| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China
2 BCCM/LMG Bacteria Collection, Department of Biochemistry, Physiology and Microbiology, University Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
3 School of Biology, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK
Correspondence
Ying Huang
huangy{at}im.ac.cn
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
-region of the 16S rRNA molecule showed that it formed a distinct phyletic line within the range of variation encompassed by the genus Streptomyces. The sharp separation of the organism from representatives of the genus Streptomyces was strengthened by the fact that its BOX-PCR and RFLP of 16S rDNA-ITS fingerprints differed from those of over 450 recognized Streptomyces species. The isolate also had a profile of phenotypic properties that readily distinguished it from the genotypically close type strains. It is evident from the combination of genotypic and phenotypic data that strain FXJ14T (=AS 4.1858T=JCM 12278T=LMG 22082T) should be classified as the type strain of a novel species of the genus Streptomyces, for which the name Streptomyces glauciniger sp. nov. is proposed.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of S. cuspidosporus AS 4.1686T and S. glauciniger FXJ14T are AY589505 and AY314782, respectively.
A scanning electron micrograph, a neighbour-joining tree based on -region sequences and dendrograms generated from RFLP and BOX-PCR results are available as supplementary material in IJSEM Online.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
; Kämpfer et al., 1991). While molecular systematic data show that, based on recognized species, the genus is clearly overspeciated (Hatano et al., 2003; Lanoot et al., 2002, 2004), polyphasic studies based on a judicious combination of genotypic and phenotypic features continue to bring us novel species and indicate that the genus Streptomyces as a whole is underspeciated (Labeda et al., 1997; S. B. Kim et al., 1998; Al-Tai et al., 1999; B. Kim et al., 2000; Kim & Goodfellow, 2002; Li et al., 2002; Saintpierre et al., 2003). The present study describes a distinct mesophilic actinomycete, strain FXJ14T, as a novel Streptomyces species based on a polyphasic approach.
Strain FXJ14T was isolated on a yeast extract/starch agar (Emerson, 1958) plate supplemented with 50 µg cycloheximide ml–1, which had been seeded with a soil suspension and incubated at 28 °C for 2 weeks. The soil sample was collected from willow woods in Nanning City, Guangxi Province, China. The isolate was maintained on Bennett's agar (Jones, 1949) slopes at 4 °C and as glycerol suspensions (20 %, v/v) at –20 °C. Biomass for chemotaxonomic and molecular systematic studies was prepared as described by Li et al. (2002).
The arrangement of hyphae and spore chains were observed on modified Bennett's agar and oatmeal agar (ISP medium 3) after 14 days at 28 °C using the coverslip technique of Kawato & Shinobu (1959). Spore chain morphology and spore surface ornamentation were observed by examining gold-coated dehydrated specimens with a model FEI QUANTA electron microscope. Cultural characteristics were observed on a number of standard media (Table 1) after 14 days at 28 °C. Strain FXJ14T was examined for a range of physiological properties using established procedures described by Williams et al. (1983) and Kämpfer et al. (1991).
|
and Lechevalier & Lechevalier (1980). Polar lipids were examined and identified using the method of Minnikin et al. (1984). Menaquinones were extracted and purified following Collins (1985) and then analysed by HPLC (Wu et al., 1989). Fatty acids were extracted, methylated and analysed by GC using the standard Sherlock MIDI (Microbial Identification) system (Sasser, 1990; Kämpfer & Kroppenstedt, 1996). DNA G+C content of the tested strain was determined using the thermal denaturation method of Marmur & Doty (1962), with Escherichia coli AS 1.365 as a control.
Genomic DNA preparation and PCR amplification of the 16S rRNA gene sequence of strain FXJ14T were performed as described by Chun & Goodfellow (1995). The PCR product was purified and directly sequenced as described by Huang et al. (2001). The resultant sequence was aligned manually using CLUSTAL X version 1.8 (Thompson et al., 1997) with available, almost complete sequences of type strains of the family Streptomycetaceae and then with corresponding sequences of representative Streptomyces species; in each case, the reference sequences were retrieved from the DDBJ/EMBL/GenBank databases. The final dataset consisted of information on 23 strains. Phylogenetic trees were inferred by using the least-squares (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Kluge & Farris, 1969) and neighbour-joining (Saitou & Nei, 1987) algorithms from the PHYLIP package version 3.5c (Felsenstein, 1993). Evolutionary distance matrices were generated following the method of Kimura (1980). The resultant unrooted tree topologies were evaluated by bootstrap analyses (Felsenstein, 1985) of the neighbour-joining method based on 1000 resamplings. A partial sequence covering the variable -region (120 nt, positions 158–277) of the 16S rRNA gene sequence of strain FXJ14T was also aligned with the corresponding nucleotide sequences of nearly 500 Streptomyces type strains retrieved from GenBank. A phylogenetic tree based on these partial sequences was generated using the neighbour-joining algorithm (Saitou & Nei, 1987).
