16S rRNA gene was amplified

from the extracted genomic DNA using the universal eubacterial 16S rRNA gene forward primer 5′-AGAGTTTGATCCTGGCTCAG-3′ (Escherichia coli positions 8–27) and the actinomycetes-specific reverse primer 5′-CCGTACTCCCCAGGCGGGG-3′ (ACT878r) (Farris & Olson, 2007). With an objective of finding the number of polymorphic groups among the isolated actinomycetes, all the amplicons representing various isolates were subjected to ARDRA. To examine the ARDRA profile, 10 μL of the PCR product was digested with HinfI, RsaI and MspI at 37 °C for 3 h. Digested DNA samples were analysed in 2% agarose gel. The amplified product (approximately selleck compound 870 bp) was purified and cloned in the pTZ57R/T vector (InsT/Aclone™ PCR Product Cloning Kit #K1214,

MBI Fermentas). Sequencing of the rRNA gene (about 870 bp) for all the coral-associated actinomycetes was carried out in Macrogen (Seoul, Korea). The sequences obtained were matched with previously published sequences available in NCBI using blast (Altschul et al., 1997). Multiple sequence analysis was carried out using clustalx (Thompson et al., 1997) and further NJ plot (Perrière & Gouy, 1996) and PhyloDRAW (Choi et al., 2000) were used for constructing a phylogenetic tree. To selleckchem validate the reproducibility of the branching pattern, a bootstrap analysis was performed. Each actinomycete isolate was grown as a c. 2 cm colony for 10–14 days on Petri plates containing SCA. Bacteria,

on the other hand, were streaked about 1–1.5 cm from the edge of the colony being tested (Zin et al., 2007). Well-characterized Gram-positive and Gram-negative clinical microbial strains Staphylococcus aureus (ATCC 11632), Pseudomonas aeruginosa (ATCC 10145), Aeromonas hydrophila (ATCC 7966), Vibrio parahaemolyticus (ATCC 27519) and Vibrio vulnificus (ATCC 29307) were used as the indicator bacteria for antibacterial activity assay. Growth of the test organisms was evaluated after 24, 48 and 72 h, and recorded as growth, inhibition and no growth as compared with a control plate containing no actinomycetes colonies. Secondary screening was performed by agar well diffusion assay (Harald et al., 2007) with the cell-free supernatant of the actinomycete isolates to confirm the antibacterial activity. The actinomycete aminophylline strains isolated from corals were transferred aseptically into 250-mL Erlenmeyer-baffled flasks with cotton plugs, containing 50 mL of ISP2 medium, which was incubated for 3–5 days at 28 °C with agitation in a rotary shaker at 250 r.p.m. After 3 days of incubation, the culture broth was filtrated through a press to separate mycelium and supernatant. The supernatant was extracted twice with ethyl acetate, chloroform or n-butanol (2 × 100 mL). The solvent extracts were combined and evaporated to dryness under reduced pressure and the extracts obtained were weighed.