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Identification of an alkaliphilic actinomycetes that produces a PrPSc-degrading enzyme

Identification of an alkaliphilic actinomycetes that produces a PrPSc-degrading enzyme Ann Microbiol (2010) 60:349–353 DOI 10.1007/s13213-010-0049-9 ORIGINAL ARTICLE Identification of an alkaliphilic actinomycetes that produces Sc a PrP -degrading enzyme Shinji Mitsuiki & Mikako Takasugi & Yasushi Moriyama & Taiki Futagami & Masatoshi Goto & Hiroaki Kanouchi & Tatsuzo Oka Received: 22 February 2010 /Accepted: 24 March 2010 /Published online: 29 April 2010 # Springer-Verlag and the University of Milan 2010 . . Abstract An alkaliphilic actinomycete strain TOA-1, that Keywords Nocardiopsis Alkaliphilic Keratinase Sc produces a PrP -degrading keratinase, was isolated from cement of bathroom tiles. This strain produced substrate and aerial mycelia. Growth occurred in the pH range 7.5–13.0, Introduction with an optimal pH of 10.0. A salt concentration of up to 8% was tolerated, and various organic substrates were used for The genus Nocardiopsis wasproposedbyMeyer basedon growth. Strain TOA-1 contained meso-diaminopimelic acid, morphological and chemotaxonomic characteristics (Meyer no diagnostic sugars, iso-C as the major cellular fatty acid, 1976). The genus currently comprises 28 species (Li et al. 16:0 and MK-10, MK-10(H ), and MK-10(H ) as the predominant 2004a, b;Chenetal. 2008;Yangetal. 2008; Zhang et al. 2 4 menaquinones. The G+C content was 71 mol%. All these 2008; Hozzein and Goodfellow 2009). Nocardiopsis strains characteristics, together with an analysis of its phylogenetic are frequently isolated from saline and alkaline soils (Hozzein position, indicated that this strain belongs to the genus et al. 2004;Lietal. 2004a, b;Chenetal. 2008;Yang et al. Nocardiopsis.DNA–DNA hybridization with its closest 2008). During a study on the diversity of alkaline-tolerant relative N. metallicus indicated a similarity of 55%, which fungi on cement of bathroom tiles in Japan, an alkaliphilic suggested that strain TOA-1, deposited in the International actinomycetes strain TOA-1 thought to be belonged to the Patent Organism Depositary National Institute of Advanced genus Nocardiopsis was isolated that produced an abnormal Sc Industrial Science and Technology (FERM BP-08603) in prion protein (PrP ) degrading keratinase (Mitsuiki et al. Sc Japan as a patent strain, represents a novel species of the 2002, 2004, 2006). PrP is beta-sheet rich abnormal PC genus Nocardiopsis. conformer of the alpha-helix rich normal prion protein (Pr ), and is a major infectious agent of prion diseases (Prusiner et al. 1998;Weissmann 1999). There have been some reports of Sc enzymes that can degrade PrP (Langeveld et al. 2003; S. Mitsuiki (*) M. Takasugi McLeod et al. 2004), but the keratinase from strain TOA-1 Department of Applied Chemistry and Biochemistry, Sc Kyushu Sangyo University, can uniquely degrade PrP without additional chemical and Fukuoka 813-8503, Japan physical treatments (Mitsuiki et al. 2006). e-mail: mitsuiki@ip.kyusan-u.ac.jp In this study, a novel alkaliphilic actinomycetes strain of TOA-1 was identified by a polyphasic approach and Y. Moriyama appears to be a novel species of the genus Nocardiopsis. Research Institute, TOTO Ltd., Kanagawa 253-8577, Japan T. Futagami M. Goto Materials and methods Department of Bioscience and Biotechnology, Kyushu University, Fukuoka 812-8581, Japan Strain and culture conditions H. Kanouchi T. Oka Strain TOA-1 was isolated from cement of bathroom tiles in Department of Veterinary Physiology, Kagoshima University, Japan. Isolation was done at 30°C by using agar plates made Kagoshima 890-0065, Japan 350 Ann Microbiol (2010) 60:349–353 with AW medium (per 1 l of distilled water), glucose, 10 g; cellulose, pectin, chitin, xylan, pullulan and Tween 80) peptone, 5 g; K HPO , 1 g; MgSO � 7H O, 0.5 g; agar, 15 g; was detected in modified Bennett’s medium (Williams et 2 4 4 2 and Na CO , 10 g. Sodium carbonate was separately al. 1983). 