BOX-PCR fingerprinting was carried out following the method of Lanoot et al. (2004). For RFLP of 16S rDNA-ITS (internally transcribed spacer), a universal primer set (PA, 5'-AGAGTTTGATCCTGGCTCAG-3'; BL235R, 5'-GCGCCCTTAAAAACTTGG-3') was used to amplify both the 16S rRNA gene and the adjacent 16S–23S rDNA ITS region in one PCR. Digested PCR products, using restriction enzymes BstUI and HaeIII, were separated on 8 % polyacrylamide gels. The fingerprinting patterns were compared with corresponding in-house databases containing more than 450 recognized Streptomyces species using the software package BIONUMERICS version 2.5 (Applied Maths).
Morphological and chemical features of strain FXJ14T were consistent with its assignment to the genus Streptomyces (Williams et al., 1989; Manfio et al., 1995). The organism formed an extensively branched substrate mycelium, aerial hyphae that carried smooth-surfaced spores in spiral spore chains (see Supplementary Fig. A in IJSEM Online) and a greyish aerial spore mass on several standard media (Table 1
). It contained major amounts of LL-diaminopimelic acid in whole-organism hydrolysates, hexa- and octahydrogenated menaquinones with nine isoprene units [MK-9(H6, H8)] as predominant isoprenologues and diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides as typical polar lipids (phospholipid type II sensu Lechevalier et al., 1977), but lacked characteristic sugars and mycolic acids. The fatty acid profile comprised mainly saturated straight-chain and iso- and anteiso-branched-chain fatty acids (fatty acid type 2c sensu Kroppenstedt, 1985).
The assignment of strain FXJ14T to the genus Streptomyces was also supported by 16S rRNA gene sequence data. An almost complete 16S rRNA gene sequence (1428 nt) was determined for the organism. Preliminary phylogenetic analysis, which included available, almost full-length sequences of type strains of the family Streptomycetaceae, showed that strain FXJ14T fell within the evolutionary radiation encompassed by the genus Streptomyces (data not shown). Despite the fact that the organism was found to be closely associated with some members of the genera Kitasatospora and Streptacidiphilus in a comparison of 16S rRNA gene sequences using a standard nucleotide–nucleotide BLAST search (Altschul et al., 1997) against DDBJ/EMBL/GenBank, specific nucleotide signatures of these two genera (Zhang et al., 1997; Kim et al., 2003) were absent in strain FXJ14T. It is clear from Fig. 1 that the tested strain forms a distinct phyletic line in the 16S rRNA gene sequence Streptomyces tree. Sequence similarity values between strain FXJ14T and its nearest neighbours, namely Streptomyces rimosus subsp. rimosus JCM 4667T, Streptomyces malaysiensis ATB-11T, Streptomyces sparsogenes NRRL 2940T and Streptomyces yeochonensis CN 732T, were 97·3 (39 nt differences at 1428 sites), 97·2 (40 nt differences at 1415 sites), 97·2 (40 nt differences at 1425 sites) and 97·2 % (40 nt differences at 1425 sites), respectively. Values in this range are well below the range recorded for many recognized Streptomyces species (S. B. Kim et al., 1998; Al-Tai et al., 1999; B. Kim et al., 2000; Sembiring et al., 2000; Kim & Goodfellow, 2002; Li et al., 2002; Saintpierre et al., 2003), nor are the relationships between the tested strain and the type strains supported by good bootstrap levels. A number of phenotypic properties also separated strain FXJ14T from its most closely related type strains (Table 2).
