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Caldimonas taiwanensis sp. nov., a amylase producing bacterium isolated from a hot spring
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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,, Jo-Shu Changb, Ching-Hsiang Chiua, Shu-Chen Changc, Wen-Chieh Chena, Chii-Ming Jianga a
Department of Seafood Science, National Kaohsiung Marine University, No. 142, Hai-Chuan Rd. Nan-Tzu, Kaohsiung City 811, Taiwan b Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan c Tajen 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 growth were 55 1C and pH 7. Strain On1T grew well in minimal medium containing starch as the sole 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) as Caldimonas taiwanensis sp. nov. r 2005 Elsevier GmbH. All rights reserved. Keywords: Caldimonas taiwanensis; b-Proteobacteria; Hot spring; Amylase; Taxonomy
Introduction Starch is a major polysaccharide reserve in plants. Starch hydrolysis creates a lot of important products with different physical and chemical properties for food and industries [13]. Amylases are enzymes which hydrolyse starch molecules to give diverse products Corresponding author. Tel.: +886 8 733 06 04; fax: +886 8 733 86 00. E-mail address: [email protected] (W.-M. Chen).
0723-2020/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2005.02.008
including dextrins and progressively smaller polymers composed of glucose units [1,7,10]. Amylolytic enzymes play an important role in the biogeochemical cycle of carbon and they are also among the most important enzymes in current biotechnological-based food, detergent, and pharmaceutical industries [16]. Due to the extensive industrial importance of amylase, there is ongoing interest in the isolation of effective bacterial strains producing amylases well suited to new industrial applications. Since hydrolysis of starch is known to proceed at a much faster rate at high temperatures, the
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thermophilic amylases are apparently preferable to practical applications. Several efforts were made on identifying thermophilic bacteria producing extracellular amylases, which are shown to have significant industrial importance [2,3,14]. In the present study, a novel bacterium was isolated from a hot spring located in southern Taiwan. The characteristics of this isolate were compared with its nearest neighbor.
Materials and methods Isolation and growth condition In May 2004, a sample (1000 ml) of water was collected from a Sih-Chong-Si hot spring at Pingtung, located in the southern part of Taiwan. The temperature of the spring water ranged from 50 to 60 1C, and the pH was approximately 8.0. The water sample was diluted with sterile distilled water (121 1C, 15 min) and spread onto plates of SD medium incubated at 55 1C. The SD medium (l1) consisted of 10 g starch, 2 g (NH4)2SO4, 0.7 g K2HPO4, 0.3 g KH2PO4, 0.5 g MgSO4 7H2O, 0.01 g FeSO4 7H2O and 15 g agar. Amylase production was determined by the development of a colorless halo zone surrounding bacterial colonies when Lugol solution (1% iodine in 2% potassium iodide [w/v]) was added to the plates. A bacterial strain, designated On1T, was obtained and shown to express amylase activities.
was performed on LB medium under the Oxoid AnaeroGen system.
16S rDNA sequencing and phylogenetic analysis Amplification and sequence analysis of the 16S rRNA gene was performed as described previously [5]. The sequence was compared with others available in GenBank and Ribosomal Database Project II. The multiplesequence alignment including strain On1T and its closest relatives were performed using the BioEdit software [12]. The phylogenetic reconstruction was inferred by using the neighbor-joining method [20]. A bootstrap analysis (confidence values estimated from 1000 replications of each sequence) was performed using the CLUSTAL w 1.7 program [23]. A phylogenetic tree was drawn using the TREEVIEW program [18]. Sequence identities were calculated using the BioEdit software [12].
Determination of the DNA base composition DNA was prepared as described by Pitcher et al. [19] and degraded enzymatically into nucleosides as described by Mesbah et al. [15]. The obtained nucleoside mixture was then separated by high-performance liquid chromatography using a Waters Symmetry Shield C8 column thermostated at 37 1C. The solvent was 0.02 M NH4H2PO4 (pH 4.0) with 1.5% acetonitrile. Nonmethylated lambda phage DNA (Sigma) was used as the calibration reference.
DNA–DNA hybridizations Morphological and physiological tests Strain On1T was grown on Luria-Bertani Broth (LB Broth, Miller; Difco) for 24 h at 55 1C. The cell morphology was observed by a light microscope (Zeiss Axioskop) at 1000 magnification. The motility of exponential-phase cells was examined by the hanging drop method. The flagella stain was performed using SoptTest Flagella Stain (Difco). The Gram stain was performed using the Gram Stain Set S (Difco). Poly-bhydroxybutyrate granule accumulation was observed by light microscopy after being stained with Sudan black [9]. The pH range for growth was determined by measuring optical densities of the cultures grown in LB medium, whose pH was adjusted to values between 3.0 and 11.0 with appropriate biological buffer [6]. For the determination of the temperature range for growth, cells were incubated in LB medium at temperatures between 15 and 80 1C and the cell growth was determined by measuring optical density of the culture with respect to time. Tolerance to NaCl was determined by adjusting the salinity of LB medium to values between 0% and 3.0% NaCl. Anaerobic cultivation
DNA–DNA hybridizations were performed with photobiotin-labeled probes as described by Ezaki et al. [8]. The hybridization temperature was 50 1C and the reaction was carried out in 50% formamide. Each value was the mean of two hybridization experiments.
Fatty acid methyl ester analysis Cells were grown on Tryptic Soy Agar (TSA) (Difco) at 55 1C. Cellular fatty acids were analyzed as methyl esters by GC according to the instructions of Microbial Identification System (MIDI; Microbial ID) [21].
