Streptomyces brevispora sp. nov. and Streptomyces laculatispora sp. nov., actinomycetes isolated from soil

The genus Streptomyces encompasses nearly 600 described species (Euzéby, 2011), members of which are the source of a broad range of secondary metabolites, notably antibiotics (Bérdy, 2005; Goodfellow & Fiedler, 2010). The subgeneric classification of the genus, while complex, is being improved by the application of genotypic and phenotypic procedures (Lanoot et al., 2005; Goodfellow et al., 2007; Rong & Huang, 2010). These procedures are also effective in the reassignment of misclassified species of the genus Streptomyces (Tamura et al., 2008; Kumar & Goodfellow, 2010) and in the recognition of new members of the taxon isolated from diverse natural habitats (Semêdo et al., 2004; Xu et al., 2006; Xiao et al., 2009; Zhao et al., 2010).

The present investigation was designed to establish the taxonomic positions of two Streptomyces strains isolated from a hay meadow soil sample. A polyphasic taxonomic study based on a combination of genotypic and phenotypic procedures showed that isolates BK160^T and BK166^T should be recognized as two novel species of the genus Streptomyces, Streptomyces brevispora sp. nov. and Streptomyces laculatispora sp. nov., respectively.

Strains BK160^T and BK166^T were isolated on starch-casein agar (Küster & Williams, 1964), supplemented with cycloheximide and nystatin (at 25 µg ml⁻¹), which had been inoculated with a pre-heated (55 °C for 20 min) soil suspension and incubated at 28 °C for 21 days. The soil sample was collected from Palace Leas meadow hay plot 6 (Atalan et al., 2000) at Cockle Park Experimental Farm, Northumberland, UK (National Grid reference NZ 200913). The organisms were maintained on oatmeal agar slopes (International Streptomyces Project - ISP medium 3; Shirling & Gottlieb, 1966) at 4 °C and as mixtures of mycelial fragments and spores in 20 % (v/v) glycerol at −80 °C. Biomass for the chemotaxonomic and molecular systematic studies was grown in shake flasks of tryptone-yeast extract broth (ISP medium 1; Shirling & Gottlieb, 1966) for 7 days at 28 °C, harvested by centrifugation and washed twice in distilled water; cells for the chemotaxonomic studies were freeze-dried.

Genomic DNA was extracted from the isolates and PCR amplification and 16S rRNA gene sequencing achieved using procedures described previously (Kim et al., 1996). The resultant, almost complete 16S rRNA gene sequences (1439 to 1457 nt) were aligned manually against corresponding sequences of representatives of the genus Streptomyces using mega 4 software (Tamura et al., 2007). Phylogenetic trees were inferred by using the maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Fitch, 1971) and neighbour-joining (Saitou & Nei, 1987) tree-making algorithms drawn from the mega 4 (Tamura et al., 2007) and phyml (Guindon & Gascuel, 2003) packages. The Jukes & Cantor (1969) model was used to generate evolutionary distance matrices for the neighbour-joining data. Topologies of the resultant trees were evaluated by bootstrap analysis (Felsenstein, 1985) of the neighbour-joining method based upon 1000 replicates using mega 4 software. Kitasatospora griseola JCM 3339^T (GenBank accession no. U93320) was used as the outgroup.

Isolates BK160^T and BK166^T formed a distinct subclade in the Streptomyces 16S rRNA gene tree together with the type strain of Streptomyces drozdowiczii, a taxon which was supported by all tree-making algorithms and by a bootstrap value of 74 % (Fig. 1). The isolates shared a 16S rRNA gene sequence similarity of 99.9 %, a value which corresponded to 2 nt differences over 1434 locations. Isolate BK166^T is the strain that is most closely related to S. drozdowiczii NRRL B-24297^T; the two organisms share a 16S rRNA gene similarity of 99.4 %, a value equivalent to 7 nt differences over 1434 locations. Similarly, isolate BK160^T shares a 16S rRNA similarity of 99.4 % with the type strain of S. drozdowiczii, a value equivalent to 9 nt differences over 1439 sites. The S. drozdowiczii subclade is loosely associated with the Streptomyces niveus subclade, which also contains the type strains of Streptomyces laceyi and Streptomyces spheroides; the latter two species have been considered to be synonyms of S. niveus (Tamura et al., 2008).

