rnpB, encoding the RNA subunit of RNase P (Vioque, 1997), was used as a loading and transfer control. All probes were 32P-labeled with a Ready-to-Go DNA labeling kit (Amersham Biosciences) using [α-32P]dCTP. Images of radioactive filters Olaparib in vivo and gels were obtained and quantified with a Cyclone storage phosphor system and optiquant image analysis software (Packard). AHLs were added to Anabaena sp. PCC7120 cultures to evaluate possible effects on growth and nitrogen metabolism of the cyanobacterial filaments both in solid and liquid media. We selected saturated and substituted representatives of short- (C4, OC4 and OHC4-HSL), middle- (C10, OC10 and OHC10-HSL) and long-chain AHLs (C12, OC12 and OHC12-HSL). A first experiment was carried out in solid media,
as described in Materials and methods. Growth inhibition halos surrounding the wells were observed after 7 days for OC10-HSL and OC12-HSL in cultures subjected to nitrogen step-down (transferred to nitrogen-free BG110 medium) (Fig. 1). OC10-HSL also inhibited growth in the presence of combined nitrogen (BG110+NH4+, data not shown). These observations suggested that at least these two AHLs could have an effect on heterocyst differentiation or maturation, which was further investigated. AHLs were also added to liquid cultures under nondiazotrophic conditions (BG110C+NH4+) selleck chemicals and to cultures subjected to nitrogen step-down to study the effect on growth and heterocyst differentiation. None of the tested AHLs showed
cytotoxic effects in liquid cultures subjected to step-down after 20 h of exposure. Moreover, no effect on heterocyst differentiation and distribution pattern was found in step-down cultures for any of the tested AHLs after Alcian blue staining and microscope observation (data not shown). The discrepancy between the inhibitory effects obtained for OC10 and OC12-HSL IMP dehydrogenase in solid plates (Fig. 1) and in liquid cultures could be derived from the longest period of incubation of solid plates or could also be due to the higher initial cell concentration in the liquid cultures compared with plates resulting in a higher AHL-acylase activity (Romero et al., 2008) that would diminish the effect of initial AHL concentration. Possible effects of AHLs on heterocyst differentiation were also tested with Anabaena sp. PCC7120 strain CSEL4a (Olmedo-Verd et al., 2006). This strain expresses gfp gene under the control of ntcA promoter, the master regulator of nitrogen assimilation, which also controls the early phases of heterocyst differentiation (Herrero et al., 2004). Expression of gfp in this strain is induced in specific cells upon nitrogen step-down, indicating the induction of ntcA during heterocyst differentiation (Olmedo-Verd et al., 2006).