Unbalancing FurB (Zur)-mediated homeostasis in Anabaena sp. PCC7120: Consequences on heterocyst development and biofilm formation
Unbalancing FurB (Zur)-mediated homeostasis in Anabaena sp. PCC7120: Consequences on heterocyst development and biofilm formation
Total RNA was prepared from Anabaena PCC7120 and Δzur cultures. The Δzur strain is a deletion-insertion mutant with a C.S3 cassette interrupting furB gene (Napolitano et al., 2012). Three independent cultures of each strain were set up by diluting a pre-inoculum from late exponential phase until an OD750 of 0.18 in final volume of 80 ml of BG11 in 250 ml Erlenmeyer flasks. Cultures were grown at 28ºC under constant illumination of 30 μE m-2 s-1 in an orbital shaker at 130 r.p.m., unless otherwise stated. Δzur cultures were supplemented with streptomycin and spectinomycin 2 μg.ml−1 . When cultures reached mid-exponential phase of growth (OD750 0.7), 25 ml of sample were collected and processed for RNA extraction following the method previously described (Sarasa-Buisan et al., 2022). Briefly, cyanobacterial cells were rapidly harvested by centrifugation at 4000 x g for 5 min at 4ºC and transferred to a 2 ml-eppendorf and collected by centrifugation at 12000 × g for 5 minutes at 4°C. Supernatant was carefully removed and cell pellet was resuspended in 600 µl of 50 mM Tris-HCl pH 8, 100 mM EDTA and 130 µl of chloroform and mixed thoroughly by inversion. The suspension was centrifuged at 12000 x g for 5 min and the cell phase was collected and flash frozen in liquid nitrogen for storage at -80ªC. Frozen pellets were suspended in 300 µl resuspension buffer (0.3 M sucrose, 10 mM sodium acetate, pH 4.5) and 100 µl 250 mM Na2-EDTA (pH 8.0), 400 µl lysis buffer (2% SDS, 10 mM sodium acetate, pH 4.5) and 1 ml of acid phenol at 65 °C. Samples were vortexed during 30 seconds and incubated at 65 °C for 2.5 min (this step was repeated three times). The suspension was centrifuged at 12000 x g for 5 min and the aqueous (upper) phase was collected and sequentially extracted with Trizol, Trizol-Chloroform and Chloroform. Finally, 2 volumes of precooled absolute ethanol were added and RNA was precipitated O/N at -80. For the DNAseI treatment, the pellet was washed with precooled 70% ethanol, dissolved in H2O and treated with RNase-free DNaseI (Roche). The absence of DNA in the RNA samples was checked by Real-Time PCR, using oligonucleotides for the housekeeping gene rnpB (Vioque et al., 1992). RNA was quantified spectrophotometrically using a SPECORD® PLUS Analytik Jena spectrophotometer RNA seq was performed by STAB VIDA Lda (Lisbon, Portugal) from total RNAs extracted and processed from two RNA biological replicates of Anabaena sp. PCC7120 and ΔfurB cultures.The library construction of cDNA molecules was carried out using a Ribosomal Depletion Library Preparation Kit;NEBNext rRNA Depletion Kit followed by Roche KAPAHyperprep; following the manufacturer instructions.