| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Instituto de Microbiologia Prof. Paulo de Góes1 , Universidade do Brasil, UFRJ, CCS, Bloco I, Ilha do Fundão, Rio de Janeiro (RJ), 21941-590, Brazil
Instituto de Biologia2 , Universidade do Brasil, UFRJ, CCS, Bloco I, Ilha do Fundão, Rio de Janeiro (RJ), 21941-590, Brazil
3 CPQBA/UNICAMP, CP 6171 Campinas (SP), 13081-970, Brazil
Correspondence
R. R. R. Coelho
rosalie{at}micro.ufrj.br
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
The GenBank accession number for the 16S rDNA gene sequence of Streptomyces drozdowiczii strain M7aT is AF529199.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
), and also a source of a wide range of other types of bioactive compounds for biotechnological applications (Okami & Hotta, 1988; Bull et al., 1992). For several years, our laboratory has been interested in isolating actinomycetes from Brazilian soils, with the purpose of selecting strains for biotechnological applications, mainly enzyme production. Using the dispersion and differential centrifugation technique (Hopkins et al., 1991), a cellulolytic actinomycete strain (M7aT) was isolated from a Mata Atlântica Forest soil sample in Rio de Janeiro (Brazil). The isolate showed high cellulolytic activity at temperatures of 70–100 °C, as measured by the release of reducing sugars after 60 min incubation in a reaction mixture containing 500 µl 1 % carboxymethylcellulose plus 700 µl 0·05 M citrate/phosphate buffer (pH 7·0) and 100 µl crude enzyme preparation (Semêdo et al., 2000). Preliminary taxonomic characterization, based upon analysis of morphological features (aerial hyphae with rectiflexibiles spore chains) and chemotaxonomic data (the presence of LL-diaminopimelic acid and the absence of diagnostic cell wall sugar, cell wall chemotype I sensu Lechevalier & Lechevalier, 1970), revealed that the strain belonged to the genus Streptomyces (Semêdo et al., 2001). Numerical taxonomic analysis performed according to Williams et al. (1983a, b) was not successful in classifying this strain in any of the known major or minor cluster groups nor as any other described cellulolytic species. Thus, in the present investigation, cellulolytic strain M7aT was further characterized to establish whether it represents a novel species in the genus Streptomyces.
Streptomycete strain M7aT was isolated from Mata Atlântica Forest soil (Mendanha Forest, Rio de Janeiro, RJ, Brazil) using starch/casein agar plates (pH 7·0) supplemented with 100 µg amphotericin B ml–1, and seeded with inocula from the dispersion and differential centrifugation technique (Hopkins et al., 1991), as described elsewhere (Semêdo et al., 2001). Spores from pure cultures were stored in 20 % glycerol (v/v) at –20 °C.
Spore chain morphology and spore ornamentation were examined by light microscopy and TEM (EM 900; Zeiss) analyses of cultures grown on malt extract/yeast extract agar for 14 days (Shirling & Gottlieb, 1966). Aerial spore-mass colour, substrate mycelial pigmentation and production of diffusible pigments were recorded after incubation at 28 °C for 14 days on inorganic salts/starch agar and glycerol/asparagine agar (Shirling & Gottlieb, 1966). Melanin production was observed on tyrosine agar medium and peptone/yeast extract/iron agar (Shirling & Gottlieb, 1966). Other physiological and biochemical characteristics were determined according to Williams et al. (1983a).
For fatty acid and chemical analyses, cells were grown in glycerol/peptone/mineral salts medium (Paim et al., 1990) in a shaker at 140 r.p.m. for 10 days at 28 °C. Biomass was harvested by centrifugation at 13 200 g for 30 min and the pellet was rinsed with distilled water prior to lyophilization. For molecular systematic analyses, cells were grown on modified Bennett's agar (Jones, 1949) as described previously, washed in 0·1 M NaCl/0·1 M EDTA (pH 8·0) buffer and stored at –20 °C.
Cell wall amino acids were analysed after acid hydrolysis of the cell mass using 6 M HCl. Hydrolysed material was dried, resuspended in the appropriate buffer and injected into an automatic analyser (High Performance amino acid analyser, system 6300; Beckman), according to the manufacturer's instructions. Amino acids were identified automatically by comparison with retention times of a standard mixture.
Fatty acid analysis was performed by GLC using the transmethylation technique described by Lepage & Roy (1984). Fatty acids were chromatographed as methyl esters on a Supelcowax 10 (Supelco) capillary column (30 m, internal diameter 0·32 mm) packed with polyethylene glycol. Analyses were performed on a Varian 3800 GC equipped with a flame-ionization detector. Helium was used as carrier gas at a flow rate of 1·3 ml min–1. Injection port and detector were kept at 250 °C. The column temperature was held at 200 °C for 10 min, then increased stepwise at 4 °C min–1 until a plateau of 240 °C was reached; the column was held at this temperature for 15 min. Peak areas were automatically integrated by the Saturno software (Varian) and expressed as percentages. Fatty acid identities were determined by comparing retention times with those of a standard mixture (Supelco) of known composition.
