Streptomyces sannurensis sp . nov . , a new alkaliphilic member of the genus Streptomyces isolated from Wadi Sannur in Egypt

The taxonomic position of an actinomycete isolated from a soil sample collected from Wadi Sannur in Egypt was established using a polyphasic approach. The isolate, which was designated WS 51 T , was shown to have chemical and morphological properties typical of streptomycetes. An almost complete 16S rDNAgene sequence of the strain was generated and compared with corresponding sequences of representative streptomycetes. The resultant data confirmed the classification of the strain in the genus Streptomyces but also showed that it formed a distinct phyletic line within the 16S rDNAStreptomyces gene tree. The organism was most closely associated to the type strains of Streptomyces hygroscopicus, Streptomyces malaysiensis and Streptomyces yatensis but was readily separated from them using a range of phenotypic properties. It is proposed that strain WS 51 T (= CCTCC 001032 T = DSM 41834 T ) be classified in the genus Streptomyces as Streptomyces sannurensis sp. nov.


INTRODUCTION
The genus Streptomyces encompasses aerobic, Grampositive actinomycetes that form an extensively branched, substrate mycelium, aerial hyphae which typically differentiate into chains of spores, have LLdiaminopimelic acid but lack major characteristic sugars in whole-organism hydrolysates (wall chemotype 1 sensu: Lechevalier and Lechevalier, 1970) and contain DNA rich in guanine plus cytosine (Williams et al., 1989;Manfio et al., 1995).The taxon contains more than 600 validly described species, representatives of which have been assigned to lumpy groups based on 16S rDNAsequence data (Bull et al., 2005).It is evident that the genus Streptomyces remains underspeciated (Sembiring et al., 2000;Manfio et al., 2003) though several validly *Corresponding author.E-mail: hozzein29@yahoo.comdescribed species have become subjective synonyms of previously described species (Lanoot et al., 2002(Lanoot et al., , 2004;;Hatano et al., 2003;Kumar and Goodfellow, 2010).It is apparent that the description of Streptomyces species needs to be based on a combination of phenotypic and genotypic properties (Kim et al., 2004;Saintpierre-Bonaccio et al., 2004) and even on a multilocus phylogeny (Guo et al., 2008).
Novel streptomycetes are still in great demand as a source of new commercially significant bioactive compounds (Bérdy, 1995;Holtzel et al., 2003;Xu et al., 2004).In the course of a screening programme designed to isolate novel bioactive actinomycetes from environmental samples collected from Wadi Sannur in the Eastern Desert of Egypt, an actinomycete, designated WS 51, was isolated and provisionally assigned to the genus Streptomyces.The aim of the present study was to determine the taxonomic status of this isolate using genotypic and phenotypic procedures.The resultant data indicate that the organism should be classified as a new Streptomyces species for which the name Streptomyces sannurensis sp.nov. is proposed.

Isolation and maintenance of the organism
Strain WS 51 T was isolated from a soil suspension used to inoculate a medium A agar plate which was incubated at 28°C for 14 days; the soil sample was collected from the Eastern Desert of Egypt at Wadi Sannur.The isolation medium, which had been recommended by Sato et al. (1983) for the isolation of alkalitolerant and alkaliphilic microorganisms, contained (g/L) glucose, 10.0; peptone, 5.0; yeast extract, 5.0; K2HPO4, 1.0; MgSO4.7H20, 0.2; Na2CO3, 10 g; agar, 15; distilled water, 1 L; the sodium carbonate was sterilised separately and added to the basal medium; the resultant preparation was adjusted to pH 10.5 using NaHCO3-Na2CO3 buffer (Sambrook and Russell, 2001).A preliminary test was carried out to confirm its requirement for alkalinity.It was unable to grow below pH 7.0.The isolate was maintained on modified Bennett's agar (Jones, 1949) at pH 10.0 and as hyphae and spores in 20% glycerol at -20°C.

Chemotaxonomy
Biomass for the chemical analyses was prepared by growing the strain in shake flasks of medium A broth (Sato et al., 1983) at 200 rpm for 7 days at 28°C.Cultures were checked for purity, harvested by centrifugation, washed three times with distilled water and freeze-dried.Determination of the isomeric form of diaminopimelic acid and the whole-organism sugar was done as described by Hasegawa et al. (1983), and Staneck and Roberts (1974), respectively.Polar lipids and fatty acids were extracted and detected as previously described (Minnikin et al., 1977;Sutcliffe, 2000), while isoprenoid quinone were extracted, purified and identified by HPLC as described by Collins (1985).

Phenotypic tests
The organism was examined for a broad range of phenotypic tests using media and methods described by Williams et al. (1983); all media were adjusted to pH 10.0 and incubated at 28°C for 14 days.

