Bioactive potential of a new strain of Streptomyces sp. PP14 isolated from Canadian soil

1 Laboratoire de Biologie des Systèmes Microbiens (LBSM), Ecole Normale Supérieure de Kouba, B.P. 92, 16 050 Vieux-Kouba, Algiers, Algeria. 2 Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, Alberta, T6G 2P5, Canada. 3 Département de Biologie, Faculté des sciences Agronomiques, Vétérinaires et Biologiques, Université Saâd Dahleb, Blida, Algeria. 4 Field Crop Development Centre, Alberta Agriculture and Rural Development, 6000 C & E Trail, Lacombe, Alberta, T4L 1W1, Canada.


INTRODUCTION
Actinomycetes, filamentous bacteria that naturally inhabit soils, continue to serve as an important source of various kinds of antimicrobial molecules.It has been estimated that approximately two-thirds of natural antibiotics have been isolated from these mycelial bacteria, and about 75% are produced by members of the genus *Corresponding author.E-mail: noureddine_bouras@yahoo.fr.Tel: + 213 29 75 11.Fax: + 213 28 20 67.
Streptomyces (Buckingham, 1997;Newman et al., 2003).The search for new antibiotics continues to be of greatest importance in screening programs around the world because of the increase of resistant pathogenic bacteria and toxicity of some used antibiotics (Berdy, 1989).As with every screening program, the probability of finding bioactive molecules with desired properties depends on the number and diversity of strains isolated and screened (Mazza et al. 2003).Moreover, one of the strategies for enhancing the likelihood of obtaining particular strains and secondary bioactive metabolites is to analyze uncommon ecosystems which exist under extreme conditions and to consider geographical regions that have been poorly studied in the past.
Canadian soils are exposed to an extreme climate and could represent particular ecosystems.Extreme environments have been found by many investigators to be inhabited by microorganisms which are especially adapted to these ecological niches (Sabaou et al., 1998).Many of these organisms may represent new taxa and thus can provide a valuable resource for use in future biotechnological processes (Aguilar, 1996;Bouras et al., 2006;Boubetra et al., 2013;Meklat et al., 2013).As in any isolation program, the strains have to be identified.Physiological characteristics together with morphological and chemical features are sufficient for accurate identification of many genera and species, but may fail to correctly identify to the species level, strains that have the similar phenotypic characteristics.The introduction of molecular tools has markedly improved the identification techniques.The 16S rRNA sequence-based phylogenetic analysis is widely used to determine taxonomic positions of many microorganisms.Assisted by the polymerase chain reaction (PCR) technique, the 16S rRNA sequence of investigated microorganisms can be easily obtained and compared with the sequences available in public databases.
As part of our program to study the extremophilic actinobacteria, we described the isolation and characterization of a new bacterial strain from Canadian soil, and its identification by conventional and molecular methods.In addition, we reported on production and extraction of three compounds which shows antimicrobial activities against mycotoxigenic and phytopathogenic fungi, Grampositive and negative bacteria and yeasts.