2 3 sterilized and then added to the sterile medium. Strain TOA-1 cultivated on AW plates was used for microscopic Chemotaxonomy observation of sporophores, spore-chains and the spore surface by light- and scanning-electron microscopy (Hitha- Amino acid and sugar analyses of whole cell hydrolysates chi, S-4700). Growth characteristics of strain TOA-1 culture were performed as described by Hasegawa et al. (1983) and were studied on ISP media (Kroppenstedt 1992). Staneck and Roberts (1974). Fatty acid methyl esters were prepared from 50 mg of wet cells. Mixtures of fatty acid Physiology methyl esters were prepared and analyzed with the MIDI system (Hewlett Packard) (Sasser 1990). Menaquinones All physiological tests were carried out at 30°C and at were extracted, purified and identified by HPLC pH 10.0 unless otherwise specified. The effect of (Collins 1985). different temperatures and pH conditions on growth and the tolerance of strain TOA-1 to salt were determined by 16S rRNA sequence determination and phylogenetic using plates made with AW medium. Production of analysis melanoid pigments was tested on an ISP 7 plate (Shirling and Gottlie 1966). Utilization of carbon source (L- Genomic DNA extraction, PCR-mediated amplification of arabinose, D-xylose, D-glucose, D-fructose, L-rhamnose, 16S rRNA genes, and sequence determination were raffinose, sucrose, mannitol and inositol) was examined performed as previously described (Mitsuiki et al. 2002). using ISP 9 as a basal medium (Shirling and Gottlie 1966). Phylogenetic tree analysis was performed using the MEGA Decomposition of substrate (starch, casein, carboxymethyl software version 4.0 (Tamura et al. 2007). Table 1 Physiology of TOA-1 T T Characteristics TOA-1 N. metallicus DSM 44598 N. listeri DSM 40297 and the phylogenetically most closely related species of the Aerial mycerium + − + genus Nocardiopsis Utilization of L-arabinose + + + D-xylose + + + D-glucose + + + D-fructose + + + L-rhamnose + + + Raffinose + −− Sucrose + + + Mannitol + + − Inositol + −− Decomposition of Starch + + − Casein + + + Carboxymethyl cellulose + + − Pectin + −− Chitin + + + Xylan + + − Pullulan − + − Tween 80 + + − All three strains utilized L- Growth at arabinose, D-xylose, D-glucose, 10°C − ++ D-fructose, L-rhamnose and su- 45°C + −− crose, and decomposed casein and chitin pH 12 + −− 5% NaCl + + + + Substrate utilized/decom- posed, − substrate not utilized/ 10% NaCl − + − decomposed Ann Microbiol (2010) 60:349–353 351 Determination of DNA-DNA similarities and G+C content The temperature range for growth was 15–45°C, with optimalgrowthoccurring at 30°C.Goodgrowthwas DNA-DNA hybridization studies were carried out by using shown at NaCl concentration up to 8%. No melanoid the thermal renaturation method (Yassin et al. 1993). The pigments were produced. All the tested carbon sources guanine-plus-cytosine (G+C) content of the DNA was could be utilized for growth by strain TOA-1. Starch, casein, determined by HPLC (Mesbah et al. 1989). carboxymethyl cellulose, pectin, chitin, xylan and Tween 80 could be decomposed, while pullulan could not be. These characteristics of strain TOA-1 and of the phylogenet- Results and discussion ically closely related typestrains of the species N. metalicus and N. listeri are given in Table 1 (Evtushenko et al. 2000; Morphological characteristics Hozzein et al. 2004;Chenet al. 