DNA–DNA relatedness values between the isolates and between them and the type strain of S. drozdowiczii were determined using the nitrocellulose membrane filter hybridization procedure described by Seldin & Dubnau (1985). DNA probes were labelled using the non-radioactive digoxigenin (DIG) High Prime System (Roche), hybridized DNA visualized using a DIG luminescent detection kit (Roche), and DNA–DNA relatedness quantified using a densitometer (Bio-Rad). The resultant data showed that all of the strains belong to different genomic species as they shared DNA–DNA relatedness values between 25.6 and 54.5 % (Table 1); values well below the 70 % cut-off point recommended for the assignment of strains to the same genomic species (Wayne et al., 1987). The two most closely related strains, isolates BK160^T and BK166^T, shared a DNA–DNA relatedness value of 54.5 %.

Isolates BK160^T and BK166^T were examined for chemical markers considered to be characteristic of strains of the genus Streptomyces. Standard procedures were used to determine the isomers of diaminopimelic acid (Hasegawa et al., 1983), predominant menaquinones (Collins, 1985), muramic acid type (Uchida et al., 1999) and diagnostic whole-cell sugars (Hasegawa et al., 1983), using appropriate controls. Fatty acids from the isolates and from S. drozdowiczii NRRL B-24297^T were extracted, methylated and analysed by GC using the standard Sherlock Microbial Identification (MIDI) system, version 5 (MIDI, 1999; Sasser, 1990). The DNA base composition of the isolates was determined using the procedure described by Gonzalez & Saiz-Jimenez (2002).

Isolates BK160^T and BK166^T contained major amounts of ll-diaminopimelic acid but lacked diagnostic sugars in whole-organism hydrolysates (wall chemotype I sensu Lechevalier & Lechevalier, 1970), contained N-acetylated muramic acid, and containined hexa- and octahydrogenated menaquinones with nine isoprene units [MK-9 (H₆, H₈)] as predominant isoprenologues in a ratio of 5 : 1 and 4 : 2, respectively. The cellular fatty acid profiles consisted mainly of saturated straight-chain and iso- and anteiso-branched-chain components (Table 2), hence all three strains belong to fatty acid type 2c sensu Kroppenstedt (1985); the fatty acid composition of the S. drozdowiczii strain was found to be similar to that recorded by Semêdo et al. (2004). The DNA G+C content of each of the isolates was 69.2 %. All of these properties support the classification of the strains in the genus Streptomyces (Williams et al., 1983; Manfio et al., 1995; Anderson & Wellington, 2001).

The isolates were examined for cultural and morphological features following growth on several standard media for three weeks at 28 °C. Cultural properties were sought using glucose-yeast extract-malt extract (GYM; DSMZ medium 65) and modified Bennett’s (Jones, 1949) agars, and on oatmeal, inorganic salts-starch, glycerol-asparagine and tyrosine agars (ISP media 3, 4, 5 and 7; Shirling & Gottlieb, 1966). Spore arrangement and spore surface ornamentation were observed by examining gold-coated, dehydrated preparations from the ISP 3 cultures, using a scanning electron microscope (Cambridge Stereoscan 240 instrument) and the procedure described by O’Donnell et al. (1993).

Isolate BK160^T formed a branched white substrate mycelium and sparse white aerial hyphae which differentiated into straight to flexuous (Rectiflexibilis) chains of smooth-surfaced spores on oatmeal agar (Fig. 2a). The organism grew well on ISP 7, did not grow on GYM agar and grew poorly on all of the other media. Similarly, isolate BK166^T produced an extensive branched light-brown substrate mycelium and abundant greyish aerial hyphae which differentiated into straight to flexuous chains of square-like smooth-surfaced spores on oatmeal agar (Fig. 2b). This organism grew well on all remaining media producing light-brown soluble pigments on ISP 3, ISP 5, ISP 7 and modified Bennett’s agars.

The isolates and the type strain of S. drozdowiczii were examined for a range of phenotypic properties using media and methods described by Williams et al. (1983). It can be seen from Table 2 that strains BK160^T and BK166^T can be readily distinguished from one another and from S. drozdowiczii NRRL B-24297^T using a combination of phenotypic properties and by quantitative differences in fatty acid composition (Table 2). Thus, isolate BK166^T, unlike BK160^T, hydrolysed allantoin, grew on l–arabinose and l-rhamnose as sole carbon sources, and was tolerant of kanamycin sulphate (30 µg ml⁻¹) and streptomycin sulphate (16 µg ml⁻¹). Unlike the two isolates, the type strain of S. drozdowiczii hydrolysed pectin and degraded cellulose, but not xanthine or xylan. Additional phenotypic properties are cited in the species descriptions.

In conclusion, the genotypic and phenotypic data show that isolates BK160^T and BK166^T represent two novel species of the genus Streptomyces. It is, therefore, proposed that these organisms be recognized as Streptomyces brevispora sp. nov. and Streptomyces laculatispora sp. nov., respectively.