Soil sample and bacterial strain isolation
A canola rhizospheric soil sample (5 to 10 cm of depth) was collected aseptically from an Edmonton field, Alberta province, western Canada.The sample was placed in sterile polyethylene bag, closed tightly and stored at 4°C until analysis.A novel strain, PP14, was selectively isolated by serial dilution agar plating method.One gram of soil was dried at 110°C during 30 min (to increase a chance to isolate Gram-positive bacteria such as actinomycetes, by destroying Gram-negative bacteria which are not able to sporulate), followed by phenol treatment (1.5%) during 15 min (to select actinomycete strains which are resistant to chemicals).The sample was aseptically added to 9 ml sterile deionized water.The suspension was vortexed and diluted.Aliquots (0.2 ml) of each dilution were spread on the surface of chitin-B vitamins agar medium (Hayakawa and Nonomura, 1987) consisting of (per liter of deionized water): 2 g of chitin, 0.35 g K2HPO4, 0.15 g KH2PO4, 0.2 g MgSO4.7H2O,0.3 g NaCl, 0.02 g CaCO3, 10 mg FeSO4.7H2O, 1 mg ZnSO4.7H2O, 1 mg MnCl2.4H2O and 18 g agar.The pH was adjusted to 7.2 prior to autoclaving.The B vitamins including thiamine-HCl, riboflavin, niacin, pyrodoxin-HCl, inositol, capantothenate, p-aminobenzoic acid (0.5 mg/l for each) and biotin (0.25 mg/l) were added to the autoclaved medium.The antifungal cycloheximide (50 mg/l) and nystatin (30 mg/l) were used to inhibit development of invasive fungi.The following antibacterials were also added to the isolation medium as selective agents: rifampicin (5 mg/l), erythromycin, streptomycin, novobiocin (10 mg/l), chloramphenicol, kanamycin and penicillin (25 mg/l).These antibiotics were chosen on the basis of good results obtained previously during our work on the selective isolation of extremophile actinomycetes (Sabaou et al., 1998;Zitouni et al., 2004aZitouni et al., , 2005;;Badji et al., 2011).The plates were incubated at 30°C for 21 days.After isolation, strain PP14 was stored on agar slant of ISP2 medium (1% malt extract, 0.4% yeast extract and 0.4% glucose) at 4°C.

Morphological and cultural characteristics
Morphological and cultural characteristics of strain PP14 were determined by naked-eye examination according to the method described by Shirling and Gottlieb (1966).Cultural characteristics were studied on various International Streptomyces Project (ISP) media: yeast extract-malt extract agar (ISP2), oatmeal agar (ISP3), inorganic salts-starch agar (ISP4), nutrient agar and Bennett media at 30°C for 21 days.Colors of aerial and substrate mycelia were determined with the ISCC-NBS centroid color charts (Kelly and Judd 1976).Furthermore, the micromorphology and sporulation were observed on the same media by optical microscopy.

Chemical analysis of cellular constituents
Strain PP14 was grown on ISP2 broth for five days at 30°C.Mycelia were harvested by centrifugation, at 7000 g for 10 min, and washed by deionized water.These were then used for chemical (chemotaxonomic) analyses of diaminopimelic acid (DAP) isomers and glycin according to the procedure of Becker et al. (1964), and analysis of whole-cell sugars according to the procedure of Lechevalier and Lechevalier (1970).Phospholipids were extracted and analyzed according to the procedure of Minnikin et al. (1977).

Physiological characteristics
Carbohydrate assimilation as sole carbon source was determined as described by Goodfellow et al. (1971).The production of melanoid pigments was tested on peptone-yeast extract-iron agar (ISP6) and tyrosine agar (ISP7) media (Shirling and Gottlieb, 1966).

DNA preparation, PCR amplification and 16S rRNA sequencing
DNA extraction was realized according to the method of Liu et al. (2000).The strain PP14 was grown at 30°C for 7 days on a rotary shaker (250 rpm) in a 500 ml flask containing 100 ml of ISP2 medium.PCR amplification of the 16S rRNA of the strain was performed using two primers: 27f (5´-AGAGTTTGATCCTGGCTCAG-3´) and 1492r (5´GGTTACCTTGTTACGACTT-3´).The PCR amplification was conducted using a GeneAmp PCR System 9700 Thermocycler (Applied Biosystems, Foster City, CA) in a 25-μl volume containing 1 U of Platinum Taq DNA polymerase, 2 mM MgCl2, 1 × PCR buffer (20 mM Tris-HCl pH 8.4 at 25°C, 50 mM KCl) (Invitrogen Life Technologies, Carlsbad, CA), 0.4 μM each forward and backward primer, 0.2 mM each dNTP, and and 1 µl (500 ng) of the purified DNA.
The amplification cycle consisted of an initial denaturation step at 98°C for 3 min, after which Taq polymerase was added, followed by 30 amplification cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 2 min and a final extension step of 72°C for 10 min.PCR product (10 μl of reaction mix per loading well) was resolved on ethidium bromide-stained 1% agarose gel electrophoresis in 1× Tris acetate-EDTA buffer (40 mM Tris acetate, 1 mM EDTA, pH 8.3), and was visualized under ultraviolet (UV) fluorescence using a Syngene BioImaging System (Synoptics Inc., Frederick, MD).The PCR products obtained were submitted to Department of Medical Genetics at the University of Alberta (Edmonton, Alberta, Canada).The same primers as above and an automated sequencer were used for this purpose.The obtained sequence was compared for similarity level with available sequences in the public databases such as GenBank (using NCBI Blast), as well as with EzTaxon, a web-based tool for the identification of prokaryotes based on 16S rRNA gene sequences from type strains (Chun et al., 2007).