2008). These character- istics are different from those of other two Nocardiopsis Morphological analysis of strain TOA-1 indicated that its species. substrate mycelium was colorless and that no soluble pigments were produced on any medium. The spores were Chemotaxonomic characteristics between 10 and 50 in number, with dimensions of 0.4–0.6× 0.8–1.2 μm, and were straightly chained, rod-shaped, Whole-cell hydrolysates contained meso-diaminopimelic smooth-surfaced and non-motile. These morphological acid as the only diamino acid of the cell wall peptidogly- characteristics are consistent with those described for the can. Diagnostic sugars such as arabinose, xylose and Nocardiopsis species (Kroppenstedt 1992). madurose (Lechevalier et al. 1971) or rhamnose (Labeda et al. 1984) were not detected. The major fatty Physiological characteristics acid detected was iso-C (42.84%). The predominant 16:0 menaquinones were MK-10(H ) and MK-10. In addition, Sc Strain TOA-1 was able to produce PrP -degrading there were detectable amounts of MK-9(H ) and MK-9(H ). 2 4 keratinase. This ability has not been reported for any All these characteristics are typical of the genus Nocar- other Nocardiopsis strain.Growthoccurredinthe pH diopsis (Grund and Kroppenstedt 1990; Kroppenstedt range 7.5–13.0, with optimal growth occurring at pH 10.0. 1992). Fig. 1 Phylogenetic dendro- 100 Nocardiopsis metallicus DSM 44598 gram based on 16S rRNA gene Strain TOA-1 sequence analysis, showing the Nocaridiopsis listeri DSM 40297 phylogenetic position of TOA-1 compared with the other species Nocardiopsis alba DSM 43377 of the genus Nocardiopsis. Bar: Nocardiopsis umidischolae DSM 44362 inferred nucleotide substitution per 100 nucleotides Nocardiopsis lucentensis DSM 44048 63 Nocardiopsis aegyptica DSM 44442 Nocardiopsis dassonvillei DSM 43111 82 Nocardiopsis synnemataformans DSM 44143 Nocardiopsis benisuefensis DSM 44686 100 Nocardiopsis halototerans DSM 44410 Nocardiopsis quinghaiensis DSM 44739 Nocardiopsis xinjiangensis DSM 44589 98 T Nocardiopsis salina DSM 44839 Nocardiopsis composta DSM 44551 Nocardiopsis halophila DSM 44494 99 T Nocardiopsis baichengensis DSM 44845 Nocardiopsis chromatogenes DSM 44844 92 T Nocardiopsis gilva DSM 44841 Nocardiopsis rhodophaea DSM 44843 Nocardiopsis rosea DSM 44842 0.005 352 Ann Microbiol (2010) 60:349–353 Hozzein WN, Li WJ, Ali MI, Hammouda O, Mousa AS, Xu LH, Jiang Phylogenetic analysis CL (2004) Nocardiopsis alkaliphila sp. nov., a novel alkaliphilic actinomycete isolated from desert soil in Egypt. Inst J Syst The sequence of the 16S rRNA gene of strain TOA-1 Bacteriol 54:247–252 (AY027776), which consists of 1,515 bp, was almost Kroppenstedt RM (1992) The genus Nocardiopsis. In: The Prokaryotes. A handbook on the biology of bacteria: ecophysiology, isolation, completely determined. A preliminary comparison of the identification, applications. Academic, New York, pp 1139–1156 sequence with other sequences in GenBank indicated that Labeda DP, Testa RT, Lechevalier MP, Lechevalier HA (1984) the closest phylogenetic neighbors of strain TOA-1 were Saccharothrix: a new genus of the Actinomycetales related to members of the genus Nocardiopsis. Pairwise similarity Nocardiopsis. Int J Syst Bacteriol 34:426–431 Langeveld JPM, Wang JJ, Van de Wiel DFM, Shin GC, Jan GG, values ranged from 92.5% (N. rhodophaea DSM 44843 ) Bossers A, Shin JCH (2003) Enzymatic degradation of prion to 98.8% (N. metallicus DSM 44598 ). The phylogenic tree protein in brain stem from infected cattle and sheep. J Infect Dis constructed on the basis of published sequences is shown 188:1782–1789 Fig. 1. This tree indicates that strain TOA-1, N. metallicus Lechevalier HA, Lechevalier MP, Gerber NN (1971) Chemical T T composition as a criterion in the classification of actinomycetes. DSM 44598 and N. listeri DSM 40297 , form one of Adv Appl Microbiol 14:47–72 several Nocardiopsis subclusters. The similarity of each of Li WJ, Kroppenstedt RM, Wang D, Tang SK, Lee JC, Park DJ, Kim CJ, these type strains to strain TOA-1 was calculated as 98.8 Xu LH, Jiang CL (2004a) Five novel species of the genus and 97.5%, respectively. Strain TOA-1 probably belongs to Nocardiopsis isolated from hypersaline soils and emended descrip- tion of Nocardiopsis salina. Inst J Syst Bacteriol 56:1089–1096 a novel species. Although a sequence similarity as high as Li WJ, Park DJ, Tang SK, Wang D, Lee JC, Xu LH, Kim CJ, Jiang 98.8% suggested that strain TOA-1 might be a novel CL (2004b) Nocardiopsis salina sp. nov., a novel halophilic species, sequence similarity values of ≧97% have been actinomycete isolated from saline soil in China. Inst J Syst reported to be of limited usefulness in species differentia- Bacteriol 54:1805–1809 McLeod AH, Murdoch H, Dickinson J, Dennis MJ, Hall GA, Buswell tion (Stackebrandt and Goebel 1994). The G+C content was CM, Carr J, Taylor DM, Sutton JM, Raven ND (2004) 71 mol%, which lies within the range for the genus Proteolytic inactivation of the bovine spongiform encephalopathy Nocardiopsis (Grund and Kroppenstedt 1990). The DNA– agent. Biochem Biophys Res Commun 317:1165–1170 DNA relatedness of strain TOA-1 to N. metallicus DSM Mesbah M, Premachandran U, Whitman WB (1989) Precise mea- T T surement of the G+C content of deoxyribonucleic acid by high- 44598 and N. listeri DSM 40297 were 55 and 49%, performance liquid chromatography. Int J Syst Bacteriol 39:159– respectively. This value lies well below 70% that is recommended as the cut-off point for species identity Meyer J (1976) Nocardiopsis, a new genus of the order Actino- (Wayne et al. 1987). mycetales. Inst J Syst Bacteriol 26:487–493 Mitsuiki S, Sakai M, Moriyama Y, Goto M, Furukawa K (2002) Purification and some properties of a keratinolytic enzyme from Acknowledgment This work was supported by Grants-in-Aid for an alkaliphilic Nocardiopsis sp. TOA-1. Biosci Biotechnol Scientific Research (C) (20580106). Biochem 66:164–167 Mitsuiki S, Ichikawa M, Oka T, Moriyama Y, Sakai M, Sameshima Y, Goto M, Furukawa K (2004) Molecular characterization of a keratinolytic enzyme from an alkaliphilic Nocardiopsis sp. TOA- References 1. Enzyme Microb Technol 34:482–489 Mitsuiki S, Hui Z, Matsumoto D, Sakai M, Moriyama Y, Furukawa K, Sc Chen YG, Cui XL, Kroppenstedt RM, Stackebrandt E, Wen ML, Xu Kanouchi H, Oka T (2006) Degradation of PrP by keratinolytic LH, Jiang CL (2008) Nocardiopsis quinghaiensis sp. nov., protease from Nocaridiopsis sp. TOA-1. Biosci Biotechnol isolated from saline soil in China. Inst J Syst Bacteriol 58:699– Biochem 70:1246–1248 705 Prusiner SB, Scott MR, DeArmond SJ, Cohen FE (1998) Prion protein Collins MD (1985) Isoprenoid quinone analysis in bacterial classifi- biology. Cell 93:337–348 cation and identification. In: Goodfellow M, Minnikin DE (eds) Sasser M (1990) Identification of bacteria by gas chromatography of Chemical methods in bacterial systematics. London, Academic, cellular fatty acid. Technical Note 101. MIDI, Newark pp 267–287 Shirling EB, Gottlie D (1966) Methods for characterization of Evtushenko LI, Taran VV, Akimov VN, Kroppenstedt RM, Tiedje JM, Streptomyces species. Int J Syst Bacteriol 16:313–340 Stackebrandt E (2000) Nocardiopsis tropica sp. nov., Nocar- Stackebrandt E, Goebel BM (1994) Taxonomic note: a place for diopsis trehalosi sp. nov., nom. rev. and Nocardiopsis dassonvil- DNA-DNA reassociation and 16S rDNA sequence analysis in the lei subsp. albirubida