For the isolation of Actinobacteria, four different culture media were used, namely, starch casein nitrate agar (Küster & Williams, 1964), Gause 1 agar (Atlas & Park, 2000), manila clam (Ruditapes philippinarum) extract agar (formulated in this study), and jewfish (Argyrosomus argentatus) extract agar (formulated in this study).
Detailed compositions of these media are given in Supporting Information, Table S1. The manila clam and jewfish extract agar were used to provide complex undefined nutrients of marine origin for bacterial growth. Isolated strains MG-132 in vitro were maintained on International Streptomyces project 2 medium (ISP-2M; Shirling & Gottlieb, 1966) prepared in 50% v/v artificial seawater (Sealife, Marinetech, Tokyo, Japan). Aliquots (100 μL) from the original and 10-times-diluted samples in sterile seawater were spread on the above-mentioned isolation media, and the plates were incubated at 25 °C for 2 weeks. Actinobacteria-like colonies with a powdery consistency were picked up and spread on ISP-2M medium. Purified strains were preserved at −80 °C in 50% artificial seawater (v/v) with Selleck LY2109761 15% glycerol (v/v). Isolated strains were identified on the basis of their partial 16S rRNA gene sequences. Fresh colonies grown on ISP-2M were transferred
to sterile microtubes. The template DNA for 16S rRNA gene amplification from these cells was prepared using the Prepman™ Ultra reagent (Applied Biosystems, CA). A pair of universal primers – 27f and 1492r – was used to amplify the portion of the 16S rRNA gene corresponding to the positions 8–1492 in Escherichia coli 16S rRNA gene (Brosius et al., 1978). The amplified fragments were directly sequenced using a BigDye Terminator® v3.1 Cycle Sequencing Kit and an ABI Prism 3100® Genetic Analyzer (Applied Biosystems). The partial sequences were determined using 27f and 536R primers. The atgc program (Genetyx, Tokyo, Japan) was used for sequence editing and assembly. The hmgr gene was amplified using the primers pHMGF (5′-GGGCATCGCCGCGGACCCTCCTCGACGAGCG-3′) and pHMGR (5′-GCGATGACGGCGAGGCGGCGGGCGTTCTC-3′) and PCR parameters (4 min
Isotretinoin at 95 °C for primary denaturation, 30 cycles consisting of 30 s at 95 °C, 30 s at 60 °C, 1 min at 72 °C, and 10 min at 72 °C for extension) as described by Sigmund et al. (2003). The reaction mixture contained 1.25 μL of dimethyl sulfoxide, 1 μM of each primer, 50 ng of genomic DNA, 12.5 μL of 2 × Go-Taq® green master mix (Promega, Madison, WI), and water to a final volume of 25 μL. Amplified fragments were purified using the QIAquick PCR purification kit (Qiagen, CA), and the purified PCR fragments were cloned using the TOPO TA cloning kit (Invitrogen, CA) according to the manufacturer’s instructions. The cloned fragments were then sequenced using plasmid-based M13 primers. Assembled 16S rRNA gene and HMGR sequences were compared with those available in the DNA Data Bank of Japan using blast (Altschul et al.