Phylogenetic analysis
Phylogenetic and molecular evolutionary analyses were conducted using software included in MEGA version 4.0 (Tamura et al., 2007) package.The 16S rRNA sequence of strain PP14 was aligned using the CLUSTAL W program (Thompson et al., 1994) against corresponding nucleotide sequences of representatives of the genus Streptomyces.Evolutionary distance matrices were generated as described by Jukes and Cantor (1969) and a phylogenetic tree was inferred by the neighbour-joining method (Saitou and Nei, 1987).Tree topologies were evaluated by bootstrap analysis (Felsenstein 1985) based on 1000 re-samplings of the neighbourjoining dataset.

Antimicrobial assay
The first screening of antimicrobial activity of the isolated actionmycete was determined by the streak assay method against several microorganisms.The strain was first cultivated in a straight line on ISP2 (containing 12 g/l agar) plates, of 90 mm diameter, and incubated for 10 days at 30°C.After the growth of the strain, the target microorganisms were seeded in streaks perpendicular to those of actinomycetes cultivation.The antimicrobial activity was evaluated, by measuring the distance of inhibition between target microorganism and actinomycetes colony margins, after incubation at 30°C for 24 h for bacteria and yeasts, and 48 h for filamentous fungi.

Kinetics of antimicrobial products of strain PP14
In the investigation of the culture medium that would enable optimal production of antimicrobial activities, the cultivation of the strain was carried out in two liquid culture media: ISP2 (composition mentioned above) and Bennett (0.1% meat extract, 0.1% yeast extract, 0.2% peptone and 1% glucose).The pH of each medium was adjusted to 7.2 prior to autoclaving.Each flask containing 100 ml of each medium (ISP2 and Bennett) was inoculated with 5% v/v of strain pre-culture grown in ISP2 medium for 72 h at 30°C under agitation of 250 rpm.The inoculated flasks were incubated at 30°C for 10 days with shaking at 250 rpm.The aliquots were collected each day by centrifuging (Microlitre Centrifuges, Heraeus Instruments, Biofuge) 4 ml of homogenized culture broth in Eppendorf tubes for 10 min at 16000 ×g.The antimicrobial activities were daily assayed for a duration of 10 days, against ten target microorganisms (which represent the most sensitive microorganisms): five filamentous fungi (A.carbonarius M333, A. niger, P. expansum, P. glabrum and M. ramannianus NRRL 1829), one yeast (S. cerevisiae ATCC 4226) and four bacteria (B.subtilis ATCC 6633, M. luteus ATCC 9314, E. cloacae E10 and E13) by the conventional agar diffusion method (wells technique).Wells (10 mm in diameter diameter) made in the ISP2 agar plates (containing 10 g/l of agar) were filled with 200 µl of the cell-free supernatant samples.Accordingly, the amounts of the active compounds present in the culture filtrate were estimated by measuring the diameters of inhibition.