subsp. nov., comb. nov. Int J Syst Evol present species definition in bacteriology. Int J Syst Bacteriol Microbiol 50:73–81 44:846–849 Grund E, Kroppenstedt RM (1990) Chemotaxonomy and numerical Staneck JL, Roberts GD (1974) Simplified approach to identification taxonomy of the genus Nocardiopsis Meyer 1976. Int J Syst of aerobic actinomycetes by thin-layer chromatography. Appl Bacteriol 40:5–11 Microbiol 28:226–231 Hasegawa T, Takizawa M, Tanida S (1983) A rapid analysis for Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular chemical grouping of aerobic actinomycetes. J Gen Appl Micro- Evolutionary Genetics Analysis (MEGA) software version 4.0. biol 29:319–322 Mol Biol Evol 24:1596–1599 Hozzein WN, Goodfellow M (2009) Nocardiopsis arabia sp. nov., a Wayne LG, Brenner DJ, Colwell RR, Grimont PAD, Kandler O, halotolerant actinomycete isolated from a sand-dune soil. Inst J Krichevsky MI, Moore LH, Moore WEC, Murray RGE, Syst Bacteriol 58:2520–2524 Stackebrandt E, Starr MP, Trüper HG (1987) Report of the ad Ann Microbiol (2010) 60:349–353 353 hoc committee on reconciliation of approaches to bacterial isolated from alkali lake soil in China. Inst J Syst Bacteriol systematics. Int J Syst Bacteriol 37:463–464 58:1542–1546 Weissmann C (1999) Molecular genetics of transmissible spongiform Yassin AF, Wohlfarth EA, Jahnke KD, Schaal KP, Truper HG (1993) encephalopathies. J Biol Chem 274:3–6 A new actinomycete species, Nocardiopsis lucentensis sp. nov. Williams ST, Goodfellow M, Alderson G, Wellington EMH, Sneath Int J Syst Bacteriol 43:266–271 PHA, Sackin MJ (1983) Numerical classification of Streptomyces ZhangX,Zhang LP,YangR,Shi N, Lu Z, Chen WX,Jiang CL, and related genera. J Gen Microbiol 129:1743–1813 Xu LH (2008) Nocardiopsis ganjiahuensis sp. nov., isolated Yang R, Zhang LP, Guo LG, Shi N, Lu Z, Zhang X (2008) from a soil from Ganjiahu, China. Inst J Syst Bacteriol 58:195– Nocardiopsis valliformis sp. nov., an alkaliphilic actinomycete 159 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Identification of an alkaliphilic actinomycetes that produces a PrPSc-degrading enzyme

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Springer Journals
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Copyright © 2010 by Springer-Verlag and the University of Milan
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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Abstract

Ann Microbiol (2010) 60:349–353 DOI 10.1007/s13213-010-0049-9 ORIGINAL ARTICLE Identification of an alkaliphilic actinomycetes that produces Sc a PrP -degrading enzyme Shinji Mitsuiki & Mikako Takasugi & Yasushi Moriyama & Taiki Futagami & Masatoshi Goto & Hiroaki Kanouchi & Tatsuzo Oka Received: 22 February 2010 /Accepted: 24 March 2010 /Published online: 29 April 2010 # Springer-Verlag and the University of Milan 2010 . . Abstract An alkaliphilic actinomycete strain TOA-1, that Keywords Nocardiopsis Alkaliphilic Keratinase Sc produces a PrP -degrading keratinase, was isolated from cement of bathroom tiles. This strain produced substrate and aerial mycelia. Growth occurred in the pH range 7.5–13.0, Introduction with an optimal pH of 10.0. A salt concentration of up to 8% was tolerated, and various organic substrates were used for The genus Nocardiopsis wasproposedbyMeyer basedon growth. Strain TOA-1 contained meso-diaminopimelic acid, morphological and chemotaxonomic characteristics (Meyer no diagnostic sugars, iso-C as the major cellular fatty acid, 1976). The genus currently comprises 28 species (Li et al. 16:0 and MK-10, MK-10(H ), and MK-10(H ) as the predominant 2004a, b;Chenetal. 2008;Yangetal. 