Biochemical analysis Characteristics presented in the API 20NE and API ZYM (bioMe´rieux) microtest systems were determined according to the recommendations of the manufacturers. For carbon substrate metabolisation tests, Biolog GNII microtiter test plates were used. Early log-phase cultures were used as inoculum for the test plate (150 ml well1). The plates were incubated at 55 1C and
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examined after 24 h and 48 h to allow for the development of a purple color indicative of substrate oxidation.
Antimicrobial susceptibility testing The susceptibility to antimicrobial agents was determined by disk diffusion. The density of the bacterial suspension from exponential-phase culture was adjusted with sterile saline to the turbidity corresponding to 0.5 of the McFarland standard and then spread onto LB medium for further incubation at 55 1C. The following antimicrobial disks (Dispens-O-Susceptibility Test Disks, Difco) were used: ampicillin (10 mg), cefotaxime (30 mg), chloramphenicol (30 mg), gentamicin (10 mg), kanamycin (30 mg), nalidixic acid (30 mg), novobiocin (30 mg), penicillin G (10 mg), rifampin (5 mg), streptomycin (10 mg) and tetracycline (30 mg). Susceptibility was defined as zones 43 mm, resistance of zones of o1 mm, and moderate resistance of zones of 1–3 mm to the edge of a disk.
Results and discussion
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in public databases (GenBank and Ribosomal Database Project II) revealed that strain On1T belonged to the Burkholderiales group of the b-Proteobacteria. The highest similarity values were obtained towards the genus Caldimonas, Leptothrix, Schlegelella and Tepidimonas. In order to determine the phylogenetic position, the 16S rDNA sequence of strain On1T was compared with those related species within the b-Proteobacteria. The 16S rDNA sequence of strain On1T clustered together with C. manganoxidans strain HST (97.0% similarity) [22], T. aquatica strain CLN-1T (95.1% similarity) [11] and T. ignava strain SPS-1037T (95.0% similarity) [17] (Fig. 1). The similarity levels towards other bacterial species belonging to the Burkholderiales group of the b-Proteobacteria were lower than 95%. Subsequently, whole-genome DNA–DNA hybridization experiment was preformed between strain On1T and the type strain of its nearest phylogenetic neighbor, C. manganoxidans strain HST. 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%.
Phenotypic and morphological characteristics
Fatty acid compositions
A bacterial strain, designated as On1T, was isolated from water of a hot spring in southern Taiwan. Strain On1T grew well aerobically in a minimal medium containing starch as the sole carbon source, and its extracellular products displayed amylase activity. Vitamins and yeast extract were not needed for growth. The strain also grew well on complex medium (LB, TSB and NB) and formed light-yellow pigmented colonies on the surface of agar plates. The colony morphology was circular, entire, and convex shape. The size of colonies was approximately 1.0–2.5 mm in diameter on LB agar after 48 h of incubation at 55 1C. Strain On1T grew at temperatures ranging from 35 to 60 1C, pH values between 6.0 and 8.0, and NaCl concentrations between 0.2% and 1.0%. Optimal growth condition (as determined by measuring optical densities) was around 55 1C, pH 7.0 and 0.5% NaCl. The strain could not grow under anaerobic condition by using the Oxoid AnaeroGen system. Light microscopic examination revealed that cells of strain On1T were Gram-negative rods. Cells were nonspore-forming and motile by means of single polar flagella. The sizes of rods were approximately 0.6–0.8 mm in diameter and 1.2–2.2 mm in length. Polyb-hydroxybutyrate granules were present.
The major cellular fatty acid components of strain On1T were 16:0 (30.4%), 18:1 o7c (20.0%) and summed feature 3 (31.3%) which comprised 16:1o7c or 15:0 iso 2OH or both. Strain On1T had a fatty acid profile similar to that of C. manganoxidans strain HST which also contained the predominant fatty acid 16:0, 18:1 o7c and summed feature 3. However, strain On1T contained a less amount of fatty acid 17:0 cyclo (1.9%), and thus clearly differentiate from C. manganoxidans strain HST. Table 1 shows the details of the fatty acid composition of C. manganoxidans strain HST and strain On1T grew under identical growth conditions (TSA and 55 1C).
Phylogenetic analysis Comparison of the 16S rRNA gene sequence of strain On1T (GenBank nucleotide sequence accession number is AY845052) with available 16S rRNA gene sequences
Biochemical analysis Strain On1T exhibited following biochemical characteristics as determined using the results of API 20NE, API ZYM and Biolog GNII microtest galleries: catalase, nitrate reduction; indole production; gelatin hydrolysis, alkaline phosphatase, C4 esterase, C8 lipase, leucine arylamidase, acid phosphatase, naphthol-AS-BIphosphohydrolase, a-glucosidase; assimilation of glucose, mannitol, maltose, gluconate, adipate, citrate, D-fructose, methyl pyruvate, b-hydroxybutyric acid, D,L-lactate, L-aspartic acid, L-proline, D-sorbitol, Dtrehalose, turanose, mono-methyl succinate, a-keto glutaric acid, urocanic acid, glycerol, arabitol, Lglutamic acid, acetic acid, cis-aconitic acid, formic acid, a-hydroxybutyric acid, a-keto valeric acid, D-saccharic acid, succinic acid, D-alanine, glycyl-L-glutamic acid,
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66
96
100
99
1%
Chitinimonas taiwanensis cf T(AY323827) T Wautersia taiwanensis LMG 19424 (AF300324) Schlegelella thermodepolymerans K14T (AY152824) Leptothrix mobilis Feox-1T (X97071) Leptothrix cholodnii CCM 1827 (X97070) Roseateles depolymerans 61AT (AB003623) Leptothrix discophora AT CC 43182T (L33975) Sphaerotilus natans 565 (Z18534) 67 T Ideonella dechloratans CCUG 30898 (X72724) T (D88007) Alcaligenes latus IAM 12599 67 T Rubrivivax gelatinosus AT CC 17011 (D16213) 86 T Aquabacterium parvum B6 (AF035052) T 98 Aquabacterium citratiphilum B4 (AF035050) 69 T Aquabacterium commune B8 (AF035054) 100 Caldimonas taiwanensis On1T (AY 845052) Caldimonas manganoxidans JC M 10698T (AB008801) Tepidimonas ignava SPS-1037T (AF177943) 100 T Tepidimonas aquatica CLN-1 (AY324139) Brachymonas denitrificans AS-P1T (D14320) T Hydrogenophagaflava DSM 619 (AB021420) T 75 Comamonastestosteroni AT CC 11996 (M11224) T Delftia acidovorans ACM 489 (AF078774) Polaromonas vacuolata 34-PT (U14585) 90 T Rhodoferax fermentans FR 2 (D16211) T Acidovorax facilis CCUG 2113 (AF078765) T 69 Variovorax paradoxus DSM 66 (AJ420329) 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
Compound
C. manganoxidans HST
10:0 3OH 12:0 14:0 15:1 o6c 16:0 17:0 cyclo 17:0 18:1 o7c 18:0 Summed feature 3b
4.4 3.6 3.0 1.1 27.3 19.4 4.1 20.0 12.3
C. taiwanensis On1T 6.3 4.3 2.9 30.4 1.9 20.0 2.1 31.3
a Values are shown as a percentage of the total fatty acid content for each strain. All strains grew at 55 1C for 48 h on TSA medium, and 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. b Summed feature 3 comprises 16:1o7c or 15:0 iso 2OH or both.