Production, extraction and detection of antimicrobial compounds
For the production of antimicrobial compounds, fermentations were carried out in 500-ml Erlenmeyer flasks each containing 100 ml of the optimal medium (ISP2).The inoculated cultures were incubated at 30°C under constant agitation of 250 rpm.The extraction of active compounds took place on the day of optimal production (after 7 days of fermentation).The culture broth was centrifuged for 20 min at 8000 ×g to remove the mycelium.The cell-free supernatant was divided into 3 equal volumes of 80 ml, and then each was extracted with an equal volume of organic solvent.A range of extraction solvents was screened for effectiveness, including ethyl acetate, dichloromethane and n-butanol.Each organic extract was evaporated to dryness under vacuum on a Rotavapor (Laborota 4000).The resulting dry extracts were recuperated in 1 ml of methanol and subjected to biological assay (paper disk of 6 mm in diameter, Institut Pasteur) against B. subtilis ATCC 6633 (30 μl per disk) and M. ramannianus NRRL 1829 (40 μl per disk).Inhibition zones were expressed as diameter and measured after incubation at 37°C for 24 h for B. subtilis ATCC 6633, and at 28°C for 48 h for M. ramannianus NRRL 1829.
After that, the organic solvent which gave the highest inhibition diameter (n-butanol) was subsequently used for further experiments.The culture broth was centrifuged to remove the biomass.The cell-free supernatant (40 ml) was extracted with an equal volume of n-butanol and the butanolic layer was dehydrated with Na2SO4 and concentrated to dryness using a Rotavapor.The crude extract was recuperated in 1 ml of methanol and bio-assayed by paper disk diffusion method (as described above) against four filamentous fungi (A.carbonarius M333, A. niger, P. expansum and P. glabrum), one yeast (Saccharomyces cerivisea ATCC 4226) and three bacteria (M.luteus ATCC 9314 and E. cloacae E10 and E13).The butanolic extract was analyzed by thin layer chromatography (TLC).For this, 30 μl (B.subtilis) or 40 μl (F.oxysporum f. sp.lini) of butanolic extract was spotted onto 20 × 20-cm silica gel plates (Merck Art.5735, Kiesselgel 60HF 254-365, Darmstaadt, Germany), and then developed with solvent systems: ethyl acetate / methanol (100:15 v/v), and n-butanol/acetic acid/deionized water (60:20:20 v/v/v).The developed TLC plates were air-dried overnight at 40°C to remove all traces of solvents.The separated compounds were visualized as colored spots and also under ultraviolet (UV) at 254 (absorbance) and at 365 nm (fluorescence).The active spots were detected by bioautography (Betina 1973).The TLC plates were then placed in a plastic bioassay dish (23 × 23 × 2.2 cm 3 , Fisher Scientific Labosi) and overlaid with 150 ml/plate of ISP2 medium (containing 7 g/l of agar) seeded with B. subtilis or Fusarium oxysporum f. sp.lini as target microorganisms.After the agar had set, the dish was incubated at 30°C.After 48 h, reddish brown-colored fungal growth or opaque bacterial growth was visible.Clear areas due to inhibition of microorganism growth indicated the location of antimicrobial compounds on the TLC plates.The retention factor (Rf) values of each active spot were recorded.