2008; Zhang et al. 2 4 menaquinones. The G+C content was 71 mol%. All these 2008; Hozzein and Goodfellow 2009). Nocardiopsis strains characteristics, together with an analysis of its phylogenetic are frequently isolated from saline and alkaline soils (Hozzein position, indicated that this strain belongs to the genus et al. 2004;Lietal. 2004a, b;Chenetal. 2008;Yang et al. Nocardiopsis.DNA–DNA hybridization with its closest 2008). During a study on the diversity of alkaline-tolerant relative N. metallicus indicated a similarity of 55%, which fungi on cement of bathroom tiles in Japan, an alkaliphilic suggested that strain TOA-1, deposited in the International actinomycetes strain TOA-1 thought to be belonged to the Patent Organism Depositary National Institute of Advanced genus Nocardiopsis was isolated that produced an abnormal Sc Industrial Science and Technology (FERM BP-08603) in prion protein (PrP ) degrading keratinase (Mitsuiki et al. Sc Japan as a patent strain, represents a novel species of the 2002, 2004, 2006). PrP is beta-sheet rich abnormal PC genus Nocardiopsis. conformer of the alpha-helix rich normal prion protein (Pr ), and is a major infectious agent of prion diseases (Prusiner et al. 1998;Weissmann 1999). There have been some reports of Sc enzymes that can degrade PrP (Langeveld et al. 2003; S. Mitsuiki (*) M. Takasugi McLeod et al. 2004), but the keratinase from strain TOA-1 Department of Applied Chemistry and Biochemistry, Sc Kyushu Sangyo University, can uniquely degrade PrP without additional chemical and Fukuoka 813-8503, Japan physical treatments (Mitsuiki et al. 2006). e-mail: mitsuiki@ip.kyusan-u.ac.jp In this study, a novel alkaliphilic actinomycetes strain of TOA-1 was identified by a polyphasic approach and Y. Moriyama appears to be a novel species of the genus Nocardiopsis. Research Institute, TOTO Ltd., Kanagawa 253-8577, Japan T. Futagami M. Goto Materials and methods Department of Bioscience and Biotechnology, Kyushu University, Fukuoka 812-8581, Japan Strain and culture conditions H. Kanouchi T. Oka Strain TOA-1 was isolated from cement of bathroom tiles in Department of Veterinary Physiology, Kagoshima University, Japan. Isolation was done at 30°C by using agar plates made Kagoshima 890-0065, Japan 350 Ann Microbiol (2010) 60:349–353 with AW medium (per 1 l of distilled water), glucose, 10 g; cellulose, pectin, chitin, xylan, pullulan and Tween 80) peptone, 5 g; K HPO , 1 g; MgSO � 7H O, 0.5 g; agar, 15 g; was detected in modified Bennett’s medium (Williams et 2 4 4 2 and Na CO , 10 g. Sodium carbonate was separately al. 1983). 2 3 sterilized and then added to the sterile medium. Strain TOA-1 cultivated on AW plates was used for microscopic Chemotaxonomy observation of sporophores, spore-chains and the spore surface by light- and scanning-electron microscopy (Hitha- Amino acid and sugar analyses of whole cell hydrolysates chi, S-4700). Growth characteristics of strain TOA-1 culture were performed as described by Hasegawa et al. (1983) and were studied on ISP media (Kroppenstedt 1992). Staneck and Roberts (1974). Fatty acid methyl esters were prepared from 50 mg of wet cells. Mixtures of fatty acid Physiology methyl esters were prepared and analyzed with the MIDI system (Hewlett Packard) (Sasser 1990). Menaquinones All physiological tests were carried out at 30°C and at were extracted, purified and identified by HPLC pH 10.0 unless otherwise specified. The effect of (Collins 1985). different temperatures and pH conditions on growth and the tolerance of strain TOA-1 to salt were determined by 16S rRNA sequence determination and phylogenetic using plates made with AW medium. Production of analysis melanoid pigments was tested on an ISP 7 plate (Shirling and Gottlie 1966). Utilization of carbon source (L- Genomic DNA