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1 Department of Pharmacy, University of Rajshahi, Rajshahi-6205, Bangladesh
2 Laboratory of Molecular Biotechnology, Department of Biotechnology, The University of Tokyo, Yayoi 113-8657, Japan
3 Department of Molecular Biology and Biotechnology, Institute for Biomedical Research, UNAM, A.P. 70228, Mexico, D.F., 04510, Mexico
4 Department of Zoology, Institute of Biology, UNAM, México, D.F., 04510, Mexico
Correspondence
María Elena Flores
mefc{at}servidor.unam.mx
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ABSTRACT |
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 TOP ABSTRACT IntroductionREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Streptomyces bangladeshensis strain AAB-4T (=LMG 22738T=NRRL B-24326T) is AY750056.
A neighbour-joining dendrogram is available as a supplementary figure in IJSEM Online.
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Introduction |
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TOPABSTRACTIntroductionREFERENCES |
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and classified in the family Streptomycetaceae on the basis of morphology and cell-wall chemotype. Streptomycetes are Gram-positive, aerobic micro-organisms with DNA G+C contents of 69–78 mol% (Korn-Wendish & Kutzner, 1992); they produce extensive branching substrate and aerial mycelia that develop into chains of spores by the formation of cross-walls in the multinucleate aerial filaments (Anderson & Wellington, 2001). The streptomycetes are widely used in industry because of their ability to produce numerous chemical compounds, including enzymes, antitumour agents and (in the main) antibiotics (Bérdy, 1995). During routine screening for antibiotic-producing organisms from soil samples from different regions of Bangladesh, an actinomycete was isolated that produced the antibiotic bis-(2-ethylhexyl)phthalate. This strain, designated AAB-4T, demonstrated a colonial morphology consistent with its assignment to the genus Streptomyces. The aim of the present investigation was to determine the taxonomic position of this organism, and the results suggest that it should be recognized as a novel species of the genus Streptomyces, for which the name Streptomyces bangladeshensis sp. nov. is proposed.
Strain AAB-4T was isolated on yeast extract–glucose agar (Shirling & Gottlieb, 1966), using serial dilutions, and was selected by means of its antibacterial activity. The strain was maintained on Czapek–Dox (Shirling & Gottlieb, 1966) alkaline (pH 8·0) slants at 4 °C. Strain AAB-4T was deposited in the Northern Regional Research Center Culture Collection (Peoria, IL, USA) as strain NRRL B-24326T and in the BCCM/LMG Bacteria Collection (Ghent, Belgium) as strain LMG 22738T.
Growth and sporulation of strain AAB-4T were observed on standard media (Table 1); aerial spore-mass colour, pigmentation of substrate mycelium and the production of diffusible pigments were recorded following incubation of the strain at 37 °C for 4–7 days. Peptone/yeast extract/iron agar and tyrosine agar (Shirling & Gottlieb, 1966) were used to score the production of melanin pigments.
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. Basal mineral salts agar (Hopwood, 1967) with 1 % (w/v) sole carbon sources was used to assess for carbon utilization. Antibiotic resistance was determined at 37 °C on YEME medium (Shirling & Gottlieb, 1966), using the disc method (Al-Tai et al., 1999). The ability of strain AAB-4T to inhibit the growth of different bacteria was detected by using the plug technique (Beur et al., 1966; Barry, 1980). Strain AAB-4T spores were inoculated onto Czapek–Dox agar plates and agar discs (8 mm) of a 7-day-old culture grown at 37 °C were transferred to nutrient agar plates (Shirling & Gottlieb, 1966) that had previously been seeded with different test organisms (as listed in Table 3). The plates were kept overnight at 4 °C and then incubated at 37 °C for 24 h. Tolerance of the novel strain to 0·2, 2, 4 and 7 % NaCl was determined on ISP9 medium (Shirling & Gottlieb, 1966).
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. The 16S rRNA gene was amplified from purified DNA of the strain as described previously (Petrosyan et al., 2003) using TripleMaster DNA polymerase (Eppendorf). The amplified fragment was purified from the gel by using a QIAquick Gel extraction kit (Qiagen) and then sequenced directly with an ABI Prism BigDye Terminator v2.0 Cycle Sequencing kit (Applied Biosystems). The sequences were obtained with a model 310 Genetic Analyser automated sequencer (Applied Biosystems).
For the identification of bis-(2-ethylhexyl)phthalate, mycelium from 7-day-old culture grown in Czapek–Dox broth (pH 8·0) (Shirling & Gottlieb, 1966) was separated by filtration and the culture filtrate was extracted twice with ethyl acetate. The organic fraction was evaporated under reduced pressure and the antibiotic was separated and purified by using chromatographic techniques. The pure compound was identified by using one-dimensional (1H and 13C) and two-dimensional NMR data.
The colonial morphology of strain AAB-4T was consistent with its assignment to the genus Streptomyces (Williams et al., 1989). The strain formed a highly branched substrate mycelium and aerial hyphae that differentiated into long Rectiflexibiles spore-chains. On standard media, the colour of the substrate mycelium was beige and that of the aerial spore mass was yellow (Table 1
). Strain AAB-4T contained LL-diaminopimelic acid, as determined according to the methodology of Staneck & Roberts (1974).
To confirm that strain AAB-4T was a streptomycete, we sequenced the almost-complete 16S rRNA gene from this micro-organism and compared it with the 16S rRNA gene sequences of previously described streptomycetes. Strain AAB-4T has a high percentage of nucleotide sequence similarity to Streptomyces thermoviolaceus NRRL B-12374T (98 %), Streptomyces thermodiastaticus NRRL B-5316T (98 %) and Streptomyces longisporus NRRL B-5336T (97 %). These values correspond to 20–30 nt differences out of 1421 positions. Nucleotide similarity values within this range have been reported for several Streptomyces species with validly published names and which can be separated from each other on the basis of DNA relatedness data and phenotypic properties (Kim et al., 1999, 2000).
