Mary Josephine Punitha et al, J. Global Trends Pharm Sci, 2017; 8(4): 4342 - 4354 An Elsevier Indexed Journal ISSN-2230-7346 Journal of Global Trends in Pharmaceutical Sciences BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF ACTINOMYCETES ISOLATED FROM MARINE SOIL SAMPLE OF KANYAKUMARI COAST C. Johny1, S. Jeeva2, and S. Mary Josephine Punitha3* 1Research Scholar, Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakkamangalam, Kanyakumari District, Tamil Nadu, India, 2Asst. Professor, Department of Microbiology, Udaya College of Arts and Science, Vellamodi, Kanyakumari District, Tamil Nadu, India, 3*Associate Professor, Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakkamangalam, Kanyakumari District, Tamil Nadu, India, *Corresponding author E-mail: [email protected] ARTICLE INFO ABSTRACT The main focus of this study was to isolate and identify the predominant Key Words actinomycetes from coastal area of Kanyakumari, Tamilnadu by cultural, microscopic, biochemical characterization and by 16S rDNA sequencing. About 16S rDNA sequencing, 52 actinomycete colonies were isolated by systematic serial dilution and plating Streptomyces sp, technique and the size, shape and margin of the two selected predominant Nocardiopsis actinomycete colonies were observed on starch casein agar after 7 to 14 days of dassonvillei, accession incubation. Based on experimental studies and genetic DNA sequence analysis, numbers, EMBL it was concluded that present strains A1 and A2 were identified as Streptomyces sp. and Nocardiopsis dassonvillei respectively and the 16s rRNA sequences of A1 and A2 were submitted to GenBank under accession numbers KU174216 and AB896798. The strains were submitted as Streptomyces sp. N56 and Nocardiopsis dassonvillei and the data are simultaneously made available to EMBL in Europe and the DNA Data Bank of Japan. INTRODUCTION: The morphological, physiological, chemical, genetical, numerical and ecological and molecular characteristics are molecular biology techniques have been the major characteristics used in taxonomy responsible for rapidly changing views on for the classification and identification of how bacteria could be classified and micro-organisms [1]. Actinomycetes form a identified [4,5]. Actinomycetes are highly distinct evolutionary line of organisms since valued for their unparalleled ability to they show marked chemical and produce biologically-active secondary morphological diversity [2]. They are gram metabolites and they perform significant positive, free living, saprophytic bacteria, biogeochemical roles in terrestrial soils. At which was characterized by the formation of least 4607 patents have been issued on substances and aerial mycelium on solid actinomycete related products and processes media, presence of spores and a high GC [6] and there are approximately 32,500 content of DNA (60-70 mol%) [3]. The natural products reported from microbial application of new and reliable biochemical, sources (Antibase data base) including about 4342 Mary Josephine Punitha et al, J. Global Trends Pharm Sci, 2017; 8(4): 4342 - 4354 1000 derived from marine microbes [7]. microscopic, biochemical characterization Many actinomycetes grow on the common and by 16S rDNA sequencing. bacteriological media used in the laboratory, MATERIAL AND METHODS such as nutrient agar, trypticase soy agar, Screening of actinomycetes from marine blood agar, and even brain-heart infusion soil sample agar. Morphological characters of The organisms which were actinomycetes are still widely used for predominantly present in the coastal area of characterizing genera, for example, the Kanyakumari, Tamilnadu throughout the presence or absence of spores on the year withstanding the physicochemical substrate mycelium or the formation of variations were screened and used for further zoospores in specialized spore vesicles or study. As the physicochemical parameter sporangia. The ability to produce motile varies, the number and type of bacterial and spores is more wide spread in the fungal colonies were also varied but few actinomycetes. The colonies of actinomycetes colonies were isolated actinomycetes have pastel colors, soil-like throughout the year. Hence this study was odor, hard and stick into agar [8]. focused with actinomycetes. The The isolation and characterization of predominant actinomycetes were separately Actinomycetes were performed in different streaked, subcultured, ensured for their biochemical methods [9]. The tests generally axenicity and maintained in starch caesine used are starch hydrolysis, Triple Sugar Iron agar slant (Himedia, Mumbai, India). (TSI) agar test, citrate utilization test, indole Cultural characterization test, methyl red test, vogus-proskauer Colony characterization (Acetone Production) test, Catalase test [10]. The selected actinomycetes colonies Physiological and biochemical were streaked on starch casein agar and characteristics were analysed as described by incubated for 7 to 14 days. The isolated Thornley [11]; Barrow and Feltham [12]. colonies were noted for their size, shape and Identification of actionmycetes to genus margin. level was made possible in a fast and Aerial mass color accurate manner. Even though, using only To check the growth condition and microscopic, cultural and biochemical aerial mass color of the selected isolates, the techniques is not enough to ascertain the pure actinomycetes colonies were streaked organism. To overcome the severe on starch casein agar (Himedia, Mumbai, limitations of culture- dependent methods in India), actinomyctes isolation agar discovering bacterial diversity [13, 14, 15, (Himedia, Mumbai, India) and yeast extract 16], molecular biological techniques have agar (Himedia, Mumbai, India) and become increasingly popular [17]. incubated at room temperature for 7 to 14 Knowledge of the phylogenetic structure days. Filtered sea water was used for media within the genus Actinomyces has improved preparation. After incubation, the aerial mass in recent years because of the advent of 16s color was noted [26]. rRNA sequencing [18, 19, 20]. Comparative Pigmentation 16s rRNA sequence analysis has been The pure isolates were streaked on applied to studies of natural microbial tryptone yeast extract agar (Himedia, communities and has resulted in the Mumbai, India) and peptone yeast extract discovery of unexpectedly high levels of iron agar (Himedia, Mumbai, India) and biodiversity [21, 22, 13, 23, 24, 25]. The incubated at room temperature for 7 to 14 phylogenetic analysis of microbes further days. After incubation, the color of mature helps in selecting potential candidates for sporulating aerial mycelium and reverse side biotechnological applications. Hence, the pigment were observed and recorded [26]. present study was focused to identify the The production of melanoid pigments (i.e. actinomycetes isolated from coastal area of greenish brown, brownish black or distinct Kanyakumari, Tamilnadu by cultural, brown pigment modified by other colours) was also noted [27]. 4343 Mary Josephine Punitha et al, J. Global Trends Pharm Sci, 2017; 8(4): 4342 - 4354 Morphological characterization Applied Biosystems model 3730XL Morphological observations were automated DNA sequencing system made by slide culture technique. One cm2 (Applied BioSystems, USA). starch casein agar blocks were cut from the BLAST analysis: BLASTN (optimized for petriplate and the actinomycetes isolates megablast) searches were manipulated with were inoculated individually on to the the sequences of isolated strains A1 and A2. respective agar blocks. The inoculated agar The corresponding sequences of blocks were placed on the center of the representative species were used for individual glass slides and covered with phylogenetic analyses. coverslips. The setup was placed inside a Construction of phylogenetic tree sterile petriplate containing moistened cotton The Maximum parsimony (MP) tree and incubated at room temperature for 7 was obtained using the Subtree-Pruning- days. After incubation, the morphological Regrafting (SPR) algorithm [30] with search observations were made with the light level 0 in which the initial trees were microscope. The slides were examined under obtained by the random addition of microscope of 100X. Active purified isolates sequences (10 replicates). Evolutionary of actinomycetes were characterized by analyses were conducted in MEGA6 [31]. comparing their morphology of spore RESULT AND DISCUSSION bearing hypae with the actinomycetes Screening of Actinomycetes from marine morphologies as described in Bergey’s soil sample manual [28]. Marine ecosystem covers almost Biochemical characterization 70% of the earth surface [32]. Soil is the The biochemical characterization of most extensively studied ecological niche actinomycete colonies were analyzed [33]. Since organisms present in these following the directions given by Bergey’s marine environments are extremely rich manual of systematic bacteriology [29]. sources of bioactive compounds [34, 35, 36], Genotypic characterization attempts were made to isolate the native 16s rDNA sequencing microbes present in the coastal area of Colonies were picked up with a Kanyakumari, Tamil Nadu [37]. The sterilized toothpick from mother culture organisms which withstand the plate and suspended in 0.5 ml of sterilized physicochemical variations throughout the saline in a 1.5 ml centrifuge tube. The year were screened and used for samples were centrifuged at 10,000 rpm for identification