extraction, PCR-mediated amplification of arabinose, D-xylose, D-glucose, D-fructose, L-rhamnose, 16S rRNA genes, and sequence determination were raffinose, sucrose, mannitol and inositol) was examined performed as previously described (Mitsuiki et al. 2002). using ISP 9 as a basal medium (Shirling and Gottlie 1966). Phylogenetic tree analysis was performed using the MEGA Decomposition of substrate (starch, casein, carboxymethyl software version 4.0 (Tamura et al. 2007). Table 1 Physiology of TOA-1 T T Characteristics TOA-1 N. metallicus DSM 44598 N. listeri DSM 40297 and the phylogenetically most closely related species of the Aerial mycerium + − + genus Nocardiopsis Utilization of L-arabinose + + + D-xylose + + + D-glucose + + + D-fructose + + + L-rhamnose + + + Raffinose + −− Sucrose + + + Mannitol + + − Inositol + −− Decomposition of Starch + + − Casein + + + Carboxymethyl cellulose + + − Pectin + −− Chitin + + + Xylan + + − Pullulan − + − Tween 80 + + − All three strains utilized L- Growth at arabinose, D-xylose, D-glucose, 10°C − ++ D-fructose, L-rhamnose and su- 45°C + −− crose, and decomposed casein and chitin pH 12 + −− 5% NaCl + + + + Substrate utilized/decom- posed, − substrate not utilized/ 10% NaCl − + − decomposed Ann Microbiol (2010) 60:349–353 351 Determination of DNA-DNA similarities and G+C content The temperature range for growth was 15–45°C, with optimalgrowthoccurring at 30°C.Goodgrowthwas DNA-DNA hybridization studies were carried out by using shown at NaCl concentration up to 8%. No melanoid the thermal renaturation method (Yassin et al. 1993). The pigments were produced. All the tested carbon sources guanine-plus-cytosine (G+C) content of the DNA was could be utilized for growth by strain TOA-1. Starch, casein, determined by HPLC (Mesbah et al. 1989). carboxymethyl cellulose, pectin, chitin, xylan and Tween 80 could be decomposed, while pullulan could not be. These characteristics of strain TOA-1 and of the phylogenet- Results and discussion ically closely related typestrains of the species N. metalicus and N. listeri are given in Table 1 (Evtushenko et al. 2000; Morphological characteristics Hozzein et al. 2004;Chenet al. 2008). These character- istics are different from those of other two Nocardiopsis Morphological analysis of strain TOA-1 indicated that its species. substrate mycelium was colorless and that no soluble pigments were produced on any medium. The spores were Chemotaxonomic characteristics between 10 and 50 in number, with dimensions of 0.4–0.6× 0.8–1.2 μm, and were straightly chained, rod-shaped, Whole-cell hydrolysates contained meso-diaminopimelic smooth-surfaced and non-motile. These morphological acid as the only diamino acid of the cell wall peptidogly- characteristics are consistent with those described for the can. Diagnostic sugars such as arabinose, xylose and Nocardiopsis species (Kroppenstedt 1992). madurose (Lechevalier et al. 1971) or rhamnose (Labeda et al. 1984) were not detected. The major fatty Physiological characteristics acid detected was iso-C (42.84%). The predominant 16:0 menaquinones were MK-10(H ) and MK-10. In addition, Sc Strain TOA-1 was able to produce PrP -degrading there were detectable amounts of MK-9(H ) and MK-9(H ). 2 4 keratinase. This ability has not been reported for any All these characteristics are typical of the genus Nocar- other Nocardiopsis strain.Growthoccurredinthe pH diopsis (Grund and Kroppenstedt 1990; Kroppenstedt range 7.5–13.0, with optimal growth occurring at pH 10.0. 