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Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, AP 70228, UNAM, 04510, México DF, Mexico
Correspondence
María Elena Flores
mefc{at}servidor.unam.mx
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ABSTRACT |
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The GenBank accession number for the 16S rDNA sequence of Streptomyces mexicanus CH-M-1035T is AF441168.
A fuller phylogenetic tree showing the placement of Streptomyces mexicanus CH-M-1035T within the genus Streptomyces (over 100 species included) can be found in IJSEM Online (http://ijs.sgmjournals.org).
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). Currently, more attention is paid to the thermophilic streptomycetes, due to their potential importance in microbial technology. These micro-organisms grow well at 50 °C (Kim et al., 1999), and can be distinguished from mesophilic streptomycetes on the basis of numerical phenotypic analyses (Goodfellow et al., 1987; O'Donnell et al., 1993). However, the thermophilic streptomycetes fall into distinct evolutionary clades, upon analyses based on 16S rDNA sequence data (Kim, D. et al., 1996; Kim, S. B. et al., 1998; Kim, B. et al., 1999).
During a routine screening of strains isolated from a sugar-cane field in the State of Morelos, Mexico, an actinomycete producing thermostable xylanolytic enzymes was isolated. This strain, designated CH-M-1035T, was shown to present a colonial morphology consistent with its assignment to the genus Streptomyces (Flores et al., 1996, 1997). In the present investigation, strain CH-M-1035T was the subject of a polyphasic taxonomic study, to establish whether it represented a novel species of the genus Streptomyces.
Strain CH-M-1035T was isolated on minimal medium containing 2 g KH2PO4 l-1, 1·4 g (NH4)2SO4 l-1 and 5 g oat spelt xylan l-1 (pH 6·5). The strain was maintained on minimal medium agar containing oat spelt xylan, as described previously (Flores et al., 1997), at 4 °C and as a spore suspension in YEME supplemented with 40 % (v/v) glycerol (Hopwood et al., 1985) at -20 °C. Strain CH-M-1035T has been deposited in the DSMZ culture collection (Braunschweig, Germany) as strain DSM 41796T, in the Industrial Culture Collection UNAM-48/WFCC (Mexico DF, Mexico) as strain BM-B-384T and in the Northern Regional Research Center culture collection (Peoria, IL, USA) as strain NRRL B-24196T.
Growth and sporulation of strain CH-M-1035T 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 7 days and, for some media, at 50 °C for 4 days. Peptone/yeast extract/iron agar and tyrosine agar (Shirling & Gottlieb, 1966) were used to score for the production of melanin pigments. Spore-chain morphology and spore-surface ornamentation were examined by scanning and transmission electron microscopy of 5-day-old cultures grown on minimal medium with 1 % (w/v) fructose. Samples were processed according to Kellenberger's technique (Nun, 1975). Whole-cell carbohydrates of the strain and the isomeric form of the diaminopimelic acid were determined according to Staneck & Roberts (1974).
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. The degradation tests were carried out at 37 °C as described by Williams et al. (1983). Growth on sole carbon sources was examined on carbon utilization medium as recommended by Shirling & Gottlieb (1966). Antibiotic resistance was determined at 37 °C on modified Bennett's agar (MBA) by the disc method (Al-Tai et al., 1999). The ability of strain CH-M-1035T to inhibit the growth of three bacterial, one yeast, one yeast-like fungus and two fungal strains was detected by using the plug technique. The bacterial, yeast and fungal strains were inoculated onto Difco nutrient, Sabouraud glucose and potato glucose agar plates, respectively. Agar discs (8 mm) of a 7-day-old culture of strain CH-M-1035T that had been grown on MBA at 37 °C were placed centrally onto the seeded cultures and the preparations were then incubated at 37 °C for 24 h (bacteria), 29 °C for 48 h (yeast) and 29 °C for 5 days (fungi). Growth of strain CH-M-1035T at temperatures between 5 and 60 °C was determined on standard media (Shirling & Gottlieb, 1966). Tolerance of the novel strain to NaCl was determined on MBA at concentrations up to 13 %. The pH requirements of the strain were tested on sodium-phosphate-buffered MBA and on minimal agar medium containing 2·64 g (NH4)2SO4 l-1, 1·00 g MgSO4.7H2O l-1 and 1·00 g KCl l-1 supplemented with 1 % fructose (sterilized separately) and corresponding buffers (O'Donnell et al., 1993). In the case of minimal medium, the buffering components were filter- or autoclave-sterilized separately to avoid chemical modifications and mixed into the cooled agar medium immediately before pouring. When necessary, K2HPO4 was added to the media to a final concentration of 0·07 % as a phosphate supplement.