Identification of strain PP14
Strain PP14 was obtained with the addition of antibiotics in the medium as selective agents.The strain was characterized based on its cultural properties (color of aerial mycelium, substrate mycelium and diffusible pigments) and chemotaxonomic characters.The description of strain morphology and the growth on various media, at 30°C for 21 days, is summarized in Table 1.The non-fragmented substrate mycelium was brown or greenish-yellow.The well-developed aerial mycelium tended to be white to gray and differentiated into straight [rectus-flexibilis (RF)] chains of spores.Mature spore masses belonged to the grey color series.The strain showed good growth on ISP4 and Bennett, and moderate growth on ISP3 and ISP2 media, and poor growth on nutrient agar, after 7 days of incubation at 30°C.Abundant greenish-yellow to brown soluble pigment was produced, after 21 days of incubation, on all media used.Endospores, sclerotic granules, synnemata and flagellated spores were not observed.The physiological characteristic studies revealed that strain PP14 did not produce melanoid pigments on any of the media used (ISP6 and ISP7).The strain utilized glucose, mannose, raffinose, saccharose, fructose, arabinose and mannitol as sole carbon sources, but not ramnose, galactose, xylose or myo-inositol.The cellwall peptidoglycan of strain PP14 contained only LLdiaminopimelic acid isomer and glycine, indicating that strain has a cell-wall chemotype I which is characteristic of the Streptomyces genus (Lechevalier and Lechevalier, 1970).The whole-cell hydrolysates contained galactose, ribose, rhamnose, with traces of mannose and xylose, but no characteristic sugars were detected indicating that the strain has a sugar type C. The phospholipid profile was of type II sensu (Lechevalier et al., 1977), characterized by the presence of phosphatidyl-ethanolamine, diphosphatidyl-glycerol and phosphatidyl-inositol as diag- nostic phospholipids.The obtained morphological and chemo-taxonomical characteristics strongly indicated that strain PP14 belonged to the genus Streptomyces.
The 16S rRNA sequence (1282 nucleotides) of strain PP14 has been determined and deposited in the Gen-Bank database under the accession number GQ374176.1.Phylogenetic relation of strain PP14 sequence with 16S rRNA of the highest 27 similar bacteria was carried out.The phylogenetic tree obtained by applying the neighbor joining method is illustrated in Figure 1.Comparison with sequences of the reference species of bacteria contained in genomic database banks exhibited a similarity level of only 98.3% with members of the S. violaceoruber species group (S.violaceoruber, S. violaceolatus, S. tricolor, S. humiferus, S. coelescens and S. anthocyanicus) which are the most closely related species.The physiological tests of strain PP14 and those of the S. violaceoruber clade are summarized in Table 2. † Data for the reference species were taken from Williams et al. (1983Williams et al. ( , 1989) ) and Duangmal et al. (2005).

Antimicrobial activities
The antimicrobial activity of strain PP14 against various target microorganisms is shown in Table 3. Strain PP14 exhibited a very strong activity against tested Grampositive bacteria (M.luteus), Gram-negative bacteria (K.pneumoniae E40 and E. coli E16, E52 and E195); however, almost no activity was observed for other tested Gram-negative bacteria.In addition, the strain exhibited a strong activity against B. subtilis, E. cloacae E10 and E13.An anti-yeast activity was also observed against S. cerevisiae but the growth of C. albicans was not affected.Moreover, an interesting antifungal activity was recorded against all mycotoxigenic and phytopathogenic fungi tested: a very strong activity against F. oxysporum f. sp.lini, F. culmorum, F. graminearum, A. carbonarius and P. expansum, and strong activity against F. moniliforme, F. solani, M. ramannianus, A. flavus, A. niger and P. glabrum.

Kinetics of antimicrobial products of strain PP14 in two different media
During the time course of fermentation in ISP2 broth and Bennett media, the antimicrobial activity was evaluated, by measuring the diameter of inhibition zone.The antifungal activity (against A. carbonarius, A. niger, P. expansum, P. glabrum, M. ramannianus and S. cerevisiae) and the antibacterial activity (against B. subtilis, M. luteus, E. cloacae E10 and E13) of strain PP14 was detected on day 3 of fermentation, but increased slowly between days 4 and 6, and reached a maximum always after 6, 7 or 8 days of fermentation; however, the anti-microbial activity was strongly decreased after day 8.The antimicrobial activity (evaluated by measuring the distance of inhibition) is given at the time of maximal values during 10 days of fermentation as reported in Table 4.

Production, extraction and detection of antimicrobial compounds
Table 5 shows the antimicrobial activity of different organic solvents (ethyl acetate, dichloromethane and nbutanol) obtained on the day of optimal production rate (after 7 days of fermentation), and the obtained results showed clearly that n-butanol is the best organic solvent for extraction.Moreover, the butanolic extract was also tested by measuring the distance of inhibition (mm) against eight other target-microorganisms.The obtained results are as follows: 24 mm for M. luteus, 17 mm for E. cloacae E13, 16 mm for E. cloacae E10, 52 mm for S. cerivisea, 33 mm for P. expansum, 23 mm for P. glabrum, 23 mm for A. niger and 39 mm for A. carbonarius.
On silica gel thin layer chromatography, the n-butanol extract, obtained on the day of optimal production rate (after 7 days), migrated and gave three bioautographic spots, which were active against B. subtilis and F. oxysporum f. sp.lini.One spot showed a yellow color: 14A (Rf = 0.81 in ethyl acetate/methanol, 100:15 v/v), with two non-colored spots: 14B (Rf = 0.72) and 14C (Rf = 0.60) exhibited a strong antibacterial and antifungal activities.Furthermore, Table 5 shows that the best organic solvent for extraction was n-butanol.