L-ornithine,
g-amino butyric acid, inosine, thymidine, 2amino ethanol, 2,3-butanediol, D,L-a-glycerol phosphate, glucose-1-phosphate, glucose-6-phosphate.
The following biochemical characteristics were absent: oxidase, glucose fermentation, arginine dihydrolase, urease, esculin hydrolysis, b-galactosidase, C14 lipase, valine arylamidase, cystine arylamidase, trypsin,a-chymotrypsin,a-galactosidase, b-glucuronidase, a-mannosidase, a-fucosidase, N-acetyl-b-glucosaminidase activity; assimilation of arabinose, mannose, N-acetyl-glucosamine, caprate, malate, phenyl-acetate, tween 40, tween 80, cellobiose, N-acetyl-Dglucosamine, L-asparagine,a-cyclodextrin, adonitol, i-erythritol, L-fucose, D-galactose, gentiobiose, m-inositol, a-Dlactose, lactulose, D-melibiose,b-methyl D-glucoside, psicose, D-raffinose, L-rhamnose, sucrose, xylitol, D-galactonic acid lactone, D-galacturonic acid, D-glucosaminic acid, D-glucuronic acid, g-hydroxybutyric acid, r-hydroxy phenylacetic acid, itaconic acid, a-keto butyric acid, malonic acid, propionic acid, quinic acid, sebacic acid, bromo succinic acid, succinamic acid, glucuronamide, alaninamide, Lalanine, L-alanyl-glycine, glycyl-L-aspartic acid, L-histidine, hydroxy L-proline, L-phenylalanine, L-pyroglutamic acid, L-serine, D-serine, L-threonine, D,L-carnitine, uridine, phenyl ethylamine and putrescine. Strain On1T was susceptible to all antibiotics tested, including ampicillin, cefotaxime, chloramphenicol,
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Table 2. The difference in phenotypic and biochemical characteristics between strain On1T and its closed neighbora Characteristic
C. manganoxidans HST
C. taiwanensis On1T
Optimum growth temperature (1C) Optimum growth pH Cell size (mm) Oxidase Catalase Utilization of: Galactose Malate Malonate Sucrose Acetate Fructose Trehalose G+C content (mol%)
50
55
8–9
7
0.5–0.7 2.2–3.5 + +
0.6–0.8 1.2–2.2 — w
+ + + + — — — 66.2
— — — — + + + 65.9
The data of C. manganoxidans HST was obtained from Takeda et al. [22].
419
citrate, whereas can not utilize arabinose, mannose, Nacetyl-glucosamine, caprate, malate, and phenyl-acetate are not utilized. The major fatty acid components were 16:0, 18:1 o7c and summed feature 3 (16:1o7c or 15:0 iso 2OH or both [about 31%]). The G+C content of its DNA is 65.9 mol%. The type strain, On1T, was isolated from water of SihChong-Si hot spring at Pingtung located near the very southern part of Taiwan. The type strain has been deposited in BCRC (Bioresource Collection and Research Center, Food Industry Research and Development Institute, PO Box 246, Hsinchu, Taiwan, 30099) as BCRC 17405T and in BCCM/LMG Bacteria Collection (Laboratorium voor Microbiologie, Universiteit Gent, Gent, Belgium) as LMG 22827T. Accession: The DDBJ/EMBL/GenBank accession number for the 16S rDNA sequence of the strain On1T is AY845052.
Acknowledgements
a
gentamicin, kanamycin, nalidixic acid, novobiocin, penicillin G, rifampin, streptomycin and tetracycline. Table 2 indicates the difference in the phenotypic and biochemical characteristics between strain On1T and C. manganoxidans strain HST. The foregoing results show that strain On1T was readily distinguished from its nearest phylogenetic neighbor C. manganoxidans strain HST in terms of fatty acid compositions (Table 1), phenotypic and biochemical characteristics (Table 2), as well as DNA–DNA reassociation experiments. Thus, strain On1T unambiguously represents a novel Caldimonas species for which we propose the name Caldimonas taiwanensis sp. nov.