Extraction of genomic DNA, PCR amplification and sequencing of 16S rDNA from strain M7aT were carried out as described previously (Pitcher et al., 1989; Lane, 1991; Lane et al., 1985; Stackebrandt et al., 1992). PCR was carried out in a thermal cycler model 480 (Perkin Elmer/Cetus). Amplified fragments were purified using the Concert kit (Life Technologies) and sequenced directly using an ABI Prism dye terminator cycle sequencing reaction kit (Applied Biosystems) in an automatic sequencer (ABI model 310; Applied Biosystems). The 16S rDNA sequence obtained was analysed by CHECK-CHIMERA (Maidak et al., 1997) and subjected to a homology search against data from the Ribosomal Database Project (Maidak et al., 1997) and GenBank (BLAST-N, Altschul et al., 1997). Sequences retrieved were aligned using CLUSTAL_W (Thompson et al., 1994). Evolutionary distance matrices and trees were generated using the Tamura–Nei (Tamura & Nei, 1993) and neighbour-joining (Saitou & Nei, 1997) algorithms with a pairwise deletion option, as implemented in the MEGA version 2.1 software package (Kumar et al., 2001). The resultant unrooted tree topologies were evaluated by bootstrap analyses based on 5000 replications according to Kumar et al. (2001).
Strain M7aT produced aerial hyphae with long rectiflexibiles spore chains (RF type). Spore surface was smooth. In inorganic salts/starch agar and glycerol/asparagine agar media, strain M7aT produced grey aerial spore-mass, with non-distinctive substrate mycelia colour.
The predominant fatty acids found in whole cell methanolysates were iso-16 : 0 (22 % total fatty acids), iso-15 : 0 (19 %), anteiso-15 : 0 (18 %), iso-17 : 0 (10 %), anteiso-17 : 0 (7 %), 16 : 0 (6 %) and iso-14 : 0 (6 %), which are consistent with patterns obtained from Streptomyces spp. The major amino acids found in cell wall hydrolysates were alanine (predominant), followed by glycine, glutamic acid and leucine.
A partial 16S rDNA sequence of strain M7aT (1191 nt) was determined. Comparisons with other 16S rDNA sequences available in GenBank using BLAST searches were used to select related sequences for constructing a multiple alignment. Except for Streptomyces cellulosae (only 870 nt), all other sequences represented nearly full-length sequences equivalent in size to that of strain M7aT. The phylogenetic tree produced is shown in Fig. 1. Strain M7aT and Streptomyces laceyi C7654T produced a consistent cluster with a high bootstrap value. The number of scored nucleotide identities and the overall percentage of similarity calculated by pairwise analysis indicates a sequence similarity of 98·4 % between these organisms. All the other 29 related Streptomyces sequences shared 95·1–98·0 % similarity with M7aT and were recovered in completely distinct branches (Fig. 1).
|
). Compared with Streptomyces celluloflavus (Locci, 1989), M7aT differed in a number of characteristics, including gelatin degradation, penicillin G resistance, L-arabinose utilization and melanin production. Compared with Streptomyces cellulolyticus (Li, 1997), differences were observed in melanin production, utilization of meso-inositol, raffinose and D-xylose, one nitrogen source (L-cysteine), growth on 7 % NaCl, and resistance to penicillin G, vancomycin and kanamycin. Strain M7aT differed from Streptomyces cellulosae in some characteristics, including adenine degradation, utilization of meso-inositol, D-mannitol and phenylalanine, spore chain type, melanin production, growth at 45 °C, and growth in the presence of phenol (0·1 %) and sodium azide (0·01 %). S. laceyi also showed some differences in relation to strain M7aT, including degradation of xanthine, antimicrobial activity against Bacillus subtilis, growth in the presence of 0·01 % sodium azide, and growth in L-histidine, L-valine and D-mannitol.
|
, 2001), isolated from Mata Atlântica soil, Brazil, is clearly distinct from other phylogenetically and phenotypically related Streptomyces species described in the literature, and thus represents a novel species. Therefore, it is proposed that strain M7aT should be included in the genus Streptomyces as a representative of a novel species, Streptomyces drozdowiczii sp. nov.
Description of Streptomyces drozdowiczii sp. nov.
Streptomyces drozdowiczii (droz.do.wicz'i.i. N.L. masc. adj. drozdowiczii referring to Adam Drozdowicz, a soil microbiologist who worked in Brazil).
Branched, grey coloured aerial mycelium is produced on inorganic salts/starch agar, with rectiflexibiles spore chains. The substrate mycelium has no distinctive pigment, but a diffusible yellow–brown pigment is produced. Oval spores, which are borne in chains on the tip of the aerial mycelium, have smooth surfaces. Melanin is produced on tyrosine agar and peptone/yeast extract/iron agar. A detailed description of phenotypic features is presented in Table 1
. Cell wall hydrolysates contain LL-diaminopimelic acid. The predominant amino acids in the cell wall hydrolysate are alanine (major), glycine, glutamic acid and leucine. The predominant fatty acids found in whole cell methanolysates are iso-16 : 0 (22 %), iso-15 : 0 (19 %), anteiso-15 : 0 (18 %), iso-17 : 0 (10 %), anteiso-17 : 0 (7 %), 16 : 0 (6 %) and iso-14 : 0 (6 %). Isolated from the Brazilian Atlantic forest soil (Mendanha Forest, Rio de Janeiro, RJ, Brazil). The species description is based on a single strain and hence serves as the type strain description. The type strain is M7aT (=CBMAI 0498T=CIP 107837T=NRRL B-24297T).
