Streptomyces synnematoformans sp. nov., a novel actinomycete isolated from a sand dune soil in Egypt

Novel streptomycetes remain a focus of attention as a unique source of new secondary metabolites (Dietera et al., 2003; Baur et al., 2006), including structurally diverse antibiotics isolated from marine streptomycetes (Fiedler et al., 2005). A consequence of this interest is that the number of species assigned to the genus Streptomyces Waksman and Henrici 1943 continues to rise (Al-Bari et al., 2005; Kim et al., 2006); the genus currently encompasses over 500 species with validly published names (Euzéby, 1997). Most of the recently described Streptomyces species have been delineated using polyphasic taxonomic approaches (Bouchek-Mechiche et al., 2006; Kim et al., 2006; Xu et al., 2006).

An interesting organism was isolated during a routine screening programme for novel bioactive actinomycetes from environmental samples from different regions of Egypt. The strain, designated S155^T, had a colonial morphology typical of members of the genus Streptomyces. The aim of the present investigation was to establish the taxonomic position of this isolate using a combination of genotypic and phenotypic procedures. It is apparent from the results that the organism should be recognized as representing a novel species of the genus Streptomyces.

Strain S155^T was isolated on an M1 agar plate (Mincer et al., 2002) which had been inoculated with a soil suspension and incubated at 28 °C for 2 weeks. The soil sample had been collected from a sand dune at Borg El-Arab in Egypt. The organism was maintained on modified Bennett's agar slopes (Jones, 1949) and as glycerol suspensions of hyphae and spores (20 %, v/v) at −20 °C. Biomass for chemotaxonomic and molecular systematic studies was grown in shake flasks of modified Bennett's broth (Jones, 1949) at 28 °C for 7 days, harvested by centrifugation and washed twice with distilled water. Biomass for chemotaxonomic studies was freeze-dried and that for the molecular systematic work was stored at −20 °C.

Extraction of chromosomal DNA, PCR-mediated amplification of the 16S rRNA gene and direct sequencing of the purified products were carried out using a standard procedure (Kim et al., 2000). The resultant 16S rRNA gene sequence was aligned manually with corresponding sequences of the type strains of available Streptomyces species retrieved from the DDBJ/EMBL/GenBank databases and then with representatives of phylogenetically close Streptomyces species highlighted by using blast (Altschul et al., 1997). Multiple alignment and phylogenetic analysis were achieved using the clustal_x 1.8 program (Thompson et al., 1997) and mega version 3.1 software (Kumar et al., 2004); phylogenetic trees were inferred using the least-squares (Fitch & Margoliash, 1967), maximum-parsimony (Kluge & Farris, 1969) and neighbour-joining (Saitou & Nei, 1987) tree-making algorithms. Evolutionary distance matrices were generated for the least-squares and neighbour-joining methods after Jukes & Cantor (1969) and the unrooted tree topologies were evaluated by bootstrap analysis (Felsenstein, 1985) of the neighbour-joining dataset based on 1000 resamplings. The root position of the neighbour-joining tree was estimated using Streptomyces scabrisporus KM-4927^T as the outgroup. It was evident from the comprehensive phylogenetic analyses that strain S155^T formed a distinct phyletic line towards the periphery of the Streptomyces 16S rRNA gene tree (data not shown).

The isolate was examined for a range of chemotaxonomic and morphological properties considered to be characteristic of the genus Streptomyces. Colonial features, notably aerial spore mass colour, substrate mycelial pigmentation and the colour of diffusible pigments, were recorded on glycerol-asparagine agar (ISP medium 5), inorganic salts-starch agar (ISP medium 4), oatmeal agar (ISP medium 3), tryptone-yeast extract agar (ISP medium 1) and yeast extract-malt extract agar (ISP medium 2), as described by Shirling & Gottlieb (1966), and on modified Bennett's agar (Jones, 1949) and nutrient agar (Waksman, 1961); all plates were incubated at 28 °C for 2 weeks. Spore arrangement and spore surface ornamentation were observed by examining gold-coated, dehydrated preparations from a 14-day-old culture grown on modified Bennett's agar (Jones, 1949) at 28 °C, using a Cambridge Stereoscan 240 scanning electron microscope and the procedure described by O'Donnell et al. (1993). Standard chromatographic procedures were used to extract and analyse the isomeric forms of diaminopimelic acid (Hasegawa et al., 1983), fatty acids (Sutcliffe, 2000), isoprenoid quinones (Minnikin et al., 1984; Collins, 1985), polar lipids (Minnikin et al., 1984) and whole-organism sugars (Staneck & Roberts, 1974).

