Consistently, the rhlG mRNA level assayed by qRT-PCR was 2.6-fold fold higher in PDO100 than in PAO1 at 20 h of growth (Additional file 1: Figure S1). These results were surprising since they indicated that the prrhlG activity was inhibited by the Rhl QS system. To further investigate this point, we first added C4-HSL at a final TGF-beta inhibitor concentration of 10 μM to the PPGAS medium when inoculating P. aeruginosa PDO100(pAB134). This led
to luminescence levels similar to those of PAO1(pAB134) (Figure 2C), confirming that C4-HSL has a negative effect on the prrhlG activity. prrhlGactivity is induced under hyperosmotic stress We previously showed that hyperosmotic stress (0.5 M NaCl in PLM63 or PPGAS medium) abolishes rhamnolipid production and inhibits the transcription Captisol cost of genes involved in rhamnolipid
synthesis (rhlAB, rhlC) and in C4-HSL synthesis (rhlI) [17, 18]. In PPGAS culture, we observed by qRT-PCR performed on the same mRNA extraction as in  that the amount of rhlG mRNA was 3.7-fold higher after 20 h of growth in hyperosmotic condition (0.5 M NaCl in PPGAS medium) (Additional file 1: Figure S1). This observation was confirmed using the prrhlG::luxCDABE fusion: the luminescence indeed increased until 24 h of growth in hyperosmotic condition, while it decreased in the absence of NaCl from 16 h (Figure 3A). The delay in luminescence increase observed in the presence of NaCl probably corresponded to the growth lag due to the hyperosmotic condition (Figure 3A). We previously observed that the presence of the osmoprotectant glycine betaine during hyperosmotic stress in PPGAS medium did not improve growth, but at least partially prevented the down-regulation of rhlAB, rhlC, and
rhlI genes and partially restored rhamnolipid production . Similarly, glycine betaine prevented the increase of prrhlG activity under hyperosmotic stress, the prrhlG activity being even lower in the presence of 0.5 M NaCl and glycine betaine than in regular PPGAS (Figure 3A). Figure 3 Transcriptional activity of rhlG under hyperosmotic stress. Promoter activity was followed by measuring luminescence from strains Sodium butyrate harbouring pAB134, which contain rhlG::luxCDABE transcriptional fusion. Activity was www.selleckchem.com/products/cobimetinib-gdc-0973-rg7420.html measured in P. aeruginosa PAO1 wildtype with or without NaCl (respectively white and black squares) and supplemented with 1 mM GB in presence of NaCl (black circles) (A). Hyperosmotic stress effect on rhlG activity was followed in PA6358 (rpoN mutant, diamonds) compared to wildtype (squares) during the same set of experiments (B). Hyperosmotic stress effect on prrhlG activity was followed in PAOU (algU mutant, triangles) compared to wildtype (squares) during the same set of experiments (C). Activity is expressed in Relative Units of Luminescence per 0.5 second in function of time growth. Gain for luminescence detection was automatically set for each experiment.