Description of Caldimonas taiwanensis sp. nov. Caldimonas taiwanensis (tai.wan.en’sis N.L. fem. adj. Taiwanensis, of Taiwan, where the type strain was isolated). Cells are Gram-negative with rod shape, 0.6–0.8 mm in diameter and 1.2–2.2 mm in length. They occur singly and are motile by single polar flagella. Polyb-hydroxybutyrate granules are stored as reserve material. Growth occurs at temperatures ranging from 35 to 60 1C with an optimum at 55 1C and pH 7.0. They are positive for catalase, nitrate reduction, indole production and gelatin hydrolysis, but are negative for oxidase, glucose fermentation, urease, arginine dihydrolase, esculin hydrolysis, and b-galactosidase. They utilize glucose, mannitol, maltose, gluconate, adipate and
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).
References [1] P. Bernfeld, Amylases a- and b- methods, Enzymol 1 (1955) 149–158. [2] C. Bertoldo, G. Antranikian, Starch-hydrolyzing enzymes from thermophilic archaea and bacteria, Curr. Opin. Chem. Biol. 6 (2002) 151–160. [3] A. Burhan, U. Nisa, C. Go¨khan, C. O¨mer, A. Ashabil, G. Osman, Enzymatic properties of a novel thermostable, thermophilic, alkaline and chelator resistant amylase from an alkaliphilic Bacillus sp. isolate ANT-6, Process 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 freshwater pond for shrimp culture, Syst. Appl. Microbiol. 27 (2004) 43–49. [5] W.M. Chen, S. Laevens, T.M. Lee, T. Coenye, P. De Vos, M. Mergeay, P. Vandamme, Ralstonia taiwanensis sp. nov., isolated from root nodules of Mimosa species and sputum of a cystic fibrosis patient, Int. J. Syst. Evol. Microbiol. 51 (2001) 1729–1735. [6] A.P. Chung, F. Rainey, M.F. Nobre, J. Burghardt, M.S. da Costa, Meiothermus cerbereus sp. nov., a new slightly thermophilic species with high levels of 3-hydroxy fatty acids, Int. J. Syst. Bacteriol. 47 (1997) 1225–1230. [7] Y.C. Chung, T. Kobayashi, H. Kanai, T. Akiba, T. Kudo, Purification and properties of extracellular amylase from the hyperthermophilic archeon Thermococccus profundus DT5432, Appl. Environ. Microbiol. 61 (1995) 1502–1506.
ARTICLE IN PRESS 420
W.-M. Chen et al. / Systematic and Applied Microbiology 28 (2005) 415–420
[8] T. Ezaki, Y. Hashimoto, E. Yabuuchi, Fluorometric DNA–DNA hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains, Int. J. Syst. Bacteriol. 39 (1989) 224–229. [9] P.C. Fahy, G.C. Persley, Plant bacterial diseases: a diagnostic guide, Academic Press, New York, 1983. [10] W.M. Fogarty, Microbial amylases, In: W.M. Fogarty (Ed.), Microbial Enzymes and Biotechnology, Applied Science, London, 1983, pp. 1–92. [11] M. Freitas, F.A. Rainey, M.F. Nobre, A.J. Silvestre, M.S. daCosta, Tepidimonas aquatica sp. nov., a new slightly thermophilic beta-proteobacterium isolated from a hot water tank, Syst. Appl. Microbiol. 26 (2003) 376–381. [12] T.A. Hall (Ed.), BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT, Nucleic Acids Symp. Ser. 41 (1999) 95–98. [13] J.M. Khire, Production of moderately halophilic amylase by newly isolated Micrococcus sp. 4 from a salt-pan, Lett. Appl. Microbiol. 19 (1994) 210–212. [14] E. Le´veˆque, Sˇ. Janecek, B. Haye, A. Belarbi, Thermophilic archaeal amylolytic enzymes, Enz. Microbiol. Technol. 26 (2000) 3–14. [15] M. Mesbah, U. Premachandran, W.B. Whitman, Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography, Int. J. Syst. Bacteriol. 39 (1989) 159–167. [16] B.R. Mohapatra, U.C. Banerjee, M. Bapuji, Characterization of a fungal amylase from Mucor sp. associated
[17]
[18]
[19]
[20]
[21]
[22]
[23]
with the marine sponge Spirastrella sp., J. Biotechnol. 60 (1998.) 113–117. C. Moreira, F.A. Rainey, M.F. Nobre, M.T. da Silva, M.S. da Costa, Tepidimonas ignava gen. nov., sp. nov., a new chemolithoheterotrophic and slightly thermophilic member of the beta-Proteobacteria, Int. J. Syst. Evol. Microbiol. 50 (2000) 735–742. R.D. Page, TreeView: an application to display phylogenetic trees on personal computers, Comput. Appl. Biosci. 12 (1996) 357–358. D.G. Pitcher, N.A. Saunders, R.J. Owen, Rapid extraction of bacterial genomic DNA with guanidium thiocyanate, Lett. Appl. Microbiol. 8 (1989) 151–156. N. Saitou, M. Nei, The neighbour-joining method: a new method for reconstructing phylogenetic trees, Mol. Biol. Evol. 4 (1987) 406–425. M. Sasser, Identification of bacteria by gas chromatography of cellular fatty acids (MIDI technical note 101), Newark, MIDI Microbial ID, 1990. M. Takeda, Y. Kamagata, W.C. Ghiorse, S. Hanada, J. Koizumi, Caldimonas manganoxidans gen. nov., sp. nov., a poly(3-hydroxybutyrate)-degrading, manganese-oxidizing thermophile, Int. J. Syst. Evol. Microbiol. 52 (2002) 895–900. Thompson, T.J. Gibson, F. Plewniak, F. Jeanmougin, D.G. Higgins, The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools, Nucleic Acids Res. 25 (1997) 4876–4882.