The chemical and morphological properties of the isolate are in line with its classification in the genus Streptomyces. The organism forms an extensively branched substrate mycelium. At maturity, when adequate aerial mycelium is produced, the aerial hyphae differentiate into short, straight to flexuous chains of smooth-surfaced spores. Synnemata-like structures were formed from hyphae that wrapped together to form long ropes (see Supplementary Fig. S1 in IJSEM Online). Sclerotia, zoospores or sporangia were not observed. Growth was moderate to abundant on all tested media. Aerial mycelium was light greyish red to blackish red and the substrate hyphae varied from dark red to reddish black. No diffusible pigments were produced except on inorganic salts-starch agar medium (ISP medium 4) (deep greyish red).

The strain contained major amounts of ll-diaminopimelic acid, glucose and galactose, but no diagnostic sugars, in whole-organism hydrolysates (wall chemotype I sensu Lechevalier & Lechevalier, 1970), tetra-, hexa- and octahydrogenated menaquinones as major isoprenologues (menaquinone type 4b sensu Kroppenstedt, 1985) and a polar lipid pattern consisting of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides and unidentified phospholipids (phospholipid type II sensu Lechevalier et al., 1977). The fatty acid profile was composed mainly of saturated straight-chain and iso- and anteiso-branched-chain components. The major fatty acid components were iso-C_16 : 0, anteiso-C_15 : 0, iso-C_14 : 0, anteiso-C_17 : 0, iso-C_15 : 0 and iso-C_15 : 0 2-OH (fatty acid type 2c sensu Kroppenstedt, 1985). This chemical profile and the morphological properties of strain S155^T are clearly consistent with its assignment to the genus Streptomyces (Williams et al., 1989; Manfio et al., 1995).

It is evident from Fig. 1⇓ that strain S155^T forms a distinct phyletic line. It is most closely related to the type strains of Streptomyces thermoalcalitolerans (97.1 %), Streptomyces ruber (97 %), Streptomyces rubiginosus (97.0 %) and Streptomyces roseiscleroticus (96.9 %); it also shares 96.8 % 16S rRNA similarity with the type strains of Streptomyces cellulosae, Streptomyces lavendulae subsp. lavendulae, Streptomyces ochraceiscleroticus and Streptomyces rimosus subsp. rimosus. DNA–DNA relatedness studies were not carried out between strain S155^T and its closest phylogenetic neighbours as representatives of Streptomyces species with high 16S rRNA gene sequence similarities are known to share low DNA–DNA relatedness (Kim & Goodfellow, 2002; Manfio et al., 2003; Kim et al., 2006; Xu et al., 2006), that is, values well below the 70 % cut-off point recommended for the delineation of genomic species (Wayne et al., 1987).

Standard media and methods were used to determine the ability of the organism to degrade organic compounds (Williams et al., 1983) and to utilize various sole carbon sources (Shirling & Gottlieb, 1966). Similarly, growth and tolerance properties were recorded on modified Bennett's agar following incubation for 14 days, as described by Gordon & Mihm (1962). Gram (Hucker's modification; Society of American Bacteriologists, 1957) and Ziehl–Neelsen (Gordon, 1967) stains were carried out on cells grown on modified Bennett's agar for 7–21 days at 28 °C.

It is evident from Table 1⇓ that strain S155^T can be distinguished from the type strains of its most immediate phylogenetic neighbours of the genus Streptomyces by using a combination of phenotypic properties. Additional phenotypic properties are cited in the species description. In this regard, the morphological features and degradation of organic compounds were very significant in distinguishing strain S155^T from its phylogenetic neighbours. It was reported in previous studies that differential morphological and pigmentation features are especially significant for the delineation of members of phylogenetically related Streptomyces species (Labeda & Lyons, 1991; Labeda et al., 1997; Kim et al., 2000; Manfio et al., 2003).

It is clear from the genotypic and phenotypic data that strain S155^T should be recognized as the type strain of a novel species in the genus Streptomyces. The name proposed for this taxon is Streptomyces synnematoformans sp. nov.