1992). Fig. 1 Phylogenetic dendro- 100 Nocardiopsis metallicus DSM 44598 gram based on 16S rRNA gene Strain TOA-1 sequence analysis, showing the Nocaridiopsis listeri DSM 40297 phylogenetic position of TOA-1 compared with the other species Nocardiopsis alba DSM 43377 of the genus Nocardiopsis. Bar: Nocardiopsis umidischolae DSM 44362 inferred nucleotide substitution per 100 nucleotides Nocardiopsis lucentensis DSM 44048 63 Nocardiopsis aegyptica DSM 44442 Nocardiopsis dassonvillei DSM 43111 82 Nocardiopsis synnemataformans DSM 44143 Nocardiopsis benisuefensis DSM 44686 100 Nocardiopsis halototerans DSM 44410 Nocardiopsis quinghaiensis DSM 44739 Nocardiopsis xinjiangensis DSM 44589 98 T Nocardiopsis salina DSM 44839 Nocardiopsis composta DSM 44551 Nocardiopsis halophila DSM 44494 99 T Nocardiopsis baichengensis DSM 44845 Nocardiopsis chromatogenes DSM 44844 92 T Nocardiopsis gilva DSM 44841 Nocardiopsis rhodophaea DSM 44843 Nocardiopsis rosea DSM 44842 0.005 352 Ann Microbiol (2010) 60:349–353 Hozzein WN, Li WJ, Ali MI, Hammouda O, Mousa AS, Xu LH, Jiang Phylogenetic analysis CL (2004) Nocardiopsis alkaliphila sp. nov., a novel alkaliphilic actinomycete isolated from desert soil in Egypt. Inst J Syst The sequence of the 16S rRNA gene of strain TOA-1 Bacteriol 54:247–252 (AY027776), which consists of 1,515 bp, was almost Kroppenstedt RM (1992) The genus Nocardiopsis. In: The Prokaryotes. A handbook on the biology of bacteria: ecophysiology, isolation, completely determined. A preliminary comparison of the identification, applications. Academic, New York, pp 1139–1156 sequence with other sequences in GenBank indicated that Labeda DP, Testa RT, Lechevalier MP, Lechevalier HA (1984) the closest phylogenetic neighbors of strain TOA-1 were Saccharothrix: a new genus of the Actinomycetales related to members of the genus Nocardiopsis. Pairwise similarity Nocardiopsis. Int J Syst Bacteriol 34:426–431 Langeveld JPM, Wang JJ, Van de Wiel DFM, Shin GC, Jan GG, values ranged from 92.5% (N. rhodophaea DSM 44843 ) Bossers A, Shin JCH (2003) Enzymatic degradation of prion to 98.8% (N. metallicus DSM 44598 ). The phylogenic tree protein in brain stem from infected cattle and sheep. J Infect Dis constructed on the basis of published sequences is shown 188:1782–1789 Fig. 1. This tree indicates that strain TOA-1, N. metallicus Lechevalier HA, Lechevalier MP, Gerber NN (1971) Chemical T T composition as a criterion in the classification of actinomycetes. DSM 44598 and N. listeri DSM 40297 , form one of Adv Appl Microbiol 14:47–72 several Nocardiopsis subclusters. The similarity of each of Li WJ, Kroppenstedt RM, Wang D, Tang SK, Lee JC, Park DJ, Kim CJ, these type strains to strain TOA-1 was calculated as 98.8 Xu LH, Jiang CL (2004a) Five novel species of the genus and 97.5%, respectively. Strain TOA-1 probably belongs to Nocardiopsis isolated from hypersaline soils and emended descrip- tion of Nocardiopsis salina. Inst J Syst Bacteriol 56:1089–1096 a novel species. Although a sequence similarity as high as Li WJ, Park DJ, Tang SK, Wang D, Lee JC, Xu LH, Kim CJ, Jiang 98.8% suggested that strain TOA-1 might be a novel CL (2004b) Nocardiopsis salina sp. nov., a novel halophilic species, sequence similarity values of ≧97% have been actinomycete isolated from saline soil in China. Inst J Syst reported to be of limited usefulness in species differentia- Bacteriol 54:1805–1809 McLeod AH, Murdoch H, Dickinson J, Dennis MJ, Hall GA, Buswell tion (Stackebrandt and Goebel 1994). The G+C content was CM, Carr J, Taylor DM, Sutton JM, Raven ND (2004) 71 mol%, which lies within the range for the genus Proteolytic inactivation of the bovine spongiform encephalopathy Nocardiopsis (Grund and Kroppenstedt 1990). The DNA– agent. 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Annals of Microbiology – Springer Journals

Published: Apr 29, 2010

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