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,, Jo-Shu Changb, Ching-Hsiang Chiua, Shu-Chen Changc, Wen-Chieh Chena, Chii-Ming Jianga a
Department of Seafood Science, National Kaohsiung Marine University, No. 142, Hai-Chuan Rd. Nan-Tzu, Kaohsiung City 811, Taiwan b Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan c Tajen 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 growth were 55 1C and pH 7. Strain On1T grew well in minimal medium containing starch as the sole 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) as Caldimonas taiwanensis sp. nov. r 2005 Elsevier GmbH. All rights reserved. Keywords: Caldimonas taiwanensis; b-Proteobacteria; Hot spring; Amylase; Taxonomy
Introduction Starch is a major polysaccharide reserve in plants. Starch hydrolysis creates a lot of important products with different physical and chemical properties for food and industries [13]. Amylases are enzymes which hydrolyse starch molecules to give diverse products Corresponding author. Tel.: +886 8 733 06 04; fax: +886 8 733 86 00. E-mail address: [email protected] (W.-M. Chen).
0723-2020/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2005.02.008
including dextrins and progressively smaller polymers composed of glucose units [1,7,10]. Amylolytic enzymes play an important role in the biogeochemical cycle of carbon and they are also among the most important enzymes in current biotechnological-based food, detergent, and pharmaceutical industries [16]. Due to the extensive industrial importance of amylase, there is ongoing interest in the isolation of effective bacterial strains producing amylases well suited to new industrial applications. Since hydrolysis of starch is known to proceed at a much faster rate at high temperatures, the
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thermophilic amylases are apparently preferable to practical applications. Several efforts were made on identifying thermophilic bacteria producing extracellular amylases, which are shown to have significant industrial importance [2,3,14]. In the present study, a novel bacterium was isolated from a hot spring located in southern Taiwan. The characteristics of this isolate were compared with its nearest neighbor.
Materials and methods Isolation and growth condition In May 2004, a sample (1000 ml) of water was collected from a Sih-Chong-Si hot spring at Pingtung, located in the southern part of Taiwan. The temperature of the spring water ranged from 50 to 60 1C, and the pH was approximately 8.0. The water sample was diluted with sterile distilled water (121 1C, 15 min) and spread onto plates of SD medium incubated at 55 1C. The SD medium (l1) consisted of 10 g starch, 2 g (NH4)2SO4, 0.7 g K2HPO4, 0.3 g KH2PO4, 0.5 g MgSO4 7H2O, 0.01 g FeSO4 7H2O and 15 g agar. Amylase production was determined by the development of a colorless halo zone surrounding bacterial colonies when Lugol solution (1% iodine in 2% potassium iodide [w/v]) was added to the plates. A bacterial strain, designated On1T, was obtained and shown to express amylase activities.
was performed on LB medium under the Oxoid AnaeroGen system.
16S rDNA sequencing and phylogenetic analysis Amplification and sequence analysis of the 16S rRNA gene was performed as described previously [5]. The sequence was compared with others available in GenBank and Ribosomal Database Project II. The multiplesequence alignment including strain On1T and its closest relatives were performed using the BioEdit software [12]. The phylogenetic reconstruction was inferred by using the neighbor-joining method [20]. A bootstrap analysis (confidence values estimated from 1000 replications of each sequence) was performed using the CLUSTAL w 1.7 program [23]. A phylogenetic tree was drawn using the TREEVIEW program [18]. Sequence identities were calculated using the BioEdit software [12].
Determination of the DNA base composition DNA was prepared as described by Pitcher et al. [19] and degraded enzymatically into nucleosides as described by Mesbah et al. [15]. The obtained nucleoside mixture was then separated by high-performance liquid chromatography using a Waters Symmetry Shield C8 column thermostated at 37 1C. The solvent was 0.02 M NH4H2PO4 (pH 4.0) with 1.5% acetonitrile. Nonmethylated lambda phage DNA (Sigma) was used as the calibration reference.
DNA–DNA hybridizations Morphological and physiological tests Strain On1T was grown on Luria-Bertani Broth (LB Broth, Miller; Difco) for 24 h at 55 1C. The cell morphology was observed by a light microscope (Zeiss Axioskop) at 1000 magnification. The motility of exponential-phase cells was examined by the hanging drop method. The flagella stain was performed using SoptTest Flagella Stain (Difco). The Gram stain was performed using the Gram Stain Set S (Difco). Poly-bhydroxybutyrate granule accumulation was observed by light microscopy after being stained with Sudan black [9]. The pH range for growth was determined by measuring optical densities of the cultures grown in LB medium, whose pH was adjusted to values between 3.0 and 11.0 with appropriate biological buffer [6]. For the determination of the temperature range for growth, cells were incubated in LB medium at temperatures between 15 and 80 1C and the cell growth was determined by measuring optical density of the culture with respect to time. Tolerance to NaCl was determined by adjusting the salinity of LB medium to values between 0% and 3.0% NaCl. Anaerobic cultivation
DNA–DNA hybridizations were performed with photobiotin-labeled probes as described by Ezaki et al. [8]. The hybridization temperature was 50 1C and the reaction was carried out in 50% formamide. Each value was the mean of two hybridization experiments.
Fatty acid methyl ester analysis Cells were grown on Tryptic Soy Agar (TSA) (Difco) at 55 1C. Cellular fatty acids were analyzed as methyl esters by GC according to the instructions of Microbial Identification System (MIDI; Microbial ID) [21].