DISCUSSION
Various selective media and pretreatment procedures were applied to assess the optimal conditions for the isolation of rare actinomycetes and non-common strains of Streptomyces from different habitats (Naikpatil and Rathod, 2011).The pretreatment procedures could be realized, for example, by using the electromagnetic waves, by dry (or wet) heating of soil samples or by using chemicals such as antibiotics or phenol, etc.Several studies  (Aouiche et al., 2011); † Each value represents the average of two measurements; the inhibition distance of 1 to 8 mm was considered as weak, 9 to 14 mm as moderate, 15 to 24 mm as strong and 25 to 50 mm as very strong activity.The target microorganisms without accession number resulted from our laboratory collection.studies reported that these techniques are extremely useful in the isolation of rare strains of actinomycetes (Kim et al., 1994;Naikpatil and Rathod, 2011).
The strain PP14 was first recognized on the basis of its morphological features.This strain was obtained with addition of 7 antibiotics as selective agents.The screen-ing, isolation and characterization of new strains of actinomycetes producing new potential antibiotics has been a major area of research by our group for many years (Sabaou et al., 1998;Lamari et al., 2002;Zitouni et al., 2004bZitouni et al., , 2005;;Boudjella et al., 2006;Bouras et al., 2007Bouras et al., , 2008;;Aouiche et al., 2011;Badji et al., 2011;Meklat et   *The antimicrobial activity on ISP2 medium (evaluated by measuring the distance of inhibition) is given on the day of optimal production rate (after 7 days of fermentation).† Value include the diameter of wells (10 mm), and represents the average of two measurements.al., 2012;Mokrane et al., 2013).The use of many antibiotics in combination with other selective agents (such as phenol) may lead in the future to the establishment of an adequate method for the selection of some species of actinomycete.The characterization of Streptomyces species is mainly based on the color of aerial and substrate mycelia, the soluble pigment and the shape and ornamentation of spore surface because of their stability (Shirling and Gottlieb, 1972;Holt et al., 1989).
The description of growth and some phenotypic characteristics of strain PP14 are summarized in Table 1.
On the basis of its morphological and chemical properties, strain PP14 was classified in the genus Streptomyces.Modern Streptomyces identification systems are based on 16S rRNA sequence data, which have provided precious information on systematic, and then have been used to identify several newly isolated Streptomyces (Lee et al., 2005;Kim et al., 2006) Bouizgarne et al. (2009) and Loqman et al. (2009).Moreover, the obtained results showed that strain PP14 could also be distinguished from the members of S. violaceoruber clade by several characters such as the color of aerial and substrate mycelia, the production of diffusible pigments and melanoid pigments; the use of xylose, rhamnose, galactose and meso-inositol as sole carbon source (Table 2).These results strongly suggest that strain PP14 is a new genomic species.
The obtained results in Table 3 showed the broad antimicrobial spectrum of strain PP14 against various target microorganisms especially against some phytopathogenic and mycotoxigenic fungi and multi-resistant bacteria.A broad-spectrum antibacterial activity was observed against multi-resistant bacteria such as K. pneumoniae (E40), A. baumanii (E16), E. coli (E52) and E. coli (E195) which are known to be resistant respectively to 11, 8, 10 and 9 antibiotics among 15 tested antibiotics (Aouiche et al. 2011).However, the strain was moderately active against E. cloacae (E10) and E. cloacae (E13), which are resistant respectively to 11 and 12 tested antibiotics.In addition, the strain exhibited strong activity against Grampositive bacterium, M. luteus (ATCC 9314), and yeast S. cerevisiae (ATCC 4226), and weak activity against Gramnegative bacterium S. enterica (E32) resistant to 9 tested antibiotics.Furthermore, strain PP14 showed a strong activity against phytopathogenic fungi such as F. oxysporum f. sp.lini (Foln 3-5), F. culmorum and F. gramenearum; and against mycotoxigenic fungi such as Aspergillus carbonarius and A. niger (both producers of ochratoxin A) and Penicillium expansum.However, the activity of the strain was moderate to F. moniliforme, F. solani, P. glabrum, M. ramannianus and A. flavus (producer of dangerous aflatoxins B1 and B2).
In general, Gram-negative bacteria are more resistant to antibacterial compounds than Gram-positive bacteria.Several studies showed that the outer cell membrane in Gram negative bacteria (double membranes) contains many protective mechanisms against antibiotics (Gupta, 2011).The activity of Streptomyces species against Gram-positive bacteria has been widely published (Park et al., 2009) but it seems that the activity against Gramnegative bacteria, yeasts and fungi has been rarely reported (Sabaratnam and Traquair, 2002;Baginski et al., 2005;Nguyen et al., 2010).Antitumoral activity has also been noted in several strains and species of Streptomyces (Pamboukian and Candida, 2004).
Table 4 shows the time of maximal antimicrobial activity (obtained always after 7, 8 or 9 day of fermentation) of strain PP14 obtained from two different culture media.Complex media have often been used for production of antibiotics, and the obtained results showed that ISP2 medium was generally better than Bennett medium to generate bioactive compounds (Boudjella et al., 2006).This variation reflects the importance of medium optimization to attain higher productivity.The chromogenic reactions of these compounds were negative with FeCl 3 , naphtoresorcinol-H 2 SO 4 , ninhydrine, formaldehyde-H 2 SO 4 and Dragendorff reagents, suggesting the absence of phenol, osidic residues, free amine groups, polycyclic aromatics and alkaloids.

