Turbo rnase

For DNA isolation, bacterial strains were grown overnight in 7 mL of Tryptic Soy Broth (TSB) at 28 °C with shaking. Cells were harvested by centrifugation and resuspended in 500 µl TE buffer (50 mM Tris/HCl, 40 mM EDTA, pH 8.0). Afterwards, the cell lysis and nucleic acids extraction were carried out according to the protocol proposed by a Joint Genome Institute for bacterial DNA isolation using CTAB [64 ] followed by the RNA digestion using Turbo RNase (Ambion). DNA quantity and quality were assessed first using a NanoDrop Spectrophotometer and later with agarose gel electrophoresis.

For DNA extraction, 10 mL of the dense Arthrospira culture cultivated at 20 °C was centrifuged, and the collected biomass was carefully washed 3 times with sterile distilled water prior to nucleic acid extraction. Afterwards, the cell lysis and nucleic acids extraction were carried out according to the protocol proposed by the Joint Genome Institute for bacterial DNA isolation using CTAB [56 ] followed by RNA digestion using Turbo RNase (Ambion). The DNA quantity and quality were assessed first using a NanoDrop Spectrophotometer and later with agarose gel electrophoresis.

Total RNA from Anabaena was isolated as described in reference 69 (link), and trace DNA in the samples was removed by treatment with Turbo RNase (Ambion) following the manufacturer’s instructions. Northern blot assays were performed as described in reference 70 (link), with 4 μg RNA loaded per lane, and electrophoresed in denaturing 1% agarose formaldehyde gels. DNA probes were internal gene fragments generated by PCR using Anabaena genomic DNA and primer pairs all0087-22/all0087-23 (mreB), all0086-13/all0086-14 (mreC), and all0085-8/all0085-9 (mreD). The rpnB gene, which was used for normalization, was amplified from plasmid pT7-7120 (71 (link)) with the primers Universal and Reverse. Probes were labeled by annealing the PCR-generated fragments to oligonucleotides complementary to the coding strand (all0087-8/all0087-23 for mreB, all0086-6/all0086-14 for mreC, and all0085-5/all0085-9 for mreD) and polymerization catalyzed by the Klenow fragment of DNA polymerase (Thermo Fisher) in the presence of [α-³²P]dCTP (Perking-Elmer). Radioactive areas in Northern blot hybridization membranes were visualized and quantified with a Cyclone storage phosphor system (Packard).