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. The 16S rRNA gene of strain CH-M-1035T was amplified from purified DNA using the primers 5'-AGAGTTTGATCCTGGCTCAG-3' and 5'-GGTTACCTTGTTACGACTT-3' (Weisburg et al., 1991), and Pwo DNA polymerase (Roche Molecular Biochemicals). The PCR conditions used were: an initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 1·5 min, annealing at 57 °C for 1·5 min and extension at 72 °C for 2 min, with a final extension at 72 °C for 10 min. The amplified fragment was purified by gel electrophoresis and cloned into pMOSBlue (Amersham Pharmacia Biotech). Randomly picked individual clones were sequenced with universal primers T7 and -21, and with internal primers, using an ABI Prism BigDye Terminator Cycle Sequencing kit (Applied Biosystems). The sequences were obtained with a model 310 Genetic Analyzer automated sequencer (Applied Biosystems).
The 16S rDNA sequence of strain CH-M-1035T generated in this work (1450 nt; GenBank accession no. AF441168) was aligned with the 16S rDNA sequences of other streptomycetes retrieved from the EMBL/GenBank dataset using the PILEUP algorithm included in the GCG software package, version 7 (Devereux et al., 1984); the resulting alignment was adjusted manually. The sequences corresponding to the primers used for amplification of the 16S rRNA gene were omitted from the alignment. The resulting alignment contained 1303 nt from 102 species. Phylogenetic analysis of strain CH-M-1035T with its nearest neighbours was carried out using PAUP*, version 4.0b10 (Swofford, 2002). To determine which model of sequence evolution best fitted our dataset, a nested likelihood ratio test was performed using MODELTEST, version 3.04 (Posada & Crandall, 1998). Phylogenetic relationships were inferred using the maximum-likelihood approach (Felsenstein, 1981). Ten random taxon addition heuristic searches with tree bisection–reconnection (TBR) branch swapping were conducted. The robustness of the inferred tree was evaluated using 1000 bootstrap replicates (Felsenstein, 1985).
The colonial morphology of strain CH-M-1035T was consistent with its assignment to the genus Streptomyces (Williams et al., 1989). It formed a highly branched substrate mycelium and aerial hyphae which differentiated into long Rectiflexibiles chains carrying 10 or more rounded (mean diameter 0·88 µm), smooth-surfaced spores (Fig. 1). The spores were green upon examination under a light microscope. The colour of the substrate mycelium was generally beige or appeared yellow-brown when modified with soluble pigments, the aerial spore mass was grey-coloured with an appreciable greenish tint, and light yellow diffusible pigments were produced on several standard media (Table 1). Strain CH-M-1035T contained LL-diaminopimelic acid but no characteristic sugars were found in the strain.
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) that strain CH-M-1035T was closely related to the thermophilic streptomycetes that belong to the Streptomyces thermodiastaticus clade (Kim et al., 1998). Strain CH-M-1035T shares high percentage nucleotide sequence similarity values with Streptomyces thermocoprophilus B19T (=DSM 41700T; 98·6 %), Streptomyces thermoviolaceus subsp. thermoviolaceus DSM 40443T (98·7 %), S. thermodiastaticus JM 4840 (99·2 %) and S. thermoviolaceus subsp. apingens DSM 41392T (99·2 %). These values correspond to 11–20 nt differences out of 1450 nt positions. Nucleotide similarity values within this range have been reported for several described species of Streptomyces (Kim et al., 1999, 2000) which can be separated from each other on the basis of DNA relatedness data and phenotypic properties (Goodfellow et al., 1987; Kim et al., 1999, 2000). A fuller phylogenetic tree showing the placement of strain CH-M-1035T within the genus Streptomyces (over 100 species included) can be found as supplementary data in IJSEM Online (http://ijs.sgmjournals.org).