|
|
-region of the 16S rRNA gene sequence showed that the organism was loosely clustered with the type strains of Streptomyces indigoferus and Streptomyces herbaricolor (Supplementary Fig. B). However, the latter two strains can be distinguished readily from strain FXJ14T using phenotypic properties (Table 2). Although RFLP of 16S rDNA ITS fingerprints revealed that strain FXJ14T was most closely associated with the type strain of Streptomyces cuspidosporus (Supplementary Fig. C), the two organisms were distinguished from one another on the basis of almost complete 16S rRNA gene sequence analysis (Fig. 1), sharing a relatively low sequence similarity of 96·9 %. With respect to BOX-PCR fingerprints, strain FXJ14T had a unique pattern when compared with corresponding data on 473 Streptomyces type strains (Supplementary Fig. D shows a partial dendrogram, including the neighbouring type strains), thereby confirming its distinct position in the genus Streptomyces. Based on a combination of genotypic and phenotypic data, strain FXJ14T merits recognition as the type strain of a novel species in the genus Streptomyces, for which we propose the name Streptomyces glauciniger sp. nov.
Description of Streptomyces glauciniger sp. nov.
Streptomyces glauciniger (glau'ci.ni.ger. L. adj. glaucus greenish grey; L. adj. niger black; N.L. masc. adj. glauciniger greenish black, referring to the colour of colony reverse on modified Bennett's agar).
Aerobic, Gram-positive mesophilic actinomycete that forms an extensively branched substrate mycelium and aerial hyphae that differentiate into long spiral spore chains with 15–20 cylindrical spores per chain. Spore surface is smooth. Soluble pigments are not produced, nor are melanin pigments formed on peptone/yeast extract/iron or tyrosine agars. Additional cultural characteristics on various agar media are given in Table 1. Growth occurs at 10–35 °C and pH 5·0–10·0, but not at 40 °C or at pH 4·0 or 11·0. Growth also occurs in the presence of phenol (0·1 %, w/v) but not in the presence of NaCl (5 %, w/v), novobiocin (5 µg ml–1) or streptomycin (10 µg ml–1). In addition to the properties listed in Table 2, the organism degrades adenine, casein, hypoxanthine, starch and xanthine, but not cellulose or elastin. It uses dextrin, D-galactose, D-glucose (all at 1 %, w/v), sodium acetate and sodium citrate (both at 0·1 %, w/v), but not D-maltose (1 %, w/v), as sole carbon sources for energy and growth. Nitrate is reduced. Gelatin is not liquefied. It shows antimicrobial activity against strains of Bacillus subtilis and Candida albicans, but not against strains of Aspergillus niger, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa or Staphylococcus aureus. Cell wall is of type I, phospholipid type II and menaquinone MK-9(H6, H8). The fatty acid profile is composed of iso-C16 : 0 (25·4 %), anteiso-C17 : 0 (17·4 %), anteiso-C15 : 0 (16·7 %), iso-C15 : 0 (9·5 %), C16 : 0 (7·7 %), iso-C17 : 0 (3·8 %), iso-C14 : 0 (3·5 %), iso-C17 : 1
9c (3·3 %), anteiso-C17 : 19c (3·0 %), iso-C16 : 1 (2·9 %), C15 : 0 (2·8 %) and C17 : 0 cyclo (2·7 %). DNA G+C content is 67·0 mol%.
The type strain, FXJ14T (=AS 4.1858T=JCM 12278T=LMG 22082T), was isolated from soil in a willow wood collected in Nanning City, Guangxi Province, China.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.
Chun, J. & Goodfellow, M. (1995). A phylogenetic analysis of the genus Nocardia with 16S rRNA gene sequences. Int J Syst Bacteriol 45, 240–245.
Collins, M. D. (1985). Isoprenoid quinone analysis in classification and identification. In Chemical Methods in Bacterial Systematics, pp. 267–287. Edited by M. Goodfellow & D. E. Minnikin. London: Academic Press.
Emerson, R. (1958). Mycological organization. Mycologia 50, 589–621.[CrossRef]
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376.[CrossRef][Medline]
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
Felsenstein, J. (1993). PHYLIP (Phylogenetic Inference Package), version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle, USA.
Fitch, W. M. & Margoliash, E. (1967). Construction of phylogenetic trees: a method based on mutation distances as estimated from cytochrome c sequences is of general applicability. Science 155, 279–284.
