FIG. 2.
In vivo validation of the RsaLPAO model. (A) Measurement of 3OC12-HSL produced by P. aeruginosa PAO1 and its rsaL mutant derivative (white bars) and by the same strains carrying either the pBBR1-MCS5 plasmid, as a control, or its derivatives expressing RsaLPAO or the indicated mutant proteins (gray bars). 3OC12-HSL was extracted from the spent-growth medium of each strain and quantitatively measured as described in Materials and Methods, using the P. putida SM17(prsaL220) strain, in which β-galactosidase activity (given in Miller units [M.u.]) is proportional to the levels of 3OC12-HSL. Standard deviations (error bars) are based on the mean values for three parallel cultures. (B) Western hybridization performed with anti-six-His tag antibody (Sigma) on the cellular soluble fractions derived from the same cultures in which 3OC12-HSL production was measured. The protein expressed in each sample is indicated above its lane.