound C. manganoxidans C. taiwanensis esculin hydrolysis, b-galactosidase, C14 lipase, valine HST On1T arylamidase, cystine arylamidase, trypsin,a-chymotrypsi- 10:0 3OH 4.4 6.3 n,a-galactosidase, b-glucuronidase, a-mannosidase, a-fuco- 12:0 3.6 4.3 sidase, N-acetyl-b-glucosaminidase activity; assimilation of 14:0 3.0 2.9 arabinose, mannose, N-acetyl-glucosamine, caprate, malate, 15:1 o6c 1.1 phenyl-acetate, tween 40, tween 80, cellobiose, N-acetyl-D- 16:0 27.3 30.4 glucosamine, L-asparagine,a-cyclodextrin, adonitol, i-ery- 17:0 cyclo 19.4 1.9 thritol, L-fucose, D-galactose, gentiobiose, m-inositol, a-D- 17:0 4.1 lactose, lactulose, D-melibiose,b-methyl D-glucoside, psicose, 18:1 o7c 20.0 20.0 D-raffinose, L-rhamnose, sucrose, xylitol, D-galactonic acid 18:0 2.1 Summed feature 12.3 31.3 lactone, D-galacturonic acid, D-glucosaminic acid, D-glu- 3b curonic acid, g-hydroxybutyric acid, r-hydroxy phenylacetic acid, itaconic acid, a-keto butyric acid, malonic acid, aValues are shown as a percentage of the total fatty acid content for propionic acid, quinic acid, sebacic acid, bromo succinic eachstrain. All strains grew at 55 1C for 48 hon TSA medium, and acid, succinamic acid, glucuronamide, alaninamide, L- then the fatty acid composition was analyzed. Values for fatty acid present at level of less than 1.0% in the strain are not given. alanine, L-alanyl-glycine, glycyl-L-aspartic acid, L-histidine, bSummed feature 3 comprises 16:1o7c or 15:0 iso 2OH or both. hydroxy L-proline, L-phenylalanine, L-pyroglutamic acid, L-serine, D-serine, L-threonine, D,L-carnitine, uridine, phenyl L-ornithine, g-amino butyric acid, inosine, thymidine, 2- ethylamine and putrescine. T amino ethanol, 2,3-butanediol, D,L-a-glycerol phos- Strain On1 was susceptible to all antibiotics tested, phate, glucose-1-phosphate, glucose-6-phosphate. including ampicillin, cefotaxime, chloramphenicol, ARTICLE IN PRESS W.-M. Chen et al. / Systematic and Applied Microbiology 28 (2005) 415–420 419

Table 2. The difference in phenotypic and biochemical citrate, whereas can not utilize arabinose, mannose, N- characteristics between strain On1T and its closed neighbora acetyl-glucosamine, caprate, malate, and phenyl-acetate are not utilized. The major fatty acid components were Characteristic C. manganoxidans C. taiwanensis HST On1T 16:0, 18:1 o7c and summed feature 3 (16:1o7c or 15:0 iso 2OH or both[about 31%]). TheG+C content of its Optimum growth 50 55 DNA is 65.9 mol%. temperature (1C) The type strain, On1T, was isolated from water of Sih- Optimum growth 8–9 7 Chong-Si hot spring at Pingtung located near the very pH southern part of Taiwan. The type strain has been Cell size (mm) 0.5–0.7 2.2–3.5 0.6–0.8 1.2–2.2 deposited in BCRC (Bioresource Collection and Re- Oxidase + — searchCenter, Food Industry Researchand Develop- Catalase + w ment Institute, PO Box 246, Hsinchu, Taiwan, 30099) as Utilization of: T Galactose + — BCRC 17405 and in BCCM/LMG Bacteria Collection Malate + — (Laboratorium voor Microbiologie, Universiteit Gent, T Malonate + — Gent, Belgium) as LMG 22827 . Sucrose + — Accession: The DDBJ/EMBL/GenBank accession Acetate — + number for the 16S rDNA sequence of the strain Fructose — + On1T is AY845052. Trehalose — + G+C content 66.2 65.9 (mol%) Acknowledgements aThe data of C. manganoxidans HST was obtained from Takeda et al. [22]. W.