16S rDNA sequencing and phylogenetic analysis
Biomass for the 16S rDNA sequencing study was prepared as described previously for chemotaxonomy.Genomic DNA was extracted from the biomass as described by Orsini and Romano-Spica (2001).PCR-mediated amplification of 16S rDNA, purification of the PCR products and sequencing of the products were carried out following the procedure of Cui et al. (2001).The resultant 16S rDNAgene sequence was aligned manually against available corresponding streptomycete sequences retrieved from the DDBJ, EMBL and GenBank databases.Phylogenetic trees were inferred for the test strain and its nearest neighbours using the leastsquares (Fitch and Margoliash, 1967), maximum-likelihood (Felsenstein, 1981) and neighbour-joining (Saitou and Nei, 1987) tree-making algorithms from the PHYLIP suite of programs (Felsenstein, 1993).The distance model of Jukes and Cantor (1969) was used to generate an evolutionary distance matrix.The resultant tree topologies were evaluated by bootstrap analyses of the neighbour-joining method based on 1000 resamplings using the SEQBOOT and CONSENSE programs from the PHYLIP package (Felsenstein, 1993).The Clustal X program version 1.8 (Thompson et al., 1997) was used for the multiple alignment and the phylogenetic analyses.

DNA base composition
The DNA base composition of genomic DNA isolated from strain WS 51 T according to the procedure described by Hopwood et al. (1985) was determined using the thermal denaturation method (Mandel and Marmur, 1968) and a Shimadzu UV-visible spectrophotometer.

RESULTS AND DISCUSSION
There are some studies on the alkaliphilic actinomycetes (Mikami et al., 1982;Groth et al., 1997;Jones et al., 1998;Hozzein et al., 2004), few of them were on their applications, as production of alkaline enzymes (Horikoshi, 1999), bioactive substances (Tsujibo et al., 1990) and enzyme inhibitors (Bahn et al., 1998).But few reports were found on description of new species of the genus Streptomyces (Li et al., 2005;Dastager et al., 2008), which is very important for discovery of novel secondary metabolites.
The classification of the organism in the genus Streptomyces was underpinned by the nucleotide (nt) sequence data analysis which showed that it forms a distinct phyletic line in the 16S rDNAStreptomyces gene tree.Comparison of the almost complete 16S rDNAgene sequence of strain WS 51 T (1494 nt) with those of its  (Saitou and Nei, 1987) showing relationship between strain WS 51 T and its closest relatives; and representatives of the genus Streptomyces based on almost complete 16S rDNA sequences.The asterisks denote branches that were also recovered using the least-squares (Fitch and Margoliash, 1967) and maximum-likelihood (Felsenstein, 1981) treeing algorithms.The numbers at the nodes indicate the level of bootstrap support (%) based on a neighbour-joining analysis of 1000.
closest relatives and representatives of the genus Streptomyces is shown in Figure 1.The organism is most closely, but nevertheless loosely, associated with the type strains of Streptomyces yatensis (97.6%),Streptomyces malaysiensis (97.3%) and Streptomyces hygroscopicus (97.1%).DNA:DNA relatedness studies  were not carried out between these strains as representatives of other Streptomyces species with similarly low 16S rDNAsequence similarities show DNA relatedness values well below 80% (Sembiring et al., 2000;Kim and Goodfellow, 2002;Manfio et al., 2003), the cut-off point recommended for the recognition of genomic species of Streptomyces (Labeda and Lyons, 1992;Labeda, 1993Labeda, , 1998)).Strain WS 51 T can also be distinguished from its three closest phylogenetic neighbours using a range of phenotypic properties, notably by using key morphological features (Table 1).Indeed, S. hygroscopicus, S. malaysiensis and S. yatensis all belong to the Streptomyces violaceusniger clade, a well circumscribed group of streptomycetes (Sembiring et al., 2000;Saintpierre et al., 2003).The cultural characteristics of the tested strain are shown in Table 2.
It is evident that strain WS 51 T can be distinguished from representatives of closely related validly described Streptomyces species using a combination of genotypic and phenotypic properties.It is, therefore, proposed that this organism be given species status in the genus Streptomyces as S. sannurensis sp.nov.

Description of S. sannurensis sp. nov.
S. sannurensis (san.nur.en'sis.N.L. max.sannurensis) pertaining to Sannur, a valley (wadi) in the Eastern Desert of Egypt, the source of the isolate.Aerobic, nonacid fast, Gram-positive actinomycete which forms an extensively branched substrate mycelium that carries abundant aerial hyphae which differentiate into long, open-looped chains of smooth-surfaced spores.The colour of the aerial spore mass varies from white to yellowish white to reddish brown and that of the substrate mycelium from yellowish brown/grayish yellow to reddish brown.A yellowish brown soluble pigment is formed on modified Bennett's agar.It grows in condition of temperature ranging from 10 to 45°C and pH from 7.0 to 12.0, though growth is scant at the lowest temperature and pH values.It grows optimally at pH 9.5 to 10 and temperature range of 28 to 30°C.

Figure 1 .
Figure1.Neighbour-joining tree(Saitou and Nei, 1987) showing relationship between strain WS 51 T and its closest relatives; and representatives of the genus Streptomyces based on almost complete 16S rDNA sequences.The asterisks denote branches that were also recovered using the least-squares(Fitch and Margoliash, 1967) and maximum-likelihood(Felsenstein, 1981) treeing algorithms.The numbers at the nodes indicate the level of bootstrap support (%) based on a neighbour-joining analysis of 1000.

Table 1 .
Characters distinguishing strain WS 51 T from the type strains of phylogenetically closely related Streptomyces species.

Table 2 .
Cultural characteristics of strain WS 51 T .