Kadiri Sunanda Kumari et al. / Journal of Pharmacy Research 2011,4(12),4678-4680 Research Article Available online through ISSN: 0974-6943 www.jpronline.info Taxonomy, Identification and Biological Activities of a Novel Isolate of Streptomyces albus Kadiri Sunanda Kumari* and Vidavalur Siddaiah Department of Organic Chemistry & FDW, Andhra University, Visakhapatnam 530 003, India Received on:20-08-2011; Revised on: 15-09-2011; Accepted on:10-11-2011 ABSTRACT Various isolates of streptomycetes species were isolated from different soil sediments of Visakhapatnam Coast. Preliminary screening by cross-streak method showed that 19 isolates exhibited good antimicrobial activity. Among these 19 isolates tested, isolate MB201 was the most active one and thus was selected for identification. Morphological, cultural, physiological, biochemical characteristics and biological properties as well as enzymatic activities and cell wall composition suggested that the isolate belonged to the genus streptomyces. The 16S ribosomal DNA amplification for phylogenetic study revealed that the isolate was highly related to Streptomyces albus (~99%), so it was designated as Streptomyces albus gangavarams. This new strain produced antimicrobial agents active against Gram +ve and Gram –ve bacteria and fungi. KEY WORDS: Streptomyces, S.albus INTRODUCTION: Numerous classifications were devised to accommodate the increasing number Morphological characterization: of streptomyces species, most of them based on a few subjectively chosen The spore chain morphology of the selected isolate MB 201 of 14 day old morphological and pigmentation properties which were rarely studied under organism was examined by the scanning electron microscope. standardized growth conditions (Atalan et al., 2000). Biochemical, nutritional and physiological characters had also been used in streptomycetes taxonomy, Physiological, biochemical and cultural properties: but usually had been applied to only selected species (Kutzner et al., 1989 and Media used were those recommended by the International Streptomyces Project Schlegel, 1992). The genus Streptomyces was classified in the family (ISP) (Shirling and Gottlieb, 1969) and by Waksman (1969). Mycelium was streptomycetaceae that includes also a number of other taxa (Waksman, 1961). observed after incubation at 28 0C for 2 weeks. Colors were determined accord- Several investigations have been done on screening new isolates of actino- ing to Prauser (1964). Reduction of nitrate and production of melanoid pig- mycetes able to produce antimicrobial agents (Augustine et al., 2005 and Lee ment were determined by the method of ISP (Shirling and Gottlieb, 1969). et at., 2005). Streptomyces have been the most fruitful source of microorgan- Carbohydrate utilization was determined by growth on carbon utilization me- isms for all types of bioactive metabolites that have important applications dium ISP 9 supplemented with 1% carbon sources at 28 0C. Liquefaction of in human medicine as anti-viral and anti-cancer compounds and in agriculture gelatin was evaluated by the method of Waksman (1969 ). Hydrolysis of starch fields as herbicides, insecticides and anti parasitic compounds (Watve et al., and milk were evaluated by using the media of Gordon et al., 1974. All the 2001). cultural characteristics were recorded after 2 weeks. Chemotaxonomic analysis: In the present work, a new isolate capable of producing a broad-spectrum For analysing the cell wall composition, the cells were obtained after incuba- antimicrobial agent against Gram positive and Gram negative bacteria, yeast tion at 28 0C for 3 days in yeast extract-glucose broth (pH 7.0) containing 10g/ and fungi was tested for its taxonomic profile. This included the study of some l of yeast extract and 10g/l of glucose. Isomers of diaminopimelic acid in the morphological, cultural, physiological, biochemical and biological properties whole-cell hydrolysates were determined by thin-layer chromatography ac- as well as enzymatic activities and cell wall chemotype. cording to the method of Hasegawa et al., 1983. Whole cell sugars were analyzed according to the method of Becker et al., 1965. MATERIALS AND METHODS: Sample Collection Enzyme activities: Marine sediments samples were collected from Visakhapatnam Coast. Several Proteolytic and amylolytic activities were performed according to the method samples were randomly collected from different localities using a core sampler of Nitsch and Kutzner (1969). at depths between 1-20m. Care was taken to see that the points of collection had as widely varying characteristics as possible with regard to the organic Antimicrobial activities: matter, particle size and color of the soil. The antimicrobial activities of the isolate (MB201) culture