The 16S rRNA gene sequence of strain AAB-4T generated in this work (1421 nt; GenBank accession no. AY750056) was aligned with the 16S rRNA gene sequences of other streptomycetes obtained from the EMBL/GenBank dataset. The alignment of the sequences was carried out using CLUSTAL W software (Thompson et al., 1994) and was adjusted manually. The alignment contained 1428 nt from 21 species. To determine which model of sequence evolution best fitted our dataset, a nested likelihood-ratio test was performed using the MODELTEST program, version 3.04 (Posada & Crandall, 1998). Phylogenetic relationships were inferred using the maximum-likelihood method (Felsenstein, 1981). Fifty random-taxon-addition heuristic searches with the tree bisection–reconnection branch-swapping option were conducted using PAUP* 4.0b10 software (Swofford, 2002). Genetic distances among species were estimated by using the Kimura method (Kimura, 1980). The relationships among taxa were also established by neighbour joining with the MEGA program (Kumar et al., 2001). The robustness of the neighbour-joining and maximum-likelihood trees was evaluated using bootstrapping with 1000 and 10 000 replicates, respectively (Felsenstein, 1981).
Nucleotide frequencies for the 16S rRNA gene sequence dataset were 0·225 (A), 0·255 (C), 0·336 (G) and 0·182 (T). The heterogeneity of nucleotide frequencies across taxa was tested using the ‘basefreq’ option implemented in PAUP* (
2=1·230, P=1·0). The result indicates that rRNA gene nucleotide frequencies were not significantly heterogeneous across taxa, which is advantageous because the maximum-likelihood inference method performs optimally when nucleotide frequencies are homogeneous (Omilian & Taylor, 2001). The likelihood-ratio test indicated that the best model to fit the 16S rRNA gene dataset was Tamura–Nei (TrN) (Tamura & Nei, 1993), with an equal rate of substitution and a proportion of invariable sites of 0·9035. A maximum-likelihood analysis using this model yielded a single tree with a –ln score of 2646·11. The maximum-likelihood tree showed that strain AAB-4T is a sister species of S. longisporus with a bootstrap value of 52 % (Fig. 1). The tree resulting from the neighbour-joining analysis yielded the same relationship between S. bangladeshensis and Streptomyces longisporus, with a low bootstrap value (34 %). The neighbour-joining dendrogram showed that the clade composed by both species was a sister clade to the species S. thermoviolaceus and S. thermodiastaticus (see Supplementary Figure, available in IJSEM Online).
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, strain AAB-4T differs from S. longisporus in spore colour, soluble pigment formation, growth on L-arabinose, fructose, xylose and sucrose, and, less strikingly, on glucose, glycerol, raffinose, galactose, melezitose and lactose as sole carbon sources. The differences between AAB-4T and S. thermodiastaticus and S. thermoviolaceus include such morphological traits as spore-chain shape and colour, pigmentation of substrate mycelium, and growth on rhamnose, m-inositol and lactose (for S. thermoviolaceus) and L-arabinose, D-xylose, mannitol, rhamnose, m-inositol and trehalose (for S. thermodiastaticus). The physiological properties of strain AAB-4T are presented in the species description below.
When grown in Czapek–Dox broth (pH 8·0) medium, strain AAB-4T produced the antimicrobial agent bis-(2-ethylhexyl)phthalate, previously isolated from Streptomyces melanosporofaciens (Kim et al., 1991). The compound was identified by 1H and 13C NMR and showed significant activity against Gram-positive and -negative bacteria and some fungi (Table 3). However, these two micro-organisms are different with respect to their phenotypic characteristics (Kim et al., 1990) and their 16S rRNA gene sequences (GenBank accesssion no. AJ391837).
The genotypic and phenotypic data suggest that strain AAB-4T should be recognized as a novel species of the genus Streptomyces, for which we propose the name Streptomyces bangladeshensis sp. nov.
Description of Streptomyces bangladeshensis sp. nov.
Streptomyces bangladeshensis (ban.gla.desh.en'sis. N.L. masc. adj. bangladeshensis belonging to Bangladesh, the source of the soil from which the organism was isolated).
Aerobic, Gram-positive, moderately thermophilic actinomycete. Forms highly branched substrate mycelium and aerial hyphae that differentiate into long Rectiflexibiles chains of eight to ten spores. Aerial spore-mass colour is yellow–green. Substrate mycelium is beige on standard media. Yellowish diffusible pigments are formed on Czapek–Dox agar. Melanin pigments are not produced on peptone/iron or tyrosine agars. Positive for H2S production. Utilizes glucose, sucrose, m-inositol, mannitol, mannose, maltose, fructose, L-arabinose, rhamnose, glycerol, raffinose and trehalose as sole carbon sources. Growth occurs at 20–50 °C, at pH 6·0–11·0 and in the presence of 2 % (w/v) NaCl, neomycin sulfate (50 µg ml–1) and penicillin (10 IU ml–1). Produces bis-(2-ethylhexyl)phthalate, an antimicrobial agent.
The type strain is AAB-4T (=LMG 22738T=NRRL B-24326T). Isolated from soil from Natore, Bangladesh.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTIntroductionREFERENCES |
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Anderson, A. S. & Wellington, E. M. H. (2001). The taxonomy of Streptomyces and related genera. Int J Syst Evol Microbiol 51, 797–814.[Abstract]
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