Culturing attempts of the associated bacterial community from Moorea producens JHB

Efforts to culture Mor1 separately from filaments of M. producens JHB used a variety of solid media containing 2 % agar, as listed in Table 2. Intact or cut filaments of M. producens JHB were placed onto each media type. For some culturing trials, M. producens JHB filaments were freeze-dried and ground up and then added to the media. Additionally, associated bacteria were washed from the surface of M. producens JHB filaments using the following protocol: 2 g of biomass was placed into 10 mL of 0.45 M NaCl, 10 mM KCl, 7 mM Na₂SO₄, 0.5 mM NaHCO₃, and 10 mM EDTA. Added to this was 0.1 mL filter-sterilized Rapid Multienzyme Cleaner (3 M). The sample was then incubated for 2 h at room temperature while shaking at 80 rpm. The sample was vortexed and then centrifuged at 300 × g for 15 min. An aliquot of the supernatant (50–100 μL) containing associated bacteria was then plated onto the various types of media.

Solid media utilized for culturing heterotrophic bacteria associated with M. producens JHB sheaths

Bacterial colonies that grew on these plates were isolated and grown overnight in liquid media. DNA was extracted from the overnight cultures using the Wizard Genomic DNA Purification Kit (Promega). PCR was performed on the DNA samples using the selA primers (Table 1), and the 16S rRNA 27 F and 1492R primers [34]. PCR was carried out in 25 μL volumes, containing 12.5 μL of 2x Taq Master Mix, 0.5 μL MgCl₂ (25 mM), 1.0 μL of each primer (10 μM), 1.0 μL of DNA template, and 9 μL sterile water. The amplification conditions were as follows: initial denaturation at 95 °C for 4 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, followed by a final extension step at 72 °C for 1 min. The 16S rRNA PCR products were then cloned into the pCR 4-TOPO Vector (Invitrogen TOPO TA Cloning Kit) using the standard protocol, followed by sequencing. The obtained sequences were analyzed using BLASTn.