Biochemical analysis Characteristics presented in the API 20NE and API ZYM (bioMe´rieux) microtest systems were determined according to the recommendations of the manufacturers. For carbon substrate metabolisation tests, Biolog GNII microtiter test plates were used. Early log-phase cultures were used as inoculum for the test plate (150 ml well1). The plates were incubated at 55 1C and
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examined after 24 h and 48 h to allow for the development of a purple color indicative of substrate oxidation.
Antimicrobial susceptibility testing The susceptibility to antimicrobial agents was determined by disk diffusion. The density of the bacterial suspension from exponential-phase culture was adjusted with sterile saline to the turbidity corresponding to 0.5 of the McFarland standard and then spread onto LB medium for further incubation at 55 1C. The following antimicrobial disks (Dispens-O-Susceptibility Test Disks, Difco) were used: ampicillin (10 mg), cefotaxime (30 mg), chloramphenicol (30 mg), gentamicin (10 mg), kanamycin (30 mg), nalidixic acid (30 mg), novobiocin (30 mg), penicillin G (10 mg), rifampin (5 mg), streptomycin (10 mg) and tetracycline (30 mg). Susceptibility was defined as zones 43 mm, resistance of zones of o1 mm, and moderate resistance of zones of 1–3 mm to the edge of a disk.
Results and discussion
417
in public databases (GenBank and Ribosomal Database Project II) revealed that strain On1T belonged to the Burkholderiales group of the b-Proteobacteria. The highest similarity values were obtained towards the genus Caldimonas, Leptothrix, Schlegelella and Tepidimonas. In order to determine the phylogenetic position, the 16S rDNA sequence of strain On1T was compared with those related species within the b-Proteobacteria. The 16S rDNA sequence of strain On1T clustered together with C. manganoxidans strain HST (97.0% similarity) [22], T. aquatica strain CLN-1T (95.1% similarity) [11] and T. ignava strain SPS-1037T (95.0% similarity) [17] (Fig. 1). The similarity levels towards other bacterial species belonging to the Burkholderiales group of the b-Proteobacteria were lower than 95%. Subsequently, whole-genome DNA–DNA hybridization experiment was preformed between strain On1T and the type strain of its nearest phylogenetic neighbor, C. manganoxidans strain HST. 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%.
Phenotypic and morphological characteristics
Fatty acid compositions
A bacterial strain, designated as On1T, was isolated from water of a hot spring in southern Taiwan. Strain On1T grew well aerobically in a minimal medium containing starch as the sole carbon source, and its extracellular products displayed amylase activity. Vitamins and yeast extract were not needed for growth. The strain also grew well on complex medium (LB, TSB and NB) and formed light-yellow pigmented colonies on the surface of agar plates. The colony morphology was circular, entire, and convex shape. The size of colonies was approximately 1.0–2.5 mm in diameter on LB agar after 48 h of incubation at 55 1C. Strain On1T grew at temperatures ranging from 35 to 60 1C, pH values between 6.0 and 8.0, and NaCl concentrations between 0.2% and 1.0%. Optimal growth condition (as determined by measuring optical densities) was around 55 1C, pH 7.0 and 0.5% NaCl. The strain could not grow under anaerobic condition by using the Oxoid AnaeroGen system. Light microscopic examination revealed that cells of strain On1T were Gram-negative rods. Cells were nonspore-forming and motile by means of single polar flagella. The sizes of rods were approximately 0.6–0.8 mm in diameter and 1.2–2.2 mm in length. Polyb-hydroxybutyrate granules were present.
The major cellular fatty acid components of strain On1T were 16:0 (30.4%), 18:1 o7c (20.0%) and summed feature 3 (31.3%) which comprised 16:1o7c or 15:0 iso 2OH or both. Strain On1T had a fatty acid profile similar to that of C. manganoxidans strain HST which also contained the predominant fatty acid 16:0, 18:1 o7c and summed feature 3. However, strain On1T contained a less amount of fatty acid 17:0 cyclo (1.9%), and thus clearly differentiate from C. manganoxidans strain HST. Table 1 shows the details of the fatty acid composition of C. manganoxidans strain HST and strain On1T grew under identical growth conditions (TSA and 55 1C).
Phylogenetic analysis Comparison of the 16S rRNA gene sequence of strain On1T (GenBank nucleotide sequence accession number is AY845052) with available 16S rRNA gene sequences
Biochemical analysis Strain On1T exhibited following biochemical characteristics as determined using the results of API 20NE, API ZYM and Biolog GNII microtest galleries: catalase, nitrate reduction; indole production; gelatin hydrolysis, alkaline phosphatase, C4 esterase, C8 lipase, leucine arylamidase, acid phosphatase, naphthol-AS-BIphosphohydrolase, a-glucosidase; assimilation of glucose, mannitol, maltose, gluconate, adipate, citrate, D-fructose, methyl pyruvate, b-hydroxybutyric acid, D,L-lactate, L-aspartic acid, L-proline, D-sorbitol, Dtrehalose, turanose, mono-methyl succinate, a-keto glutaric acid, urocanic acid, glycerol, arabitol, Lglutamic acid, acetic acid, cis-aconitic acid, formic acid, a-hydroxybutyric acid, a-keto valeric acid, D-saccharic acid, succinic acid, D-alanine, glycyl-L-glutamic acid,
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66
96
100
99
1%
Chitinimonas taiwanensis cf T(AY323827) T Wautersia taiwanensis LMG 19424 (AF300324) Schlegelella thermodepolymerans K14T (AY152824) Leptothrix mobilis Feox-1T (X97071) Leptothrix cholodnii CCM 1827 (X97070) Roseateles depolymerans 61AT (AB003623) Leptothrix discophora AT CC 43182T (L33975) Sphaerotilus natans 565 (Z18534) 67 T Ideonella dechloratans CCUG 30898 (X72724) T (D88007) Alcaligenes latus IAM 12599 67 T Rubrivivax gelatinosus AT CC 17011 (D16213) 86 T Aquabacterium parvum B6 (AF035052) T 98 Aquabacterium citratiphilum B4 (AF035050) 69 T Aquabacterium commune B8 (AF035054) 100 Caldimonas taiwanensis On1T (AY 845052) Caldimonas manganoxidans JC M 10698T (AB008801) Tepidimonas ignava SPS-1037T (AF177943) 100 T Tepidimonas aquatica CLN-1 (AY324139) Brachymonas denitrificans AS-P1T (D14320) T Hydrogenophagaflava DSM 619 (AB021420) T 75 Comamonastestosteroni AT CC 11996 (M11224) T Delftia acidovorans ACM 489 (AF078774) Polaromonas vacuolata 34-PT (U14585) 90 T Rhodoferax fermentans FR 2 (D16211) T Acidovorax facilis CCUG 2113 (AF078765) T 69 Variovorax paradoxus DSM 66 (AJ420329) 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
Compound
C. manganoxidans HST
10:0 3OH 12:0 14:0 15:1 o6c 16:0 17:0 cyclo 17:0 18:1 o7c 18:0 Summed feature 3b
4.4 3.6 3.0 1.1 27.3 19.4 4.1 20.0 12.3
C. taiwanensis On1T 6.3 4.3 2.9 30.4 1.9 20.0 2.1 31.3
a Values are shown as a percentage of the total fatty acid content for each strain. All strains grew at 55 1C for 48 h on TSA medium, and 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. b Summed feature 3 comprises 16:1o7c or 15:0 iso 2OH or both.