-M. Chen was supported by a grant from the National Science Council, Taipei, Taiwan, Republic of China (NSC 93-2320-B-022-001 and -003). gentamicin, kanamycin, nalidixic acid, novobiocin, penicillin G, rifampin, streptomycin and tetracycline. Table 2 indicates the difference in the phenotypic and References biochemical characteristics between strain On1T and C. manganoxidans strain HST. T [1] P. Bernfeld, Amylases a- and b- methods, Enzymol The foregoing results show that strain On1 was 1 (1955) 149–158. readily distinguished from its nearest phylogenetic [2] C. Bertoldo, G. Antranikian, Starch-hydrolyzing enzymes T neighbor C. manganoxidans strain HS in terms of fatty from thermophilic archaea and bacteria, Curr. Opin. acid compositions (Table 1), phenotypic and biochem- Chem. Biol. 6 (2002) 151–160. ical characteristics (Table 2), as well as DNA–DNA [3] A. Burhan, U. Nisa, C. Go¨ khan, C. O¨ mer, A. Ashabil, G. reassociation experiments. Thus, strain On1T unam- Osman, Enzymatic properties of a novel thermostable, biguously represents a novel Caldimonas species for thermophilic, alkaline and chelator resistant amylase from which we propose the name Caldimonas taiwanensis an alkaliphilic Bacillus sp. isolate ANT-6, Process sp. nov. Biochem. 38 (2003) 1397–1403. [4] S.C. Chang, J.T. Wang, P. Vandamme, J.H. Hwang, P.S. Chang, W.M. Chen, Chitinimonas taiwanensis gen. nov., sp. nov., a novel chitinolytic bacterium isolated from a Description of Caldimonas taiwanensis sp. nov. freshwater pond for shrimp culture, Syst. Appl. Micro- biol. 27 (2004) 43–49. Caldimonas taiwanensis (tai.wan.en’sis N.L. fem. adj. [5] W.M. Chen, S. Laevens, T.M. Lee, T. Coenye, P. De Vos, Taiwanensis, of Taiwan, where the type strain was M. Mergeay, P. Vandamme, Ralstonia taiwanensis sp. isolated). Cells are Gram-negative withrod shape, nov., isolated from root nodules of Mimosa species and 0.6–0.8 mm in diameter and 1.2–2.2 mm in length. They sputum of a cystic fibrosis patient, Int. J. Syst. Evol. occur singly and are motile by single polar flagella. Poly- Microbiol. 51 (2001) 1729–1735. b-hydroxybutyrate granules are stored as reserve mate- [6] A.P. Chung, F. Rainey, M.F. Nobre, J. Burghardt, M.S. rial. Growthoccurs at temperatures ranging from 35 to da Costa, Meiothermus cerbereus sp. nov., a new slightly thermophilic species with high levels of 3-hydroxy fatty 60 1C withan optimum at 55 1C and pH 7.0. They are acids, Int. J. Syst. Bacteriol. 47 (1997) 1225–1230. positive for catalase, nitrate reduction, indole produc- [7] Y.C. Chung, T. Kobayashi, H. Kanai, T. Akiba, T. tion and gelatin hydrolysis, but are negative for oxidase, Kudo, Purification and properties of extracellular amy- glucose fermentation, urease, arginine dihydrolase, lase from the hyperthermophilic archeon Thermococccus esculin hydrolysis, and b-galactosidase. They utilize profundus DT5432, Appl. Environ. Microbiol. 61 (1995) glucose, mannitol, maltose, gluconate, adipate and 1502–1506. ARTICLE IN PRESS 420 W.-M. Chen et al. / Systematic and Applied Microbiology 28 (2005) 415–420

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