, 2009). Cells from a liquid exponentially growing culture of Anabaena sp. PCC7120 in BG110C+NH4+ were harvested by filtration, washed and resuspended TSA HDAC in BG110C at a concentration of 5 μg chlorophyll a (Chl a) mL−1 and 100 μL of the suspension was spread on top of BG110+NH4+ or BG110 plates. Small holes were made in the centre of each plate and filled with 100 μL of 100 μM AHL or acetonitrile (as control). Growth was checked after

7 days of incubation at 30 °C with light. Synthetic AHLs were also added to liquid cultures of Anabaena sp. PCC7120 both under nondiazotrophic conditions (BG110C+NH4+ medium) and during nitrogen step-down. Anabaena sp. PCC7120 was grown to exponential phase in BG110C+NH4+ [cultures with about 5 μg Chl a mL−1; Chl a levels were determined in methanolic

extracts (Mackinney, 1941)]. The cells were filtered, washed with BG110C, inoculated in fresh BG110C+NH4++AHL (100 μM) or BG110C+AHL (100 μM) and bubbled with air or phosphatase inhibitor library CO2-enriched air with a final Chl a concentration of 4 μg mL−1. The AHLs used were: N-butyryl-homoserine lactone (C4-HSL), N-(3-oxobutyryl)-l-homoserine (OC4-HSL), N-(3-hydroxybutyryl)-l-homoserine (OHC4-HSL), N-decanoyl-l-homoserine (C10-HSL) N-(3-oxodecanoyl)-l-homoserine lactone (OC10-HSL), N-(3-hydroxydecanoyl)-l-homoserine (OHC10-HSL), N-dodecanoyl-l-homoserine (C12-HSL) OC12-HSL and N-(3-hydroxydodecanoyl)-l-homoserine (OHC12-HSL) (unsubstituted AHLs were purchased from Sigma-Aldrich, all other AHLs were kindly provided by Prof. Miguel Cámara from the University of Nottingham). AHL stock solutions of 1 mg mL−1 were prepared in acetonitrile. Parallel control assays were carried out using equal amounts of acetonitrile (AHL solvent). In nitrogen step-down cultures, the differentiation of heterocysts was monitored by Alcian blue staining of polysaccharides in the heterocyst envelope LY294002 (Olmedo-Verd et al., 2006). To further evaluate the lethal effect observed for OC10-HSL in ammonium-grown nondiazotrophic cultures

of Anabaena sp. PCC7120 (BG110C+NH4+), different concentrations of this signal (0.01, 0.1, 1, 10, 25, 50, 75 and 100 μM) as well as OC12-tetramic acid (100 μM) were also assayed. The effect of OC10-HSL (100 μM) was also tested in cultures with nitrate as combined nitrogen source (BG11C). OD600 nm of the cultures was measured at different time points after treatment (Kuznetsova et al., 2008). Biomass (200 mL, 2–3 μg mL−1 Chl a) from BG110C+NH4+ aerated cultures of Anabaena sp. PCC7120 was harvested, washed and resuspended in fresh BG110C at a Chl a concentration of 2 μg mL−1 to induce the differentiation of heterocysts. Cultures of 20 mL were established in flasks supplemented with AHLs (100 μM) or acetonitrile as control. After 20 h of incubation at 30 °C, 120 r.p.m.