Hasegawa, T., Takizawa, M. & Tanida, S. (1983). A rapid analysis for chemical grouping of aerobic actinomycetes. J Gen Appl Microbiol 29, 319–322.[CrossRef]
Hatano, K., Nishii, T. & Kasai, H. (2003). Taxonomic re-evaluation of whorl-forming Streptomyces (formerly Streptoverticillium) species by using phenotypes, DNA–DNA hybridization and sequences of gyrB, and proposal of Streptomyces luteireticuli (ex Katoh and Arai 1957) corrig., sp. nov., nom. rev. Int J Syst Evol Microbiol 53, 1519–1529.
Huang, Y., Qi, W., Lu, Z., Liu, Z. & Goodfellow, M. (2001). Amycolatopsis rubida sp. nov., a new Amycolatopsis species from soil. Int J Syst Evol Microbiol 51, 1093–1097.[Abstract]
Jones, K. L. (1949). Fresh isolates of actinomycetes in which the presence of sporogenous aerial mycelia is a fluctuating characteristic. J Bacteriol 57, 141–145.
Kämpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of fatty acid patterns of coryneform bacteria and related taxa. Can J Microbiol 42, 989–1005.
Kämpfer, P., Kroppenstedt, R. M. & Dott, W. (1991). A numerical classification of the genera Streptomyces and Streptoverticillium using miniaturized physiological tests. J Gen Microbiol 137, 1831–1891.
Kawato, M. & Shinobu, R. (1959). On Streptomyces herbaricolor sp. nov., supplement: a single technique for microscopical observation. Mem Osaka Unit Lib Arts Educ B Nat Sci 8, 114–119.
Kim, S. B. & Goodfellow, M. (2002). Streptomyces avermitilis sp. nov., nom. rev., a taxonomic home for the avermectin-producing streptomycetes. Int J Syst Evol Microbiol 52, 2011–2014.[Abstract]
Kim, S. B., Falconer, C., Williams, E. & Goodfellow, M. (1998). Streptomyces thermocarboxydovorans sp. nov. and Streptomyces thermocarboxydus sp. nov., two moderately thermophilic carboxydotrophic species from soil. Int J Syst Bacteriol 48, 59–68.
Kim, B., Al-Tai, A. M., Kim, S. B., Somasundaram, P. & Goodfellow, M. (2000). Streptomyces thermocoprophilus sp. nov., a cellulase-free endo-xylanase-producing streptomycete. Int J Syst Evol Microbiol 50, 505–509.[Abstract]
Kim, S. B., Lonsdale, J., Seong, C.-N. & Goodfellow, M. (2003). Streptacidiphilus gen. nov., acidophilic actinomycetes with wall chemotype I and emendation of the family Streptomycetaceae (Waksman and Henrici 1943AL) emend. Rainey et al. 1997. Antonie van Leeuwenhoek 83, 107–116.[CrossRef][Medline]
Kim, S. B., Seong, C. N., Jeon, S. J., Bae, K. S. & Goodfellow, M. (2004). Taxonomic study of neutrotolerant acidophilic actinomycetes isolated from soil and description of Streptomyces yeochonensis sp. nov. Int J Syst Evol Microbiol 54, 211–214.
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]
Kluge, A. G. & Farris, F. S. (1969). Quantitative phyletics and the evolution of anurans. Syst Zool 18, 1–32.
Kroppenstedt, R. M. (1985). Fatty acid and menaquinone analysis of actinomycetes and related organisms. In Chemical Methods in Bacterial Systematics, pp. 173–199. Edited by M. Goodfellow & D. E. Minnikin. London: Academic Press.
Labeda, D. P., Lechevalier, M. P. & Testa, R. T. (1997). Streptomyces stramineus sp. nov., a new species of the verticillate streptomycetes. Int J Syst Bacteriol 47, 747–753.
Lanoot, B., Vancanneyt, M., Cleenwerck, I., Wang, L., Li, W., Liu, Z. & Swings, J. (2002). The search for synonyms among streptomycetes by using SDS-PAGE of whole cell proteins. Emendation of the species Streptomyces aurantiacus, Streptomyces cacaoi subsp. cacaoi, Streptomyces caeruleus and Streptomyces violaceus. Int J Syst Evol Microbiol 52, 823–829.[Abstract]
Lanoot, B., Vancanneyt, M., Dawyndt, P., Cnockaert, M., Zhang, Z., Huang, Y., Liu, Z. & Swings, J. (2004). BOX-PCR fingerprinting as a powerful tool to reveal synonymous names in the genus Streptomyces. Emended descriptions of the species Streptomyces cinereorectus, S. fradiae, S. tricolor, S. colombiensis, S. filamentosus, S. vinaceus and S. phaeopurpureus. Syst Appl Microbiol 27, 84–92.[CrossRef][Medline]
Lechevalier, H. A. & Lechevalier, M. P. (1980). The chemotaxonomy of actinomycetes. In Actinomycete Taxonomy, Special Publication 6, pp. 277–284. Edited by A. Dietz & D. W. Thayer. Arlington, VA: Society of Industrial Microbiology.