Conclusion
The obtained findings give support to the biotechnological use of the rare and extremophile actinomycetes as sources of interesting bioactive natural molecules against mycotoxigenic and phytopathogenic fungi, Gram-positive and Gram negative bacteria and yeasts.Furthermore, these antimicrobial molecules could be beneficial to undertake a more comprehensive search for rare forms of antibiotic-producing organisms in extremophile biosystems; however, further studies will be necessary to determine the structures of the active compounds.

Figure 1 .
Figure 1.Neighbor-joining tree, based on 16S rRNA gene sequences showing the relationships between strain PP14 and the most close 27 type strain species of Streptomyces.The numbers at the nodes indicate the percentages of bootstrap support based on neighbor-joining analyses of 1000 resampled data sets; only values over 45% are given.Bar: 0.002 nucleotide substitution per nucleotide position.

Table 1 .
Growth and some phenotypic characteristics of strain PP14 on different media at different incubation time.

Table 2 .
Physiological characteristics of strain PP14 in comparison with those of closest phylogenetic neighbours (S. violaceoruber clade).

Table 3 .
Antimicrobial activity of Streptomyces sp.PP14 against various microorganisms on solid ISP2 medium.
*Resistant to multiple antibiotics

Table 4 .
Antimicrobial activity of Streptomyces sp.PP14 against various microorganisms on ISP2 and Bennett liquid media.The antimicrobial activity (evaluated by measuring the distance of inhibition) is given at the time of maximal values during 10 days of fermentation.†Value include the diameter of wells (10 mm), and represents the average of two measurements. *

Table 5 .
Effect of different organic solvents on antimicrobial activity of Streptomyces sp.PP14 against B. subtilis ATCC 6633 and M. ramannianus NRRL 1829.
. The 16S rRNA sequence of strain PP14 was compared with those of other Streptomyces species; it showed a sequence similarity of only 98.3% with members of the Streptomyces violaceoruber species clade (S. violaceoruber, S. violaceolatus, S. tricolor, S. humiferus, S. coelescens and S. anthocyanicus) which are the most closely related spe-