L-ornithine,
g-amino butyric acid, inosine, thymidine, 2amino ethanol, 2,3-butanediol, D,L-a-glycerol phosphate, glucose-1-phosphate, glucose-6-phosphate.
The following biochemical characteristics were absent: oxidase, glucose fermentation, arginine dihydrolase, urease, esculin hydrolysis, b-galactosidase, C14 lipase, valine arylamidase, cystine arylamidase, trypsin,a-chymotrypsin,a-galactosidase, b-glucuronidase, a-mannosidase, a-fucosidase, N-acetyl-b-glucosaminidase activity; assimilation of arabinose, mannose, N-acetyl-glucosamine, caprate, malate, phenyl-acetate, tween 40, tween 80, cellobiose, N-acetyl-Dglucosamine, L-asparagine,a-cyclodextrin, adonitol, i-erythritol, L-fucose, D-galactose, gentiobiose, m-inositol, a-Dlactose, lactulose, D-melibiose,b-methyl D-glucoside, psicose, D-raffinose, L-rhamnose, sucrose, xylitol, D-galactonic acid lactone, D-galacturonic acid, D-glucosaminic acid, D-glucuronic acid, g-hydroxybutyric acid, r-hydroxy phenylacetic acid, itaconic acid, a-keto butyric acid, malonic acid, propionic acid, quinic acid, sebacic acid, bromo succinic acid, succinamic acid, glucuronamide, alaninamide, Lalanine, L-alanyl-glycine, glycyl-L-aspartic acid, L-histidine, hydroxy L-proline, L-phenylalanine, L-pyroglutamic acid, L-serine, D-serine, L-threonine, D,L-carnitine, uridine, phenyl ethylamine and putrescine. Strain On1T was susceptible to all antibiotics tested, including ampicillin, cefotaxime, chloramphenicol,
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Table 2. The difference in phenotypic and biochemical characteristics between strain On1T and its closed neighbora Characteristic
C. manganoxidans HST
C. taiwanensis On1T
Optimum growth temperature (1C) Optimum growth pH Cell size (mm) Oxidase Catalase Utilization of: Galactose Malate Malonate Sucrose Acetate Fructose Trehalose G+C content (mol%)
50
55
8–9
7
0.5–0.7 2.2–3.5 + +
0.6–0.8 1.2–2.2 — w
+ + + + — — — 66.2
— — — — + + + 65.9
The data of C. manganoxidans HST was obtained from Takeda et al. [22].
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citrate, whereas can not utilize arabinose, mannose, Nacetyl-glucosamine, caprate, malate, and phenyl-acetate are not utilized. The major fatty acid components were 16:0, 18:1 o7c and summed feature 3 (16:1o7c or 15:0 iso 2OH or both [about 31%]). The G+C content of its DNA is 65.9 mol%. The type strain, On1T, was isolated from water of SihChong-Si hot spring at Pingtung located near the very southern part of Taiwan. The type strain has been deposited in BCRC (Bioresource Collection and Research Center, Food Industry Research and Development Institute, PO Box 246, Hsinchu, Taiwan, 30099) as BCRC 17405T and in BCCM/LMG Bacteria Collection (Laboratorium voor Microbiologie, Universiteit Gent, Gent, Belgium) as LMG 22827T. Accession: The DDBJ/EMBL/GenBank accession number for the 16S rDNA sequence of the strain On1T is AY845052.
Acknowledgements
a
gentamicin, kanamycin, nalidixic acid, novobiocin, penicillin G, rifampin, streptomycin and tetracycline. Table 2 indicates the difference in the phenotypic and biochemical characteristics between strain On1T and C. manganoxidans strain HST. The foregoing results show that strain On1T was readily distinguished from its nearest phylogenetic neighbor C. manganoxidans strain HST in terms of fatty acid compositions (Table 1), phenotypic and biochemical characteristics (Table 2), as well as DNA–DNA reassociation experiments. Thus, strain On1T unambiguously represents a novel Caldimonas species for which we propose the name Caldimonas taiwanensis sp. nov.