Lechevalier, M. P., De Bièvre, C. & Lechevalier, H. A. (1977). Chemotaxonomy of aerobic actinomycetes: phospholipid composition. Biochem Syst Ecol 5, 249–260.[CrossRef]
Li, W., Lanoot, B., Zhang, Y., Vancanneyt, M., Swings, J. & Liu, Z. (2002). Streptomyces scopiformis sp. nov., a novel streptomycete with fastigiate spore chains. Int J Syst Evol Microbiol 52, 1629–1633.[Abstract]
Lonsdale, J. T. (1985). Aspects of the biology of acidophilic actinomycetes. PhD thesis, University of Newcastle, Newcastle upon Tyne, UK.
Manfio, G. P., Zakrzewska-Czerwinska, J., Atalan, E. & Goodfellow, M. (1995). Towards minimal standards for the description of Streptomyces species. Biotechnologia 7–8, 242–253.
Marmur, J. & Doty, P. (1962). Determination of base composition of deoxyribonucleic acid from its denaturation temperature. J Mol Biol 5, 109–118.[Medline]
Minnikin, D. E., O'Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated procedure for the extraction of isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.[CrossRef]
Saintpierre, D., Amir, H., Pineau, R., Sembiring, L. & Goodfellow, M. (2003). Streptomyces yatensis sp. nov., a novel bioactive streptomycete isolated from a New-Caledonian ultramafic soil. Antonie van Leeuwenhoek 83, 21–26.[CrossRef][Medline]
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Sasser, M. (1990). Identification of Bacteria by Gas Chromatography of Cellular Fatty Acids. Technical Note 101. Newark, DE: Microbial ID.
Sembiring, L., Ward, A. C. & Goodfellow, M. (2000). Selective isolation and characterisation of members of the Streptomyces violaceusniger clade associated with the roots of Paraserianthes falcataria. Antonie van Leeuwenhoek 78, 353–366.[CrossRef][Medline]
Shirling, E. B. & Gottlieb, D. (1968). Cooperative description of type cultures of Streptomyces. III. Additional species description from first and second studies. Int J Syst Bacteriol 18, 279–392.
Shirling, E. B. & Gottlieb, D. (1969). Cooperative description of type cultures of Streptomyces. IV. Species descriptions from the second, third and fourth studies. Int J Syst Bacteriol 19, 391–512.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Williams, S. T., Goodfellow, M., Alderson, G., Wellington, E. M. H., Sneath, P. H. A. & Sackin, M. J. (1983). Numerical classification of Streptomyces and related genera. J Gen Microbiol 129, 1743–1813.[Medline]
Williams, S. T., Goodfellow, M. & Alderson, G. (1989). Genus Streptomyces Waksman and Henrici, 1943, 339AL. In Bergey's Manual of Systematic Bacteriology, vol. 4, pp. 2452–2492. Edited by S. T. Williams, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.
Wu, C., Lu, X., Qin, M., Wang, Y. & Ruan, J. (1989). Analysis of menaquinone compound in microbial cells by HPLC. Microbiology (English translation of Mikrobiologiya) 16, 176–178.
Zhang, Z., Wang, Y. & Ruan, J. (1997). A proposal to revive the genus Kitasatospora (Omura, Takahashi, Iwai, and Tanaka 1982). Int J Syst Bacteriol 47, 1048–1054.
This article has been cited by other articles:
|
|
 |
|
  W. Sun, Y. Huang, Y.-Q. Zhang, and Z.-H. Liu Streptomyces emeiensis sp. nov., a novel streptomycete from soil in China Int J Syst Evol Microbiol, July 1, 2007; 57(7): 1635 - 1639. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Gu, M. Pasciak, H. Luo, A. Gamian, Z. Liu, and Y. Huang Ruania albidiflava gen. nov., sp. nov., a novel member of the suborder Micrococcineae Int J Syst Evol Microbiol, April 1, 2007; 57(4): 809 - 814. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
