*3.3. CuSO4*·*5H2O Assisted Differential Genomic DNA (gDNA) Extraction of M. producens*

Prior to bacteria genomic DNA (gDNA) isolation, the cyanobacteria cells were treated with cycloheximide (5 mg L−1, overnight) to kill eukaryotic cells, protozoa, and fungi. Thereafter, the cells were rinsed several times with filtered sterile seawater (12 times, 45 μ) and left overnight in phosphate-poor autoclaved seawater [29]. For detaching filaments, clusters of Moorea biomass were submerged in phosphate buffered saline (PBS, pH 7.4). The resulting filaments were rinsed with Millipore water and pooled together, and aliquots were weighed to afford sufficient biomass for gDNA extraction and genome sequencing. Thereafter, the cyanobacteria were treated with copper sulphate powder (CuSO4·5H2O) and left to stand for 10 min, 30 min, and 60 min, respectively, to kill any available bacteria. The control was untreated. For removing the bacteria, biomass of pooled filaments was placed in an Eppendorf tube, freeze-dried with liquid nitrogen, and thawed slowly. The tube contents were sonicated (pulsar 30% of maximal amplitude, 10 min) and the process was repeated 5 times. Representative filaments were stained with acridine to observe the presence of any associated bacteria using a LEICA DMIRB microscope (LEICA DMIRB, Mannheim, Germany) with Media Cybernetics image analysis software Image-Pro Plus 7 (MEDIA CYBERNETICS, Rockville, MA, USA).

Aliquots of the biomass (0.25 g) were weighed in sterile MoBio microfuge tubes into which was added 100 μL of sodium dodecyl sulfate (SDS) and 0.5 mL of RNAse free TE buffer (50 mM Tris HCl, 20 mM EDTA, pH 8.0). The specimen was crushed and homogenized using a bench-top bead-based homogenizer (PowerLyzer™, 5 min). The resulting homogenate was transferred into an Eppendorf tube, freeze-dried in liquid nitrogen, and thawed slowly. The tube contents were sonicated (pulsar 30% maximal amplitude, 10 min) and the process was repeated five times [43]. The sample was centrifuged (10,000× *g*, 3 min) and bacterial gDNA were extracted following the MoBio kit for soil bacteria; the residue was retained. The DNeasy PowerMax Soil Kit used here was formerly sold by MOBIO as PowerMax Soil DNA Isolation Kit, and this is currently sold by Qiagen (QIAGEN, Valencia, CA, USA). The residue was re-extracted for bacterial gDNA until no traces of the gDNA were detected on an electrophoresis gel.

To the bacteria-free residue was added 1 mL of Lysis buffer (TE buffer with RNase) and heated (55 ◦C, 1 h) prior to adding 300 μL of freshly prepared lysozyme at 50 mg/mL. The mixture was heated (55 ◦C, 30 min). Into the contents was added 100 μL of a solution containing 450 μL of 10% sarcosyl and 20 μL of 20 μg/mL proteinase k and incubated (55 ◦C, 30 min). To each of the contents in a 2-mL Eppendorf tube was added 3 volumes of phenol: chloroform: isoamyl alcohol (25:24:1) and the contents of the tubes were mixed with inversion and centrifuged (10,000× *g*, 5 min). The resulting gDNA was isolated and purified following conventional standard methods [44]. Purity and integrity of the gDNA was confirmed using a combination of spectrophotometry (NanoDrop) and agarose gel electrophoresis [44].

PCR amplification of the purified gDNA was done according to the protocol of Nubel et al. [29]. The forward primer is CYA106F (5 -CGGACGGGTGAGTAACGCGT GA-3 ), whereas the reverse primer is an equimolar mixture of CYA781Ra (5 -GACTACTG GGGTATCTAATCCCATT-3 ) and CYA781Rb (5 -GACTACAGGGGTATCTAATCCCTTT-3 ) [29]. Preparatory columns (Invitrogen PCR Preps) were used to clean the PCR products. PCR products were sequenced by Genius labs, UK, and the sequences were annotated using Bioedit 7. Alignment of the sequences and phylogeny of the aligned sequences utilized the MEGA X program [45]. For obtaining gDNA of the cyanobacterium, homogenized *L. majuscula* pellets were exhaustively extracted for bacteria gDNA. The resulting bacterial gDNA of mixed species was degenerated by CuSO4·5H2O and therefore it did not generate a 16S rDNA sequence.

#### *3.4. Isolation of Bacteria from M. producens' Filaments*

Preparation of *M. producens* to detach filaments and to determine its (*M. producens*) gDNA using CuSO4·5H2O is described in Section 3.3. In another experiment, filaments from cyanobacteria biomass that was treated with CuSO4·H2O were plated onto marine agar to determine the assemblage of bacteria associated with the filaments on a cyanobacterial sheath. These experiments were repeated for a near-dead *M. producens* mat. Bacteria isolated from the live and dead filaments were designated as LFB and DFB, respectively, prior. These bacterial isolates were identified using 16S rDNA partial gene sequence. Additional filaments from the live and near-dead *M. producens* were thoroughly washed and stained with acridine orange and nigrosin to establish association of the filaments with heterotrophic bacteria.
