For each AHL, one flask was incubated under standard aerobic conditions. Another flask was incubated with an anaerobic atmosphere by injecting argon for 3 min and adding 10 μM 3-(3,4dichlorophenyl)-1,1-dimethylurea
(DCMU) to inhibit photosynthesis and therefore oxygen (O2) production (Rippka & Stanier, 1978) to avoid a possible inhibition of nitrogenase activity derived from the formation of abnormal heterocyst cell walls during maturation or the damage from other mechanisms responsible for maintaining low O2 concentration within the heterocysts. After 1-h incubation at 30 °C, 2 mL of acetylene was injected. Samples of 1 mL from the air in the sealed flask were taken at different times during 20 h starting 15 min after acetylene injection to determine the concentration of the ethylene produced PLX4032 chemical structure using a GC-MS (HP 5890 series II) equipped with injector, column (Porapak Q) and flame
ionization detector (kept at 100, 80 and 150 °C, respectively). The detected signals were processed with the computing integrator PYE Unicam DP88. The equipment was calibrated with known concentrations of ethylene. To determine the nitrogenase activity of the cultures per unit Chl a, the following formula was used: nitrogenase activity=nmol ethylene in sample × 14 mL/2 ×μg Chl check details a mL−1; where 14 was the atmosphere volume in 17-mL flasks and 2 the volume of culture in the flask. C10-HSL was also added to BG110C cultures of Anabaena sp. PCC7120 with mature heterocysts (24 h after nitrogen step-down) and the nitrogenase
activity then measured as described before. To assess a possible effect of AHLs on the expression of genes involved in nitrogen fixation, Northern hybridization was carried out with probes for the nifH and fdxH genes. Samples of 50 mL were taken at 0, 3, 6, 20 and 24 h after nitrogen step-down. Cells were filtered, washed and resuspended in 1 mL of Tris 50 mM/EDTA 100 mM, centrifuged and the pellet was frozen in liquid nitrogen before RNA extraction. RNA from whole filaments was extracted in Selleckchem Paclitaxel the presence of ribonucleoside–vanadyl complex as described previously (Muro-Pastor et al., 2002). For Northern analysis, 30 μg of RNA was loaded per lane and electrophoresed in 1% agarose denaturing formaldehyde gels. Transfer and fixation to Hybond-N+ membranes (Amersham Biosciences) were carried out using 0.1 M NaOH. Hybridization was performed at 65 °C according to the recommendations of the manufacturer of the membranes. The nifH and fdxH probes were fragments of these genes amplified by PCR. The nifH probe was amplified using plasmid pCSAV60 (containing the nifH gene cloned in pGEM-T vector) as a template and oligonucleotides NH-1 (corresponding to positions −334 to −314 with respect to the translation start of nifH) and NH-4 (complementary to nucleotides +884 to +863 with respect to the translation start of nifH) (Valladares et al., 2007).