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), on the basis of a numerical classification using the simple matching coefficient (Goodfellow et al., 1987). The numerical analysis showed that strain CH-M-1035T was phenotypically most closely related to S. thermocoprophilus B19T, another xylanolytic organism (Kim et al., 2000). However, strain CH-M-1035T and S. thermocoprophilus B19T could be separated on the basis of differences in melanin production, growth temperature, tolerance to NaCl, utilization of carbon sources and substrate degradation (Table 2). Moreover, separation of these two strains, as well as of strain CH-M-1035T from other members of the clade, in the 16S-rDNA-based phylogenetic analysis was supported by significant bootstrap values (Fig. 2). The genotypic and phenotypic data suggest that strain CH-M-1035T should be recognized as a novel species of the genus Streptomyces, for which we propose the name Streptomyces mexicanus.
Strain CH-M-1035T grew well on simple minimal media containing ammonium sulphate, potassium phosphate and a wide range of carbohydrates; it is also important to mention that strain CH-M-1035T grows well with xylan as sole carbon source (Flores et al., 1997). The novel strain grew well up to 55 °C, although it did not form spores at elevated temperatures. Growth of strain CH-M-1035T occurred in media with pH values between 4·3 and 8·0, and in the presence of 6 % (w/v) NaCl. Strain CH-M-1035T was resistant to ampicillin, but sensitive to erythromycin, kanamycin, gentamicin, thiostrepton, tetracycline, nalidixic acid, chloramphenicol and rifampicin. It did not produce antibiotics that were active against Escherichia coli JM 109, ‘Sarcina lutea’ NRRL-B-1018, Bacillus subtilis ATCC 6633, Aureobasidium sp. CH-M-1018, Aspergillus flavipes ATCC 16795T, Trichoderma viridae or Pichia pastoris GS 115.
Description of Streptomyces mexicanus sp. nov.
Streptomyces mexicanus (mex.i.ca'nus. N.L. adj. mexicanus belonging to Mexico, 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 which differentiate into long Rectiflexibiles chains of 10 or more green, smooth spores. Spores are spherical, about 0·88 µm in diameter, or slightly oval. Aerial spore-mass colour is grey with a slight green tint. Substrate mycelium is beige on standard media. Yellowish diffusible pigments are formed on yeast-extract, malt-extract and oatmeal agar. Melanin pigments are not produced on peptone/iron or tyrosine agars. Positive for H2S production. Degrades arbutin, starch, xylan, adenine, casein, hypoxanthine and L-tyrosine, but not guanine or testosterone. Utilizes L-arabinose, fructose, glucose, raffinose, mannitol, mannose, xylose, galactose, maltose, glycerol, lactose, cellobiose, trehalose and sodium acetate as sole carbon source, but not meso-inositol, melezitose, L-rhamnose, sorbitol or sucrose. Growth occurs between 20 and 55 °C and from pH 4·3 to 8·0, and in the presence of 6 % (w/v) NaCl and ampicillin (100 µg ml-1). Growth is inhibited in the presence of chloramphenicol, erythromycin, gentamicin, nalidixic acid sodium salt, kanamycin sulphate, rifampicin, tetracycline hydrochloride and thiostrepton, and in the presence of 7 % (w/v) NaCl. No antimicrobial activity is shown against Escherichia coli JM 109 or against representative strains of Aspergillus flavipes, Aureobasidium sp., Bacillus subtilis, Pichia pastoris, 'Sarcina lutea' or Trichoderma viridae. Isolated from a soil sample obtained at a sugar-cane field in the State of Morelos, Mexico. The type strain is CH-M-1035T (=DSM 41796T =BM-B-384T =NRRL B-24196T).
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ACKNOWLEDGEMENTS |
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