broth were exam- ined by agar diffusion method and expressed as diameter of the inhibition Isolation of Streptomyces species: zones according to the agar plate diffusion method (Wu, 1984). The test About 1gm of sample was transferred to a sterile Erlenmeyer (EM) Flask organisms were obtained from MTCC, Chandigarh. Assay plates were pre- containing 50ml sterile water. The flasks were shaken on rotary shaker for 30 pared by inoculating 20ml of Mueller- Hinton agar medium with test organism. Agar cups (6mm diameter) were filled with 100 µl of mycelia-free culture min for the detachment of the spore chains, if any. The flasks were kept aside 0 for 30 min to settle down the particulate matter. The clear supernatant was filtrate in triplicate and the plates were incubated at 37 C for 24 hrs. diluted with sterile water. These dilutions (10-1 - 10-3) were used as inoculm. One ml of each of these dilutions were inoculated into sterile Petri plates contain- PCR amplification and sequencing of 16 S rDNA: 0 The 16 S rDNA gene was amplified by PCR (Weisburg et al., 1991) using ing sterile medium and incubated at 28 C for 2-3 weeks. For isolation starch 1 1 1 casein agar medium, potassium tellurite agar medium, actinomyces isolation primers fP 1 (5 - AGAGTTTGATCCTGGCTCAG-3 ) and rP 2 (5 - agar medium were used. ACGGCTACCTTGTTACGACTT-31). Amplication was performed in a total volume of 50 µl containing 30-50 µg DNA, 100 µM of each primer, 10 µM dNTP, 10x buffer (10mMTris-HCL, pH 8.0, 500 mM KCl, 20mM MgCl ) and *Corresponding author. 2 1.5U Taq DNA polymerase. PCR was performed under the following condi- Kadiri Sunanda Kumari tions : 4 min at 94 0C, followed by 35 cycles of 1min at 94 0C, 1min at 58 0C, Department of Organic Chemistry & 2 min at 72 0C. FDW, Andhra University, Visakhapatnam 530 003, India Data Analysis: (Tel): 0891-2844683, The sequences determined in this study were aligned and the nucleotide simi- Mobile: 9441571261 larity values were calculated from the alignment (Thompson et al., 1994). The evolutionary distance matrices were constructed using the algorithm of E-mail: [email protected], Jukes and Cantor (1969) and evolutionary trees for the datasets were inferred [email protected] from the neighbour – joining method (Saitou and Nei 1987 and Kumar et al., 2001). Journal of Pharmacy Research Vol.4.Issue 12.December 2011 4678-4680 Kadiri Sunanda Kumari et al. / Journal of Pharmacy Research 2011,4(12),4678-4680 RESULTS AND DISCUSSION not produce tyrosine and could reduce nitrate. Well growth was recorded at a temperature range 20 to 37 0C and at pH range of 6-8. The utilization of Isolation of Streptomyces species: various carbohydrates by the isolate MB 201 suggests a good pattern of carbon A total of 3 different marine sediment samples were collected and used for assimilation. Inoisitol was poorly utilized while fructose, arabinose, mannitol screening and isolation of streptomycetes. A total of 51 streptomycetes were and rhamnose were well utilized. isolated from the marine sediments of Visakhapatnam Coast. Preliminary Enzyme activities: screening by cross-streak method was carried out for all the 51 isolates. After The isolate MB 201 showed moderate activ- preliminary screening, 18 isolates showed antimicrobial (antibacterial, anti- ity of enzyme like amylase, gelatinase and fungal) activity. (Table 1). Among these 18 isolates tested, the most active protease. isolate (MB 201) was selected for identification. Soluble pigment Nil Faint yellow Nil Nil Nil Nil Nil Nil Chemotaxonomic Analysis: Table 1: Activity of Promising Isolates by Cup Plate Method Analysis of the whole-cell hydrolysate of the Selected Inhibition zone (mm) isolate for the detection of diamino- pimelic isolates Bacteria Filamentous fungi & yeast acid (DAP) showed the presence of Type –I S.a B.s E.c P.v C.a S.c A.n A.a cell wall characterized by LL-DAP. Hydroly- sis of cells for sugar analysis revealed no di- Spore mass Poor, white Moderate, white Moderate, white Poor, white Moderate, white Poor, white Moderate, white Poor, white MB 101 13 13 10 9 - - - - agnostic sugars. 102 10 12 09 10 103 14 15 11 11 - - - - 107 - - - - 8 10 11 11 Antimicrobial Activities: 111 10 11 12 11 - - - - 114 - - - - 13 15 12 12 Table 3 showed abroad spectrum antimicro- 120 9 9 10 11 - - - - bial activities against Gram+ve and Gram- 201 16 15 13 12 17 16 14 14 ve bacteria, yeasts and fungi. 236 9 7 7 8 - - - - 306 11 12 9 9 - - - - Aerial mycelia Moderate, white Abundant, white Abundant, white Good, white Good, white Good, white Good, white Moderate, white 309 15 16 13 13 - - - - Analysis of 16S rDNA gene sequence: 312 10 10 12 12 - - - - 16S rDNA gene sequence analysis was car- 346 - - - - 9 10 11 10 401 12 10 10 13 - - - - ried out to elucidate the taxonomic