Description of Caldimonas taiwanensis sp. nov. Caldimonas taiwanensis (tai.wan.en’sis N.L. fem. adj. Taiwanensis, of Taiwan, where the type strain was isolated). Cells are Gram-negative with rod shape, 0.6–0.8 mm in diameter and 1.2–2.2 mm in length. They occur singly and are motile by single polar flagella. Polyb-hydroxybutyrate granules are stored as reserve material. Growth occurs at temperatures ranging from 35 to 60 1C with an optimum at 55 1C and pH 7.0. They are positive for catalase, nitrate reduction, indole production and gelatin hydrolysis, but are negative for oxidase, glucose fermentation, urease, arginine dihydrolase, esculin hydrolysis, and b-galactosidase. They utilize glucose, mannitol, maltose, gluconate, adipate and
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).
References [1] P. Bernfeld, Amylases a- and b- methods, Enzymol 1 (1955) 149–158. [2] C. Bertoldo, G. Antranikian, Starch-hydrolyzing enzymes from thermophilic archaea and bacteria, Curr. Opin. Chem. Biol. 6 (2002) 151–160. [3] A. Burhan, U. Nisa, C. Go¨khan, C. O¨mer, A. Ashabil, G. Osman, Enzymatic properties of a novel thermostable, thermophilic, alkaline and chelator resistant amylase from an alkaliphilic Bacillus sp. isolate ANT-6, Process 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 freshwater pond for shrimp culture, Syst. Appl. Microbiol. 27 (2004) 43–49. [5] W.M. Chen, S. Laevens, T.M. Lee, T. Coenye, P. De Vos, M. Mergeay, P. Vandamme, Ralstonia taiwanensis sp. nov., isolated from root nodules of Mimosa species and sputum of a cystic fibrosis patient, Int. J. Syst. Evol. Microbiol. 51 (2001) 1729–1735. [6] A.P. Chung, F. Rainey, M.F. Nobre, J. Burghardt, M.S. da Costa, Meiothermus cerbereus sp. nov., a new slightly thermophilic species with high levels of 3-hydroxy fatty acids, Int. J. Syst. Bacteriol. 47 (1997) 1225–1230. [7] Y.C. Chung, T. Kobayashi, H. Kanai, T. Akiba, T. Kudo, Purification and properties of extracellular amylase from the hyperthermophilic archeon Thermococccus profundus DT5432, Appl. Environ. Microbiol. 61 (1995) 1502–1506.
ARTICLE IN PRESS 420
W.-M. Chen et al. / Systematic and Applied Microbiology 28 (2005) 415–420
[8] T. Ezaki, Y. Hashimoto, E. Yabuuchi, Fluorometric DNA–DNA hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains, Int. J. Syst. Bacteriol. 39 (1989) 224–229. [9] P.C. Fahy, G.C. Persley, Plant bacterial diseases: a diagnostic guide, Academic Press, New York, 1983. [10] W.M. Fogarty, Microbial amylases, In: W.M. Fogarty (Ed.), Microbial Enzymes and Biotechnology, Applied Science, London, 1983, pp. 1–92. [11] M. Freitas, F.A. Rainey, M.F. Nobre, A.J. Silvestre, M.S. daCosta, Tepidimonas aquatica sp. nov., a new slightly thermophilic beta-proteobacterium isolated from a hot water tank, Syst. Appl. Microbiol. 26 (2003) 376–381. [12] T.A. Hall (Ed.), BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT, Nucleic Acids Symp. Ser. 41 (1999) 95–98. [13] J.M. Khire, Production of moderately halophilic amylase by newly isolated Micrococcus sp. 4 from a salt-pan, Lett. Appl. Microbiol. 19 (1994) 210–212. [14] E. Le´veˆque, Sˇ. Janecek, B. Haye, A. Belarbi, Thermophilic archaeal amylolytic enzymes, Enz. Microbiol. Technol. 26 (2000) 3–14. [15] M. Mesbah, U. Premachandran, W.B. Whitman, Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography, Int. J. Syst. Bacteriol. 39 (1989) 159–167. [16] B.R. Mohapatra, U.C. Banerjee, M. Bapuji, Characterization of a fungal amylase from Mucor sp. associated
[17]
[18]
[19]
[20]
[21]
[22]
[23]
with the marine sponge Spirastrella sp., J. Biotechnol. 60 (1998.) 113–117. C. Moreira, F.A. Rainey, M.F. Nobre, M.T. da Silva, M.S. da Costa, Tepidimonas ignava gen. nov., sp. nov., a new chemolithoheterotrophic and slightly thermophilic member of the beta-Proteobacteria, Int. J. Syst. Evol. Microbiol. 50 (2000) 735–742. R.D. Page, TreeView: an application to display phylogenetic trees on personal computers, Comput. Appl. Biosci. 12 (1996) 357–358. D.G. Pitcher, N.A. Saunders, R.J. Owen, Rapid extraction of bacterial genomic DNA with guanidium thiocyanate, Lett. Appl. Microbiol. 8 (1989) 151–156. N. Saitou, M. Nei, The neighbour-joining method: a new method for reconstructing phylogenetic trees, Mol. Biol. Evol. 4 (1987) 406–425. M. Sasser, Identification of bacteria by gas chromatography of cellular fatty acids (MIDI technical note 101), Newark, MIDI Microbial ID, 1990. M. Takeda, Y. Kamagata, W.C. Ghiorse, S. Hanada, J. Koizumi, Caldimonas manganoxidans gen. nov., sp. nov., a poly(3-hydroxybutyrate)-degrading, manganese-oxidizing thermophile, Int. J. Syst. Evol. Microbiol. 52 (2002) 895–900. Thompson, T.J. Gibson, F. Plewniak, F. Jeanmougin, D.G. Higgins, The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools, Nucleic Acids Res. 25 (1997) 4876–4882.