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Bull, A. T., Goodfellow, M. & Slater, J. H. (1992). Biodiversity as a source of innovation in biotechnology. Annu Rev Microbiol 46, 219–252.[CrossRef][Medline]
Hopkins, D. W., MacNaughton, S. J. & O'Donnell, A. G. (1991). A dispersion and differential centrifugation technique for representatively sampling microorganisms from soil. Soil Biol Biochem 23, 217–225.[CrossRef]
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.
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). MEGA2: Molecular Evolutionary Genetics Analysis software. Tempe, AZ: Arizona State University.
Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics, pp. 115–175. Edited by E. Stackebrandt & M. Goodfellow. Chichester: John Wiley.
Lane, D. J., Pace, B., Olsen, G. J., Stahl, D. A., Sogin, M. L. & Pace, N. R. (1985). Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci U S A 82, 6955–6959.
Lechevalier, M. P. & Lechevalier, H. (1970). Chemical composition as a criterion in the classification of aerobic actinomycetes. Int J Syst Bacteriol 20, 435–443.
Lepage, G. & Roy, C. C. (1984). Improved recovery of fatty acid through direct transesterification without prior extraction or purification. J Lipid Res 25, 1391–1396.[Abstract]
Li, X. (1997). Streptomyces cellulolyticus sp. nov., a new cellulolytic member of the genus Streptomyces. Int J Syst Bacteriol 47, 443–445.
Locci, R. (1989). Streptomyces and related genera. In Bergey's Manual of Systematic Bacteriology, vol. 4, pp. 2451–2506. Edited by S. T. Williams, M. E. Sharp & J. G. Holt. Baltimore: Williams & Wilkins.
Maidak, B. L., Olsen, G. J., Larsen, N., Overbeek, R., McCaughey, M. J. & Woese, C. R. (1997). The RDP (Ribosomal Database Project). Nucleic Acids Res 25, 109–111.
Manfio, G. P., Atalan, E., Zakrzewska-Czerwinska, J., Mordarski, M., Rodríguez, C., Collins, M. D. & Goodfellow, M. (2003). Classification of novel soil streptomycetes as Streptomyces aureus sp. nov., Streptomyces laceyi sp. nov. and Streptomyces sanglieri sp. nov. Antonie van Leeuwenhoek 83, 245–255.[CrossRef][Medline]
Okami, Y. & Hotta, K. (1988). Search and discovery of new antibiotics. In Actinomycetes in Biotechnology, pp. 37–67. Edited by M. Goodfellow, S. T. Williams & M. Mordarski. London: Academic Press.
Paim, S., Linhares, L. F., Mangrich, A. S. & Martin, J. P. (1990). Characterization of fungal melanins and soil humic acids by chemical analysis and infrared spectroscopy. Biol Fertil Soils 10, 72–76.[Medline]
Pitcher, D. G., Saunders, N. A. & Owen, R. J. (1989). Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8, 151–156.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Semêdo, L. T. A. S., Gomes, R. C., Bon, E. P. S., Soares, R. M. S., Linhares, L. F. & Coelho, R. R. R. (2000). Endocellulase and exocellulase activities of two Streptomyces strains isolated from a forest soil. Appl Biochem Biotechnol 84–86, 267–276.[Medline]
Semêdo, L. T. A. S., Linhares, A. A., Gomes, R. C., Manfio, G. P., Alviano, C. S., Linhares, L. F. & Coelho, R. R. R. (2001). Isolation and characterization of actinomycetes from Brazilian tropical soils. Microbiol Res 155, 291–299.[Medline]
Shirling, E. B. & Gottlieb, D. (1966). Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16, 313–340.
Stackebrandt, E., Liesack, W. & Witt, D. (1992). Ribosomal RNA and rDNA sequence analyses. Gene 115, 255–260.[CrossRef][Medline]
Tamura, K. & Nei, M. (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 10, 512–526.[Abstract]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL_W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Williams, S. T., Goodfellow, M., Alderson, G., Wellington, E. M. H., Sneath, P. H. A. & Sackin, M. J. (1983a). Numerical classification of Streptomyces and related genera. J Gen Microbiol 129, 1743–1813.[Medline]
Williams, S. T., Goodfellow, M., Wellington, E. M. H., Vickers, J. C., Alderson, G., Sneath, P. H. A., Sackin, M. J. & Mortimer, A. M. (1983b). A probability matrix for identification of some streptomycetes. J Gen Microbiol 129, 1815–1830.[Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
